CN1118571C - Protein with thrombocytopoietic factor active - Google Patents

Abstract

The present invention relates to a thrombopoietin (TPO) polypeptide or a derivative thereof having the biological activity of specifically stimulating or increasing platelet production and containing 1-332 of the amino acid sequence of SEQ ID NO: 6, involving a DNA molecule encoding a TPO polypeptide, generating the Methods for said polypeptides, antibodies specifically immunoreactive with said polypeptides, and methods for treating platelet disorders such as thrombocytopenia with said polypeptides.

Description

protein with thrombopoietin activity

This application is a continuation-in-part of the now pending U.S. Patent Application No. unknown filed on January 31, 1995, the latter application being the now pending U.S. Patent Application No. 08 filed on December 22, 1994 /361,811, which is a continuation-in-part of the now-pending U.S. Patent Application No. 08/320,300, filed October 11, 1994, which is a now-pending July 20, 1994 A continuation-in-part of U.S. Patent Application No. 08/278,083 filed April 1, 1994, which is a continuation-in-part of now-abandoned U.S. Patent Application No. 08/221,020, filed April 1, 1994, a now-discontinued A continuation-in-part of abandoned US Patent Application No. 08/212,164, filed March 14, 1994. field of invention

The present invention relates to a novel protein having the activity of stimulating or increasing platelet production or enhancing the proliferation and differentiation of megakaryocyte progenitor cells in a specific manner in vivo, a DNA sequence encoding said protein and a method for its preparation. Background of the invention

Megakaryocytes are large, cytoplasmic, multinucleated cells that produce platelets and are found primarily in the bone marrow. Megakaryocytes originate from multipotent hematopoietic stem cells in the bone marrow. Primitive pluripotent stem cells are programmed to differentiate into megakaryocyte progenitor cells, which commit to the megakaryocyte lineage. Megakaryocyte progenitors proliferate and divide into megakaryocytes. Megakaryocytes undergo further polyploidy and cytoplasmic maturation, and finally release their anucleated cytoplasmic fragments (ie, platelets) into the circulation. A mature megakaryocyte forms an average of 2,000-4,000 platelets. Although the mechanism of platelet formation is unknown in some detail, it is thought that megakaryocytes, typically located on the albumin (albuminal) surface of the sinusoidal endothelium of the bone marrow, produce cytoplasmic processing that extends into the sinusoidal space, whereby Megakaryocytes fragment platelets.

There are still many doubts about the mechanism of platelet production, although it is considered possible that megakaryocytes exist in the cortex of the bone marrow venous sinus, and the cytoplasm passes through the cortex to produce cord-like protrusions on the inner wall of the vein, whereby platelets are released.

There is thought to be a specific function in the generation and regulation of megakaryocyte hematopoietic platelet production. In healthy humans and animals, although it is known that after administration of anti-platelet antibodies to, for example, healthy animals, the number of platelets decreases rapidly, then initially spikes higher than usual, but eventually returns to normal levels, so that the number of effective platelets is maintained at a certain level . Also, it is known that a decrease in the number of platelets (thrombocytopenia) or an increase in the number of platelets (thrombocytosis) is seen clinically even when the number of erythrocytes and blood cells is at a normal level.

Incidentally, the most important function of platelets is the formation of thrombus in the mechanism of hemostasis. If the hemostatic mechanism is not functioning properly due to decreased platelet count, a bleeding tendency will result.

Specific regulatory mechanisms have been demonstrated for megakaryopoiesis and thrombopoiesis. Platelets are maintained in effective numbers in healthy humans and normal animals. However, it is known that when anti-platelet antibodies are administered to animals, platelet counts rapidly decrease only for a short period of time, then begin to rise and temporarily exceed normal levels, and eventually return to normal levels. It is also known in the clinical field that a decrease in the number of platelets (thrombocytopenia) or an increase in the number of platelets (thrombocytosis) can be seen even when the number of red and white blood cells is normal. However, so far there have been no reports of successful isolation and identification of specific regulators involved in platelet production, such as, for example, erythropoietin during erythrocyte formation.

The most important function of platelets is the formation of blood clots in the hemostatic mechanism. Bleeding tendencies occur when the normal function of the hemostatic mechanism is disrupted by thrombocytopenia. In the field of radiotherapy and chemotherapy for cancer, platelet count reduction due to myelosuppression is a fatal complication; such patients are given platelet transfusions to prevent bleeding tendencies. Platelet transfusions are also used in patients after bone marrow transplantation or in patients with aplastic anemia.

Platelets for this type of platelet transfusion can be prepared from the blood of healthy donors by platelet apheresis, but the half-life of these transfused platelets is short and there is a possibility of bacterial contamination. Platelet transfusion may also expose patients to harmful viruses (such as human immunodeficiency virus HIV or various hepatitis viruses), induce major histocompatibility antigens ( HLA) have specific antibodies or cause the risk of graft-versus-host disease (GVHD).

Therefore, it would be highly beneficial if intrinsic platelet formation could be stimulated in thrombocytopenic patients while simultaneously reducing the dependence on platelet transfusions. Additionally, being able to correct or prevent thrombocytopenia in cancer patients undergoing radiation or chemotherapy would make such treatments safer, potentially increasing the intensity of treatment and further improving the desired anticancer effects.

For all of the above reasons, a great deal of research has been devoted to the isolation and identification of specific regulators involved in the regulation of megakaryocyte and platelet production. According to the results of in vitro studies, the regulatory factors controlling megakaryocyte production are roughly classified into the following two factors (see Williams et al., J. Cell. Physiol., vol. 110, p 101-104, 1982). Megakaryocyte colony-stimulating factor (Meg-CSF) is a regulator that stimulates the proliferation and differentiation of CFU-MK in semi-solid media to form megakaryocyte colonies. Another regulatory factor called megakaryocyte potentiating factor (Meg-Pot), megakaryocyte stimulating factor, platelet production stimulating factor, etc. mainly acts on immature or mature megakaryocytes, thereby enhancing their differentiation and maturation. In some cases, Meg-Pot can be detected together with Meg-CSF. In addition, because platelet counts are increased when serum or plasma collected from animals with experimentally induced thrombocytopenia is administered to otherwise normal animals, it suggests the presence of a promoting factor capable of promoting platelet production in vivo, called thrombopoietin (TPO) .

In recent years, some cytokines whose genes have been cloned have been examined for their ability to stimulate megakaryocytopoiesis and thrombopoiesis. Human IL-3 stimulates the formation of human megakaryocyte colonies (Bruno et al., Exp.Hamatol., vol.16, p371-377, 1988) and at least elevates platelet counts in monkeys (Donahue et al., Science, vol. 241, p1820, 1988). However, because IL-3 acts on the proliferation and differentiation of all hematopoietic cells, it is distinct from the specific regulators that control megakaryocytopoiesis and thrombopoiesis. Human IL-6 does not exhibit Meg-CSF activity, but it acts on immature megakaryocytes and promotes their differentiation into mature megakaryocytes (Williams et al., Exp.Hamatol., vol.18, p.69, 1990). In vivo administration of IL-6 can induce platelet production, promote the maturation of primate bone marrow megakaryocytes and promote to a higher ploidy, but also produce some side effects, such as weight loss, induction of acute phase proteins (Asano et al., Blood, vol.75 , p1602-1605, 1990; Stahl et al., Blood, vol.78, p1467-1475, 1991). Human IL-11 has no Meg-CSF activity, but has Meg-Pot activity, and promotes platelet production in mice (Neben et al., Blood, vol.81, p901-908, 1993). In addition, human LIF significantly increased the number of primate platelets (Mayer et al., Blood, vol.81, p3226-3233, 1993), but its effect on megakaryocytes in vitro was weak (Burstein et al., J.Cell Physiol., vol.153, p305-312, 1992).

Although these cytokines are expected to be used clinically as platelet potentiating factors, their functions are not specific to the megakaryocyte lineage, and they can cause side effects. Therefore, it is clinically necessary to develop a platelet increasing factor that is specific to the megakaryocyte-platelet system and causes less side effects.

Meg-CSF, Meg-Pot or TPO activity has been found in the serum, plasma or urine of thrombocytopenic patients or animals or in the culture supernatant of certain cultured human cell lines. However, it remains unclear whether these activities are due to the presence of a single factor or a combination of several factors, or whether these factors differ from known cytokines.

Hoffman et al found that serum from patients with aplastic anemia and amegakaryocytic thrombocytopenic purpura contained Meg-CSF activity, which significantly increased megakaryocyte colony formation (Hoffman et al., N.Eng.J.Med. , vol. 305, p533-538, 1981). Thereafter, Mazur et al. also reported that the activity of Meg-CSF present in serum of patients with aplastic anemia was different from that of IL-3 and GM-CSF (Mazur et al., Blood, vol. 76, p290-297, 1990). Similar Meg-CSF activity has been found in the sera of cancer patients receiving extensive cytotoxic chemotherapy and bone marrow transplantation patients (Mazur et al., Exp. Hematol., vol. 12, p624-628, 1984; de Alarcon and Schinieder, Prog. Clin. Bio. Res., vol. 215, p335-340, 1986). According to Hoffman et al., Meg-CSF has been purified from the serum of hypomegakaryocytic thrombocytopenia patients with an apparent molecular weight of 46,000 (Hoffman et al., J. Clin Invest., vol.75, p1174-1182, 1985), but further studies found that the substance was not present at a level of purity that allowed accurate amino acid sequencing (Hoffman Blood, vol.74, p1196-1212, 1989). Substances with TPO-like activity, which were partially purified from the plasma of patients with thrombocytopenia or the urine of patients with idiopathic thrombocytopenic purpura (ITP), enhanced the incorporation of ⁷⁵ Se-selenomethionine into newly formed platelets in mice, The apparent molecular weight of the plasma-derived factor was determined to be 40,000 (Grossi et al., Hematologica, vol. 72, p291-295, 1987; Vannucchi et al., Leukemia, vol. 2, p236-240, 1988).

Meg-CSF activity and TPO-like activity have also been detected in urine samples from patients with aplastic anemia and severe ITP (Kawakita et al., Br.J.Haemtol., vol.48, p609-615, 1981; Kawakita et al. al., Blood, vol. 556-560, 1983). Kawakita et al. further reported that the activity of Meg-CSF seen in urine extracts of aplastic anemia patients was shown to have an apparent molecular weight of 45,000 by gel filtration under dissociated conditions (Kawakita et al., Br.J.Haematol. , vol 62, p715-722, 1986). Purification of Meg-CSF from a similar urine sample was also reported by Erikson-Miller et al., but provided no information on its structure (Erikson-Miller et al., "Blook Cell Growth Factors: their Present and future use inhematology and oncology" ed. by Murphy, AlphaMed Press, Dayton, Ohio, p204-220, 1992). Tumer et al. have purified megakaryocyte-stimulating factor (MSF) with Meg-CSF activity from the urine of bone marrow transplantation patients and cloned its gene (Turner et al., Blood, vol.78, p1106-279a, 1991, ( abstr., supplement. 1)). The MSF has a molecular weight of 28,000-35,000. The identity of this factor with Meg-CSF, which has been detected so far in serum and plasma samples of thrombocytopenic patients, and its thrombocytopenic activity remain to be elucidated.

People also purified a substance with TPO-like activity and a molecular weight of 32,000 from the culture supernatant of a human embryonic kidney-derived cell line (HEK cells), and extensively tested its biological and biochemical properties, but Its structure is still unclear (McDonald et al., J.Lab.Clin.Med., vol.106, p162-174, 1985; McDonald, Int.J. Cell Cloning, vol.7, p139-155, 1989). In contrast, other investigators reported that the major activity of the ex vivo enhanced megakaryocyte maturation process produced in the conditioned medium of HEK cells was due to known cytokines, namely IL-6 and EPO (Withy et al. ., J. Cell. Physiol., vol. 15, p362-372, 1992).

Evatt et al. reported factors of animal origin. They found TPO-like activity in rabbit plasma that could promote the incorporation of ⁷⁵ Se-selenomethionine into newly formed platelets in rabbits with thrombocytopenia induced by antiplatelet serum injection ( Evatt et al., J. Lab. Clin. Med., vol. 83, p364-371, 1974). Besides, a large number of similar research results were reported from the 1960s to the 1970s (eg, Odell et al., Proc. Soc. Biol. Med., vol. 108, P428-431, 1961; Evatt and Levin, J .Clin.Invest., vol.48, p1615-1626, 1969; Harker, Am.J.Physiol., vol.218, p1376-1380, 1970; Shreiner and Levin, J.Clin.Invest., vol.49, p1709-1713, 1970; Penington, Br. Med. J. vol.1, p606-608, 1970). Evatt et al. and Hill and Levin have partially purified TPO-like activity from thrombocytopenic rabbit plasma (Evatt et al., Blood, vol.54, p377-388, 1979; Hill and Levin, Exp.Hematol., vol.14 , p752-759, 1986). After this, the purification work of this factor was continued, and the monitoring of its activity in promoting differentiation and maturation of megakaryocytes in vitro (i.e., Meg-Pot activity) showed that the apparent molecular weight of the activity was 40,000-46,000 (Keller et al., Exp. Hematol., vol.16, p262-267, 1988; Hill et al., Exp. Hematol., vol.20, p354-360, 1992). Since IL-6 activity was undetectable in the plasma of rabbits with severe acute thrombocytopenia induced by administration of antiplatelet serum, it was suggested that this TPO-like activity arises from a factor other than II-6 (Hill et al., Blood , vol. 80, p346-351, 1992).

Tayrien and Rosenberg have also purified a factor with an apparent molecular weight of 15,000 from thrombocytopenic rabbit plasma and culture supernatants of HEK cells that stimulates the production of platelet factor 4 in a rat megakaryocyte cell line, but they have not Provides information on its structure (Tayrien and Rosenberg. J. Biol. Chem., vol. 262, p3262-3268, 1987).

In addition, Nakeff found Meg-CSF activity in the serum of mice with thrombocytopenia induced by administration of antiplatelet serum (Nakeff, "Experimental Hematology Today" ed. by Baum and Ledney, Springer-Verlag, NY, p111-123, 1977) . On the other hand, thrombocytopenic rabbit serum can promote megakaryocyte maturation (keller et al., Exp.Hematol., vol.16, p262-267, 1988; Hill et al., Exp.Hematol., vol.17, p903 -907, 1989), and stimulated the morphological change of megakaryocytes into platelets (Leven and Yee, Blood, vol.69, p1046-1052, 1989), but there was no measurable Meg-CSF activity.

Miura et al. detected Meg-CSF activity in platelet-reduced rabbit plasma provided by sublethal whole-body irradiation (Miura et al., Blood, vol.63, p1060-1066, 1984), which indicates that Meg-CSF is induced in vivo - CSF activity is associated with megakaryocyte reduction, but not thrombocytopenia, since this activity is not altered after transfusion of platelets (Miura et al., Exp. Hematol., vol. 16, p139-144, 1988). Mazur and South have detected Meg-CSF activity in sublethally irradiated dog serum, and reported that the factor has an apparent molecular weight of 175,000 by gel filtration (Mazur and South, Exp.Hematol., vol.13, p1164-1172, 1985). In addition, other researchers (such as Straneva et al., Exp. Hematol., vol.15, p657-663, 1987) have also reported factors derived from serum, plasma and urine.

Thus, as mentioned above, conditional activities that stimulate megakaryopoiesis and thrombopoiesis have been found in biological samples taken from thrombocytopenic patients and animals, but because they are present in very small amounts in natural sources such as blood and urine, they are not The isolation, biochemical and biological identification and characterization of these factors have not yet been completed. Summary of the invention

The object of the present invention is to isolate and identify a TPO protein from a natural source, which has the activity of stimulating or increasing platelet production and/or promoting the proliferation and differentiation of megakaryocyte progenitor cells in vivo (hereinafter referred to as "TPO activity"); The object of the invention is also to isolate the gene encoding the TPO protein and to provide a method for homogeneous and mass production of said protein by recombinant DNA technology. Successfully achieving the above objectives will replace current platelet transfusions or reduce the frequency of platelet transfusions; said new protein will also be used in the treatment and diagnosis of platelet disorders.

Therefore, the present invention relates to:

(i) the DNA sequence that the coding of purification and separation has TPO activity protein, it is selected from following sequence:

(a) the DNA sequence shown in SEQ ID NO 194, 195 and 196 or its complementary strand; and

(b) DNA sequences or fragments thereof which hybridize under stringent conditions to the DNA sequences defined in (a); and

(c) DNA sequences that would hybridize to the DNA sequences defined in (a) and (b) if there were no degeneracy of the genetic code.

(ii) produce the method for protein with TPO activity, it comprises the following steps:

culturing prokaryotic or eukaryotic host cells transformed or transfected with said DNA sequence in a manner capable of expressing said protein, under suitable nutritional conditions;

isolating the protein product of interest obtained by expressing said DNA sequence,

(iii) A protein product obtained by expressing said DNA sequence in a prokaryotic or eukaryotic host cell.

The present invention further relates to pharmaceutical compositions containing an effective amount of said protein having TPO activity and a method of treating platelet disorders, especially thrombocytopenia, comprising administering said protein to a patient suffering from said disorder. Brief description of the drawings

Figure 1 shows Sephacryl S-200HR gel filtration chromatography of Phenyl Sepharose 6 FF/LS F2 derived from XRP.

Fig. 2 shows the Capcell PakC1 reverse phase chromatography of YMC-pack CN-AP TPO active fraction, said fraction is derived from the low molecular weight TPO sample (Sephacryl S-200HRF3) of XRP.

Figure 3 shows the SDS-PAGE analysis of Capcell PakC1 TPO active fraction (FA) derived from XRP low molecular weight TPO samples.

Figure 4 shows the peptide map of rat TPO separated by SDS-PAGE on C18 reverse phase HPLC. The peptide fragments shown were obtained by complete hydrolysis with three proteases.

Fig. 5 shows the activity of TPO derived from XRP in the rat CFU-MK assay system.

Fig. 6 shows the construction of expression vector pEF18S.

Fig. 7 shows TPO activity in the culture supernatant of COS1 cells into which pEF18S-A2X was introduced in the rat CFU-MK assay system.

Fig. 8 shows TPO activity in the culture supernatant of COS1 cells into which pEF18S-HL34 was introduced in the rat CFU-MK assay system.

Fig. 9 shows TPO activity in the culture supernatant of COS1 cells into which pHT1-231 was introduced in the rat CFU-MK assay system.

Figure 10a shows TPO activity in the culture supernatant of COS1 cells into which pHTF1 was introduced in the rat CFU-MK assay system.

Figure 10b shows TPO activity in the culture supernatant of COS1 cells into which pHTF1 was introduced in the M-07e assay system.

Figure 11 shows the restriction map of the λHGT1 clone and the construction of pHGT1 and pEFHGTE.

(E: EcoRI, H: Hind III, S: SalI)

Figure 12a shows TPO activity in the culture supernatant of COS1 cells into which pEFHGTE was introduced in the rat CFU-MK assay system.

Figure 12b shows TPO activity in the culture supernatant of COS1 cells into which pEFHGTE was introduced in the M-07e assay system.

Figure 13a shows TPO activity in the culture supernatant of COS1 cells into which pHT1-211#1, pHT1-191#1 or pHT1-171#2 were introduced in the rat CFU-MK assay system.

Figure 13b shows TPO activity in the culture supernatant of COS1 cells in which pHT1-163#2 was primed in the rat CFU-MK assay system.

Figure 14 shows the TPO activity in the culture supernatant of COS1 cells in the M-07e assay system, wherein pHT1-211#1, pHT1-191#1, pHT1-171#2 or pHT1-163#2 were introduced in COS1 cells.

Figure 15 shows the chromatogram of reverse phase chromatography (Vydac C4 column) used to purify human TPO from the culture supernatant of CHO cells into which pDEF202-hTPO-P1 was introduced so that TPO can be expressed .

Fig. 16 is a photograph showing separation by SDS-PAGE of human TPO purified from the culture supernatant of CHO cells into which pDEF202-hTPO-P1 has been introduced so that TPO can be expressed.

Figure 17 is a chromatogram of reverse phase chromatography used to purify human TPO from E. coli that has been introduced into pCFM536/h6T(1-163) to enable expression of TPO.

Figure 18 is a picture of human TPO variant h6T(1-163) isolated and purified from E.coli by SDS-PAGE, where E.coli has been introduced into pCFM536/h6T(1-163) to enable Express TPO.

Figure 19 shows the elution profile of hTPO163 purified on a Superdex 75pg column from culture supernatants prepared as a starting material by transfection of the human TPO expression plasmid pDEF202-hTPO163 into CHO cells. Protein content was detected at 220nm.

Figure 20 shows the SDS-PAGE analysis of standard hTPO163 eluted from a Superdex 75pg column after purification of hTPO163 from the culture supernatant prepared as a starting material by transfecting the human TPO expression plasmid pDEF202-hTPO163 into CHO cells , hTPO163 was stained with silver on the gel.

Fig. 21 shows the structure of expression vector pSMT201.

Fig. 22 shows TPO activity detected by M-07e assay in the culture supernatant of COS7 cells into which ?GL-TPO, N3/TPO or 09/TPO had been introduced and then expressed.

Figure 23 shows TPO activity detected by the M-07e assay in culture supernatants of COS7 cells into which an insertion or deletion derivative of human TPO has been introduced and expressed.

Figure 24 shows the increase in platelet counts in mice after intravenous and subcutaneous injection of TPO.

Figure 25 shows that after subcutaneous injection of TPO, the number of platelets in mice increased in a dose-dependent manner.

Figure 26 shows that TPO induces an increase in platelet counts after treatment of mice with 5-FU to induce thrombocytopenia.

Figure 27 shows that after treatment of mice with pyrimidine nitrosourea hydrochloride induced thrombocytopenia, TPO can induce an increase in platelet count.

Figure 28 shows that TPO can induce an increase in platelet counts in thrombocytopenic mice following bone marrow transplantation.

Figure 29 shows that TPO induces an increase in platelet counts after X-ray irradiation of mice to induce thrombocytopenia.

Figure 30 shows a dose-dependent increase in platelet counts following administration of truncated TPO (amino acids 1-163 in SEQ ID NO: 6).

Figure 31 shows that after administration of pyrimidine nitrosourea hydrochloride to induce thrombocytopenia, administration of truncated TPO (amino acids 1-163 in SEQ ID NO: 6) causes an increase in platelet count.

Figure 32 shows that increasing the concentration of Mpl-X added to a human megakaryocyte culture system increases the blockage of megakaryocyte development.

Figure 33 depicts TPO derivatives [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹³³ ]TPO(1-163), [Met ^-2 , Lys ^-1 , Ala ¹ expressed in E. coli , Val ³ , Pro ¹⁴⁸ ]TPO(1-163) and [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹¹⁵ ]TPO activity of TPO(1-163), the activity was determined by M-07e detection.

Figure 34 depicts TPO derivatives [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹²⁹ ]TPO(1-163), [Met ^-2 , Lys ^-1 , Ala ¹ expressed in E. coli , Val ³ , Arg ¹⁴³ ]TPO(1-163), [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Leu ⁸² ]TPO(1-163), [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Leu ¹⁴⁶ ]TPO(1-163) and [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ⁵⁹ ]TPO(1-163) TPO activity, which was determined by M-07e detection. Detailed description of the invention

Specifically, the present invention provides 1-332 thrombopoietin (TPO) polypeptides or derivatives thereof having the biological activity of specifically stimulating or increasing platelet production and comprising the amino acid sequence of SEQ ID NO:6. Illustrative polypeptides include those polypeptides consisting of the following amino acid sequences: 1-163 of the amino acid sequence of SEQ ID NO: 6; 1-232 of the amino acid sequence of SEQ ID NO: 6; 1-151 of the amino acid sequence of SEQ ID NO: 6; Mature amino acid sequences of SEQ ID NO: 2, 4 and 6; include those polypeptides missing 1-6 N-terminal amino acids. Polypeptides further illustrated by the present invention include [Thr ³³ , Thr ³³³ , Ser ³³⁴ , Ile ³³⁵ , Gly ³³⁶ , Tyr ³³⁷ , Pro ³³⁸ , Tyr ³³⁹ , Asp ³⁴⁰ , Val ³⁴¹ , Pro ³⁴² , Asp ³⁴³ , Tyr ³⁴⁴ , Ala ³⁴⁵ , Gly ³⁴⁶ , Val ³⁴⁷ , His ³⁴⁸ , His ³⁴⁹ , His ³⁵⁰ , His ³⁵¹ , His ³⁵² , His ³⁵³ ] TPO, [Asn ²⁵ , Lys ²³¹ , Thr ³³³ , Ser ³³⁴ , Ile ³³⁵ , Gly ³³⁶ , Tyr ³³⁷ , Pro ³³⁸ , Tyr ³³⁹ , Asp ³⁴⁰ , Val ³⁴¹ , Pro ³⁴² , Asp 343, Tyr ³⁴⁴ , Ala ³⁴⁵ , Gly ³⁴⁶ , Val ³⁴⁷ , His ³⁴⁸ , His ³⁴⁹ , His ³⁵⁰ , His ^{351 , His 352 ,} His ³⁵³ []T Asn ²⁵ ]TPO and [Thr ³³ ]TPO.

Other polypeptides of the present invention also include derivatives of TPO polypeptides

[ΔHis ³³ ]TPO(1-163), [ΔArg ¹¹⁷ ]TPO(1-163), [ΔGly ¹¹⁶ ]TPO(1-163); [His ³³ , Thr ^33' , Pro ³⁴ ]TPO(1-163) , [His ³³ , Ala ^33' , Pro ³⁴ ]TPO(1-163), [His ³³ , Gly ^33' , Pro ³⁴ , Ser ³⁸ ]TPO(1-163), [Gly ¹¹⁶ , Asn ^116' , Arg ¹¹⁷ ]TPO(1-163), [G1y ¹¹⁶ , Ala ^116' , Arg ¹¹⁷ ]TPO(1-163), [Gly ¹¹⁶ , Gly ^116' , Arg ¹¹⁷ ]TPO(1-163), [Ala ¹ , Val ³ , Arg ¹²⁹ ]TPO(1-163), [Ala ¹ , Val ³ , Arg ¹³³ ]TPO(1-163), [Ala ¹ , Val ³ , Arg ¹⁴³ ]TPO(1-163), [Ala ¹ , Val ³ , Leu ⁸² ]TPO(1-163), [Ala ¹ , Val ³ , Leu ¹⁴⁶ ]TPO(1-163), [Ala ¹ , Val ³ , Pro ¹⁴⁸ ]TPO(1-163), [Ala ¹ , Val ³ , Arg ⁵⁹ ]TPO (1-163), and [Ala ¹ , Val ³ , Arg ¹¹⁵ ]TPO (1-163).

TPO polypeptides of the invention also include those covalently linked to a polymer, preferably polyethylene glycol. The TPO polypeptides of the present invention may further include amino acids [Met ^-2 -Lys ^-1 ], [Met ^-1 ] or [Gly ^-1 ]. The DNA provided by the present invention includes DNA encoding the above-mentioned TPO polypeptides and derivatives, and is provided in the form of cDNA, genomic DNA and produced DNA as appropriate.

The present invention also provides a method for producing the above TPO polypeptide, the steps of which include expressing the polypeptide encoded by the DNA of the present invention in a suitable host, and isolating said TPO polypeptide. Where the expressed TPO polypeptide is a Met ^-2 -Lys ^-1 polypeptide, said method can further comprise the step of cleaving Met ^-2 -Lys ^-1 from said isolated TPO polypeptide.

The invention also provides methods of producing TPO polypeptides, such as glutathione-S-transferase (GST) fusion polypeptides. DNA encoding the N-terminal GST polypeptide, thrombin recognition peptide, and TPO polypeptide are introduced into a suitable host, and the fusion polypeptides are isolated and treated with thrombin to remove the GST moiety. The resulting TPO polypeptide has a [Gly ^-1 ] structure.

The present invention additionally provides prokaryotic or eukaryotic host cells transformed or transfected with the DNA sequence of the present invention, which is transfected or transformed to be able to express in said host cells a biological activity that specifically stimulates or increases platelet production in the form of peptides.

The pharmaceutical composition of the present invention includes an effective amount of TPO polypeptide or derivatives and a pharmaceutical carrier, and is easy to treat platelet disorders, especially thrombocytopenia, such as thrombocytopenia induced by chemotherapy, radiotherapy or bone marrow transplantation. The invention provides corresponding treatment methods.

Finally, the present invention provides antibodies that specifically immunoreact with the above-mentioned TPO polypeptides and derivatives thereof. Such antibodies are useful in methods for the analysis and quantification of TPO polypeptides of the invention.

According to the present invention, there is provided a novel DNA sequence encoding a protein having TPO activity (hereinafter referred to as "DNA sequence of the present invention"). The DNA sequence of the present invention includes the DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 in the attached sequence listing.

The DNA sequence of the present invention also includes partially modified (substitution, deletion, insertion or addition) variants encoding the amino acids shown in the aforementioned SEQ ID NO: 2, 4 or 6, provided that such modification does not impair TPO activity. That is, DNA sequences encoding TPO derivatives are also included in the present invention.

In other words, the DNA sequence of the present invention includes a DNA sequence encoding a protein molecule whose amino acid sequence is substantially the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. In addition, the phrase "the amino acid sequence is substantially the amino acid sequence shown in SEQ ID NO: 2, 4 or 6" means that said amino acid sequence includes those represented by SEQ ID NO: 2, 4 or 6, and those represented by Wherein represented by SEQ ID NO: 2, 4 or 6 which is partially modified (such as substitution, deletion, insertion, increase, etc.), the premise of the latter is that said modification does not impair TPO activity.

Furthermore, the DNA sequence of the present invention essentially includes a DNA sequence encoding a protein with TPO activity.

The expression "DNA sequence encoding an amino acid sequence" includes all DNA sequences in which degeneracy may exist in the nucleotide sequence.

The DNA sequence of the present invention also includes the following sequences:

(a) DNA sequences represented by SEQ ID NO: 7, 194, 195 and 196 or their complementary strands,

(b) a DNA sequence or a fragment thereof which hybridizes under stringent conditions to the DNA sequence defined in (a), or

(c) DNA sequences that would hybridize to the DNA sequences defined in (a) and (b) if there were no degeneracy of the genetic code.

In other words, the DNA sequences of the present invention also include the following sequences:

(a) DNA sequence integrated into the following vectors and encoding the amino acid sequence of the protein with TPO activity: the vector pEF18S-A2α carried by E.coli DH5 strain (the deposit number is FERMBP-4565), the vector pHT1 carried by E.coli DH5 strain -231 (the deposit number is FERM BP-4564), the vector pHTF1 carried by the E.coli DH5 strain (the deposit number is FERM BP-4617) and the vector pHGT1 carried by the E.coli DH5 strain (the deposit number is FEMR BP-4616); or

(b) A DNA sequence that hybridizes to the DNA sequence defined in (a) or a fragment thereof under stringent conditions and encodes an amino acid sequence having TPO activity.

"Stringent" hybridization conditions as described herein refer to the conditions applied in the examples illustrating PCR amplification of the DNA of the invention using denatured and/or single sequence oligonucleotide primers (probes) (see also For example Molecular Cloning, Chapter 11 and 12, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989; Current Protocols in Molecular Biology, Unit 2.10, Ausubel et al., Eds., Current Protocols, USA, 1993).

The DNA sequence of the present invention also includes a DNA sequence encoding a protein with TPO activity, including a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 6 at positions 1-163.

Such DNA sequences can also have restriction enzyme digestion sites and/or additional DNA sequences added at the start, stop and middle positions to facilitate the construction of easy expression vectors. When using a non-mammalian host, preferred codons for gene expression may be incorporated into the host.

An example of the DNA sequence of the present invention is a cDNA molecule obtained by preparing mRNA from mammalian (including human) cells, and then screening desired cDNA from a cDNA library prepared by a known method in a conventional way. Sources of mRNA in this case include rat hepatocyte-derived cell line McA-RH8994, HTC cells, H4-II-E cells, rat liver, kidney, brain and small intestine, human liver, and the like.

Another example of a DNA sequence of the present invention is a genomic DNA molecule obtained by conventional screening from genomic libraries prepared by known methods from mammalian (including human) cells. Sources of genomic DNA in this case include preparations of chromosomal DNA from humans, rats, mice, and the like.

The DNA sequence encoding TPO derivatives can be prepared by using known site-directed mutagenesis to modify the above cDNA sequence, thereby partially modifying the corresponding amino acid sequence, thereby preparing from the above cDNA sequence encoding a protein with TPO activity.

The amino acid sequence or DNA sequence of the protein with TPO activity of the present invention has been deduced, so the DNA sequence encoding the partially modified amino acid sequence can be easily prepared by chemical synthesis.

The DNA sequences of the present invention are useful substances for the large-scale production of proteins having TPO activity using various recombinant DNA techniques.

Likewise, the DNA sequences of the present invention are also useful as labeled probes for isolating genes encoding TPO-related proteins, as well as cDNA and genomic DNA encoding TPO in other mammalian species. It can also be used for gene therapy in humans and other mammals. In addition, the DNA sequence of the present invention is also used to develop transgenic mammals that can be used as eukaryotic hosts for large-scale production of TPO (Palmiter et al., Science, vol. 222, p809-814, 1983).

The present invention also provides a vector integrated with the aforementioned DNA sequence encoding a protein with TPO activity, a host cell transformed with said vector and a method for producing a protein with TPO activity, said method comprising culturing said host cell, isolating and purifying Expressed protein with TPO activity. Examples of host cells used in the above case include prokaryotic cells such as E. coli and the like, eukaryotic cells such as yeast, insects, mammalian cells and the like. Illustrative examples of mammalian cells include COS cells, Chinese Hamster Ovary (CHO) cells, C-127 cells, Baby Hamster Kidney (BHK) cells, and the like. Illustrative examples of yeast include baker's yeast (Saccharomyces cerevisiae), methanol-assimilating yeast (Pichia pastoris). Illustrative examples of insect cells include cultured cells of silkworms and the like.

Regarding vectors for transforming the above-mentioned host cells, pKC30 (Shimatake H. and M. Rosenberg, Nature, 292, 128-132, 1981), pTrc99A (Amann E. et al., Gene, 69, 301-315, 1988) etc. can be used to transform E.coli cells. pSV2-neo (Southern and Berg, J. Mol. Appl. Genet., 1, 327-341, 1982), pCAGGS (Niwa et al., Gene, 108, 193-200, 1991), pcDL-SRα296 (Takebe.et al., Mol. Cell. Biol., 8, 466-472, 1988) etc. can be used to transform mammalian cells. pG-1 (Schena M. and Yamamoto K.R., Science, 241, 965-967, 1988) etc. can be used to transform yeast cells. The transfer vector used to construct the recombinant virus, such as pAc373 (Luckow et al., Bio/Technology, 6, 47-55, 1988), can transform silkworm cells.

Each of the above-mentioned vectors may contain an origin of replication, a selection marker, a promoter, etc., and in a vector for eukaryotic cells, an RNA splicing site, a polyadenylation signal, etc., as necessary.

Regarding the origin of replication, sequences from eg SV40, adenovirus, bovine papilloma virus, etc. can be used in vectors for transformation of mammalian cells. Sequences from ColEI, R factors, F factors, etc. can be used in vectors for transformation of E. coli cells. Sequences from 2μm DNA, ARS1, etc. can be used in vectors to transform yeast cells.

As for the promoters for gene expression, those derived from, for example, retroviruses, polyomaviruses, adenoviruses, SV40, etc. can be used in vectors for transformation of mammalian cells. Promoters from bacteriophage lambda, such as trp, Ipp, Lac or tac promoters, can be used as vectors for transformation of E. coli cells. ADH, PHO5, GPD, PGK or MAFα promoters can be used to transform baker's yeast cells, and AOX1 promoter can be used to transform methanol assimilating yeast cells. The promoter from nuclear polyhedrosis virus can be used as a vector to transform silkworm cells.

Typical examples of selectable markers used in mammalian cell vectors include neomycin (neo) resistance gene, thymidine kinase (TK) gene, dihydrofolate reductase (DHFR) gene, E. coli xanthine-guanine Phosphoribosyltransferase (Ecogpt) gene, etc. Illustrative examples of selectable markers for use in E. coli cell vectors include kanamycin resistance gene, ampicillin resistance gene, tetracycline resistance gene, etc. Examples of selectable markers for use in yeast cells include Leu2, Trp1, Ura3 and other genes.

A protein having TPO activity can be produced by appropriately combining the above-mentioned host-vector system by transforming a suitable host cell with the recombinant DNA obtained by inserting the gene of the present invention at a suitable site in the above-mentioned vector, culturing the resulting transformant, and then The desired polypeptide is isolated and purified from the resulting cells or medium or filtrate. Commonly used methods and processes can be used in combination in the above methods.

When expressing a gene of interest, the original signal sequence can be modified or replaced with a signal sequence derived from another protein to obtain a uniform N-terminus of the expressed product. N-terminal homogenization can be performed by modifying (substituting or adding) amino acid residues at the N-terminus or its adjacent positions. For example, in the case of using E.coli as a host cell for expression, the Lys residue can be further complemented by the addition of a met residue.

The novel protein having TPO activity of the present invention (hereinafter referred to as "the protein of the present invention") includes proteins each containing the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. TPO derivatives whose amino acid sequence is partially modified (substitution, deletion, insertion or addition) are also included in the present invention, provided that the activity of TPO is not destroyed by the modification.

In other words, the protein of the present invention includes protein molecules whose amino acid sequence is substantially the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.

The expression "amino acid sequence is substantially the amino acid sequence shown (or expressed) in SEQ ID NO: 2, 4 or 6" herein means that said amino acid sequence includes those shown in SEQ ID NO: 2, 4 or 6 sequence, and the sequence shown in SEQ ID NO: 2, 4 or 6 which has been partially modified (such as substitution, deletion, insertion or addition, etc.), in the case of the latter, provided that said modification does not destroy TPO activity.

The protein of the present invention includes the protein comprising positions 7-151 of the amino acid sequence shown in SEQ ID NO: 6 and having TPO activity. Also included in the protein of the present invention is a protein having TPO activity and containing the amino acid sequence 1-163 shown in SEQ ID NO:6.

Examples of other TPO derivatives of the present invention include derivatives whose in vivo stability and persistence are improved by amino acid modification (substitution, deletion, insertion or addition), wherein at least one potential glycosylation is due to amino acid deletion or Derivatives altered by addition, or derivatives in which at least one Cys residue is deleted or replaced by other amino acid residues such as Ala or Ser residues.

Preferably, the protein of the invention is characterized in that it is isolated and purified from a host cell transformed with a recombinant vector containing a cDNA molecule, a genomic DNA molecule or a DNA fragment obtained by chemical synthesis.

When using bacteria such as E.coli as a host to effectively carry out intracellular expression, a protein in which the initial methionine residue is added to the N-terminal side of the protein molecule having TPO activity can be obtained, and this protein is also included in the present invention . Depending on the host used, the produced protein with TPO activity can or cannot be glycosylated, each of which is included in the protein of the present invention.

The proteins of the present invention also include naturally occurring TPO active proteins, which are purified and isolated from natural sources, such as TPO active cell culture medium or human urine, serum and plasma.

Methods of purifying TPO from the aforementioned natural sources are also included in the present invention. This type of purification can be accomplished by one or a combination of the following steps commonly used in protein purification, such as ion exchange chromatography, lectin affinity chromatography, triazine dye affinity chromatography, hydrophobic interaction chromatography, gel filtration layer Analysis, reverse phase chromatography, heparin affinity chromatography, sulfated gel chromatography, hydroxyapatite chromatography, isoelectric focusing chromatography, metal chelation chromatography, preparative electrophoresis, isoelectric focusing gel Electrophoresis, etc. Purification methods utilizing the physicochemical properties of TPO that can be inferred from the examples in this specification combined with certain techniques are also included in the present invention. In addition, antibody affinity chromatography, in which an antibody capable of recognizing TPO is used, can also be applied. Moreover, TPO has been found to be a ligand for Mpl (de Sauvage et al., Nature 369:533-538 (1994); Bartley et al., Cell 77:1117-1124 (1994); Kaushansky et al., Nature 369: 565-568 (1994)), whereby TPO can be purified using an affinity gel column in which Mpl has been coupled to a resin. More precisely, an example of said column is an Mpl-X column made by coupling a resin to the extracellular region of Mpl (Mpl-X), said MpL-X being produced by using CHO cells Produced by recombinant DNA technology as a host (Bartley, et al. (1994) cited above).

As disclosed herein, a TPO polypeptide of the invention is further characterized in that it binds the Mpl receptor and specifically binds its extracellular (soluble) domain.

Also included in the present invention are proteins encoded by the DNA portion complementary to the protein-coding strand of the human cDNA or genomic DNA sequence of the TPO gene, i.e. Tramontano et al. (Nucl. Acids. Res., vol. 12, p5049-5059 , 1984) published "complementary reverse protein".

Also included in the invention are proteins of the invention labeled with a detectable label such as ¹²⁵ I labeling or biotinylation, thus providing for use in solid samples (such as tissue) and liquid samples (such as blood, urine, etc.) Possible reagent supplies for detection and quantification of TPO or TPO receptor expressing cells.

The biotinylated protein of the invention is used in its conjugation with immobilized streptavidin to remove megakaryocytes from bone marrow in allogeneic bone marrow transplantation. It is also used in combination with immobilized streptavidin to enrich autologous or allogeneic megakaryocytes upon autologous or allogeneic bone marrow transplantation. The combination of TPO and toxins such as ricin, diphtheria toxin, etc. or radioactive isotopes can be used for anti-tumor therapy and conditioning bone marrow transplantation.

The present invention also provides nucleic acid material for use in a hybridization process to detect human TPO gene and/or its related gene family on chromosome when labeled with a detectable label including radioactive or non-radioactive label such as biotin. position on the map. Such nucleic acid substances are also used to demonstrate human TPO gene disorders at the DNA level, and can be used as genetic markers to demonstrate adjacent genes and their disorders.

Also included in the present invention are pharmaceutical compositions comprising a therapeutically effective amount of the protein of the present invention together with practically effective diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. As used herein, the term "therapeutically effective amount" refers to an amount that provides an effective effect on the particular disease and route of administration. Such compositions can be used in the form of liquid, lyophilized or dry formulations, which contain diluents selected from various buffers (such as Tris-HCl, acetate and phosphate) with different pH values and ionic strengths to prevent Additives expressing adsorption such as albumin and gelatin, surfactants such as Tween 20, Tween 80, Pluronic F68 or cholate, solubilizers such as glycerol or polyethylene glycol, antioxidants such as ascorbic acid or sodium metabisulfite, Preservatives such as sodium ethylmercury thiosalicylate, benzyl alcohol or parabens, and carriers or tonicity agents such as galactose or mannitol. Also used compounds include proteins of the invention covalently linked to polymers such as polyethylene glycol, proteins chelated with metal ions, proteins incorporated into granular formulations, or on their surface, containing polymer compounds such as polylactic acid, poly Glycolic acid or hydrogels, or protein incorporation into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, ghosts, or protoplasts. Such compositions will function according to the physical conditions, solubility, stability, rate of in vivo release and in vivo clearance of the TPO. The choice of composition can be dictated by the physicochemical properties of the TPO active protein used. Also included in the present invention are granular compositions wherein the particles are coated with a polymer such as poloxamer, poloxamine, TPO is bound to an antibody to a tissue-specific receptor, ligand or antigen or a ligand to a tissue-specific receptor . Other examples of compositions of the invention are granular forms with protective coatings and containing protease inhibitors or penetration enhancers for various routes of administration, such as parenteral, pulmonary, nasal and oral administration.

The pharmaceutical composition containing the protein of the present invention can be administered several times a day, and the usual dosage is 0.05μg-1mg/kg body weight (TPO protein) according to the patient's condition, gender, and route of administration.

Depending on the patient's symptoms, sex and route of administration, the pharmaceutical composition containing the protein of the present invention can be administered every day in an amount of 25,000-500,000,000 active ingredients per kilogram of body weight (relative activity detected by the M-07e assay method to be given below) 1 to several times, 1-7 days per week.

The present inventors have confirmed that, regarding the C-terminal site of human TPO, even when the amino acid sequence shown in SEQ ID NO: 6 is deleted to the 152nd amino acid residue, it can still maintain its activity; for the N-terminal site, when the amino acid residue The activity is still maintained when the base is deleted to the sixth position.

Accordingly, proteins having TPO activity, containing the amino acid sequence of 7-151 of SEQ ID NO: 6 and modified (substitution, deletion, insertion or addition) in other parts are also preferably used as active ingredients of the present invention. More preferred TPO derivatives have the amino acid sequence 1-163 of SEQ ID NO:6.

Compositions also included in the present invention contain, in addition to the proteins of the present invention, at least one other hematopoietic factor, such as EPO, G-CSF, GM-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF and SCF.

The protein of the present invention is used alone or in combination with other hematopoietic factors to treat various thrombocytopenic diseases. Examples of other hematopoietic factors include EPO, G-CSF, GM-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 8. IL-9, IL-10, IL-11, IL-12, IL-13, LIF and SCF.

A number of diseases characterized by platelet disorders (eg, thrombocytopenia) caused by impaired platelet production or shortened platelet survival (due to destructive accumulation of platelets) can be treated with the proteins of the invention. For example, it can be used in patients with congenital Fanconi anemia or thrombocytopenia due to chemotherapy, radiotherapy, myelodysplastic syndrome, acute myeloid leukemia, aplastic crisis after bone marrow transplantation, To promote the recovery of platelets. It can also be used to treat thrombocytopenia caused by the production or decrease of TPO. Thrombocytopenia due to shortened platelet or megakaryocyte survival includes idiopathic thrombocytopenic purpura, aplastic anemia, acquired immunodeficiency syndrome (AIDS), disseminated intravascular coagulation syndrome, and thrombotic thrombocytopenia . In addition, the protein of the present invention is also used in autologous reinfusion of platelets, where TPO is administered to the patient prior to surgery to increase the patient's own platelet count, and the thus increased platelets are used as transfused platelets at the time of surgery.

Another use of the protein of the present invention is to treat diseases caused by temporary loss or damage of platelets caused by chemical or pharmaceutical drugs or treatment methods. TPO can be used to promote the release of new "intact" platelets in such patients.

The invention further relates to antibodies specific for TPO. The protein of the present invention can be used as an antigen, and the corresponding antibodies include monoclonal antibodies, polyclonal antibodies and chimeric antibodies prepared by conventional methods, that is, "recombinant antibodies". To prepare such an anti-TPO antibody, human TPO itself can be used as an antigen, or alternatively, a partial peptide of human TPO can be used as an antigen. When using antibodies directed against such peptide antigens, specific antigenic regions can be used to define epitopes. On the other hand, when an antibody against the antigenic protein (TPO) itself is used, the antigenic region can be clarified by analyzing the epitope of the antibody. In this case, these antibodies each having such a defined epitope can be used for fractionation, detection, quantitative analysis and purification of various TPOs different in their properties (such as the kind of added sugar chain, the length of peptide chain, etc.).

The TPO peptide containing the amino acid sequence selected above can be synthesized and covalently linked with a suitable carrier protein, such as albumin, KLH (keyhole-hemocyanin), etc., to prepare an immunoreactive antigen. In addition, multiple antigenic peptide (MAP) type peptides were produced as antigens according to the method of Tam (Tam, Proc. Natl. Acad. Sci. USA. 85:5409-5413, 1988). Then, immunize mammals, birds, etc. with the prepared antigen and adjuvant, etc., which are usually used to make them produce antibodies, such as rabbits, mice, rats, goats, sheep, chickens, hamsters, horses, guinea pigs, etc. Antisera and cells are recovered from the immunized animals, whereby polyclonal antibodies are obtained. When a peptide is used as an antigen, a specific antigenic region is used to define an epitope. In this case, immunological engineering was used to screen and identify the antigenic regions of various TPOs having different molecular weights. For example, peptide deletion regions can be screened and identified. Alternatively, antibody genes or parts thereof are cloned from cells expressing desired antibodies to obtain antibody molecules expressed by genetic engineering methods.

In contrast to polyclonal antibodies, which usually contain multiple antibodies specific for multiple antigenic determinants (epitopes), monoclonal antibodies are specific for only a single epitope on an antigen. Antibodies specific for TPO are used to improve the selectivity and stability of antigen-antibody reaction-assisted diagnostic and analytical assays, and for the isolation and purification of TPO. Additionally, such antibodies can be used to neutralize or remove TPO from serum. Monoclonal antibodies are also used to detect and quantify TPO in eg serum or whole blood.

The anti-human TPO antibodies of the present invention can be used as ligands in affinity chromatography for the purification and isolation of human TPO. To immobilize antibodies for use in affinity chromatography, any conventional method for immobilizing various enzymes can be used. For example, a method using a CNBr-activated Sepharose 4B carrier (Pharmacia Fine Chemicals Co.) or the like can be used. In order to actually purify human TPO using the immobilized anti-human TPO antibody, the immobilized anti-human TPO antibody is packed into a column, and a liquid containing human TPO is passed through the column. Through this operation, a large amount of human TPO was adsorbed to the surface of the carrier in the column. As for the solvent for elution, for example, usable are Gly-HCl buffer (pH 2.5), NaCl solution, propionic acid, dioxane, 1,2-ethylene glycol, chaotropic salts, guanidine hydrochloride, urea Su and so on. Elution with the above-mentioned solvents can elute human TPO with high purity.

The antibody of the present invention can be used to detect human TPO by immunochemical quantitative assay, especially enzyme-linked immunoassay by solid-phase sandwich method.

The advantage of monoclonal antibodies is that they can be produced by hybridoma cells in the culture medium without any other immunoglobulin molecules. Monoclonal antibodies can be prepared from the culture supernatant of hybridoma cells or from the ascites of mice induced by intraperitoneal injection of hybridomas. The hybridoma technique originally disclosed by Kohler and Milstein (Eur. J. Immunol. 6:511-519, 1976) has been widely used to form hybridoma cells with high levels of monoclonal antibodies directed against specific antigens.

In order to separate the desired antibody from the resulting antibody-containing substance, one step or a combination of multiple steps commonly used for protein purification (affinity chromatography such as protein A affinity chromatography, protein G affinity chromatography, Avid gel chromatography, anti-immunoglobulin immobilized gel chromatography, etc., as well as cation exchange chromatography, anion exchange chromatography, lectin affinity chromatography, dye adsorption chromatography, hydrophobic interaction chromatography, gel permeation layer analysis, reverse phase chromatography, hydroxyapatite chromatography, fluoroapatite chromatography, metal chelation chromatography, isoelectric point chromatography, etc.). In addition to this, an antigen affinity purification method can also be used, in which a gel carrier or membrane chemically linked to human TPO protein itself or a peptide containing an antigen region or a part thereof or a molecule capable of recognizing a desired antibody is prepared, Antibody-containing substances are added thereto, so that the prepared desired antibodies are adsorbed to the carrier or the surface of the membrane, and then the adsorbed antibodies are eluted under suitable conditions and recovered.

The present invention also allows the use of endogenous DNA sequences encoding TPO polypeptides for mass production of polypeptide products. For example, host cells are transformed using in vitro and in vivo homologous recombination procedures to provide expression or enhance expression of the polypeptide. Preferred host cells include human cells (such as liver, bone marrow cells, etc.) into which a promoter or enhancer sequence has been inserted using flanking sequences homologous to the target region of the genome of the cell, thereby completing or enhancing the expression of the TPO polypeptide. Expression (see e.g. U.S. Letters Patent 5,272,071, PCT WO 90/14092, WO 91/06666 and WO 91/09955).

The present invention also provides a method for producing the above TPO polypeptide, comprising expressing the polypeptide encoded by the DNA of the present invention in a suitable host, and isolating said TPO polypeptide. When the expressed TPO polypeptide is a Met ^-2 -Lys ^-1 polypeptide, such methods further comprise the step of cleaving Met ^-2 -Lys ^-1 from said isolated TPO polypeptide.

The present invention also provides a method for producing a TPO polypeptide with a [Gly ^-1 ] structure, comprising introducing a linker between the DNA 5' encoding the glutathione-S-transferase (GST) polypeptide and the TPO polypeptide coding sequence into a suitable host Cells in which the GST and TPO polypeptides are separated by DNA encoding a thrombin recognition polypeptide, the GST-TPO expression product is isolated, and the expressed polypeptide is treated with thrombin to remove the GST amino acid, and the resulting TPO polypeptide has a [Gly ^-1 ] structure .

The present invention will be described in detail below. (A) Purify rat TPO, analyze the partial amino acid sequence of the purified rat TPO and analyze

Biological Characteristics of Purified Rat TPO

The inventors of the present invention made the most attempt to purify rat TPO protein having activity of promoting proliferation and differentiation of rat CFU-MK. In this purification study, numerous trials and failed attempts were made on the purification process such as selection of various natural supply sources and selection of gel and separation mode for chromatography. Finally, the present inventors succeeded in purifying a protein with TPO activity from the plasma of rats with thrombocytopenia induced by X-ray or gamma-ray irradiation, based on the following reference examples and Example 1 of determining the partial amino acid sequence of the purified protein and the rat CFU-MK assay described in 2, will utilize TPO activity as a marker.

The biological characteristics of plasma-derived rat TPO were also examined in Example 3.

The main points of the process from the purification of rat TPO to the determination of the partial amino acid sequence of the purified protein are listed below.

(i) Plasma samples were prepared from about 1,100 rats with thrombocytopenia induced by X-ray or γ-ray irradiation, and subjected to Sephadex G-25 chromatography, anion exchange chromatography (Q-Sepharose FF) and lectin chromatography sequentially (WGA-Agarose), so as to obtain the TPO active fraction adsorbed by WGA-Agarose.

(ii) Next, the thus obtained active fraction adsorbed by WGA-Agarose was subjected to triazine dye affinity chromatography (TSK AF-BLUE 650MH), hydrophobic interaction chromatography (Phenyl Sepharose 6FF/LS) and gel Filtration chromatography (Sephacryl S-200HR). Because this TPO activity is divided into 4 peaks by Sephacryl S-200HR gel filtration (F1 is the highest molecular weight fraction, followed by F2, F3 and F4), so concentrate the TPO active fractions F2 and F3 respectively, and obtain respectively for subsequent High molecular weight TPO sample F2 and low molecular weight TPO sample F3 in the purification step.

(iii) Low molecular weight TPO sample F3 was subjected to preparative reverse phase chromatography (YMC-PackPROTEIN-RP), reverse phase chromatography (YMC-Pack(N-AP) and another reverse phase chromatography (CapcellPack C1) in sequence. When the TPO active fraction thus obtained was used for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and after extracting the TPO active substance from the gel, it was obtained under non-reducing conditions from the equivalent The presence of TPO activity was confirmed in bands with an apparent molecular weight of approximately 17,000-19,000.

(iv) Next, all TPO active fractions were subjected to SDS-PAGE under non-reducing conditions and transferred to PVDF membranes. The protein on the PVDF membrane was hydrolyzed into peptide fragments by complete restriction enzymes, and then the partial amino acid sequence of the rat TPO protein was determined. The rat TPO gene was cloned according to the amino acid sequence information of the two peptide fragments.

(v) So far, the high molecular weight TPO sample F2 obtained by Sephacryl S-200 HR was purified in the same manner as the low molecular weight TPO sample F3. When the TPO active fraction obtained by the last step of reverse phase chromatography (Capcell Pack C1) is applied to SDS-PAGE, and the TPO active substance is extracted from the gel, under non-reducing conditions, the equivalent apparent molecular weight is about 17,000 TPO activity was confirmed in the band at -22,000. (B) Characterization of rat TPO-producing cells, preparation of mRNA and construction of rat cDNA library

Since the above-mentioned purified rat TPO was derived from plasma, it was necessary to screen an organ or cell as a source of mRNA for cDNA cloning. Therefore, TPO activity in various organs and cell culture supernatants was screened based on the biochemical and biological properties of rat plasma-derived TPO. As a result, almost identical to rat plasma-derived TPO activity equivalent to that of TPO (Example 4).

On the other hand, expression vector pEF18S was constructed from expression vectors pME18S and pEFBOS (Example 5). The construction of this vector can provide the possibility of easy cDNA cloning using high-efficiency expression vectors with multiple cloning sites that can be used to integrate inserts.

In addition to the above-mentioned expression vectors, pUC and pBR of plasmid vectors and lambda-based phage vectors are mainly used to construct cDNA libraries.

McA-RH8994 cells were cultured, homogenized by adding guanidine thiocyanate solution, and then subjected to CsCl density gradient centrifugation to obtain total RNA (Example 6).

RNA can also be prepared by the hot phenol method, an acid guanidinium phenol chloroform method, etc.

After purifying poly(A) ⁺ RNA from total RNA with oligo-deoxythymidylate-fixed latex particles, use oligo-deoxythymidylate as a primer, add restriction enzyme NotI recognition sequence, and synthesize the first First cDNA strand, and then use RNase H and E.coli DNA polymerase I to synthesize the second cDNA strand. Add an EcoRI connector to the double-stranded cDNA obtained, and connect the obtained cDNA to the mammalian cell expression vector pEF18S (digested with NotI and EcoRI) constructed in Example 5, and then transform the connected cDNA into E.coli DH5 Competent cells of the strain to construct a cDNA library (Example 7). (C) Preparation (cloning) of rat TPO cDNA fragment by PCR

The DNA sequence was deduced from the partial amino acid sequence of rat TPO purified from rat plasma, and degenerate primers for polymerase chain reaction (PCR) were synthesized. The primers used can also be obtained from amino acid sequences other than the positions of the primers used here. Highly degenerate primers without inosine can also be used. In addition, primers with reduced degeneracy can be designed using codons that are commonly used in rats (Wada et al., Nucleic Acids Res., vol 18, p2367-2411, 1990).

When plasmid DNA was extracted from the entire portion of the cDNA library prepared above and PCR was performed using the extracted DNA as a template, a band of about 330 bp was detected, which was determined to be the DNA encoding the rat TPO portion in the subsequent amino acid sequence analysis Fragment (A1 Fragment) (Example 8). (D) PCR screening of rat TPO cDNA, sequence of rat TPO cDNA and demonstration of TPO activity

The cDNA library prepared above was divided into small pools each containing about 10,000 clones, and plasmid DNA was extracted from every 100 small pools. When PCR was performed using each plasmid DNA pool as a template and using primers newly synthesized based on the nucleotide sequence of the A1 fragment, bands considered to be specific were detected in 3 small pools. One of the 3 small pools was subdivided into subpools, each containing about 900 clones, and plasmid DNA was purified from the 100 subpools, and PCR was performed in the same manner. As a result, specific bands were detected in 3 subpools. One of these pools was subdivided into subpools, each containing 40 clones, and each clone was finally screened by PCR in the same manner. As a result, a clone pEF18S-A2α apparently encoding rat TPO cDNA was isolated (Examples 9 and 10).

When the nucleotide sequence of the clone was analyzed, the result showed that the clone encoded a partial amino acid sequence of a protein purified from rat plasma, thus confirming that the clone most likely contained rat TPO cDNA (Example 10).

When the plasmid DNA was purified from the clone pEF18S-A2α obtained above and transfected into COS1 cells, TPO activity was found in the culture supernatant of the transfected cells. As a result, it was confirmed that the clone pEF18S-A2α contained cDNA encoding rat TPO (Example 11). (E) Detection of TPO mRNA in various rat tissues

The expression of TPO mRNA in rat tissue was analyzed by PCR, and the specific expression in rat brain, liver, small intestine and kidney was detected (embodiment 12). (F) Construction of human cDNA library

Based on the results of Examples 4 and 12, the liver was selected as the starting tissue for human TPO cDNA cloning. Therefore, a cDNA library was constructed using commercially available mRNA derived from normal human liver. As in the case of the rat library, cDNA was synthesized using pEF-18S as a vector in the same manner, and a cDNA library of direct cloning was obtained using restriction enzymes NotI and EcoRI. The library prepared by introducing vector-ligated cDNA into E. coli DH5 contained approximately 1,200,000 clones (Example 13). (G) Preparation (cloning) of human TPO cDNA fragment by PCR

Several primers for PCR were synthesized based on the nucleotide sequence of clone pEF18S-A2α encoding rat TPO cDNA. When cDNA was synthesized using commercially available mRNA derived from normal human liver, and PCR was performed using the above primers and using cDNA as a template, a band of about 620 bp was observed. Analysis of its nucleotide sequence proved that this clone contained a DNA fragment with about 86% homology to rat TPO cDNA, thus proving that this was most likely the portion of the gene encoding human TPO (Example 14). (H) PCR screening of human TPO cDNA, sequence of human TPO cDNA and confirmation of TPO activity

The human cDNA library prepared above was amplified, divided into small pools each containing approximately 100,000 clones, and plasmid DNA was extracted from each of the 90 small pools. When PCR was performed using the plasmid molecules of each pool as a template and using newly synthesized primers based on the nucleotide sequence of the human TPO fragment obtained in Example 14, possible bands were detected in 3 small pools. One of these small pools was divided into subpools, each containing 5,000 clones, and plasmid DNA was purified from each of the 90 subpools. When PCR was performed in the same manner using the plasmid DNA of each pool as a template, possible bands were detected in 5 subpools. When one of these pools was subdivided into subpools, each containing 250 clones, plasmid DNA was purified from each of the 90 subpools and subjected to PCR, and possible bands were detected in 3 subpools. Furthermore, 90 colonies were isolated from one of these subpools, and PCR was performed from the plasmid DNA purified from each colony to finally obtain a clone named HL34 (Example 15).

When the nucleotide sequence of the plasmid DNA in this clone was analyzed, it was confirmed that this clone contained cDNA having about 84% homology to the nucleotide sequence of rat TPO cDNA (Example 16).

When the plasmid DNA of this clone was purified and transfected into COS1 cells, TPO activity was found in the culture supernatant of the transfected cells. As a result, it was confirmed that the plasmid clone contained cDNA encoding human TPO (Example 17).

However, this cDNA appeared to be an artifact of the cloning process, as no stop codon was found in this clone, and there was a poly A tail-like sequence at its 3' end. Therefore, an expression vector encoding an amino acid sequence not corresponding to a poly A tail-like sequence was constructed. When the thus constructed vector was expressed in COS1 cells, TPO activity was found in the resulting culture supernatant (Example 18).

A DNA fragment of the 3' terminal region of human TPO was obtained by PCR to analyze the structure of the full-length cDNA.

Determination of the nucleotide sequence of this fragment showed that it partially overlaps with the cDNA carried by the clone HL34 obtained in Example 15. It is also expected that the full-length human TPO cDNA may contain its open reading frame and encode a protein of 353 amino acids. Thus, this indicates that human TPO contains a 353 amino acid sequence including a 21 amino acid signal sequence (Example 19).

In addition to the above cloning methods, pUC or pBR-based plasmid vectors, lambda phage-based vectors, etc. can also be used to obtain human TPO cDNA by colony hybridization or plaque hybridization using rat TPO cDNA fragments as probes. clone. When designing degenerate probes, inosine can be used to reduce the degree of degenerate heterogeneity. When an assay that detects TPO activity in a specific manner or with high sensitivity is available, it is possible to express clones using an expression library as used in the present invention.

However, since, as described in Example 15 below, the amount of human TPO-encoding RNA in normal human liver seems to be very low (1:3×10 ⁶ calculated from the results of this Example 15), the synthetic oligo Hybridization screening using nucleotides or rat or human TPO cDNA fragments as probes is difficult, because the number of colonies or plaques to be processed becomes very large, and the sensitivity and specificity of hybridization methods are not as good as PCR methods. In fact, the present inventors have used the rat TPO cDNA fragment as a probe to hybridize 2×10 ⁶ clones in the normal human liver cDNA library prepared in Example 13, but failed to obtain human TPO cDNA clones. (I) Reconstruction of cDNA derived from normal human liver

Because the clone HL34 obtained in Example 15 may contain incomplete cDNA, a commercially available normal human liver-derived poly(A) ⁺ RNA preparation was used to re-construct the cDNA library to obtain a complete human TPO cDNA by linking the vector The library (hTPO-F1) prepared by introducing cDNA into E. coli DH5 contained 1.0×10 ⁶ transformants (Example 20). (J) Screening TPO cDNA clones, sequencing and expressing human TPO cDNA and confirming TPO activity

According to the nucleotide sequence (SEQ ID NO: 3) of the partial cDNA obtained in embodiment 14 and the nucleotide sequence (SEQ ID NO: 196) of the people TPO complete cDNA predicted in embodiment 19, synthesize the primers that are used for PCR .

The human liver cDNA library (hTPO-F1) constructed in Example 20 was divided into 3 small libraries (library numbers 1-3#). PCR was performed using the plasmid DNA prepared from each small pool as a template and using the synthesized primers. As a result, when the plasmid DNA prepared from the 3# mini-pool was used, a DNA fragment with the expected size was amplified. The 3# small pool was then divided into sub-pools, each containing 15,000 transformants, and screened by PCR as described above. As a result, amplification of DNA with the expected size was found in 6 of the 90 small pools. When these positive small pools were divided into subpools, each containing 1,000 clones, plasmid DNA was extracted in the same manner and PCR was performed, no DNA amplification was observed. This is considered to be due to the low recovery rate of plasmid DNA caused by the weaker growth of the clone of interest than other clones. Therefore, the original 3# pool was plated in 100 LB plates at an inoculation volume growing 4,100 colonies per LB plate, and replica plates were prepared from each of the plates so inoculated. When DNA extracted from the colonies grown on the plate was subjected to PCR in the same manner as above, amplification of the expected band was observed in one of the 100 subpools.

Duplicate filters were prepared from plates of this subpool and colony hybridization was performed with a labeled probe (EcoRI/BamHI fragment of plasmid pEF18S-HL34). As a result, a single signal considered positive was observed, and colonies were collected from the original plate and plated again on LB plates. DNA samples were prepared from a total of 50 colonies grown on the plate, and PCR was performed to finally obtain a clone named pHTF1 (Example 21). When screening cDNA clones, methods such as hybridization, PCR, and expression cloning are mainly used, and these methods are used in combination to improve the efficiency and sensitivity of screening, or reduce labor intensity. When the nucleotide sequence of the clone pHTF1 thus obtained was determined, it was shown that the clone pHTF1 had an open reading frame, and the amino acid sequence of the protein thought to be encoded by the open reading frame was identical to that of the deduced human TPO (SEQ ID NO: 6) Exactly. The nucleotide sequence differed from a deduced sequence (SEQ ID NO: 196) at 3 positions, but these differences did not result in amino acid changes. The human TPO protein has been shown to contain 353 amino acid residues with a 21 amino acid signal sequence. When plasmid DNA was prepared from the thus obtained clone pHTF1 and transfected into human COS1 cells, TPO activity was found in the culture supernatant (Example 23). (K) Screening of human TPO chromosomal DNA, sequencing and expression of human TPO by plaque hybridization

Chromosomal DNA, confirming TPO activity

A genomic library donated by T. Yamamoto (the Gene Research Center, Tohoku University) was inoculated on 18 NZYM plates at an inoculum size of 30,000 phage particles per plate, and two plates were prepared from each of the 18 plates prepared. Photocopied filters. A human TPO cDNA fragment (base numbers 178-1,025 in SEQ ID NO: 7) was amplified by PCR and purified. Plaque hybridization was performed using ³² P-labeled purified fragments as probes. As a result, 13 positive signals were obtained. Plaques were collected from the original plate and re-inoculated onto NZYM plates at an inoculation size of 1,000 plaques per plate. Two replica filters were prepared from each of the resulting plates, and plaque hybridization was performed under the same conditions as above. As a result, positive signals were detected on all filters of the 13 groups. A single plaque was recovered from each of the resulting plates to prepare phage DNA. Phage DNA samples prepared from 13 clones were checked for the presence of cDNA coding regions by PCR. 5/13 clones appeared to contain the complete amino acid coding region predicted from the cDNA. Therefore, one of these clones (clone λHGT1) was selected and analyzed by Southern blot (using the probes described above). Since a single band of about 10 kb was observed in the case of Hind III digestion, clone λHGT1 was digested with Hind III and subjected to agarose gel electrophoresis. The 10 kb band was excised and purified from the gel, subcloned into the cloning vector pUC13, and finally a clone named pHGT1 was obtained (Example 24).

When determining the nucleotide sequence of the clone pHGT1 thus obtained, it was found that the chromosomal DNA carried by the clone contained the complete coding region of the protein predicted in Example 19, and the nucleotide sequence of the coding region was the same as the predicted nucleotide sequence ( SEQ ID NO: 196) matches exactly.

In addition, there are 4 introns in the region corresponding to the exon encoding the amino acid, and the nucleotide sequence is different from the complete cDNA sequence (SEQ ID NO: 7) carried by the clone pHTF1 obtained in Example 21.

When the nucleotide sequences of the remaining 4 clones among the 5 chromosomal DNA clones selected separately by screening were determined, 2 of the 4 clones were found to be identical to the clone pHGT1, and the other 2 clones were found to be identical to the clone pHGT1 except as observed with the clone pHTF1 was also identical to clone pHGT1 with a different nucleotide in the 3' UTR (Example 25).

The EcoRI fragment in the thus obtained plasmid clone pHGT1 was ligated with the expression vector pEF18S to obtain the human TPO expression plasmid pEFHGTE. When plasmid DNA (pEFHGTE) was prepared and transfected into COS1 cells, TPO activity was found in the culture supernatant (Example 26). (L) Preparation of deletion derivatives of human TPO, expression of the derivatives in COS1 cells and confirmation of TPO activity The results of Examples 18 and 22 show that human TPO protein can still express even after removing the 3 amino acids at its C-terminal its biological activity. Therefore, deletion derivative experiments were performed for further analysis of the biologically active fractions. A series of expression plasmids were prepared by PCR using the DNA of the plasmid clone pHT1-231 obtained in Example 18 as a template and synthetic oligonucleotides as primers.

The resulting expression plasmid contains the DNA encoding the human TPO deletion derivative, which lacks the C-terminal region of the TPO protein, that is, the deletion derivative encodes amino acid sequences at positions 1-211, 1-191, 1-171 and 1-163. When plasmid DNA was prepared from individual clones and transfected into COS1 cells, TPO activity was detected in each culture supernatant (Example 27).

Design a series of derivatives with deletion of C-terminal amino acid residues to position 151 and other derivatives with deletion of N-terminal side amino acid residues to positions 6, 7 and 12, and detect their activity after expression in COS1 cells to further detail When the region having TPO activity was analyzed, when the amino acid residue on the N-terminal side was deleted up to position 7 or the amino acid residue on the C-terminal side was deleted up to position 151, no TPO activity was detected (Examples 28 and 29).

Derivatives of proteins with TPO biological activity can be obtained by modifying (deleting, replacing, inserting or adding) the cDNA encoding the protein. Methods for performing modifications such as PCR, site-directed mutagenesis, and chemical synthesis. (M) Expression of human TPO cDNA and purification of TPO in CHO cells

Construct expression vector pHTP1, it can express the DNA (embodiment 30) of the human TPO deduced amino acid sequence of coding as shown in SEQ ID NO:6 in animal cell.

The TPO cDNA region in pHTP1 was used to construct the vector pDEF202-hTPO-P1 expressed in CHO cells (Example 31).

After transfecting CHO cells with the above-mentioned vectors followed by selection of the transfected cells, transfected cells in which the expression vector carrying the human TPO cDNA had been integrated into the chromosome of the CHO cells were obtained (Example 32).

In Example 32, a CHO cell line producing human TPO (CHO28-30 cells, anti-25nM MTX) was cultivated on a large scale, which was prepared by transfecting CHO cells with the human TPO expression plasmid pDEF202-hTPO-P1 ( Example 55).

Human TPO was purified from 100 liters of culture supernatant (Example 56).

Human TPO can be purified from the culture supernatant of Example 55 (Example 57) in different ways. (N) Express human TPO cDNA in X63.6.5.3. cells and confirm activity

The TPO cDNA region encoded in the plasmid pBLTEN prepared in Example 30 was used to construct the expression vector BMCGSneo-hTPO-P1 (Example 33) used in X 63.6.5.3. cells.

When X63.6.5.3. cells were transformed with the above-mentioned vectors, the expression vectors encoding human cDNA in the resulting transformed cells had been integrated into their chromosomes. The transformed cells were cultured, and TPO activity was detected in the culture supernatant (Example 34). (O) Mass expression of human TPO in COS1 cells, purification of TPO, determination of molecular weight and description of its biological characteristics

The expression vector pHTP1 prepared in Example 30 was transfected into COS1 cells to obtain a large amount (about 40 liters in total) of culture supernatant containing the expression product (Example 35).

TPO was purified from 7 liters of the serum-free culture supernatant of COS1 cells prepared in Example 35 containing TPO derived from the expression vector pHTP1. TPO with high activity can be obtained by means of hydrophobic interaction chromatography, cation exchange column chromatography, WGA column chromatography and reverse phase column chromatography (Example 36).

TPO thus partially purified from COS1 cell culture supernatants was subjected to molecular weight detection and biological characterization (Examples 37 and 38). (P) Expression of human TPO in E.coli

Construction of vector pGEX for expressing a fusion protein of glutathione-S-transferase and human TPO (amino acid residues 1-174) (referred to as "GST-TPO(1-174)") in E. coli -2T/hT(1-174). In this case, the human TPO cDNA nucleotide portion (approximately half of the 5' side region) was exchanged with the preferred codons of E. coli (Example 39).

GST-TPO(1-174) was expressed in E. coli, the resulting cells were lysed, and then GST-TPO(1-174) contained in the pellet fraction was dissolved. Next, after various steps of checking TPO refolding conditions, purification conditions (glutathione affinity chromatography, cation exchange columns, etc.) The sequenced proteins were partially purified. This protein has been shown to exhibit TPO activity in the rat CFU-MK assay system (Examples 40 and 41).

Same parent, construct the vector pCFM536/h6T(1-163) expressing mutant human TPO protein in E.coli, in said mutant protein (referred to as "h6T(1-163)"), Ser at position 1 and Ala at position 3 is replaced by Ala and Val respectively, and a Lys and a Met are added at positions -1 and -2 respectively. The entire part of the human TPO cDNA nucleotide sequence (corresponding to 1-163 amino acid residues) carried by the above vector was exchanged with the preferred codon of E. coli (Example 42).

h6T(1-163) is expressed in E. coli. The resulting cells were lysed, then h6T(1-163) contained in the pellet fraction was dissolved, and the refolding conditions of the protein were examined. Thus, the protein containing the designed amino acid sequence of TPO was successfully partially purified. The TPO activity of this protein has been demonstrated in the rat CFU-MK assay system (Examples 43 and 44).

In addition, a vector pCFM536/hMKT(1-163) for expressing a mutant human TPO protein in E. coli was constructed. Lys and Met were added to the 1 and -2 positions, respectively. hMKT(1-163) was expressed in the same manner as described in Example 43 in E.coli, and the expressed protein was subjected to SDS-PAGE, transferred to a PVDF membrane, and then N-terminal amino acid sequence analysis was performed to confirm that the protein contained Designed amino acid sequences (Example 52).

In addition, the vector pCFM536/hMKT(1-332) for expressing the mutant human TPO protein in E.coli was constructed. Lys was added at position -1 of TPO (amino acids 1-332) and Met was added at position -2. hMKT(1-332) was expressed in E. coli in the same manner as in Example 42, and the above expression results were confirmed by Western blotting using the anti-human TPO peptide antibody prepared in Example 45 given below (Example Example 66). (Q) Preparation of anti-TPO peptide antibody and anti-TPO peptide antibody column

A rabbit polyclonal anti-TPO peptide antibody was prepared using a synthetic peptide (corresponding to three partial regions of the rat TPO amino acid sequence determined in Example 10). These antibodies have been shown to recognize both rat and human TPO molecules. Furthermore, a peptide corresponding to the 6-part region of the human TPO amino acid sequence shown in SEQ ID NO: 6 (or SEQ ID NO: 7) was synthesized, and then used to prepare a rabbit polyclonal anti-TPO peptide antibody. The resulting antibody was shown to recognize human TPO (Example 45).

It is possible to purify TPO by affinity column chromatography using a column packed with certain molecules with binding affinity for TPO (eg anti-TPO antibody, TPO receptor, etc.). Therefore, an anti-TPO antibody column was first prepared by binding the anti-TPO peptide antibody obtained in Example 45 (Example 46). (R) Purification, molecular weight determination and biological characterization of human TPO expressed in COS1 cells using an anti-TPO peptide antibody column

The culture supernatant of COS1 cells transfected with the expression vector pHTP1 was used as the starting material to obtain partially purified TPO, which was then applied to the anti-TPO antibody column. Since TPO activity was found in the adsorbed fraction, this fraction was further subjected to reverse phase column chromatography to purify the protein and examine its molecular weight and biological characteristics (Example 47). (S) Confirmation of the biological activity of a partially purified sample of human TPO expressed by COS1 cells

Check the bioactivity of the purified TPO active fraction in Example 36 to determine whether it functions to increase the number of platelets in vivo (Example 48), said active fraction is transfected with the expression vector pHTP1 The culture supernatant of COS1 cells was purified to the TPO sample of the CapcellPak C1 300A column step.

Similarly, the biological activity of the crude TPO fraction obtained by passing the 33 liters of culture supernatant prepared from transfected COS1 cells carrying the expression vector pHTP1 through a cation exchange column was checked to confirm that it can increase platelet counts in vivo (implementation Example 49). (T) Expression of human TPO chromosomal DNA in CHO cells and confirmation of its activity

The vector pDEF202-ghTPO for expression of human TPO chromosome in CHO cells was constructed (Example 50).

When this vector is introduced into CHO cells, transformed cells are obtained in which the DNA-encoded expression vector of human TPO chromosome has been integrated into its chromosome. When this cell clone was cultured, TPO activity was detected in the culture supernatant (Example 51). (U) Partial purification of mutant human TPO expressed in E.coli and confirmation of its activity

Refolding of mutant human TPO derived from clone pCFM536/h6T(1-163) encoding the nucleotide sequence of human TPO and expressed in E. coli using guanidine hydrochloride and glutathione to determine h6T thus obtained (1-163) acts to increase platelet count in vivo (Example 53).

The mutant human TPO derived from the clone pCFM536/h6T(1-163) encoding the human TPO nucleotide sequence and expressed in E. coli was refolded using N-lauroyl sarcosine and copper sulfate, Cation exchange chromatography was then performed to determine that h6T(1-163) thus obtained raised platelet numbers in vivo (Example 54).

Alternatively, mutant human TPO h6T(1-163) can be refolded and purified by other methods (Examples 60 and 61). (V) Expression of human TPO cDNA in insect cells and identification of TPO activity

A recombinant virus was prepared for expression of human TPO in insect cells (Example 58) and expressed in insect cells Sf21, followed by identification of TPO activity in the culture supernatant (Example 59). (W) express human TPO (1-163 amino acid) in CHO cell and purify this TPO

The expression vector pDEF202-hTPO163 expressing human TPO protein in CHO cells was constructed, and said protein (referred to as "hTPO163") has amino acids 1-163 of the human TPO amino acid sequence shown in SEQ ID NO:7. The expression vector was transfected into CHO cells, and the hTPO163-producing CHO cell line thus obtained was cultured on a large scale. hTPO163 was purified from culture supernatants (Examples 62-65). (X) Preparation of Alternative Derivatives of Human TPO

Derivatives in which Arg-25 and Glu-231 in human TPO were replaced by Asn and Lys respectively (referred to as "N3/TPO" and His-33 was replaced by Thr (referred to as "09/TPO") were prepared.

Plasmids encoding each derivative were transfected into COS7 cells and then cultured. TPO activity was detected in the culture supernatant (Example 67).

Derivatives in which one amino acid in h6T(1-163) was replaced by other amino acids were prepared by using the E. coli expression system. TPO activity was found in each derivative (Example 94). (Y) Preparation of insertion or deletion derivatives of human TPO

An amino acid was inserted into or deleted from hTPO163 to prepare its insertion or deletion derivative, and then the plasmid encoding the derivative was transfected into COS7 cells, and TPO activity was detected in the culture supernatant of COS7 cells (Example 68) .

The method (in vitro assay system) of the present invention for detecting TPO activity is described below as "Reference Example". (Z) Preparation and purification of anti-human TPO antibody

Human TPO peptide fragments were used to prepare polyclonal antibodies (Example 69), which were then used for Western blot analysis (Example 70) and construction of anti-TPO antibody affinity columns (Example 71). (AA) In vivo activity of human TPO

Purified human TPO was administered to mice in which thrombocytopenia was induced, and changes in platelet numbers were monitored relative to controls (Examples 72-79). The effective use of pharmaceutical compositions in the treatment of thrombocytopenia is also described (Examples 80-89). (BB) Relationship between TPO activity and MLP receptor binding

In the assay provided, TPO activity is defined by its specific binding to the Mpl receptor (Example 93). Reference Example A. Rat Megakaryocyte Progenitor Cell Assay (Rat CFU-MK Assay) System (Liquid Culture System)

Megakaryocytes incorporate and store serotonin into cytoplasmic density granules through an active energy-dependent process (Fedorko, Lab. Invest., vol. 36, p310-320, 1977). This phenomenon has been observed in smaller mononuclear acetylcholinesterase-positive cells, which are thought to be localized between CFU-MK and recognizable megakaryocytes (Bricker and Zuckerman, Exp. Hematol., vol. 12 , p672, 1984). The amount of serotonin incorporated increases with the increase in megakaryocyte size (Schick and Weinstein, J, Lab. Clin. Med., vol. 98, p607-615, 1981). In addition, it is known that the above phenomenon occurs specifically only in megakaryocytes among bone marrow cells (Schick and Weinstein, J, Lab. Clin. Med., vol. 98, p607-615, 1981). In the assay system herein, highly enriched rat CFU-MK cells (ie, the Gp II b/III a ⁺ CFU-MK fraction described below) were cultured in the presence of the test sample, and the incorporation To ¹⁴ C-serotonin in megakaryocytes ( ¹⁴ C-creatine sulfate hydroxytryptamine, ¹⁴ C-5HT).

The advantage of this assay system is the ability to reduce the direct effects of contaminated cells (e.g., the formation of Meg-CSF activity of contaminated cells stimulated by something other than the factor of interest, or the production of a factor by contaminated cells that is associated with The combined effect of the factor of interest), because the percentage of CFU-MK in the cells of the GpII b/III a ⁺ CFU-MK fraction was quite high (see [Assay method] below) and the number of contaminating cells was low. In addition, suitable culture conditions can be maintained for a relatively long period of time because the total number of cells seeded per well is small. Another advantage is that many larger volumes of mature megakaryocytes grown by CFU-MK in the presence of active samples during culture can be examined under a phase-contrast microscope, thus providing a possible quantitative determination of the presence and extent of said activity. The results of the quantitative judgment are in good agreement with the results obtained based on the quantitative determination of the incorporation of ¹⁴ C-serotonin. Therefore, the reliability of quantitative determination can be further improved by joint application of quantitative judgment. test methods

The method of Miyazaki et al. (Exp. Hematol., vol. 20, p855-861, 1992) was slightly modified to prepare highly enriched rat CFU-MK cells (Gp II b /III a ⁺ CFU-MK fraction).

Briefly, femurs and tibiae were removed from Wister rats (male, 8-12 weeks old) to prepare the bone marrow cell suspensions. Suspension medium for preparing bone marrow cell suspension (this solution contains 13.6mM trisodium citrate, 11.1mM glucose, 1mM adenosine, 1mM theophylline, 10mM HEPES (pH7.25), 0.5% bovine serum albumin (hereinafter Referred to as "BSA") (Path-O-Cyte 4; Seikagaku Kogyo Co., Ltd.) and Hanks' balanced salt solution without Ca ²⁺ and without Mg ²⁺ (hereinafter referred to as "HATCH solution") in Levine and Fedoroko Minor modifications were made on the basis of the reported megakaryocyte isolation medium (Levine and Fedoroko, Blood, vol.50, p713-725, 1977). The bone marrow cell suspension prepared in this way was spread on the Percoll discontinuous density gradient solution (density : 1.050g/ml/1.063g/ml/1.082g/ml), the gradient solution was prepared by diluting with HATCH solution, and centrifuged at 400×g for 20 minutes at 20°C. After centrifugation, 1.063g/ml and Cells between the 1.082g/ml density layers. After washing, the cells recovered were suspended in Iscove's modified Dulbeceo medium (hereinafter referred to as "IMDM" medium) containing 10% fetal calf serum (hereinafter referred to as "FCS"). ), placed in a 100mm tissue culture plastic plate, and cultivated at 37°C for 1 hour in a 5% CO ₂ incubator. After incubation, recover non-adherent cells, and then culture in a plastic plate at 37°C for 1 hour. In HATCH Non-adherent cells were suspended in a solution to which the mouse monoclonal anti-rat platelet Gp II b/III a antibody P55 antibody had been adsorbed (Miyazaki et al., Thromb. Res., vol.59, p941-953, 1990 ) in a 100mm cytology culture dish for 1 hour. After incubation, rinse thoroughly with HATCH solution to remove non-adhered cells, separate the remaining cells adsorbed on the immobilized P55 antibody by pipetting, and collect them. Generally , 3-4×10 ⁵ cells were obtained from one rat. The cell fraction thus obtained contained highly enriched rat CFU-MK (hereinafter referred to as "Gp IIb/III a ⁺ CFU-MK fraction"), Known that this cell part contains about 5-10% CFU-MK according to the detection result of the colony assay system under the existence situation of the rat IL-3 of saturating concentration.The hybridoma that can produce aforementioned P55 antibody has been in February, 1994 Deposit No. 14, the deposit number is FERM BP-4563 (National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Minstry of International Trade and Industry).

Next, the Gp II b/III a ⁺ CFU-MK fraction thus obtained was suspended in IMDM medium containing 10% FCS, and dispersed in a 96-well tissue culture plate at a ratio of 10 ⁴ cells/well. Standard samples (described in detail below) or samples to be tested were added to each well, so the final medium volume was adjusted to 200 μl/well. The plates thus prepared were placed in a CO _incubator at 37°C for 4 days. On the fourth day of incubation, 0.1 μCi (3.7 kBq) ¹⁴ C-serotonin was added to each well 3 hours before the completion of incubation, and the final incubation was continued at 37°C. After incubation for 3 hours, the plate was centrifuged at 1,000 rpm for 3 minutes, and the resulting supernatant was removed by aspiration. A 200 [mu]l portion of PBS containing 0.05% EDTA was added to each well, the plate was centrifuged, and the resulting supernatant was discarded to wash the plate. Repeat the washing step once. Add 200 μl of 2% Triton X-100 to each well of the resulting cell pellet, and shake the plate on a plate mixer for about 5-10 minutes to completely lyse the cells. 150 μl of the cell lysate thus obtained was transferred to a commercial cap-shaped solid scintillator (Ready Cap; Beckman), and then placed in an oven at 50° C. overnight to dry the lysate. The next day, put the Ready Cap into a glass vial and detect ^{the 14} C radioactivity with a liquid scintillation counter.

In this case, when the acetylcholinesterase activity in the aforementioned cell lysate was detected according to the method of Ishibashi and Burstein (Blood, vol.67, p1512-1514, 1986), it was possible to obtain the incorporation of ¹⁴ C-5-hydroxytryptamine. Almost identical results were measured. standard sample:

First, blood plasma of thrombocytopenic rats for preparing standard samples was prepared in the following manner.

Normal male Wister rats (7-8 weeks old) were intravenously injected with the above-mentioned P55 antibody twice (at an interval of about 24 hours) at a dose of 0.5 mg/animal to induce thrombocytopenia. 24 hours after the second medication, blood was collected through the abdominal aorta under ether anesthesia, and 1ml of 3.8% (v/v) trisodium citrate solution was added to the used 10ml syringe as an anticoagulant. The blood sample thus collected was dispensed into a plastic centrifuge tube and centrifuged at 1,200 x g for 10 minutes to recover a plasma fraction. The plasma fraction thus collected was centrifuged again at 1,200 xg for 10 minutes. The resulting plasma fractions were recovered, handled carefully so as not to contain cells, platelets, etc. therein, and pooled (plasma obtained in this manner is hereinafter referred to as "TRP"). The Ca processing TRP (standard sample C), WGA-Agarose column active fraction (standard sample W) or Phenyl Sepharose 6FF/LS column active fraction obtained by TPO according to the method of Example 1 will thoroughly carry out the IMDM medium of sufficient volume Dialyzed and used as assay standard.

In the rat TPO purification method described in Example 1, standard sample C was used in the early stage, but standard sample W was used after the middle stage, and then standard sample P was used instead. The specific activity of standard sample C was tentatively set as 1, and the relative activities of standard samples W and P were calculated according to this definition. Determine the relative activity of each sample to be tested by comparing the dose-response curve of the standard sample with the dose-response curve of the sample to be tested, and the relative activity of the sample to be tested is defined as n, where n represents that its activity is higher than that of the standard sample C times. B. Colony Assay System

In this assay system, bone marrow cells are cultured in a semi-solid medium in the presence of a test sample, and Meg-CSF activity is detected by counting the number of megakaryocyte colonies formed by the proliferation and differentiation of CFU-MK. Measurement method: (a) When using unseparated rat bone marrow cells, etc.

A final volume of 1 ml of IMDM medium containing unisolated rat bone marrow cells, cells from each isolation step of GpII b/III a ⁺ CFU-MK from Assay System A above or GpII b/III a ⁺ CFU- The cells of the MK fraction and 10% FCS, 2mM Gln, 1mM sodium pyruvate, 50μM 2-mercaptoethanol and 03% agar (AGAR NOBLE, DIFCO) were added to a 35mm tissue culture plastic plate, and after solidification at _room temperature, the Incubate at 37°C in an incubator. In general, the number of cells in each plate is adjusted to 2-4×10 ⁵ in the case of isolated bone marrow cells; in the case of Percoll density gradient or depletion of adherent cells, the number of cells is 2-5 ×10 ⁴ ; or in case of Gp II b/III a ⁺ CFU-MK fraction, cell number 0.5-2×10 ³ . After culturing for 6-7 days, the agar plates were separated from the plates and placed on glass slides (76mm x 52mm). To dry each agar plate, a 50-mesh nylon mesh and filter paper were placed sequentially over the gel. The agar slide thus dried was heat-fixed on a heating plate at 50° C. for 5 minutes, and soaked in acetylcholinesterase stain prepared according to Jackson’s method (Blood, vol.42, p413-421, 1973) for 2-4 hours . When it was confirmed that the megakaryocytes were fully stained, the agar slides were washed with water, dried, and then stained with Harris hematoxylin solution for 30 seconds, washed with water, and air-dried. Megakaryocyte colonies were defined as having 3 or more tightly clustered acetylcholinesterase-positive cells. (b) The case of using unisolated mouse bone marrow cells

Colony assays were performed in the same manner as in (a) above using 2-4 x 10 ⁵ cells per plate. (c) When using human bone marrow cells or human cord blood cells

Human bone marrow cells or umbilical cord blood cells can be used directly, or as an enriched CFU-MK fraction in the manner described below.

First, bone marrow fluid or cord blood was plated on Lymphoprep (Daiichi Kagaku Co., Ltd) and centrifuged, and the resulting interface leukocyte fraction was recovered. Cells linked to biotinylated monoclonal antibodies specific for human surface antigens (CD2, CD11c and CD19) were removed from the above recovered cell fraction using avidin-linked magnetic beads. The cells removed by the magnetic bead method are mainly B cells, T cells, macrophages and some granulocytes. The remaining cells were stained with FITC-labeled anti-CD34 antibody and PE-labeled anti-HLA-DR antibody, and then CD34 and HLA-DR positive fractions were recovered with a cell sorter (eg, ELITE produced by COULTER). CFU-MK cells were enriched in this fraction (hereinafter referred to as "CD34 ⁺ DR ⁺ CFU-MK fraction"). Colony assays for human cells can be performed in the same manner as described above for the case of rat bone marrow cells, with the exception of using 3-5 x ¹⁰³ CD34 ⁺ DR ⁺ CFU-MK fraction cells per plate and using 12.5% A mixture of human AB plasma and 12.5% FCS was used instead of 10% FCS. The culture time for the formation of megakaryocyte colonies is 12-14 days. To detect human megakaryocytes, megakaryocytes were immunostained by the alkaline phosphatase anti-alkaline phosphatase antibody method using a mouse monoclonal antibody specific for the megakaryocyte surface antigen Gp II b/III a (see, e.g., Teramura et al. al., Exp.Hematol., vol.16, p843-848, 1988), counting the colonies each containing 3 or more megakaryocytes. C. Assay system utilizing a cell line of human megakaryoblasts (M-07e assay)

Human megakaryoblast cell line M-07e cells proliferate in response to GM-CSF, IL-3, SCF, IL-2, etc. (Avanzi et al., J. Cell. Physiol., vol.145, p458-464, 1990). Because these cells also respond to TPO, they were used in an assay system that replaced the rat CFU-MK assay system. test methods:

M-07e cells maintained in the presence of GM-CSF were recovered, thoroughly washed, and suspended in IMDM medium containing 10% FCS. The resulting M-07e cell suspension was dispersed in a 96-well tissue culture plate at a ratio of 10 ⁴ cells per well, and standard samples or samples to be tested were added to each well, so the final volume was adjusted to 200 μl/well. The plates thus prepared were placed in a 5% CO ₂ incubator and incubated at 37°C for 3 days. After 3 days of culture, 1 μCi (37 KBq) ³ H-thymidine was added to each well 4 hours before the completion of culture. After the culture is completed, the cells are collected on a glass fiber filter with a cell harvester, and ³ H radioactivity is detected with a liquid scintillation counter (for example, Beta Plate produced by Pharmacia). D. Assay system utilizing mouse primary B cell line (Ba/F3 assay)

The original mouse B cell line Ba/F3 is considered to proliferate in response to IL3, I4, etc. (Palacios et al., Cell, vol. 41, p727-734). Because the substrain BF-TE22 can not only respond to IL3 and IL4, but also respond to TPO to produce cell proliferation, so this cell can be used as an assay system to replace the rat CFU-MK assay, or the cell line auxiliary assay of human megakaryocytes ( M-07e assay) is as follows. Briefly, BF-TE22 grown in Iscove’s modified DMF medium (IMDM: GIBCO) containing 1 ng/ml mouse IL-3 was recovered, washed 3 times with IMDM, and resuspended in IMDM medium containing 10% FCS middle. Then, the cell suspension was dispersed in each well of a 96-well tissue culture plate at a density of 1×10 ⁴ cells/well, and standard TPO solution or samples to be tested were added to each well, and the final volume was adjusted to 200 μl/well. The resulting plates were incubated for 2-3 days in a 5% _CO2 incubator. On day 2 or 3, 1 μCi (37 KBq) ³ H-thymidine was added to each well, and culture was continued for 4 hours. Finally, the cells were collected on the glass fiber filter membrane with a cell harvester, and the ³ H radioactivity was detected with a liquid scintillation counter. In this assay system, almost all the cells cultured in the medium lacking TPO activity died, so no ³ H-thymidine incorporation. However, cells cultured in media containing TPO activity exhibited active proliferation and thymidine incorporation in a TPO concentration-dependent manner. In addition, the assay results were consistent with the assay results of M-07e and CFU-MK.

The following examples further illustrate the invention.

Example 1-1

    Purification induced by administration of antiplatelet antibodies

Preparation of Rat TPO in Thrombocytopenic Rat Plasma Plasma of Thrombocytopenic Rats Induced by Administration of Anti-Platelet Antibodies

TRP was prepared as a purified source from approximately 1,000 rats according to the method described in the aforementioned Reference Example A Rat Megakaryocyte Progenitor Cell (CFU-MK) Assay System. Purification of rat TPO from TRP

First, TRP from about 1,000 rats was purified on the assumption that the TPO content in TRP was at most 1:3×10 ⁶ , and as a result, slightly less than 1 pmole of partially purified rat TPO was obtained. Despite the difficulty in purifying TPO in general, attempts to analyze its partial amino acid sequence resulted in a three-part amino acid sequence, but with very low accuracy. These sequences are identical or very similar to the amino acid sequences of serine protease inhibitors (SPIs) produced in the liver. From this result, it is unclear whether TPO has a similar structure to serine protease inhibitors, or whether the sequence is derived from contaminating cells other than TPO. The TPO gene was cloned from the rat cDNA library using these unidentified sequences, but the effective TPO coding gene could not be obtained. Therefore, extensive research was carried out on the assumption that this failure was due to the low purity and insufficient content of the analyzed samples. In order to obtain the purified final sample required for amino acid analysis, purification was carried out according to the methods described in Examples 1-2 below. Surprisingly, from the estimation of the results of Examples 1-2, the TPO content in TRP or XRP (to be described below) is very small, only 1:10 ⁸ -10 ⁹ of the total plasma protein, therefore, for TPO It is quite difficult to perform complete purification.

Example 1-2 Purification of rat TPO from plasma of rats with thrombocytopenia induced by whole-body X-ray or γ-ray irradiation [Preparation of plasma of rats with thrombocytopenia induced by whole-body X-ray or γ-ray irradiation]

Normal male Wister rats (aged 7 to 8 weeks) were exposed to sublethal doses (6 to 7Gy) of X-rays or γ-rays to make them suffer from thrombocytopenia. Rat blood was collected on the 14th day of irradiation and a plasma fraction (hereinafter referred to as "XRP") was prepared in the same manner as the aforementioned method for preparing TRP.

In this case, XRP (about 8 L) produced from a total of about 1,100 rats was used as a source for purification. Purification of rat TPO by XRP

Since the total protein content in the plasma sample of 1,100 rats reached 493,000 mg, it could not be processed at one time. Thus, the plasma sample was divided into 11 fractions, each corresponding to about 100 rats, in the following purification steps (1) to (4). In the subsequent purification steps (5) to (7), the samples were divided into 6 batches, and in the purification step (8) and subsequent steps, samples crudely purified from the whole plasma of about 1,100 rats were used. A typical example of each purification step in the case of the samples being (XW9) and (XB6) is described below.

In all purification steps, TPO activity was determined using the rat CFU-MK detection system described in the reference examples. Unless otherwise noted, all purification steps were performed at 4°C, except for reverse phase chromatography and Superdex 75 pg gel filtration in the presence of surfactant, which was performed at room temperature. Protein assays were performed using a Coomassie dye-binding assay (a reagent system manufactured by PIERCE, cat. no. 23236X) or a method using bicinchoninic acid (a reagent system manufactured by PIERCE, cat. no. 23225) .

The summary of the purification is shown in Table 1.

         Table 1 Purification of TPO in Rat Plasma

Total Egg Relative Total Activity Activity

White matter Activity Recycling

(mg) (mg) (%)

                                                                      

CA processing Plasma/G25 480300 2 864600 100 <ion exchange> Q-SEPHAROSE FF 314400 9 704000 313 <outer source condensate> WGA-Agarose 15030 132 1987000 230 <trione dyes> TSK-Gel AF-Blue 650MH 4236 9055555 3834000 443<hydrophobic> Phenyl-Sepharose 6FF/LS 2762 847 2339000 271<gel filtration> S-200HRF3 (low molecular weight TPO) 51 20000 0 0 0 10

S-200HR F2 (high molecular weight TPO) 262 7838 2055000 238

  Low molecular weight TPO (obtained from S-200HR F3)

Step Step Total Eggs Relatively Activated Activation

White matter Activity Recycling

(mg)      (％)<凝胶滤过>    S-200HR F3(低分子量TPO)    50.3300   20000      1007000   116<反相>        YMC Protein-RP制备柱       2.8540    130000     371000    43<反相>        YMC CN-AP                  0.3730    800000     298400 35<reverse phase> Cepcell pak Cl 0.0396 4890000 193600 22<electrophoresis> 15% SDS-PAGE 0.0017 5 030 0 9 2

  (under non-reducing conditions)

High molecular weight TPO (from S-200HR F2)

Total Egg Relative Total Activity

White matter Activity Recycling

(mg) ( %) <Gel filtering) S-200HRF2 (high molecular weight TPO) 257.0000 7840 2015000 233 <nende> YMC Protein-RP preparation column 11.4000 227000 2588000 300 <Gel filtering> SuperDex 75pg/Chaps 1.1660 17500000000 2041000 236 <Dainer> YMC CN-AP 0.6200 700000 434000 50 <nexial Phase> Capcell Pak CL 0.0630 000000 1260000 146 <Electric Swimming> 15 % SDS-PAGE 0.0030 3300000 990000 117

(Under non-reducing conditions) (1) Calcium chloride treatment, centrifugation and protease inhibitor treatment of rat plasma in the case of sample number XW9

Stored at -80°C, XRP corresponding to about 100 rats (742 ml; protein concentration, 54.8 mg/ml; total protein, 40,686 mg) was melted and dispensed into polypropylene tubes (manufactured by Nalgere). Calcium chloride powder was added to each tube to a final concentration of 100 mM. After overnight incubation at 4°C, the resulting mixture was centrifuged at 8,000 rpm for 60 minutes. A protease inhibitor p-APMSF ((p-aminodiphenyl)methanesulfonyl fluoride hydrochloride, produced by Wako Pure Chemical Industries, Lte., Cat. No. 010-10393)) was added to the obtained TPO-active The supernatant (742ml; protein concentration, 54.9mg/ml; total protein, 40,740mg) was adjusted to a final concentration of 1mM. The obtained sample was used in the following Sephadex G-25 column buffer exchange step.

In this way, calcium chloride/p-APMSF for all XRP was accomplished by dividing the total XRP from 1,100 rats into 11 fractions each equal to approximately 100 rat XRP. deal with. The resulting 11 samples were processed separately in the subsequent Sephadex G-25 column step. (2) Sephadex G-25<buffer exchange> in the case of sample number XW9

The supernatant (742ml; protein concentration, 54.9mg/ml; total protein, 40,740mg) obtained after the calcium treatment step (1) was added to a SephadexG-25 column (produced by Pharmacia Biotech, Cat. No. 17-0033-03; diameter, 113 cm; bed height, 47 cm), the column had previously been equilibrated with 20 mM Tris-HCl, pH 8. Elution was performed with the same buffer. The 1,300 ml eluate prior to protein elution was discarded. When UV absorption was measured in the eluent, the distillate was stored until the conductance reached 500 μS/cm, thereby finding the TPO active protein fraction exchanged with 20 mM Tris-HCl, pH 8 (1,377 ml; protein concentration, 27.56 mg/ml ; total protein, 37.882 mg; protein yield, 93%). The relative activity of TPO in the fraction was 23.

As a result of carrying out the Sephadex G-25 column processing of all the samples, a total of 21,117ml of Sephadex G-25 column containing TPO activity fractions (total protein, 480,300mg; average relative activity, 2; total activity, 864,600). (3) Q-Sepharose FF (strong anion exchange chromatography) in the case of sample number XW9

The TPO active fraction (1,377ml; protein concentration, 27.5mg/ml; total protein, 37,841mg; relative activity, 2.3) obtained by Sepharose G-25 treatment in the above step (2) was added at a flow rate of 40ml/min. On the Q-Sepharose FF column (produced by PharmaciaBiotech, catalog number 17-0510-01; diameter 5cm; bed height 27cm), and with 20mM Tris-HCl (pH8) elution, the fraction F1 that passes column is stored (3,949ml ; protein concentration, 0.98 mg/ml; total protein, 3,870 mg; relative activity, 0).

In the next step, the buffer was exchanged to 20 mM Tris-HCl (pH 8) containing 175 mM NaCl to elute fraction F2 (4,375 ml; protein concentration, 5.36 mg/ml) containing TPO activity.

Finally, fraction F3 (1,221 ml; protein concentration, 3.9 mg/ml; total protein, 4,783 mg; relative activity, 3.8) was eluted with 20 mM Tris-HCl containing 1,000 mM NaCl. The total protein in fraction F2 containing TPO activity was 23,440 mg, and the protein yield of F2 by this step was 61.9%. In addition, the relative activity of TPO increased to 6.8.

As a result of adding all the Sephadex G-25 containing TPO active fractions to Q-Sepharose FF, 35,842ml Q-Sepharose FF containing TPO active fraction F2 (total protein, 314,384mg; average relative activity, 9; total activity, 2,704,000). (4) Wheat Germ Agglutinin (WGA)-agarose <Lectin Affinity Chromatography> in the case of sample number XW9

The TPO-containing active fraction F2 obtained in the above-mentioned step (3) will be processed by Q-Sepharose FF and divided into three parts, and added to the WGA-Sepharose column (produced by HonenCorp., the number is 800273, diameter 5cm) with a flow rate of 5ml/min , bed height 22.5cm). and eluted with Dulbecco's isotonic phosphate buffered saline (DPBS). The columned fraction F1 (9,336 ml; protein concentration, 2.30 mg/ml; total protein, 21,407 mg, relative activity 6.9) was stored.

Then, the buffer was exchanged with 20 mM sodium phosphate buffer (pH 7.2 ), store the resulting eluate and concentrate it with an ultrafiltration device (OmegaUltrosette, with a nominal molecular weight cut-off of 8,000; produced by Filtron) to obtain fraction F2 (2,993ml; protein concentration, 0.376 mg/ml).

The total protein in fraction F2 containing TPO activity was 1,125 mg, and the protein yield of F2 by this step was 4.8%. In addition, the relative activity of TPO increased to 101. Fraction F2 thus obtained was stored at -80°C.

As a result of adding all fractions of Q-Sepharose FF TPO activity F2 to WGA-agarose: a total of 33,094ml of fraction F2 of WGA-agarose TPO activity (total protein, 15,030mg; average relative activity, 132; total activity, 1,987,000). (5) TSK gel AF-BLUE 650MH <triazine dye affinity chromatography> in the case of sample batch number XB6

The fraction of the TPO activity adsorbed by the WGA-agarose with the part number XW8 was mixed with the TPO activity-containing fraction of the WGA-agarose adsorbed by the part number XW9 obtained from the XRP of equal to 215 rats in the above step (4). Fraction F2 was pooled to form a sample with batch number XB6 (5,947 ml; protein concentration, 0.388 mg/ml; total protein, 2,319 mg; relative activity, 150).

A portion of 5,974 ml of the mixed sample was mixed with 1000 ml of 0.85 molar NaCl (296.76 g in total) to generate 6,132 ml of a solution with a final NaCl concentration of 0.822 M, and the resulting solution was added to the solution at a flow rate of 7 ml/min. On the TSK gel AF-BLUE 650MH column (TOSOH CORP, catalog number 08705; diameter 5cm; bed height 23cm) that has been equilibrated with 20mM sodium phosphate buffer (pH7.2) containing 1M NaCl in advance.

After this process was completed, the protein was eluted at a flow rate of 10 ml/min with 20 mM sodium phosphate buffer (pH 7.2) containing 1 M NaCl. The resulting eluate was stored and concentrated with an ultrafiltration device (Omega Ultrasette, nominal molecular weight cut-off of 8,000) to obtain the column-passed fraction F1 (543ml; protein concentration, 2.05mg/ml; total protein, 1,112mg; relative activity , 31).

In the next step, the elution buffer was changed to 2M NaSCN to obtain the TPO activity-containing fraction F2 (1,427ml; protein concentration, 0.447mg/ml) adsorbed by the eluted TSK gel AF-BLUE 650MH.

The total protein in fraction F2 containing TPO activity was 638 mg, and the protein yield of F2 by this procedure was 27.5%. In addition, the relative activity of TPO increased to 1,500.

As a result of adding the TPO activity-containing fraction F2 adsorbed by WGA-agarose to TSK gel AF-BLUE 650MH, a total of 10,655ml of TPO activity-containing fraction F2 of TSK gel AF-BLUE 650MH was obtained ( Total protein, 4,236 mg; mean relative activity, 905; total activity, 3,834,000). (6) Phenyl Sepharose 6FF/LS<Hydrophobic Interaction Chromatography> In the case of the sample is XB6 batch

Part of 1,424ml TSK gel AF-BLUE 650MH containing TPO active fraction F2 (1,424ml; protein concentration, 0.447mg/ml; total protein, 638mg; relative activity, 1,500) was mixed with 1.5 moles of ammonium sulfate powder per 1000ml ( The total amount is 282.2 g) and mixed to obtain 1,581 ml of a solution having a final concentration of ammonium sulfate of 1.35 M.

The resulting solution was added to a Phenyl Sepharose 6FF (Low Sub) column (Phomacia Biotech, catalog number 17-0965- 05; diameter 5cm; bed height 10cm). After this operation was completed, elution was performed at a flow rate of 10 ml/min with 36 mM sodium phosphate buffer (pH 7.2) containing 0.8 M ammonium sulfate. The resulting eluate (about 3,160ml) was stored and concentrated with an ultrafiltration device (Omega Ultrasette, nominal molecular weight cut-off 8,000), thereby obtaining fraction F1 (485ml; protein concentration, 0.194mg/ml; total protein, 94.2mg; relative activity, 0).

In the next step, the elution buffer was changed to 20 mM sodium phosphate buffer (pH 7.2) to obtain fraction F2 (about 3,500 ml) containing TPO activity. The eluted fractions were concentrated using an ultrafiltration device (Omega Ultrasette, nominal molecular weight cut-off 8.000) and a sample was taken for determination. At this stage, the protein concentration and total protein of fraction F2 containing TPO activity were 1.45 mg/ml and 319 mg, respectively, and the protein yield of F2 through this step was 50.0%. The relative activity of TPO was 1,230.

As a result of adding the F2 fraction containing TPO activity of each batch of TSK gel AF-BLUE 650MH to Phenyl Sepharose 6FF/LS, a total of 1,966ml of fraction F2 containing TPO activity of PhenylSepharose 6FF/LS (total protein, 2,762 mg; mean relative activity, 847; total activity, 2,339,000). (7) Sephacryl S-200 HR <Gel Filtration Chromatography> In the case of the sample is XB6 batch (see Figure 1)

Fraction F2 of Phenyl Sepharose 6FF/LS with TPO activity (217ml; protein concentration, 1.45mg/ml; total protein, 315mg; relative activity, 1,230) was mixed with 144.8ml of 5M NaCl solution to obtain 362ml of a solution with a final NaCl concentration of 2M , and the resulting solution was concentrated to about 50 ml using an ultrafiltration device using a YM3 membrane (76 mm in diameter, produced by Amicon Corp.).

To this solution was added the same volume (50ml) of 8M urea to obtain 100ml of a solution containing 1M NaCl and a final concentration of 4M urea. The solution was concentrated to about 80 ml to prepare a sample of 88.78 ml, which was then applied to a Sephacryl S-200 HR column (manufactured by Pharmaci Biotech, catalog number 17-0584-01; diameter 7.5 cm, bed height 100 cm).

Thereafter, elution was efficiently performed with DPBS at a flow rate of 3 ml/min, and the eluate after discarding useless 1,200 ml of the eluate was collected in 60 polypropylene tubes in 45 ml portions. One assay was performed in every two tubes and the rest were stored at -85°C until all samples from 1,100 rats had completed Sephacryl S-200HR treatment. Based on the assay results, the fractions of Lot XB6 are grouped as follows: (F1) tube number 1-15 (fraction at the edge of useless eluent; molecular weight equal to or greater than

   at 94,000) (F2) tube number 16-26 (molecular weight, 94,000 to 33,000) (F3) tube number 27-44 (molecular weight, 33,000 to 3,000) (F4) tube number 45-55 (molecular weight, less than or equal to 3,000)

In this way, all batches of F2 fractions containing TPO activity obtained from Phenyl Sepharose 6FF/LS were added to Sephacryl S-200HR for treatment, assay and storage at -85°C. After all the batches had completed the S-200HR treatment and before subsequent reverse phase chromatography (YMC-Pack PROTEIN-RP), the samples were thawed and ultrafiltered using a YM3 membrane (76 mm in diameter, manufactured by Amicon Corp.) Concentration was performed to obtain two fractions of samples as follows. The concentrated sample containing TPO active fraction F2 obtained by Sephacryl S-200HR treatment is hereinafter referred to as "high molecular TPO sample F2", and the similarly treated F3 is called "low molecular TPO sample F3".

High molecular weight TPO sample F2 and low molecular weight TPO sample F3 are for the convenience of storing fractions in different elution regions in gel filtration chromatography, and the terms "high molecular weight" and "low molecular weight" therefore do not imply their real molecular weights.

Polymer TPO sample F2 low-molecular TPO sample F3 molecular weight 94,000-33,000 33,000-3,000 total volume 3,420ml 6,480ml total volume (concentrated after) 293ml 280ml total protein 262mg 51.3mg average relative activity 7,838 20,000 total activity 2,055,000 1,020,000

Low-molecular TPO sample F3 and high-molecular TPO sample F2 were subjected to subsequent purification steps, respectively.

The purification of low molecular weight TPO sample F3 is described in steps (8)-(11) below. (8) YMC-Pack PROTEIN-RP<Reverse Phase Chromatography>

The low-molecular-weight TPO sample F3 (total protein, 50.3mg; protein concentration, 0.184mg/ml; relative activity, 20,000; total activity, 1,007,000, total volume, 274ml) obtained in the above step (7) was mixed with solvent A (0.025 % trifluoroacetic acid (TFA)) and solvent B (1-propanol containing 0.025% TFA) were mixed to obtain a total volume of 508.63 ml, and the final concentrations of propanol, TFA and protein were 20%, 0.012% and 0.0989 mg/ ml of solution. The insoluble matter generated during the preparation of the solution was separated by centrifugation, and the supernatant was divided into two parts of 254.3ml (25.2mg protein) and added to a column equipped with YMC-PackPROTEIN-RP (manufactured by YMC) at a flow rate of 2ml/min. , catalog number A-PRRP-33-03-15; diameter 3 cm; bed height 7.5 cm), the column had been pre-equilibrated with 30% B. The centrifuged pellet was washed with 20 ml of 20 mM sodium acetate (pH5. 5) Dissolve and apply to the column.

After the sample was applied to the column, about 50 ml of solvent (solvent A:B=321) was used to pass through the column to obtain the column fraction. Next, start the developing process (elution with a linear gradient solution from 30% B to 45% B within 120 minutes), and collect eluate from 36 polypropylene tubes with 10 ml per tube. This process was repeated for the remaining samples, collected in the same collection tubes, thus obtaining a total of 36 fraction tubes each containing 20 ml of eluate. The column fraction was directly concentrated to 20 ml using an ultrafiltration device using a YM3 membrane (76 mm in diameter, produced by Amicon Corp.).

Take each 0.1ml of each column fraction and the 20ml fraction of tubes 1 to 36, mix with 20μl 5% BSA, evaporate to dryness, dissolve in 0.25ml IMDM assay culture medium, and then measure to find out the fraction containing TPO activity. point. Results TPO activity was found in fractions from tubes 17 to 27 (propanol concentration range of 36.0 to 43.0%), which were taken together as the TPO active grade of YMC-Pack PROTEIN-RP derived from low molecular weight TPO sample F3 Sub-F2 use. This fraction was stored at -85°C until used in the subsequent YMC-Pack CN-AP step. YMC-Pack PROTEIN-RP derived from low molecular weight TPO sample F3 contains TPO active fraction F2

Total volume 220ml

  Protein Concentration                                              

  Total protein  

Relative Activity 130,000

  Total activity                                                                                     371,000(9) YMC-Pack CN-AP<Reverse Phase Chromatography>

The YMC-PackPROTEIN-RP of the low-molecular-weight TPO sample F3 source that obtains in above-mentioned step (8) contains TPO active fraction F2 214.9ml (total protein, 2.79mg; Protein concentration, 0.0130mg/ml; Relative activity, 130,000; Total activity, 36,300) was mixed with 0.6 ml of 50% glycerol and concentrated to 1.8 ml. The concentrate is adjusted to a volume of 5 ml with less than or equal to 20% propanol and about 6% glycerol.

The concentrate was divided into 5 aliquots for use in the following column runs (1 ml and 0.555 mg protein for each run). Add the divided sample (at a speed of 0.6ml/min) to a column (produced by YMC, catalog number AP-513; diameter 6mm; bed height 250mm) covered with YMC-PackCN-AP, using 0.1% TFA as solvent A and 0.05% TFA containing 1-propanol as solvent B, the column was pre-equilibrated with 15% B. After addition, the concentration of propanol was increased from 15% B to 25% B, and the concentration of B was increased linearly from 25% to 50% within 65 minutes. After the last operation (No. 5) was completed, 1 ml of the solution containing the same fraction with the protein removed was applied to the column and treated in the same way to obtain the TPO activity still present in the column. Since the same polypropylene tube was used to store the eluate from the 6 runs, a total of 44 test tubes containing 7.2 ml eluate each were obtained.

30 μl (1/240 of each fraction) of each fraction obtained above was mixed with 20 μl 5% BSA, evaporated to dryness, then dissolved in 0.24 ml IMDM assay medium for assay to determine the fraction containing TPO activity. Results Strong TPO activity was found in the fractions from tubes 28 to 33 (37.0 to 42.0% propanol range) which were combined to serve as the main TPO active fraction of the YMC-Pack CN-AP derived from the low molecular weight TPO sample F3 Sub-FA. Low molecular weight TPO sample F3 source YMC-Pack CN-AP main TPO active fraction FA

Total volume 43.20ml

  Protein concentration             0.00863mg/ml

  Total Protein                 0.373mg

Relative Activity 800,000

Total activity 298,400(10)Capcell Pak C1 300<final reversed-phase chromatography>

43.12ml (total protein, 0.372mg; protein concentration, 0.00863mg/ml; relative activity, 800,000; total activity, 297,500) was mixed with 0.2ml 50% glycerin and concentrated to obtain 0.1ml glycerol solution.

This solution was mixed with 2 ml of solvent A (0.1% TFA): solvent B (1-propanol with 0.05% TFA) = 85:15 (15% B) to prepare 2.1 ml of about 14% propanol, about Sample of 4.8% glycerol and 0.177 mg/ml protein. The prepared solution was added to a Capcell Pak C1 300A column (manufactured by Shiseido Co., Ltd.; catalog number C1 TYPE: SG 300A; diameter 4.6 mm, bed height 250 mm) equilibrated with 15% B in advance, and the column was heated for 65 minutes. A linear gradient from 27% B to 38% B was used internally at a flow rate of 0.4 ml/min. The eluate was collected in 0.6 ml portions into 72 polypropylene tubes.

3 µl of each fraction obtained (1/200 of each fraction) was mixed with 20 µl of 5% BSA, the matrix was changed to 225 µl of IMDM assay medium, and the 75-fold diluted fraction was assayed.

1 μl of each fraction (1/600 of the fraction) was used for electrophoresis, evaporated to dryness and treated with 10 μl of non-reducing sample buffer at 95° C. for 5 minutes for SDS-PAGE. The processed samples were then subjected to SDS-PAGE using 15-25% SDS-polyacrylamide precast gels (manufactured by Daiichi Pure Chemicals Co., Ltd), and 2D-Silver Stain II "DAIICHI" (produced by Daiichi Pure Chemicals Co. , Ltd., production, catalog number 167997; hereinafter referred to as "silver staining kit") staining with a silver staining kit). [DAIICHI]-III low molecular weight marker (manufactured by Daiichi Pure Chemicals Co., Ltd.; catalog number 181061; hereinafter referred to as "DPC III") was used as a molecular weight marker.

As a result of the above assay, significant TPO activity was found in the fractions from tubes 35 to 43 (30.0 to 32.5% propanol concentration range). Fractions from tubes 36 to 42 (30.5 to 32.0% propanol concentration range) were combined to serve as the main TPO active fraction FA. The results are shown in Figure 2.

When the protein content deduced from chromatography was compared with the assay results, the resulting fraction was found to have a total protein of 39.6 µg, a protein concentration of 9.4 µg/ml, a relative activity of 4,890,000, and a total activity of 193,600. Examination of the SDS-PAGE profiles of the TPO active fractions from tubes 36 to 42 showed the presence of bands that correlated staining intensity with activity intensity. Furthermore, the apparent molecular weight of this non-reduced band was found to be 17,000 to 19,000 when compared to the reduced standard molecular weight protein on the same gel, suggesting that this band is likely to be TPO. (11) Example of FA analysis of fractions containing TPO activity extracted from electrophoresis gel <15% SDS-PAGE>

In the 4,200 μl low molecular weight TPO sample F3 source TPO activity fraction FA (total protein, 39.6 μg; protein concentration, 9.4 μg/ml; relative activity, 4,890,000; total activity, 193,600) obtained in the above step (10), the 5.5 μl (1/764 of the fraction) for active protein extraction and 2.5 μl (1/1680 of the fraction) for silver staining were transferred to respective sample tubes, evaporated to dryness, and 10 μl for SDS-PAGE The non-reducing sample buffer was reacted at 37° C. for 1 hour, and then allowed to stand at room temperature for 18 hours, thereby performing SDS reaction.

Prestained Low Range Marker (manufactured by Bio-Rad Laboratories, Inc., 161-0305) and the aforementioned DPC III were used as molecular weight markers. 15% SDS-PAGE of these samples was performed at 4° C. using microslab gels according to a commonly used method (Laemmli, Nature, vol. 227, pp. 680-685, 1970). After the electrophoresis is completed, use a knife to quickly cut off the part of the gel to be stained with silver and place it in the fixative solution, and then use the aforementioned silver staining kit for staining.

In addition, the gels of all molecular weight ranges used to detect the activity were cut into 34 pieces with a knife, and the electric width of each gel was 1.5-2.5mm, and it was used to slightly improve the Kobayashi method (Kobayashi, M., Seikagaku (Biochemistry) , vol.59, no.9, 1987, published by the Japanese Biochemistay Society) crushed. Each gel piece crushed into small pieces was mixed with 0.3 ml of extraction buffer (20 mM Tris-HCl, pH 8, 500 mM NaCl, 0.05% BSA), and extracted by shaking at 4°C for 6 hours.

500 mM potassium phosphate (pH 6.8) was added thereto to make the final concentration 20 mM. After shaking at 4°C for 1 hour, the resulting mixture was transferred to an Ultra Free C3GV 0.22 μm filter unit (manufactured by Millipore Corp., UFC3 OGV OS), and centrifuged at 1,000×g (4,000 rpm) for 15 minutes to remove precipitated SDS , to recover the resulting supernatant. The filtrate was transferred to an Ultra Free C3-LGC ultrafiltration device (nominal cut-off molecular weight 10,000, manufactured by Millipore Corp., UFC3 LGC 00), and centrifuged at 3,000×g (7,000 rpm). When the volume of the concentrated solution reaches about 50 μl, add 300 μl of 20 mM sodium phosphate buffer (pH 7.2), and perform ultrafiltration again.

Repeat ultrafiltration with 300 μl 20 mM sodium phosphate buffer twice to remove residual SDS. Thereafter, a similar procedure was repeated to exchange for a test culture solution to prepare a sample (300 μl) which was then sterilized and used for TPO activity measurement.

Three protein bands were clearly detected by silver staining showing proteins with apparent molecular weights of approximately 17,000 to 19,000, 14,000 to 11,000 based on the DPC III molecular weight marker.

In the aforementioned step (10), a band having an activity intensity correlated with the staining density and an apparent molecular weight of about 17,000 to 19,000 was observed in the electrophoresis of the TPO active fraction on the Capcell Pak C1 column. In the experiment of this step (11), TPO activity was found in a band with an apparent molecular weight of about 17,000 to 19,000 (see FIG. 3 ).

Based on the above results, it has been confirmed that the TPO active protein in the active fraction in the Capcell Pak C1 300A column was purified to a detectable level on the electrophoresis gel. The amount of probable TPO protein (apparent molecular weight about 17,000 to 19,000) in the total TPO active fraction was determined to be about 1.7 μg based on the silver staining intensity of the band obtained by 15% SDS-PAGE.

The purification of the polymeric TPO sample F2 is described in the following steps (12) to (15). (12) YMC-Pack PROTEIN-RP<Reverse Phase Chromatography>

The polymer TPO sample F2 (total protein, 257mg; protein concentration, 0.894mg; relative activity, 7,840; total activity, 2,015,000; total volume, 287ml) obtained in the above step (1) was mixed with 95.8ml (1/3 of the sample volume) of solvent B (1-propanol with 0.025% TFA) were mixed to obtain a total volume of 383 ml, with final concentrations of propanol, TFA, and protein of approximately 25%, 0.006%, and 0.671 mg/ml, respectively. A solution of 0.025% TFA was used as solvent A. The insoluble material formed during the preparation of the input sample was separated by centrifugation, and the resulting supernatant was divided into six 62.3ml (42.8mg protein) portions at a flow rate of 2ml/min. On a column with YMC-Pack PROTEIN-RP (manufactured by YMC, catalog number A-PRRP-33-03-15; diameter 3 cm; bed height 7.5 cm). The precipitate from centrifugation was dissolved in 10 ml of 20 mM sodium acetate (pH 5.5) containing 5 mM CHAPS and added to the column.

After the sample addition was completed, about 50 ml of solvent (solvent A:B=3:1) was passed through the column to obtain a column fraction. Next, start a stepwise change elution method (in 120 minutes with a linear gradient of 30% B to 45% B solvent for elution), the eluate was collected in 24 polypropylene tubes with 15 ml per tube tube. This procedure was repeated with the same collection tubes for 6 separate samples, resulting in a total of 24 fraction tubes each containing 90 ml of eluate. The column fraction and tube 1 fraction were combined and directly concentrated to 90 ml with an ultrafiltration device using a YM3 membrane (76 mm diameter, manufactured by Amicon Corp.).

Take 0.3ml of the mixture of the column fraction and tube 1 fraction and the fractions from tube 2 to tube 24, mix with 10μl 5% BSA, evaporate to dryness, dissolve in 0.3ml IMDM assay culture medium, and then measure to find out the content of Fractions with TPO activity. Results TPO activity was found in fractions from tubes 10 to 15 (34.0 to 39.5% propanol concentration range), which were combined as YMC-Pack PROTEIN-RP TPO active fraction F2 from polymer TPO sample F2 . This fraction was stored at -85°C until used in the subsequent YMC-Pack CN-AP step. YMC-Pack PROTEIN-RP TPO active fraction F2 derived from polymer TPO sample F2

Total volume 540ml

  Protein concentration             0.021mg/ml

  Total Protein                                                

Relative Activity 227,000

  Total activity                     2,588,000(13) Seperdex 75pg <Gel filtration chromatography in the presence of CHAPS>

The YMC-PackPROTEIN-RP TPO active fraction F2 (total protein, 11.3mg; Protein concentration, 0.021mg/ml; Relative activity, 227,000; Total activity, 2,565,000 of the YMC-PackPROTEIN-RP TPO active fraction F2 (total protein, 11.3mg) that obtains in the polymer TPO sample F2 source that obtains in above-mentioned step (12) ) of 538.2ml was mixed with 0.6ml 50% glycerol and concentrated by evaporation. 18 ml of 20 mM CHAPS was added thereto, followed by stirring, followed by incubation at 4°C for 41 hours. Thereafter, the first sample was added to a column (manufactured by Pharmacia Biotech, catalog number 17-1070-01; diameter 2.6 cm, bed height 60 cm) covered with HiLoad26/60 Superdex 75pg, with DPBS containing 5mM CHAPS at 1ml/ The flow rate of min is eluted. 4 ml of each sample (protein concentration 0.466 mg/ml; protein, 1.86 mg) was applied to the column.

In this example, the Superdex 75 column procedure was repeated 6 times by either the entire YMC-Pack PROTEIN-RP TPO active fraction or 6 fractions. The eluate from each run was collected in 45 test tubes in 5 ml aliquots, so that 45 fractions each containing 30 ml eluate were finally obtained after completion of 6 column runs.

Get 0.1ml of each fraction obtained and mix with 10μl of 5% BSA, evaporate to dryness, dissolve in 0.25ml IMDM assay culture medium, and measure to find out the fraction containing TPO activity. As a result, TPO activity was found in the fractions from tubes 13 to 31 (molecular weight range from 78,000 to 3,000), and the fractions from tubes 13 to 31 were combined as the Superdex 75pg TPO active fraction F2 from which the polymer TPO sample F2 was derived. Superdex 75pg TPO active fraction F2 derived from polymer TPO sample F2

Total volume 540ml

  Protein concentration                 0.00216mg/ml

Total protein 1.17mg

Relative Activity 1,750,000

  Total activity                                                                         2,041, 000(14) YMC-Pack CN-AP

513.2ml (total protein, 1.11mg; protein concentration, 0.00216mg/ml ; relative activity, 1,750,000; total activity, 1,943,000) was mixed with 1/10 volume of solvent B (1-propanol containing 0.05% TFA) and mixed with solvent A (0.1% TFA) and solvent B Add it to a column (manufactured by YMC, catalog number AP-513, diameter 6 mm, bed height 250 mm) covered with YMC-Pack CN-AP equilibrated with 15% B in advance. After this process was complete, the propanol concentration was increased from 15% B to 25% B with a linear gradient from 25% B to 50% B over 65 minutes.

Before starting the YMC-Pack CN-AP column chromatography, 1/20 of the total loaded sample was used for the experimental run to confirm proper recovery of activity. Thereafter, the remaining 19/20 was split into 2 samples for two runs. In other words, the column operation was repeated three times. Since the same 44 polypropylene tubes were used to store the eluate from the three operations, 44 test tubes each containing 3.6 ml of the eluate were obtained.

5 μl (1/720 of each fraction) of each fraction obtained was mixed with 0.25ml IMDM assay culture fluid, and then measured to find out the TPO active fraction. As a result, strong TPO activity was found in fractions from tubes 24 to 30 (propanol concentration ranging from 36.0 to 42.0%), which were combined as the YMC-Pack CN-AP for the F2 source of the polymeric TPO sample Main TPO active fraction FA. YMC-Pack CN-AP TPO active fraction FA derived from polymer TPO sample F2

Total Volume 25.20ml

  Protein concentration             0.0246mg/ml

  Total Protein             0.620mg

Relative Activity 700,000

  Total activity                                               434,000(15) Capcell Pak C1 300A<Final reverse phase chromatography>

24.66ml (total protein, 0.606mg; protein concentration, 0.0246mg/ml; Relative activity, 700,000; total activity, 424,000) was mixed with 0.4 ml of 50% glycerol and concentrated by evaporation.

In this way, a 2 ml concentrated sample containing n percent concentration of propanol, 10% glycerol and a protein concentration of 0.303 mg/ml was obtained. Add solvent A (0.1% TFA) and solvent B (containing 0.05% TFA) to a Capcell Pak C1 300A-coated column (manufactured by Shiseido Co., Ltd., catalog number Cl TYPE: On SG 300A; diameter 4.6 mm; bed height 250 mm), the process was carried out at a flow rate of 04 ml/min, during which a linear gradient from 27% B to 38% was increased in 65 minutes. The eluate was collected in 72 polypropylene test tubes at 0.6 ml per tube.

0.75 μl (1/800 of each fraction) of the fractions obtained in each tube was mixed with 20 μl 5% BSA, the matrix was converted into 225 μl IMDM assay culture medium, and then the 300-fold diluted fraction was measured.

2 μl of each fraction (1/300 of the fraction) was sampled for electrophoresis, evaporated to dryness and treated with 10 μl of non-reducing sample buffer at 95° C. for 5 minutes for SDS-PAGE. The treated samples were then used for SDS-PAGE using 15-25%, SDS-polyacrylamide precast gels (manufactured by Daiichi Pure Chemicals Co., Ltd.), and stained with the silver staining kit described above. The aforementioned DPC III was used as a molecular weight marker.

As a result of the above assay, significant TPO activity was found in the fractions from tubes 33 to 39 (propanol concentration ranging from 29.5 to 31.5%). Of these fractions, fractions from tubes 34 to 39 (propanol concentration range 30.0 to 31.5%) were combined as the main TPO active fraction FA. Examination of the SDS-PAGE pattern of the major TPO active fraction revealed a band with apparent molecular weights ranging from 17,000 to 19,000 under non-reducing conditions, the staining density of which correlates with the intensity of activity, which is consistent with the previously described The situation of the TPO active fraction derived from the low-molecular-weight TPO sample F3 in step (10) is similar. <Example 2> Analysis of the Amino Acid Sequence of the Purified Rat TPO Part

According to Iwamatsu (Iwamatsu et al., "Shin Kiso Seikagaku Jikken-ho (New Basic Biochemical Experiments)", vol4, pp.33-84, pub. Maruzeu; Iwamatsu, A., Seikagaku (Biochemistry), vol.63, no .2, pp.139-143, 1991; Iwamatsu, A., Electrophoresis, vol.13, pp.142-147, 1992) method, to obtain in the purification step (10) by embodiment 1, exist in Capcell Amino acid sequence analysis of rat TPO candidate proteins in the TPO active fraction FA of PakC1 300A column. That is, samples were subjected to SDS-PAGE and electrotransferred to polyvinylidene fluoride (PVDF) membranes. After the protein was reduced and S-alkylated on the PVDF membrane, the protein that had been treated above was subjected to a thorough and step-by-step in situ restriction enzyme digestion with three proteases to digest or peptide fragments, in each The resulting peptides are isolated by the digestion step and purified by reverse-phase chromatography, and their amino acid sequence is analyzed by high-sensitivity amino acid sequencing. This process is described in detail below. Example of analysis of TPO candidate proteins in Capcell Pak C1 300A TPO fraction FA derived from low molecular weight TPO sample F3 (1) Concentrated Capcell Pak C1 300A column TPO fraction FA (tube numbers 36 to 42)

4,200 μl of the Capcell Pak C1 300A column TPO active fraction FA (tube numbers 36 to 42) derived from the low-molecular-weight TPO sample F3 obtained in the purification step (10) of Example 1 (total protein, 39.6 μg; protein concentration, 4,151 μl (98.8% of the total fraction) of 9.4 μg/ml; relative activity, 4,890,000; total activity, 193,600) was used for amino acid sequence analysis. The total protein deduced from the chromatogram was 39.1 μg, of which about 1.6 μg was the TPO candidate protein that was silver-stained after SDS-PAGE, and the apparent molecular weight was about 17,000 to 19,000.

The sample was mixed with glycerol and concentrated by evaporation to obtain 5 µl of a glycerol solution. A non-reducing buffer for SDS-PAGE and 1M Tris-HCl (pH8) were added thereto to adjust its pH, thereby obtaining about 25 μl of a solution containing 200 mM Tris-HCl (pH8.0), 50 mM Tris-HCl (pH6.8) , 1.1% SDS, 2mM EDTA, 0.02% bromophenol blue and 30% glycerol.

The resulting samples were maintained at room temperature for 14 hours and then treated at 60°C for 5 minutes to produce an efficient and proper SDS reaction without excessive heating. (2) Electrophoresis

Micro-Slab gels (4.0% acrylamide stacking gel and 15% acrylamide separating gel) were prepared, and SDS-PAGE was performed at room temperature with a steady current of 12.5 mA and then 17.5 mA for 2 hours. Prestained Low Range Marker (Bio-Rad, 161-0305) and DOCIII were used as molecular weight markers. Immediately after electrophoresis, the obtained protein molecules were transferred to a PVDF membrane (see steps below).

A portion of the sample was used in another gel using 15-25% polyacrylamide precast (Multi Gel 15/25, Daiichi Pure Chemicals Co. .Ltd production, catalog number 211072) electrophoresis. When the resulting gel was stained with the aforementioned silver staining kit, it was confirmed that the molecular weight of the desired TPO protein band was about 19,000 under reducing conditions, and the purity of the TPO candidate protein on the Capcell Pak C1 300A column was 100%. few. In addition, since the mobility of this band differs under non-reducing and reducing conditions, it is suggested that the candidate protein contains at least one disulfide bond. (3) Transfer the protein on the PVDF membrane and detect the band by electrophoretic blotting

Protein transfer was carried out on a PVDF membrane (ProBlott, produced by Applied Biosystems, catalog number 400994) at a steady current of 160 mA (11 to 17 V) using a semi-dry transfer device (Model KS-8460, produced by Marysol) for 1 hour. A solution composed of 0.3M Tris and 20% methanol (pH 10.4) was used as a positive electrolyte, a solution composed of 25mM Tris and 20% methanol (pH 10.4) was used as a transfer membrane solution, and a solution composed of 25mM Tris, 40mM aminocaproic acid and A solution consisting of 20% methanol (pH 10.4) was used as the catholyte.

When the resulting transferred membrane was stained with Ponceau S staining solution (0.1 g Ponceau S and 1 ml acetic acid in 100 ml water), multiple bands were detected, one of which was confirmed to be that of a candidate protein with a molecular weight of about 19,000. This band was excised for use in the subsequent peptide generation step. (4) Generation of peptide fragments, zymogram analysis of peptides and amino acid sequence analysis

In order to thoroughly fragment TPO candidate proteins that were transferred and S-alkylated after reduction on PVDF membranes, the following three proteases were used for step-by-step restriction enzymatic hydrolysis. First digestion: Lysyl endopeptidase (Achromobacter lyticus m497-1, produced by Wako Pure Chemical Industries, Ltd., catalog number 129-02541). Second digestion: Endoprotease Asp-N (manufactured by Boehringer-Mannheim Corp., catalog number 1054589) Third digestion: trypsin-TPCK (manufactured by Worthington Biochemical, catalog number 3740)

The peptide fragments obtained by each enzymatic digestion were recovered, added to a Wakosil-II 5C18 C18 reversed-phase column (manufactured by Wako Pure Chemical Industries, Ltd., 2.0 mm in diameter, 150 mm in length), and heated at a flow rate of 0.25 ml/min at 30° C. Column temperature, using solvent A (0.05% TFA) and solvent B (isopropanol: acetonitrile = 7:3, 0.02% TFA) to elute B with a linear gradient from 1% to 50% B within 30 minutes. In this way, zymographic analysis of the peptides was performed (see Figure 4). The resulting peptides were recovered and subjected to Edman degradation using a gas-phase amino acid sequencer (PPSQ-2, manufactured by Shimadzu Corp.). Thereafter, each amino acid recovered sequentially from the sequencer, said amino acid having its N-terminus coupled to PTH, was identified by C18 reverse-phase column chromatography eluted with the same solvent. Results are summarized below. Amino acid sequence fragment of the peptide fragment obtained by the first digestion with lysyl endopeptidase Amino acid sequence AP3 (K)XYYESZ (X is A, S, G, M or Q, Z is E or K)

(SEQ ID NO: 184) AP6 (K)XRAAZ (X is E or Q, Z is E or N) (SEQ ID NO: 185) AP7 (K) AGXCSG (cannot be fully identified) (SEQ ID NO: 186) AP8 (K) XPVPPACDPRLL (X is I, T or S) (SEQ ID NO: 187) AP12 (K) DSFLADVK (SEQ ID NO: 188) AP5 (cannot be identified) AP20 (cannot be identified) AP21 (cannot be identified )AP23 (cannot be identified) Amino acid analysis of peptide fragments obtained by a second digestion with the endoprotease Asp-N

Fragment Amino Acid Sequence

ASP2 (cannot be authenticated)

ASP2 (cannot be authenticated)

ASP6 (cannot be authenticated)

  ASP11 (unidentified) Amino acid analysis of peptides obtained from the third digestion with trypsin-TPCK

Fragment Amino Acid Sequence

TP2 (K or R) TLPTXAVP (SEQ ID NO: 189)

TP3 (K or R)TLPTXAVP

In the above sequences, the N-terminal brackets show amino acid residues that can be deduced from exhaustive enzymatic digestion. (5) Homology analysis of the obtained amino acids <homology search>

In order to know whether the obtained amino acid sequence is contained in a known reported protein or whether a protein has a similar sequence, it was analyzed with the sequence analysis software Mac Vector (Kodak International Biotechnologies, Inc.). For information on known proteins or known genes, the Entrez Release 6 database (National Cenfer fbr Biotechnology Informatio, National Library of Medicine, National Institute of Health, USA, published on August 15, 1993) was used. The database includes the following: Entrez Release 6 database NCBI-GenBank, August 15, 1993 (Release 78.0) EMBL, July 15, 1993 (Release 35.0 plus updates) DDBJ, July, 1993 SWISS-PROT, April, 1993 (Release 25.0) PIR, June 30, 1993(Release 37.0) PDB, April, 1993PRF, May, 1993dbEST, July 15, 1993(Release, 1.10) U.S. and European patents

The result was the sequence (K)DSFLADVK of AP12 with the internal sequence KDSFLDVK of rat corticosteroid-binding globulin (CBG) precursor [PR database accession number A40066; Smithand Hammond; "Rat corticosteroid-binding globulin: primary structure and messenger ribonucleic acid levels in the liver under different physiological conditions.", Mol. Endocrinol., (1989), 3, 420-426] exactly coincides.

Further examination revealed a KQYYESE sequence (SEQ ID NO: 193) with high similarity to the (K)XYYESZ (X is A, S, G, M or Q, Z is E or K) sequence of AP3 contained in the rat CBG amino acid in sequence. In rat CBG, the sequences corresponding to AP12 and AP3 are joined together to form the internal amino acid sequence KDSFLADVKQYYESE (SEQ ID NO: 190).

As for the amino acid sequences of fragments other than AP12 and AP3, no proteins or genes whose similarity should be considered were found. <Example 3> Analysis of Biological Properties of TPO Derived from Thrombocytopenic Rat Plasma (1) In Rat CFU-MK Assay System (Liquid Culture System)

As a typical example, Fig. 5 shows the TPO sample purified from the plasma part of the thrombocytopenic rat (by the TPO active fraction from the YMCPack Protein-RP column described in the purification step (8) of Example 1-2). Part F2) dose-response curve. When the cultured cells were periodically observed under a microscope, the generation and maturation of megakaryocytes, ie an increase in cell size, was identified, possibly in conjunction with an increase in the number of megakaryocytes. A particularly striking change was the formation of megakaryocyte protrusions on day 4, the last day of culture (almost no protrusion formation of megakaryocytes could be recognized on day 3). Such protrusion formation is called cytoplasmic protrusion formation (Leven and Yee, Blood, vol.69, pp.1046-1052, 1987) or proplatelet protrusion formation (Topp et al., Blood, vol.76, pp.912- 924, 1990), the latter is considered to be a platelet precursor structure further differentiated from mature megakaryocytes and is considered to be in the final stage of megakaryocyte differentiation currently observed in vitro. Since such morphological changes were observed at high frequency with TPO samples alone, it is possible that this factor alone is capable of stimulating CFU-MK proliferation and differentiation, giving rise to mature megakaryocytes and eventual release of platelets. (2) In the colony assay system

Rat plasma-derived TPO stimulated the formation of megakaryocyte colonies when tested in samples of partially purified TPO from thrombocytopenic rat plasma using a colony assay system using unisolated rat bone marrow cells, Cell or Gp II b/III a ⁺ CFU-MK fractions per isolation/concentration step. Compared to megakaryocyte colonies induced by other cytokines such as rat IL-3, mouse GM-SF, or human EPO, TPO-induced megakaryocyte colonies are characterized by a smaller number of megakaryocytes per colony, but Each megakaryocyte is larger, implying premature maturation. In addition, the Meg-CSF activity of TPO can thus be considered megakaryocyte-specific since TPO produces little or no colonies of other cell lines. Based on these facts, it is clear that TPO has biological properties different from other cytokines such as rat IL-3, mouse GM-CSF and human EPO, and has unique Meg-CSF activity.

Partially purified TPO samples from the plasma of thrombocytopenic rats also showed Meg-CSF activity against CD34 ⁺ , DR ⁺ cell fractions derived from human bone marrow cells or human cord cells and significantly increased the expression of human megakaryocyte colonies. number, indicating that the factor has no species specificity. <Example 4> Specificity of rat TPO-producing cells (1) Screening of rat TPO-producing organs

In order to ensure the source of mRNA used for rat TPO gene cloning, screening and identification of rat TPO producing organs were performed. First, bone marrow, lung, liver, and spleen were regularly obtained from rats suffering from thrombocytopenia by administration of P55 antibody, and their cells were cultured (lung and liver were slices of organs), and the rat CFU-MK assay system was used to detect the produced culture supernatant. However, this initial attempt did not yield significant results. A further attempt was later made to culture hepatocytes prepared by collagenase perfusion of livers of rats with thrombocytopenia induced by administration of P55 antibodies, in view of a report indicating a relationship between rat livers and TPO production (Siemensma et al., J.Lab.Clin.Med., vol.86, pp.817-833, 1975). The resulting culture supernatant was applied to a WGA-Agarose column and the adsorbed fraction was fractionated on a Vydac phenyl reversed-phase column at the same position as the rat plasma-derived TPO activity in the rat CFU-MK assay system A very similar activity was found at . A weaker but identical activity was also found in normal rat hepatocyte culture supernatants. These results strongly suggest that the liver is one of the TPO-producing organs. (2) Screening of TPO-producing cell lines in rats

Based on the above results, screening of rat TPO-producing cell lines was performed. First, each of the 20 rat liver-derived cell lines was cultured in its own subculture medium until the cells reached nearly confluence, and the culture was replaced with the same respective medium medium supplemented with 5% FCS Base, continue to culture for 3 days. When each of the resulting culture supernatants was partially purified as described in step (1) above to determine the presence of TPO production, significant TPO activity was found in three rat liver parenchymal cell-derived cell lines, which indicated that Three cell lines are called McA-RH8994 cells (ATCC deposit number CRL 1602; Becker et al., "Oncodevelopmental Gene Expression" ed. by Fishman and Sell, Academic Press, NY.pp.259-270, 1976; purchased from Dainippon Pharmaceutical Co., Ltd.), H4-II-E cells (ATCC deposit number CRL 1548; Pitot et al., Nat. Cancer Inst. Monogr., vol.13, pp 229-245, 1964; purchased from Dainippon Pharmaceutical Co., Ltd.) and HTC cells (Thompson et al., Proc Natl. Acad. Sci. USA, vol.56, pp.296-303, 1966; purchased from Dainippon Pharmaceutical Co., Ltd.). (3) Detailed analysis of TPO in McA-RH8994 cells, H4-II-E cells and HTC cells

active

The biochemical and biological properties of TPO-active proteins secreted by the three rat cell lines were examined and compared with rat plasma-derived TPO.

McA-RH8994 cells were suspended in α-MEM(-) liquid medium containing 10% FCS and transferred into 175 cm ² plastic tissue culture flasks, each containing 1×10 ⁶ cells. After culturing at 37°C in a 5% _CO2 incubator for 3 days, the medium was changed to IMDM medium containing 5% FCS, and the culture was continued for 3 days, and the resulting culture supernatant was collected. H4-II-E cells were suspended in Dulbecco's modified Eagle liquid medium (glucose 4.5g/l, hereinafter referred to as "DMEM") containing 10% FCS and moved into ^175cm plastic tissue culture flasks, each bottle Contains 5×10 ⁵ cells. After culturing at 37°C in a 5% _CO2 incubator for 3 days, the medium was replaced with IMDM medium containing 5% FCS, and the culture was continued for 3 days, and the resulting culture supernatant was recovered. Similarly, HTC cells were suspended in DMEM liquid medium containing 5% FCS and transferred into 175 ^cm2 plastic tissue culture flasks, each containing 2.5× ¹⁰⁵ cells. After culturing at 37 °C for 3 days in a 5% _CO2 incubator, the medium was changed to IMDM medium containing 5% FCS and culture was continued for 3 days. The resulting culture supernatant was recovered.

TPO derived from each cell line was partially purified according to the method for purifying TPO by XRP described in Example 1-2 using 2 liters of supernatants obtained from the culture of the three cell lines respectively. An overview of their results is described below.

First, each culture supernatant was concentrated about 6-fold with an ultrafiltration device and the medium was exchanged with 20 mM Tris-HCl buffer (pH 8.0) with a Sephadex G-25 column. The resulting eluate was applied to a Q-Sepharose FF column, the column was washed with 20 mM Tris-HCl (pH 8.0), and then the adsorbed fraction was eluted with 20 mM Tris-HCl (pH 8.0) containing 175 mM NaCl. The resulting eluted fraction was applied to a WGA-Sepharose column, the column was washed with PBS, and the adsorbed fraction was eluted with 20 mM sodium phosphate (pH 7.2) containing 0.2M GlcNAc and 0.15M NaCl. The resulting eluate was applied to a TSK-gel AF-BLUE 650MH column, the column was washed with 20 mM sodium phosphate (pH 7.2) containing 1M NaCl, and the adsorbed fraction was eluted with 2M NaSCN. Gained eluate is added on the Phenyl-Sepharose 6FF/LS column, and this column is washed with the 50mM sodium phosphate (pH7.2) containing 1.5M ammonium sulfate earlier, then with the 0.8M ammonium sulfate containing 36mM sodium phosphate, then The adsorbed fraction was eluted with 20 mM sodium phosphate (pH 7.2). The resulting adsorbed fraction was concentrated and subjected to fractional distillation using a reverse phase Vydac Protein C4 column (manufactured by The Separations Group, catalog number 214TP51015; diameter 1 cm; bed height 15 cm). That is to add the sample to the C4 column pre-balanced with 20% B, use 0.1% TFA in developing solvent A and 1-propanol containing 0.05% TFA in developing solvent B, through a linear gradient from 20% B to 40% B, effective elution was performed at a flow rate of 1 ml/mm. Results Every TPO active protein derived from these cell lines was eluted at 1-propanol concentrations of 30-43%.

When the rat CFU-MK assay system was used to detect the TPO activity in the samples of each purification step, the results showed that the TPO activity of these three cell lines had a similar pattern to that of the XRP-derived TPO (see Examples 1-2). Table 2 shows the relative activity, activity yield, etc. in each purification step detected by the rat CFU-MK assay system. In addition, when the TPO activity of the eluate from the final reversed-phase column was detected with the rat CFU-MK system, the TPO activity of the three cell lines and the XRP-derived TPO were found in the same peak region. Elution pattern of Meg-CSF activity when colony assay of reversed-phase column TPO active fraction was performed with non-adherent cells obtained in the deadherent step of isolation and concentration of rat GpIIb/IIIa ⁺ CFU-MK The elution pattern was very similar to that of TPO activity detected with the rat CFU-MK assay system (see Table 3). In addition, similar to the case of XRP-derived TPO, the TPO activity of each of the three cell lines did not form or rarely formed colonies of the other cell lines.

Each of the stocked active fractions from the reverse phase column was applied to the SDS-PAGE column as described in Examples 1-2. When proteins were extracted from the resulting gels and TPO activity was detected using the rat CFU-MK assay system, the apparent The molecular weights were 17,000 to 22,000, 33,000 to 39,000, and 31,000 to 38,000, respectively, and HTC cell-derived TPO exhibited apparent molecular weights of 17,000 to 22,000 and 28,000 to 35,000.

Thus, these results show that the TPO proteins produced by McA-RH8994 cells, H4-II-E cells and HTC cells are equivalent in their biological properties to XRP-derived TPO, although their biochemical properties differ slightly from each other in apparent molecular weight. There are different. A remarkable finding of the present invention is the possibility that the TPO-active protein molecules contained in blood may be the product of partial digestion at specific or unconventional sites in the cells in which they are produced or on them after their secretion. It also suggests that there may be TPO genes (mRNA and cDNA) of different lengths.

Table 2 Purification of rat cell line TPO Cell line McA-RH8994 HTC H4-II-E step TP ^* 1 RA ^*2 TA ^*3 TP RA TA TP RA TA

(mg) (mg) (mg) Cultivation Line 4806 8 38450 5760 5 28800 4087 5 20440Sephadex G25 4824 167180 6458 12 77500 40-setharose FF 1973 20 39460 15 4668218320WGA -AGA -AGAAARARARARARARARAARAARAARARARARARARARARARARARARAARAARARARARAARAARAARAARAARAARAAROSARAAROSAROSAROSAROSAROSAROSAROSAAROSAROSAROSAROSAROSAROSAROSAROSAROSAROSARADADE 132.0 250 33000 80.0 90 7200TSK-凝胶AF-Blue 5.0 12500 62500 9.2 7500 69000 5.4 150 810650MHPhenyl-Sepharose 14.7 500 7350 13.1 2500 32750 11.4 170 19406FF/LSVydac Protein C4 0.23 - - 0.75 - - 0.11 - - ^* 1，总蛋白质; ^*2 , relative activity; ^*3 , total activity

                                                                                            

The exciting rats giant cells set into the formation of glycol XRP mcA-RH8994 episodes HTC settled H4-II-E set 30.0-32.6 % 0 33 4 1832.6-34.7 % 0 132 19 5034.7-36.7 % 67 290 23036.7-38.8.8 % 70 214 183338.8-40.9 % 2 15 5 2840.9-43.0 % 0 4 0 4 <Example 5> Construction carrier (PEF18S) for CDNA library (PEF18S)

The construction of cDNA fragments can be easily integrated into the vector, and the vector can establish a high expression efficiency of the cloned cDNA for the cloning and expression of TPO cDNA. That is, the expression vector pEF18S was constructed from the expression vector pEF18S by replacing the SRα promoter with the elongation factor 1α (EF1α) promoter known as a highly expressed promoter (see FIG. 6 ). The promoter of elongation factor 1α was obtained by partially digesting 1 μg of the expression vector pEF-BOS with restriction enzymes Hind III and EcoRI (Mizushima et al., Nucleic Acids Res., vol.18, p.5322, 1990), the digest was used for electrophoresis on a 2% agarose gel (produced by FMC BioProducts) to separate a DNA fragment of about 1,200bp, and was purified with the Prep-A-Gene DNA Purification Kit (produced by Bio-Rad Laboratories, Inc. It is a test kit for selective purification of DNA, which is based on the method development reported by Willis et al. in Bio Techniques, vol.9, pp.92-99, 1990, which helps form matrix DNA by porous silica Adsorption is effective for selective purification of DNA) to purify the DNA fragments. Part 100ng of the resulting DNA fragment was ligated with 50ng of the expression vector pME18S (Liu et al., Proc.Natl.Acad.Sci.USA, vol.90, pp.8957-8961, 1993) that had been digested with Hind III and EcoRI in advance. Commercially available host strains - high competent (Competent High) E.coli DH5 cells (Toyobo Co., Ltd. production; a kind of through Hanahan et al., J.Mol.Biol., vol.166, pp.557 -558, the competent E.coli strain prepared by the improved method of 1983) was transformed with the constructed vector obtained. After culturing the transformed cells, 12 colonies were randomly picked and plasmid DNA was purified from each colony. A total of 10 clones containing the desired plasmid (pEF18S) were obtained by analyzing the restriction enzyme digestion pattern of these DNA fractions. Thereafter, the selected one clone was cultured to prepare a large amount of plasmid DNA.

Purification of plasmid DNA was performed essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laborordorg Press, 1989). Cultivate the obtained clone pEF18S overnight at 37°C in 50 ml LB medium (1% Becto-tryptone, 0.5% Becto-yeast extract, 0.5% NaCl) containing 50 μg/ml ampicillin, and centrifuge the collected Cells were suspended in 4ml TEG-lysozyme solution (25mM Tris-HCl, pH8, 10mM EDTA, 50mM glucose, 0.5% lysozyme). Add 8ml of 0.2N NaOH/1% SDS solution to it first, and then add 6ml of 3M potassium/5M acetate solution to thoroughly suspend the cells. After the suspension was centrifuged, the resulting supernatant was treated with phenol/chloroform (1:1) mixed with the same volume of isopropanol, followed by centrifugation. The resulting pellet was dissolved in TE solution (10mM Tris-HCl, pH 7.5, 1mM EDTA) and treated with RNase, then treated with phenol/chloroform (1:1), and precipitated with ethanol. The resulting precipitated pellet was redissolved in TE solution, and then NaCl and polyethylene glycol 3,000 were added to the final concentrations of 0.63M and 7.5%, respectively. After centrifugation, the pellet was dissolved in TE solution and precipitated with ethanol. In this way, about 300 µg of the plasmid DNA was obtained. Among them, 100 μg was completely digested with EcoRI and NotI and subjected to 0.8% agarose gel (manufactured by FMC BioProducts) electrophoresis, and the recovered vector fragment was purified with Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad Laboratocies, Inc.) to obtain 55 μg of plasmid DNA for use in subsequent cDNA library construction. <Example 6> mRNA purification of McA-RH8994 cells

McA-RH8994 cells showing relatively high activity in the colony assay of Example 4 were selected as materials for rat TPO cDNA cloning and the following experiments were performed.

Isolation of total RNA was performed essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). That is, McA-RH8994 cells were grown to confluence in 15 petri dishes with a diameter of 90 mm. After removing the liquid matrix, the cells in each dish were thoroughly suspended in 0.8ml 5M guanidine solution (5M guanidine thiocyanate, 5mM sodium citrate (pH7.0), 0.1M β-mercaptoethanol, 0.5% sarcosyl sulfate Sodium (Sodium sarcosylsulfate)), the resulting suspension in all Petri dishes was collected in one test tube, and the total volume was adjusted to 20 ml with guanidine solution. The resulting mixture which became viscous due to cell destruction was repeatedly pipetted about 20 times with a 20 ml syringe equipped with an 18G needle (later changed to a 21G needle) until the mixture became almost viscous. With 18ml of 5.7MCsCl-0.1M EDTA (pH7.5) as a buffer layer in a centrifuge tube of a Beckman SW 28 rotor, about 20ml of the aforementioned mixture was lightly mixed in such a way that the layers were not destroyed. Lightly placed on the buffer layer, the tube was almost full. The prepared test tubes were then centrifuged at 25,000 rpm for 20 hours at 20°C. The resulting precipitated pellet was washed twice with a small amount of 80% ethanol and dissolved in TE solution, then extracted with phenol/chloroform (1:1), and then extracted with 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol. Ethanol precipitation. About 2.5 mg of total RNA was obtained in this way from about ¹⁰⁸ cells.

Poly(A) ⁺ RNA was purified from total RNA using Oligotex ^TM -dT30 (Super) (Oligotex ^TM -dT30 (Super) used was produced by Japan Synthetic Rubber/Nippon Roche; oligo dT molecules were immobilized on latex particles by covalent bonding , and the purification of poly(A) ⁺ RNA can be carried out by using the method similar to the oligodT column that is generally used for this purpose.The result is obtained 20 μg poly(A) ⁺ RNA from about 500 μg total RNA.<Example 7 >Construction of rat cDNA library

Using Time Saver™ cDNA Synthesis Kit (produced by Pharmacia; cDNA kit based on the improved method of Okayama and Berg, Mol. Cell. Biol., vol. 2, pp. 161-170, 1982) and DIRECTIONAL CLONING TOOLBOX, from A double-stranded cDNA having an EcoRI recognition site at its 5' end and a NotI recognition site at its 3' end was synthesized from the 5 μg poly(A) ⁺ RNA obtained in Example 6 (the DIRECTIONALCLONING TOOLBOX used was produced by Pharmacia; It is a set of primers 5'-AACTGGAAGAATTCGCGGCCGCAGGAA(T) ₁₈ -3'(SEQ ID NO: 15) containing a NotI recognition sequence for cDNA synthesis and a linker 5'-AACTGGAAGAATTCGCGGCCGCAGGAA(T) for adding an EcoRI recognition sequence 5'- AATTCGGCACGAG-3' (SEQ ID NO: 16) and 5'-CTCGTGCCG-3' (SEQ ID NO: 17)). The synthesized cDNA was ligated with 1.2 μg of the expression vector pEF18S (see Example 5) digested with EcoRI and NotI in advance, and transformed into 8.4 ml of the aforementioned highly competent E.coliDH5 (produced by Toyobo Co., Ltd. )middle. As a result, 5.3 x 10 ⁵ transformants were obtained. <Example 8> Preparing (cloning) rat TPO cDNA fragments by PCR

The McA-RH8994 cDNA library of 530,000 clones constructed in Example 7 was cultured overnight in a 50 ml LB matrix containing 50 μg/ml ampicillin, and was produced with QIAGEN-tip 100 (produced by DIAGEN, an anion-exchange silica gel used for DNA purification). column) The McA-RH8994 cDNA library plasmid was purified from cells collected by centrifugation. About 200 μg of plasmid DNA was obtained.

Two antisense nucleotide primers AP8-1R and AP8-2R corresponding to the amino acid sequence of the peptide fragment AP8 described in Example 2 and corresponding to the plasmid vector pEF-18S used to prepare the cDNA library were synthesized (see FIG. 6 ). 2 sense nucleotide primers EF1α-1 and EF1α-2 of the first intron of human elongation factor 1α. Synthesis of these primers was efficiently performed by using 394 DNA/RNA Synthesizer (produced by Applied Biosystems; a synthesizer based on the β-cyanoethylamdite method), and an OPC column (produced by Applied Biosystems; It was efficiently purified on a reverse-phase silica column for the purification of synthetic DNA containing trityl groups. Each DNA molecule thus synthesized and purified was dissolved in TE solution to a final concentration of 50 µM and stored at -20°C until use. Synthetic oligonucleotides used in the procedures described below were synthesized and purified in the same manner.

Primers AP8-1R and AP8-2R are mixed primers containing 17 consecutive nucleotides as reported by Takahashi et al (Proc.Natl.Acad.Sci.USA, vol.82, pp.1931-1935, 1985), so Said nucleotides incorporate deoxyinosine.

Based on Uetsuki et al. (J.Biol.Chem., vol.264, pp.5791-5798, 1989) reported genome sequence 1491-1512 and 1513-1532 nucleotide sequence synthesis containing 21 and 20 nucleotide primers EF1α-1 and EF1α-2. AP8: Ile Pro Val Pro Pro Ala Cys Asp Pro Arg Leu Leu

(SEQ ID NO: 18)

Thr

(SEQ ID NO: 19)

Ser

(SEQ ID NO: 20) AP8-1R: 3′-ACG CTG GGG GCI GAI GA-5′ (SEQ ID NO: 21)

A A A A T A A A (SEQ ID NO: 22)

T T T (SEQ ID NO: 23)

C C (SEQ ID NO: 24) AP8-2R: 3′-GGG GGG CGG ACG CTG GG-5′ (SEQ ID NO: 25)

A A A A A A A A (SEQ ID NO: 26)

T T T T T (SEQ ID NO: 27)

C C C (SEQ ID NO: 28) EF1a-1: 5′-GGA TCT TGG TTC ATT GTC AAG-3′ (SEQ ID NO: 29) EF1a-2: 5′-CCT CAG ACA GTG GTT CAA AG-3 '(SEQ ID NO: 30)

3 μg of the McA-RH8994 cDNA library plasmid was used as a template, AP8-1R (500 pmol) and EF1α-1 (100 pmol) as primers, and GeneAmp ^™ PCR kit with AmpliTaq ^™ DNA polymerase (manufactured by Takara Shuzo Co., Ltd., for A set of thermostable Taq polymerase for PCR, reaction buffer and dNTP), PCR was carried out by GeneAmp ^TM PCRsystem 9600 (manufactured by Perkin-Elmer, a thermal cycler for PCR) (volume of 100 μl; heated at 95° C. for 2 minutes , 35 cycles were performed, each cycle consisting of denaturation at 95°C for 1 min, annealing at 40°C for 1 min, synthesis at 72°C for 1 min, and a final incubation at 72°C for 7 min). To improve the specificity of the amplified DNA fragments, a further PCR was performed (100 μl volume; 35 cycles of heating at 95°C for 2 minutes, each cycle including denaturation at 95°C for 1 minute and annealing at 45°C for 1 minute and synthesis at 72°C for 1 minute, and finally incubation at 72°C for 7 minutes), this PCR used 1 µl of the resulting PCR solution as a template and EF1α-2 (100 pmol) and AP8-2R (500 pmol) as primers.

The obtained reaction solution was subjected to 2% agarose gel (manufactured by FMC BioProducts) electrophoresis to separate a DNA fragment of about 330 bp as the main product of PCR, and said fragment was purified using the aforementioned Prep-A-Gene DNA Purification Kit (Bio- Rad Laboratories, Inc.) for further purification. The resulting purified DNA fragment was subcloned into PCR™ II vector (vector for TA cloning of PCR products, manufactured by Invitrogen) using T4 DNA ligase (manufactured by Life Technologies). Since the thermostable polymerase used in PCR has the activity of terminal transferase, using the property of this enzyme to add a deoxyadenylic acid to the 3' end of the DNA amplified by PCR allows the amplified DNA fragment to be directly subcloned into 5'-dT cohesive ends in the PCR ^TM II vector. Plasmid DNA molecules were purified from 28 clones randomly selected from the generated clones using QLAGEN-tip100 (manufactured by DLAGEN), and were purified using Taq Dye Deoxy ^™ Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems, a method based on Sanger et al. Dideoxy method Proc.Natl.Acad.Sci.USA, vol.74, pp.5463-5467, 1977, the kit that uses fluorescent dye in the nucleotide sequencing that aids in PCR), by 373A DNA sequencer (a A fluorescent sequencer, produced by Applied Biosystems) was used to determine its nucleotide sequence.

When a DNA fragment encoding the three amino acid residues (Ile/Thr/Ser)-Val-Pro adjacent to the AP8-2R primer in the AP8 amino acid sequence shown above is selected from the resulting DNA fragments and sequenced over its entire length , the fragment contains 261bp cDNA. The 173rd to 175th positions of the cDNA fragment encode methionine, and the encoding frame is consistent with the amino acid frame of AP8. Since the sequence at positions 173 to 175 inserted into the DNA fragment is consistent with that of Kozak (Kozak, M., Cell, vol.44, pp.238-292, 1986), it appears that the cDNA fragment contains a Translation initiation region, encoding the N-terminus of rat TPO protein. The nucleotide sequence of the A1 fragment excluding the vector sequence and its deduced amino acid sequence are shown in the Sepuence Listing (SEQ ID NO: 1) attached below. <Example 9> Screening rat TPO cDNA by PCR

The cDNA library described above was divided into small libraries of about 10,000 clones, cultured overnight in 1 ml of the aforementioned LB medium containing 50 μg/ml ampicillin, and then used an automatic plasmid isolation device PI-100 (VER-3.0, Kurabo Industries , Ltd. production; is an automatic plasmid DNA extraction device based on the improved method of the alkaline SDS method disclosed in Molecular Cloning, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Separately from this step, the following two oligonucleotides were synthesized and purified based on cDNA fragment A1. 5'CGAGGGTGTACCTGGGTCCTG3' (SEQ ID NO: 31) (SEQ ID NO: a section of sense sequence from the 1st to 17th position of SEQ ID NO: 1 sequence; CGAG represents a linker sequence) 5'CAGAGTTAGTCTTGCGGTGAG3' (SEQ ID NO: 32) (SEQ ID NO: an antisense sequence from the 212th to the 232nd position of the 1 sequence)

Using 1/30 volume of each of the plasmid DNA samples obtained in the above step as a template, the synthesized oligonucleotides as primers, and GeneAmp ^™ PCR Reagent Kit with AmpliTaq ^™ DNA polymerase (Takara Shuzo Co., Ltd. .production), PCR was performed by GeneAmp ^™ PCR System 9600 (manufactured by PERKIN-ELMER) (30 cycles each including denaturation at 94°C for 30 seconds, annealing at 66°C for 30 seconds and synthesis at 72°C for 1 minute). As a result, 236bp specific bands were detected in 3 of the 100 small libraries detected. When one of the 3 small pools was subdivided into subpools of about 900 clones to extract plasmid DNA and PCR was performed in the same manner, specific bands were detected in 3 of the 100 examined subpools. When one of the 3 subpools was subdivided into pools of 40 clones and the same screening process was performed, specific bands were found in 3 of the 100 tested pools. One of these candidate small pools was cultured on LB plates (medium containing 15% agar) supplemented with 50 μg/ml ampicillin, and each formed colony was subjected to plasmid DNA extraction and PCR in the same manner. As a result, positive bands were detected in 2 of the 100 clones tested. <Example 10> Sequencing of rat TPO cDNA

Purification of plasmid DNA was performed essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Each of the 2 clones obtained in Example 9 was cultured overnight in 50 ml LB medium containing 50 μg/ml ampicillin, and about 300 μg of plasmid DNA was obtained after purification in the same manner as described in Example 6 .

The resulting plasmid DNA was sequenced in the same manner as described in Example 8 to determine the entire nucleotide sequence of the A1-containing fragment using Taq Dye ^Deoxy Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems). It turned out that the nucleotide sequences of the two clones were identical, which indicated that they were the same clone. One of the clones was selected, and the plasmid it carried was called pEF18S-A2α. Its nucleotide sequence and its deduced amino acid sequence are shown in Sequence Listing (SEQ ID NO: 2).

The sequence shown in Sequence Listing (SEQ ID NO: 2) has notable features. The sequence downstream of the 172bp 5'untranslated region encodes an amino acid sequence rich in hydrophobic amino acids, which is considered to be a protein secretion signal sequence containing 21 amino acids starting with methionine. The protein contains 126 amino acid residues, a 3' untranslated sequence of 1,025 nucleotides, and a poly A tail following a stop codon (TAA). This protein contains a sequence corresponding to the amino acid sequence AP8 analyzed in Example 2 (from amino acids 1 to 12 in SEQ ID NO: 2), but does not contain a consensus sequence of N-glycosylation. The sequence of nucleotides 1624-1629 immediately adjacent to the end of the 3' untranslated sequence differs from a consensus sequence but appears to be a potential polyadenylation sequence.

The carrier pEF18S-A2α carried by E.coli strain DH5 has been stored in the National Research Institute of Bioscience and Human Technology, Division of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, by the inventor on February 14, 1994, storage number FERM BP-4565. <Example 11> Expression of rat TPO cDNA and confirmation of TPO activity in COS1 cells

According to the following slightly modified DEAE-dextran method that includes chloroquine treatment (Sompayrac et al., Proc. Natl. Acad. Sci. USA, vol.78, pp.7575-7578, 1981; Nucl. Acids Res., vol.11, pp.1295-1308, 1983) The plasmid pEF18S-A2α was transfected into COS1 cells. Suspend COS1 cells (ATCC CRL 1650) in the aforementioned DMEM medium containing 10% FCS, place them in a 100mm plastic tissue culture dish, and culture them at 37°C in a 5% CO ₂ incubator until the cells reach about 40 % fusion stage. Independently of this, pEF18S-A2α (10 μg) dissolved in 30 μl of HBS (21 mM HEPES, 145 mM NaCl, pH 7.1) was added to 4 ml of DMEM medium supplemented with 500 μg/ml of DEAE-dextran (manufactured by Pharmacia), 80 µM chloroquine (manufactured by Sigma Chemical Co.), 8% (v/v) HBS and 9% (v/v) Nu-Serum (manufactured by Collaborative Research, Inc.). The resulting mixture was added to the COS1 cells cultured above that had been washed twice with DMEM medium, and culture was continued at 37° C. for 5 hours in a 5% CO ₂ incubator. Thereafter, aspirate the culture supernatant in the culture dish, and after washing the cells left in _the dish twice with DMEM medium, add 15 ml of DMEM medium containing 10% FCS. After culturing for 3 to 5 days, the resulting culture supernatant was collected.

The resulting culture supernatant was extensively dialyzed against IMDM medium and evaluated with the rat CFU-MK detection system. TPO activity was found to be dose-dependent in the culture supernatant of COS1 cells expressing the plasmid pEF18S-A2[alpha] (see Figure 7). Similar to XRP-derived TPO, many megakaryocytes formed elongated cytoplasmic protrusions in 4-day culture. In contrast, no TPO activity was found in the culture supernatant of COS1 cells transfected with pEF18S alone (Fig. 7). In the M-07e assay system, it was also found that the proliferation-enhancing activity of M-07e cells in the culture supernatant of COS1 cells expressing the plasmid pEF18S-A2α was dose-dependent, but in the culture supernatant of COS1 cells transfected only with pEF18S was not found in the supernatant. These results indicate that A2α contains a cDNA encoding a protein with TPO activity.

Next, partially purified TPO samples were prepared to test the ability of this TPO activity to promote platelet production in vivo. First, COS1 cells transfected with plasmid pEF18S-A2α were cultured for 3 days in serum-free medium supplemented with 0.2 mg BSA, thereby obtaining approximately 5.8 liters of serum-free culture supernatant. To the serum-free culture supernatant was added the protease inhibitor p-APMSF to a final concentration of 1 mM, and the mixture was filtered through a 0.22 μm filter. To the resulting 5,793 ml filtrate (protein concentration, 0.229 mg/ml; total protein, 1,326 mg; relative activity, 1,000; total activity, 1,326,000) was added 1000 ml of 0.85 moles (total 288 g) of NaCl to obtain 5,849 ml containing 0.822M NaCl The solution. The prepared solution was added to a TSK-gel AF-BLUE 650MH column (manufactured by Tosoh Corp., catalog number 08705; Diameter 5cm, bed height 6cm). After sample loading, 7,900ml of 20mM sodium phosphate (pH 7.2) containing 1M NaCl was used as the eluent to pass through the column. Store the eluate and concentrate it with an ultrafiltration device (Omega Ultrasette, nominal molecular weight cut-off 8,000, produced by Filtron) to obtain the column fraction F1 (460ml; protein concentration, 2.11mg/ml; total protein, 973mg; relative activity, 16.3) . In the next step, the eluting solution was changed to 2M NaSCN, and the TPO active fraction F2 (2840ml) of the eluted TSK-gel AF-BLUE 650MH was adsorbed by an ultrafiltration device (76mm in diameter, produced by Amicon Corp.) with a YM3 membrane Concentrate to 6.81ml. The total protein in the TPO active fraction F2 was 12.5 mg, and the protein yield of this step F2 was 0.62%. TPO relative activity was calculated to be 240. Next, add F2 to HiLoad 26/60Superdex 200pg (produced by Pharmacia Biotech, catalog number 17-1071-01; diameter 2.6cm; bed height 60cm) and add 20mM sodium acetate (pH 5.5) containing 50mM NaCl at 1ml/ The flow rate of min is expanded. After development started, TPO activity was found in the eluate ranging from 194 to 260 ml after loading. These eluates were stored to obtain Superdex 200pg TPO active fraction F2 (66ml; protein concentration, 0.112mg/ml; total protein, 7.41mg; relative activity, 142,860; total activity, 1,058,600). Next, prepare buffer A (20mM sodium acetate, pH 5.5) and buffer B (20mM sodium phosphate, pH 7.2, add 500mM NaCl), add the TPO active fraction to the previously used 100% A equilibrated strong cation exchange column RESOURCE S (manufactured by Pharmacia Biotech, catalog number 17-1178-01; diameter 0.64 cm; bed height 3 cm). Thereafter, elution was performed using a linear gradient from 100% A to 100% B in 40 minutes at a flow rate of 0.3 ml/min. TPO activity was detected in a wide range of eluents (from 5%B to 32%B). These eluates were stored and concentrated with an ultrafiltration device (25 mm in diameter, produced by Amicon Corp.) with a YM3 membrane to obtain RESOURCE S TPO active fraction F2 (1.65 ml; protein concentration, 4.74 mg/ml; total protein, 7.82 mg ; relative activity, 71,400; total activity, 558,600).

Partially purified TPO samples were subcutaneously administered (474 μg (100 μl)/mouse/day) for 5 consecutive days to 1CR male mice (9 weeks old) whose platelet count had been measured on the day before the administration. As a control, BSA (200 µg (100 µl)/animal/day) or buffer (100 µl/animal/day) as a solvent for partially purified TPO was administered in the same manner. On the second day after the last administration, blood was collected from the heart of each mouse, and the number of platelets was measured with a hemocytometer (F800, manufactured by Toa Iyo Denshi). In mice administered with partially purified TPO, the number of platelets increased about 2.14 times compared to the number of platelets before administration. Compared with the control group, the increase in the number of platelets in the partially purified TPO-administered mice was about 1.74-fold higher than that of BSA-administered mice and about 1.9-fold higher than that of buffer-administered mice. These results show that TPO promotes platelet production in vivo. In addition, since the amount of an immunosuppressive acidic protein (IAP), known as the mouse acute phase protein, was not elevated in mice administered partially purified TPO, this strongly suggests that TPO differs from IL-6 and IL-11. <Example 12> Detection of TPO mRNA in rat tissue

In order to localize the tissues expressing rat TPO mRNA in rats, RNA was extracted from different rat tissues. A total of 6 rats were used for X-ray irradiation in the same manner as described in Example 1, and different tissues (brain, thymus, lung, liver, heart, spleen, small intestine, etc.) were excised from the rats on day 11 to 14 after irradiation , kidney, testis and bone marrow cells) and were immediately frozen in liquid nitrogen. Total RNA extraction was efficiently performed by using RNA isolation reagent ISOGEN (manufactured by Wako Pure Chemical Industries, Ltd). The amount of ISOGEN used was determined according to the weight of each frozen tissue sample, and the tissue sample to which the reagent had been added was processed with a homogenizer ( ^{Physcotron_NS} -60, produced by NITI-ON Medical & Physical Instruments MFG.Co., Ltd.) until Completely comminuted tissue was obtained (approximately 45 to 60 seconds at 10,000 rpm). Total RNA was extracted from the tissue homogenate using a method based on the acid guanidine phenol chloroform method of Chomczynski et al. (Anal. Biochem., vol. 162, pp. 156-159, 1987). As a result, 1.1 to 5.6 mg of total DNA was obtained from each tissue.

Using Oligotex ^™ -dT30 (Supper) (manufactured by Japan Synthetic Rubber/NipponRoche), 20 µg of poly(A) ⁺ RNA was purified from about 500 µg of total RNA.

First-strand cDNA was synthesized from 1 μg of poly(A) ⁺ RNA obtained from each tissue using random primers. That is, 1 µg of poly(A) ⁺ RNA was dissolved in 10 µl of sterile water, incubated at 70°C for 15 minutes, and then rapidly cooled. Add 75pmoles random primer (TakaraShuzo Co., Ltd.), 10U RNase inhibitor (Bochringer-Mannheim Coop.), 50mM Tris-HCl (pH 8.3), 75mM of KCl, 3mM MgCl ₂ and 200U SuperScript ^TM II (- a reverse transcriptase produced by Life Technologies). The prepared solution (total volume 20 µl) was incubated at room temperature for 1 hour, and the resulting reaction solution was incubated at 70°C for 10 minutes to inactivate the enzyme, and then stored at -20°C until use.

New primers for PCR were synthesized based on the rat TPO cDNA sequence obtained in Example 10. The sequence of the synthesized primer is as follows: rTPO-I: 5'-CCT GTC CTG CTG CCT GCT GTG-3' (SEQ ID NO: 33) (positions 347 to 367 in SEQ ID NO: 2) rTPO-N: 5'-TGA AGT TCG TCT CCA ACA ATC-3' (SEQ ID NO: 34) (antisense primer corresponding to positions 1005 to 1025 in SEQ ID NO: 2).

Using 1/10 volume of each of the synthesized cDNA solutions as templates, 1 µM of each of the synthesized rTPO-I and rTPO-N as primers, and GeneAmp ^™ PCR Reagent Kit with AmpliTaq ^™ DNA polymerase (Takara Shuzo Co. , Ltd.), using GeneAmp ^TM PCR System 9600 (manufactured by PERKIN-ELMER) to perform PCR in a volume of 100 μl, heating at 95°C for 2 minutes, and repeating 30 cycles, each cycle including denaturation at 95°C for 1 minute, heating at 57°C Annealing at 72°C for 1 minute and synthesis at 72°C for 1 minute, followed by a final incubation at 72°C for 7 minutes. When each of the resulting reaction solutions was electrophoresed with 2% agarose gel (manufactured by FMCBioProducts) and the amplified bands were detected, it was found that TPO mRNA specific sex strips. These results indicate that expression of TPO mRNA in rats occurs in these organs, although the amount of expression cannot be determined. When taking into account the possibility of a similar expression pattern in humans and the results of Example 4, it appears that liver is a suitable starting material for obtaining human TPO cDNA. <Example 13> Construction of cDNA library derived from human normal liver

Based on the results of Example 12, liver was selected as a starting material for cloning human TPO cDNA. ^According to the same method as described in Example 7, poly(A) ⁺ RNA (produced by Clontech Production, a product based on the acid guanidine phenol chloroform method of Chomczynski et al.) synthesized a double-stranded cDNA with an EcoRI recognition site at its 5' end and a NotI recognition site at its 3' end. The synthesized cDNA was ligated with 1.2 μg of the aforementioned expression vector pEF18S that had been digested with EcoRI and NotI in advance, and transformed into 8.4 ml of the aforementioned highly competent E.coli DH5 (manufactured by Toyobo Co., Ltd.) middle. As a result, 1.2 x 10 ⁶ transformants were obtained. <Example 14> Preparation (cloning) of human TPO cDNA fragment by PCR

Using random primers, cDNA first strand was synthesized from μg of commercially available normal human liver-derived poly(A) ⁺ RNA (manufactured by Clontech). That is, 1 μg poly(A) ⁺ RNA was dissolved in 10 μl sterile water, incubated at 70° C. for 15 minutes, and then rapidly cooled. 75pmoles of random primers (Takara Shuzo Co., Ltd), 10U RNase inhibitor (Boehringer-Mannheim Corp.), 50mM Tris-Hcl (pH8.3), 75mM KCl, 3mM MgCl ₂ and 200U reverse transcriptase SuperScript were added thereto ^TM II (Life Technologies). The resulting solution (total volume 20 μl) was incubated at 37°C for 1 hour, and the resulting reaction solution was incubated at 70°C for 10 minutes to inactivate the enzyme, and then stored at -20°C until use.

Primers for PCR were synthesized according to the rat TPO cDNA sequence (SEQ ID NO: 2). The sequence of the synthesized primer is as follows: rTPO-AIN: 5'-ATG GAG CTG ACT GAT TTG CTC-3' (SEQ ID NO: 35) (positions 173 to 193 in SEQ ID NO: 2) rTPO-N: 5' - TGA AGT TCG TCT CCA ACA ATC-3' (SEQ ID NO: 36) (antisense primer corresponding to positions 1005 to 1025 in SEQ ID NO: 2).

Using 1/10 volume of the synthetic cDNA solution as a template, 1 µM of each of the synthesized rTPO-AIN and rTPO-N as primers, and GeneAmp ^™ PCR Reagent Kit with AmpliTaq ^™ DNA Polymerase (manufactured by Takara Shuzo Co., Ltd.), using GeneAmp ^™ PCR System 9600 (manufactured by PERKIN-EIMER) performed PCR in a volume of 100 μl, heated at 95° C. for 2 minutes, and repeated 35 cycles, each cycle including denaturation at 95° C. for 1 minute, annealing at 40° C. for 1 minute, and heating at 72° C. °C for 1 minute, and finally incubated at 72°C for 7 minutes.

The resulting reaction solution was subjected to 2% agarose gel (FMC BioProducts) electrophoresis to isolate a DNA fragment of about 620bp as the main product of PCR, which was then purified using the aforementioned Prep-A-Gene DNA purification kit (Bio-Rad Laboratories.Inc.) for purification. The nucleotide sequence of the purified DNA fragment was directly determined by a 373A DNA sequencer (manufactured by Applied Biosystems) using the aforementioned Taq DyeDeoxy ^™ Terminatex Cycle Sequencing Kit (manufactured by Applied Biosystems). The determined nucleotide sequence of the exclusion primer portion and the amino acid sequence deduced therefrom are shown in SequenceListing (SEQ ID NO: 3).

The DNA fragment excluding the primer sequence was 580 bp long. When compared, the human cDNA showed 80% homology to the nucleotide sequence of the rat cDNA, which indicated that this DNA fragment encoded a portion of the human TPO cDNA. <Example 15> Screening of human TPO cDNA clones by PCR

Primers corresponding to human TPO for PCR were synthesized based on SEQ ID NO:3. The synthesized primer sequence is as follows: hTPO-I: 5'-TTG TGA CCT CCG AGT CCT CAG-3' (SEQ ID NO: 37) (60th to 80th in SEQ ID NO: 3) hTPO-J: 5' - TGA CGC AGA GGG TGG ACC CTC-3' (SEQ ID NO: 38) (antisense primer corresponding to positions 479 to 499 in SEQ ID NO: 3).

The human cDNA library constructed in Example 13 was amplified, and divided into multiple small libraries (each containing about 100,000 clones), cultivated overnight in 1 ml of the aforementioned LB medium containing 50 μg/ml ampicillin, and used Plasmid DNA extraction was performed with an automatic plasmid isolation device PI-100 (VER-3.0, manufactured by Kurabo Industries, Ltd.). The extracted DNA was dissolved in TE solution.

Using 5% of the extracted DNA sample as a template, 1 µM of each of the synthesized oligonucleotides (hTPO-I and hTPO-J) as a primer, and GeneAmp ^™ PCR Reagent Kit with AmpliTaq ^™ DNA Polymerase (Takara Shuzo Co., Ltd. production), PCR was performed by GeneAmp ^™ PCR System 9600 in a volume of 20 μl (a total of 35 cycles, each cycle including denaturation at 95°C for 1 minute, annealing at 59°C for 1 minute and synthesis at 72°C for 1 minute, and finally at 72°C incubation for 7 minutes). As a result, specific bands were detected in 3 of the 90 small pools used. One of the 3 small pools was divided into subpools, each containing approximately 5,000 clones, and plasmid DNA was purified from the 90 subpools to perform PCR in the same manner. As a result, specific bands were detected in 5 sub-pools. One of these 5 small pools was subdivided into small pools, each containing 250 clones, and plasmid DNA was extracted from 90 small pools. When these extracted samples were subjected to PCR in the same way, specific bands were detected in three small pools. One of the three small pools was further divided into sub-pools, each containing 30 clones. Plasmid DNA was purified from 90 sub-pools, and PCR was carried out in the same way. As a result, specific bands were detected in 3 subpools. One of the candidate small pools was cultured on the aforementioned LB plate containing 50 μg/ml of ampicillin, and each of the 90 colonies formed was subjected to plasmid DNA extraction and PCR in the same manner. As a result, clone HL34 was finally obtained. <Example 16> Sequencing of human TPO cDNA

Purification of plasmid DNA was performed essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Clone HL34 was cultured overnight in 50 ml LB medium containing 50 μg/ml ampicillin, and the cells collected by centrifugation were suspended in 4 ml of the TEG-lysozyme solution described above. Add 8ml of 0.2N NaOH/1% SDS solution to it, and then add 6ml of 3M potassium/5M acetate solution to completely suspend the cells. After the suspension was centrifuged, the resulting supernatant was treated with phenol/chloroform (1:1), mixed with an equal volume of isopropanol, and centrifuged. The resulting pellet was dissolved in TE solution and treated with RNase, then treated with phenol/chloroform (1:1), and precipitated with ethanol. The resulting precipitated pellet was redissolved in TE solution, followed by the addition of NaCl and polyethylene glycol 3,000 to their final concentrations of 0.63M and 7.5%, respectively. After centrifugation, the pellet was dissolved in TE solution and precipitated with ethanol. In this way, about 300 µg of pEF18S-HL34 plasmid DNA was obtained.

The purified plasmid DNA was applied to the aforementioned 373A DNA sequencer (manufactured by Applied Biosystems) to determine its complete nucleotide sequence using the aforementioned Taq Dye Deoxy ^™ TerminaterCycle Sequencing Kit (manufactured by Applied Biosystems). The measured nucleotide sequence and the amino acid sequence deduced therefrom are shown in SequenceListing (SEQ ID NO: 4). In this example, an oligonucleotide synthesized based on the nucleotide sequence of SEQ ID NO: 3 and a synthetic oligonucleotide designed based on an internal sequence obtained by sequence analysis were used as primers in nucleotide sequencing.

The result confirmed that the plasmid clone pEF18S-HL34 comprises a cDNA fragment of 861bp, and contains the amino acid sequence AP8 (the first to the 12th amino acid in SEQ ID NO: 4) and TP2/TP3 (SEQ ID NO: The 157th to 162nd amino acid in 4) have highly homologous sequences. This DNA fragment appears to encode an open reading frame starting at position 25, but without a stop codon, and contains a 76-base poly-A tail sequence at its 3' end. Its amino acid sequence containing 253 amino acid residues just before the poly A tail sequence showed 84% identity with the corresponding part of rat TPO cDNA (the amino acid sequence part including 147 residues shown in SEQ ID NO: 2) homology. As a result, the clone was found to be a DNA fragment encoding a human cDNA portion corresponding to rat TPO cDNA. Due to the absence of a stop codon and the presence of a poly-A tail sequence at its 3' end, it appears that this clone is not a complete cDNA but an artifact generated during cDNA library construction. <Example 17> expressing human TPO cDNA in COS1 cells and confirming TPO activity

According to the method of Example 11, the obtained plasmid clone pEF18S-HL34 was transfected into COS1 cells. That is, transfection was performed with 10 µg of plasmid DNA by the DEAE-dextran method including chloroquine treatment. COS1 cells were cultured at 37°C for 3-5 days, and then the supernatant was collected.

The resulting culture supernatant was extensively dialyzed against IMDM medium and evaluated with the rat CFU-MK assay system. In the culture supernatant of COS1 cells expressing the plasmid pEF18S-HL34, TPO activity was detected in a dose-dependent manner (Fig. 8). After 4 days in culture, many megakaryocytes formed growing cytoplasmic protrusions. In contrast, no TPO activity was found in the culture supernatant of COS1 cells expressing only pEF18S (Fig. 8). In the M-07e assay system, M-07e cell proliferation enhancing activity was found only in the culture supernatant of COS1 cells expressing it by transfection of the plasmid pEF18S-HL34. These results indicate that pEF18S-HL34 contains a gene encoding a protein having TPO activity. Additionally, this also indicates that human TPO acts on rat megakaryocyte progenitors (no species specificity). <Example 18> Expression of human TPO-deleted cDNA and confirmation of TPO activity

The clone HL34 obtained in Example 15 contains a cDNA with a poly-A tail-like continuous sequence at its 3' end, which does not exist in the rat TPO cDNA, so it seems to be an artificial product of the experiment. Results A cDNA molecule with the poly A tail sequence removed was prepared to see if the protein expressed from the deleted cDNA had TPO activity. The deletion cDNA was prepared by PCR. The primer sequences used for PCR were as follows, and a restriction enzyme recognition site was added to the 5' end of each primer (EcoRI was added to hTPO5, NotI and two stop codons TAA and TGA were added to hTPO3). hTPO5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3' (SEQ ID NO: 170) (positions 1 to 21 of SEQ ID NO: 4) hTPO3: 5'-TTT GCG GCC GCT CAT TAT TCG TGT ATC CTG TTCAGG TAT CC-3' (SEQ ID NO: 171) (antisense primer corresponding to positions 757 to 780 in SEQ ID NO: 4).

Using 1 μg of plasmid DNA of the plasmid clone pEF18S-HL34 obtained in Example 16 as a template, 10 μM each of the synthesized hTPO5 and hTPO3 as primers, and GeneAmp ^™ PCR Reagent Kit with AmpliTaq ^™ DNA Polymerase (manufactured by Takara Shuzo Co., Ltd. ), using GeneAmp ^™ PCR System 9600 (manufactured by PERKIN-ELMER), PCR was performed in a volume of 100 μl, repeating 15 cycles, each cycle including denaturation at 95°C for 1 minute, annealing at 65°C for 1 minute and synthesis at 72°C for 1 minute , and finally incubated at 72°C for 7 minutes. The obtained band of about 800 bp was digested with restriction enzymes EcoRI and NotI, purified, and then subcloned into expression vector pEF18S previously treated with the same restriction enzymes. Five clones containing a DNA fragment of about 800 bp were selected from the resulting transformants, and a large amount of plasmid DNA was prepared according to the method described in Example 5. The full length of the amplified region of about 800 bp of each plasmid was subjected to nucleotide sequence analysis to find that it was completely consistent with the nucleotide sequence analyzed in Example 16 (positions 1 to 780 in SEQ ID NO: 4) sex.

The obtained plasmid clone was designated pHT1-231. The carrier pHT1-231 carried by E.coli DH5 has been preserved by the inventor on February 14, 1994 in the National Institute of Bioscience and Human Technology, Department of Industrial Science and Technology, Ministry of International Trade and Industry of Japan, and its preservation number is FERM BP-4564.

The obtained plasmid was transfected into COS1 cells according to the method in Example 11. That is, transfection was performed with 10 µg of plasmid DNA by the DEAE-dextran method including chloroquine treatment. COS1 cells were cultured at 37°C for 3-5 days, and the supernatant was collected.

The resulting culture supernatant was extensively dialyzed against IMDM medium and evaluated using the rat CFU-MK assay system. TPO activity was found in the culture supernatant of COS1 cells expressing plasmid pHT1-231 (Figure 9). During 4 days in culture, many megakaryocytes formed growing cytoplasmic processes. In contrast, no TPO activity was found in the culture supernatant of COS1 cells expressing only the plasmid pEF18S (Fig. 9). In the M-07e assay system, M-07e cell proliferation enhancing activity was found only in the culture supernatant of COS1 cells expressing plasmid pHT1-231. These results show that pHT1-231 contains a cDNA encoding TPO activity. <Example 19> Prepare human TPO cDNA 3' end region by PCR

Since the clone HL34 prepared in Example 15 contained a cDNA containing a poly(A) tail sequence, this indicated that its 3' end was incomplete. An attempt was therefore made to obtain the full length 3' terminal region by PCR. Based on the sequence determined in Example 16, the following 4 primers on the 5' side for PCR were synthesized: hTPO-H: 5'-AGC AGA ACC TCT CTA GTC CTC-3' (SEQ ID NO: 39) (SEQ ID NO: 574-594 of 4) hTPO-K: 5'-ACA CTG AAC GAG CTC CCA AAC-3' (SEQ ID NO: 40) (SEQ ID NO: 595-615 of 4) hTPO- N: 5'-AAC TAC TGG CTC TGG GCT TCT-3' (SEQ ID NO: 41) (SEQ ID NO: 660-680 of 4) hTPO-O: 5'-AGG GAT TCA GAG CCA AGA TTC- 3' (SEQ ID NO: 42) (positions 692-712 of SEQ ID NO: 4)

The following 3' side primers containing mixed nucleotides at four bases at the 3' end were synthesized to amplify cDNA starting from the poly(A) portion. Anchor primers were also synthesized without mixed nucleotides. hTPO 3 mix: 5'-TAG CGG CCG C(T) ₁₇ G GGG-3' (SEQ ID NO: 43)

A AAA-3' (SEQ ID NO: 44)

C TTT-3' (SEQ ID NO: 45)

CCC-3' (SEQ ID NO: 46) hTPO 3 anchor: 5'-TAG CGG CCG C(T) ₁₁ -3' (SEQ ID NO: 47)

As in Example 14, cDNA first-strand strands were synthesized from 1 µg of commercially available normal human liver-derived poly(A) ⁺ RNA (manufactured by Clontech), except that 0.5 µg of the oligo dT primer (which was included in the in the TimeSaver ^™ cDNA Synthesis Kit produced by Pharmacia). Using 1/10 volume of the synthetic cDNA solution as a template, 20 µM of hTPO-H primer and 10 µM of hTPO 3mix primer, and GeneAmp ^™ PCR Reagent Kit with AmpliTaq ^™ DNA Polymerase (manufactured by Takara Shuzo Co., Ltd.), using GeneAmp ^™ PCR System 9600 (PERKIN-ELMER) performed PCR in a volume of 50 μl, and heated at 96°C for 2 minutes, repeating 10 cycles, each cycle including heat denaturation at 96°C for 1 minute, annealing at 48°C for 1 minute and heating at 72°C Synthesis was performed for 1 min, followed by a final incubation at 72°C for 7 min.

Use 1/10 volume of the solution formed by the first PCR as a template, 50 μl of 20 μM hTPO-K primer and 10 μM hTPO 3mix for the second PCR, heat at 96 ° C for 2 minutes, repeat 10 cycles, each Each cycle consisted of heat denaturation at 96°C for 1 minute, annealing at 63°C for 1 minute and synthesis at 72°C for 1 minute, and finally incubation at 72°C for 7 minutes.

Use 1/10 of the volume of the second PCR formation solution as a template, 20mM hTPO-N primers and 10μM hTPO 3mix primers in a volume of 50μl for the third PCR, heat at 96°C for 2 minutes, repeat 10 cycles, each cycle includes Heat denaturation at 96°C for 1 minute, annealing at 63°C for 1 minute, synthesis at 72°C for 1 minute, and finally incubation at 72°C for 7 minutes.

Use 1/10 volume of the third PCR forming solution as a template, 20 μM hTPO-O primer and 10 μM hTPO 3mix primer in a volume of 50 μl for the fourth PCR, heat at 96 ° C for 2 minutes, repeat 10 cycles, each cycle This included heat denaturation at 96°C for 1 minute, annealing at 63°C for 1 minute, synthesis at 72°C for 1 minute, and a final incubation at 72°C for 7 minutes.

Use 1/10 volume of the fourth PCR forming solution as template, 50 μl 20 μM hTPO-O primer and 20 μM hTPO 3 anchor primer to carry out the fifth PCR, heat at 96 ° C for 2 minutes, repeat 10 cycles, each cycle includes Heat denaturation at 96°C for 1 minute, annealing at 58°C for 1 minute, synthesis at 72°C for 1 minute, and finally incubation at 72°C for 7 minutes.

The resulting reaction solution was subjected to 2% agarose gel electrophoresis (manufactured by FMC Bio-Products) to separate a DNA fragment of about 600 bp as the main product of PCR, which was then purified using the aforementioned Prep-A-Gene DNA Purification Kit (Bio-Products). -Rad Laboratories, Inc.) for purification. The nucleotide sequence of the purified DNA fragment was directly determined by a 373A DNA sequencer (manufactured by Applied Biosystems) using the Taq Dye Deoxy ^™ Terminateer CycleSequening Kit (manufactured by Applied Biosystems) described above. The determined nucleotide sequence and the amino acid sequence deduced therefrom are shown in Sequence Listing (SEQ ID NO: 5).

This DNA fragment has a nucleotide sequence encoding 130 amino acids from primer hTPO-O followed by a sequence of more than 180 nucleotides at the 3' end (nucleotides after position 577 cannot be determined) . The 30 amino acid sequence from glycine is consistent with the 203-232 amino acid sequence in SEQ ID NO:4. The 1-94th nucleotide sequence is also consistent with the 692-785th nucleotide sequence in SEQID NO:4.

The sequence analysis results of the cDNA fragment in Example 17 and the fragment amplified by PCR in this example make it reasonable to estimate that the human TPO protein consists of 353 amino acids containing a 21 amino acid signal sequence shown in SEQ ID NO:6. composition. <Example 20> Construction of cDNA library derived from human normal liver

Since the clone HL34 obtained in Example 15 contained a poly A tail sequence directly on the 3' end of its open reading frame without a stop codon and seemed thus to be an artifact of cDNA synthesis, 5 μg of commercially available human liver-derived A poly(A) ⁺ RNA preparation (manufactured by Clontech) was used to reconstruct a cDNA library. cDNA synthesis was performed with SuperScript ^™ LambdaSystem and λ Cloning Kit for cDNA synthesis and SuperScript ^™ II RNase H ^- (both produced by LIFETECHNOLOGIES). Poly(A) ⁺ RNA was subjected to thermal denaturation, and then added to 20 μl of the reaction solution (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl ₂ , 1mM DTT, 1mM dNTP mix, 200U SuperScript ^TM II RNase H ^- ), and then incubated at 37°C for 60 minutes. After the second strand of cDNA was synthesized (incubate at 16°C for 2 hours in a 150 μl reaction solution containing 25 mM Tris-Hcl (pH 8.3), 100 mM KCl, 5 mM MgCl ₂ , 250 μM dNTP mix, 5 mM DTT, 40 U E.coli DNA polymerase I, 2U E.coli RNase H and 10U E.coli DNA ligase), 10U T4 DNA polymerase was added, and the resulting mixture was incubated at 16°C for 5 minutes. The reaction solution was heated at 65° C. for 10 minutes, extracted with the same volume of phenol/chloroform and used with a SizeSep ^™ 400 spin column (a spin column attached to the TimeSaver ^™ cDNA Synthesis Kit produced by Pharmacia for removal of low-molecular-weight DNA) cDNA molecules less than 400bp in length were removed. After adding EcoRI adapter (attached to Directional Cloning Toolbox produced by Pharmacia), the treated samples were digested with NotI and reloaded on SizeSep ^™ 400 spin column to remove low molecular weight DNA. Ligate the synthesized 1.3 μg double-stranded cDNA with EcoRI recognition sequence at its 5' end and NotI recognition sequence at its 3' end with the expression vector pEF18S digested with EcoRI and NotI, and then transform into 9.2ml high competent E.coli DH5 (manufactured by Toyobo Co., Ltd.). The obtained human liver cDNA library (hTPO-F1) contained 1.0×10 ⁶ transformants. <Example 21> Screening of TPO cDNA clones from human liver cDNA library hTPO-F1

The corresponding primers for human TPO cDNA for PCR were synthesized based on Sequence ID Nos: 3 and 6 shown in SEQUENCE LISTING. The sequence of the synthesized primer is as follows: hTPO-I: 5'-TTG TGA CCT CCG AGT CCT CAG-3' (SEQ ID NO: 48) (60-80 in SEQ ID NO: 3) hTPO-KU: 5' - AGG ATG GGT TGG GGA AGG AGA-3' (SEQ ID NO: 49) (antisense primer corresponding to sequence 901-921 in SEQ ID NO: 6).

The human liver cDNA library hTPO-F1 (1.0×10 ⁶ transformants) constructed in Example 20 was divided into 3 small pools (bank numbers 1-3), and the small pools were frozen. PCR was carried out using plasmid DNA prepared from each small pool as a template and 1 µM of each of the synthesized oligonucleotides (hTPO-I and hTPO-KU) as primers. Using GeneAmp ^™ PCRReagent Kit with AmpliTaq ^™ DNA Polymerase (manufactured by Takara Shzuo Co., Ltd.), PCR was performed in a volume of 100 µl by GeneAmp ^™ PCR System9600 (manufactured by PERKIN-ELMER) (35 cycles in total, each cycle including Denaturation for 1 minute, annealing at 59°C for 1 minute and synthesis at 72°C for 1 minute; final incubation at 72°C for 7 minutes). As a result, when the plasmid DNA prepared from the small pool No. 3 was used, a DNA fragment having the desired size was amplified. Then the No. 3 small pool was divided into sub-small pools, each sub-small pool contained 15,000 transformants, these sub-small pools were cultured overnight in 1 ml LB medium containing 50 μg/ml ampicillin, and then the automatic plasmid isolation device P1 -100 to extract plasmid DNA. The extracted DNA was dissolved in TE solution, and 5% of the resulting solution was used as a template to perform PCR using the same primers as described above and under the same conditions. As a result, amplification of DNA with the expected size was found in 6 of 90 small pools. When one of these positive pools was subdivided into sub-pools so that each sub-pool contained 1,000 clones, plasmid DNA was extracted and PCR was performed in the same manner as described above, and no amplification of DNA was observed. On electrophoretic gels of DNA fragments amplified by serial PCR, the density of bands becomes thinner as subpools are further subdivided. This appears to be due to low recovery of plasmid DNA due to poor production of the clone of interest. Therefore, another screen was performed using the colony hybridization method using the original library No. 3.

Small library No. 3 was spread on 100 LB agar plates with a diameter of 15 cm, and the inoculation strength was such that 4,100 colonies were grown on each LB agar plate. After preparing a duplicate plate from each inoculated plate, one of the duplicate plates was incubated at 37°C for 6 hours to recover colonies growing on the plate and to extract a plasmid DNA sample. When these DNA samples were subjected to PCR in the same manner as described above, amplification of a band equivalent to the expected size was observed in one of 100 subpools. Two replicate filters were made from the sub-pool plate using BIODYNE ^(TM) A TRANSFER MEMBRANE (manufactured by PALL). Denaturation of these filters was performed by sequentially immersing them in 10% SDS for 10 min, 0.5N NaOH/1.5M NaCl for 10 min, and 0.5M Tris-Hcl (pH 8.0)/1.5M NaCl for 10 min, followed by Air-dried for 30 minutes, and then baked in a vacuum oven at 80°C for 1 hour. The baked filter was rinsed with 6*SSC (prepared by diluting a 20*SSC stock solution consisting of 175.3 g NaCl and 88.2 g sodium citrate in 1 liter of water) supplemented with 1% SDS. Pre-hybridization of the washed filter was carried out by incubating at 42°C for 30 minutes while shaking in a 30 ml reaction solution consisting of 50% formamide, 5×SSC, 5×Denhardt's solution (derived from Prepared in 50×Denhardt's solution containing 5g Ficoll, 5g polyvinylpyrrolidone and 5g bovine serum albumin fraction V in 500ml water), 1% SDS and 20μg/ml salmon sperm DNA. After prehybridization, the reaction solution was replaced with 30 ml of a hybridization solution containing the same components, mixed with a probe labeled with [α- ^32P ]dCTP (manufactured by Amersham), and then incubated at 42°C for 20 hours with shaking. The labeled probe used in this experiment was the EcoRI/BamHI fragment of plasmid pEF18S-HL34, which was prepared by purifying the part from the 5' end to the 458th base of SEQ ID NO: 4 and labeling the purified part , and the labeling of the purified fraction was carried out by random primer technique using Megaprime DNA Labeling System (based on the method disclosed in Anal. Biochem., 132, 6-13, 1983, kit produced by Amersham). The treated filters were rinsed in 2×SSC/0.1% SDS solution at 42°C for 30 minutes, then in 0.2×SSC/0.1% SSD solution at 42°C for 30 minutes. The filter was then subjected to autoradiography at -70°C for 16 hours using an intensifying screen and X-OMAT ^™ AR5 film (manufactured by Eastman Kodak). As a result, a single signal considered positive was observed. Colonies approximately corresponding to this signal were collected from the original plate and replated on 10 cm LB agar plates. Fifty colonies grown on the plate were respectively cultured, and DNA samples of these colonies were subjected to PCR under the same conditions as described above using the aforementioned primers hTPO-I and hTPO-KU. As a result, amplification of a band equivalent to the expected size was found in only one clone, called pHTF1. <Example 22> Determination of the nucleotide sequence of human TPO cDNA clone pHTF1

Plasmid DNA was purified essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). The clone pHTF1 was grown overnight in LB medium containing 50 μg/ml ampicillin. The resulting cells were collected by centrifugation, and then suspended in 4 ml of the aforementioned TFG-lysozyme solution. To this solution was added 8 ml of 0.2N NaOH/1% SDS solution, followed by 6 ml of 3M potassium/5M acetic acid solution to completely suspend the cells. After the suspension was centrifuged, the resulting supernatant was treated with phenol/chloroform (1:1), mixed with an equal volume of isopropanol, and centrifuged. The obtained particles were dissolved in TE solution, then treated with RNase and phenol/chloroform (1:1), followed by ethanol precipitation. The resulting precipitate was redissolved in TE, and then NaCl and polyethylene glycol 3,000 were added thereto to make final concentrations of 0.63M and 7.5%, respectively. After centrifugation, the pellet was dissolved in TE solution and precipitated with ethanol. In this way, 300 µg of plasmid DNA pHTF1 was obtained.

The plasmid DNA thus purified was used in the above-mentioned 373A DNA sequencer (manufactured by Applied Biosystems) to determine the entire nucleotide sequence using the above-mentioned Taq Dye Deoxy ^™ Terminateer CycleSequening Kit (manufactured by Applied Biosystems). The thus determined nucleotide sequence and deduced amino acid sequence are listed in the Sequence Listing (SEQ ID NO: 7). In the determination of the nucleotide sequence, oligonucleotides synthesized based on the nucleotide sequence of SEQ ID NO: 6 and the internal sequence obtained by its sequence reaction analysis were used as primers for the determination of the nucleotide sequence.

As a result, it was confirmed that the clone pHTF1 contained a cDNA fragment of 1.721bp, and was consistent with the amino acid sequence AP8 (amino acid numbers 1 to 12 in SEQ ID NO: 7) and TP2/TP3 (amino acid in SEQ ID NO: 7) analyzed in Example 2. Numbers 157 to 162) have a high degree of homology. This DNA fragment appears to contain a 101 base 5' non-coding region, an open reading frame consisting of 353 amino acid residues (starting with the Met residue encoded at nucleotides 102 to 104, nucleotide-encoded Gly residue termination), followed by a stop codon (TAA), a 531-base 3' non-coding region, and a 30-base polyA tail sequence, the amino acids thought to be encoded by the open reading frame The sequence is completely consistent with the predicted human TPO amino acid sequence shown in SEQ ID NO:6. The cDNA sequence of pHTF1 is larger than the deduced cDNA sequence shown in SEQ ID NO: 6, containing 77 additional bases on the 5' side and 347 bases on the 3' side before its polyA tail sequence. And the nucleotide sequence differs from SEQ ID NO: 6 at 3 positions. That is, A (position 84) in SEQ ID NO: 7, A (position 740) and G (position 1198) are respectively C, T and A in SEQ ID NO: 6. Only the mutation at position 740 is included in the protein coding region, but since both A and T are the third base of the Thr codon, this mutation will not cause an amino acid change. Although the reason for these base substitutions was not clear at the time, analysis of the plasmid clone pHTF1 confirmed that the human TPO protein consists of 353 amino acid residues with a 21 amino acid signal sequence. The estimated molecular weight of the mature protein after removal of the signal sequence is 35,466.

The carrier pHTF1 carried by E.coli strain DH5 was deposited by the inventor on March 24, 1994 under the accession number No. FERM BP-4617 in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan. <Example 23> expressing human TPO cDNA clone in COS1 cells and confirming TPO activity

According to the method of Example 11, COS1 cells were transfected with the thus obtained plasmid clone pHTF1, that is, through the DEAE-dextran method including chloroquine treatment, and 10 μg of plasmid DNA was used to complete the transfection. The transfected COS1 cells were cultured at 37°C for 3 days, and then the culture supernatant was collected.

The culture supernatant was extensively dialyzed against IMDM medium and then assessed with the rat CFU-MK detection system. TPO activity was detected in a dose-dependent manner in supernatants of COS1 cells expressing plasmid pHTF1 ( FIG. 10 a ). In contrast, there was no TPO activity in the culture supernatant of COS1 cells expressing only plasmid pEF18S (Fig. 10a). Similar results were obtained with the M-07e detection system. The supernatant of COS1 cells expressing the plasmid pHTF1 in it significantly increased the proliferation of M-07e cells in a dose-dependent manner (Fig. 10b). These results indicate that pHTF1 contains a gene encoding a protein having TPO activity. <Example 24> Cloning of human TPO chromosomal DNA

Human TPO chromosomal DNA was cloned using human TPO cDNA as a probe. The genome library used in the cloning was donated by Professor T. Yamamoto from the Gene Research Center, Tohoku University (Sakai et al reported the library in J. Biol. Chem., 269, 2173-2182, 1994, by using It was constructed by partially digesting human chromosomal DNA with restriction enzyme Sau 3AI, and then ligating the partially digested product to the BamHI site of phage vector λEMBL 3 manufactured by Stratagene). The library was screened using human TPO cDNA as a probe essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Using E.coli LE392 as _a host, inoculate _the library on 15- cm plates, one plate contains 30,000 phage particles at this inoculum size. Two replica filters were prepared from each of the 18 plates thus prepared using BIODYNETMA TRANSFER MEMBRANE (manufactured by PALL). Filter membranes were soaked in 0.5N NaOH/1.5M NaCl for 10 minutes, then in 0.5M Tris-Hcl (pH 8.0)/1.5M NaCl for 10 minutes, then air-dried for 30 minutes, and then dried in a vacuum oven at 80 °C for 1.5 hours to denature it. The filter thus treated was incubated at 42°C for 1 hour in a 500 ml reaction solution containing 50% formaldehyde, 5 x SSC, 5 x Denhardt's solution, 1% SDS and 20 µg/ml salmon sperm DNA to complete prehybridization. In order to be used as a probe, a human TPO cDNA fragment (bases 178 to 1,025 in SEQ ID NO: 7) was amplified by PCR, and then purified, using the Random Primer DNALabelling kit (by Takara Shuzo in Anal. Biochem, 132, 6-13 , DNA labeling cassette produced by the random primer method disclosed in 1983) with ³² P-labeled purified fragments. In this PCR, the primer sequences used were as follows: hTPO-I: 5'-TTG TGA CCT CCG AGT CCTCAG-3' (SEQ ID NO: 50) (positions 178 to 198 in SEQ ID NO: 7) hTPO-N : 5'-AGG GAA GAG CGT ATA CTG TCC-3' (SEQ ID NO: 51) (antisense primer corresponding to the sequence from position 1,005 to 1,025 in SEQ ID NO: 7).

Using isotope-labeled probes, hybridize at 42° C. for 20 hours in 500 ml of reaction solution containing the same components as the prehybridization solution. The obtained filter membrane was washed three times with 2×SSC/0.1% SDS solution for 5 minutes at room temperature, and then washed once with 0.1×SSC/0.1% SDS solution for 1 hour at 68°C. The filter was then subjected to autoradiography at -70°C for 16 hours using an intensifying screen and X-OMAT ^(TM) AR5 film (manufactured by Eastman Kodak). As a result, 13 positive signals were obtained. Plaques approximately equivalent to each positive signal were collected on the original plate and re-plated on 15-cmNZYM plates at an inoculation size of 1,000 plaques per plate. Two replica membranes were prepared from each resulting plate to perform hybridization under the same conditions as above. As a result, positive signals were detected on all filters of the 13 groups. One plaque was recovered from each resulting plate to prepare phage DNA using the plate lysate method described in Molecular Cloning. The phage DNA samples thus prepared from the 13 clones were examined for the presence or absence of the cDNA coding region by PCR using primers containing the following sequences. hTPO-L: 5'-GGC CAG CCA GAC ACC CCG GCC-3' (SEQ ID No: 52) (positions 1 to 21 in SEQ ID NO: 6) hTPO-F: 5'-ATG GGA GTC ACG AAG CAG TTT-3' (SEQ ID No: 53) (antisense primer corresponding to sequence 127 to 147 in SEQ ID NO: 6) hTPO-P: 5'-TGC GTT TCC TGA TGC TTG TAG-3' (SEQ ID No: 54) (positions 503 to 523 in SEQ ID NO: 6) hTPO-V: 5'-AAC CTT ACC CTT CCT GAG ACA-3' (SEQ ID No: 55) (equivalent to Antisense primers for sequences at positions 1,070 to 1,090)

When PCR was performed with these primer combinations, 5 of the 13 clones appeared to contain the complete amino acid coding region predicted from the cDNA. The chromosomal DNA molecules contained in these 5 clones have a similar length of about 20kb, and preliminary restriction enzyme analysis shows that the structure is almost the same. Therefore, one such clone (clone λHGT1) was screened and then analyzed by Southern blot. That is, µg DNA of clone λHGT1 was completely digested with restriction enzyme EcoRI or Hind III, subjected to 0.8% agarose gel electrophoresis, and then the band on the gel was transferred to BIODYNE ^™ ATRANSFER MEMBRANE (manufactured by PALL). The resulting filter membrane was air dried for 30 minutes and then baked in a vacuum oven at 80°C for 2 hours. The pretreatment was accomplished by incubating the filter thus treated at 42°C for 1 hour in 50 ml of a reaction solution containing 50% formaldehyde, 5×SSC, 5×Denhardt’s solution, 1% SDS and 20 μg/ml salmon sperm DNA. hybridize. For use as a probe, a human TPO cDNA fragment (bases 178 to 1,025 in SEQ ID NO: 7) was amplified by PCR, purified, and labeled with ³² P using a Random Primer DNA Labeling Kit (manufactured by Takara Shuzo) Purified fragments. Using the isotope-labeled probe, hybridization was carried out at 42° C. for 20 hours in 50 ml of a reaction solution containing the same components as the prehybridization solution. The resulting filter was washed three times with 2×SSC/0.1% SDS solution for 5 minutes at room temperature, and then washed once more with 0.1×SSC/0.1% SDS solution for 1 hour at 68°C. The filter was then subjected to autoradiography at -70°C for 16 hours using an intensifying screen and X-OMAT ^™ AR5 film (manufactured by Eastman Kodak). As a result, in the case of Hind III digestion, a single band of about 10 kb was observed. Therefore, 10 µg of DNA of clone λHGT1 was digested with Hind III, followed by electrophoresis on 0.8% agarose gel, and a 10 kb band was excised from the gel, purified using Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad), Then, it was subcloned into cloning vector pUC13 (manufactured by Pharmacia) which had been digested with Hind III in advance. In this case, highly competent E. coli DH5 produced by TOYOBO was used as the host strain.

Among the obtained clones, a clone containing a 10 kb Hind III fragment, called pHGT1, was screened.

The restriction map of phage clone λHGT 1 is shown in Figure 11. <Example 25> Determine the nucleotide sequence of human TPO chromosome clone pHGT1

Clone pHGT1 was grown and plasmid DNA was purified essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). Clone pHGT1 was cultured overnight in 50 ml LB medium containing 50 μg/ml ampicillin. The resulting cells were collected by centrifugation, and then suspended in 4 ml of the aforementioned TEG-lysozyme solution. To this solution, 8ml of 0.2N NaOH/1% SDS solution was first added, and then 6ml of 3M potassium/5M acetic acid solution was added to completely suspend the cells. After the suspension was centrifuged, the resulting supernatant was treated with phenol/chloroform (1:1), mixed with the same volume of isopropanol, and then centrifuged. The resulting particles were dissolved in TE solution and treated with RNase, then phenol/chloroform (1:1), followed by ethanol precipitation. The obtained particles were redissolved in TE solution, and then NaCl and polyethylene glycol 3,000 were added thereto so that the final concentrations thereof became 0.63M and 7.5%, respectively. After centrifugation, the particles were dissolved in TE solution and precipitated with ethanol. By this method, about 300 µg of plasmid DNA pHGT1 was obtained.

Using the above-mentioned Taq Dye Deoxy ^™ Terminateer Cycle Sequencing Kit (manufactured by Applied Biosystems), the plasmid DNA thus purified was used in the above-mentioned 373A DNA sequencer (manufactured by Applied Biosystems) to determine the protein-coding sequence predicted from the cDNA nucleotide sequence. Nucleotide sequences surrounding the region. The thus determined nucleotide sequence and deduced amino acid sequence (SEQ ID NO: 8) are listed in the Sequence Listing. In this case, the oligonucleotide used for cDNA nucleotide sequence analysis in Example 22 and the synthetic oligonucleotide designed on the basis of the internal sequence obtained by its sequence reaction analysis were used as the nucleotide sequence determination primers in .

As a result, it was found that the chromosomal DNA carried by the plasmid clone pHGT1 contained the entire coding region of the amino acid sequence deduced from SEQ ID NO:6, and that the nucleotide sequence of the coding region was completely identical to that of SEQ ID NO:6. In addition, the region corresponding to the amino acid-coding exon counted from the 5' side contained four introns with lengths of 231 bp, 286 bp, 1932 bp and 236 bp (Fig. 11). There is also a nucleotide sequence different from the cDNA nucleotide sequence of SEQ ID NO: 7 at 3 positions, and the 3 positions are identical to those described in Example 22 (between SEQ ID NO: 6 and 7 different locations). That is, A (position 84), A (position 740) and G (position 1198) in SEQ ID NO: 7 are C, T and A in SEQ ID NO: 8, respectively. Thus, it was revealed that the nucleotide sequence of the human TPO cDNA clone pHTF1 obtained in Example 21 was different from the nucleotide sequence of the human chromosomal DNA clone pHGT1 at 3 positions. Therefore, in order to analyze whether these mutations in the cDNA clone pHTF1 sequence reflected the chromosomal DNA sequence, the nucleotide sequences of the other 4 clones among the 5 chromosomal clones obtained individually by screening were determined. Sequence analysis was performed by direct nucleotide sequencing using phage DNA samples prepared according to the plate lysate method described in Molecular Clonmg. With the sequence primer used in Example 22 (synthesized on the basis of the sequence of the aforementioned 3 mutation positions in the nucleotide sequence that can be analyzed), through the aforementioned 373A DNA sequencer (manufactured by Applied Biosystems) and the aforementioned Taq Dye Deoxy ^™ TerminaterCycle Sequencing cartridge (manufactured by Applied Biosystems) performed sequencing of each clone. The nucleotide sequence of all four clones was found to be identical to that of SEQ ID NO:6. In these 4 clones, the nucleotides corresponding to positions 84 and 740 of SEQ ID NO: 7 were substituted by C and T, respectively. But at position 1,198, there is a G in two clones and an A in the other two clones. In other words, this indicates that the original chromosomal DNA originally had two types of nucleotide sequences. At present, it is unclear whether this difference in nucleotide sequence is due to homologous chromosomes or polygenicity. In addition, this suggests that differences in nucleotide sequences may be caused by ethnic differences. Because the poly(A ⁺ ) RNA purchased from Clontech is Caucasian and the chromosomal DNA is Japanese.

The inventors have deposited the carrier pHGT1 carried by E.coli strain DH5 with the registration number No. TERM BP-4616 on March 24, 1994 at the National Institute of Bioscience and Human Technology in Japan, the Agency for Industrial Science and Technology, Ministry of International Trade and Industry. <Example 26> Expressing human TPO chromosomal DNA in COS1 cells and determining the activity of TPO

The plasmid clone pHGT1 obtained by subcloning contained 4 EcoRI recognition sequences, 3 in the insert part and 1 in the vector. Nucleotide sequence analysis showed that a DNA fragment of about 4.3 kb contained the coding region of the complete human TPO protein, which was between the EcoRI recognition sequence closest to the 5' side of the insert and the EcoRI recognition sequence in the vector. Therefore, this fragment was ligated with EcoRI-treated expression vector pEF18S, and four human TPO expression plasmids pEFHGTE#1-4 were obtained (see FIG. 11 ). By preparing these plasmid DNAs, expression experiments were performed. Plasmid DNA was purified substantially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989), whereby about 250 µg of plasmid DNA was obtained.

The clone pEFHGTE #1-4 thus obtained was used to transfect COS1 cells according to the method of Example 11. That is, transfection was performed from 10 µg of plasmid DNA by the DEAE-dextran method including chloroquine treatment. Transfected COS1 cells were incubated at 37°C for 3 days, and then the culture supernatant was collected.

Culture supernatants were extensively dialyzed against IMDM medium and then assessed with the rat CFU-MK detection system. In the supernatant of COS1 cells expressing 4 clones (pEFHGTE #1-4), the TPO activity was detected in a dose-dependent manner. In contrast, there was no TPO activity in the supernatant of COS1 cells expressing plasmid pEF18S alone. Representative data obtained with pEFHGTE #1 is given in Figure 12a. Similar results were obtained in the M-07e detection system. The supernatants of COS1 cells expressing 4 clones (pEFHGTE #1-4) significantly increased the growth of M-07e cells in a dose-dependent manner. Representative data obtained with pEFHGTE#1 are presented in Figure 12b.

These results indicated that plasmid clone pEFHGTE#1-4 harbored functional human TPO chromosomal DNA. <Example 27> Preparation of human TPO-deficient DNA and expression in COS1 cells and determination of TPO activity

The results in Example 18 demonstrate that human TPO can exhibit its biological activity even after removal of its third carboxyl group (its carboxyl third). Therefore, to further estimate the biologically active moiety, deletion derivative experiments were performed. In this example, the ability of TPO-deficient derivatives lacking the carboxy-terminal number 20, 40, 60 or 68 of the TPO protein (amino acids 1-231) encoded from pHT1-231 to exert the biological activity of TPO in vitro was tested. amino acids. The shortest derivative (amino acids 1-163) still contains the amino acid sequence corresponding to TP2/3 of the rat plasma TPO described in Example 2. Using the DNA of the plasmid clone pHT1-231 obtained in Example 18 as a template and the synthesized oligonucleotide as a primer, a deletion plasmid was prepared by PCR. The sequences of the primers used in PCR are as follows: hTPO-5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3' (SEQ ID NO: 56) (by adding an EcoRI recognition sequence to SEQ ID NO: 4 It is obtained on the sequence of positions 1 to 21 of the 21; it is identical to the sequence described in Figure 9); hTPO-S: 5'-TTT GCG GCC GCT CAT TAG CTG GGG ACA GCT GTGGTG GGT-3' (SEQ ID NO : 57) (corresponding to the antisense primer of the sequence of position 555 to 576 in SEQ ID NO: 4, its preparation method is by adding two stop codons TAA and TGA and a Not I recognition sequence, in order to be used for preparing coding Deletion derivative of amino acid residues at positions 1 to 163); hTPO-4: 5'-TTT GCG GCC GCT CAT TAC AGT GTG AGG ACT AGAGAG GTT CTG-3' (SEQ ID NO: 58)

(An antisense primer corresponding to the sequence of positions 576 to 600 in SEQ ID NO: 4, which is prepared by adding two stop codons TAA and TGA and a Not I recognition sequence to prepare coding positions 1 to 171 Deletion derivatives of amino acid residues); hTPO-30: 5'-TTT GCG GCC GCT CAT TAT CTG GCT GAG GCA GTGAAG TTT GTC-3' (SEQ ID NO: 59) (equivalent to position 636 in SEQ ID NO: 4 antisense primers to the sequence of 660, prepared by adding the two stop codons TAA and TGA and a NotI recognition sequence for the preparation of deletion derivatives encoding amino acid residues from positions 1 to 191); and hTPO-2: 5'-TTT GCG GCC GCT CAT TAC AGA CCA GGA ATC TTGGCT CTG AAT-3' (SEQ ID NO: 60) (antisense primer corresponding to sequence 696 to 720 in SEQ ID NO: 4 , prepared by adding two stop codons, TAA and TGA, and a NotI recognition sequence for the preparation of deletion derivatives encoding amino acid residues from positions 1 to 211).

Using 1 μg of the plasmid DNA of the clone pHT1-231 obtained in Example 18 as a template, each 10 μM of hTPO-5 (for the 5' side) and hTPO-2, -3, -4 and -S (for the 3' side) as a primer and GeneAmp ^™ PCR Kit (manufactured by Takara Shuzo) with AmpliTaq ^™ DNA polymerase in a volume of 100 μl, using GeneAmp ^™ PCRSystem 9600 (manufactured by PERKIN-ELMER) was repeated for a total of 20 cycles, each Each cycle includes denaturation at 95°C for 1 minute, annealing at 65°C for 1 minute and synthesis at 72°C for 1 minute, followed by incubation at 72°C for 7 minutes to complete the PCR. Desired bands obtained by each PCR were digested with restriction enzymes EcoRI and NotI, and the resulting digests were subjected to 1% agarose gel (manufactured by FMC BioProducts) electrophoresis to separate bands of desired sizes amplified by each PCR. Major DNA fragments. The DNA fragment thus isolated was purified with Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad), and then subcloned into expression vector pEF18S previously treated with the same restriction enzymes. In this case, highly competent E. coli DH5 manufactured by TOYOBO was used as a host strain. From the resulting transformants from each PCR, 4 to 5 clones containing an insert of the desired size were screened to prepare plasmid DNA essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Prass, 1989) sample. In a series of the above manipulations, plasmid DNA samples were obtained from the following deletion derivatives: a deletion derivative encoding amino acid residues from position 1 to 163 (pHT1-163 #1-5), a deletion encoding amino acid residues from position 1 to 171 Derivatives (pHT1-171 #1-4), deletion derivatives encoding amino acid residues from position 1 to 191 (pHT1-191 #1-4) and deletion derivatives encoding amino acid residues from position 1 to 211 (pHT1-211 #1-4). Nucleotide sequence analysis was performed on the full-length amplified region of each plasmid DNA, and it was found to be completely consistent with the nucleotide sequence shown in SEQ ID NO:4.

According to the method of Example 11, COS1 cells were transfected with the clone thus obtained. Namely: Transfection was performed with each 10 μg of plasmid DNA samples by the DEAE-dextran method including chloroquine treatment. The transfected COS1 cells were cultured for 3 days, and then the culture supernatant was recovered.

Culture supernatants were dialyzed extensively against IMDM medium and then evaluated with the rat CFU-MK detection system. TPO activity was detected in a dose-dependent manner in the supernatants of COS1 cells expressing pHT1-211, pHT1-191, pHT1-171 or pHT1-163, respectively. Representative data obtained with pHT1-211#, pHT1-191# and pHT1-171#2 are presented in Figure 13a, while data obtained with pHT1-163#2 are presented in Figure 13b. Similar results were obtained in the M-07e detection system. The supernatants of COS1 cells expressing pHT1-211, pHT1-191, pHT1-171 or pHT1-163, respectively, significantly increased the proliferation of M-07e cells. Representative data obtained with pHT1-211#1, pHT1-191#1, pHT1-171#2 are shown in Figure 14.

These results indicate that human TPO retains its in vitro activity even if the carboxy-terminal half above Ser (position 163) is deleted, and strongly demonstrates that the biologically active part of human TPO is located at the half bounded by Ser (position 163) within the amino terminus. <Example 28> Preparation of C-terminal deleted human TPO and its expression and activity in COS1 cells

In order to analyze the region necessary for the human TPO protein to exhibit its activity, the deletion derivative obtained from Example 27 was further deleted from the C-terminal amino acid, and then the TPO activity expressed in COS1 cells was examined. Using the plasmid obtained in Example 16 to clone pEF18S-HL34, an expression plasmid was prepared by deleting the nucleotide sequence encoding the C-terminal amino acid residue starting from Ser 163. Construction of these deletion plasmids was efficiently performed using PCR. The primer sequences prepared for PCR are as follows: hTPO-5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3' (SEQ ID NO: 61) (by adding the EcoRI recognition sequence to SEQ ID NO: 4 prepared from the sequence of positions 1 to 21 in the sequence; the same sequence is listed in Example 18); hTPO-150: 5'-TTT GCG GCC GCT CAT TAG AGG GTG GAC CCTCCT ACA AGC AT-3' (SEQ ID NO: 62)

(corresponding to the antisense primer of the sequence of positions 514 to 537 in SEQ ID NO: 4, prepared by adding two stop codons TAA and TGA and a NotI recognition sequence used to prepare the deletion mutant, the deletion mutant Amino acid residues encoding positions 1 to 150); hTPO-151: 5'-TTT GCG GCC GCT CAT TAG CAG AGG GTG GACCCT CCT ACA A-3' (SEQ ID NO: 63) (corresponding to SEQ ID NO: 4 Antisense primers for the sequence at positions 518 to 540, prepared by adding the two stop codons TAA and TGA and a NotI recognition sequence for the generation of deletion derivatives encoding amino acid residues at positions 1 to 151) ; hTPO-153: 5'-TTT GCG GCC GCT CAT TAC CTG ACG CAG AGGGTG GAC CC-3' (SEQ ID NO: 64) (antisense primer corresponding to the sequence of positions 526 to 546 in SEQ ID NO: 4, Prepared by adding two stop codons TAA and TGA and a NotI recognition sequence for the preparation of deletion derivatives encoding amino acid residues from positions 1 to 153); hTPO-154: 5'-TTT GCG GCC GCT CAT TAC CGC CTG ACG CAGAGG GTG GA-3' (SEQ ID NO: 65) (antisense primer corresponding to the sequence of positions 529 to 549 in SEQ ID NO: 4, by adding two terminations used to prepare deletion derivatives codons TAA and TGA and a NotI recognition sequence, the deletion derivatives encode amino acid residues from positions 1 to 154); hTPO-155: 5'-TTT GCG GCC GCT CAT TAG GCC CGC CTG ACGCAG AGG GT-3 '(SEQ ID NO: 66) (An antisense primer corresponding to the sequence of positions 532 to 552 in SEQ ID NO: 4, identified by adding two stop codons TAA and TGA and a NotI recognition sequence for making deletion derivatives prepared, the deletion derivatives encode amino acid residues from positions 1 to 155); hTPO-156: 5'-TTT GCG GCC GCT CAT TAT GGG GCC CGC CTGACG CAG AG-3' (SEQ ID NO: 67) (corresponding to Antisense primers for the sequence of positions 535 to 555 in SEQ ID NO: 4 prepared by adding the two stop codons TAA and TGA and a NotI recognition sequence for the preparation of deletion derivatives encoding position 1 to 156 amino acid residues); hTPO-157: 5'-TTT GCG GCC GCT CAT TAG GGT GGG GCC CGCCTG ACG CA-3' (SEQ ID NO: 68) (corresponding to one of positions 538 to 558 in SEQ ID NO: 4 The antisense primer for the sequence was prepared by adding the two stop codons TAA and TGA and a NotI recognition sequence used to prepare the deletion derivatives encoding the amino acid residues in positions 1 to 157).

Using 1 μg of the plasmid DNA of the cloned pEF18S-HL34 obtained in Example 16 as a template, 10 μM each of the oligonucleotides thus synthesized (hTPO-5 for the 5' side, hTPO-150, -151, -153, - 154, -155, -156 and -157 for the 3' side) as primers for PCR. Using GeneAmp ^™ PCR Kit (manufactured by Takara Shuzo) containing AmpliTaq ^™ DNA polymerase, a total of 20 cycles of denaturation at 95°C for 1 minute per cycle were repeated using GeneAmp ^™ PCR System 9600 (manufactured by PERKIN-ELMER), 66 Annealing for 1 minute at 72°C and synthesis for 1 minute at 72°C followed by a final incubation of 7 minutes at 72°C completed the PCR reaction in a volume of 100 μl. Desired bands obtained from each PCR were digested with restriction enzymes EcoRI and NotI, and then the resulting digests were subjected to 1% agarose gel (manufactured by FMC BioProducts) electrophoresis to separate major DNAs having a desired size from each PCR fragment. The isolated DNA fragment was purified using Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad), and then subcloned into expression vector pEF18S previously treated with the same restriction enzymes. In this case, highly competent E. coli DH5 produced by TOYOBO was used as a host strain. From the resulting transformants from each PCR experiment, 3 to 5 clones were screened for inserts of the desired size to prepare plasmid DNA samples essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) . Through a series of operations as described, plasmid DNA was prepared from the following deletion derivatives: deletion derivatives encoding amino acid residues from position 1 to 150 (pHT1-150#21, 22 and 25), Deletion derivatives (pHT1-151#16, 17 and 18), deletion derivatives encoding amino acid residues from position 1 to 153 (pHT1-153#1 to 5), deletion derivatives encoding amino acid residues from position 1 to 154 ( pHT1-154#1 to 5), encoding the deletion derivative of amino acid residues 1 to 155 (pHT1-155#1 to 5), encoding the deletion derivative of amino acid residues 1 to 156 (pHT1-156#1 to 5) and deletion derivatives encoding amino acid residues at positions 1 to 157 (pHT1-157#1 to 5).

In these purified plasmid DNA samples, pHT1-50#21, 22 and 25 and pHT1-151# were detected by using a Taq Dye Deory ^™ Terminater Cycle sequencing cartridge (Applied Biosystems) with a 373A DNA sequencer (manufactured by Applied Biosystems). 16, 17 and 18, thus confirming that there is no substitution in the expected TPO cDNA sequence in the full-length nucleotide sequence.

According to the method of Example 11, the obtained clones were used to transfect COS1 cells, respectively. Namely, transfection was accomplished with each 10 μg of plasmid DNA samples by the DEAE-dextran method including chloroquine treatment, and the culture supernatant was recovered after 3 days of culture. The resulting culture supernatants were incubated with IMDM as previously described and dialyzed, then evaluated with the M-07e detection system. TPO activity was detected in COS1 cell culture supernatants transfected with corresponding clones encoding C-terminal deletion derivatives consisting of amino acids 1 to 151, amino acids 1 to 153, amino acids 1 to 154, respectively. Amino acids, 1 to 155 amino acids, 1 to 156 amino acids or 1 to 157 amino acids. All of these derivatives contain a Cys residue at position 151. However, no TPO activity was detected in the culture supernatant of COS1 cells transfected with a clone encoding a C-terminal side deletion derivative consisting of amino acids 1-150, and the Cys residue at position 151 of the derivative was also blocked. missing. <Example 29> Preparation of N-terminal deletion human TPO and its expression and activity in COS1 cells

In order to analyze the region necessary for TPO protein to exhibit its activity, the N-terminal amino acid of the deletion derivative obtained in Example 28 was further deleted, and then the expression of TPO activity of the obtained deletion derivative was detected. Using the plasmid clone pEF18S-HL34 obtained in Example 16 and the plasmid clone pHT1-163 obtained in Example 27, expression plasmids were prepared by deleting the nucleotide sequence encoding the N-terminal amino acid residue following the signal sequence. Construction of these deletion plasmids was efficiently performed using PCR. The primer sequences prepared for use in PCR are as follows: hTPO-5: 5'-TTT GAA TTC GGC CAG CCA GAC ACC CCG GCC-3' (SEQ ID NO: 69) (by adding the EcoRI recognition sequence to SEQ ID NO : prepared from the sequence of positions 1 to 21 in 4; the same sequence listed in Example 18); hTPO3: 5'-TTT GCG GCC GCT CAT TAT TCG TGT ATC CTG TTCAGGIAICC-3' (SEQ ID NO: 70 ) (antisense primer corresponding to the sequence of positions 757 to 780 in SEQ ID NO: 4, prepared by adding two stop codons TAA and TGA and a NotI recognition sequence; the same sequence synthesized was used to prepare coding position 1 to the deletion derivative of 231 amino acid residues); hTPO-S: 5'-TTT GCG GCC GCT CAT TAG CTG GGG ACA GCT GTGGTG GGT-3' (SEQ ID NO: 71) (corresponding to position in SEQ ID NO: 4 Antisense primer for sequence 555 to 576; used to generate deletion derivatives encoding amino acid residues 1 to 163); hTPO-13: 5'-AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT CACAGC AGA CTG AGC CAG TG -3' (SEQ ID NO: 72) (positions 124 to 173 in SEQ ID NO: 4; used to prepare derivatives with amino acid residues deleted from positions 1 to 12); hTPO-13R: 5'-CAT GGG AGT CAC GAA GCA GTT TAC TGG ACAGCG TTA GCC TTG CAG TTA G-3' (SEQ ID NO: 73) (corresponding to the antisense primers of the sequences of positions 64 to 87 and 124 to 148 in SEQ ID NO: 4, used to prepare Derivative of amino acid residues from positions 1 to 12 deleted); hTPO-7: 5'-TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGTGAC TCC CAT GTC CTT C-3' (SEQ ID NO: 74) (SEQ ID NO: positions 106 to 154 in 4, for the preparation of derivatives with amino acid residues deleted from positions 1 to 6); hTPO-7R: 5'-TTT ACT GAG GAC TCG GAG GTC ACA GGA CAGCGT TAG CCT TGC AGT TAG -3' (SEQ ID NO: 75) (antisense primers corresponding to the sequences of positions 64 to 87 and 106 to 129 in SEQ ID NO: 4, used to prepare derivatives in which the amino acids of positions 1 to 6 have been deleted); hTPO-8: 5'-GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT GACTCC CAT GTC CTT CAC A-3' (SEQ ID NO: 76) (positions 109 to 157 in SEQ ID NO: 4; used to make deletions and hTPO-8R: 5'-CAG TTT ACT GAG GAC TCG GAG GTC GGA CAGCGT TAG CCT TGC AGT TAG-3' (SEQ ID NO: 77) (corresponding to Antisense primers for the sequences of positions 64 to 87 and 109 to 132 in SEQ ID NO: 4; used to prepare derivatives in which the amino acid residues at positions 1 to 7 have been deleted).

(1) Preparation of derivatives (pHT13-231) in which amino acid residues at positions 1 to 12 are deleted

Use 1.4 μ g of the plasmid DNA of clone pEF18S-HL34 obtained in Example 18 as a template and each 5 μ M of the oligonucleotides synthesized therefrom (hTPO-13 and hTPO-3 in one combination, hTPO-13 in another combination -5 and hTPO-13R) were used as primers for PCR. Using GeneAmp ^™ PCR Kit containing AmpliTaq ^™ DNA polymerase (manufactured by Takara Shuzo), GeneAmp ^™ PCR System 9600 (manufactured by PERKIN-ELMER), after denaturation at 95°C for 5 minutes, a total of 30 cycles were repeated, each Cycling consisted of denaturation at 95°C for 1 minute, annealing at 65°C for 1 minute and synthesis at 72°C for 1 minute, and a final incubation at 72°C for 7 minutes to complete the PCR reaction in a volume of 100 μl. Each PCR product was subjected to electrophoresis on a 1.2% agarose gel (manufactured by FMC BioProducts) to separate a major DNA fragment of an expected size which was subsequently purified with a Prep-A-Gene DNA purification kit (manufactured by Bio-Rad), and then dissolved. in 15 μl TE buffer. Thereafter, a second PCR was performed using 1 µl each of the resulting solution as a template.

A second PCR was done with 5 μM each of the synthetic primers (hTPO-5 and hTPO3). Using GeneAmp ^™ PCR Kit containing AmpliTaq ^™ DNA polymerase (manufactured by Takara Shuzo), GeneAmp ^™ PCR System 9600 (manufactured by PERKIN-ELMER), after denaturation at 95°C for 5 minutes, a total of 30 cycles were repeated, each cycle The PCR reaction was completed in a volume of 100 μl by including denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute and synthesis at 72°C for 1 minute, and finally incubation at 72°C for 7 minutes. Each PCR product was subjected to 1.2% agarose gel (manufactured by FMC BioProducts) electrophoresis to separate the main DNA fragment of the expected size which was subsequently purified by the Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad), and then dissolved in 15 μl TE buffer. After digestion with restriction enzymes EcoRI and NotI, the resulting solution was extracted with the same volume of phenol/chloroform, followed by ethanol precipitation. After centrifugation, the resulting pellet was dissolved in 15 µl of TE buffer, and then subcloned into expression vector pEF18S previously treated with the same restriction enzymes. In this case, highly competent E. coli DH5 manufactured by TOYOBO was used as a host strain. From the resulting transformants, 45 clones were screened for inserts of the expected size to prepare plasmid DNA samples essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). In this way, it is possible to obtain a plasmid DNA encoding a deletion derivative (pHT13-231) of a protein molecule in which the amino acids at positions 1 to 12 have been deleted from the original amino acid residues at positions 1 to 231 Residues. PCR of these 45 clones using primers hTPO-5 and hTPO3 confirmed that in 8 clones, there were inserts of about the expected size. The sequences of three of the clones were detected using a Taq Dye Deoxy ^™ Terminateer Cycle Sequencing Kit (Applied Biosystems) with a 373A DNA sequencer (manufactured by Applied Bisystems), thereby obtaining a clone pHT13-231#3, which contained the designed TPO cDNA and the designed Desired are no deletions such as substitutions in the entire nucleotide sequence. (2) Preparation of a derivative (pHT7-163) in which amino acid residues at positions 1 to 6 are deleted

Since although the 231st amino acid was designed as the C-terminal of the TPO protein used to prepare the deletion derivative in the above step (1), it was found that the TPO activity could still be expressed when the C-terminal amino acid was even further deleted, so in this case Below, the 163rd amino acid was used as the C-terminus for the preparation of the deletion derivative. For the same reason, amino acid 163 was also used as the C-terminal in the subsequent step (3). Use 1.4 μg of plasmid DNA of clone pHT1-163 obtained in Example 27 as a template, and each 5 μM of synthetic oligonucleotides (hTPO-7 and hTPO-S combined, hTPO-5 and hTPO-7R combined) as primers for PCR . Using the GeneAmp ^™ PCR kit with AmpliTaq ^™ DNA polymerase (manufactured by Takara Shuzo), through the GeneAmp ^™ PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 µl, after denaturation at 95°C for 5 minutes, a total of 30 repetitions were performed. Each cycle includes denaturation at 95°C for 1 minute, annealing at 65°C for 1 minute and synthesis at 72°C for 1 minute, followed by a final incubation at 72°C for 7 minutes to complete the PCR reaction. Each PCR product was electrophoresed on a 1.2% agarose gel (manufactured by FMC BioProducts) to separate a major DNA fragment having a desired size, followed by purification with a Prep-A-Gene DNA purification kit (manufactured by Bio-Rad), and then dissolved. in 15 μl TE buffer. The second PCR was performed using 1 µl each of the solutions thus prepared as a template.

A second PCR was performed with 5 μM each of the synthetic primers (hTPO-5 and hTPO-S). Using GeneAmp ^™ PCR Kit with AmpliTaq ^™ DNA Polymerase (manufactured by Takara Shuzo), through GeneAmp ^™ PCR System 9600 (manufactured by PERKIN-ELMER) in a volume of 100 µl, after denaturation at 95°C for 5 minutes, a total of 30 repetitions Cycles, each cycle including denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute and synthesis at 72°C for 1 minute, followed by a final incubation at 72°C for 7 minutes complete the PCR reaction. Each PCR product was electrophoresed on a 1.2% agarose gel (manufactured by FMC BioProducts) to separate a major DNA fragment having a desired size, followed by purification with a Prep-A-Gene DNA purification kit (manufactured by Bio-Rad), and then dissolved. in 15 μl TE buffer. After digestion with restriction enzymes EcoRI and NotI, the resulting solution was extracted with the same volume of phenol/chloroform followed by ethanol precipitation. After centrifugation, the resulting pellet was dissolved in 15 µl of TE buffer, and then subcloned into the expression vector pEF18S that had been previously digested with the same restriction enzymes. In this case, highly competent E. coli DH5 (manufactured by TOYOBO) was used as a host strain. From the resulting transformants, 30 clones containing an insert of the desired size were screened to prepare plasmid DNA samples essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). In this way, a plasmid DNA encoding a deletion derivative (pHT7-163) of a protein molecule in which the original amino acid residues at positions 1 to 163 of SEQ ID NO: 4 have been deleted can be obtained Amino acid residues in positions 1 to 6. When these clones were subjected to PCR using primers hTPO-5 and hTPO-S, it was confirmed that all clones contained inserts of about the expected size. Of these, 2 clones, pHT7-163 #4 and #29, were obtained by detecting the sequences of 3 of them by using a 373A DNA sequencer (manufactured by Applied Biosystems) using a Taq DyeDeoxy ^™ Terminateer Cycle Sequencing Kit (Applied Biosystems) , both of which contain the designed TPO cDNA sequence and the desired deletion without substitution in the entire nucleotide sequence. (3) Preparation of derivatives (pHT8-163) in which amino acid residues 1-7 have been deleted

A derivative (pHT8-163) in which amino acid residues 1 to 7 were deleted (pHT8-163) was prepared in substantially the same manner as in the above example for preparing a derivative in which amino acid residues 1 to 6 were deleted (pHT7-163). PCR was carried out using 1.4 µg of plasmid DNA of clone pHT1-163 obtained in Example 27 as a template and 5 µM of synthetic oligonucleotides (hTPO-8 and hTPO-S combined, hTPO-5 and hTPO-8R combined) as primers. Using GeneAmp ^™ PCR kit with AmpliTaq ^™ DNA polymerase (manufactured by TakaraShuzo), through GeneAmp ^™ PCR System9600 (manufactured by PERKIN-ELMER) in a volume of 100 µl, after denaturation at 95°C for 5 minutes, a total of 30 cycles were repeated, Each cycle included denaturation at 95°C for 1 minute, annealing at 65°C for 1 minute and synthesis at 72°C for 1 minute, followed by a final incubation at 72°C for 7 minutes to complete the PCR reaction. Each PCR product was subjected to electrophoresis on 1.2% agarose gel (manufactured by FMC BioProducts) to separate the main DNA fragment with the expected size, followed by purification with the Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad), and then dissolved in 15 μl TE buffer. Thereafter, a second PCR was performed using 1 µl of each prepared solution as a template.

A second PCR was performed with 5 μM of each of the synthesized primers (hTPO-5 and hTPO-S). Using GeneAmp ^™ PCR Reagent Kit (manufactured by Takara Shuzo) with AmpliTaq ^™ DNA polymerase, using GeneAmp ^™ PCR System 9600 (manufactured by PERKIN-ELMER), after denaturation at 95°C for 5 minutes, repeat 30 cycles, each cycle including Denaturation for 1 minute at 60°C, annealing for 1 minute at 60°C and synthesis for 1 minute at 72°C followed by a final incubation at 72°C for 7 minutes complete the PCR reaction. Each PCR product was subjected to 1.2% agarose gel (manufactured by FMC BioProducts) electrophoresis to separate the main DNA fragment with the expected size, followed by purification with the Prep-A-Gene DNA Purification Kit (manufactured by Bio-Rad), and then dissolved in 15 μl TE buffer. After digestion with restriction enzymes EcoRI and NotI, the resulting solution was extracted with the same volume of phenol/chloroform followed by ethanol precipitation. After centrifugation, the resulting pellet was dissolved in 15 µl of TE buffer, and then subcloned into the expression vector pEF18S that had been previously digested with the same restriction enzymes. In this example, highly competent E. coli DH5 (manufactured by TOYOBO) was used as a host strain. From the resulting transformants, 30 clones containing an insert of the expected size were screened to prepare plasmid DNA samples essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). In this way, a plasmid DNA encoding a deletion derivative (pHT8-163) of a protein molecule in which 1 has been deleted from the original amino acid residues 1-163 of SEQ ID NO:4 can be obtained Amino acid residue at position -7. PCR of these clones using primers hTPO-5 and hTPO-S confirmed that all clones contained inserts of approximately the expected size. The sequences of 3 of the clones were detected by using a 373A DNA sequencer (manufactured by Applied Biosystems) of the Taq Dye Deoxy ^™ Terminateer Cycle Sequemng Box (Applied Biosystems), thereby obtaining 2 clones, pHT8-163 #33 and #48, respectively Contains the designed TPO cDNA sequence and no expected deletions such as substitutions in the entire nucleotide sequence. (4) Expression of deletion derivatives in COS1 cells and confirmation of TPO activity

According to the method of Example 11, COS1 cells were transfected with the deletion clones thus obtained, respectively. That is, by using the DEAE-dextran method including chloroquine treatment, transfection was completed with 10 μg of each plasmid DNA sample, and after culturing for 3 days, the culture supernatant was recovered. The culture supernatant thus obtained was thoroughly dialyzed against the aforementioned IMDM medium, and then evaluated with the M-07e detection system.

As a result, weak but significant TPO activity was detected in the culture supernatant of COS1 cells transfected with the clone encoding the deletion derivative consisting of amino acids 7-163. However, no TPO activity was detected in the culture supernatant of COS1 cells transformed with clones encoding derivatives consisting of amino acids 8-163 or amino acids 13-231. <Example 30> Preparation of full-length human TPO cDNA plasmid (pHTP1)

An expression vector containing the entire amino acid coding region of the hypothetical human TPO cDNA in SEQ ID NO: 6 was constructed for expression in mammalian cells.

To prepare a DNA fragment covering the entire coding region of human TPO cDNA, PCR was carried out in the following manner.

The nucleotide sequences of the primers used are as follows: hTPO-1: 5'-TTG TGA CCT CCG AGT CCT CAG-3' (SEQ ID NO: 78) (positions 105 to 125 in SEQ ID NO: 6); SA: 5'-CAG GTA TCC GGG GAT TTG GTC-3' (SEQ ID NO: 79) (antisense primer relative to the sequence at positions 745-765 in SEQ ID NO: 6); and hTPO-P: 5'-TGC GTT TCC TGA TGC TTG TAG-3' (SEQ ID NO: 80) (positions 503 to 523 in SEQ ID NO: 6); and hTPO-KO: 5'-GAG AGA GCG GCC GCT TAC CCT TCC TGA GACAGA TT- 3' (SEQ ID NO: 81) (generated by adding a restriction enzyme NotI recognition sequence and the GAGAGA sequence (SEQ ID NO: 82) to the antisense sequence relative to the sequence at positions 1066 to 1086 in SEQ ID NO: 6 prepared sequences).

The first PCR was performed using 300 ng of the clone pEF18S-HL34 obtained in Example 16 as a template. Using 0.5 μM each of the primers hTPO-1 and SA and 1 unit of Vent R ^™ DNA polymerase (manufactured by New England BioLabs), PCR was performed (30 cycles were repeated in total, each cycle including incubation at 96° C. for 1 minute, at 96° C. 62° C. for 1 minute and 72° C. for 1 minute, followed by a final incubation at 72° C. for 7 minutes) The final concentrations of the reaction solution components were as follows: 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 20 mM Tris-HCl (pH 8.8), 2 mM MgSO ₄ , 0.1% Triton X-100 and 200 μM dNTP mix.

1 mg of a commercially available poly(A) ⁺ RNA preparation (manufactured by Clontech) obtained from normal human liver was heated at 70°C for 10 minutes, rapidly cooled on ice, and mixed with 10 mM DTT, 500 µM dNTP, 25 ng of random primers (manufactured by Takara Shuzo prepared), 10 units of RNase inhibitor (manufactured by Boehringer-Mannheim) and 200 units of SuperScript ^™ II RNaseH ^- (manufactured by LIFE TECHNOLOGIES) were mixed, followed by incubation at 37°C for 1 hour to synthesize cDNA. Thereafter, using 1/20 volume of the reaction solution of synthesized cDNA as a template, 2.5 μM each of primers hTPO-P and hTPO-KO, and 2.5 units of AmpliTaq ^™ DNA polymerase (manufactured by Takara Shuzo) was used for the second PCR (a total of 30 repetitions) cycle, each cycle consisting of incubation for 1 minute at 96°C, 1 minute at 58°C and 1 minute at 72°C).

The resulting first and second PCR solutions were subjected to 1% agarose gel electrophoresis to separate the respective major DNA fragments containing the expected size, followed by the Prep-A-Gene DNA purification kit (purified by Bio-Rad) purification. Thereafter, a third PCR was carried out using the solutions thus obtained as templates, respectively. The reaction was carried out with 1 unit of Vent R ^™ DNA polymer (manufactured by NewEngland BioLabs) (heating at 96°C for 2 minutes, followed by a total of 3 cycles of incubation at 96°C for 2 minutes and incubation at 72°C for 2 minutes). minutes, followed by an additional incubation at 72°C for 7 minutes). The resulting reaction solution was mixed with 1 μM each of hTPO-1 and hTPO-KO, heated at 96 °C for 2 min, and then subjected to 25 cycles of reaction, each cycle including 1 min at 96 °C, 1 min at 62 °C and 1 min at 72 °C incubation, and finally at 72°C for another 7 minutes. The resulting reaction solution was extracted with the same volume of water-saturated phenol-chloroform followed by the same volume of chloroform, and the extract was treated with ethanol precipitation (2.5 volumes of ethanol containing 0.3 M sodium acetate and 0.5 μl of glycogen manufactured by Boehriger-Mannheim) , to recover DNA. The recovered DNA was digested with restriction enzymes BamHI and NotI, then subjected to 1% agarose gel electrophoresis, and the thus-isolated main band with the expected size was purified with a Prep-A-Gene DNA purification kit (manufactured by Bio-Rad), and the The pBluescriptII SK ⁺ vector (manufactured by Stratagene), which had been digested with restriction enzymes BamHI and NotI, was ligated, and then transformed into highly competent E.coliDH5 (manufactured by TOYOBO). From the resulting clones, 4 clones were screened to prepare plasmid DNA samples. The sequence of the purified plasmid DNA sample was detected by using a 373A DNA sequencer (manufactured by Applied Biosystems) of the Taq Dye Deoxy ^™ Terminateer Cycle Sequencing Kit (Applied Biosystems), thereby obtaining a clone, pBLTP, containing the designed TPO cDNA sequence, at There were no substitutions in the nucleotide sequence in the region between BamHI and NotI.

The clone pBLTP was digested with restriction enzymes EcoRI and BamHI, electrophoresed on 1% agarose gel, and a band having a high molecular weight thus isolated was purified using a Prep-A-Gene DNA purification kit (manufactured by Bio-Rad). In the same manner, pEF18S-HL34 was treated with restriction enzymes to purify a DNA fragment of about 450 bp. The respective DNA samples thus obtained were ligated and then transformed into highly competent E. coli DH5 (manufactured by TOYOBO). Plasmid DNA samples were prepared from the resulting colonies to obtain clone pBLTEN containing the human TPO cDNA insert. The thus-obtained pBLTEN was digested with restriction enzymes EcoRI and NotI, subjected to 1% agarose gel electrophoresis, and a DNA fragment of about 1,200 bp thus isolated was purified with a Prep-A-Gene DNA transformation kit (manufactured by Bio-Rad), Then, it was ligated with the expression vector pEP18S digested with the same restriction enzyme in advance and transformed into highly competent E. coli DH5 (manufactured by TOYOBO). Plasmid DNA samples were prepared from the resulting clones to obtain clones containing the insert pHTP1 of the entire human TPO cDNA coding region. Plasmid DNA was prepared in large quantities from this clone and used in the following experiments. Plasmid DNA was prepared essentially as described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989). <Example 31> Construction of mammalian expression plasmid pDEF202-hTPO-P1 so as to express human TPO in Chinese hamster ovary (CHO) cells

1 mg of plasmid pMG1 containing the mouse dihydrofolate reductase (DHFR) minigene was digested with restriction enzymes EcoRI and BamHI, and subjected to agarose gel electrophoresis to separate a fragment (about 2.5 kb) containing the mouse DHFR minigene. The isolated DNA fragments were dissolved in 25 ml of a reaction solution consisting of 50 mM Tris-HCl (pH 7.5), 7 mM MgCl ₂ , 1 mM 2-mercaptoethanol and 0.2 mM dNTR mixture, mixed with 2 units of klenow fragments, and then incubated at room temperature for 30 minutes to form blunt ends on both ends of the DNA fragments. After phenol/chloroform treatment and ethanol precipitation, the resulting fragment was dissolved in 10 ml of TE solution (10 mM Tris-HCl (pH 8.0)/1 mM EDTA). Mammalian expression vector pEF18S was digested with restriction enzyme Smal, dephosphorylated with alkaline phosphatase (manufactured by Takara Shuzo), and then ligated with a DNA fragment containing the mouse DHFR minigene with T4 DNA ligase (manufactured by Takara Shuzo) to obtain The expression vector pDEF202 was obtained.

Next, the constructed expression vector pDEF202 was digested with restriction enzymes EcoRI and SpeI, and then subjected to agarose gel electrophoresis to separate a larger DNA fragment. Using T4 DNA ligase (manufactured by Takara Shuzo), human TPO cDNA obtained by digesting plasmid pHTP1 containing human TPO cDNA (clone P1) with restriction enzymes EcoRI and SpeI was ligated to this linearized pDEF202 vector to obtain Plasmid pDEF202-hTPO-P1 of human TPO cDNA. The constructed plasmid contains SV40 origin of replication, human elongation factor 1-α-promoter, and SV40 early polyadenylation signal for transcription of human TPO cDNA, mouse DHFR minigene, and pUC18-origin of replication and β- Lactamase gene (Amp ^r ). <Example 32> Expression of human TPO in CHO cells

CHO cells were maintained in a 6 cm diameter tissue culture dish (Falcon) in α-minimal essential medium (α-MEM(-), supplemented with hypoxanthine and thymidine) containing 10% fetal calf serum (FCS) (A DHFR strain; Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, vol. 77, p4216 (1980)). CHO cells were transformed with the pDEF202-hTPO-P1 plasmid according to the following calcium phosphate method (CellPhect, manufactured by Pharmacia). 10 mg of the plasmid pDEF202-hTPO-P1 prepared in Example 31 was mixed with 120 ml of buffer A and 120 ml of water, and the resulting mixture was incubated at room temperature for 10 minutes. The solution was mixed with another 120 ml of buffer B and left at room temperature for a further 30 minutes. This DNA mixture was added to the medium and incubated for 6 hours in a _CO2 incubator. After 6 hours, the culture was washed twice with serum-free α-MEM(-), treated with α-MEM(-) containing 10% DMSO for two minutes, and then incubated in non-serum containing 10% dialyzed FCS Incubate in selection medium (α-MEM(-) supplemented with hypoxanthine and thymidine) for 2 days. After 2 days of culture, cells were selected in selection medium (α-MEM(-)) containing 10% dialyzed FCS. To select transformed cells with a DHFR-positive phenotype, cells were treated with trypsin/EDTA solution, and then suspended in selection medium. Divide cells from 6 cm tissue culture dishes into five 10 cm tissue culture dishes or twelve 24-well tissue culture dishes and culture in selective medium. Medium was exchanged at 2-day intervals. Human TPO activity in the medium of CHO cells with a DHFR-positive phenotype was detected by the CFU-MK assay, the M-07e assay or the Ba/F3 assay. Cells secreting human TPO into the medium were further screened in selection medium supplemented with 25 nM methotrexate to isolate cell clones producing high amounts of human TPO.

In this example, CHO cells were also transfected by co-transfecting CHO cells with pHTP1 and pMG1 plasmids.

The applicant has deposited the CHO strain (CHO-DUKXB11) transfected with the plasmid pDEF202-hTPO-P1 with the deposit number FERM BP-4988 on January 31, 1995 at the National Institute of Bioscience and Human Technology, Industrial Science and Technology Division, Ministry of International Trade and Industry. <Example 33> Construction of recombinant vector BMCGSneo-hTPO-P1 for X63.6.5.3 cells

1 mg of the mammalian expression vector pBMCGSneo was digested with restriction enzymes XhoI and NotI, followed by agarose gel electrophoresis to separate the vector DNA fraction. This DNA fragment was ligated with human TPO cDNA (clone P1) obtained by digesting plasmid pBLTEN containing human TPO cDNA with restriction enzymes XhoI and NotI with T4 DNA ligase to obtain the desired expression vector pBMCGSneo-hTPO-P1. The expression plasmid contains the early promoter of cytomegalovirus, part of the intron derived from the rabbit β-globin gene and the polyadenylation region for transcription of human TPO cDNA transcription, human β-globin gene, 69% bovine papillomavirus 1 gene, thymidine kinase promoter and polyadenylation region, phosphotransferase I gene (neomycin resistance gene) and pBR322 origin of replication and β-lactamase gene (Amp ^r ), human TPO cDNA was ligated in the cell Downstream site of the megavirus promoter. <Example 34> expressed in X63.6.5.3 cells

X63.6.5.3 cells were cultured in Dulbecco's minimal essential (DME) medium containing 10% fetal bovine serum. X63.6.5.3 cells were transfected with the BMCGSneo hTPO plasmid by electroporation as described below.

20 mg of the plasmid BMCGSneo-hTPO-P1 obtained in Example 33 was added to 750 μl of DME medium containing 10 ⁷ cells, and then the mixture was placed in an electroporation cell with a gap of 4 mm, and placed at 4° C. for 10 minutes. The sample cell was then put into an electroporation device (BTX600, manufactured by BTX) so as to complete gene transfer under the conditions of 380V, 25mF and 24mm. After gene transfer, the sample pool was placed at 4°C for another 15 minutes, and then the cells were washed once with DME containing 10% fetal bovine serum. The transfected cells were suspended in 50 ml of DME containing 10% fetal bovine serum, dispersed into each well of a 96-well tissue culture dish, and then cultured in a _CO incubator for 2 days. After 2 days of culture, the medium was replaced with DME containing 10% fetal bovine serum and 1 mg/ml G418 (manufactured by GIBSCO), and then every 3 days. Human TPO activity in transformant media was detected using CFU-MK, M-07e or Ba/F3 assays. The transformant secreting human TPO into the medium was cloned twice by limiting dilution cloning to establish a human TPO producing cell line. <Example 35> Large-scale expression of human TPO in COS1 cells

Transfection of COS1 cells was accomplished as described in Example 11 by the DEAE-dextran method including chloroquine treatment. COS1 cells (ATCC CRL1650) were cultured in a collagen-coated 175 cm ² culture flask in MDM containing 10% (v/v) FCS at 37°C in a 5% CO ₂ incubator until the cells were 100% confluent. In order to coat the culture flasks with collagen, 25 ml of a collagen solution (Cellmatrix type 1-C, manufactured by Lwaki) whose concentration was adjusted to 0.3 mg/ml with 1 mM HCl was added to each culture flask of 175 cm ² . After standing at room temperature for 1 hour, the collagen solution was recovered, and the thus-treated flask was washed once with 20 to 50 ml of PBS. By mixing 20 ml of IMDM containing 250 µg/ml DEAE-dextran (manufactured by Pharmacia), 60 µM chloroquine (manufactured by Sigma), and 10% (v/v) Nu-Serum (manufactured by Collaborative) with the plasmid that had been dissolved in 500 µl of PBS , the resulting mixture was added to the COS1 cells contained in the above 175cm ² culture flask (before transfection, the cells had been washed once with IMDM), and then placed in a 5% CO ₂ incubator at 37°C Cells were incubated for 3 hours to complete transfection. Thereafter, the culture supernatant was removed by suction, the cells were washed once with IMDM, mixed with 50 ml of serum-free medium, and then cultured at 37°C for 5 days in a 5% _CO2 incubator to recover the culture supernatant. In one operation, 100-260 culture flasks of 175 ^cm2 were used and 5 to 13 liters of culture supernatant were recovered. By adding 5 μg/ml insulin (manufactured by Sigma), 5 μg/ml transferrin (manufactured by Sigma), 10 μM monoethanolamine (manufactured by Wako Pure Chemical Industries), 20 nM sodium selenite (manufactured by Sigma) and 200 μg/ml BSA (fatty acid-free high-purity bovine albumin, manufactured by Nichirei) was used to prepare a serum-free medium. <Example 36> Purification of human full-length TPO expressed in COS1 cells (obtained from expression plasmid pHTP1.P1 clone) and its biological activity

The activity and purification of human full-length TPO expressed in COS1 cells (from the expression plasmid pHTP1 ) is described below. In order to detect the platelet-increasing activity, a partially purified product was prepared and then purified to obtain a high-purity product. In the following preparation steps, the TPO activity was mainly detected with the M-07e detection system. Similar TPO activity was found in the rat CFU-MK assay system. Upon completion of these assays, human serum albumin (HSA) was separately added to each sample to achieve a final concentration of 0.02 to 0.05%.

First, in 5% _CO2 , in 1000 ml of serum-free IMDM medium containing O.2 g BSA, 5 mg bovine insulin, 5 mg human transferrin, 0.02 mM monoethanolamine and 25 nM sodium selenite, according to Ohashi and The method of Sudo (Hideya Ohashi and Tadashi Sudo, Biosci.Biotech.Biochem.58 (4), 758-759, 1994), with the COS1 cell of plasmid pHTP1 transfection, cultivates 5 days at 37 ℃, obtains 7 liters of thus without Serum culture supernatant. The proteolytic enzyme inhibitors p-APMSF and Pefabloc SC (4(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (manufactured by Merk, product number 24839) were added to the supernatant of the serum-free culture to an concentration of about 1 mM. Final concentration, then filter with 0.2 μ m filter to recover the supernatant.With an ultrafiltration unit (Omega Ultrasette, normal molecular weight cut-off is 8,000, manufactured by Filtron; or PLGC Pellicon Cassetle, normal molecular weight cut-off is 10,000, manufactured by Millipore) The supernatant was concentrated about 10 times, and 723 ml of the concentrated product (protein concentration: 338 mg/ml; total protein 2,445 mg; relative activity 43,000; total activity: 105,100,000) was mixed with 1.6 mol of ammonium sulfate per 1000 ml (total 288 g) to 804 ml of a solution containing a final concentration of 1.5 M ammonium sulfate was obtained, which was then applied to a Macro-PrepMethyl HIC column (diameter 5 cm, bed height 9 cm, manufactured by Bio-Rad, product number 156-0080) at a flow rate of 10 ml/min. , the column has been pre-equilibrated with 20mM sodium citrate buffer (pH 5.2) containing 1.5M ammonium sulfate. After loading, elute with 20mM sodium citrate buffer (pH 5.2) containing 1.25M ammonium sulfate to The column fraction F1 (2,384 ml; protein concentration, 0.864 mg/ml; total protein 2,061 mg; relative activity; 6,000) was obtained.

Next, the eluent was changed to sodium citrate, pH 5.8, to collect fraction F2 (1,092 ml; protein concentration 0.776 mg/ml; total protein 847 mg; relative activity, 150,000).

F2 (1,081 ml) of the Macro-Prep Methyl HIC column thus obtained was applied to an SP Sepharose Fast Flow column (diameter 3 cm, bed height 10 cm, manufactured by Pharmacia Biotech, product number 17-0729-01) at a flow rate of 10 ml/min, The column was pre-equilibrated with 20 mM sodium citrate buffer (pH 5.8). After loading, it was eluted with 20 mM sodium citrate buffer (pH 5.6) containing 110 mM NaCl to obtain F1 fraction (2,262 ml; protein concentration 0.270 mg/ml; total protein 610 mg, relative activity 30,000). Next, the elution solution was changed to 20 mM sodium citrate buffer (pH 5.4) containing 400 mM NaCl to collect the F2 fraction (856 ml; protein concentration, 0.189 mg/ml; total protein 162 mg, relative activity 300,000). Then, the eluent was changed to 20mM sodium citrate buffer (pH5.2) containing 1000mM NaCl to collect the eluted F3 fraction (370ml; protein concentration 0.034mg/ml; total protein 12.6mg; relative activity 150,000 ).

The main TPO active fraction F2 (845ml) of the SP Sepharose Fast Flow column step is mixed with 1-propanol with a final concentration of about 10%, and then used for LA-WGA columns (diameter 2cm, bed height 14 cm, manufactured by Honen, product number WG-007), which had been pre-equilibrated with 20 mM sodium citrate buffer (pH 5.4) containing 400 mM NaCl. After sample loading, the F1 fraction (64.4ml; protein concentration: 0.0178mg/ml; total protein 1.15mg, relative activity : 17,220). Then the elution solution was changed to 20mM sodium citrate buffer (pH 6.1) containing 0.4M GlcNAc and 10% 1-propanol to collect the F2 fraction (45ml; protein concentration 0.0104mg/ml; total protein 0.470mg; active 675,000).

The main TPO active F2 fraction (340ml) of LA-WGA column operation is mixed with about 0.005% TFA of final concentration, then through YMC-Pack CN-AP (diameter 6mm, bed height 250mm, YMC manufactures, product number AP-513 )) column chromatography. That is, use 0.1% TFA as developing agent A, 1-propanol containing 0.05% TFA as developing agent B, with the flow rate of 0.6ml/min, the sample is passed through the YMC-Pack CN-AP column equilibrated with 15% B in advance. After loading, increase the propanol concentration from 15%B to 25%B. Elution was accomplished with a linear gradient increasing in concentration from 25% B to 50% B over 65 minutes while collecting the eluate every 1.5 ml (2.5 minutes) in polypropylene tubes.

0.5 μl (1/3,000 fraction) of the eluate in each tube was mixed with HSA, concentrated by ultrafiltration to obtain 0.25 ml of IMDM detection culture solution containing 0.05% HSA, to identify the TPO active fraction by detection. As a result, strong TPO activity (relative activity about 630,000 to 4,800,000) was found in test tubes No. 24 to 30 (propanol concentration between 35.0 and 42.0%), which were collected as TPO active fraction FA (13.5 ml).

Use 0.1% TFA as developing agent A, 1-propanol containing 0.05% TFA as developing agent B, make the FA fraction obtained by YMC-Pack CN-AP column through Capcell Pak CI 300A (diameter 4.6mm, bed height 150mm , manufactured by Shiseido, product number CI TYPE: SG 300A) column chromatography. The fraction FA part (8.9ml) obtained by the operation of the YMC-Pack CN-AP column was mixed with 0.3ml glycerol, concentrated by centrifugal evaporation, then mixed with 2.5ml 10% B, and the resulting solution was mixed with 0.4ml/min The flow rate was through a Capcell Pak CI300A column pre-equilibrated with 20% B. After loading the sample, first eluted with 20% B for 5 minutes, and then eluted with a linear gradient from 20% B to 40% B for 50 minutes, while collecting the eluate every 1 ml (2.5 minutes) in a polypropylene test tube.

1 µl (1/1,000 fraction) of the eluate in each tube was mixed with HSA, concentrated by ultrafiltration to obtain 0.25 ml of IMDM detection solution containing 0.05% HSA, to identify the TPO active fraction by detection. As a result, strong TPO activity (relative activity about 3,000,000 to 22,500,000) was found in test tubes Nos. 20 to 23 (propanol concentration between 28.5 and 32.5%), which were collected as high activity fractions. <Example 37> Detection of the molecular weight of human TPO expressed in COS1 cells

The molecular weight of human full-length TPO expressed in COS1 cells (from expression plasmid pHTP1, F1 clone) was presumed to be larger than that estimated from the size of the peptide chain due to the addition of sugar chains. As a result, in the first step, a partially purified TPO fraction was prepared from the culture supernatant containing human full-length TPO expressed in COS1 cells (from the expression plasmid pHTP1, F1 clone) by the following method. That is, using developing agent A (0.1% trifluoroacetic acid (TFA)) and developing agent B (1-propanol containing 0.05% TFA), 0.3 ml of culture supernatant was used to pre-equilibrated with 25% B YMC-Pack PROTEIN-RP (diameter 0.46cm, bed height 15cm, manufactured by YMC, product number A-PRRP-33-46-25) column, 25% B passed through the column with a flow rate of 0.4ml/min for 5 minutes, and then Separation was accomplished using a linear density gradient from 25% B to 50% B over 50 minutes. TPO activity was found in eluates with propanol concentrations ranging from 34.5% to 43.5%, which were evaporated to dryness by centrifugal evaporation to obtain partially purified samples.

Next, after protein extraction from the SDS-PAGE electrophoresis gel performed under the non-reducing conditions described in Example 1, TPO activity was detected using the M-07e detection system, or as will be described in Example 45 below. The Western assay, based on reduction-treated DPC III labeling, measures molecular weight. As a result, TPO activity was found in a wide molecular weight range of about 69,000 to 94,000, thereby confirming a change in molecular weight. In addition, after digesting its N-glycosidic bond type sugar chains with N-glycanase (manufactured by Genzyme, product number: 1472-00), the apparent expression of TPO was measured based on the reduction-treated DPC III labeling. The molecular weight is 36,000 to 40,000, which is larger than the molecular weight of about 35,000 estimated from the size of the peptide chain. Therefore, it is also strongly suggested that there is an O-glycosidic bond type sugar chain.

Using the same method, the molecular weight of human full-length TPO (from expression plasmid pHTP1, P1 clone) expressed in COS1 cells was found to be 63,000 to 83,000. <Example 38> Biological characteristics of human TPO

The effect of human TPO on human and rat hematopoietic cells was primarily tested using culture supernatants of COS1 cells transfected with pHTF1.

In a colony assay using a CD34 ⁺ DR ⁺ cell fraction prepared from human cord blood, human TPO clearly stimulated the formation of megakaryocyte colonies. For example, 11.5 megakaryocyte colonies were formed from 6,000 CD34 ⁺ DR ⁺ cells in the presence of 10% (v/v) culture supernatant of COS1 cells transfected with pHT1-231. In order to examine the effect of human TPO on human megakaryocyte progenitor cells in peripheral blood, CD34 ⁺ cells were prepared from human peripheral blood as follows. Cells adhered to plastic discs were removed from leukocyte fractions obtained from human peripheral blood using Ficoll-Paque density gradients. Subsequently, SBA (soybean agglutinin) positive cells were removed from non-adherent cells by knockout using AIS Microselecter SBA (Asahi Medical). The resulting cells were cultured on AISMicroselecter CD34, and when CD3 ⁺ cells were cultured in liquid medium in the presence of the culture supernatant of transfected COS1 cells, the adsorbed cells were collected, CD34 ⁺ cells, and a large volume of Selective proliferation of GpIIb/IIIa ⁺ cells in which ploidy gains were ^also found. These results strongly suggest that human TPO has a selective effect on progenitor cells of the human megakaryocyte lineage and stimulates their proliferation and differentiation.

Human TPO induced significant numbers of megakaryocyte colonies in a colony assay using Gp II b/IV a ⁺ highly enriched for CFU-MK from rat bone marrow cells and Gp II b in front Formed non-adherent cells from the /III a ⁺ cell step. For example, in the presence of 20% (v/v) transfected COS1 cell culture supernatant, 34.5 megakaryocyte colonies were formed from 1,000 Gp II b/III a ⁺ cells, and 20,000 formed non-adherent Attached cells formed 28.5 megakaryocyte colonies. Additionally, although the shaped non-adherent cell fraction contained various hematopoietic progenitor cells rather than CFU-MK, megakaryocyte colonies formed only in the presence of human TPO, strongly illustrating the role of human TPO in the megakaryocyte lineage Progenitor cells are selected. In the presence of 20% (v/v) of the culture supernatant of transfected COS1 cells, Gp II b/III a ⁺ cells obtained from rat bone marrow were cultured in liquid medium for 3 to 5 days. A ploidy increase was clearly observed on day 5 of culture. <Example 39> Construction of a fusion protein (hereinafter referred to as "GST-TPO( 1-174)") Vector

To facilitate the expression of human TPO in E. coli, an artificial gene encoding human TPO and containing E. coli preferred codons was prepared. The nucleotide sequence of this DNA contains an amino-terminal Met codon (ATG) at position -1 for translation initiation in E. coli. Prepare synthetic oligonucleotides 1 to 12:

1:5-CTAGAAAAAACCAAGGAGGTAATAAATAATGAGCCC

GGCTCCGCCAGCTTGTGACCTTCGTGTTCTGTCT-3' (SEQ ID NO: 83);

2:5-CAGTTTAGACAGAACACGAAGGTCACAAGCTGGCGG

AGCCGGGCTCATTATTTATTACCTCCTTGGTTTTTT-3' (SEQ ID NO: 84);

3: 5-AAACTGCTTCGCGACTCTCACGTGCTGCACTCTCG

TCTGTCCCAGTGCCCGGAAGTTCACCCGCTGCCG-3' (SEQ ID NO: 85);

4:5-CGGGGTCGGCAGCGGGTGAACTTCCGGGGACTGGG

ACAGACGAGAGTGCAGCACGTGAGAGTCGCGAAG-3' (SEQ ID NO: 86);

5:5-ACCCCGGTTCTGCTTCCGGCTGTCGACTTCTCCCCT

GGGTGAATGGAAAACCCAGATGGAAGAGACCAAA-3' (SEQ ID NO: 87);

6:5-CTGAGCTTTGGTCTCTTCCATCTGGGTTTTTCCATT

CACCCAGGGAGAAGTCGACAGCCGGAAGCAGAAC-3' (SEQ ID NO: 88);

7:5-GCTCAGGACATCCTGGGTGCAGTAACTCTGCTTCT

GGAAGGCGTTATGGCTGCACGTGGCCAGCTTGGC-3' (SEQ ID NO: 89);

8:5-GGTCGGGCCAAGCTGGCCACGTGCAGCCATAACGC

CTTCCAGAAGCAGAGTTACTGCACCCAGGATGTC-3' (SEQ ID NO: 90);

9:5-CCGACCTGCCTGTCTTCCCTGCTTGGCCAGCTGTC

TGGCCAGGTTCGTCTGCTGCTCGGCGCTCTGCAG-3' (SEQ ID NO: 91);

10:5-CAGAGACTGCAGAGCGCCGAGCAGACGAACCTGGC

CAGACAGCTGGCCAAGCAGGGAAGACAGGCA-3' (SEQ ID NO: 92);

11:5-TCTCTGCTTGGCACCCAGCTGCCGCCACAGGGCCG

TACCACTGCTCACAAGGATCCGAACGCTATCTTCC-3' (SEQ ID NO: 93) and

12:5-AAGACAGGAAGATAGCGTTCGGATCCTTGTGAGCA

GTGGTACGGCCCTGTGGCGGCAGCTGGGTGCCAAG-3' (SEQ ID NO: 94) Then, in a solution consisting of 1 mM ATP, 10 mM Tris-acetate, 10 mM magnesium acetate and 50 mM potassium acetate, each of 2-11 Phosphorylation of synthetic oligonucleotides. In order to make these synthetic oligonucleotides into 6 double-stranded DNA fragments, these synthetic oligonucleotides were divided into 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, and 11 and 12 For binding, each set of bindings was annealed in a solution consisting of 10 mM Tris/HCl (pH 7.5), 10 mM _MgCl2 and 50 mM NaCl. Next, the three double-stranded DNA fragments of 1 and 2, 3 and 4, and 5 and 6, and the other three double-stranded DNAs of 7 and 8, 9 and 10, and 11 and 12 were treated with T4 ligase (manufactured by Life Technology) Fragments, the two reaction solutions were retreated with T4 ligase in the same manner. The DNA fragment obtained by the ligation reaction was digested with BamHI (manufactured by Boehringer-Mannheim), then subjected to 2% agarose gel electrophoresis to recover a fragment of about 390 to 400 bp, followed by purification using the Prep-A-Gene DNA purification kit, and then subcloning into pUC18 digested with XbaI and BamHI in advance (using E.coli DH5α as host). From these obtained clones, a clone having an artificial gene encoding human TPO and containing the following E. coli preferred codons was screened by nucleotide sequence analysis and named pUC18(XB)(1-123). The DNA sequence of the coding strand is listed in the sequence listing (SEQ ID NO: 9).

10 1 20 30 40 50 60

CTAGAAAAAA CCAAGGAGGT AATAAATAAT GAGCCCGGCT CCGCCAGCTT GTGACCTTCG

  TTTTTT GGTTCCTCCA TTATTTATTA CTCGGGCCGA GGCGGTCGAA CACTGGAAGC

wxya _

70 80 3 90 100 110 120

TGTTCTGTCT AAACTGCTTC GCGACTCTCA CGTGCTGCAC TCTCGTCTGT CCCAGTGCCC

ACAAGACAGA TTTGACGAAG CCCTGAGAGT GCACGACGTG AGAGCAGACA GGGTCACGGG

4

130 140 150 5 160 170 180

GGAAGTTCAC CCGCTGCCGA CCCCGGTTCT GCTTCCGGCT GTCGACTTCT CCCTGGGTGA

CCTTCAAGTG GGCGACGGCT GGGGCCAAGA CGAAGGCCGA CAGCTGAAGA GGGACCCACT

6

190 200 210 220 7 230 240

ATGGAAAACC CAGATGGAAG AGACCAAAGC TCAGGACATC CTGGGTGCAG TAACTCTGCT

TACCTTTTGG GTCTACCTTC TCTGGTTTCG AGTCCTGTAG GACCCACGTC ATTGAGACGA

8

250 260 270 280 290 9 300

TCTGGAAGGC GTTATGGCTG CACGTGGCCA GCTTGGCCCG ACCTGCCTGT CTTCCCTGCT

AGACCTTCCG CAATACCGAC GTGCACCGGT CGAACCGGGC TGGACGGACA GAAGGGACGA

10

310 320 330 340 350 360

TGGCCAGCTG TCTGGCCAGG TTCGTCTGCT GCTCGGCGCT CTGCAGTCTC TGCTTGGCAC

ACCGGTCGAC AGACCGGTCC AAGCAGACGA CGAGCCGCGA GACGTCAGAG ACGAACCGTG

11 370 380 390 400 410 420

CCAGCTGCCG CCACAGGGCC GTACCACTGC TCACAAGGAT CCGAACGCTA TCTTCC

GGTCGACGGC GGTGTCCCGG CATGGTGACG AGTGTTCGTA GGCTTGCGAT AGAAGGACAG AA

12 BamHI

Single-strand cDNA was synthesized from 1 μg of poly(A) ⁺ RNA derived from human normal liver with oligo dT primer, and then PCR was performed using 0.1 volume of cDNA synthesis reaction solution as a template. Using hTPO-C and hTPO-Z (EcoRI) as primers, a PCR reaction was performed in a volume of 100 μl (incubated at 96°C for 2 minutes, repeated for 30 cycles, each cycle including incubation at 95°C for 1 minute, 58°C 1 min and 1 min at 72°C, with a final incubation of 7 min at 72°C). The human TPO cDNA fragment obtained in this way was digested with BamHI and EcoRI, then subjected to 2% agarose gel electrophoresis to recover a fragment of about 600bp, then purified with the Prep-A-Gene DNA purification kit, and then subcloned into In pUC18 digested with EcoRI and BamHI (E. coli DH5α as host). The clone encoding the correct human TPO nucleotide sequence was screened out by nucleotide sequence analysis and named pUC18(BE)(124-332). The oligonucleotide sequences of the primers used in the PCR reaction were as follows: hTPO-C: 5'-GGA GGA GAC CAA GGC ACA GGA-3' (SEQ ID NO: 95) (positions 329 to 349 of the pHT1 clone); and hTPO-Z(EcoRI): 5'-CCG GAA TTC TTA CCC TTC CTG AGA CAGATT-3' (SEQ ID NO: 96) (by adding an EcoRI recognition sequence to the antisense primer corresponding to positions 1,143 to 1,163 of the pHTF1 clone prepared in).

The clone pUC18(BE)(124-332) was digested with BamHI and EcoRI, and then subjected to 2% agarose gel electrophoresis to recover the C-terminal side fragment of human TPO cDNA of about 600bp, followed by Prep-A-Gene DNA purification kit Purified and then subcloned into pUC18(XB)(1-123) predigested with EcoRI and BamHI (using E. coli DH5α as host). The clone obtained by this method was called pUC18(XE)(1-332).

Since the examples of measuring in vitro human TPO activity by preparing and expressing C-terminal deletion constructs of various human TPO cDNAs have confirmed that human TPO activity exists in human TPO peptide fragments of amino acid residues 1 to 163, the construct An expression vector containing the peptide fragment. In this example, the DNA sequence encoding GST-TPO(1-174) was expressed.

Since the restriction enzyme SacI recognizes the nucleotide sequence corresponding to positions 681-686 (amino acid residues 173 and 174) of the pHTF1 clone, two synthetic oligonucleotides were used to introduce the two ends using the recognition site of the restriction enzyme. a. That is, in a solution consisting of 10 mM Tris/HCl (pH 7.5), 10 mM MgCl ₂ and 50 mM NaCl, using a DNA ligation kit (manufactured by Takara Shuzo), annealing of two synthetic oligonucleotides SSE1 and SSE2 will thereby The obtained double-stranded DNA fragment was introduced into pUC18(XE)(1-332) from which about 480 bp human TPO cDNA C-terminal fragment had been removed by SacI and EcoRI digestion, thereby obtaining clone pUC18(XS)(1-174)( E. coli DH5α was used as host). The sequences of the synthetic oligonucleotides used herein are as follows:

SSE1: CTAATGAG (SEQ ID NO: 97);

SSE2: AATTCTCATTAGAGCT (SEQ ID NO: 98);

SacI SSE1 EcoRI

5'-CTAATGAG-3' (SEQ ID NO: 99)

3'-TCGAGATTACTCTTTAA-5' (SEQ ID NO: 100)

SSE2

The clone PUC18(XS)(1-174) obtained above was digested with XbaI and EcoRI, and then subjected to 2% agarose gel electrophoresis to recover a fragment of about 600bp encoding the 1-174 amino acid residues of human TPO, followed by Prep - The fragment was purified by A-Gene DNA purification kit, and then subcloned into pBluescript II SK ⁺ (manufactured by Stratagene) (using E. coli DH 5α as a host) digested with XbaI and EcoRI in advance. The clone obtained by this method was called pBL(XS)(1-174). Using the same method, the clone pBL(XS)(1-174) was digested with XbaI and Hind III to recover an approximately 550bp fragment encoding amino acid residues 1-174 of human TPO, and then purified with the Prep-A-Gene DNA purification kit The fragment was then subcloned into pCFM536 (EP-A-136490) previously digested with Xbal and Hind III (using E. coli DH5α as host). The clone obtained by this method was called pCFM536/hT(1-174).

In order to efficiently express GST-TPO(1-174), the following experiments were performed using PGEX-2T (Pharmacia), which is an expression vector for glutathione-S-transferase (GST) fusion protein. Use pCFM536/hT(1-174) as template, and two PCR primers GEX1 and GEX3 to carry out PCR reaction (incubate at 96°C for 2 minutes, repeat 22 cycles in total, each cycle includes incubation at 95°C for 1 minute, 41°C for 1 minute and 72°C for 1 minute, and finally at 72°C for 7 minutes). The human TPO coding fragment obtained by this method was digested with NaeI and EcoRI, and then subjected to 2% agarose gel electrophoresis to recover a fragment of about 550bp, followed by purifying the fragment with the Prep-A-GeneDNA purification kit, and cloning it into a pre- In the pGEX-2T digested with EcoRI and SmaI (using E.coli DH5α as the host), the clone encoding the correct human TPO nucleotide sequence was screened out by nucleotide sequence analysis, which was called pGEX-2T/hT(1- 174) to prepare a transformant for expressing GST-TPO(1-174). The sequences of the primers used in the PCR reaction are as follows: GEX1: 5'-ATC GCC GGC TCC GCC AGC TTG TGA C-3' (SEQ ID NO: 101) (by adding the NaeI recognition sequence to 21 of SEQ ID NO: 10 prepared at position 39); and GEX3: 5'-GCC GAA TTC TCA TTA GAG CTC GTT CAG TGT-3' (SEQ ID NO: 102) (corresponding to the reverse of the sequence at position 523-549 in SEQ ID NO: 10 sense primers).

The expression plasmid contains DNA sequence encoding GST protein connected with thrombin recognition peptide and human TPO (1-174 amino acids). The DNA sequences encoding the thrombin recognition peptide and human TPO (amino acids 1-174) are listed in the sequence listing (SEQ ID NO: 10). <Example 40> Expression of GST-TPO (1-174) in E.coli

The transformant prepared in Example 39 was cultured overnight at 37°C on a shaker with 60 ml of LB medium containing 50 μg/ml of ampicillin, and 25 ml of the resulting culture solution was added to 1,000 ml of LB medium containing 50 μg/ml of ampicillin. In culture medium, grow on a shaker at 37°C until the OD at _A600 reaches 0.7 to 0.8. Then, IPTG was added to the culture medium to reach a final concentration of 0.1 mM, and cultured with shaking for another 3 hours to induce the expression of GST-TPO(1-174). <Example 41> Purification of GST-TPO (1-174) expressed in E.coli and proof of its biological activity

The frozen cells of 5.9 grams of recombinant bacterial strains, which produce GST-TPO ( 1-174). The suspension is centrifuged and GST-TPO(1-174) is recovered in the pelleted fraction, removing most of the contaminating proteins, cellular components, and the like. Next, the resulting precipitated fraction containing GST-TPO(1-174) was suspended in 5 ml of water, and then 6 ml of 1M Tris buffer (pH 8.5), 120 ml of 10M urea and 16 ml of water were added thereto with stirring. After stirring for 5 minutes to dissolve the contents, the resulting solution was equally divided into 4 parts to complete the following steps (1)-(4).

(1) Dilute a sample 10 times with 20mM Tris buffer (pH8.5). Reduced glutathione and oxidized glutathione were added thereto to concentrations of 5 mM and 0.5 mM, respectively, followed by overnight culture at 4°C. The resulting mixture was centrifuged to recover GST-TPO(1-174) as a supernatant, diluted 2-fold with 20 mM sodium citrate at pH 5.5, then adjusted to pH 5.5 with acetic acid, and GST-TPO(1-174) 174) solution was added to SP Sepharose Fast Flow (manufactured by Pharmaeia Biotech, product number 17-0729-01) cation exchange column equilibrated with 20 mM sodium citrate (pH 5.5) in advance. After washing the column with 20 mM sodium citrate buffer (pH 5.5), GST-TPO(1-174) was eluted with 20 mM sodium citrate buffer (pH 5.5) containing 500 mM NaCl. 129 ml of the eluate was mixed with 2.6 ml of 1M Tris buffer (pH 8.5), and the resulting mixture at pH 8.1 was used on a Glutathione Sepharose 4B (manufactured by Pharmacia Biotech, article number 17-0756-01) column to adsorb GST- TPO (1-174). After washing the column with PBS, GST-TPO(1-174) was eluted with 20 mM Tris buffer containing 10 mM reduced glutathione. The resulting eluate was mixed with 37 NIH units of thrombin, placed at room temperature for 4 hours, diluted 10-fold with PBS, then used on a Glutathione Sepharose4B column to adsorb digested GST, and reclaim TPO in the non-adsorbed fraction (1 -174).

The recovered non-adsorbed fraction was diluted 3-fold with 20 mM sodium citrate buffer pH 5.5, and then used on a SP Sepharose Fast Flow cation exchange column pre-equilibrated with the same buffer, using a linear density gradient in the same buffer 0-500mM NaCl to elute the desired substance.

(2) Existing all operations of step (1) under the condition of 0.1% polysorbate (polysorbate 80).

(3) Dilute a sample 10-fold with 20mM Tris buffer at pH 8.5. Reduced glutathione and oxidized glutathione were added thereto to make final concentrations of 5 mM and 0.5 mM, respectively, followed by culturing overnight at 4°C. The resulting mixture was centrifuged to collect GST-TPO(1-174) as a supernatant, which was then diluted 2-fold with 20 mM sodium citrate buffer at pH 5.5, and the pH was adjusted to 5.5 with acetic acid. Add GST-TPO (1-174) solution to SP Sepharese Fast Flow cation exchange resin pre-equilibrated with 20mM sodium citrate buffer (pH5.5). After washing the resin with 20 mM sodium citrate buffer (pH 5.5), GST-TPO(1-174) was eluted with 20 mM sodium citrate buffer (pH 5.5) containing 500 mM NaCl. Use 1M Tris buffer at pH 8.5 to adjust the pH of the eluate to 8, mix it with 320 NIH units of thrombin to remove the GST polypeptide sequence through cleavage at the thrombin recognition peptide linker, put it at room temperature for 4 hours, and use pH 5.5 The 20mM sodium citrate buffer was diluted 5 times, and then added to the SP Sepharose Fast Flow cation exchange column pre-equilibrated with the same buffer, and the desired substance was eluted with a linear density gradient of 0-500mM NaCl in the same buffer.

(4) Complete all the operations in step (3) under the condition of 0.1% polysorbate.

Each fraction eluted by SP Sepharose Fast Flow cation exchange column chromatography at a NaCl density of about 200-400 mM was thoroughly dialyzed against IMDM medium, and then evaluated with the rat CFU-MK detection system. When the fraction was analyzed by SDS-PAGE in the presence of a reducing agent, a band corresponding to a molecular weight of 19 kd (purity 1-20%) was detected as a major protein band of the fraction. After N-terminal sequence analysis of this band by SDS-PAGF and transfer through PVDF membrane as described in Example 1, it was confirmed that this band contained a protein expressed as a pGEX-2T/hT(1-174)-derived fusion protein. TPO sequence. The deduced amino acid sequence of the resulting TPO is [Gly ^-1 ]TPO, given the sequence of the thrombin recognition peptide linker and the known activity of thrombin on the linker. <Example 42> Construction of a vector for expressing human TPO (amino acids 1-163) substituted at position 1 (Ser->Ala) and position 3 (Ala->Val) in E.coli

The clone pUC18(XE)(1-332) prepared in Example 39 was digested with XbaI and EcoRI, and a fragment of about 1000 bp was recovered by 2% agarose gel electrophoresis, which encoded the 1-332 amino acid residues of human TPO , followed by purification of the fragment with a Prep-A-Gene DNA purification kit, and then subcloning into pBluescript II SK ⁺ (manufactured by Stratagene) digested with XbaI and EcoRI in advance (using E.coli DH5α as a host). The clone obtained in this way was called pBL(XE)(1-332). Next, the region from its BamHI recognition sequence to amino acid 163 (366-489) of clone pBL(XE)(1-332) was replaced with the dominant codon of E.coli. Prepare the synthetic oligonucleotides 13-20 shown below and then phosphorylate each pair of synthetic oligonucleotides 13 and 14, 15 and 16, and 17 and 18 in the same tube containing a solution made of 0.1 Composition of mM ATP, 10 mM Tris-acetic acid, 10 mM magnesium acetate and 50 mM potassium acetate. After adding 1/10 volume of a solution consisting of 100 mM Tris/HCl (pH 7.5), ₁₀₀ mM MgCl and 500 mM NaCl, the resulting solution was heated in a boiling water bath for 3 minutes and then cooled to room temperature to obtain double-stranded DNA fragments. Next, the resulting 3 pairs of double-stranded DNA fragments: 13 and 14, 15 and 16, 17 and 18 were ligated using a DNA ligation kit (manufactured by Takara Shuzo), and using the thus ligated product as a template, the synthesized oligonucleotides 19 and 20 Complete PCR for primers. The resulting PCR product was digested with BamHI and Hind III, and then subjected to 2% agarose gel electrophoresis to recover a fragment of about 130 bp with the Prep-A-Gene DNA purification kit. The fragment was subcloned into pBL(XE)(1-332) previously digested with BamHI and Hind III (using E. coli DH5 as host). A clone containing the following nucleotide sequence was screened out from the obtained clones by sequence analysis, and named as pBL(XH)(1-163):

13: 5′-GATCCGAACGCTATCTTCCTGTCTTTCCAGCACCTGCTGCGT-3′

(SEQ ID NO: 103);

14: 5′-TTTGCCACGCAGCAGGTGCTGGAAAGACAGGAAGATAGCGTTCG-3′

(SEQ ID NO: 104);

15: 5′-GGCAAAGTTCGTTTCCTGATGCTGGTTGGCGGTTTCTACCCTG-3′

(SEQ ID NO: 105);

16: 5′-ACGCACAGGGTAGAACCGCCAACCAGCATCAGGAAACGAAC-3′

(SEQ ID NO: 106);

17: 5′-TGCGTTCGTCGGGCGCCGCCAACCACTGCTGTTCCGTCTTAATGAA-3′

(SEQ ID NO: 107);

18: 5′-AGCTTTCATTAAGACGGAACAGCAGTGGTTGGCGGCGCCCGACGA-3′

(SEQ ID NO: 108);

19: 5'-AAGGATCCGAACGCTATCTTCCTG-3' (SEQ ID NO: 109); and

20: 5'-AGAAGCTTTCATTAAGACGGAACA-3' (SEQ ID NO: 110).

In order to increase the expression quantity of human TPO (position 1-163) and prevent protease from hydrolyzing the N-terminus, an expression for expressing mutant human TPO (position 1-163) (hereinafter referred to as "h6T(1-163)") was constructed Carrier, the mutant human TPO is at position 1 (Ser becomes Ala) and position 3 (Ala becomes Val) of human TPO (position 1-163) ([Ala ¹ , Val ³ ]TPO (1-163)) There are substitutions while encoding Lys at position -1 and Met at position -2 ([Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ ]TPO(1-163)). The following 4 kinds of synthetic oligonucleotides were prepared, and the synthetic oligonucleotides were 2- 9 and 3-3 phosphorylation. In order to make these synthetic oligonucleotides _form double-stranded DNA fragments, each pair of single-stranded DNA fragments 1-9 and 2- 9 and 3-3 and 4-3 annealed. The resulting two pairs of double-stranded DNAs consisting of 1-9 and 2-9 and 3-3 and 4-3 were then treated with a DNA ligation kit (manufactured by Takara Shuzo). Subclone the DNA fragments obtained by the ligation reaction into pBL(XH)(1-163) digested with XbaI and NruI in advance, and use sequence analysis to screen for clones that are correctly substituted by the following synthetic oligonucleotides (use E.coliDH5α as host) and named pBL(XH)h6T(1-163):

1-9: 5′-CTAGAAAAAACCAAGGAGGTAATAAATAATGAAAGCACCTGTACCA-3′

(SEQ ID NO: 111);

2-9: 5′-CAGGTGGTACAGGTGCTTTCATTATTTATTACCTCCTTGGTTTTTT-3′

(SEQ ID NO: 112);

3-3: 5′-CCTGCATGTGATTTACGGGTCCTGTCTAAACTGCTGCG-3′

(SEQ ID NO: 113);

4-3: 5′-CGCAGCAGTTTAGACAGGACCCGTAAATCACATG-3′

(SEQ ID NO: 114);

10 1-9 20 30 40

CTAGAAAAAA CCAAGGAGGT AATAAATAAT GAAAGCACCT

TTTTTT GGTTCCTCCA TTATTTATTA CTTTCGTGGA

XbaI 2-9

50 60 3-3 70 80

GTACCACCTG CATGTGATTT ACGGGTCCTG TCTAAACTGC TGCG

CATGGTGGAC GTACACTAAA TGCCCAGGAC AGATTTGACG ACGC

4-3

(SEQ ID NO: 115)

Clone pBL(XH)(1-163) was digested with XbaI and HindIII to recover a fragment of about 500bp. The fragment encodes amino acid residues 1-163 of mutant human TPO, and the fragment is then purified with a Prep-A-Gene DNA purification kit, and then cloned into pCFM536 (EP-A-136490) (EP-A-136490) digested with XhaI and Hind III in advance ( E. coli JM109 previously transformed with pMW1 (ATCC No. 39933) was used as a host). The clone obtained by this method was named pCFM536/h6T(1-163). The E. coli strain containing the expression vector was used as a transformant for expressing mutant human TPO, namely h6T(1-163). The expression plasmid contains the DNA sequence (SEQ ID NO: 11) shown in the sequence listing. <Example 43> Expression of h6T(1-163) in E.coli

Expression of the expression plasmid pCFM536 is controlled by its own lambda PL promoter under the control of the cI857 repressor gene. Using 60ml of LB medium containing 50μg/ml of ampicillin and 12.5μg/ml of tetracycline, the transformant obtained in Example 42 was cultured overnight at 30°C on a shaker, and 25μl of the resulting culture solution was added to 1000ml of 50μg In LB medium with ampicillin/ml, culture on a shaker at 37°C until the OD value at _A600 reaches 1.0-1.2. Thereafter, about 330 ml of LB medium heated to 65° C. was added to the culture solution so that the final temperature of the medium was 42° C., and shaking culture was continued at 42° C. for 3 hours to induce expression of h6T(1-163). <Example 44> Purification of h6T(1-163) expressed in E.coli and confirmation of its biological activity

3.6 g of frozen cells of the recombinant strain producing h6T(1-163) were suspended in 10 ml of water, and crushed with a high-pressure crusher. The suspension is centrifuged, the pellet is recovered and most of the contaminating proteins, cellular components, etc. are removed. The recovered precipitate part containing h6T(1-163) was suspended in 7ml water, and 3ml Tris buffer solution (pH8.5) was added while stirring, then urea (final concentration, 8M), guanidine hydrochloride (final concentration, 6M), sodium N-lauroyl sarcosinate (final concentration, 2%). After stirring at room temperature for 5-20 minutes to dissolve the contents, the resulting solution was diluted 10-fold with 20 mM Tris buffer at pH 8.5, and incubated overnight at 4°C to refold the protein. In this example, glutathione and copper sulfate were used as additives in addition to air oxidation. Upon centrifugation, h6T(1-163) was recovered in the supernatant. When each recovered fraction was analyzed by SDS-PAGE in the presence of a reducing agent, a major protein band (purity, 30-40%) corresponding to a molecular weight of 18 kd was detected for each fraction. N-terminal sequence analysis of this band by SDS-PAGE followed by transfer with PVDF membranes as described in Example 1 demonstrated that this band contained TPO expressed as pCMF536/h6T(1-163)-derived mutant TPO sequence. Fractions were dialyzed extensively against IMDM medium and assessed with the rat CFU-MK detection system, and TPO activity in a dose-dependent manner was found in all fractions. <Example 45> Preparation of anti-TPO peptide antibody and detection of TPO by SDS-PAGE and Western analysis

The successful preparation of anti-TPO peptide antibodies has made it possible to detect the TPO protein by immunological methods. As a result, 3 regions that seemed to be relatively useful as antigens were screened from the determined amino acid sequence of TPO (see below), and with each screened region, according to Tam (Proc. 1988) to synthesize four-chain multiple antigenic peptide (MAP) type peptides, and then immunize 2 rabbits with 100 μg of various synthesized peptides 8 times to obtain corresponding antiserum samples. Amino acid sequences contained in synthetic peptide antigens

(a) Rat TPO(9-28) (peptide RT1 region)

PRLLNKLLRDSYLLH R RLSQ (SEQ ID NO: 116)

(In the amino acid residue determined from the gene, the R at position 24 is originally S.)

(b) Rat TPO (46-66) (peptide RT2 region)

FSLGEWKTQTEQSKAQDILGA (SEQ ID NO: 117)

(c) Rat TPO (163-180) (peptide RT4 region)

SRTSQLLTLNKFPNR LLD (SEQ ID NO: 118)

(Among the amino acid residues determined from the gene, LLD at positions 178-180 was originally TSG.)

Next, among these antiserum samples, the anti-RT1 peptide and anti-RT2 peptide were first subjected to protein A (PROSEP-A; manufactured by Bioprocessing Ltd., product number 8427) column chromatography to prepare IgG fractions (2,344 mg, respectively). and 1,920 mg), each fraction of 54 mg and 32 mg, respectively, was biotinylated by coupling it with active biotin (NHS-LC-Biotin II; manufactured by PIERCE, product number 21336). The sample containing recombinant TPO was subjected to SDS-PAGE, and then electroblotted on PVDF or nitrocellulose membrane with the same method as described in the examples, using these biotinylated antibodies as primary antibodies, and completed Western by conventional methods. analyze.

That is, the blotted membrane was washed with a solution consisting of 20mM Tris-HCl and 0.5M NaCl (pH7.5) (TBS) for 5 minutes, and washed twice with 0.1% Tween 20 (TTBS) containing TBS for 5 minutes each time. , and then treated with a blocking agent (BlockAce; manufactured by Dainippon Pharmaceutical, product number uk-B25) for 60 minutes. Next, the membrane was treated with a TTBS solution containing 10 µg/ml of biotinylated anti-TPO peptide antibody, 0.05% BSA and 10% BlockAce for 60 minutes, and then washed twice with TTBS for 5 minutes each. Thereafter, the membrane was treated for 30 minutes with a solution obtained by diluting 5,000-fold alkaline phosphatase-labeled avidin (manufactured by Leinco Technologies, product number A108) from a TTBS solution containing 10% BlockAce, and washed with TTBS for 2 5 minutes each time, washed with TBS for 5 minutes, and then developed with an alkaline phosphatase substrate (manufactured by Bio-Rad, product number 170-6432). The Western blot analysis described above was performed at room temperature.

As a result, it was able to recognize not only various recombinant rat TPO proteins expressed in COS1 cells, but also various recombinant human TPO proteins expressed in COS1 cells and E. coli cells (illustratively, as in COS1 cells Expression of plasmid pHTP1- or plasmid pHTF1-derived human TPO, and products of glycosidic bond-cleavage and thrombin digestion of GST-TPO(1-174) expressed in E.coli and in E.coli Mutant TPO expressed: h6T(1-163), all of which are described in the Examples). In addition, these results also made it possible to analyze various types of recombinant human TPO.

In addition to the above, the possibility of producing anti-human TPO peptide antibodies was also demonstrated. Antibodies obtained by these techniques can not only be used for Western analysis, but also can be used for purification of TPO through antibody columns and commonly used antibody-assisted immunological methods.

Six regions expected to have relatively suitable antigenicity in the human TPO amino acid sequence shown in SEQ ID NO: 7 were screened (see Table 4). Tetrameric multiple antigenic peptide (MAP) peptides were synthesized, each monomer corresponding to a different domain. Two rabbits were immunized 8 times with each peptide at a dose of 100 mg/time.

In contrast to this, monomeric peptides in which Cys residues have been bonded to the C-terminus of each peptide region were also synthesized. This peptide was used as a test antigen to determine antibody titers by enzyme immunoassay. As a result, it was confirmed that the above-prepared sera had increased antibody titers, and therefore these sera were used as antisera.

Antibodies were prepared by immunizing two rabbits with each antigenic peptide to produce antisera against the peptide. According to different individual rabbits, the obtained two kinds of anti-HT1 peptide antibodies were respectively called anti-HT1-1 peptide antibody and anti-HT1-2 peptide antibody.

The purification of the anti-HT1-1 peptide antibody will be exemplified below.

First, HT1 monomeric peptides that had been bonded to Cys residues were coupled to 12 ml of Sulfo-Liak coupling gel (Pierce, Cat. No. 44895). Briefly, antigen-containing peptide solutions were coupled to gels equilibrated with 6 volumes (relative to gel volume) of coupling buffer (50 mM Tris, 50 mM EDTA-NapH 8.5) for 15 min. Then after 30 minutes of incubation, the gel was washed with 3 volumes (relative to the volume of the gel) of coupling buffer. Add the coupling buffer containing 0.05M L-Cys-HCl to the gel at the amount of 1ml/ml gel and block unreacted groups for more than 15 minutes. After 30 minutes of incubation, the gel was washed with 8 volumes (relative to the gel volume) of coupling buffer. All coupling reactions were performed at room temperature. Therefore, the peptide was covalently bound to the gel at a coupling rate of 28.3%, and then an antigenicity containing 0.8 mg peptide/ml gel was prepared.

76.7ml (protein content 3620mg) containing anti-HT1-1 peptide antibody antiserum (extracted from a rabbit, a total of 78.4ml) was added to the 50mM solution containing 150mM NaCl and 0.05% sodium azide in advance The antigen column was equilibrated with phosphate buffer (pH 8), then washed with the same buffer to obtain 105.9 ml of effluent fraction (protein content 3680 mg). The adsorbed fraction was then eluted with 0.1M citrate buffer (pH 3.0), followed by neutralization with 21.1 ml of 0.1M carbonate buffer (pH 9.9), followed by ultrafiltration (with Amicon YM30 membrane) Concentration gave purified anti-HT1-1 peptide antibody in 50 mM phosphate buffer (pH 8) containing 150 mM NaCl and 0.05% _NaN3 .

Similarly, the following antibodies were prepared: anti-HT1-2 peptide antibody (60.0 mg); anti-HT2-1 peptide antibody (18.8 mg); anti-HT2-2 peptide antibody (8.2 mg); Anti-HT3 to -HT6 peptide antibodies can be prepared in a similar manner.

3 mg affinity purified anti-HT1-1 peptide antibody, anti-HT1-2 peptide antibody, anti-HT2-1 peptide by conjugation with activated biotin (Pierce, NHS-LC-Biotin II, Cat. No. 21336) Antibody or anti-HT2-2 peptide antibody biotinylated.

After SDS-PAGE of the partially purified recombinant human TPO standard from the culture supernatant of CHO cells in which the gene encoding the amino acid sequence listed in Table 4 was introduced and expressed, Western blotting (in nitrocellulose filter or PVDF), it was found that all of the above-mentioned purified antibodies could recognize and detect human TPO. Table 4 human TPO amino acid sequence human TPO (amino acid number 8-28) peptide HT1 region included in the tetrameric MAP type synthetic peptide antigen: DLRVLSKLLRDSHVLHSRLSQ (SEQ ID NO: 119) human TPO (amino acid number 47-62) peptide HT2 Region: SLGEWKTQMEETKAQD (SEQ ID NO: 120) human TPO (amino acid number 108-126) peptide HT3 region: LGTQLPPQGRTTAHKDPNA (SEQ ID NO: 121) human TPO (amino acid number 172-190) peptide HT4 region: NELPNRTSGLLETNFTASA (SEQ ID NO: 122) Human TPO (amino acid number 262-284) peptide HT5 region: SLPPNLQPGYSPSPTHPPTGQYT (SEQ ID NO: 123) human TPO (amino acid number 306-332) peptide HT6 region: PSAPPTPTSPLLNTSYTHSQNLSQEG (SEQ ID NO: 124) <Example 46> preparation Anti-TPO Peptide Antibody Column

Since the anti-rat TPO peptide antibody obtained in Example 45 can recognize rat and human TPO molecules, the immunoglobulin (IgG) fractions of the anti-RT1 peptide antibody and the anti-RT2 peptide antibody were combined with chemical The activated gel supports were linked to prepare anti-TPO peptide antibody columns. Two kinds of antibodies used as materials were prepared from two rabbit sera, respectively. That is, the anti-RT1 peptide antibodies prepared from two rabbits were named anti-RT1-1 peptide antibody and anti-RT1-2 peptide antibody, and the anti-RT2 peptide antibodies prepared from the other two rabbits were named anti-RT2 -1 peptide antibody and anti-RT2-2 peptide antibody. Since these antibodies were used for the preparation of antibody columns, a total of 5 columns were obtained, that is, 2 anti-RT1 peptide antibody columns (hereinafter referred to as "anti-RT1-1 antibody column" and "anti-RT1-2 antibody column"). ”), 2 anti-RT2 peptide antibody columns (hereinafter referred to as “anti-RT2-1 antibody column” and “anti-RT2-2 antibody column”) and one by combining anti-RT1-2 peptide antibody with anti-RT2 -1 peptide antibody was mixed, and then the mixture was coupled with gel to prepare an antibody column (anti-RT1-2+2-1 mixed antibody column).

Dissolve each antibody in a solution of 50 mM sodium phosphate and 0.15 M NaCl (pH 8.0) to a final concentration of 5 mg/ml (or for an anti-RT1+2 mixed antibody column, anti-RT1-2 peptide antibody and anti- - 2.5 mg/ml for each of the RT2-1 peptide antibodies), 2.31 ml of the resulting solution was mixed with 1.54 ml of swollen formyl-activated gel (Formyl-Cellulofine, manufactured by Chisso), and the reaction was carried out at 4° C. for 2 hours. coupling reaction. 1.1 ml of a 10 mg/ml reducing agent (trimethylamine borane (TMAB) solution, manufactured by Seikagaku kogyo, product number 680246) solution was added thereto, followed by further coupling reaction at 61 pm, followed by centrifugation to recover the gel fraction . 10 ml of purified water was added to the gel, and the gel fraction recovered by centrifugation was repeated 4 times to remove unreacted antibody molecules. Next, the resulting gel was mixed with 4.6 ml of blocking solution (0.2 M sodium phosphate and 1 M ethanolamine, pH 7.0) and 1.1 ml of reducing agent solution, and treated at 4 °C for at least 2 h to block the activity of unreacted gel group. Thereafter, the gel was washed with purified water and DPBS, centrifuged with a centrifuge, placed in a small cylindrical tube, washed with 3M potassium thiocyanate solution, and 0.1M Gly-HCl (pH2.5) solution, equilibrated with DPBS, Then store.

Anti-RT1-1 Antibody Column, Anti-RT1-2 Antibody Column, Anti-RT2-1 Antibody Column, Anti-RT2-2 Antibody Column and Anti-RT1-2-2-1 Mixed Antibody Column for 5 Anti-TPO In peptibody gels, the coupling efficiencies of the corresponding IgG fractions were 97.4%, 95.4%, 98.4%, 98.3% and 99.4%, respectively. Additionally, the numbers of IgG fractions coupled per gel volume were: 5.6 mg/ml gel, 5.8 mg/ml gel, 5.7 mg/ml gel, 5.7 mg/ml gel and 2.9 mg/ml gel. As a result, the number and efficiency of conjugation did not significantly vary depending on the source of the antibody and the difference in the antigen, and the experiments shown in Example 47 below were carried out using these antibody columns. <Example 47> TPO obtained from the culture supernatant (obtained by transfecting COS1 cells with the expression vector pHTP1) was purified using an anti-TPO antibody column and reverse-phase column chromatography, and then its biological activity was determined

In vitro assays were performed with the M-07e detection system to evaluate the following purified samples. The TPO partial transformation sample obtained from the culture supernatant obtained by transfecting COS1 cells with the expression vector pHTP1 in Example 35 (fractions other than the main TPO fraction, that is, washing Macro- Fractions F1 and F2 after the Prep Methyl HIC column and the fractions F1 and F3 of the SP Sepharose Fast Flow column) were mixed to prepare a sub-collection pool fraction of TPO (5,463.79ml; protein concentration, 0.490mg/ ml; total protein, 2,676 mg; relative activity 12,100; and total activity 32,380,000), then concentrated with an ultrafiltration device (Omega Ultraset, normal molecular weight cutoff is 8,000, manufactured by Filtron), and the solvent of the concentrated fraction was changed to DPBS, and then A small-volume sample was treated with an ultrafiltration device (manufactured by Amicon) equipped with a YM-10 membrane to make it a 120.2 ml DPBS solution containing 0.05% NaN ₃ . Next, all five kinds of anti-TPO peptide antibody gels obtained in Example 46 were mixed to make an antibody column (diameter 1.6 cm, bed height 4.8 cm, called anti-RT1+2 mixed antibody column), and TPO The pooled fraction was fed to the column at a flow rate of 0.033 ml/min. After loading the sample, it was eluted with DPBS, and the eluate was collected until the UV absorption value was very small, and then the thus collected eluate was concentrated using an ultrafiltration device (manufactured by Amicon) equipped with a YM-10 membrane to obtain a column grade Fraction F1 (82.62ml; protein concentration, 14.3mg/ml; total protein 1.184mg; relative activity 67,500; total activity 79,900,000). Next, the column-adsorbed F2 fraction (92.97 ml; protein concentration, 0.12 mg/ml; total protein, 11.1 mg; relative activity, 257,100, Total activity, 2,860,000). Although the column fraction F1 still contained TPO activity, the relative activity of TPO adsorbed on the further purified antibody column increased by about 20 times. That is, use 0.1% TFA as developer A, 1-propanol containing 0.05% TFA as developer B, mix the F2 fraction obtained from the antibody column with 1/10 volume of developer B, and mix the mixture with 0.4ml The flow rate per minute is used for a Capcell Pak CI 300A column equilibrated with 20% B in advance, thereby completing Capcell Pak CI300A (manufactured by Shiseido, article number CI TYPE: SG300A, diameter 4.6mm, bed height 150mm, and a diameter 4.6mm, bed High 35mm front column connection) column chromatography. After complete loading, elution was performed with 20% B for 5 minutes, followed by a linear density gradient from 20% B to 40% B for 50 minutes, and the eluate was collected in polypropylene tubes in 1 ml (2.5 minutes) aliquots.

The eluate of 2 μl (1/500 of the fraction) in each test tube was mixed with HSA, concentrated by ultrafiltration to obtain 0.25ml IMDM detection culture solution containing 0.02% HSA for identifying the TPO active fraction through detection. As a result, strong TPO activity was found in the samples of test tube Nos. 20 to 23 (propanol concentration in the range of 28.5-32.5%). In addition, after analyzing these samples by Western blot as described in Example 45, the presence of TPO was confirmed, based on the DPC III molecular weight marker, and its apparent molecular weight was 60,000-70,000 after detection under reducing conditions. Other molecules with molecular weights of 32,000-43,000 and 20,000-30,000, respectively. <Example 48> Determination of the biological activity of TPO obtained from the culture supernatant obtained by transfecting COS1 cells with the expression vector pHTP1 and purified by Capcell Pak CI 300A column

Collect the TPO active fraction purified in Example 36, i.e. the No. 20-23 test tube of the Capcell Pak CI 300A column (the concentration of propanol is in the range of 28.5% and 32.5%), mix with 0.21ml glycerol, and then concentrate through centrifugal evaporation . The resulting concentrate was mixed with 0.21 ml of 6M guanidine hydrochloride solution, diluted to 1 ml with DPBS, and carried out with DPBS containing 0.01% HSA using a Sephadex G25 column (NAP-10, manufactured by PharmaciaBiotech, product number 17-0854-01). Buffer exchange, then with 1.1ml DPBS containing 0.01% HSA, finally obtain TPO active fraction FA (2.6ml). In order to test the TPO bioactivity of this sample, an in vivo assay was performed. That is, the active fraction was subcutaneously administered to ICR male mice (8 weeks old), and each group included 4 animals at a dose of 100 μl per animal per day for 5 days (the total activity measured by the M-07e detection system was 110,000). As a control, 100 µl of DPBS containing 0.01% HSA was subcutaneously administered in the same manner. Before the administration and on the day after the last administration, blood was collected from the fundus to measure the platelet count with a microcytometer (F800, manufactured by Toa Lyo Denshi). In the TPO treatment group, compared with before the administration, the platelet count increased by an average of about 1.42 times after the administration, and was 1.23 times higher than that of the control group, and there was a significant difference between them (p<0.05, Studeut t-test). On the basis of these results, it was confirmed that the aforementioned human TPO produced by mammalian cells has the ability to increase platelet count in vivo. <Example 49> Determination of biological activity of crude TPO fraction obtained from 33 liters of culture supernatant obtained by transfecting COS1 cells with expression vector pHTP1 and partially purified by cation exchange column

(1) In vitro assays were performed with the M-07e detection system to evaluate the following purified samples. In the same manner as described in Example 36, 33 liters of a serum-free culture supernatant obtained by transfecting COS1 cells with the expression vector pHTP1 was then filtered through a 0.22 µm filter to recover the supernatant. It was concentrated 10-fold with an ultrafiltration device (PLGC pellicon Cassette, normal molecular weight cut-off 10,000, manufactured by Millipore), and then mixed with protease inhibitor p-APMSF at a final concentration of 1 mM to obtain a sample of 2,018 ml (protein concentration 3.22 mg /ml; total protein 6,502 mg, relative activity 66,000; total activity, 429,100,000). Then concentrate with an ultrafiltration device (Omega Ultraset, normal molecular weight cutoff is 30,000, manufactured by Filtron) to obtain fractions with a molecular weight of 30,000 or more (volume, 1,190 ml; protein concentration, 2.54 mg/ml; total protein 3,020 mg , relative activity 82,500; total activity, 249,000,000) and fractions with a molecular weight of 30,000 or less (volume 2,947ml; protein concentration 0.471mg/ml; total protein 1,402mg; relative activity, 4,500, total activity 6,310,000). In fractions with a molecular weight of 30,000 or less, TPO activity was present, suggesting that the culture supernatant may contain it during its expression or secretion from animal cells. TPO whose molecular weight has been reduced. On the other hand, exchange the buffer of molecular weight 30,000 or higher fraction with 20mM sodium citrate buffer (pH 6.1), and add to the pre-treated 20mM sodium citrate buffer (pH 6.1) at a flow rate of 5ml/min. Equilibrated column of SP Sepharose Fast Flow (diameter 2.6 cm, bed height 29 cm, manufactured by Pharmacia Biotech, product number: 17-0729-01). After loading the sample, the column was washed and eluted with 20 mM sodium citrate buffer (pH 6.1). Next, use 20mM sodium citrate buffer (pH6.1) as developer A and a solution composed of developer A and 1M NaCl as developer B, with a flow rate of 3ml/min, with a linear gradient of 0%B-50% B was eluted for 215 minutes, and then 50% B-100% B was eluted for 20 minutes, and the eluate was collected once every 30 ml (10 minutes) in a polypropylene test tube. When each eluate was tested with the M-07e detection system to determine the activity profile, it was found that TPO activity was found over a wide range. As a result, the fraction including the passing fraction eluted with NaCl at a concentration of 50 mM or less was collected as the F1 fraction, and the main TPO fraction eluted with 50-1,000 mM NaCl was collected as the F2 fraction. The volume of F1 fraction is 1,951ml (protein concentration, 2.05mg/ml; total protein, 3,994mg, relative activity; 13,500; total activity 53,900,000), F2 is 649.8ml (protein concentration 1.11mg/ml; total protein 721mg; relative activity activity 268,000; total activity, 193,000,000).

(2) Determine the biological activity of SP Sepharose Fast Flow fraction F2

2.5 ml of the SP Sepharose Fast Flow fraction separated in the above step (1) was buffer exchanged with 3.5 ml of DPBS containing 0.01% HSA through a Sephadex G 25 column (NAP-25, manufactured by Pharmacia Biotech, article number 17-0852-01) F2, the TPO biological activity of the sample (protein concentration, 1.46 mg/ml) was determined by in vivo assay. That is, the active fraction was subcutaneously administered to ICR male mice (8-week-old), 100 μl per animal per day for 5 days (total activity measured by the M-07e detection system: 123,000) consisting of 4 animals. As a control, 100 µl of DPBS containing 0.01% HSA was subcutaneously administered in the same manner. Before the administration and the next day after the administration, blood was collected from the fundus to measure the platelet count with a microcytometer (F800, manufactured by Toa Lyo Deushi). In the group administered with TPO, compared with the value before administration, the platelet count increased by an average of about 1.29 times after the application, and was 1.12 times higher than that of the control group, and there was a significant difference between them (p＜0.05, Student t-test ). These results demonstrate that the aforementioned human TPO produced by mammals has the ability to increase platelet count. <Example 50> Construction of a recombinant vector for expressing human TPO chromosomal DNA in CHO cells, pDEF202-ghTPO

The vector pDEF202 was digested with restriction enzymes KpnI and SpeI, followed by agarose gel electrophoresis to separate a fragment containing the mouse DHFR minigene and SV40 polyadenylation signal, and ligated with T4 DNA ligase (manufactured by Takara Shuzo) to obtain expression Vector pDEF202-ghTPO, the resulting fragment contains vector DNA obtained by removing the region containing the SV40 polyadenylation signal from plasmid pEFHGTE by treating with restriction enzymes KpnI and SpeI. This plasmid contains the SV40 origin of replication, the human elongation factor 1-α promoter for transcription of the SV40 early polyadenylation region of the human TPO gene, the mouse DHFR minigene, the pUC18 origin of replication, and the β-lactamase gene (Amp ^r ), and human TPO chromosomal DNA is connected to the downstream site of human elongation factor 1-α promoter. <Example 51> Expression of human TPO chromosomal DNA in CHO cells

CHO cells (a dhfr- strain; Uvlaub and Chasin, Proc. Natl. Acad. Sci USA, vol. 77, p4216, 1980) were grown, and the resulting cells were transfected by the calcium phosphate method (Cellphect, manufactured by Pharmacia).

That is, 10 µg of the plasmid pDEF202-ghTPO prepared in Example 50 was mixed with 120 µl of buffer A and 120 µl of H ₂ O, and the resulting mixture was incubated at room temperature for 10 minutes. This solution was then mixed with 120 µl of buffer B and incubated for an additional 30 minutes at room temperature. The prepared DNA solution was placed on a plate and incubated in a _CO2 incubator for 6 hours. After removing the medium and washing the resulting plate twice with α-MEM(-), α-MEM(-) containing 10% dimethylsulfoxide was added to the plate and treated at room temperature for 2 minutes. Next, a non-selective medium (α-MEM(-) op.cit., supplemented with hypoxanthine and thymidine) containing 10% dialyzed fetal bovine serum was added, followed by culturing for 2 days, and a medium containing 10% dialyzed fetal calf serum was added. Selection medium (α-MEM(-), without hypoxanthine and thymidine) with bovine serum completes the selection. Treat the cells with trypsin, and disperse the cells treated in a 6cm-diameter plate into 5 10cm-diameter plates or 20 24-well plates, and continue to culture the cells while changing the selection medium every 2 days to complete choose. When human TPO activity was measured using the Ba/F3 assay, human TPO activity was present in the supernatant of plates or wells where cell proliferation was observed.

In this example, CHO cells can also be transfected by co-transfecting CHO with pEFHGTE and pMG1. <Example 52> Construction of a vector for expressing human TPO protein (amino acid residues 1-163) (hereinafter referred to as "hMKT(1-163)") in E.coli and determining its expression, in which TPO In the protein, a Lys residue and a Met residue are added at the -1 and -2 positions, respectively

In order to express a protein whose 1- and 3-amino acid residues are identical to those of the human TPO protein (1-163 amino acid residues), the following synthetic oligonucleotides 1-13, 2-13 , 3-3 and 4-3, according to the same method as described in Example 42, the vector pCFM536/hMKT (1-163) used to express hMKT (1-163) in E.coli was constructed, also known as [ Met ^-2 , Lys ^-1 ]TPO (1-163):

1-13: 5′-CTAGAAAAAACCAAGGAGGTAATAAATAATGAAATCTCCTGCACCA-3′

(SEQ ID NO: 125);

2-13: 5′-CAGGTGGTGCAGGAGATTTCATTATTTATTACCTCCTTGGTTTTTT-3′

(SEQ ID NO: 126);

3-3: 5′-CCTGCATGTGATTTACGGGTCCTGTCTAAACTGCTGCG-3′

(SEQ ID NO: 127);

4-3: 5′-CGCAGCAGTTTAGACAGGACCCGTAAATCACATG-3

(SEQ ID NO: 128);

10 1-13 20 30 40

CTAGAAAAAA CCAAGGAGGT AATAAATAAT GAAATCTCCT

TTTTTT GGTTCCTCCA TTATTTATTA CTTTAGAGGA XbaI 2-13

50 60 3-3 70 30

GCACCACCTG CATGTGATTT ACGGGTCCTG TCTAAACTGC TGCG

CGTGGTGGAC GTACACTAAA TGCCCAGGAC AGATTTGACG ACGC

4-3

(SEQ ID NO: 129)

The expression plasmid contains the DNA sequence (SEQ ID NO: 12) shown in the sequence listing. Thereafter, expression of hMKT(1-163) was induced by the same method as described in Example 43.

The thus obtained expressed protein was subjected to SDS-PAGE, then transferred to PVDF membrane, and analyzed by N-terminal amino acid sequence, it was confirmed that the protein contained the amino acid sequence of expressed hMKT(1-163). <Example 53> Human TPO obtained from human TPO expressed in E.coli was refolded with guanidine hydrochloride and glutathione, h6T(1-163), then purified and its biological activity determined

Suspend 1.2 g of frozen cells of the h6T(1-163)-producing recombinant strain prepared in Example 43 in 3 ml of water, and crush with a high-pressure crusher. The suspension is centrifuged to recover the pellet and remove most of the contaminating proteins, cellular components, etc. The h6T(1-163)-containing precipitate fraction thus recovered was suspended in water to a final volume of 4 ml. 1 ml of 1M Tris buffer (pH 8.5) was added to the suspension with stirring, and then 20 ml of 8M guanidine hydrochloride was added, followed by stirring at room temperature for 5 minutes to dissolve the contents. Dilute the resulting solution 10 times with 20mM Tris buffer (pH8.5), dissolve 5mM reduced glutathione and 0.5mM oxidized glutathione in the diluted solution while stirring, and place the resulting solution at 4°C Incubate overnight. After centrifugation, h6T(1-163) was recovered in the supernatant. The resulting 160 ml supernatant was concentrated with a YM10 ultrafiltration membrane (manufactured by Amicon), and then buffered with DPBS to obtain fractions with a final volume of 3.4 ml (protein concentration, 2.18 mg/ml). After exhaustive dialysis of this fraction against IMDM medium and evaluation with the rat CFU-MK detection system, strong TPO activity was found (relative activity, 58,000).

The TPO active fraction prepared above was tested in vivo. That is, the active fraction was subcutaneously administered to ICR male mice (7 weeks old), 4 animals per group, 170 μl per animal per day for 5 consecutive days (the total activity measured by the rat CFU-MK detection system was 22,000) . As a control, 170 µl of DPBS was subcutaneously administered to 6 animals in the control group in the same manner. Before the administration and on the second day and 3 days after the administration, blood was collected from the fundus to measure the platelet count with a microcytometer (F800, manufactured by Toa Lyo Denshi). In the TPO-administered group, the platelet count increased about 2.15-fold on average on the second day after the administration, and remained unchanged at 2.13-fold even after the end of the administration, compared with the value before the administration. The platelet counts in the TPO-treated group were 1.73 and 1.80 times higher than those in the control group on the 2nd and 3rd day after administration, and there was a significant difference between them (p<0.001, Student t-test). These results indicate that human TPO produced by E. coli and prepared by the method described above has the ability to increase platelet count in vivo. <Example 54> Using N-lauroyl sarcosinate and copper sulfate to refold the human TPO expressed in E.coli, h6T(1-163), then purify and determine its biological activity

Suspend 0.6 g of frozen cells of the h6T(1-163)-producing recombinant strain prepared in Example 43 in 3 ml of water, crush with a high-pressure crusher, and then centrifuge to recover the precipitated part. After the precipitated part was suspended in 3.1ml of water, 0.19ml of 1M Tris buffer (pH9.2), 11.25μl of 1MDTT, 38μl of 0.5M EDTA and 0.38ml of 10% sodium deoxycholate solution were added to the suspension while stirring, The resulting mixture was stirred at room temperature for 40 minutes. It is then centrifuged to recover the pellet and remove most of the contaminating proteins, cellular components, etc. The resulting precipitate was suspended in 4 ml of water, and the precipitate containing h6T(1-163) was recovered by centrifugation. The recovered precipitate was suspended in 3.8ml of water, 0.2m of 1M Tris buffer (pH8) and 1ml of 10% N-lauroyl sarcosinate were added to the suspension while stirring, then stirred at room temperature for 20 minutes to dissolve the contents. The resulting solution was mixed with 5 μl of 1% copper sulfate and stirred overnight (approximately 20 hours) at room temperature. The resulting solution was centrifuged to recover the supernatant portion containing h6T(1-163), 5ml of the supernatant was mixed with 5ml of water and 10ml of 20mM Tris buffer (pH7.7), and then mixed with 2.6g of 20-50 mesh Dowex1- The X4 chloride form was centrifuged to exchange the resin for mixing, followed by stirring for 90 minutes. The ion exchange resin was removed with a glass filter to recover a resin-unadsorbed fraction, which was then centrifuged to recover a supernatant. The resulting supernatant was used on a SP Sepharose Fast Flow cation exchange column equilibrated with 20mM Tris buffer (pH7.7) in advance, and eluted with the same buffer by a linear gradient of 0-500mM NaCl. Each fraction eluted by SP Sepharose Fast Flow cation exchange column chromatography was thoroughly dialyzed to IMDM medium, and was evaluated with a rat CFU-MK detection system, and a strong TPO activity was found in the elution fraction with a NaCl concentration of about 100mM ( relative activity, 19,000,000). The fraction was concentrated 1.6 times (2.5 ml, protein concentration, 25 µg/ml) using an ultrafiltration device (Ultra Free CL, normal molecular cutoff 5,000, manufactured by Millpore, product number UFC4LCC25). When the concentrated fractions were analyzed by SDS-PAGE in the presence of reducing agent, a major band corresponding to TPO was detected (purity, 70-80%).

The active fraction of TPO prepared by the method described above was tested by an in vivo assay. That is, the active fraction was subcutaneously administered to ICR male mice (8 weeks old), and each group consisted of 4 animals, 100 µl per animal per day for 5 consecutive days (total activity measured by the rat CFU-MK detection system was 47,500). As a control, 100 µl of 20 mM Tris buffer (pH 7.7) containing 100 mM NaCl was subcutaneously administered in the same manner. Before the administration and the day after the administration, blood was collected from the fundus to measure the platelet count with a microcytometer (F800, manufactured by Toa Lyo Denshi). In the group using TPO, compared with before administration, after administration, the platelet count increased by about 1.73 times on average, and was 1.59 times higher than that of the control group, there was a significant difference between the two (p<0.01, Student t- test). These results demonstrate that human TPO produced by E. coli and prepared by the method described above has the ability to increase platelet count in vivo. <Example 55> Large-scale cultivation of CHO cells

The human TPO-producing CHO cell line (CHO28-30 cells, 25 nM MTX resistance) obtained by transfecting the human TPO expression plasmid pDEF202-hTPO-P1 into the CHO cells in Example 32 was cultured on a large scale by the following method. CHO cells were cultured and proliferated in DMEM/F-12 medium (GIBCO) containing 25 nM MTX and FCS. After detaching the cells with trypsin solution, 1×10 ⁷ cells were inoculated in a spinner flask (ex Falcon, Falcon 3000) containing 200 ml of the same medium, and then cultured at 37°C for 3 days at 1 rpm. After culturing for 3 days, the culture supernatant was removed by suction, and then the adhered CHO cells were rinsed with 100 ml of PBS, and 200 ml of DMEM/F-12 medium (GIBCO) containing neither 25 nM MTX nor 10% FCS was added to Cells were grown in flasks at 37°C for 7 days at 1 rpm. After 7 days of culture, the culture supernatant was recovered as a starting material for the subsequent purification process. The above process was done with 500 spinner flasks to obtain 100 L of culture supernatant. <Example 56> Purification of human TPO from TPO-producing CHO cell line

(1) Filter about 100 L of the serum-free culture supernatant obtained in Example 55 through a 0.22 μm filter to obtain the filtrate, and then filter it on an ultrafiltration device (RLTK Pelliconcassette, normal molecular weight cutoff is 30,000, ex Millipore) concentrate. After replacing the solvent of the concentrate with pure water, protease inhibitors p-APMSF (Wako Pure Chemicals) and Pefabloc SC (Marck) were added to the resulting aqueous solution at final concentrations of 1 mM and 0.35 mM, respectively, to obtain 3628 ml of a molecular weight of 30,000 or Fraction greater than 30,000 (protein concentration: 1.60 mg/ml; total protein 5805 mg; relative activity: 1,230,000: total activity: 7,149,000,000). This fraction was then analyzed by Western blot as described in Example 45. As a result, the TPO protein contained has a molecular weight between 66,000 and 100,000. In a more detailed examination, it was found that the fractions passed through the column contained TPO species with a molecular weight lower than that of the TPO mentioned above.

Use an ultrafiltration device (PLGC Pellicon cassette, normal molecular weight cut-off 10,000, Millipone) to concentrate the ultrafiltrate that contains the TPO of molecular weight not more than 30,000 separately, to obtain the low molecular weight TPO fraction of 1901ml (protein concentration: 0.36mg/ml; Total protein: 684 mg; relative activity: 245,500; total activity: 167,900,000). This indicates that low molecular weight TPO species were produced during cell culture.

Next, 764 g of ammonium sulfate and 144.5 ml of 0.5 M sodium citrate buffer (pH 5.5) were added to 3614 ml of the fraction containing TPO with a molecular weight of 30,000 or greater than 30,000 to obtain 4089 ml of 1.41 M (final concentration) sodium sulfate and 17.7 Solution in mM (final concentration) sodium citrate buffer. Centrifugation removes undissolved material from the solution, resulting in a clear solution.

Add the clarified solution to the Macro-Prep Methyl HIC column (Bio-Rad, article number: 156-0080; Diameter 5cm, bed height 24.5cm). After the sample was loaded, it was eluted with 20 mM sodium citrate buffer (pH 5.5) containing 1.2 M sodium sulfate. The eluted fraction was then concentrated on an ultrafiltration device (exFitron. Omega Ultrasette, normal molecular weight cut-off 8,000) to obtain column fraction F1 (4455ml; protein concentration: 0.400mg/ml; total protein: 1780mg; relative activity: 1,831,000 ).

Next, 20 mM sodium citrate (pH 6.0) was passed through the column as an elution buffer to obtain an eluate, which was then concentrated using the same ultrafiltration device (i.e., Omega Ultrasette, normal molecular weight cutoff 8,000) to collect the F2 fraction (1457 ml; protein concentration: 0.969 mg/ml; total protein: 1411 mg; relative activity: 1,715,000). In F2, the presence of a protein with a molecular weight of 66,000 to 100,000 was confirmed by SDS-PAGE. The protein was confirmed to be TPO by Western blot analysis.

Then, the F2 eluted from the Macro-Prep Methyl HIC column was added to the SP Sepharose FastFlow (Pharmacia Bioteck, article number 17-0729) equilibrated with 20mM sodium citrate buffer (pH6. -01; diameter 5cm, bed height 12cm). After sample loading, elution was carried out with 20 mM sodium citrate buffer (pH 6.0) containing 50 mM NaCl. The eluate was collected and named F1 fraction (3,007; protein concentration: 0.226 mg/ml; total protein 679 mg; relative activity: 88,830). Thereafter, the elution buffer was replaced with 20 mM sodium citrate buffer (pH 5.4) containing 750 mM NaCl to collect the eluted F2 fraction (931 ml; protein concentration: 0.763 mg/ml; total protein: 710 mg; relative activity: 5,558,000), and then concentrated to 202 ml with an ultrafiltration device (Filtron, Omega (Ultrasette, normal molecular weight cut-off 8,000; and Amicon, YN3 membrane).

Then, the concentrated F2 fraction (197ml) containing TPO activity was added to a Sephacryl S-200HR gel filtration column (Pharmacia Biotech, product number 17-0584-05; diameter 7.5cm, bed height 100cm) at a flow rate of 3ml/min. ). Elution volumes 1,200-1785ml; 1785-2010ml, 2010-2280ml and 2280-3,000ml were collected respectively to obtain fractions F1 (585ml; protein concentration: 1.00mg/ml; total protein: 589ml, relative activity: 4,118,000), F2 (225ml, protein concentration: 0.263mg/ml; total protein: 59.2mg; relative activity: 2,509,000), F3 (270ml; protein concentration: 0.119mg/ml, total protein: 32.1mg; relative activity: 2,535,000) and F4 (720ml ; protein concentration: 0.0467 mg/ml; total protein: 33.6 mg; relative activity: 1,155,000). Thus, fractionated TPO activity has a broad molecular weight range as determined by gel filtration. After analyzing the separated fractions by SDS-PAGE and Western blot, it was found that F1 mainly contained TPO molecular species with a molecular weight of 66,000 to 1,000,000; 32,000 to 42,000. Moreover, all TPO molecules have TPO activity.

Based on the results of N-terminal amino acid sequence analysis of TPO molecules with a molecular weight of 66,000 to 100,000, it was confirmed that the TPO molecule has the amino acid sequence of the protein encoded by the human TPO gene.

In addition, with N-glycanase (ex Genzyme, product number: 1472-00), neuraminidase (Nakarai-Tesqu, product number 242-29SP), endo-α-N-acetylgalactosaminidase (Seikagaku Kogyo, product number: 100453) and O-glucosidase (Boehringer Mannheim Biochemicala, product number 1347101) alone or in combination, the enzyme digestion experiment of TPO molecules with a molecular weight of 66,000-100,000, followed by SDS-PAGF analysis . As a result, it was found that, as expected from its theoretical molecular weight, the polypeptide part of TPO had a molecular weight of about 36,000 and was a glycoprotein having N- and O-linked sugar chains. <Example 57> Purification of human TPO from human TPO-producing CHO cell line

(1) 211.4 g of ammonium sulfate was added to 1 L of the serum-free culture supernatant of CHO cells obtained by the method in Example 55, and then, the mixture was passed through a 0.2 µm filter (exGelman Science, product number 12992). The resulting filtrate was used at a flow rate of 15 ml/min on a Macro-Prep Methyl HIC column (Bio-Rad, product number 156-0081, dia. 50mm, bed height 90mm). After sample loading, 450ml of 20mM acetate buffer (pH5.6) containing 1.2M ammonium sulfate was used for elution. The eluted fraction was applied to a reverse phase Vydac C4 column (The Separations Group, product number: 214BTP54, diameter 4.6 mm, bed height 250 mm) at a flow rate of 0.75 ml/min. Wash the column for 15 minutes with 10 mM Tris buffer (pH6.4) containing 5% ethanol (referred to as "developing agent A"), and use developing agent A to contain 10 mM Tris buffer (pH6.4) containing 94% ethanol ( A 66-minute linear gradient, referred to as "developer B"), accomplished elution. The resulting chromatograms are listed in FIG. 15 . As a result of SDS-PAGE analysis, the molecule (molecular weight 65,000-100,000) that was considered to be TPO and corresponded to the fraction with a retention time of 68-72 minutes after application was a single band on the SDS-PAGE gel (see Fig. 16). Further Western analysis indicated that the resulting protein was TPO.

N-terminal amino acid analysis of the protein sample showed that the protein had the amino acid sequence of the protein encoded by the human TPO gene. <Example 58> Preparation of recombinant virus for expressing human TPO in insect cells

Plasmid pHTP1 prepared in Example 30 was digested with restriction enzymes EcoRI and NotI, then subjected to 1% agarose gel electrophoresis to a band of about 1200 bp, followed by purification with Prep-A-Gene DNA purification kit. The purified DNA was then ligated with transfer vector pVL1393 (Invitrogen) pretreated with the same restriction enzymes, and then transformed into highly competent E. coli DH5 (ToyoBoseki). The clone pVL1393/hTPO containing the full-length coding region of human TPO cDNA was screened from the resulting colonies. Plasmid DNA was prepared from the clone according to the method described in Molecular Cloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989), and then transfected into insect cells Sf21 (Invitragen) with the BaculoGold ^™ transfection cassette, and the transfected cells were The virus-containing supernatant was recovered by culturing in Sf-900 medium (Lifetechnology) for 4 days at 27°C. The supernatant was diluted 1:10 ⁹ to 1:10 ⁵ , and approximately 7×10 ⁵ Sf21 cells were infected with 1 ml of the diluted supernatant in -35 mm (diameter) Shale for 1 hour at 27°C. After removing the supernatant, 1% hot agarose in Sf-900 medium was added to the culture to solidify, followed by culturing at 27°C for 6 days in a humidified atmosphere. A formed plaque was detected and virus was released from the plaque into 200 μl of Sf-900 medium. The virus clone obtained in the 24-well plate was used to infect Sf21 cells to propagate the virus. Virus-containing fluids from individual plaques were treated with phenol/chloroform followed by ethanol precipitation to recover viral DNA, which was then used as a template to perform PCR with primers specific for human TPO cDNA. The recombinant virus containing human TPO cDNA was screened on the basis of amplifying specific DNA fragments. Sf21 cells were then infected with the supernatant containing the recombinant virus carrying the human TPO cDNA to propagate the virus. <Example 59> Expression of human TPO in Sf21 insect cells and identification of TPO activity

Cultivate Sf21 cells in a ^175cm culture flask to reach about 80% fusion, then infect with the recombinant virus carrying human TPO cDNA prepared in Example 58 at 27°C for 1 hour, and then place the resulting cells in Sf-900 medium cultured at 27°C for 4 days to recover the culture supernatant. The resulting culture supernatant was replaced with IMDM medium on a NAP ^™ -5 column (Pharmacia). In rat CFU-MK assay and M-07e assay, significant dose-dependent TPO activity was detected in the resulting supernatant. Recombinant human TPO expressed in Sf21 cells was also identified by Western analysis as described in Example 45. <Example 60> The variant human TPO obtained from the clone pCFM536/h6T(1-163), h6T(1-163), was purified by refolding h6T(1-163) with N-lauroyl sarcosinate and copper sulfate -163), said clone carries the human TPO base sequence expressed in E.coli.

30 g of the frozen recombinant microorganisms producing h6T(1-163) prepared in Example 43 were suspended in 300 ml of water, crushed with a high-pressure crushing device (10,000 psi, Rannie High Pressure Laboratory), and then centrifuged to recover the precipitated part. The precipitated portion was suspended in 90 ml of water, and water was added with stirring until a total of 150 ml was reached. Then, 9 ml of 1M Tris buffer (pH 9.2), 540 μl of 1M DTT, 1.8 ml of 0.5M EDTA and 18 ml of 10% sodium oxycholate were added to the mixture with stirring, followed by stirring at room temperature for 30 minutes. The pellet was recovered by centrifugation and most of the contaminating proteins and microbial components were removed. 180 ml of DTT was added to the precipitate to obtain a suspension, and the precipitated fraction containing h6(1-163) was recovered by centrifugation. To the resulting precipitate was added 300 ml of water to obtain a suspension, and water was added thereto with stirring so that the total amount of the liquid was 570 ml. Add 30ml of 1M Tris buffer (pH8), 150ml of 10% sodium N-lauroyl sarcosine to the mixture at room temperature and stir for 20 minutes to dissolve h6T(1-163). 750 µl of 1% copper sulfate was further added thereto, and the mixture was stirred overnight (about 20 hours) at room temperature. After the supernatant fraction containing h6T (1-163) was recovered by centrifugation, 750ml of water and 1500ml of 20mM Tris buffer (pH7.7) were added to the 750ml supernatant, followed by adding 3ml of polysorbate (pH7.7) while stirring. Nikko Chemicals). 600 g of Dowex 1-X4 (20-50 mesh, chloride form) ion exchange resin was added to the resulting solution and stirred at room temperature for 90 minutes. After recovering the non-adsorbed fraction with a glass filter, the ion exchange resin was washed with 750 ml of 20 mM Tris buffer (pH 7.7). The combined solution of unadsorbed and washed fractions was adjusted to pH 9.2 with 2N NaOH, and then used in Q-Sepharose Fast pre-equilibrated with 20 mM Tris buffer (pH 9.2) containing 0.1% polysorbate Flow anion exchange column (ID5cm×10cm) to recover unadsorbed fraction. The pH of the obtained unadsorbed fraction was adjusted to 7.2 with hydrochloric acid, and added to the SP Sepharose FastFlow column (ID 5cm×10cm) equilibrated with 20mM Tris buffer solution (pH7.2) containing 0.1% polysorbate in advance, and then Elution was performed with a linear gradient of 0 M to 500 mM NaCl in the same buffer. The eluted fraction was analyzed by SDS-PAGE to collect the TPO fraction, to which trifluoroacetic acid was added to a concentration of 0.1%. After the resulting solution was applied to a Capcell Pak 5 μm 300A C1 column (ID 2.1 cm x 5 cm x 2, Shiseido), reverse phase HPLC was performed using a linear gradient elution method with increasing concentrations of 1-propanol in 0.1% trifluoroacetic acid. The eluates were analyzed on SDS-PAGE without reducing agent. As a result, three fractions were fractionated according to the elution position in reversed-phase HPLC, and each fraction was diluted 3-fold with 0.1% trifluoroacetic acid. In the same manner, each eluted fraction was applied to the above-mentioned reverse-phase HPLC column. The resulting three TPO fractions, named Fr.S-a, Fr.S-b and Fr.S-c according to the order of elution on reverse phase HPLC, were analyzed on SDS-PAGE in the absence of reducing agent (see Figure 1.7) . As a result, a single band was detected at a molecular weight of about 18 KDa (Fr. S-a), about 19 KDa (Fr. S-b) or 18 KDa (Fr. S-c) (see FIG. 18 ). After amino acid analysis, the determined amino acid components are almost consistent with the corresponding theoretical values estimated from the sequence data. In addition, the results of N-terminal amino acid analysis showed that they were the expected sequences. The amount of protein in each fraction determined by amino acid analysis was 0.64 mg (Fr.S-a), 1.81 mg (Fr.S-b) or 3.49 mg (Fr.S-c). After each fraction was fully dialyzed against IMDM medium, the M-07e assay was performed. As a result, the relative activity of each fraction was about 1,620,000 (Fr.S-a), 23,500,000 (Fr.S-b) or 746,000,000 (Fr.S-c). <Example 61> Refolding and Purifying h6T(1-163) of Human TPO, h6T(1-163) from Clone pCFM536/h6T Carried in E.coli Using Guanidine Hydrochloride and Cys-Cystine Expressed human TPO base sequence

Add 50 g of the frozen recombinant microorganism producing h6T(1-163) prepared in Example 43 to 500 ml of water and suspend it, crush it with a high-pressure crushing device (10,000 psi, Rannie High Pressure Laboratory), and then centrifuge to recover the precipitated part. The resulting precipitate was suspended in water, and water was added with stirring to bring the liquid volume to 250 ml. Thereafter, 15 ml of 1M Tris buffer (pH 9.2), 900 µl of 1M DTT, 3 ml of 0.5M EDTA and 30 ml of 10% sodium deoxycholate were added thereto and stirred at room temperature for 30 minutes. After centrifugation, the pellet is recovered while most of the contaminating proteins, microbial components, etc. are removed. Add 300ml of 5mM DTT to the pellet, suspend it, and then centrifuge to recover the pellet containing h6T(1-163). The recovered pellet fraction containing h6T(1-163) was suspended in water to a total volume of 104 ml. 20ml of 1M Tris buffer (pH8.5) was added to the suspension, and then 376ml of 8M guanidine hydrochloride was added with stirring, followed by stirring at room temperature for 10 minutes to dissolve h6T(1-163). Add 2500ml 20mM Tris buffer (pH8.5) containing 0.1% polysorbate to it, then add 2000ml 20ml Tris buffer (pH8.5) containing 1M guanidine hydrochloride while stirring, then add 5mM Cys and 0.5 mM cystine (cystin). The solution thus obtained was incubated at 4°C overnight, h6T(1-163) in the supernatant was recovered by centrifugation, and then concentrated using a Prep Scale UF cartridge PLDC ultrafiltration membrane (Millipore). The buffer of the concentrate was replaced with 20 mM Tris buffer (pH 9.2) containing 0.1% polysorbate until the final volume reached 1,000 ml. Add the resulting solution to a Q-Sepharose Fast Flow anion exchange column (ID5cm×10cm) equilibrated with 20mM Tris buffer (pH9.2) containing 0.1% polysorbate in advance, and then use a linear gradient in the same buffer 0M to 500mM NaCl elution. Fractions (240 ml) eluting at about 20 mM to about 150 mM NaCl were recovered. The resulting fraction was diluted 4-fold with 20mM Tris buffer (pH7.2) so that the total volume reached 960ml, then the pH was adjusted to 7.2 with acetic acid, and then added to 20mM Tris pre-treated with 0.1% polysorbate Buffer (pH7.2) equilibrated SP Sepharose Fast Flow cation exchange column (ID 5cm×10cm), and then eluted with linear gradient 0M-500mM NaCl in the same buffer. The eluate was analyzed on SDS-PAGE to collect the TPO fraction. Trifluoroacetic acid was added to the TPO fraction to a final concentration of 0.1%. The resulting solution was applied to a Capcell Pak 5 μm 300A C1 column (ID 2.1 cm × 5 cm × 2, Shiseido), followed by reversed phase by linear gradient elution with increasing concentrations of 1-propanol in 0.1% trifluoroacetic acid HPLC. The eluate was analyzed by SDS-PAGE in the absence of reducing agent and then fractionated into 2 fractions according to the elution position in reverse phase HPLC. The two TPO fractions were designated as Fr.G-a and Fr.G-d, respectively, according to the elution order in reverse-phase HPLC (see FIG. 17 ). Fractions were analyzed on SDS-PAGE without reducing agent. As a result, a single band was detected at a position with a molecular weight of about 18 KDa (Fr. G-a) or about 32 KDa (Fr. G-d) (see FIG. 18 ). Furthermore, SDS-PAGE analysis of the above fractions in the presence of a reducing agent showed that a single band was detected at a position with a molecular weight of about 20 KDa in both fractions. The results of amino acid analysis of each fraction showed that the composition of each amino acid was almost consistent with the corresponding theoretical value estimated from the sequence data. In addition, the results of N-terminal amino acid analysis showed that they were the expected sequences. The protein amount of each fraction determined from the results of amino acid analysis was 2.56 mg (Fr.G-a) or 1.16 mg (Fr.G-d). After complete dialysis of each fraction against IMDM, the M-07e assay was performed. As a result, the relative activity of the TPO fraction was about 3,960,000 (Fr.G-a) or about 7,760,000 (Fr.G-d). <Example 62> Construction of recombinant vector pDEF202-hTPO163 for expressing a partial length of human TPO (amino acids 1-163) (hereinafter referred to as "hTPO163") in CHO cells

The vector pDEF202 constructed in Example 31 was treated with restriction enzymes EcoRI and SpeI, and then a larger vector fragment was recovered by agarose gel electrophoresis. This fragment was ligated with T4 DNA ligase (Takara-Shuzo) to hTPO163 cDNA prepared by treating plasmid pEF18S-hTPO163 containing hTPO163 cDNA encoding amino acids -21 to 163 (SEQ ID NO: 13) with restriction enzymes EcoRI and SpeI To obtain the expression vector pDEF202-hTPO163. This plasmid contains the replication origin of SV40, human elongation factor 1-α promoter, SV40 early polyadenylation site, mouse DHFR minigene, pUC18 replication origin and β-lactamase gene (Amp ^r ), wherein, The hTPO163 cDNA was ligated downstream of the human elongation factor 1-α-promoter site. <Example 63> Expression of hTPO163 in CHO cells

CHO cell lines (dhfr ^- strain, Urlaub and Chasin, Proc. USA, 77, p4216, 1980), and then transformed with the pDEF202-hTPO163 plasmid by the transfectum method (Seikagaku Kogyo KK).

Briefly, 10 μg of plasmid pDEF202-hTPO163 prepared in Example 62 was mixed with 240 μl of 0.3M NaCl, and then mixed with a mixture of 20 μl of transfectum and 220 μl of _H2O . The DNA solution was added dropwise to the plate, followed by incubation in a _CO incubator for 6 hours. The culture medium was removed from the plate, then washed twice with α-MEM(-), followed by the addition of 10% DMSO containing α-MEM(-), followed by incubation at room temperature for 2 minutes. Thereafter, a non-selective medium (α-MEM(-) containing hypoxanthine and thymidine) containing 10% dialyzed fetal bovine serum was added to the plate, followed by culturing for another 2 days, and then the culture medium was added to the plate containing 10% dialyzed fetal bovine serum. Selection medium (α-MEM(-) without hypoxanthine and thymidine) of bovine serum was selected. Divide the cells in each of the above 6 cm dishes into 5 10 cm dishes or 20 24-well dishes by trypsinizing the cells, and continue culturing while replacing the medium with fresh medium every 2 days , thus completing the screening. TPO activity was observed by checking the plate or well supernatant for TPO activity using the Ba/F3 assay. After splitting the cells at a cell concentration of 1:15 with selection medium containing 25 nM methotrexate, cells in which TPO activity was observed in the supernatant were transferred to plates or wells and subsequently cultured to grow and clone ammonia Methotrexate-resistant cells.

In addition, transformation of CHO cells was accomplished by co-transfecting pEF18S-hTPO163 and pMG1 into CHO cells.

The CHO strain (CHO-DUKXB11) transfected with the plasmid pDEF202-hTPO163 has been deposited by the applicant on January 31, 1995 at the Japanese National Institute of Bioscience and Human Technology, Division of Industrial Science and Technology, Ministry of International Trade and Industry, Deposit number FERM BP-4989. <Example 64> Large-scale cultivation of CHO cells

The hTPO163-producing CHO cell line obtained by transfecting the hTPO163-expression plasmid pDEF202-hTPO163 into the CHO cells in Example 63 (CHO 109 cells, cultured on a large scale in the presence of 10% dialyzed fetal bovine serum) was as follows: Selected culture [α-MEM ^- without hypoxanthine and thymidine] obtained by the above screening). CHO 109 cells were grown in DMEM/F-12 medium (GIBCO) containing 10% FCS. After collecting (or trypsinizing) cells with trypsin solution, 10× ¹⁰⁷ cells were inoculated into a Falcon spinner bottle (Falcon 3000) containing 200 ml of the same medium, and then cultured at 37°C for 3 days at 1 rpm. The medium was removed by suction and the surface of the cell culture was rinsed with 100 ml PBS. 200 ml of DMEM/F-12 medium (GIBCO) without 10% FCS was added to the cell culture, followed by culturing at 37°C at 1 rpm for 7 days. The collected culture supernatant was used as starting material for subsequent purification steps. A similar process was done in 300 spinner flasks to obtain 60 L of serum-free culture supernatant. <Example 65> Purification of hTPO163 from a hTPO163-producing CHO cell line

(1) 60 L of the serum-free culture supernatant obtained in Example 64 was filtered through a 0.22 μm filter to collect the filtrate, and then concentrated with an ultrafiltration device (Filtron, molecular weight cutoff 10,000) to obtain a concentrated fraction (600ml, 11.2mg protein/ml, total protein 6430mg). Western analysis of this fraction with an anti-HT1 peptide antibody revealed the presence of expressed hTPO163 protein with an apparent molecular weight of 20,000 to 26,000.

The resulting concentrated culture supernatant was processed on a Sephadex G-25 Frne column (Pharmacia-Biotech, Cat. No. 17-0032-02; diameter 10 cm, bed height 30 cm) pre-equilibrated with 10 mM sodium phosphate buffer (pH 6.8) solution (537 ml) to obtain a solution of protein fraction F1 (938 ml, protein concentration 4.9 mg/ml, total protein 4594 mg) in 10 mM sodium phosphate buffer (pH 6.8).

This protein fraction (929ml) from the Sephadex G-25 Fine column is added to the SP Sepharose Fast Flow column (Pharmacia-Biotech, article number: 17-0729-01; diameter 5 cm, bed height 12 cm), then eluted with 10 mM sodium phosphate buffer (pH 6.8) and then with the same buffer containing 10% ethanol. The elutions were combined into the F1 fraction (1608ml, protein concentration 2.13mg/ml, total protein 3426mg). Carry out the second elution with 10mM sodium phosphate buffer (pH6.8) containing 750mM NaCl and 25% ethanol to obtain the main hTPO163 eluate of F2 fraction (651ml, protein concentration 1.67mg/ml, total protein 1087mg ).

To the F2 fraction (200 ml) containing TPO activity from the SP Sepharose Fast Flow column, ethanol and purified water were added to prepare 300 ml of a 45% (final concentration) ethanol solution. Insoluble material resulting from the addition of ethanol was removed by centrifugation. Inject the supernatant at a flow rate of 2ml/min and pre-equilibrate with 50% solvent A (10mM sodium acetate buffer, pH6.7) plus 50% solution B (10mM sodium acetate buffer containing 90% ethanol, pH6.7) SOURCE 15RPC column (Pharmacia-Biotech, article number: 17-0727-02; diameter 2cm, bed height 20cm). The column was then washed with 55% solvent A plus 45% solvent B until the unadsorbed material was almost completely eluted, and then eluted with a flow rate of 1.5 ml/min in the following elution method: 55% B for 5 minutes; linear gradient 50 %B to 100% B over 140 minutes; 100% B 35 minutes. Fractions were collected every 5 minutes (corresponding to a volume of 7.5 ml). All fractions were subjected to SDS-PAGE followed by Western analysis to examine the elution range of hTPO163. As a result, highly purified hTPO163 with an apparent molecular weight of about 20,000 to 26,000 was found in the eluate ranging from 66% to 87% ethanol. Among these hTPO163 proteins, hTPO163 with a higher molecular weight elutes earlier from the reversed-phase column, indicating that the hydrophilicity of the glycosylated hTPO163 molecule is enhanced.

CHAPS was added to 88.8 ml of the hTPO163 eluted fraction from the SOURCE 15RPC column (i.e., the hTPO163 fraction (90 ml) eluted in the range of 68% to 86.5% ethanol), then the mixture was concentrated and washed to obtain a fraction containing about 5 2.5ml concentrate of % ethanol and 4mM CHAPS. Subsequently, the concentrate was added to a Superdex 75pg column (Pharmacia-Biotech, article number: 17-1070-01) equilibrated with 10mM sodium phosphate buffer (pH 6.8) at a flow rate of 1.5ml/min, 2.6cm in diameter, bed 60cm high). From 60 minutes after loading the sample, the eluted fractions were collected in 6 ml volumes (ie every 4 minutes). As a result, hTPO163 was eluted in the fractions from tube numbers 16 to at least 31 as determined by SDS-PAGE. This elution position corresponds to about 44,000 to about 44,000 as determined by gel filtration using standard molecular weight markers (Bio-Rad's mixture, Gel Filtration Standard, Cat. No.: 151-1901, and Calbiochem, insulin, Cat. No.: 407696). 6,000 molecular weight range. Additionally, these hTPO163 molecules appear to be eluted in an order of decreasing glycosylation. All hTPO163 species of tube numbers 16 to 31 (elution volume: 180 to 276 ml) were collected as AF fractions. Except for the FA fraction, fractions of tube numbers 16 to 18 (elution volume: 180 to 198 ml), 19 to 24 (elution volume 198 to 234 ml) and 25 to 31 (elution volume: 234 to 276 ml) were collected, respectively, Then named fractions FH, FM and FL, respectively. The FA fraction can also be prepared by combining some of the fractions FH, FM and FL. Figure 19 describes the above.

(2) Next, the hTPO163 eluted fractions FH, FM, FL and FA from Superdex 75pg in the aforementioned (1) will be described in more detail. The N-terminal amino acid sequence of each hTPO163 species described above was determined by the same method as in Example 1, but most of the N-terminal Ser residues could not be identified. This indicates the addition of an O-linked sugar to the N-terminal Ser. In addition, it was confirmed that the sequence following the N-terminal Ser was an amino acid sequence deduced from the gene sequence of hTPO. Determined by amino acid analysis (AccQ.Tag method, Wasters), the concentration of hTPO163 protein in FH, FM, FL and FA is 10.2ng/ml, 6.2ng/ml, 0.84ng/ml and 3.2ng/ml, respectively, indicating This concentration is for the peptide moiety without any sugar chains. Under non-reducing conditions, these fractions (100 ng each) were subjected to SDS- PAGE followed by silver-staining (Daiichi Pure Chemicals). As a result, each of these fractions was found to contain hTPO163 in high purity. Under reducing conditions, the apparent molecular weights of hTPO163 were 24,000-21,500, 23,000-21,000, 23,000-20,500 and 23,500 to 20,500 in FH, FM, FL and FA, respectively, calculated using DPCIII molecular weight markers (Daiichi Dure. Chemicals) as a standard (See Figure 20). In addition, in the Western analysis under reducing conditions, the FH, FM, FL and FA calculated with biotinylated SDS-APGE standards (Bio-Rad, Biotynylated SDS-PAGE Standards, Board Range: product number 161-0319) The apparent molecular weights of hTPO are 26,000 to 22,000, 25,500 to 22,000, 26,000 to 21,000 and 26,000 to 21,000, respectively. The inconsistency of molecular weight of FH, FM, FL and FA may be caused by the inconsistency of O-linked sugar chains. Therefore, the fractions FH, FM, FL and FA were analyzed on SDS-PAGE after digestion with neuraminidase (Neuraminidase, Nacalai tesque, product number: 242-29SP) and reduction with DTT. As a result, in all fractions, the apparent molecular weight was about 19,000, indicating that the inconsistency in the molecular weight of hTPO was mainly caused by the inconsistency in the amount of sialic acid in the sugar chain coupled with the hTPO163 protein, and in CHO cells, The resulting hTPO163 was expressed as a glycoprotein.

(3) The in vitro activities of the FH, FM, FL and FA fractions obtained in (2) were detected with the M-07e detection system. As a result, the relative specific activities were 511,000,000, 775,000,000, 1,150,000,000 and 715,000,000/mg hTPO163 protein (the weight of the peptide portion excluding any sugar weight). <Example 66> Construction of an E.coli vector for expressing human TPO (amino acid 1-332) (hereinafter referred to as "hMKT(1-332)") in which Lys was added at position -1 and Met was added at position -2, respectively and express hMKT(1-332)

To express the full-length amino acid sequence of human TPO in E. coli, the codons used from amino acid 164 to amino acid 332 were replaced with the E. coli preferred codons described below.

table 5

21: 5′-CTCCCGAACCGTACCAGCGGCCTGCTGGAAACCAACTTTACCGCGAG-3′

(SEQ ID NO: 130)

22: 5'-GGTAAAGTTGGTTTCCAGCAGGCCGCTGGTACGGTTCGGGAGCTCGT-3'

(SEQ ID NO: 131)

23: 5'-CGCGCGTACCACCGGCAGCGGCCTGCTGAAATGGCAGCAGGGCTTTCGT-3'

(SEQ ID NO: 132)

24: 5′-AGCCCTGCTGCCATTTCAGCAGGCCGCTGCCGGTGGTACGCGCGCTCGC-3′

(SEQ ID NO: 133)

25: 5′-GCGAAAATCCCGGGCCTGCTGACCAGACCAGCCGTAGCCTGGATCAGAT-3′

(SEQ ID NO: 134)

25: 5′-ATCCAGGCTACGGCTGGTCTGGTTCAGCAGGCCCGGGATTTTCGCACGAA-3′

(SEQ ID NO: 135)

27: 5′-CCCGGGCTATCTGAACCGTATCCATGAACTGCTGAACGGCACCCGTG-3′

(SEQ ID NO: 136)

28: 5′-GTGCCGTTCAGCAGTTCATGGATACGGTTCAGATAGCCCGGGATCTG-3′

(SEQ ID NO: 137)

29: 5′-GCCTGTTTCCGGGCCCGAGCCGTCGCACCCTGGGCGCGCCGGATATCAG-3′

(SEQ ID NO: 138)

30: 5′-ATCCGGCGCGCCCAGGGTGCGACGGCTCGGGCCCGGAAACAGGCCACGG-3′

(SEQ ID NO: 139)

31: 5′-ATCAGCTCTGGCACCAGCGATACCGGCAGCCTGCGCCGAACCTGCAGCC-3′

(SEQ ID NO: 140)

32: 5′-CAGGTTCGGCGGCAGGCTGCCGGTATCGCTGGTGCCAGAGCTGATATCCG-3′

(SEQ ID NO: 141)

33: 5′-GGGCTATAGCCCGAGCCCGACCCATCCGCCGACCGGCCAGTATACCCTGTT-3′

(SEQ ID NO: 142)

34: 5′-GGTATACTGGCCGGTCGGCGGATGGGTCGGGCTCGGGCTATAGCCCGGCTG-3′

(SEQ ID NO: 143)

35: 5′-TCCGCTGCCGCCGACCCTGCCGACCCCGGTGGTTCAGCTGCATCCGCTGC-3′

(SEQ ID NO: 144)

36: 5′-GGATGCAGCTGAACCACCGGGGTCGGCAGGGTCGGCGGCAGCGGAAACAG-3′

(SEQ ID NO: 145)

37: 5′-TGCCGGATCCGAGCGCGCCGACCCCGACCCCGACCAGCCCGCTGCTGAAGA-3′

(SEQ ID NO: 146)

38: 5′-AGCAGCGGGCTGGTCGGGGTCGGGGTCGGCGCGCTCGGATCCGGCAGCAGC-3′

(SEQ ID NO: 147)

39: 5′-CCAGCTATACCCATAGCCAGAACCTGAGCCAGGAAGGCTAATGAAGCTTGA-3′

(SEQ ID NO: 148)

40: 5′-CTTCATTAGCCTTCCTGGCTCAGGTTCTGGCTATGGGTATAGCTGGTGTTC-3′

(SEQ ID NO: 149)

41: 5'-ACGAGCTCCCGAACCGTACCA-3' (SEQ ID NO: 150)

42: 5'-CTGATATCCGGCGCGCCCAGG-3' (SEQ ID NO: 151)

43: 5'-CGGATATCAGCTCTGGCACCA-3' (SEQ ID NO: 152)

44: 5'-TCAAGCTTCATTAGCCTTCCT-3' (SEQ ID NO: 153)

21 and 22, 23 and 24; 25 and 26; 27 and 28; 29 and 30; 31 and 32; 33 and 34; 35 and 36; 37 and 38; or 39 and 40 phosphorylation. Then 1/10 volume of a solution containing 100 mM Tris/HCl (pH 7.5), 100 mM MgCl ₂ , 500 mM NaCl was added to the reaction mixture, boiled in a water bath for 3 minutes, and then allowed to cool to form double-stranded DNA. Four sets of double-stranded DNA: oligonucleotides 21 and 22/23 and 24 (combo-A); oligonucleotides 25 and 26/27 and 28 (combo-B); and oligonucleotides 31 and 32 /33 and 34 (combination-C); and oligonucleotides 35 and 36/37 and 38/39 and 40 (combination-D) were ligated with a DNA ligation cassette (Takara-Shuzo) respectively, thereafter, combination-A Concatenated with Combo-B, and similarly concatenated Combo-C with Combo-D to obtain Conjugate-1 and Conjugate-2, respectively. Using adapters-1 and -2 as templates, oligonucleotides-41 and -42 as primers for adapter-1, and oligonucleotides-43 and -44 as primers for adapter-2, Perform PCR. The PCR products of connectors-1 and -2 were digested with SacI and EcoRV and EcoRV and Hind III respectively, then electrophoresed on 2% agarose gel and purified with Prep-A-Gene DNA purification kit to recover about 240bp and 250bp respectively fragment. The resulting two fragments were subcloned into pBluescript II KS+ (Stratagene) predigested with SacI and HindIII (using E. coli DH5 as host). Among the obtained clones, a clone having the base sequence shown in Table 6 was selected by sequencing and named pBL(SH)(174-332) (see SEQ ID NO: 154).

表6CTC CCG AAC CGT ACC AGC GGC CTG CTG GAA ACC AAC TTT ACC GCG AGCLeu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser174                     180GCG CGT ACC ACC GGC AGC GGC CTG CTG AAA TGG CAG CAG GGC TTT CGTAla Arg Thr THR GLY Serg Leu LEU LEU LYS TRP GLN GLN GLN GLY PHE ARG190 200GCG AAA AAA AAA CCG GGC CTG CTG ACC AGC CGT AGC CGT AGC CTG GATGALA LYS ILEU

210 220ATC CCG GGC TAT CTG AAC CGT ATC Cat GAA CTG CTG AAC GGC ACC CGTILE PRO GLY TYR Leu Asn ARG Ile His Glu Leu asn GLY THR ARG

                                                                                                

240 250AGC TCT GGC ACC AGC GAT ACC GGC AGC CTG CCG CCG AAC CTG CAG CCGSer Ser Gly Thr Ser Asp Thr Gly Gly Ser Leu Pro

260GGC TAT AGC CCG AGC CCG ACC CAT CCG CCG ACC GGC CAG TAT ACC CTGGly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu270                                     280TTT CCG CTG CCG CCG ACC CTG CCG ACC CCG GTG GTT CAG CTG CAT CCGPhe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro

290 290 300CTG CTG CCG GAT CCG AGC GCG CCG ACC CCG ACC CCG ACC AGC CCG CTGLeu Leu Pro Asp Pro Leu Thr Pro Ser Pro Alahr Pro Thr

                                                                                                              

320 330 332TGA AGC TTG A

Next, the following synthetic oligonucleotides 45 and 46 were used as primers, and pBL(XH)h6T(1-163) prepared in Example 42 was used as a template to complete PCR, and the PCR product was digested with BamHI and SacI, followed by a 6% polymerase A fragment of about 160 bp was recovered from the gel by electrophoresis on an acrylamide gel. 45:5'-AAGGATCCGAACGCTATCTTCCTG-3' (SEQ ID NO: 155) 46:5'-GGGAGCTCGTTCAGGGTCAGAACCAGA

GAGGTACGAGACGGAACAGCAGTGGTTGG-3' (SEQ ID NO: 156)

pBL(SH)(174-332) was digested with SacI and Hind III, and then the digested product was purified with a Prep-A-Gene DNA purification kit to obtain a fragment of about 480bp. The two fragments prepared above were subcloned into pBluescript II KS+ (Stratahene) predigested with BamHI and Hind III (E.coli DH5 as host.).

Among the clones obtained, the clone with the base sequence shown in Table 7 was selected by sequencing and named as pBL(BH)(123-332) (see SEQ ID NO: 157).

Table 7

G GAT CCG AAC GCT ATC TTC CTG TCT TTC CAG CAC CTG CTG CGC CGC

Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly

123 120

AAA GTT CGT TTC CTG ATG CTG GTT GGC GGT TCT ACC CTG TGC GTT CGT

Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg

140 150

CGG GCG CCG CCA ACC ACT GCT GTT CCG TCT CGT ACC TCT CTG GTT CTG

Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu

                               

ACC CTG AAC GAG CTC

Thr Leu Asn Glu Leu

170 174

The pBL(XH)h6T(1-163) prepared in Example 42 was digested with BamHI and Hind III, and the pBL(BH)(123-332) prepared above was similarly digested to obtain a fragment of about 640bp, after which the two The fragments were joined together to obtain clone pBL(XH)h6T(1-334). In addition, pCFM536/hMKT(1-163) prepared in Example 52 was digested with XbaI and SfiI to obtain a fragment of about 270 bp, and then, this fragment was digested with the same restriction enzymes pBL(XH)h6T(1-334 ) to obtain clone pBL(XH)hMKT(1-334). After digesting pBL(XH)hMKT(1-334) with XbaI and Hind III, a fragment of about 1040bp encoding mutant human TPO amino acid 1-332 was obtained, then purified, and cloned into pCFM536 predigested with XbaI and Hind III ( EP-A-136490) (E. coli JM109 previously transformed by pMW (ATCC No. 39933) was used as a host.). The resulting clone was named pCFM536/hMKT(1-332), and the E. coli strain carrying the expression vector was used as a transformant to express the mutant protein hMKT(1-332). The expression plasmid pCFM536/hMKT(1-332) contains the DNA sequence shown in SEQ ID NO:14.

In 60 ml of LB medium containing 50 μg/ml ampicillin and 12.5 μg/ml tetracycline, culture the above transformant overnight at 30°C, and then add the culture (25 ml) to 1000 ml LB medium containing 50 μg/ml ampicillin , cultured with shaking at 36°C until the OD ₆₀₀ reached 1.0 to 1.2. About 330ml LB culture medium of 65 ℃ was added in the culture so that the final temperature of the culture reached 42 ℃, and then continued shaking culture at 42 ℃ for 3 hours to induce the expression of variant human TPO; hMKT(1-332) (also Known as [Met ^-2 -Lys ^-1 ]TPO (1-332).

The resulting cultures were directly subjected to SDS-PAGE analysis. In SDS-PAGE analysis, Multigen 15/25 (Dai-ichi Chemical Co.) was used. After electrophoresis and Coomassie blue staining, according to the transformants that induced hMKT(1-332) protein expression, a protein with specificity for induced expression was detected at a molecular weight of about 35KD on the gel. In addition, after SDS-PAGE and electroblotting (with nitrocellulose membrane), the above-expressed protein was reacted with the anti-HT1 peptide antibody prepared in Example 45. After staining, a band was detected at a molecular weight of approximately 35 KD, indicating expression of hMKT(1-332). <Example 67> Preparation of human TPO substituted derivatives, their expression in COS7 cells and identification of their activity

According to the following method, whether or not several derivatives of human TPO amino acids partially substituted with other amino acids had TPO activity was investigated.

The vector pSMT201 used in this example for expression in animal cells was constructed as follows.

First, the expression plasmid pXM-mEPORn containing the cDNA of the mouse erythropoietin receptor (obtained from Dr. D'Andrea of Dana Farbor Cancer Institute: Cell: 57, 277-285 (1989)) was treated with restriction enzymes KpnI and EcoRI, The pXM vector fragment from which the cDNA of the mouse erythropoietin receptor was removed was then recovered by electrophoresis on an agarose gel. Next, two oligonucleotides for introducing different cloning restriction sites into the above expression vectors were synthesized using a DNA synthesizer (ABI). The synthetic oligonucleotide has the following basic sequence: Primer-1: 5'-CCTCGAGGAATTCCTGCAGCCCGGGACTAGTATCGGCTACCCCCTACGACGTCCCCGACTACG

CCGGCGTCCATCACCATCACCATCACTGAGCGGCCGCC-3' (SEQ ID No: 158) Primer-2: 5'-AATTGGCGGCCCCTCAGTGATGGTGATGGTGATGAGCGCCGGCGTAGTCGGGGACGTCGTAGGGGTAGccGATACTAGTCCCGGGcTGCAGGAATTCcTcGAGG5IDAc-3') (SEQ 1

The two oligonucleotides were mixed and annealed to form a double-stranded oligonucleotide, which was then ligated with the above recovered pXM vector in the presence of T4 DNA ligase (Takara-Shuzo) to obtain expression vector pDMT201. To remove the mouse DHFR cDNA contained in pDMT201 from pDMT201, vectors pDMT201 and pEF18S were digested with NotI and HpaI, respectively, followed by agarose gel electrophoresis to recover a larger fragment from pDMT201 vector DNA and from pEF18S vector DNA A smaller fragment. These fragments were then ligated together using T4 DNA ligase (Takara-Shuzo) to obtain expression vector pSMT201. As shown in Figure 21, pSMT201 contains SV40 replication origin, enhancer sequence, adenovirus main late promoter sequence, adenovirus three-part leader sequence, splicing signal sequence, SV40 early polyadenylation site sequence, adenovirus VA RNA Gene sequence, origin of replication of pUC18 and β-lactamase gene (Amp ^r ), and the following restriction sites for ligation of the desired gene: BgIII, PstI, KpnI, XhoI, EcoRI, SmaI, SpeI and NotI sites. The pHTP carrying the full-length human TPO cDNA described in Example 30 was digested with EcoRI and SpeI to obtain a full-length human TPO cDNA fragment, which was subsequently subcloned into similarly predigested vector pSMT201 to obtain plasmid pSMT201-hTPO.

Using the thus obtained plasmid pSMT201-hTPO as a template, the plasmid βGL-TPO/pBlue was first constructed as follows, and this plasmid was used as a template for preparing the desired derivative.

In βGL-TPO/pBlue, the rabbit β-globin leader sequence was added to the 5'-untranslated site of human TPO using Annweiler's method (Annweiler et al., Nucleic Acids Research, 19:3750, 1991) . For this purpose, the following double DNA strands were chemically synthesized: 5'-AATTCCAAGATCTCACACTTGCTTTTGACACAAC

TGTGTTTACTTGCAATCCCCCAAAACAGACAGACCC-3' (SEQ ID NO: 160) and 5'-GGGTCTGTCTGTTTTGGGGGATTGCAAGTAAACA

CAGTTGTGTCAAAAAGCAAGTGTGAGATCTTGG-3' (SEQ ID NO: 161), was ligated with the segment obtained by digesting the vector Bluescript II SK+ (Toyo-boseki) with EcoRI and SmaI to obtain the vector GLp/Blue with the leader sequence inserted.

PCR was done with the following primer sequences: Sma-ATG: 5'-GGCCCGGGATGGAGCTGACTGAATTGCTC-3'

(SEQ ID NO: 162) (i.e.: sequence 102-122 of SEQ ID NO: 7 linked to the SmaI sequence); and end: 5'-TCAAGCTTACTAGTCCCTTCCTGAGACAGATTCTG-3'

(SEQ ID NO: 163) (ie: the antisense sequence of the sequence 1140-1160 of SEQ ID NO: 7 linked to the SpeI and Hind III sequences).

PCR was completed with 1 ng of plasmid pSMT201-hTPO DNA as a template and 10 μM of synthetic primers. Use TakaRa PCR amplification kit (Takara-Shuzo) and Programmable ThermalController (MJ Research) to complete PCR in a volume of 100 μl under the following conditions: 94°C for 1 minute, 55°C for 2 minutes, 72°C for 2 minutes/cycle for a total of 3 cycles ; Then 94°C for 45 seconds, 55°C for 1 minute, 72°C for 1 minute/cycle, a total of 20 cycles. The PCR product was extracted with chloroform, then ethanol-precipitated twice, and then suspended in 100 μl TE buffer.

Subsequently, PCR was generated by restriction digestion with SmaI and HindIII, extracted with phenol/chloroform, and ethanol precipitated. The resulting pellet was dissolved in 10 µl of TE buffer, and then subcloned into vector βGL/pBlue (using highly competent E. coliJM109 (Toyo-boseki) as a host) predigested with the same restriction enzymes. Of the resulting transformant cells, 20 clones were screened, and plasmid DNA was prepared from these clones essentially as described in Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)). Purified plasmid DNA was identified by sequencing using a TaqDye Deoxy ^™ Terminateer Cycle Sequencing Kit (Applied Biosystems, Inc.) and a 373A DNA Sequencer (Applied Biosystems, Inc.). As a result, it was confirmed that the plasmid encoding βGL-TPO/pBlue contained the expected TPO cDNA sequence without any substitution over its entire length. After digesting βGL-TPO/pBlue with Bg III and SpeI, the digested product was extracted with phenol/chloroform followed by ethanol-precipitation. The resulting precipitate was dissolved in 10 µl of TE buffer, and then subcloned into vector pSMT201 (highly competent E. coli JM109 (Toyo-boseki) was used as a host.) digested with the same enzyme. Plasmid DNA was prepared from transformant cells as described in Molecular Cloning (Sambrook et al., supra). As shown in Figure 21, the structure of the obtained expression vector pSMT/βGL-TPO is: the BgIII/SpeI restriction site downstream of the splicing signal sequence of the pSMT201 vector, the β-globin leader sequence is connected with human TPO cDNA. This pSMT/βGL-TPO vector was transfected into COS cells.

To prepare human TPO substituted derivatives, the Ito method was used in PCR (Ito et al., Gene, 102:67-70, 1991). Specifically, two derivatives of human TPO in which Arg-25 and His-33 of human TPO were substituted with Asn and Thr, respectively, were prepared illustratively. Accordingly, these derivatives may be referred to as [Asn ²⁵ ]TPO and [Thr ³³ ]TPO. In PCR, use the following primers:

T7: 5'-TAATACGACTCACTATAGGGCG-3' (SEQ ID NO: 164)

(corresponding to the T7 promoter region of Bluescript II SK+);

ΔBgIII: 5'-AATTCCAAGATCACACACTTGC-3' (SEQ ID NO: 165)

(used to replace the BgIII recognition sequence);

 Terminal: 5′-TCAAGCTTACTAGTCCCTTCCTGAGACAGATTCTG-3′

(SEQ ID NO: 166)

(antisense of 1140-1160 of SEQ ID NO:7 linked to SpeI and Hind III):

N3: 5′-TGGGCACTGGCTCAGGTTGCTGTGAAGGACATGGG-3′

(SEQ ID NO: 167)

(Arg25-Asn of SEQ ID NO: 7): and

O9: 5'-TGTAGGCAAAGGGGTAACCTCTGGGCA-3' (SEQ ID NO: 168)

(His-33-Thr of SEQ ID NO: 7).

Use 1ng of plasmid βGL-TPO/pBlue DNA as template and 10μg of synthetic primers to complete the first step of PCR. The primer combinations were: [1] ΔBgIII and the "end"primer; [2] T7 and N3; and [3] T7 and O9. Using TakaRa PCR Amplification Kit (Takara-Shuzo) and ProgrammableThrmal Controller (MJ Research), complete PCR in a volume of 100 μl under the following conditions: 94°C for 1 minute, 55°C for 2 minutes, 72°C for 2 minutes/cycle for a total of 3 cycles ; Then 94°C for 45 seconds, 55°C for 1 minute, 72°C for 1 minute/cycle, a total of 17 cycles. The PCR product was extracted with chloroform and precipitated twice with ethanol, and then the precipitate was dissolved in 100 μl of TE buffer. Subsequently, the resulting PCR products (1 µl each) were subjected to a second PCR. The combination of templates is: PCR product [1]/[2] and PCR product [1]/[3]. For primers, T7 and end combinations were used in both cases. After incubating at 95°C for 10 minutes, TakaRa Taq was added, and PCR was completed under the following conditions: 94°C for 1 minute, 55°C for 2 minutes and 72°C for 2 minutes/cycle for a total of 3 cycles, then 94°C for 45 seconds, 55°C for 1 and 72°C for 1 minute/cycle for a total of 9 cycles. Add 1 μl of proteinase K (5 mg/ml), 2 μl of 0.5M EDTA and 2 μl of 20% SDS to each of the obtained PCR products, incubate at 37°C for 30 minutes to inactivate Taq, extract with phenol/chloroform, and ethanol-precipitate . Thereafter, the pellet dissolved in 20 μl of sterile water was digested with BgIII and SpeI, followed by phenol/chloroform extraction and ethanol-precipitation. The resulting precipitate was dissolved in 10 μl TE buffer, and then subcloned into the expression vector pSMT201 (using highly competent E.coli JM109 as host .). Of the resulting transformed cells, two clones were screened for each case, and plasmid DNA was prepared from the clones substantially according to the method described in Molecular Cloning (Sambrook et al., supra). The purified plasmid DNA was sequenced using a 373A DNA sequencer and a Taq Dye Deoxy ^™ Terminateer Cycle Sequencing Kit (both from Applied Biosystems, Inc.).

A plasmid prepared by PCR using primers [1] and [3] contained cDNA encoding a TPO substituted derivative (O9/TPO). It was confirmed that the Thr (ACC) amino acid replaced His 33 (CAC) and the original C-terminus (Gly332) was extended by adding the amino acid sequence TSIGYPYDVPDYAGVHHHHHH (SEQ ID NO: 169). The derivative is called [Thr ³³ , Thr ³³³ , Ser ³³⁴ , Ile ³³⁵ , Gly ³³⁶ , Tyr ³³⁷ , Pro ³³⁸ , Tyr ³³⁹ , Asp ³⁴⁰ , Val ³⁴¹ , Pro ³⁴² , Asp ³⁴³ , Tyr ³⁴⁴ , Ala ³⁴⁵ , Gly ³⁴⁶ , Val ³⁴⁷ , HiS ³⁴⁸ , HiS ³⁴⁹ , HiS ³⁵⁰ , His ³⁵¹ , HiS ³⁵² , HiS ³⁵³ ] TPO.

On the other hand, a plasmid prepared by PCR using primers [1] and [2] contained cDNA encoding a TPO-substituted derivative (N3/TPO). It was confirmed that Arg25 (AGA) was replaced by Asn (AAC) amino acid and Glu231 (GAA) was replaced by Lys (AAA) and the original C-terminus (Gly332) was extended by adding the amino acid sequence TSIGYPYDVPDYAGVHHHHHH. The derivative is called [Asn ²⁵ , Lys ²³¹ , Thr ³³³ , Ser ³³⁴ , Ile 335 , Gly ³³⁶ , Tyr ³³⁷ , Pro ³³⁸ , Tyr ³³⁹ , Asp ³⁴⁰ , Val ³⁴¹ , Pro ³⁴² , ^{Asp 343 ,} Tyr ³⁴⁴ , Ala ³⁴⁵ , Gly ³⁴⁶ , Val ³⁴⁷ , His 348, ^{His 349} , His ³⁵⁰ , His ³⁵¹ , His ³⁵² , His ³⁵³ ] TPO.

The resulting clones were transfected into COS7 cells according to the DEAE-dextran method described in Example 35 including chloroquine treatment. Briefly, 40 μg of each plasmid DNA was used in transfection, and after 5 days, the culture supernatant was recovered and then concentrated to 1/20 volume with Centricon-30 (Amicon) to assess its TPO activity by M-07e assay . As a result, TPO activity was detected in a dose-dependent manner in the culture supernatants of COS7 cells transfected with plasmids encoding human TPO substituted derivatives N3/TPO and O9/TPO, respectively (see FIG. 22 ). <Example 68> Preparation of insertion or deletion derivatives of human TPO, its expression in COS7 cells and identification of TPO activity

This example is to confirm whether the insertion or deletion derivatives of hTPO163 have TPO activity.

The prepared derivatives were: His33-deletion derivative ([ΔHis ³³ ]TPO(1-163), dH33); Gly116-deletion derivative ([ΔGly ¹¹⁶ ]TPO(1-163), dG116); Arg117-deletion derivative ([ΔArg ¹¹⁷ ]TPO(1-163), dR116); Thr-insertion derivatives ([His ³³ , Thr ^33' , Pro ³⁴ ]TPO(1-163), T33'); Ala-insertion derivatives ( [His ³³ , Ala ^33' , Pro ³⁴ ]TPO(1-163), A33') and Gly-insertion derivatives ([His ³³ , Gly ^33' , Pro ³⁴ , Ser ³⁸ ]TPO(1-163), G33 '), where Thr, Ala and Gly are inserted between His33 and Pro34, respectively; and Asn-inserted derivatives ([Gly ¹¹⁶ , Asn ^116' , Arg ¹¹⁷ ]TPO(1-163), N116'), Ala-inserted derivatives ([Gly ¹¹⁶ , Ala ^116' , Arg ¹¹⁷ ]TPO(1-163), A116') and Gly-insertion derivatives ([Gly ¹¹⁶ , Gly ^116' , Arg ¹¹⁷ ]TPO(1-163), G116 '), wherein Asn, Ala and Gly are inserted between Gly116 and Arg117, respectively, all sequences are based on SEQ ID NO:13.

In these derivatives, T33' and N116' were introduced into mucin-type and Asn-binding type sugar chains, respectively.

The above derivatives were prepared by PCR using the method described by Ito et al. (Gene, 102:67-70, 1991). The primer sequences used in the PCR are as follows: dH33"5'-TGTAGGCAAAGGAACCTCTGGGCA-3' (SEQ ID NO: 172) (for the preparation of His33-deleted derivatives based on the sequence shown in SEQ ID NO: 7); T33' : 5'-TGTAGGCAAAGGAGTGTGAACCTCTGG-3' (SEQ ID NO: 173) (for the preparation of Thr-insertion derivatives containing an inserted Thr between His33 and Pro34 of the sequence shown in SEQ ID NO: 7); A33': 5'-TGTAGGCAAAGGAGCGTGAACCTCTGG-3' (SEQ ID NO: 174) (for the preparation of Ala-insertion derivatives containing inserted Ala between His33 and Pro34 of the sequence shown in SEQ ID NO: 7); G33': 5 '-TGTAGGCAAAGGTCCGTGAACCTCTGG-3' (SEQ ID NO: 175) (for the preparation of Gly-insertion derivatives containing an inserted Gly between His33 and Pro34 of the sequence shown in SEQ ID NO: 7); dG116: 5'- AGCTGTGGTCCTCTGTGGAGGAAG-3' (SEQ ID NO: 176) (for the preparation of Gly116-deleted derivatives based on the sequence shown in SEQ ID NO: 7); dR117: 5'-GTGAGCTGTGGTGCCCTGTGGAGG-3' (SEQ ID NO: 177 ) (for the preparation of Arg117-deleted derivatives based on the sequence shown in SEQ ID NO: 7); N116': 5'-AGCTGTGGTCCTGTTGCCCTGTGGAGG-3' (SEQ ID NO: 178) (for the preparation of : Asn-insertion derivative containing an inserted Asn between Gly116 and Arg117 in the sequence shown in 7); A116': 5'-AGCTGTGGTCCTAGCGCCCTGTGGAGG-3' (SEQ ID NO: 179) (for preparation of : Ala-insertion derivative containing inserted Ala between Gly116 and Arg117 in the sequence shown in 7); G116': 5'-AGCTGTGGTCCTTCCGCCCTGTGGAGG-3' (SEQ ID NO: 180) (for the preparation of : Gly-insertion derivative containing an inserted Gly between Gly116 and Arg117 in the sequence shown in 7); dRI: 5'-CGGGCTGCAGGATATCCAAGATCTCA-3' (SEQ ID NO: 181) (used to replace restriction enzyme EcoRI-recognition sequence); T7: 5'-TAATACGACTCACTATAGGGCG-3' (SEQ ID NO: 182) (corresponding to the T7 promoter region of Bluescript II SK ⁺ ); and terminal Notl 5'-GGGCGGCCGCTCAGCTGGGGACAGCTGTGGTGGG-3'

(SEQ ID NO: 183) (the antisense of the 633-653 nucleotide sequence of SEQ ID NO: 7, wherein a stop codon and a NotI recognition sequence have been added).

Use 1 ng of plasmid βGL-TPO/pBlue DNA prepared in Example 67 as a template and 10 μM of synthetic primers to complete the first step of PCR. Primer combinations were [1] dRI and terminal NotI or [2] T7 and mutant primers (dH33, A33', G33', T33', dG116, dR117, A116', G116' and N116'). In PCR, PCR was performed in a final volume of 100 μl using TakaRa PCR Amplification Kit (Takara-Shuzo) and Programmable Thermal Controller (MJ Research). In the first PCR reaction of [1], one cycle is 94°C for 1 minute, 45°C for 2 minutes and 72°C for 2 minutes, completing 3 cycles; followed by 1 cycle of 94°C for 45 seconds, 45°C for 1 minute and 72°C 1 minute at ℃ for a total of 17 cycles. On the other hand, complete the first PCR reaction of [2] under the following conditions; 94°C for 1 minute, 55°C for 2 minutes, 72°C for 2 minutes in total for 3 cycles, then 94°C for 45 seconds, 55°C for 1 minute, and 72°C A total of 17 cycles in 1 minute. Before ethanol precipitation twice, each PCR product was extracted with chloroform, and the resulting pellet was dissolved in 100 μl TE buffer.

Next, the second-step PCR was performed with 1 µl of each PCR product of [1] and [2]. The primers used were a combination of T7 and terminal NotI. After incubation at 94°C for 10 minutes, TakaRa Taq was added to each PCR reaction mixture. The second PCR was performed under the following conditions: 3 cycles of 94°C for 1 minute, 55°C for 2 minutes and 72°C for 2 minutes; followed by 9 cycles of 94°C for 45 seconds, 55°C for 1 minute and 72°C for 1 minute. 1 µl of 5 mg/ml proteinase K, 2 µl of 0.5 MEDTA and 2 µl of 2% SDS were added to each PCR product, and then the mixture was kept at 37°C for 30 minutes to inactivate Taq. The PCR product was extracted with phenol/chloroform, followed by ethanol precipitation, and the resulting precipitate was dissolved in 20 µl of sterile water. After digestion with EcoRI and NotI, the precipitate was extracted with phenol/chloroform, followed by ethanol precipitation. The resulting pellet was dissolved in 10 μl of TE buffer and then subcloned into the expression vector pEF18S predigested with the same restriction enzymes and treated with alkaline phosphatase (from calf small intestine, Boehringer-Mannheim). The host cell used was highly competent E. coli JM109 (Toyo-Boseki). From the resulting transformant, plasmid DNA was prepared basically according to the method described in Molecular Cloning (Sambook et al., Cold Spring Harbor Laboratory Press, (1989)). Thereafter, the nucleotide sequence of the purified plasmid DNA was determined using a Taq Dyc Deoxy ^™ Terminateer Cycle Sequencing Kit (Applied Biosystems) and an Applied Biosystems 373A DNA sequencer. Combined, it was confirmed that all plasmids encoding dH33, A33', T33', dG116, dR117, A116', G116' and N116' contained the expected TPO cDNA sequence and were free of any nucleotides except at the desired site sequence replacement. Furthermore, in G33', a base substitution [Pro38(CCT)→Ser(TCT)] was observed, but not at the desired position.

According to the method in Example 11, the obtained clones were transfected into COS7 cells. In summary, transfection was accomplished with 10 μg of each plasmid DNA using the DEAE-dextran method including chloroquine treatment, and after 4 to 5 days, culture supernatants were recovered and evaluated with the M-07e detection system. As a result, TPO activity was detected in a dose-dependent manner in each of the culture supernatants of COS7 cells transfected with plasmids encoding amino acid insertion or deletion derivatives (see FIG. 23 ). In contrast, there was no TPO activity in the culture supernatant of COS7 cells transfected and expressing the expression plasmid pEF18S that does not contain the cDNA encoding the TPO derivative. <Example 69> Preparation and purification of anti-human TPO peptide antibody

(1) From the human TPO amino acid sequence of SEQ ID NO: 191, screen out 6 regions that are considered relatively suitable as antigens (shown in Table 8), using Tam's method (Proc.Natl.Acad.Sci. USA, 85, 5409-5413, 1988) synthesized therefrom four-chain peptides of the multiple antigenic peptide (MAP) type. Rabbits were immunized twice with 100 μg of the peptide each time, and immunized 8 times.

Regardless of the above situation, the Cys residues and each peptide region shown in SEQ ID NO: 119-124 (respectively referred to as the peptide region HT1-6 region, which correspond to the sequence 8-28 of SEQ ID NO: 191, 47-62, 108-126, 172-190, 262-284, and partial peptides of 306-332) were combined at the C-terminus to produce single-chain peptides. Using these as detection antigens, the antibody values in the sera from the thus immunized rabbits were detected by enzyme-immunoassay, and an increase in the antibody values was found in all the tested sera. Therefore, these sera are called anti-sera.

In addition, the anti-serum was affinity-purified in the following (2) again using the above-mentioned single-chain synthetic peptide (to which a Cys residue was bonded at its C-terminus) as an antigen.

(2) Two rabbits were immunized with one of the above antigen peptides, and antibodies were prepared from the immunized rabbits. Specifically, for anti-HT1 peptide antibody, anti-HT1-1 peptide antibody and anti-HT1-2 peptide antibody were obtained from two immunized rabbits.

As an example, the purification of anti-HT1-1 peptide antibody is exemplified below.

First, 30 mg of a single-chain peptide of HT1 (to which a Cys residue is bound) was coupled to 12 ml of Sulfo Link Couling Gel (manufactured by Pierce Co., product number 44895). Specifically, antigen-containing peptide solutions were coupled to gels equilibrated with 6 gel volumes of coupling buffer (50 mM Tris, 5 mM EDTA-Na, pH 8.5) over a period of 15 minutes. Then it was allowed to stand for 30 minutes as it was, and the gel was washed with 3 gel volumes of coupling buffer. Then, the coupling buffer containing 0.05M L-cys-HCl was added to the gel at a rate of 1ml/ml-gel, so that untreated groups were blocked within 15 minutes. After standing as it was for 30 min, the gel was washed with 8 gel volumes of coupling buffer. The above coupling reaction was carried out at room temperature. In this way, peptides containing antigen regions were coupled to gels by covalent bonding at a coupling rate of 28.3% to prepare antigen peptide gels in which 0.8 mg of peptides were coupled to 1 ml of gel in an antigen column. After the whole blood was collected from each immunized rabbit, 78.4ml of anti-serum containing anti-HT1-1 peptide antibody was obtained. 76.7ml of anti-serum (3620mg of protein content) was added to the solution containing 150mMNaCl and 0.05% sodium azide in advance. The antigen column was equilibrated with 50 mM phosphate buffer (pH 8), and then washed with the same buffer. A 105.9 ml flow-through fraction was thus obtained (protein content 3680 mg). The adsorbed fraction was then eluted with 0.1M citric acid (pH 3.0), followed by immediate neutralization by adding 21.1 ml of 0.1M carbonate buffer (pH 9.9), and filtered by ultrafiltration (produced by Amicon Co. YM30 membrane) concentrated and purified in 50 mM phosphate buffer (pH 8) containing 150 mM NaCl and 0.05% sodium azide. Thus, 11.2 ml of purified anti-HT1-1 peptide antibody (protein content 77.7 mg) was obtained in the buffer. In the same manner as above, anti-HT1-2 peptide antibody (60.0 mg), anti-HT2-1 peptide antibody (18.8 mg), anti-HT2-2 peptide antibody (8.2 mg) and the like were obtained.

Anti-HT3 to anti-HT6 peptide antibodies were obtained in the same manner as above. <Example 70> Detection of TPO Western analysis

(1) In order to evaluate the anti-sera obtained in Example 69, they were tested by the following method.

SDS-polyacrylamide electrophoresis (SDS-PAGE) was carried out on standard recombinant human TPO samples purified from the culture supernatant of CHO cells according to conventional methods, and then electroblotting on PVDF or nitrocellulose membranes. A gene encoding amino acids -21 to 332 of SEQ ID NO: 6 has been introduced and expressed in the cells. After blotting, wash the membrane with 20mM Tris-HCl and 0.5M NaCl (pH7.5) (TBS) for 5 minutes, then wash with TBS (TTBS) containing 0.1% Tween20 for 5 minutes, wash twice, and then block with (Block Ace; produced by Dai-Nippon Pharmaceutical Co., product number UK-B25) for 60 minutes. Anti-HT1-1 peptide antibody, anti-HT2-1 peptide antibody, anti-HT3-2 peptide antibody, anti-HT4-1 peptide antibody, anti-HT5 Each anti-serum of one of the -2 peptide antibody and the anti-HT6-1 peptide antibody was diluted to a dilution of 1/1000. The antibody thus diluted was used as the primary antibody for Western detection. Specifically, recombinant human TPO samples processed as above were treated with anti-TPO peptide antibody, 0.05% BSA and 10% Block Acl in TTBS for 60 minutes, followed by TTBS washing for 5 minutes. Next, treat with TTBS containing anti-rabbit (ab') goat biotinylated antibody (manufactured by Celtag Co., product number L43015) as a secondary antibody, and 0.05% BSA and 10% Block Ace for 60 minutes, and then use TTBS Wash twice for 5 minutes each. Next, it was treated with alkaline phosphatase-labeled avidin (manufactured by Leinco Technologoes Co., product number A108) diluted 1/5000 in TTBS solution containing 10% Block Ace for 30 minutes, and then treated with TTBS Wash 2 times, 5 minutes each, and then wash with TBS for 5 minutes. Next, color was developed with an alkaline phosphatase substrate (manufactured by Bio-Rad Co., product number 170-6432). The above-mentioned Western detection was performed at room temperature, thereby confirming that the anti-serum can recognize and detect human TPO.

(2) 3 mg each of the anti-HT1-1 peptide antibody, anti-HT1-2 peptide antibody, anti-HT2-1 peptide antibody and anti-HT2-2 peptide antibody purified by affinity chromatography in Example 69 It was weighed and biotinylated by coupling it to activated biotin (NHS-LC-Biotin II, manufactured by Pierce Co., product number 21336). The same standard recombinant human TPO sample as used in (1) above was subjected to SDS-PAGE, then electroblotted on PVDF or nitrocellulose membrane, and the biotinylated antibodies were used as primary antibodies for conventional Western detection. Immediately after blotting, the membrane was washed with TBS for 5 minutes, then washed twice with TTBS for 5 minutes each time, and then treated with blocking agent (Block Ace) for 60 minutes. Then, they were treated with a TTBS solution containing 1 μg/ml biotinylated anti-TPO peptide antibody, 0.05% BSA and 10% Block Ace for 60 minutes, and then washed twice with TTBS for 5 minutes each. Then, it was treated with alkaline phosphatase-labeled avidin (manufactured by Leinco Technologies Co., product number A108) diluted 1/5000 in TTBS solution containing 10% BlockAce for 30 minutes, and washed with TTBS thereafter. 2 times, 5 minutes each time, and then washed with TBS for 5 minutes. Color was developed with an alkaline phosphatase substrate (manufactured by Bio-Rad Co., product number 170-6432). The above-mentioned Western detection was performed at room temperature to confirm that human TPO could be recognized and detected by the purified antibody. <Example 71> Preparation of anti-TPO peptide antibody column and detection of human TPO

It was confirmed that the anti-human TPO peptide antibody obtained in Example 69 recognized human TPO. These antibodies were respectively coupled to chromatography supports to prepare anti-TPO peptide antibody columns. As an example, the preparation of an anti-HT1-2 peptide antibody column is described below.

(1) Prepare a solution containing 50mM sodium phosphate and 0.15M NaCl (pH8.0) and 5mg/ml antibody. 0.8 ml of this solution and then 1.54 ml of swollen formyl-activated gel (Formyl-Gellulofine, manufactured by Chisso Co.) were mixed and coupled at 4°C for 2 hours. Next, 1.1 ml of a solution containing 10 mg/liter of a reducing agent (trimethylamineborane (TMAB), manufactured by Seikagaku Kogyo K.K., product number 680246) was added thereto, and further coupled thereto for 4 hours. Then centrifugation recovers only the gel fraction from the reaction mixture. 10 ml of pure water was added thereto, and only the gel portion was recovered by centrifugation. This process was repeated 4 times to remove unreacted antibody. The gel thus recovered was then treated with 2.1ml of blocking buffer (0.2M sodium phosphate, 1M ethanolamine, pH 7.0) and 1.1ml of reducing agent solution added thereto at 4°C for 2 hours to block unreacted gel. Active groups in glue. Finally, the resulting gel was washed with water, 20 mM Tris-HCl and 0.15 M NaCl (pH 8.0) using a centrifuge. The resulting gel was filled in a small column test tube, washed with 3M sodium thiocyanate and 0.1M Gly-HCl (pH2.5) solution and equilibrated with 20mM Tris-HCl and 0.15M NaCl (pH8.0). Store the resulting equilibrated column.

In the anti-HT1-2 antibody column, the coupling rate of the anti-TPO peptide antibody gel to each IgG fraction was as high as 94.2%. The amount of IgG fraction coupled to the gel per unit volume was 1.9 mg/ml-gel. In the same manner as above, a gel was prepared in which 21.8 mg of IgG was coupled per ml of the gel, the IgG not containing TPO as an antigen. This was used as a previous column (hereinafter referred to as a pre-antibody column) to remove non-specifically bound molecules from the TPO antibody column as described in (2).

(2) Another example of preparing an anti-HT1-2 antibody column is explained below.

Add the standard sample of recombinant human TPO partially purified from the CHO cell culture supernatant to the previous antibody column (containing 1ml gel) prepared in (1), and use 10 times the gel volume of 20mM Tris-HCl (pH8. 0) and 0.15M NaCl are passed through the column, and the fractions passing through the column are collected, and the gene encoding any amino acid sequence of SEQ ID NO: 119-124 has been introduced and expressed in the CHO cells. Next, the fraction adsorbed to the previous antibody column was eluted with an acidic eluent (0.1M Gly-HCl, pH 2.5) 10 times the volume of the gel. Then, add the fraction that has flowed out from the previous antibody column to the anti-HT1-2 antibody column (containing 2ml gel) prepared in (1), and use 10 times the gel volume of 20mM Tris-HCl (pH8.0 ) and 0.15M NaCl to wash the column while collecting the fractions flowing through the anti-HT1-2 antibody column. The fraction adsorbed on the anti-HT1-2 antibody column was eluted with an acidic eluent (0.1M Gly-HCl; pH 2.5) 8 times the gel volume. Analysis of these fractions by SDS-PAGE demonstrated that TPO was not adsorbed to the former antibody column, but was specifically bound to the anti-HT1-2 antibody column, and almost all TPO in the spiked sample was bound to the latter column . It was thus confirmed that in the latter column at least 200 to 300 μg/ml-gel of TPO was bound to the gel. <Example 72> Effect of TPO on increasing platelet count

Twenty 8-week-old normal ICR-strain male mice (whose blood had been collected from their orbital veins; and whose platelet count had been measured with a microcytometer F-800 manufactured by Toa Iyo Denshi) were randomly divided into four groups. One of the four groups (control-iv group) was injected intravenously with 100 μl of PBS once a day for 5 consecutive days. 100 µl of the solution (prepared by substituting the concentrated solution of TPO active fraction F2 of SPSepharose Fast Llow obtained in Example 56 with PBS passed through a NAP-25 column [PharmaciaBiotech; Product No. 17-0852-02], and then diluting with PBS , the relative activity detected by M-07e was 211,900) intravenously injected into another group (TPO-iv group), injected once a day for 5 consecutive days. The other group (control-SC group) was subcutaneously injected with 100 μl of PBS once a day for 5 consecutive days, while the remaining group (TPO-SC group) was subcutaneously injected with 100 μl in the same way as in the TPO-iv group, every day Once, for 5 consecutive days. After 6, 8, 10, 13 and 15 days from the start of the administration, blood was collected from the orbital vein of each mouse and the number of platelets was counted using a microcell count (F-890, manufactured by Toa Iyo Denshi). Figure 24 shows the changes in the number of platelets. Therefore, in the control-iv group, even on the 6th day when the number of platelets was high (day 0 on the day of administration), the number of platelets increased by 44% compared with before administration, and in the control-sc group, even at the number The highest on day 8, also only increased by 47%, on the other hand, in the TPO-iv group, on day 6, compared to the number before administration, it increased by 177%, in the TPO-sc group, in Day 6 has increased by 347%, and on day 8, the highest increase has been 493%.

From the above results, it can be confirmed that human TPO increases the number of platelets independently of the type and route of administration. <Example 73> Effect of TPO on increasing platelet count

Sixteen 11-week-old normal C3H/HeJ-strain male mice whose blood was collected from orbital veins and whose platelet count was measured with a microcytometer F-800 manufactured by Tao Iyo Denshi were randomly divided into 4 groups. Group 1 (control group) was subcutaneously injected with 100 μl PBS once a day for 5 consecutive days. Subcutaneously with 100 μl of the solution (prepared by substituting the TPO active fraction of Sepha cryl S-200HR obtained in Example 56 with PBS of NAP-25 and then diluting it with PBS, the relative activity was 83,000 as detected by M-07e) The other group (TPO-1 group) was injected once a day for 5 consecutive days. The other group (TPO-2 group) was subcutaneously injected with 100 μl of the solution (prepared by diluting the TPO active fraction used in the TPO-1 group 2-fold with PBS; the relative activity was 41,500 as detected by M-07e) once a day , for 5 consecutive days. The last group (TPO-3 group) was subcutaneously injected with 100 μl of the solution (prepared by diluting the TPO active fraction used in the TPO-2 group more than 2 times; detected by M-07e, relative activity, 20,750) once a day, 5 consecutive days.

On days 6, 8, 10 and 12 after the injection, blood was collected from the orbital vein of the mice and the number of platelets was counted with a microcytometer (F-800 type, manufactured by Toa Iyo Denshi).

Figure 25 shows the changes in the number of platelets. Therefore, in the control group, there was little change in the number of platelets, and in the TPO-injected group, the number of platelets in all groups increased to the highest degree on the 8th day after the injection. In addition, between the groups, there were dose-response differences. Thus, in the TPO-1 group, on day 6, there was already an increase of about 200%, on day 8 the increase reached about 270%, and then even though it decreased on day 12, there was still about 65- % increase and therefore significantly different from the control group (p<0.01; by Dunnet's multiple comparison test). In the TPO-2 group, although the increase was less than in the TPO-1 group, the same increase was found, about 140% and about 160% on days 6 and 8, respectively. On day 12, the number decreased to almost the same level as the control group. The increasing effect of the TPO-3 group was weaker than that of the TPO-2 group. On the 8th day when the number was the highest, there was an increase of about 110-%, which was still significantly different from that of the control group (p<0.01).

From the above results, it can be confirmed that human TPO increases the number of platelets in mice independently of the strain of the mice, and that its effect has a dose-response characteristic. <Example 74> Inhibitory effect of TPO on thrombocytopenia caused by administration of anticancer agent

Thirty 8-week-old male ICR mice were intravenously injected with 200 mg/kg 5-FU, and then randomly divided into 2 groups (15 mice in each group). The next day (the 1st day), one group (control group) was subcutaneously injected with 100 μl of PBS once a day for 5 consecutive days, while the other group (TPO group) was injected with 100 μl of the TPO active fraction used in Example 72 (via The relative activity of M-07e was 211,900) subcutaneously injected once a day for 5 consecutive days. On days 4, 6 and 8, 5 mice were randomly selected from each group, and blood was collected from orbital veins and platelets were counted with a microcytometer (E-2500 type, manufactured by Toa Iyo Denshi). Figure 26 lists the changes in the number of platelets. Therefore, in the control group, after the administration of 5-FU, the number of platelets decreased over time, and on the 6th day, the platelet number was the lowest (28% of the value before the administration of 5-FU), while on the 8th day days, increased about 1.3-fold due to decreased response. Even in the TPO group, after the administration of 5-FU, the number of platelets decreased over time, and on the 6th day, the value was lowest (about 52% of the value before the administration of 5-FU). However, on days 4 and 6, the difference between the number of platelets was small, while at day 6 it remained significantly (p<0.05; measured by Dunnet Multiple comparison test) high compared to the control group. On day 8, there was an increase of about 200% compared to the value before 5-FU administration.

From the above results, it was confirmed that human TPO suppresses the decrease in the number of platelets caused by anticancer agents. <Example 75> The therapeutic effect of TPO on thrombocytopenia

Thirty-six 7-week-old ICR male mice were intravenously injected with pyrimidinium nitriurea hydrochloride (ACNU) (50 mg/kg), and then the mice were randomly divided into 2 groups (18 mice in each group).

From the next day (the first day), one group (control group) was subcutaneously injected with 200 μl of PBS, twice a day, for several consecutive days, and the other group was injected with 200 μl of the TPO active fraction (not diluted with PBS) used in Example 73. One group (TPO group), 2 times a day, for several consecutive days, added 0.04% Tween80 to the TPO active fraction (detected by M-07e, the relative activity was 380,000).

After injection, the mice in each group were randomly divided into 3 groups - on days 5 and 12, blood was collected from one group; on days 8 and 14, from the other group; on day 10, from Collect the rest of the group. Blood was collected from the orbital vein and the number of platelets was counted with a microcytometer (Model F-800, manufactured by Toa Iyo Denshi). Figure 27 lists the changes in the number of platelets. Therefore, in the control group, after the administration of ACNU, the number of platelets decreased over time, and on days 8-10, the values were lowest (about 29% of the value before the administration of ACNU), but on days 12 and 14 , only recovered to about 49% and about 74%, respectively. On the other hand, in the TPO group, on the 5th day, it decreased to about 38% of the value before ACNU administration, and then gradually increased. Thus, on day 8, it recovered to about 63% of the value before ACNU administration, while on day 10, the number of platelets was greater than the value before ACNU administration. On days 12 and 14, there was an increase of about 300% and about 400%, respectively.

As mentioned above, while in the control group a decrease was observed on days 8-10, an increase was already observed in the TPO group, and on day 10 platelet counts were higher than before ACNU administration. Therefore, it was confirmed that human TPO can promote the recovery of thrombocytopenia caused by anticancer agents.

After also considering the results in Example 74, it is now expected that human TPO can suppress the decrease in platelet number and promote recovery from thrombocytopenia independently of the type of anticancer agent. <Example 76> After BMT, the therapeutic effect of TPO on thrombocytopenia

Forty-eight 7-week-old male mice of the C3H/HeN strain were irradiated whole body with 10 Gy of radioactive radiation, immediately followed by intravenous injection of 1 x ¹⁰⁶ bone marrow cells from mice of the same strain. The mice were then randomly divided into two groups, and from the next day (day 1), one group (control group) was injected with PBS, and the TPO active fraction used in Example 73 (detected by M-07e, relative activity 44,000) were injected into another group (TPO group), both groups were subcutaneously injected with a dose of 100ml, once a day for 20 consecutive days.

After the start of injection, on days 5, 10, 14 and 21, 6 mice were randomly selected each time, blood was collected from their orbital veins, and the number of platelets was counted with a microcytometer (E-2500 type, manufactured by Toa Iyo Denshi) .

Figure 28 lists the changes in the number of platelets. Thus, in all groups, the number of platelets decreased over time after application of BMT, and on day 10, the number was lowest (approximately 3% of the number before application of BMT). Thereafter, recovery was gradual, and on day 14, the numbers in the control group and the TPO group were about 11% and about 13%, respectively. On the 21st day, only about 37% was recovered in the control group, but about 65% was recovered in the TPO group, therefore, a significant recovery-promoting effect was observed. From the above results, it can be concluded that the human TPO prepared in Example 56 promotes the recovery of platelet count after BMT application. <Example 77> After irradiation with radiation, the therapeutic effect of TPO on thrombocytopenia

Thirty-six 8-week-old ICR strain male mice were irradiated whole body with 5Gy of X-ray, and then randomly divided into 2 groups. From the next day (the first day), one group (control group) was injected with PBS, and the other group (TPO group) was injected with the TPO active fraction used in Example 73 (detected by M-07e, the relative activity was 1,440,000). ), both groups were injected subcutaneously once a day for 3 consecutive days, and then, starting from the next day, injected subcutaneously (as detected by M-07e, the relative activity was 360,000), once a day for 7 consecutive days. After the injection started, each group was randomly divided into 3 groups again - blood was collected from one group on days 4, 11 and 12, from the other group on days 7 and 13; blood from the remaining group on days 9 and 15 collected in the next group. Blood was collected from the orbital vein and the number of platelets was counted with a microcytometer (Model F-800; manufactured by Toa Iyo Denshi). The changes in platelet counts are shown in FIG. 29 . In the control group, after irradiation with X-rays, the number of platelets decreased over time, and on day 9, the number was minimal (about 25% of the number before irradiation with X-rays). On days 11 and 13, the numbers recovered to about 38% and about 67%, respectively, and on day 15, to the values before irradiation with X-rays. In the TPO group, at day 7, the number decreased to about 24% of the number before irradiation with X-rays. Thereafter, an increase began, with the number recovering to about 82% on day 9. On day 11 and thereafter, the number of platelets exceeded the number before X-ray irradiation, and this trend was maintained by day 21. As mentioned above, in the control group, the number of which still tended to decrease on day 9, the number of platelets in the TPO group had begun to increase, and on day 11, the number of platelets exceeded the number before irradiation with X-rays, and kept increasing thereafter. On the other hand, in the control group, it took 15 days to recover to the number before irradiation with X-rays. From these results, it was concluded that the human TPO produced in Example 56 shortened the time required for recovery from the decrease in the number of platelets after X-ray irradiation, and had a therapeutic effect on thrombocytopenia after X-ray irradiation. <Example 78> Effect of hTPO163 on increasing platelet count

Fifteen healthy male mice of the C3H/HeN strain whose blood was collected from their orbital veins and whose platelet counts were measured with a microcytometer (F-800 type; manufactured by Toa Iyo Denshi) were randomly divided into four groups—one group was Control group, the rest are A, B and C groups. The first group (control group) was subcutaneously injected with PBS containing 0.1% serum from mice once a day for 5 consecutive days. Subcutaneously inject A, For groups B and C, for 5 consecutive days, the TPO active fraction was diluted with PBS containing 0.1% mouse serum.

On days 6, 8, 10 and 12 after the injection, blood was collected from the orbital vein of each mouse, and the number of platelets was counted with a microcytometer (Type F-800; manufactured by Toa Iyo Denshi). The changes in platelet counts are shown in Figure 30.

Therefore, in the control group, there was little change in the number of platelets, while in the TPO group, the increase in the number of platelets in all mice reached the highest value on day 8 after injection. In each TPO group, a dose-response relationship was observed. In group A, on days 6 and 8, there was an increase of about 88% and about 100%, respectively, and even on day 10, there was still an increase of about 97%, so all data were significantly different from the control group (p <0.01 by Dunnet's multiple comparison test). A similar increase was also observed in group B, but the degree of increase was lower than that of group A. On days 6 and 8, the increases were about 65% and about 84%, respectively, indicating a significant difference from the control group (p<0.01 or 0.05, obtained by Dunnet's multiple comparison test). The increase in group C was lower than that in group B. There was still an increase of about 31% on the 8th day when the number was highest. From the above results, it can be confirmed that hTPO163 prepared in Example 65 can increase the number of platelets, and its effect shows a dose-response relationship. <Example 79> The therapeutic effect of hTPO163 on thrombocytopenia

100 8-week-old C3H/HeN strain male mice were injected intravenously with 50 mg/kg of pyrimidine ninitrourea hydrochloride (ACNU), and then randomly divided into four groups-a control group, and groups A, B, and C, each group 25 mice. From the next day (Day 1), the control group was subcutaneously injected with 5ml/kg PBS, once a day, for several consecutive days, at about 16,000,000, about 48,000,000 and 160,000,000/kg body weight/day (relative to hTPO163 detected by M-07e) Activity meter) dose, use the TPO active fraction of the SP Sepharose Fast Flow diluted with PBS obtained in Example 65 to subcutaneously inject groups A, B, and C, once a day, for several consecutive days. Before the start of injection, each group of mice was subdivided into 5 groups, and then blood was collected on days 5, 8, 10, 12 and 14. Serum was collected from his orbital vein, and the number of platelets was counted with a microcytometer (E-2500 type; manufactured by Toa Iyo Denshi). The changes in platelet counts are shown in Figure 31. In the control group, after the administration of ACNU, the number of platelets decreased over time, reaching the lowest value (about 15-16% of the value before the administration of ACNU) on the 8th to 10th day, and on the 12th and 14th day, only Very little recovery, about 28% - about 51%, respectively. On the other hand, in group A, the number decreased to about 25% of that before ACNU administration on day 8. But thereafter, gradually increased and recovered to about 34% and about 89% on day 10 and 12, respectively. The number at 14 was greater than the number before ACNU administration.

As described above, although the lowest number of platelets was observed on day 8 in all groups, in the group administered with human TPO, the degree of decrease at the lowest number of platelets was lower than that of the control group. In the control group, even on the 10th day, there was little recovery of the platelet count, while in the TPO-administered group, there was recovery of the platelet count in all groups, albeit to varying degrees. In the group administered with 50 µg/kg, there was recovery, and on the 12th day, the number was already higher than that before the administration of ACNU. By comparison, in the control group, even on the 14th day, only about 51% of the pre-ACNU administration was recovered, thus clearly showing that hTPO163 produced in Example 65 favorably promotes recovery from decrease in platelet count. Thus, it was also demonstrated that hTPO163 has a therapeutic effect on thrombocytopenia caused by anticancer agents.

The following are examples of pharmaceutical formulations. <Example 80>

The human TPO fraction obtained in Example 56 was concentrated, sterilized and frozen at -20°C to obtain a frozen product for use as an injection preparation. <Example 81>

The human TPO fraction obtained in Example 56 was concentrated, 5 ml thereof was aseptically filled in a 10 ml vial, lyophilized at -20°C, and the vial was closed with a rubber stopper to obtain a lyophilized substance for injection preparation. <Example 82>

The human TPO fraction obtained in Example 56 was concentrated, sterile-filtered and filled in 10 ml vials to obtain a product usable as an injection preparation. <Example 83>

The hTPO163 fraction obtained in Example 65 was concentrated, aseptically processed and freeze-dried at -20°C to obtain a frozen product usable as an injection preparation. <Example 84>

The hTPO163 fraction obtained in Example 65 was concentrated, 5 ml thereof was aseptically filled in a 10 ml vial, lyophilized at -20°C, and the vial was sealed with a rubber stopper to obtain a lyophilized product usable as an injection preparation. <Example 85>

The hTPO163 fraction obtained in Example 65 was concentrated, sterile-filtered and filled in 10 ml vials to obtain a product usable as an injectable preparation. <Example 86>

The human TPO fraction obtained in Example 57 was concentrated, aseptically processed and frozen at -20°C to obtain a frozen product usable as an injection preparation. <Example 87>

The human TPO fraction obtained in Example 57 was concentrated, 5 ml thereof was filled in a 10 ml vial by aseptic manipulation, lyophilized at -20°C and sealed with a rubber stopper to obtain a lyophilized product usable as an injection preparation. <Example 88>

The human TPO fraction obtained in Example 57 was concentrated, sterile-filtered and filled in 10 ml vials to obtain a product usable as an injection preparation. <Example 89> aplastic canine plasma

In addition to whole-body radiation of 450 rads to the recipient, according to [Mazur, E. and South, K. Exp. Hematol. 13: 1164-1172 (1985); Arriaga, M., South K., Cohen J.L. and Mazur , E.M. Blood 69:486-492 (1987); Mazur, E., Basilico, D., Newton, J.L., Cohen, J.L., Charland, C., Sohl, P.A., and Narendran, A. Blood 76:1771-1782 (1990)] to produce heparinized aplastic canine plasma ("APK9") or normal canine plasma ("NK9"). <Example 90> human megakaryocyte detection

In widely known handbooks of molecular biology such as Molecular Cloning by Sambrook et al. (Eds), 2nd Edition, Cold Spring Harbor Laboratory Press (1987) and Current Protocols in Molecular Biology by Ausubel et al. (Eds), Green Associates/Wiley Interscience, Standard or other suitable methods for many of the procedures described in the following examples are provided in New York (1990).

The ability of APK9 and isolated APK9 to stimulate development of adult megakaryocytes from CD34 ⁺ progenitor cells was examined. Cells were screened for CD34 from peripheral blood cells as described (Hokom, MH, Choi, E. Nichol, JL, Hornkohl, A., Arakawa, T. and Hunt, P. Molecular Biology of Haematopiesis 3:15-31, 1994) and cultivated in the following medium: Iscove's modified Dulbecco's medium (IMDM ; GIBCO, Grand Island, NY). Also contains 2-mercaptoethanol (10 ^-4 M), pyruvate (110μg/ml), cholesterol (7.8μg/ml), adenosine, guanine, cytidine, uridine, thymidine, 2-deoxycytosine, 2-deoxyadenosine, 2-deoxyguanosine (10 μg/ml each, Sigma), human recombinant insulin (10 μg/ml), human transferrin (300 μg/ml), soybean fat (1%, BoehringerMannheim Indianapolis, IN) , human recombinant basic fibroblast growth factor ((2ng/ml), Genzyme, Cambridge, MA), human recombinant epidermal growth factor (15ng/ml), platelet-derived growth factor (10ng/ml, Amgen, Inc., Thousand Oaks, CA). CD34-selected cells were plated at 2×10 ⁵ ml in wells of Terasaki-type microtiter plates (Vanguard, Inc. Neptrne, NJ) for culture (final volume 15 μl). At 37°C, cells were cultured for 8 days in 5% CO ₂ air in a humid chamber, fixed directly into culture wells with 1% glutaraldehyde, and then mixed with monoclonal scintillation fluid (anti-GPIb, anti-GP II B, (Biodesign) and anti-GpIb (Dako, Carpinteria, CA) are incubated together.Use streptavidin-beta-galactosidase detection system (HistoMark, Kirgegaard and Perry) to make immunoreaction color development.Invert with an Blue megakaryocytes were counted with a phase microscope at 100X magnification. Results were expressed as the mean +/- standard error of the mean (SEM) of megakaryocytes per well. In some cases expressed as "megakaryocyte units/ml" Data, where the extent to which megakaryocyte development was induced for a given sample was normalized to the positive APK9 control for that experiment. 1 unit refers to the amount of substance that brought megakaryocytes to the same number of megakaryocytes as 1 μl APK9 standard. 5-10 μg/ml MPL if available -X blocks activity, its activity can be indicated by the MPL ligand (soluble MPI receptor).

In this system, APK9 has been shown to contain factors that stimulate the development of human megakaryocytes. CD34-select cells cultured with 10% NK9 for 8 days had only negligible blue megakaryocytes, while CD34-select cells cultured with 10% APK9 for 8 days had a large number of blue megakaryocytes.

Figure 32 shows that the blocking effect on the development of megakaryocytes was gradually increased when increasing concentrations of MpI-X were added to the human megakaryocyte culture system. When the concentration of MpI-X is greater than 5 μg/ml, it is completely inhibited. In this experiment, CD34-selected cells were stimulated with 5% APK9. This indicates that activity interacting with MpI-X (the putative MpI ligand) is essential for human megakaryocyte development and that MpI ligands are present in APK9.

It is also shown herein that MpI ligand activity essential for human megakaryocyte development is present in APK9. APK9 (135 ml) was diluted 6-fold in Iscove's medium and applied to MpI-X affinity column. Unbound material (effluent) was collected and concentrated to original volume before assay. Bound material was eluted with 10 ml 1M NaCl, and the 20% harvest was diafiltered and concentrated 4-fold for detection. CD34-selected cells alone did not develop into megakaryocytes in culture. Cells cultured at 5% APK9 (same harvest as used for column) developed 48+/-8 megakaryocytes/well. Cells cultured in 10% unbound material failed to develop into megakaryocytes. Cells cultured in the 10% elution pool developed 120+/-44 megakaryocytes/well. In the test, 5 μg/ml MpI-X can almost completely inhibit the activity of the loading column and the elution collection. <Example 91> Transfection of mouse or human MpI receptors into mouse cell lines A. Mouse MpI receptors

The full-length murine MpI receptor cDNA was subcloned into an expression vector containing the transcriptional promoter from LTP from the Moloney murine sarcoma tumor virus. 5 μg of this construct and 1 μg of selectable marker plasmid pWLNeo (Stratagene) were co-porated into IL-3-dependent mouse cytokine (32D, cline23; Greenberger et al, DNAS 80:2931-2936 (1983)). The cells were harvested after culturing for 5 days, and then cultured in a selection medium containing 800 µg/ml Geneticin (G418, Sigma) and 1 ng/ml murine IL-3. The surviving cells were divided into multiple pools of 2 x ¹⁰⁵ cells per pool and cultured until a sufficient number for further analysis was grown. Six cell populations were tested for surface expression of the MpI receptor by FACS analysis using polyclonal rabbit anti-peptide sera. Using the same anti-peptide serum as above, select a FACS-typed cell population. One cell clone of the parental cell line was screened by growth in 10% APK9 and Geneticin. After 35 days of selection in APK9, cells were maintained in 1 ng/ml murine IL-3. One subclone 1A6.1 was used for this work. B. Human MpI receptor

The full-length human MpI receptor sequence (Vigon, I et al, DNAS 89:5640-5644 (1992)) was subcloned into an expression vector containing the Maloney murine sarcoma tumor virus transcriptional promoter (the same vector as for the murine receptor) middle. Using the CaPO ₄ mammalian transfection cassette (Stratagene), 6 μg of this construct and 6 μg of the amphipathic retroviral packaging construct (Landu NR Littman, DR, J. Virology 66:5110-5113 (1992)) were transfected into 3 x 10 ⁶ 293 cells. The same cells were transfected 2 days later and again 4 days later. The day after the last transfection, 293 cells were cultured with an IL-3-dependent murine cell line (32D, clone23; Greenberger et al, PNAS 80:2931-2936 (1983)). After 24 hours, 32D cells were rescued and banded in a BSA gradient (Path-o-cyte; Mills, Inc.). Cells were expanded in 1 ng/ml murine IL-3 and selected for growth in 20% APK9. Cells were sorted by surface expression of cell receptors by FACS using polyclonal rabbit anti-peptide serum. These cells were then used for testing. <Example 92> 1A6.1 detection of MpI ligand

1A6.1 cells were washed to make it free of IL-3 and the test samples were serially diluted 1:1, and the cells were then placed in a Terasaki-type microtitre dish supplemented with 10% fetal calf serum (FCS), Geneticin (800 μg/ ml) and 1% penicillin/streptomycin (Gibco) in αMEM (Gibco) (1000 cells/15 μl total col/well). After 48 hours, the number of viable cells in each well was determined microscopically. 1 activity unit refers to the amount of activity that produces 200 viable cells in each well. Activity refers to MpI ligand if, in the assay, the addition of 5-10 μg/ml MpI-X completely blocks the activity. In APK9, Mpl ligand activity averaged 4400 +/- 539 units/ml aplastic plasma. Units of MpI ligand activity are defined in the 1A6.1 assay unless otherwise stated.

Assays with cells transfected with the human MpI receptor gene were performed essentially in the same manner as with 1A6.1 cells. <Example 93> Demonstration that MpI-ligand exists in the aplastic plasma of mice, dogs, pigs and human-derived sera

MpI ligands were present in aplastic plasma from murine, canine, porcine and human sera (Table 9). Plasma was collected from BDF1 mice before irradiation and 12 days after irradiation (500 rads). Plasma was tested in the 1A6.1 assay, and the results showed that the activity of 2000 units/ml was substantially completely inhibited by MpI-X (10 μg/ml). Irradiated mouse plasma was also positive in the human megakaryocyte assay with an activity of 1833 units/ml, plasma was collected from dogs before irradiation and 10 days after irradiation (450 rads). Activity was tested in the 1A6.1 assay and the human megakaryocyte assay. Activity was detectable in both assays and was completely inhibited with MpI-X (10 μg/ml). Plasma was collected from pigs before irradiation and 10 days after irradiation (650 rads). Plasma was tested in the 1A6.1 assay and the human megakaryocyte assay. In both assays, MpI ligand activity (inhibitable by 10 μg/ml MpI-X) was shown to be comparable to that seen in the plasma of aplastic dogs. Its serum was obtained from aplastic humans. The material was collected from bone marrow transplant patients. Sera from six patients tested in the 1A6.1 assay had an activity of 903 units/ml, 88% of which was due to MpI ligand (inhibitable with 10 μg/ml MpI-X). Sera from 14 aplastic patients were tested in the human megakaryocyte assay. As a group, they showed basal activity of 941 megakaryocyte units/ml, which was completely inhibited by 10 μg/ml MpI-X. Murine IL-3 data are included to illustrate 1A6.1. Assay specificity. Although the recombinant cytokine induced cell growth, it was not blocked by 10 μg/ml MpI-X.

Table 9

Species 1A6.1 cell detection Human megakaryocyte detection

(Unit/ml) (Megakaryocyte unit/ml)

Base+mpi-x matrix+MPI-X normal mice 0 +/- 0 0+/0 0 +/- 0 0 +/- 0 recycled mice 2000 0 1833 No normal dog 0 +/- 0 0 0 /-0 0+/-0 0+/-0 Aplastic Dog 4400+/-539 0+/-0 792+/-128 0+/-0 Normal Pig 0+/-0 0+/-0 0 + /-0 0+/-0 Aplastic Pig 3866+/-1136 0+/-0 1284+/-182 10+/-10 Normal Human 0+/-0 0+/-0 0+/-0 0 + /-0 aplastic people 903+/-64 114+/-33 941+/-178 0+/-0 mouse IL-3 6000+/-565 6000+/-565 not made <Example 94> preparation Human TPO derivative expressed in E.coli system

To improve bioactivity, stability and solubility, several TPO derivatives were designed and expressed in E.coli. The derivatives obtained with the amino acid sequence of the aforementioned SEQ ID NO: 11 include [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹²⁹ ] TPO(1-163) (also known as L129R ); Arg replaces the [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹³³ ]TPO(1-163) (also known as H133R) of the 133-position His, Arg replaces the 143-position Met [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹⁴³ ]TPO(1-163) (also known as M143R), Leu replacing Gly at position 82 [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Leu ⁸² ] TPO(1-163) (also known as G82L), [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Leu ¹⁴⁶ ] TPO(1-163) (also known as G146L) with Leu replacing Gly at position 146, Pro replaces [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Pro ¹⁴⁸ ]TPO(1-163) (also known as S148P) at position 148 Ser, and Arg replaces [Met ^-2 , Lys ^- at position 59 Lys ¹ , Ala ¹ , Val ³ , Arg ⁵⁹ ]TPO (1-163) (also known as K59R), Arg substituted for 115 Gln [Met ^-2 , Lys ^-1 , Ala ¹ , Val ³ , Arg ¹¹⁵ ]TPO ( 1-163) (also known as Q115R).

For the preparation of TPO derivatives, h6T(1-163) as described in Example 42 was used as a template to synthesize oligonucleotides with the following sequence: L129R: 5'-ATCTTCCGTTCCTTTCCAGCACCT-3' (for the preparation of Arg substituted for SEQ ID NO: the Arg-substituted derivative of Leu-129 in the sequence shown in 11); H133R: 5'-TCTTTCCAGCGTCTGCTGCGT-3' (for the preparation of His-133 in the sequence shown in SEQ ID NO: 11 substituted by Arg M143R: 5'-CGTTTCCTGCGTCTGGTTGGC-3' (for the preparation of Arg-substituted derivatives of Met-143 substituted by Arg in the sequence shown in SEQ ID NO: 11); G82L: 5' -GGCCAGCTTCTGCCGACCTGCCT-3' (for the preparation of Leu-substituted derivatives of Gly-82 substituted by Leu in the sequence shown in SEQ ID NO: 11); G146L: 5'-ATGCTGGTTCTGGGTTTCTACCCT-3' (for the preparation of Leu substituted SEQ ID NO: the Leu-substituted derivative of Gly-146 in the sequence shown in 11); S148P: 5'-GTTGGCGGTCCGACCCTGTGCG-3' (for the preparation of Ser in the sequence shown in SEQ ID NO: 11 replaced by Pro -148 Pro-substituted derivatives); and K59R: 5'-GAAGAGACCCGCGCTCAGGACATCC-3' (for the preparation of Arg-substituted derivatives of Lys-59 substituted by Arg in the sequence shown in SEQ ID NO: 11); Q115R : 5'-CTGCCGCCACGTGGCCGTACCAC-3' (for the preparation of an Arg-substituted derivative of Gln-115 substituted by Arg in the sequence shown in SEQ ID NO: 11).

Mutant plasmids were constructed using the Sculptor in vitro mutagenesis cassette (Amersham). The XbaI-HindIII fragment obtained from pCFM536/h6T(1-163) described in Example 42 was introduced into the Bluescript II SK(-) phagemid vector (Stratagene, California USA) digested with XbaI and HindIII to obtain Plasmid pSKTPO was then transformed into E. coli JM109. Single-stranded DNA for mutagenesis was prepared from pSKTPO with helper phage M13KO7 (Takara Shuzo, Japan). Mutations were introduced into the TPO gene using the above-mentioned oligonucleotides according to the supplier's instructions. According to the supplier's instructions, DNA sequencing kit (Applied Biosystems, USA) was used to completely sequence and analyze the mutation of TPO.

The XbaI-HindIII fragment prepared from mutated pSKTPO was introduced into pCFM536 digested with XbaI and HindIII. The plasmid pCFM536 carrying the mutated gene was transformed into E. coli #261 to obtain a transformant expressing the TPO derivative gene.

E. coli #261 transformed with the mutated pCFM536 was cultured as in Example 43 above. Purified cell pellets were collected and stored in a freezer for at least 1 day. Frozen cell pellets (3 g) were suspended in 30 ml of 20 mM Tris buffer (pH 8.5) containing 10 mM EDTA, 10 mM DTT and 1 mM PMSF. Place the suspension on ice and sonicate 5 times at 1 min (at least) intervals. Sonicated cells were harvested by centrifugation at 15000 rpm for 10 minutes.

The particles were suspended in 30ml of 10mM Tris buffer (pH 8.7) containing 8M guanidine hydrochloride, 5mM EDTA, and 1mM PMSF, and were reduced by adding 50mg of DTT. After stirring for 1-1.5 hours, the pH of the solution was adjusted to 5.0 with diluted HCl, then cooled to 4°C before dilution.

The sample solution was gradually diluted with 1.5 L of 10 mM CAPS buffer (pH 10.5) containing 30% glycerol, 3M urea, 3 mM cystamine and 1 mM L-Lys and left overnight. After stirring the samples for at least 2 days, the precipitate was removed by centrifugation at 8000 rpm for 45 minutes. The pH of the solution was adjusted to 6.8 with 6M phosphoric acid, and then diluted 2-fold. The solution was filtered with 2 sheets of #2 filter paper (φ=90mm, Toyo filter paper, Japan), and then loaded onto a CM-Sepharose Fast Flow column (2.6×10 cm) (Pharmacia). The column was washed with 400 ml of 10 mM phosphate buffer (pH 6.8) containing 15% glycerol and 1M urea, and then equilibrated with 10 mM phosphate buffer (pH 7.2) containing 15% glycerol. The protein was eluted from the column with a linear gradient from 10 mM phosphate buffer (pH 7.2) containing 15% glycerol to the same buffer containing 0.5 M NaCl at a flow rate of 1.0 ml/min. Protein eluates were monitored by absorbance at 280 nm; fractions containing TPO derivatives were identified by SDS-PAGE and collected.

After concentrating the TPO fraction (about 30 ml) to 2 ml with Centriprep 10 (Amicon), the mutated TPO was further purified by reverse-phase HPLC (Waters) with a μBondasphere C4 column (3.9×150 mm). The protein was eluted with a linear gradient from 0.05% TFA in _H2O to 30% _CH3CN and 0.02% TFA in 2-propanol at a flow rate of 0.5 ml/min over 40 minutes. Under these conditions, the TPO derivative eluted after about 30 minutes.

For bioassays, the purified TPO samples were dialyzed twice for 2 days against 1 L of 10 mM phosphate buffer. After concentration to about 0.1 mg/ml with Centriprep 10 (Amicon), the biological activity of the derivatives was evaluated with the M-07e detection system as described above. All mutants were found to have activity equivalent to the reference sample of h6T(1-163), TPO (see Figures 33 and 34). Collection list Escherichia coli (pHT1-231/DH5):

FERM BP-4564 and CCTCC-M95001 Escherichia coli (pEF18S-A2α/DH5):

FERM BP-4565 and CCTCC-M95002 Escherichia coli (pHGT1/DH5):

FERM BP-4616 and CCTCC-M95003 Escherichia coli (pHTF1/DH5):

FERM BP-4617 and CCTCC-M95004 mouse-mouse hybridoma P55:

FERM BP-4563 and CCTCC-C95001CHO28/1/1/3-C6:

FERM BP-4988 and CCTCC-C95004CHO163T-63-79-C1:

FERM BP-4989 and CCTCC-C95005

Sequence Listing (1) General information:

(i) Applicants: MIYAZAKI, Hiroshi

KATO, Takashi

OHGAMI, Kinya

IWAMATSU, Akihiro

AKAHORI, Hiromichi

KUROKI, Ryota

SHIMIZU, Toshiyuki

MUTO, Takanori

(ii) Title of invention: protein with TPO activity

(iii) Number of sequences: 197

(iv) Corresponding address:

(A) Recipient: Marshall, O′Toole, Gerstein, Murray & Borun

(B) Street: 6300 Sears Tower, 233 South Wacker Drive

(C) City: Chicago

(D) State: Illinois

(E) Country: United States

(F) Zip code: 60606-6402

(v) in computer readable form:

(A) Media type: Floppy disk

(B) Computer: IBM PC compatible

(C) Operating system: PC-DOS/MS-DOS

(D) Software: PatentIn Release#1.0, Version#1.25

(vi) Current application materials:

(A) Application number:

(B) Applicant:

(C) Classification number:

(vii) Prior application materials:

(A) Application number: JP 39090/94

(B) Application date: 14-FEB-1994

(vii) Prior application materials:

(A) Application number: JP 79842/94

(B) Application date: 25-MAR-1994

(vii) Prior application materials:

(A) Application number: JP 155126/94

(B) Application date: 01-JUN-1994

(vii) Prior application materials:

(A) Application No.: JP 167328/94

(B) Application date: 15-JUN-1994

(vii) Prior application materials:

(A) Application number: JP 227159/94

(B) Application date: 17-AUG-1994

(vii) Prior application materials:

(A) Application No.: JP 304167/94

(B) Application date: 01-NOV-1994

(vii) Prior application materials:

(A) Application number: JP UNKNOWN

(B) Application date: 28-DEC-1994

(vii) Prior application materials:

(A) Application No.: JP 193169/94

(B) Application date: 17-AUG-1994

(vii) Prior application materials:

(A) Application number: JP 298669/94

(B) Application date: 01-DEC-1994

(vii) Prior application materials:

(A) Application number: JP 193916/94

(B) Application date: 18-AUG-1994

(vii) Prior application materials:

(A) Application number: US 08/212,164

(B) Application date: 14-MAR-1994

(vii) Prior application materials:

(A) Application number: US 08/221,020

(B) Application date: 01-APR-1994

(vii) Prior application materials:

(A) Application number: US 08/278,083

(B) Application date: 20-JUL-1994

(vii) Prior application materials:

(A) Application number: US 08/320,300

(B) Application date: 11-OCT-1994

(vii) Prior application materials:

(A) Application number: US 08/361,811

(B) Application date: 22-DEC-1994

(viii) Agent/Agent Information:

(A) Name: Borun, Michael F.

(B) Registration number: 25,447

(C) Literature/Abstract No.: 01017/32425

(ix) Telecommunication information:

(A) Tel: 312/474-6300

(B) Telex: 312/474-0448 (2) Information of SEQ ID NO: 1:

(i) Sequential features:

(A) Length: 261 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D) Topology: linear

(ii) Molecular type: cDNA to mRNA

(vi) Original source:

(A) Creature: Rattus norvegicus

(B) Cell line: McA-RH8994

(xi)序列描述：SEO ID NO：1：GGTGTACCTG GGTCCTGAAG CCCTTCTTCA CCTGGATAGA TTCCTTGGCC CACCTGTCCC      60CACCCCACTC TGTGCAGAGG TACAAAAGCT CAAGCCGTCT CCATGGCCCC AGGAAAGATT     120CAGGGGAGAG GCCCCACACA GGGAGCCACT GCAGTCAGAC ACCCTGGGCA GA             172ATG GAG CTG ACT GAT TTG CTC CTG GTG GCC ATA CTT CTC CTC ACC GCA       220Met Glu Leu Thr ASP Leu Leu Val Ala Ile Leu Leu THR ALA1 5 10 15ACA ACT CTG TCC CCA GTT CCC GCC TGT GAC CC 261AR Leu Serire Pro PRO ALA CYS Asp

                                                                                                25(2) Information on SEQ ID NO: 2:

(i) Sequential features:

(A) Length: 147 amino acids

(B) type: amino acid

(D) Topology: linear

(ii) Molecule type: protein

(xi) Sequence description: SEQ ID NO: 2:

Met Glu Leu Thr Asp Leu Leu Leu Val Ala Ile Leu Leu Leu Thr Ala

-21 -20 -15 -10

Arg Leu Thr Leu Ser Ser Pro Val Pro Pro Ala Cys Asp Pro Arg Leu

-5 1 1 5 10

Leu Asn Lys Leu Leu Arg Asp Ser Tyr Leu Leu His Ser Arg Leu Ser

15 20 25

Gln Cys Pro Asp Val Asn Pro Leu Ser Ile Pro Val Leu Leu Pro Ala

30 35 40

Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Thr Glu Gln Ser Lys

45 50 55

Ala Gln Asp Ile Leu Cly Ala Val Ser Leu Leu Leu Glu Gly Val Met

60 65 70 75

Ala Ala Arg Gly Gln Leu Glu Pro Ser Cys Leu Ser Ser Leu Leu Gly

80 85 90

Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Gly Leu

95 100 105

Leu Gly Thr Gln Val Ser Pro Gln Thr Tyr Arg Asn Tyr Pro Leu Thr

110 115 120

Gln Phe Leu

125(2) Information of SEQ ID NO: 3:

(i) Sequential features:

(A) Length: 580 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D) Topology: Linear

(ii) Molecular type: cDNA to mRNA

(vi) Original source:

(A) Creature: Homo Sapiens

(F) Tissue type: Liver

(XI) Sequence description: SEQ ID NO: 3: CTC GTG GTC ATG CTT CTC CTA AGG CTA ACG CTG CTG TCC AGC CCG 48leu Val Val Met Leu THR ARG Leu Thr Leu Ser Ser Series CCT CCT GCT TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT GAC 96Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp

20 25 30TCC CAT GTC CTT CAC AGC AGA CTG AGC CAG CAGC CCA GAG GAG GAG GAG GAG CAC CCT 144SER HIS Leu His Serg Leu Val His Pro Val His Pro Val His Pro

35 40 45 TTG CCT ACA CCT GTC CTG CTG CCT CCT GTG GAC TTT AGC TTG GGA GAA 192Leu Pro Thr Le G Leu P Lu S Pro Val Ala

50                  55                  60TCG AAA ACC CAG ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GGA GCA    240Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ale65                  70                  75                  80GTG ACC CTT CTG CTG GAG GGA GTG ATG GCA GCA CGG GGA CAA CTG GGA 288Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly

85 90 95CCC ACT TGC CTC TCC CTC CTG GGG CAG CAG CGA CGA CAG GTC CGT 336PRO Thr Cys Leu Leu Leu Gln Leu Serg Gln Val ARG

100 105 110CTC CTC CTC CTT GGG GCC CTG AGC CTC CTT GGA ACC CAG NNN NNN NNN 384Leu Leu Leu GLY Ala Leu Leu Gln XAA XAA xaaa

115 120 125NNN NNN NNN NCC ACA GCT CAC CAC CAT CCC AAT GCC AGC CTG AGC 432XAA XAA XAA THR Ala His Lys ALA Ile Phe Leu Seru Seru Seru Seru Seru Leu Leu Leu Leu Leu Leu Leu Leu's Luu Leu Leu Leu's

130                 135                 140TTC CAA CAC CTG CTC CGA GGA AAG GTG CGT TTC CTG ATG CTT GTA GGA    480Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly145                 150                 155                 160GGG TCC ACC CTC TGC GTC AGG CGG GCC CCA CCC ACC ACA GCT GTC CCC 528Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro

165 175AGC AGA ACC TCT CTA GTC CTC ACA CTG AAC GAG CTC CCA AGG AGG AGG ARG THR Leu Val Leu THR Leu Pro Asn ARG THR

180 185 190TCT G 580Ser: ID of NO 4 Q (2)

(i) Sequential features:

(A) Length: 253 amino acids

(B) type: amino acid

(D) Topology: Linear

(ii) Molecule type: protein

(XI) Sequence description: SEQ ID NO: 4: Met Glu Leu Thr Glu Leu Val Val Met Leu Leu ThR ALA-21 -20 -10arg Leu sel Pro Ala Cys Asp Leu ARG. Val-5 1 1 5 5 10Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser

15 20 20 25Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala

30 35 40Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys

45 50 55ALA GLN ASP ILE Leu Gly Ala Val Thr Leu Leu GLU GLU GLY VAL MET60 70ALA Ala ARG GLN Leu GLY Pro Thr Cyser Leu Leu Gly Gly

80 85 90Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu

95 100 105Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp

110 115 120Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val

125 135arg PHE Leu Met Leu Val Gly GLY GLY SER Leu Cys Val ARG ALA140 145 150 155PRO THR THR ALA Val Pro Serg THR Leu Val Leu Thr Leu

160 165 170Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr

175 180 185Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly

190 195 200Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu

205 210 225Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile His Glu Leu220 225 Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile His Glu Leu220 220 225 ID: 230 (

(i) Sequential features:

(A) Length: 576 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D) Topology: Linear

(ii) Molecular type: cDNA to mRNA

(vi) Original source:

(A) Creature: Homo Sapiens

(F) Tissue type: Liver

(xi) Sequence description: SEQ ID NO: 5: AG GGA TTC AGA GCC AAG ATT CCT GGT CTG CTG AAC CAA ACC TCC AGG 47Gly Phe Arg Ala Lys Tle Pro Gly Leu Leu Asn Gln Thr Ser Arg

1 5 10 15TCC CTG GAC GAC CCC CCC GGA TAC CTG AGG AGG AGG AGG AGG AGG GAA CTC TTG 95SER Leu ASLN Ile

20 25 30AAT GGA ACT CGT GGA CTC TTT CCC TCC TCA CGC AGG AGG AGG AGG AGG AGGA GGA 143ASN GLY THR ARG GLY LU PRE PRO GLE

35 40 45GCC CCG GAC ATT TCC TCC TCA GGA ACA GAC ACA GGC CTG CCA CCC 191ALA Pro ASP iLe Ser Ser Ser Ser Ser Gly Ser

50 55 60AAC CTC CAG CAGA TAT TAT TAT TCT CCC CCA ACC Cat CCT CCT CCT GGA 239ASN Leu Gln Pro Gly Tyr Ser Pro ThR His Pro ThR Gly Gly Gly Gly Gly Gly Gly

65                  70                  75CAC TAT ACG CTC TTC CCT CTT CCA CCC ACC TTG CCC ACC CCT GTG GTC       297Gln Tyr Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val80                  85                  90                  95CAG CTC CAC CCC CTG CTT CCT CAC CCT TCT GCT CCA ACG CCC ACC CCT 335Gln Leu His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro

100 105 110acc AGC CTT CTT CTA ACA TCC TAC CAC CAC CAC CAG AAT CAG TCT 383thr Seru Leu asn Thr His Ser Gln Leu Ser

                                                                                                                               

    130TACAGCTCCC TTCCCTGCAG GGCGCCCCTG GGAGACAACT GGACAAGATT TCCTACTTTC     492TCCTGAAACC CAAAGCCCTG GTAAAAGGGA TACACAGGAC TGAAAAGGGA ATCATTTTTC     552ACTGTACATT ATAAACCTTC AGAA                                            576(2)SEQ ID NO：6的资料：

(i) Sequential features:

(A) Length: 353 amino acids

(B) type: amino acid

(D) Topology: linear

(ii) Molecule type: protein

(XI) Sequence description: SEQ ID NO: 6: Met Glu Leu Thr Glu Leu Val Val Met Leu Leu ThR ALA-21 -20 -10arg Leu sel Pro Ala Cys Asp Leu ARG. Val-5 1 1 5 5 10Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser

15 20 20 25Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala

30 35 40Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys

45 50 55ALA GLN ASP ILE Leu Gly Ala Val Thr Leu Leu GLU GLU GLY VAL MET60 70ALA Ala ARG GLN Leu GLY Pro Thr Cyser Leu Leu Gly Gly

80 85 90Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu

95 100 105Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp

110 115 120Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val

125 135arg PHE Leu Met Leu Val Gly GLY GLY SER Leu Cys Val ARG ALA140 145 150 155PRO THR THR ALA Val Pro Serg THR Leu Val Leu Thr Leu

160 165 170Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr

175 180 185Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly

190 195 200Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu

205 215ASP Gln Ile Pro Gly Tyr Leu asn ARG iLe His Glu Leu Leu asn Gly220 235thr ARG GLE PRO GLE PRO GLY Pro Sar ARG Thr Leu Gly Ala Pro

240 245 250Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu

255 260 255Gln Pro Gly Tyr Ser Pro Ser Pro Thr His His Pro Pro Thr Gly Gln Tyr

270 275 280Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu

285 290 295HIS Pro Leu PRO ASP Pro Sera Pro THR Pro ThR Ser300 305 315pro Leu Leu asn Tyr Tyr His Gln Gln Gln Gln Gln Gln Glu

320 325 330Gly(2) SEQ ID NO: 7 information:

(i) Sequential features:

(A) Length: 1721 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D) Topology: Linear

(ii) Molecular type: cDNA to mRNA

(vi) Original source:

(A) Creature: Homo Sapiens

(F) Tissue type: Liver

(xi) Sequence description: SEQ ID NO: 7: GCGGCACGAG GGGGGTGTCT GGCTGGCGTG GCTCCCTGTT TGGGGCCTCT CCCCTGAATC 60CTTCCTGGGG CCATGGAGGC CAGACAGACA CCCCGGCCAG A ATG GAG CTG ACT 113

                     

-21 -20GAA TTG CTC CTC GTG GTC ATG CTT CTC CTA AGG CTA ACG CTG 161GLU Leu Leu Val Val Met Leu Leu THRA Leu Thr Leu

-15 -10 -5TCC AGC CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC CTC AGT AAA CTG 209Ser Le u Ser Pro Ala Ar u Pro L Pro ys sp Ala Cys

1 5 10 15CTT CGT GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC CAGC CCA GAG 257leu ARG As His Val Leu His Serg Leu Sergs Pro Glu

20 25 30GTT CAC CAC CCTG CCT ACA CCT GTC CTG CTG CTG GCT GCT GCT GAC TTT AGC 305VAL HIS Pro Leu Pro THR Prou PRO Ala Val Ala Val ASP PHE Ser

35 40 45ttg GGA GGA TGG AAA ACC CAG ATG GAG GAG GAG GAG GAG GCA CAC ATT 353leu GLY GLY GLU TRP LYS THR Gln Met Glu ThR LYS ALA GLN ASP Ile

50 55 60CTG GGA GCA GCA GTG ACC CTG CTG CTG GAG GAG GGA GCA GCA GCA CCG GCA 401leu Gly Ala Vr Leu Leu GLU GLU GLY Val Val Met Ala Ala Ala Ala Ala Ala Ala Ala's Meru's

65                  70                  75CAA CTG GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG CAG CTT TCT GGA       449Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly80                  85                  90                  95CAG GTC CGT CTC CTC CTT GGG GCC CTG CAG AGC CTC CTT GGA ACC CAG 497Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln

100 105 110CTT CCT CCA CAG GGC AGG AGG ACC ACA GCT CAC AAG GCC AAT GCC ATC 545leu Pro GLN GLY ARG THR ALA His Lysn Ala Ile

115 120 125TTC CTG ACC TTC CAA CAA CAC CTC CGA GGA AAG GTG CGT TTC CTG 593PHE Leu Seru Leu Leu ARG GLY LYS Val ARG PHE Leu Met

130 135 140CTT GGA GGA GGG TCC ACC CTC TGC GCC CCC CCC ACC ACC ACC ACC ACC GLY GLY GLY GLE GLE GLE GLE GLE GLE

145                 150                 155GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG AAC GAG CTC CCA       689Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro160                 165                 170                 175AAC AGG ACT TCT GGA TTG TTG GAG ACA AAC TTC ACT GCC TCA GCC AGA 737Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg

180 185 190aca Act GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA TTC AGA GCC 785thr THR GLY Leu LEU LYS TRN GLN GLN ARLA LY AR -Alas

195 200 205ATT CCT GGT GGT CTG AAC CAA ACC TCC AGG TCC CTG GAC CAA ATC CCC 833ile Pro GLY Leu Asn Gln Thr Serg Serg Ser Leu asp Gln Ile Pro Pro

210 215 220GGA TAC CTG AGG AGG AGG AGG AGG ATA CAA CTC TTG AAT GGA Act CGT GGT GGT

225                 230                 235TTT CCT GGA CCC TCA CGC AGG ACC CTA GGA GCC CCG GAC ATT TCC TCA       929Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser240                 245                 250                 255GGA ACA TCA GAC ACA GGC TCC CTG CCA CCC AAC CTC CAG CCT GGA TAT 977Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr

260 265 270TCT CCC CCA ACC Cat CCT CCT CCT GGA CAG TAG TAG CTC CCT 1025SER Pro ThR His Pro ThR GLN TYR Leu Phe Prou PHE PRO

        275                 280                 285CTT CCA CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC CAC CCC CTG CTT      1073Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro Lau Leu

290 295 300CCT GAC CCT TCT GCT CCA ACG CCC ACC CCT ACC AGC CCT CTT CTA AAC As 1121Pro u Asp Pro Ser u Sh Pro T Pro Ala Prohr Thr Pro Thr

305                 310                 315ACA TCC TAC ACC CAC TCC CAG AAT CTG TCT CAG GAA GGG TAAGGTTCTC       1170Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly320                 325                 330AGACACTGCC GACATCAGCA TTGTCTCGTG TACAGCTCCC TTCCCTGCAG GGCGCCCCTG    1230GGAGACAACT GGACAAGATT TCCTACTTTC TCCTGAAACC CAAAGCCCTG GTAAAAGGGA    1290TACACAGGAC TGAAAAGGGA ATCATTTTTC ACTGTACATT ATAAACCTTC AGAAGCTATT    1350TTTTTAAGCT ATCAGCAATA CTCATCAGAG CAGCTAGCTC TTTGGTCTAT TTTCTGCAGA    1410AATTTGCAAC TCACTGATTC TCTACATGCT CTTTTTCTGT GATAACTCTG CAAAGGCCTG    1470GGCTGGCCTG GCAGTTGAAC AGAGGGACAG ACTAACCTTG AGTCAGAAAA CACAGAAAGG    1530GTAATTTCCT TTGCTTCAAA TTCAAGGCCT TCCAACGCCC CCATCCCCTT TACTATCATT    1590CTCAGTGGGA CTCTGATCCC ATATTCTTAA CAGATCTTTA CTCTTGAGAA ATGAATAAGC    1650TTTCTCTCAG AAATGCTGTC CCTATACACT AGACAAAACT GAAAAAAAAA AAAAAAAAAA    1710AAAAAAAAAA A                                                         1721(2)SEQ ID NO：8的资料：

(i) Sequential features:

(A) Length: 4506 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D) Topology: Linear

(ii) Molecular type: Genomic DNA

(vi) Original source:

(A) Creature: Homo Sapiens

(C) Tissue type: peripheral leukocytes

(xi)序列描述：SEQ ID NO：8：GAATTCAGGG CTTTGGCAGT TCCAGGCTGG TCAGCATCTC AAGCCCTCCC CAGCATCTGT      60TCACCCTGCC AGGCAGTCTC TTCCTACAAA CTTGGTTAAA TGTTCACTCT TCTTGCTACT     120TTCAGGATAG ATTCCTCACC CTTGGCCCGC CTTTGCCCCA CCCTACTCTG CCCAGAAGTG     180CAAGAGCCTA AGCCGCCTCC ATGGCCCCAG GAAGGATTCA GGGGAGAGGC CCCAAACAGC     240GAGCCACGCC AGCCAGACAC CCCGGCCAGA ATG GAG CTG ACT  G GTGAGAACAC        293

                                                                                         ,

                             -21 -20ACCTGAGGGG CTAGGGCCAT ATGGAAACAT GACAGAAGGG GAGAGAGAAA GGAGACACGC     353TGCAGGGGGC AGGAAGCTGG GGGAACCCAT TCTCCCAAAA ATAAGGGGTC TGAGGGGTGG     413ATTCCCTGGG TTTCAGGTCT GGGTCCTGAA TGGGAATTCC TGGAATACCA GCTGACAATG     473ATTTCCTCCT CATCTTTCAA CCTCACCTCT CCTCATCTAA G  AA TTG CTC CTC         525

Glu Leu Leu Leu

-15CTG GTC ATG CTC CTC CTA ACT GCA ACG CTA ACG CTG TCC CCG GCG GCG GCG GCG 573VAL MET Leu Leu ThR ALA AREU

-10 -5 1CCT CCT GAC CTC CTC CGA GTC CTC AGT AAA CTT CTT GAC TCC 621pro Pro Ala Cys ASP Leu Serg Val Leu Leu Leu ARG ARG ARG ARG

5                  10                  15CAT GTC CTT CAC AGC AGA CTG GTGAGAACTC CCAACATTAT CCCCTTTATC          672His Val Leu His Ser Arg Leu20                  25CGCGTAACTG GTAAGACACC CATACTCCCA GGAAGACACC ATCACTTCCT CTAACTCCTT     732GACCCAATGA CTATTCTTCC CATATTGTCC CCACCTACTG ATCACACTCT CTGACAAGGA     792TTATTCTTCA CAATACAGCC CGCATTTAAA AGCTCTCGTC TAGAGATAGT ACTCATGGAG     852GACTAGCCTG CTTATTAGGC TACCATAGCT CTCTCTATTT CAGCTCCCTT CTCCCCCCAC     912CAATCTTTTT CAACAG AGC CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA 961

Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr

30 35cct GTC CTG CTG CCT GCT GCT GAC TTT AGC TTG GGA GAA TGG AAA ACC 1009Pro Val Leu Pro Ala Val ASR Leu GLY GLU TRP LYS Thr

40 45 50CAG ATG GTAAGAAAGC CATCCCTAAC CTTGGCTTCC CTAAGTCCTG TCTTCAGTTT 1065Gln Met

55CCCACTGCTT CCCATGGATT CTCCAACATT CTTGAGCTTT TTAAAAATAT CTCACCTTCA    1125GCTTGGCCAC CCTAACCCAA TCTACATTCA CCTATGATGA TAGCCTGTGG ATAAGATGAT    1185GGCTTGCAGG TCCAATATGT GAATAGATTT GAAGCTGAAC ACCATGAAAA GCTGGAGAGA    1245AATCGCTCAT GGCCATGCCT TTGACCTATT CCTGTTCAGT CTTCTTAAAT TGGCATGAAG    1305AAGCAAGACT CATATGTCAT CCACAGATGA CACAAAGCTG GGAAGTACCA CTAAAATAAC    1365AAAAGACTGA ATCAAGATTC AAATCACTGA AAGACTAGGT CAAAAACAAG GTGAAACAAC    1425AGAGATATAA ACTTCTACAT GTGGGCCGGG GGCTCACGCC TGTAATCCCA GCACTTTGGG    1485AGGCCGAGGC AGGCAGATCA CCTGAGGGCA GGAGTTTGAG AGCAGCCTGG CCAACATGGC    1545GAAACCCCGT CTCTACTAAG AATACAAAAT TAGCCGGGCA TGGTAGTGCA TGCCTGTAAT    1605CCCAGCTACT TGGAAGGCTG AAGCAGGAGA ATCCCTTGAA CCCAGGAGGT GGAGGTTGTA    1665GTGAGCTGAG ATCATGCCAA TGCACTCCAG CCTGGGTGAC AAGAGCAAAA CTCCGTCTCA    1725AAAAGAAAAA AAAATTCTAC ATGTGTAAAT TAATGAGTAA AGTCCTATTC CAGCTTTCAG    1785GCCACAATGC CCTGCTTCCA TCATTTAAGC CTCTGGCCCT AGCACTTCCT ACGAAAAGGA    1845TCTGAGAGAA TTAAATTGCC CCCAAACTTA CCATGTAACA TTACTGAAGC TGCTATTCTT    1905AAAGCTAGTA ATTCTTGTCT GTTTGATGTT TAGCATCCCC ATTGTGGAAA TGCTCGTACA    1965GAACTCTATT CCGAGTGGAC TACACTTAAA TATACTGGCC TGAACACCGG ACATCCCCCT    2025GAAGACATAT GCTAATTTAT TAAGAGGGAC CATATTAAAC TAACATGTGT CTAGAAAGCA    2085GCAGCCTGAA CAGAAAGAGA CTAGAAGCAT GTTTTATGGG CAATAGTTTA AAAAACTAAA    2145ATCTATCCTC AAGAACCCTA GCGTCCCTTC TTCCTTCAGG ACTGAGTCAG GGAAGAAGGG    2205CAGTTCCTAT GGGTCCCTTC TAGTCCTTTC TTTTCATCCT TATGATCATT ATGGTAGAGT    2265CTCATACCTA CATTTAGTTT ATTTATTATT ATTATTTGAG ACGGAGTCTC ACTGTATCCC    2325CCAGGCTGGA GTGCAGTGGC ATGATCTCAA CTCACTGCAA CCTCAGCCTC CCGGATTCAA    2385GCGATTCTCC TGCCTCAGTC TCCCAAGTAG CTGGGATTAC AGGTGCCCAC CGCCATGCCC    2445AGCTAATTTT TGTATTTTTG GTAGAGATGG GGTTTCACCA TGTTGGCCAG GCTGATCTTG    2505AACTCCTGAC CTCAGGTGAT CCACCTGCCT CAGCCTCCCA AAGTGCTGGG ATTACAGGCG 2565TGAGCCACTG CACCCAGCCT TCATTCAGTT TAAAAATCAA ATGATCCTAA GGTTTTGCAG    2625CAGAAAGAGT AAATTTGCAG CACTAGAACC AAGAGGTAAA AGCTGTAACA GGGCAGATTT    2685CAGCAACGTA AGAAAAAAGG AGCTCTTCTC ACTGAAACCA AGTGTAAGAC CAGCCTGGAC    2745TAGAGGACAC GGGAGTTTTT GAAGCAGAGG CTGATGACCA GCTGTCGGGA GACTGTGAAG    2805GAATTCCTGC CCTGGGTGGG ACCTTGGTCC TGTCCAGTTC TCAGCCTGTA TGATTCACTC    2865TGCTGGCTAC TCCTAAGGCT CCCCACCCGC TTTTAGTGTG CCCTTTGAGG CAGTGCGCTT    2925CTCTCTTCCA TCTCTTTCTC AG GAG GAG ACC AAG GCA CAG GAC ATT CTG GGA     2977

Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly

60 65GCA GTG ACC CTT CTG CTG GAG GAG GGA GCA GCA CGG GGG GGA CAA CAA CTG 3025ALA Val THR Leu Leu GLU GLY Val Met Ala Ala ARG GLN Leu

70 75 80GGA CCC ACT TGC CTC CTC CTC CTC CTG GGG CAG CAG CTT GGA CAG GTC 3073G1y Pro Thr Cys Leu Leu Leu GLN Leu Seru Gln Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val

85 90 95CGT CTC CTC CTT GCC CTG CTG AGC CTC CTT GGA ACC CAG 3115arg Leu Leu Leu Gln Seru Leu Leu GLY THR GLN

100                 105                 110GTAAGTCCCC AGTCAAGGGA TCTGTAGAAA CTGTTCTTTT CTGACTCAGT CCCCCTAGAA    3175GACCTGAGGG AAGAAGGGCT CTTCCAGGGA GCTCAAGGGC AGAAGAGCTG ATCTACTAAG    3225AGTGCTCCCT GCCAGCCACA ATGCCTGGGT ACTGGCATCC TGTCTTTCCT ACTTAGACAA    3295GGGAGGCCTG AGATCTGGCC CTGGTGTTTG GCCTCAGGAC CATCCTCTGC CCTCAG        3351CTT CCT CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT CCC AAT GCC ATC      3399Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile

115 120 125TTC CTG AGC TTC CAA CAA CAC CTC CTC CGA GGA AAG GTG CGT TTC CTG ATG 3447phe Leu Seru Leu Leu ARG GLY LY ARS VE Leuu Leu's Le

    130                 135                 140CTT GTA GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC CCA CCC ACC ACA      3495Leu Val Gly G1y Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr

145                 150                 155GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG AAC GAG CTC CCA      3543Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro160                 165                 170                 175AAC AGG ACT TCT GGA TTG TTG GAG ACA AAC TTC ACT GCC TCA GCC AGA 3591Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg

180 185 190ACT ACT GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA TTC AGA GCC AAG 3639thr THR GLY Leu LEU LYS TRN GLN GLN ARALALALLALALN's

195 200 205ATT CCT GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG GAC CAA ATC CCC 3687ile Pro GLY Leu Leu Asn Gln Thr Leu ASP Gln Ile Pro Pro

210 215 220GGA TAC CTG AGG AGG AGG AGG AGG AGG GAA CTC TTG AAT GGA Act CGT GGT GGA CTC 3735GLY TYR Leu Asn

225                 230                 235TTT CCT GGA CCC TCA CGC AGG ACC CTA GGA GCC CCG GAC ATT TCC TCA      3783Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser240                 245                 250                 255GGA ACA TCA GAC ACA GGC TCC CTG CCA CCC AAC CTC CAG CCT GGA TAT 3831Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr

260 265 270TCT CCC CCA ACC Cat CCT CCT GGA CAG TAG TAG CTC TTC CCT 3879SER Pro ThR His Pro ThR Gln Tyr Thr Leu Phe Pro

275 280 285ctt CCA CCC ACC TTG CCC CCC CCT GTG GTC CAG CCC CCC CCC CTG CTG 3927Leu Pro THR Leu Pro THR Val Val Val Val Gln Leu His Pro

290 295 300CCT GAC CCT TCT CCT CCA ACG CCC ACC CCT ACC AGC CCT CTT CTA AAC As 3975Pro Le u Asp Pro Ser u Sh Pro T Ala Prohr Thr Pro Thr

305                 310                 315ACA TCC TAC ACC CAC TCC CAG AAT CTG TCT CAG GAA GGG TAAGGTTCTC       4024Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly320                 325                 330AGACACTGCC GACATCAGCA TTGTCTCATG TACAGCTCCC TTCCCTGCAC GGCGCCCCTG    4084GGAGACAACT GGACAAGATT TCCTACTTTC TCCTGAAACC CAAAGCCCTG GTAAAAGGGA    4144TACACAGGAC TGAAAAGGGA ATCATTTTTC ACTGTACATT ATAAACCTTC AGAAGCTATT    4204TTTTTAAGCT ATCAGCAATA CTCATCAGAG CACCTAGCTC TTTGGTCTAT TTTCTGCACA    4264AATTTGCAAC TCACTGATTC TCTACATGCT CTTTTTCTGT GATAACTCTG CAAAGGCCTG    4324GGCTGGCCTG GCAGTTGAAC AGAGGGAGAG ACTAACCTTG AGTCAGAAAA CAGAGAAAGG    4384GTAATTTCCT TTGCTTCAAA TTCAAGGCCT TCCAACGCCC CCATCCCCTT TACTATCATT    4444CTCAGTGGGA CTCTGATCCC ATATTCTTAA CAGATCTTTA CTCTTGAGAA ATGAATAAGC    4504TT                                                                   4506(2)SEQ ID NO：9的资料：

(i) Sequential features:

(A) Length: 416 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D) Topology: Linear

(ii) Molecular type: Synthetic DNA

(xi)序列描述：SEQ ID NO：9：CTAGAAAAAA CCAAGCAGGT AATAAATAAT GAGCCCGGCT CCGCCAGCTT GTGACCTTCG      60TGTTCTGTCT AAACTGCTTC GCGACTCTCA CGTGCTGCAC TCTCGTCTGT CCCAGTGCCC     120GGAAGTTCAC CCGCTGCCGA CCCCGGTTCT GCTTCCGGCT GTCGACTTCT CCCTGGGTGA     180ATGGAAAACC CAGATGGAAG AGACCAAAGC TCAGGACATC CTGGGTGCAG TAACTCTGCT     240TCTGGAAGGC GTTATGGCTG CACGTGGCCA GCTTGGCCCG ACCTGCCTGT CTTCCCTGCT     300TGGCCAGCTG TCTGGCCAGG TTCGTCTGCT GCTCGGCGCT CTGCAGTCTC TGCTTGGCAC     350CCAGCTGCCG CCACAGGGCC GTACCACTGC TCACAAGGAT CCGAACGCTA TCTTCC 416(2) Information of SEQ ID NO: 10:

(i) Sequential features:

(A) Length: 179 amino acids

(B) type: amino acid

(D) Topology: linear

(ii) Molecule type: protein

(XI) Sequence description: SEQ ID NO: 10: Leu Val Pro ARG GLY Serg Gly Ser,

15 20 20 25Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala

30 35 40Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys

45 50 55ALA GLN ASP ILE Leu Gly Ala Val Thr Leu Leu GLU GLU GLY VAL MET60 70ALA Ala ARG GLN Leu GLY Pro Thr Cyser Leu Leu Gly Gly

80 85 90Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu

95 100 105Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp

110 115 120Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val

125 135arg PHE Leu Met Leu Val Gly GLY GLY SER Leu Cys Val ARG ALA140 145 150 155PRO THR THR ALA Val Pro Serg THR Leu Val Leu Thr Leu

160 165 170Asn Glu Leu (2) SEQ ID NO: 11 information:

(i) Sequential features:

(A) Length: 535 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D) Topology: linear

(ii) Molecular type: Synthetic DNA

(vi) Original source:

(A) Creature: Homo Sapieng

(xi) Sequence description: SEQ ID NO: 11: CTAGAAAAAA CCAAGGAGGT AATAAATA ATG AAA GCA CCT GTA CCA CCT GCA 52

                                                                      , 

-2 1 5TGT GAT TTA CTC CTG TCT AAA CTG CTG CTG CGC GAC TCT CAC GTG 100CYS ASP Leu ARG Val Leu Leu Leu ARG ARG ARG ARS Val Val Leu

10 15 20CAC TCT CGT CGT CGT CGC TCC CAG TCC CCG GAA GAA GTT CCG CCG CCG ACC CCG 148HIS Serg Leu Serg

25 30 35GTT CTG CTG CCG GCT GCT GTC CAC CAC TCC CTG GAA TGG AAA ACC CAG 195VAL Leu Pro Ala Val ASP PHE Seru GLY GLU TRS Thr Gln

40                  45                  50ATG GAA GAG ACC AAA GCT CAG GAC ATC CTG GGT GCA GTA ACT CTG CTT       244Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu55                  60                  65                  70CTG GAA GGC GTT ATG GCT GCA CGT GGC CAG CTT GGC CCG ACC TGC CTG 292Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu

75 80 85TCT TCC CTG CTT GGC CAG CGC CGC CGC CGC CGT CGT CTG CTG CTC GGC 340SER Leu Leu GLN Leu Serg Leu Leu Leu Leu Gly Gly

90 95 100GCT CTG CAG TCT CTG CTT GCC ACC CAG CCG CCG CCA CAG GGC CCT ACC 388ala Leu Leu Leu GLY THR Gln Leu Gln GLN GLY ARG THR

105 110 115act GCT CAT CAT CCG AAC GCT ATC TTC CTG TCT TTC CAG CAC CAC CAC CAC CAC CAC CAC 436thr His LYS ASN ALA ILE PHE Leu Seru Gln His Leu

120                 125                 130CTG CGT GGC AAA GTT CGT TTC CTG ATG CTG GTT GGC GGT TCT ACC CTG       484Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu135                 140                 145                 150TGC CTT CGT CGG GCG CCG CCA ACC ACT GCT GTT CCG TCT TAATGAAAGC 533Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser

155 160TT 535(2) Information of SEQ ID NO: 12:

(i) Sequential features:

(A) Length: 535 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D) Topology: Linear

(ii) Molecular type: Synthetic DNA

(vi) Original source:

(A) Creature: Homo Sapieng

(xi) Sequence description: SEQ ID NO: 12: CTAGAAAAAA CCAAGGAGGT AATAAATA ATC AAA TCT CCT GCA CCA CCT GCA 52

                                                                              , 

-2 1 5TGT GAT TTA CTC CTG TCT AAA CTG CTG CTG CGC GAC TCT CAC GTG 100CYS ASP Leu ARG Val Leu Leu Leu ARG ARG ARG ARS Val Val Leu

10 15 20CAC TCT CGT CGT CGT CGC CAG TGC CCG GAA GAA GAA GTT CCG CCG CCG ACC CCG 148HIS Serg Leu Serg Leu Val His Pro Leu Prou THR PRO

25 30 35GTT CTG CTG CCG GCT GCT GAC GAC GAC TCC CTG GAA TGG AAA ACC CAG 196 Val Leu Pro Ala Val ASP PHE Seru GLY GLU TRS THR GLN

40                  45                  50ATG GAA GAG ACC AAA GCT CAG GAC ATC CTG GGT GCA GTA ACT CTG CTT       244Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu55                  60                  65                  70CTG GAA GGC GTT ATG GCT GCA CGT GGC CAG CTT GGC CCG ACC TGC CTG 292Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu

75 80 85TCT TCC CTG CTT GGC CAG CAG CTG TCT GGC CAG GTT CGT CTG CTG CTG CTC GGC 340SER Leu Leu Gln Leu Serg Leu Leu Leu Gly Gly

90 95 100GCT CTG CAG TCT CTG CTG CTT GGC ACC CAG CCG CCG CAG GGC CGT ACC 388Ala Leu Gln Seru Leu GLY THR GLN GLN GLN GLN GLN GLN's G

105 110 115act GCT CAT CAT CCG AAC GCT ATC TTC CTG TCT TTC CAG CAC CAC CAC CAC CAC CAC CAC 436thr His LYS ASN ALA ILE PHE Leu Seru Gln His Leu

120                 125                 130CTG CGT GGC AAA GTT CGT TTC CTG ATG CTG GTT GGC GGT TCT ACC CTG       484Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu135                 140                 145                 150TGC GTT CGT CGG GCG CCG CCA ACC ACT GCT GTT CCG TCT TAATGAAAGC 533Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser

155 160TT 535(2) Information of SEQ ID NO: 13:

(i) Sequential features:

(A) Length: 555 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D) Topology: linear

(ii) Molecular type: cDNA to mRNA

(vi) Original source:

(A) Creature: Homo Sapiens

(F) Tissue type: Liver

(xi) Sequence description: SEQ ID NO: 13: ATG GAG CTC Act Gaa TTG CTC CTC GTC ATG CTT CTC CTA Act GCA 48MET GCA 48MET GCA 48MET GLU Leu Leu Leu Leu Leu Thr Ala-21 -20 -20 -20 -10agG CTA ACG CCC CCG GCG GCT CCT CCT GCT GAC CTC CGA GTC 96ARG Leu Thr Leu Pro Ala Ala Cys ARG Val Val Val-5 10CTC CTGCCCCCCCCCC Cat GAC Cat GAC Cat GAC Cat GAC Cat GAC Cat AGA CTG AGC 144Leu Ser Lys Leu Leu Arg Asp Sar His Val Leu His Ser Arg Leu Ser

15 20 25CAG TGC CCA GAG GAG GTT CAC CCT CCT ACA CCT GTC CTG CTG CCT GCT 192GLN CYS Pro Glu Val His Pro THR Pro Val Leu Pro Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

30 35 40GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC CAG GAG GAG GAG GAG GAG GAG GAG GAG GAG 240val ASP PHE Seru GLY GLY GLY GLN MET GLN GLU Thr Lys

45                  50                  55GCA CAG GAC ATT CTG GGA GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG       288Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met60                  65                  70                  75GCA GCA CGG GGA CAA CTG GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG 336Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly

80 85 90CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT GCC CTG CAG CAG CAG CTC 384GLN Leu Serg Leu Leu Leu GLY Ala Leu Gln Seruu glu glue Glu Leu's Le leu Leu Leu Leu Leu Leu's Le leu Leu Leu Leu's

95 1005CTT GGA ACC CAG CAG CTT CCA CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT 432leu GLY THR Gln Leu Pro GLN GLY ARA His Lys Lys Lys ASP

110 115 120CCC AAT GCC ATC TTC CTG AGC TTC CAA CAA CAC CTC CGA GGA AAG GTG 480PRO Asn

125                 130                 135CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC       528Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala140                 145                 150                 155CCA CCC ACC ACA GCT GTC CCC AGC TGA                                   555Pro Pro Thr Thr Ala Val Pro Ser

                         Information for 160(2) SEQ ID NO: 14:

(i) Sequential features:

(A) Length: 1043 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D) Topology: linear

(ii) Molecular type: Synthetic DNA

(vi) Original source:

(A) Creature: Homo Sapiens

(xi) Sequence description: SEQ ID NO: 14: CTAGAAAAAA CCAAGGAGGT AATAAATA ATG AAA AGT CCT GCA CCA CCT GCA 52

                                                                              , 

-2 1 5TGT GAT TTA CTC CTG TCT AAA CTG CTG CTG CGC GAC TCT CAC GTG 100CYS ASP Leu ARG Val Leu Leu Leu ARG ARG ARG ARS Val Val Leu

10 15 20CAC TCT CGT CGT CGT CGG TGG CCG GAA GAA GAA GTT CCG CCG CCG ACC CCG 148his Serg Leu Serg Leu Val His Pro Leu Pro THR PRO

25 30 35GTT CTG CTG CCG GCT GCT GAC GAC GAC TCC CTG GAA TGG AAA ACC CAG 196 Val Leu Pro Ala Val ASP PHE Seru GLY GLU TRS THR GLN

40                  45                  50ATG GAA GAG ACC AAA GCT CAG GAC ATC CTG GGT GCA GTA ACT CTG CTT       244Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu55                  60                  65                  70CTG GAA GGC GTT ATG GCT GCA CGT GGC CAG CTT GCC CCG ACC TCC CTG 292Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu

75 80 85TCT TCC CTG CTT GGC CAG CAG CTG TCT GGC CAG GTT CGT CTG CTG CTC GCC 340SER Leu Leu Gln Leu Serg Leu Leu Leu Gly Gly

90 95 100GCT CTG CAG TCT CTG CTG CTT GGC ACC CAG CCG CCG CAG GGC CGT ACC 388Ala Leu Gln Seru Leu GLY THR GLN GLN GLN GLN GLN GLN's G

105 110 115ACT GCT CAT CAT CCG AAC GCT ATC TTC CTG TCT TTC CAG CAC CAC CAC CAC CAC 436thr His LYS ASN ALA ILE PHE Leu CLN His Leu

120                 125                 130CTG CGT GGC AAA GTT CGT TTC CTG ATG CTG GTT GGC GGT TCT ACC CTG       484Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu135                 140                 145                 150TGC GTT CGT CGG GCG CCG CCA ACC ACT GCT GTT CCG TCT CGT ACC TCT 532Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser

155 160 165CTG GTT CTG ACC CTG AAC GAG CTC CCG AAC CGT AGC GGC CTG 580leu Val Leu Thr Leu Pro ARG THR Serg Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu's's's

170 175 180GAA ACC ACC GCG AGC GCG CGT ACC GGC GGC GGC CTG CTG 628GLU Thr Ala Serg THR THR GLY Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu leu

185 195AAA TGG CAG CAG GGC TTT CGT GCG AAA AAA AAA ATC CCG GGC CTG CTG AAC CAG 676LYS TRN GLN GLN GLY PHE ALA LY Leu Leu Asn Gln Glu Asn Leu Leu's Gue

200                 206                 210ACC AGC CGT AGC CTG GAT CAG ATC CCG GGC TAT CTG AAC CGT ATC CAT       724Thr Ser Arg Ser Leu Aso Gln Ile Pro Gly Tyr Leu Asn Arg Ile His215                 220                 225                 230GAA CTG CTG AAC GGC ACC CGT GGC CTG TTT CCG GGC CCG AGC CGT CGC 772Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg

235 240 245acc CCG GCG CCG CCG GAT AGC TCT GGC AGC GGC GGC GGC AGC 820thr Leu GLY ALA PRO Aser Ser Ser Ser Ser Gly Ser

250 255 260CTG CCG CCG AAC CTG CAG CAG CCG GGC TAT AGC CCG AGC CCG ACC Cat CCG 858Le

265 270 275ccg ACC GGC CAG TAG TAT ACC CTT CCG CCG CCG CCG CCG CTG CCG ACC 916Pro ThR Gln Tyr Leu Pro Pro THR Leu Prou THR THR Leu THR Thr Leu's Pro that THR -PHR that thesia

280                 295                 290CCG GTG GTT CAG CTG CAT CCG CTG CTG CCG GAT CCG AGC GCG CCG ACC       964Pro Val Val Gln Leu His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr295                 300                 305                 310CCG ACC CCG ACC AGC CCG CTG CTG AAC ACC AGC TAT ACC CAT AGC CAG 1012Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gln

                                                                                                                                                  

330

(2) Information of SEQ ID NO: 15:

(i) Sequence features:

(A) Length: 45 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 15: AACTGGAAGA ATTCGCGGCC GCAGGAATTT TTTTTTTTTT TTTTT 45

(2) Information of SEQ ID NO: 16:

(i) Sequence features:

(A) Length: 13 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 16: AATTCGGCAC GAG 13

(2) Information of SEQ ID NO: 17:

(i) Sequence features:

(A) Length: 9 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 17: CTCGTGCCG 9

(2) Information of SEQ ID NO: 18:

(i) Sequence features:

(A) Length: 12 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: Linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 18: Ile pro Val Pro Pro Ala Cys Asp Pro Arg Leu Leu1 5 10

(2) Information of SEQ ID NO: 19:

(i) Sequence features:

(A) Length: 12 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: Linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 19: Thr Pro Val Pro Pro Ala Cys Asp Pro Arg Leu Leu1 5 10

(2) Information of SEQ ID NO: 20:

(i) Sequence features:

(A) Length: 12 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: Linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 20: Ser Pro Val Pro Pro Ala Cys Asp Pro Arg Leu Leu1 5 10

(2) Information of SEQ ID NO: 21:

(i) Sequence features:

(A) Length: 17 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(ix) Features:

(A) Name/keyword: modified-base

(B) Position: 12&15

(D) Other information: /modified_base=i

(xi) Sequence description: SEQ ID NO: 21: ACGCTGGGGG CNGANGA 17

(2) Information of SEQ ID NO: 22:

(i) Sequence features:

(A) Length: 17 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(ix) Features:

(A) Name/keyword: modified_base

(B) Position: 12&15

(D) Other information: /modified_base=i

(xi) Sequence description: SEQ ID NO: 22: ACACTAGGAT CNAANAA 17

(2) Information of SEQ ID NO: 23:

(i) Sequence features:

(A) Length: 17 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(ix) Features:

(A) Name/keyword: modified_base

(B) Position: 12&15

(D) Other information: /modified_base=i

(xi) Sequence description: SEQ ID NO: 23: ACGCTGGGTG CNGANGA 17

(2) Information of SEQ ID NO: 24:

(i) Sequence features:

(A) Length: 17 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(ix) Features:

(A) Name/keyword: modified_base

(B) Position: 12&15

(D) Other information: /modified_base=i

(xi) Sequence description: SEQ ID NO: 24: ACGCTGGGCG CNGANGA 17

(2) Information of SEQ ID NO: 25:

(i) Sequence features:

(A) Length: 17 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 25: GGGGGGCGGA CGCTGGG 17

(2) Information of SEQ ID NO: 26:

(i) Sequence features:

(A) Length: 17 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 26: GGAGGACGAA CACTAGG 17

(2) Information of SEQ ID NO: 27:

(i) Sequence features:

(A) Length: 17 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 27: GGTGGTCGTA CGCTGGG 17

(2) Information of SEQ ID NO: 28:

(i) Sequence features:

(A) Length: 17 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 28: GGCGGCCGCA CGCTGGG 17

(2) Information of SEQ ID NO: 29:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 29: GGATCTTGGT TCATTCTCAA G 21

(2) Information of SEQ ID NO: 30:

(i) Sequence features:

(A) Length: 20 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 30: CCTCAGACAG TGGTTCAAAG 20

(2) Information of SEQ ID NO: 31:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 31: CGAGGGTGTA CCTGGGTCCT G 21

(2) Information of SEQ ID NO: 32:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 32: CAGAGTTAGT CTTGCGGTGA G 21

(2) Information of SEQ ID NO: 33:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 33: CCTGTCCTGC TGCCTGCTGT G 21

(2) Information of SEQ ID NO: 34:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 34: TGAAGTTCGT CTCCAACAAT C 21

(2) Information of SEQ ID NO: 35:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 35: ATGGAGCTGA CTGATTTGCT C 21

(2) Information of SEQ ID NO: 36:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 36: TGAAGTTCGT CTCCAACAAT C 21

(2) Information of SEQ ID NO: 37:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 37: TTGTGACCTC CGAGTCCTCA G 21

(2) Information of SEQ ID NO: 38:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 38: TGACGCAGAG GGTGGACCCT C 21

(2) Information of SEQ ID NO: 39:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 39: AGCAGAACCT CTCTAGTCCT C 21

(2) Information of SEQ ID NO: 40:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 40: ACACTGAACG AGCTCCCAAA C 21

(2) Information of SEQ ID NO: 41:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 41: AACTACTGGC TCTGGGCTTC T 21

(2) Information of SEQ ID NO: 42:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 42: AGGGATTCAG AGCCAAGATT C 21

(2) Information of SEQ ID NO: 43:

(i) Sequence features:

(A) Length: 31 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 43: TAGCGGCCGC TTTTTTTTTT TTTTTTTGGG G 31

(2) Information of SEQ ID NO: 44:

(i) Sequence features:

(A) Length: 31 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 44: TAGCGGCCGC TTTTTTTTTT TTTTTTTAAA A 31

(2) Information of SEQ ID NO: 45:

(i) Sequence features:

(A) Length: 31 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 45: TAGCGGCCGC TTTTTTTTTT TTTTTTTCTT T 31

(2) Information of SEQ ID NO: 46:

(i) Sequence features:

(A) Length: 31 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 46: TAGCGGCGC TTTTTTTTTT TTTTTTTGCC C 31

(2) Information of SEQ ID NO: 47:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 47: TAGCGGCCGC TTTTTTTTTT T 21

(2) Information of SEQ ID NO: 48:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 48: TTGTGACCTC CGAGTCCTCA G 21

(2) Information of SEQ ID NO: 49:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 49: AGGATGGGTT GGGGAAGGAG A 21

(2) Information of SEQ ID NO: 50:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 50: TTGTGACCTC CGAGTCCTCA G 21

(2) Information of SEQ ID NO: 51:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 51: AGGGAAGAGC GTATACTGTC C 21

(2) Information of SEQ ID NO: 52:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 52: GGCCAGCCAG ACACCCCGGC C 21

(2) Information of SEQ ID NO: 53:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 53: ATGGGAGTCA CGAAGCAGTT T 21

(2) Information of SEQ ID NO: 54:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 54: TGCGTTTCCT GATGCTTGTA G 21

(2) Information of SEQ ID NO: 55:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 55: AACCTTACCC TTCCTGAGAC A 21

(2) Information of SEQ ID NO: 56:

(i) Sequence features:

(A) Length: 30 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 56: TTTGAATTCG GCCAGCCAGA CACCCCGGCC 30

(2) Information of SEQ ID NO: 57:

(i) Sequence features:

(A) Length: 39 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 57: TTTGCGGCCG CTCATTAGCT GGGGACAGCT GTGGTGGGT 39

(2) Information of SEQ ID NO: 58:

(i) Sequence features:

(A) Length: 42 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 58: TTTGCGGCCG CTCATTACAG TGTGAGGACT AGAGAGGTTC TG 42

(2) Information of SEQ ID NO: 59:

(i) Sequence features:

(A) Length: 42 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 59: TTTGCGGCCG CTCATTATCT GGCTGAGGCA GTGAAGTTTG TC 42

(2) Information of SEQ ID NO: 60:

(i) Sequence features:

(A) Length: 42 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 60: TTTGCGGCCG CTCATTACAG ACCAGGAATC TTGGCTCTGAA T 42

(2) Information of SEQ ID NO: 61:

(i) Sequence features:

(A) Length: 30 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 61: TTTGAATTCG GCCAGCCAGA CACCCCGGCC 30

(2) Information of SEQ ID NO: 62:

(i) Sequence features:

(A) Length: 41 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 62: TTTGCGGCCG CTCATTAGAG GGTGGACCCT CCTACAAGCA T 41

(2) Information of SEQ ID NO: 63:

(i) Sequence features:

(A) Length: 40 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 63: TTTGCGGCCG CTCATTAGCA GAGGGTGGAC CCTCCTACAA 40

(2) Information of SEQ ID NO: 64:

(i) Sequence features:

(A) Length: 38 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 64: TTTGCGGCCG CTCATTACCT GACGCAGAGG GTGGACCC 38

(2) Information of SEQ ID NO: 65:

(i) Sequence features:

(A) Length: 38 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 65: TTTGCGGCCG CTCATTACCG CCTGACGCAG AGGGTGGA 38

(2) Information of SEQ ID NO: 66:

(i) Sequence features:

(A) Length: 38 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 66: TTTGCGGCCG CTCATTAGGC CCGCCTGACG CAGAGGGT 38

(2) Information of SEQ ID NO: 67:

(i) Sequence features:

(A) Length: 38 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 67: TTTGCGGCCG CTCATTATGG GGCCCGCCTG ACGCAGAG 38

(2) Information of SEQ ID NO: 68:

(i) Sequence features:

(A) Length: 38 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 68: TTTGCGGCCG CTCATTAGGG TGGGGCCCGC CTGACGCA 38

(2) Information of SEQ ID NO: 69:

(i) Sequence features:

(A) Length: 30 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 69: TTTGAATTCG GCCAGCCAGA CACCCCGGCC 30

(2) Information of SEQ ID NO: 70:

(i) Sequence features:

(A) Length: 41 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 70: TTTGCGGCCG CTCATTATTC GTGTATCCTG TTCAGGTATC C 41

(2) Information of SEQ ID NO: 71:

(i) Sequence features:

(A) Length: 39 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 71: TTTGCGGCCG CTCATTAGCT GGGGACAGCT GTGGTGGGT 39

(2) Information of SEQ ID NO: 72:

(i) Sequence features:

(A) Length: 50 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 72: AGTAAACTGC TTCGTGACTC CCATGTCCTT CACAGCAGAC TGAGCCAGTG 50

(2) Information of SEQ ID NO: 73:

(i) Sequence features:

(A) Length: 49 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 73: CATGGGAGTC ACGAAGCAGT TTACTGGACA GCGTTAGCCT TGCAGTTAG 49

(2) Information of SEQ ID NO: 74:

(i) Sequence features:

(A) Length: 49 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 74: TGTGACCTCC GAGTCCTCAG TAAACTGCTT CGTGACTCCC ATGTCCTTC 49

(2) Information of SEQ ID NO: 75:

(i) Sequence features:

(A) Length: 48 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 75: TTTACTGAGG ACTCGGAGGT CACAGGACAG CGTTAGCCTT GCAGTTAC 48

(2) Information of SEQ ID NO: 76:

(i) Sequence features:

(A) Length: 49 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 76: GACCTCCGAG TCCTCAGTAA ACTGCTTCGT GACTCCCATG TCCTTCACA 49

(2) Information of SEQ ID NO: 77:

(i) Sequence features:

(A) Length: 48 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 77: CAGTTTACTG AGGACTCGGA GGTCGGACAG CGTTAGCCTT GCAGTTAG 48

(2) Information of SEQ ID NO: 78:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 78: TTGTGACCTC CGAGTCCTCA G 21

(2) Information of SEQ ID NO: 79:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 79: CAGGTATCCG GGGATTTGGT C 21

(2) Information of SEQ ID NO: 80:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 80: TGCGTTTCCT GATGCTTGTA G 21

(2) Information of SEQ ID NO: 81:

(i) Sequence features:

(A) Length: 35 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 81: GAGAGAGCGG CCGCTTACCC TTCCTGAGAC AGATT 35

(2) Information of SEQ ID NO: 82:

(i) Sequence features:

(A) Length: 6 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 82: GAGAGA 6

(2) Information of SEQ ID NO: 83:

(i) Sequence features:

(A) Length: 70 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence Description: SEQ ID NO: 83: CTAGAAAAAA CCAAGGAGGT AATAAATAAT GAGCCCGGCT CCGCCAGCTT GTGACCTTCG 60TGTTCTGTCT                                        

(2) Information of SEQ ID NO: 84:

(i) Sequence features:

(A) Length: 72 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence Description: SEQ ID NO: 84: CAGTTTAGAC AGAACACGAA GGTCACAAGC TGGCGGAGCC GGGCTCATTA TTTATTACCT 60CCTTGGTTTT TT                                        

(2) Information of SEQ ID NO: 85:

(i) Sequence features:

(A) Length: 69 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence Description: SEQ ID NO: 85: AAACTGCTTC GCGACTCTCA CGTGCTGCAC TCTCGTCTGT CCCAGTGCCC GGAAGTTCAC 60CCGCTGCCG                                

(2) Information of SEQ ID NO: 86:

(i) Sequence features:

(A) Length: 69 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 86: CGGGGTCGGC AGCGGGTGAA CTTCCGGGCA CTGGGACAGA CGAGAGTGCA GCACGTGAGA 60GTCGCGAAG 9

(2) Information of SEQ ID NO: 87:

(i) Sequence features:

(A) Length: 69 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 87: ACCCCGGTTC TGCTTCCGGC TGTCGACTTC TCCCTGGGTG AATGGAAAAC CCAGATGGAA 60GAGACCAAA 9 6

(2) Information of SEQ ID NO: 88:

(i) Sequence features:

(A) Length: 69 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 88: CTGAGCTTTG GTCTCTTCCA TCTGGGTTTT CCATTCACCC AGGGAGAAGT CGACAGCCGG 60AAGCAGAAC 9 6

(2) Information of SEQ ID NO: 89:

(i) Sequence features:

(A) Length: 69 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 89: GCTCAGGACA TCCTGGGTGC AGTAACTCTG CTTCTGGAAG GCGTTATGGC TGCACGTGGC 60CAGCTTGGC 9 6

(2) Information of SEQ ID NO: 90:

(i) Sequence features:

(A) Length: 69 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 90: GGTCGGGCCA AGCTGGCCAC GTGCAGCCAT AACGCCTTCC AGAAGCAGAG TTACTGCACC 60CAGGATGTC                            

(2) Information of SEQ ID NO: 91:

(i) Sequence features:

(A) Length: 69 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 91: CCGACCTGCC TGTCTTCCCT GCTTGGCCAG CTGTCTGGCC AGGTTCGTCT GCTGCTCGGC 60GCTCTGCAG 9 6

(2) Information of SEQ ID NO: 92:

(i) Sequence features:

(A) Length: 66 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 92: CAGAGACTGC AGAGCGCCGA GCAGACGAAC CTGGCCAGAC AGCTGGCCAA GCAGGGAAGA 60CAGGCA 6 6

(2) Information of SEQ ID NO: 93:

(i) Sequence features:

(A) Length: 70 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 93: TCTCTGCTTG GCACCCAGCT GCCGCCACAG GGCCGTACCA CTGCTCACAA GGATCCGAAC 60GCTATCTTCC 0 7

(2) Information of SEQ ID NO: 94:

(i) Sequence features:

(A) Length: 70 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence Description: SEQ ID NO: 94: AAGACAGGAA GATAGCGTTC GGATCCTTGT GAGCAGTGGT ACGGCCCTGT GGCGGCAGCT 60GGGTGCCAAG                                                

(2) Information of SEQ ID NO: 95:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 95: GGAGGAGACC AAGGCACAGG A 21

(2) Information of SEQ ID NO: 96:

(i) Sequence features:

(A) Length: 30 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 96: CCGGAATTCT TACCCTTCCT GAGACAGATT 30

(2) Information of SEQ ID NO: 97:

(i) Sequence features:

(A) Length: 8 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 97: CTAATGAG 8

(2) Information of SEQ ID NO: 98:

(i) Sequence features:

(A) Length: 16 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 98: AATTCTCATT AGAGCT 16

(2) Information of SEQ ID NO: 99:

(i) Sequence features:

(A) Length: 8 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 99: CTAATGAG 8

(2) Information of SEQ ID NO: 100:

(i) Sequence features:

(A) Length: 16 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 100: AATTCTCATT AGAGCT 16

(2) Information of SEQ ID NO: 101:

(i) Sequence features:

(A) Length: 25 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 101: ATCGCCGGCT CCGCCAGCTT GTGAC 25

(2) Information of SEQ ID NO: 102:

(i) Sequence features:

(A) Length: 30 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 102: GCCGAATTCT CATTAGAGCT CGTTCAGTGT 30

(2) Information of SEQ ID NO: 103:

(i) Sequence features:

(A) Length: 42 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 103: GATCCGAACG CTATCTTCCT GTCTTTCCAG CACCTGCTGC GT 42

(2) Information of SEQ ID NO: 104:

(i) Sequence features:

(A) Length: 44 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 104: TTTGCCACGC AGCAGGTGCT GGAAAGACAG GAAGATAGCG TTCG 44

(2) Information of SEQ ID NO: 105:

(i) Sequence features:

(A) Length: 42 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 105: GGCAAAGTTC GTTTCCTGAT GCTGGTTGGC GGTTCTACCC TG 42

(2) Information of SEQ ID NO: 106:

(i) Sequence features:

(A) Length: 41 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 106: ACGCACAGGG TAGAACCGCC AACCAGCATC AGGAAACGAA C 41

(2) Information of SEQ ID NO: 107:

(i) Sequence features:

(A) Length: 46 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 107: TGCGTTCGTC GGGCGCCGCC AACCACTGCT GTTCCGTCTT AATGAA 46

(2) Information of SEQ ID NO: 108:

(i) Sequence features:

(A) Length: 45 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 108: AGCTTTCATT AAGACGGAAC AGCAGTGGTT GGCGGCGCCC GACGA 45

(2) Information of SEQ ID NO: 109:

(i) Sequence features:

(A) Length: 24 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 109: AAGGATCCGA ACGCTATCTT CCTG 24

(2) Information of SEQ ID NO: 110:

(i) Sequence features:

(A) Length: 24 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 110: AGAAGCTTTC ATTAAGACGG AACA 24

(2) Information of SEQ ID NO: 111:

(i) Sequence features:

(A) Length: 46 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 111: CTAGAAAAAA CCAAGGAGGT AATAAATAAT GAAAGCACCT GTACCA 46

(2) Information of SEQ ID NO: 112:

(i) Sequence features:

(A) Length: 46 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 112: CAGGTGGTAC AGGTGCTTTC ATTATTTATT ACCTCCTTGG TTTTTT 46

(2) Information of SEQ ID NO: 113:

(i) Sequence features:

(A) Length: 38 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 113: CCTGCATGTG ATTTACGGGT CCTGTCTAAA CTGCTGCG 38

(2) Information of SEQ ID NO: 114:

(i) Sequence features:

(A) Length: 34 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 114: CGCAGCAGTT TAGACAGGAC CCGTAAATCA CATG 34

(2) Information of SEQ ID NO: 115:

(i) Sequence features:

(A) Length: 84 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 115: CTAGAAAAAA CCAAGGAGGT AATAAATAAT GAAAGCACCT GTACCACCTG CATGTGATTT 60ACGGGTCCTG TCTAAACTGC TGCG 8 4

(2) Information of SEQ ID NO: 116:

(i) Sequence features:

(A) Length: 20 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecular type: peptide

(XI) Sequence description: SEQ ID NO: 116: Pro ARG Leu Leu asn LYS Leu Leu ARG ARG ARG ARGR Leu His ARG Leu Serg Leu Serg Leu Serg Leu Serg Leu Serg Leu Serg Leu Serg Leu Serg Leu Serg Leu Serg

(2) Information of SEQ ID NO: 117:

(i) Sequence features:

(A) Length: 21 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: Linear

(ii) Molecular type: peptide

(Xi) Sequence description: SEQ ID NO: 117: PHE Ser Leu Gly Glu TRP LYS ThR Gln Thr Gln Ser Lys Ala Gln Asp Ile Leu Gly Ala1 5 10 15 20

(2) Information of SEQ ID NO: 118:

(i) Sequence features:

(A) Length: 18 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecular type: peptide

(xi) Sequence description: SEO ID NO: 118: Ser Arg Thr Ser Gln Leu Leu Thr Leu Asn Lys Phe Pro Asn Arg Leu Leu Asp1 5 5 1

(2) Information of SEQ ID NO: 119:

(i) Sequence features:

(A) Length: 21 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: Linear

(ii) Molecular type: peptide

(XI) Sequence description: SEQ ID NO: 119: ASP Leu ARG Val Leu Seru Leu Leu ARG ARG ASP Ser His Val Leu Ser Gln1 5 10 15 20

(2) Information of SEQ ID NO: 120:

(i) Sequence features:

(A) Length: 16 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecular type: peptide

(xi) Sequence Description: SEQ ID NO: 120: Ser leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp1 5 5 10 1 5

(2) Information of SEQ ID NO: 121:

(i) Sequence features:

(A) Length: 19 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 121: Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala1 5 5 10 5  

(2) Information of SEQ ID NO: 122:

(i) Sequence features:

(A) Length: 19 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 122: Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala1 5 5 10 5  

(2) Information of SEQ ID NO: 123:

(i) Sequence features:

(A) Length: 23 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecular type: peptide

(XI) Sequence description: SEQ ID NO: 123: Ser Leu Pro Pro ASN Leu Gln Pro GLY TYR Ser Pro ThR His Pro Pro THR GLN TYR THR1 5 10 15 20 20

(2) Information of SEQ ID NO: 124:

(i) Sequence features:

(A) Length: 27 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecular type: peptide

(XI) Sequence description: SEQ ID NO: 124: Pro Ser Ala Pro Thr Pro Thr Seru Leu Leu asn Thr Tyr THR HIS GLN Leu Ser1 5 10 15GLN GLU GLY25

(2) Information of SEQ ID NO: 125:

(i) Sequence features:

(A) Length: 46 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 125: CTAGAAAAAA CCAAGGAGGT AATAAATAAT GAAATCTCCT GCACCA 46

(2) Information of SEQ ID NO: 126:

(i) Sequence features:

(A) Length: 46 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 126: CAGGTGGTGC AGGAGATTTC ATTATTTATT ACCTCCTTGG TTTTTT 46

(2) Information of SEQ ID NO: 127:

(i) Sequence features:

(A) Length: 38 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 127: CCTGCATGTG ATTTACGGGT CCTGTCTAAA CTGCTGCG 38

(2) Information of SEQ ID NO: 128:

(i) Sequence features:

(A) Length: 34 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 128: CGCAGCAGTT TAGACAGGAC CCGTAAATCA CATG 34

(2) Information of SEQ ID NO: 129:

(i) Sequence features:

(A) Length: 84 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 129: CTAGAAAAAA CCAAGGAGGT AATAAATAAT GAATCTCCT GCACCACCTG CATGTGATTT 60ACGGGTCCTG TCTAAACTGC TGCG 4 8

(2) Information of SEQ ID NO: 130:

(i) Sequence features:

(A) Length: 47 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 130: CTCCCGAACC GTACCAGCGG CCTGCTGGAA ACCAACTTTA CCGCGAG 47

(2) Information of SEQ ID NO: 131:

(i) Sequence features:

(A) Length: 47 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 131: GGTAAAGTTG GTTTCCAGCA GGCCGCTGGT ACGGTTCGGG AGCTCGT 47

(2) Information of SEQ ID NO: 132:

(i) Sequence features:

(A) Length: 49 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 132: CGCGCGTACC ACCGGCAGCG GCCTGCTGAA ATGGCAGCAG GGCTTTCGT 49

(2) Information of SEQ ID NO: 133:

(i) Sequence features:

(A) Length: 49 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 133: AGCCCTGCTG CCATTTCAGC AGGCCGCTGC CGGTGGTACG CGCGCTCGC 49

(2) Information of SEQ ID NO: 134:

(i) Sequence features:

(A) Length: 50 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 134: CGCAAAATCC CGGGCCTGCT GAACCAGACC AGCCGTAGCC TGGATCAGAT 50

(2) Information of SEQ ID NO: 135:

(i) Sequence features:

(A) Length: 50 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 135: ATCCAGGCTA CGGCTGGTCT GGTTCAGCAG GCCCGGGATT TTCGCACGAA 50

(2) Information of SEQ ID NO: 136:

(i) Sequence features:

(A) Length: 47 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 136: CCCGGGCTAT CTGAACCGTA TCCATGAACT GCTGAACGGC ACCCGTG 47

(2) Information of SEQ ID NO: 137:

(i) Sequence features:

(A) Length: 47 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 137: GTGCCGTTCA GCAGTTCATG GATACGGTTC AGATAGCCCG GGATCTG 47

(2) Information of SEQ ID NO: 138:

(i) Sequence features:

(A) Length: 49 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 138: GCCTGTTTCC GGGCCCGAGC CGTCGCACCC TGGGCGCGCC GGATATCAG 49

(2) Information of SEQ ID NO: 139:

(i) Sequence features:

(A) Length: 49 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 139: ATCCGGCGCG CCCAGGGTGC GACGGCTCGG GCCCGGAAAC AGGCCACGG 49

(2) Information of SEQ ID NO: 140:

(i) Sequence features:

(A) Length: 50 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 140: ATCAGCTCTG GCACCAGCGA TACCGGCAGC CTGCCGCCGA ACCTGCAGCC 50

(2) Information of SEQ ID NO: 141:

(i) Sequence features:

(A) Length: 50 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 141: CAGGTTCGGC GGCAGGCTGC CGGTATCGCT GGTGCCAGAG CTGATATCCG 50

(2) Information of SEQ ID NO: 142:

(i) Sequence features:

(A) Length: 51 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 142: GGGCTATAGC CCGAGCCCGA CCCATCCGCC GACCGGCCAG TATACCCTGT T 51

(2) Information of SEQ ID NO: 143:

(i) Sequence features:

(A) Length: 51 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 143: GGTATACTGG CCGGTCGGCG GATGGGTCGG GCTCGGGCTA TAGCCCGGCT G 51

(2) Information of SEQ ID NO: 144:

(i) Sequence features:

(A) Length: 50 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 144: TCCGCTGCCG CCGACCCTGC CGACCCCGGT GGTTCAGCTG CATCCGCTGC 50

(2) Information of SEQ ID NO: 145:

(i) Sequence features:

(A) Length: 50 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 145: GGATGCAGCT GAACCACCGG GGTCGGCAGG GTCGGCGGCA GCGGAAACAG 50

(2) Information of SEQ ID NO: 146:

(i) Sequence features:

(A) Length: 51 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 146: TGCCGGATCC GAGCGCGCCG ACCCCGACCC CGACCAGCCC GCTGCTGAAC A 51

(2) Information of SEQ ID NO: 147:

(i) Sequence features:

(A) Length: 51 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 147: AGCAGCGGGC TGGTCGGGGT CGGGGTCGGC GCGCTCGGAT CCGGCAGCAG C 51

(2) Information of SEQ ID NO: 148:

(i) Sequence features:

(A) Length: 51 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 148: CCAGCTATAC CCATAGCCAG AACCTGAGCC AGGAAGGCTA ATGAAGCTTG A 51

(2) Information of SEQ ID NO: 149:

(i) Sequence features:

(A) Length: 51 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 149: CTTCATTAGC CTTCCTGGCT CAGGTTCTGG CTATGGGTAT AGCTGGTGTT C 51

(2) Information of SEQ ID NO: 150:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 150: ACGAGCTCCC GAACCGTACC A 21

(2) Information of SEQ ID NO: 151:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 151: CTGATATCCG GCGCGCCCAG G 21

(2) Information of SEQ ID NO: 152:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 152: CGGATATCAG CTGGCACC A 21

(2) Information of SEQ ID NO: 153:

(i) Sequence features:

(A) Length: 21 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 153: TCAAGCTTCA TTAGCCTTCC T 21

(2) Information of SEQ ID NO: 154:

(i) Sequence features:

(A) Length: 490 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(XI) Sequence description: SEQ ID NO: 154: CTC CCG AAC CGT AGC GGC GGC CTG CTG GAA ACC ACC GCG ACC 48leu Pro ARG THR Serte Leu Leu THR Ala Ser1 5 10 15GCG CGT ACC ACC GGC AGC GGC CTG CTG AAA TGG CAG CAG GGC TTT CGT 95Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg

20 25 30GCG AAA ATC CCG GGC CTG CTG CTG AAC CAG ACC AGC CGT ACC CTG GAT CAG 144ALA LYS ILE Pro GLY Leu Asn Gln Ser Leu ASP GLN -Leu ASESESN's Le

35 40 40 45ATC CCG GGC TAT CTG AAC CGT ATC CAT GAA CTG CTG AAC GGC ACC CGT 192Ile Pro Gly As Tyr Leu Leu G Asnhr Glu Ile His

50                  55                  60GGC CTG TTT CCG GGC CCG AGC CGT CGC ACC CTG GGC GCG CCG GAT ATC  240Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile65                  70                  75                  80AGC TCT GGC ACC AGC GAT ACC GGC AGC CTG CCG CCG AAC CTG CAG CCG 288Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro

85 90 95GGC TAT AGC CCG AGC CCG ACC Cat CCG CCG ACC GGC CAG Tat ACC CTG 336GLY TYR Ser Pro Thr His Pro ThR Gln Tyr THR Leu

100 105 110TTT CCG CTG CCG CCG ACC CTG CCG ACC CCG GTG GTT CAG CTG CAT CCG 384Phe Pro Leu Pro H Pro Val Thr Leu Pro Thr

115 120 125CTG CTG CCG GAT CCG AGC GCG CCG ACC CCG ACC CCG ACC AGC CCG CTG 432Leu Leu Pro Asp Pro Ser Pro Sr Pro Ala Prohr Thr Pro

130 135 140CTG AAC ACC ACC TAT AGC Cat AGC CAG AGC CTG AGC CAG GAA GGC TAA 480leu Asn Thr Tyr His Ser Gln Gln Glu Glu GLN

(2) Information of SEQ ID NO: 155:

(i) Sequence features:

(A) Length: 24 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 155: AAGGATCCGA ACGCTATCTT CCTG 24

(2) Information of SEQ ID NO: 156:

(i) Sequence features:

(A) Length: 56 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 156: GGGAGCTCGT TCAGGGTCAG AACCAGAGAG GTACGAGACG GAACAGCAGT GGTTGG 56

(2) Information of SEQ ID NO: 157:

(i) Sequence features:

(A) Length: 157 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 157: G GAT CCC AAC GCT ATC TTC CTG TCT TTC CAG CAC CTG CTG CGT GGC 46Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly

1 5 10 15AAA GTT CGT CGTC CTG ATG CTG GGC GGC GGT ACC CTG TGC GTT 94LYS Val ARG PHE Leu Met Leu Val Gly GYR Leu Cys Val Val ARG

20 25 30CGG GCG CCA ACC ACC ACT GCT GCT CCG TCT CGT ACC TCT CTG 142ARG Ala Pro ThR THR ALA Val Pro Ser Leu Val Leu Val Leu Val Leu

35 40 45ACC CTG AAC GAG CTC 157Thr Leu

50

(2) Information of SEQ ID NO: 158:

(i) Sequence features:

(A) Length: 100 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 158: CCTCGAGGAA TTCCTGCAGC CCGGGACTAG TATCGGCTAC CCCTACGACG TCCCCGACTA 60CGCCGGCGTC CATCACCATC ACCATCACTG AGCGGCCGCC                                    

(2) Information of SEQ ID NO: 159:

(i) Sequence features:

(A) Length: 108 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 159: AATTGGCGGC CGCTCAGTGA TGGTGATGGT GATGAGCGCC GGCGTAGTCG GGGACGTCGT 60AGGGGTAGCC GATACTAGTC CCGGGCTGCA GGAATTCCTC GAGGGTAC 108

(2) Information of SEQ ID NO: 160:

(i) Sequence features:

(A) Length: 70 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 160: AATTCCAAGA TCTCACACTT GCTTTTGACA CAACTGTGTT TACTTGCAAT CCCCCAAAAC 60AGACAGACCC                                                

(2) Information of SEQ ID NO: 161:

(i) Sequence features:

(A) Length: 66 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 161: GGGTCTGTCT GTTTTGGGGG ATTGCAAGTA AACACAGTTG TGTCAAAAGC AAGTGTGAGA 60TCTTGG 6 6

(2) Information of SEQ ID NO: 162:

(i) Sequence features:

(A) Length: 29 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 162: GGCCCGGGAT GGAGCTGACT GAATTGCTC 29

(2) Information of SEQ ID NO: 163:

(i) Sequence features:

(A) Length: 35 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 163: TCAAGCTTAC TAGTCCCTTC CTGAGACAGA TTCTG 35

(2) Information of SEQ ID NO: 164:

(i) Sequence features:

(A) Length: 22 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 164: TAATACGACT CACTATAGGG CG 22

(2) Information of SEQ ID NO: 165:

(i) Sequence features:

(A) Length: 22 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 165: AATTCCAAGA TCACACACTT GC 22

(2) Information of SEQ ID NO: 166:

(i) Sequence features:

(A) Length: 35 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 166: TCAAGCTTAC TAGTCCCTTC CTGAGACAGA TTCTG 35

(2) Information of SEQ ID NO: 167:

(i) Sequence features:

(A) Length: 35 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 167: TGGGCACTGG CTCAGGTTGC TGTGAAGGAC ATGGG 35

(2) Information of SEQ ID NO: 168:

(i) Sequence features:

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 168: TGTAGGCAAA GGGGTAACCT CTGGGCA 27

(2) Information of SEQ ID NO: 169:

(i) Sequence features:

(A) Length: 21 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: Linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 169:

Thr Ser Ile Gly Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Gly Val His Xaa Xaa Xaa Xaa Xaa

1 5 10 15 20

(2) Information of SEQ ID NO: 170:

(i) Sequence features:

(A) Length: 30 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 170: TTTGAATTCG GCCAGCCAGA CACCCCGGCC 30

(2) Information of SEQ ID NO: 171:

(i) Sequence features:

(A) Length: 41 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 171: TTTGCGGCCG CTCATTATTC GTGTATCCTG TTCAGGTATC C 41

(2) Information of SEQ ID NO: 172:

(i) Sequence features:

(A) Length: 24 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 172: TGTAGGCAAA GGAACCTCTG GGCA 24

(2) Information of SEQ ID NO: 173:

(i) Sequence features:

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 173: TGTAGGCAAA GCAGTGTGAA CCTCTGG 27

(2) Information of SEQ ID NO: 174:

(i) Sequence features:

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 174: TGTAGGCAAA GGAGCGTGAA CCTCTGG 27

(2) Information of SEQ ID NO: 175:

(i) Sequence features:

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 175: TGTAGGCAAA GGTCCGTGAA CCTCTGG 27

(2) Information of SEQ ID NO: 176:

(i) Sequence features:

(A) Length: 24 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 176: AGCTGTGGTC CTCTGTGGAG GAAG 24

(2) Information of SEQ ID NO: 177:

(i) Sequence features:

(A) Length: 24 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 177: GTGAGCTGTG GTGCCCTGTG GAGG 24

(2) Information of SEQ ID NO: 178:

(i) Sequence features:

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 178: AGCTGTGGTC CTGTTGCCCT GTGGAGG 27

(2) Information of SEQ ID NO: 179:

(i) Sequence features:

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 179: AGCTGTGGTC CTAGCGCCCT GTGGAGG 27

(2) Information of SEQ ID NO: 180:

(i) Sequence features:

(A) Length: 27 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 180: AGCTGTGGTC CTTCCGCCCT GTGGAGG 27

(2) Information of SEQ ID NO: 181:

(i) Sequence features:

(A) Length: 26 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 181: CGGGCTGCAG GATATCCAAG ATCTCA 26

(2) Information of SEQ ID NO: 182:

(i) Sequence features:

(A) Length: 22 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 182: TAATACGACT CACTATAGGG CG 22

(2) Information of SEQ ID NO: 183:

(i) Sequence features:

(A) Length: 34 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: other nucleic acids

(xi) Sequence description: SEQ ID NO: 183: GGGCGGCCGC TCAGCTGGGG ACAGCTGTGG TGGG 34

(2) Information of SEQ ID NO: 184:

(i) Sequence features:

(A) Length: 7 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecular type: peptide

(ix) Features:

(A) Name/keyword: misc_feature

(D) Other information:

/Comment = "The amino acid at position 2 is Ala, Ser, Gly, Met

or Gln"

(ix) Features:

(A) Name/keyword: misc_feature

(D) Other information:

/Comment = "The amino acid at position 7 is Glu or Lys"

(xi) Sequence description: SEQ ID NO: 184: Lys Xaa Tyr Tyr Glu Ser Xaa1 5

(2) Information of SEQ ID NO: 185:

(i) Sequence features:

(A) Length: 6 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: Linear

(ii) Molecular type: peptide

(ix) Features:

(A) Name/keyword: misc_feature

(D) Other information:

/Comment = "The amino acid at position 6 is Glu or Asp"

(xi) Sequence description: SEQ ID NO: 185: Lys Glx Arg Ala Ala Xaa1 5

(2) Information of SEQ ID NO: 186:

(i) Sequence features:

(A) Length: 7 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: Linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 186: Lys Ala Gly Xaa Cys Ser Gly1 5

(2) Information of SEQ ID NO: 187:

(i) Sequence features:

(A) Length: 13 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecular type: peptide

(ix) Features:

(A) Name/keyword: misc_feature

(D) Other information:

/ Comment = "The amino acid at position 2 is Ile, Thr or Ser"

(xi) Sequence description: SEQ ID NO: 187: Lys Xaa Pro Val Pro Pro Ala Cys Asp Pro Arg Leu Leu1 5 10

(2) Information of SEQ ID NO: 188:

(i) Sequence features:

(A) Length: 9 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 188: Lys Asp Ser Phe Leu Ala Asp Val Lys1 5

(2) Information of SEQ ID NO: 189:

(i) Sequence features:

(A) Length: 9 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: Linear

(ii) Molecular type: peptide

(ix) Features:

(A) Name/keyword: misc feature

(D) Other information:

/ Comment = "The amino acid at position 1 is Lys or Arg"

(xi) Sequence description: SEQ ID NO: 189: Xaa Thr Leu Pro Thr Xaa Ala Val Pro1 5

(2) Information of SEQ ID NO: 190:

(i) Sequence features:

(A) Length: 15 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 190: Lys Asp Ser Phe Leu Ala Asp Val Lys Gln Tyr Tyr G1u Ser Glu1 5 5 10 5 1

(2) Information of SEQ ID NO: 191:

(i) Sequence features:

(A) Length: 332 amino acids

(B) type: amino acid

(D) Topology: Linear

(ii) Molecular type: protein

(vi) Source:

(A) Organism: Homo Sapiens

(Xi) Sequence description: SEQ ID NO: 191: Ser Pro Ala Pro Ala Cys ASP Leu ARG Val Leu Seru Leu1 5 10 15ARG ASP Seru His Serg Leu Serg Leu Serg

20 25 25 30His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu

35 40 45Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu

50 55 60Gly Ala Val THR Leu Leu Leu Glu GLY Val Met Ala Ala ARG GLN65 70 80leu Gly Pro Thr Cys Leu Leu Leu Gln Leu Ser Gln

85 90 95Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu

100 105 110Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe

115 120 125 Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu

130 135 140VAL GLY GLY SER Leu Cys Val Val ARG ALA Pro Pro ThR THR ALA145 150 155 160VAL Pro Serg THR Leu Thr Leu Leu Pro Leu Pro ASN

165 170 175Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr

180 185 190Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys Ile

195 200 205Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly

210 215 220Tyr Lau asn ARG Ile His Glu Leu Leu asn Gly THR ARG GLY Leu PHE225 230 235 240pro Gly Pro Serg THR Leu Gly Ala ASP ILE Serly Sergly

245 250 255Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser

260 265 270Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu Phe Pro Leu

275 280 285Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro

290 295 300asp Pro Ser Ala project

325 330

(2) Information of SEQ ID NO: 192:

(i) Sequence features:

(A) Length: 1086 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D) Topology: linear

(ii) Molecular type: cDNA→mRNA

(vi) Source:

(A) Organism: Homo Sapiens

(xi) Sequence description: SEQ ID NO: 192: GGCCAGCCAG ACACCCCGGC CAGA ATG GAG CTG ACT GAA TTG CTC CTC GTG 51

                                                                                     , 

-21 -20 -15GTC ATG CTT CTC CTA ACT GCA AGG CTA ACG CTG TCC AGC CCG GCG GCT 99VAL MET Leu Leu THRA Leu Ala Ala Ala Ala Ala Ala's Le Ala's Le Ala's Le

-10 -5 1CCT GCT GCT GAC CTC CGA GTC CTC AGT AAA CTT CTT CTT GAC TCC Cat 147pro Ala Cys ASP Leu ARG Val Leu Leu Leu ARG ARG AGC AGC AGC CTCA CAGC ’s CAC CCT TTG CCT 195Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro

25ACA CCT GTC CTG CTG CCT GCT GCT GAC TTT AGC TTG GAA TGG AAA 243thr Prou Leu Pro Ala Val Ala Val Leu GLY GLU TRP LYS

40 45 50ACC CAG ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GCA GCA GCA GCA GCA GTG ACC 291thr Gln Met Glu Thr Lys Ala Gln ASLA Val Ala Val Thr Val

55 60 65CTT CTG CTG GAG GGA GGA GCA GCA GCA CGG GGG GGA CAA CTG GGA CCC Act 335leu Leu GLU GLE MET ALA ALA ARG GLN Leu GLY Pro Thr Thr

70                  75                  80TGC CTC TCA TCC CTC CTG GGG CAG CTT TCT CGA CAC GTC CGT CTC CTC      387Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu55                  90                  95                 100CTT GGG GCC CTG CAG ACC CTC CTT GGA ACC CAG CTT CCT CCA CAC GGC 415Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly

105 110 115AGG ACC ACA GCT CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC TTC CAA 483ARG THR Ala His Lys as Ala Ile Phe Leu Seru Leu Gln Gln

120 125 130CAC CTG CTC CGA GGA AAG GTG CGT TTC CTG ATG CTT GGA GGA GGG TCC 531HIS Leu ARG GLY LYS Val ARG PHE Leu Val Gly GLY GLY GLY GLE

135 140 Ar 145ACC CTC TGC CTC ACG CGG GCC CCA CCC ACC ACA GCT GTC CCC AGC AGA 579Thr Leu Th er Cys Pro Val Arg Prohr Arg Ala

150                 155                 160ACC TCT CTA GTC CTC ACA CTG AAC GAG CTC CCA AAC AGG ACT TCT GGA       627Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly165                 170                 175                 180TTG TTG GAG ACA AAC TTC ACT GCC TCA GCC AGA ACT ACT GGC TCT GGG 675Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser Cly

185 190 195CTT CTG AAG TGG CAG CAG GGA TTC AGA GCC AAG AAG AAG At GGT CTG 723leu Leu Lys Trn Gln GLN GLN GLN GLN GLN GLN GLN GLN GLN ARLN Leu Leu Leu Leu Leu leu

200 205 20AAC CAA ACC TCC AGG TCC CTG GAC CAA ATC CCC GGA TAC CTG AGG 772asn Gln Thr Serg Serg Ser

215 220 225ata CAC GAA CTC TTG Aat GGA Act CGT GGA CTT CCT GGA CCC TCA 819ile His GLU Leu Asn Gly THR Leu Pro GLY PRY PRLE PRE

230                 235                 240CGC AGG ACC CTA GGA GCC CCG GAC ATT TCC TCA GGA ACA TCA GAC ACA       867Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser Gly Thr Ser Asp Thr245                 250                 255                 260GGC TCC CTG CCA CCC AAC CTC CAG CCT GGA TAT TCT CCT TCC CCA ACC 916Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser Pro Ser Pro Thr

265 270 275cat CCT CCT ACT GGA CAG TAG TAG CTC CTC CCA CCC ACC TTG 963HIS Pro Pro ThR Gln Tyr Leu PRO PRO ThR Leu

        280                 285                 290CCC ACC CCT GTG GTC CAG CTC CAC CCC CTG CTT CCT GAC CCT TCT GCT      1011Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro Asp Pro Ser Ala

295 300 305CCA ACC ACC CCT ACC AGC CCT CTT CTA ACA TCC TAC CAC CAC 1059Pro ThR Pro Thr Seru Leu asn Thr Tyr Thr His

310 315 320TCC CAG AAT CTG TCT CAG GAA GGG TAASer Gln Asn Leu Ser Gln Glu Gly325 330

(2) Information of SEQ ID NO: 193:

(i) Sequence features:

(A) Length: 7 amino acids

(B) type: amino acid

(C) Chain type: single chain

(D) topology; linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 193: Lys Gln Tyr Tyr Glu Ser Glu1 5

(2) Information of SEQ ID NO: 194:

(i) Sequence features:

(A) Length: 1663 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D) Topology: linear

(ii) Molecular type: cDNA→mRNA

(vi) Source:

(A) Organism: Rattus norvegicus

(H) Cell line: McA-RH8994

    (xi)序列描述：SEQ ID NO：194：GGTGTACCTG GGTCCTGAAG CCCTTCTTCA CCTGGATAGA TTCCTTCGCC CACCTGTCCC      60CACCCCACTC TGTGCAGAGG TACAAAAGCT CAAGCCGTCT CCATGGCCCC AGGAAAGATT     120CAGGGGAGAG GCCCCACACA GGGAGCCACT GCAGTCAGAC ACCCTGGGCA GA ATG         175

Met

-21GAG CTG ACT GCC ATC CTC CTC CTC CTC CTC CTC CTC CTC GCA AGA THR ASP Leu Leu Val Ala Leu Leu THR Ala ARA ARG-5CT CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC TGT GAC CCC AGA CTC CTA 271Leu Thr Leu Ser Ser Pro Val Pro Pro Ala Cys Asp Pro Arg Leu Leu

1 5 10AAAAAA CTG CTG CTG CT CT GAC TCC TAC CTC CTT CAC AGC CGA CGA CTG AGT CAG 319asn LYS Leu Leu ARG As Tyr Leu His Serg Leu Serg

15 20 25TGT CCT GAC GTC AAC CCT TTG TCT ATC CCT GTC CTG CTG CCT GCT GTG 367Cys Pro Leu Asp Val le A Asn Pro Leu Ser I

30                  35                  40CAC TTT AGC CTC GGA GAA TGG AAA ACC CAG ACC GAA CAG AGC AAG GCA       415Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Thr Glu Gln Ser Lys Ala45                  50                  55                  60CAG GAC ATT CTA GGG GCA GTG TCC CTT CTA CTG GAG GGG GTG ATG GCA 463Gln Asp Ile Leu Gly Ala Val Ser Leu Leu Leu Glu Gly Val Met Ala

65 70 75GCA CGA Ga CAG TTG GAA CCC TGC CTC CTC CTC CTC CTG GGA CAG 511ALA ARG GLN Leu Pro Series Leu Leu Leu GLY GLN

80 85 90CTT TCT GTT CGC CGC CTC CTC CTC CTC CTG CTG GGC CTC CTA 559leu Serg Leu Leu Leu GLN GLN GLN GLN GLN Leu Leu Leu

95 1005GGA ACC CAC GTA AGT CCC CAG ACC TAC TAC TAC CCT CTT CAG 607Gly Thr Gln Val Serg ARG Asn Tyr Prou Thr Gln

110                 115                 120TTC CTC TAAGGACCTG GGAAAAGACA ACGGATTCTA GATTCTAGGT GTCTTCAGTG        663Phe Leu125TATGAAAGCT GGTCTATACG GAGTGATGCT TCTCACCCAC AATACCTGGG TGCTGGCAGT     723AAATCTTTCC ACCTTAGTGA GAAGAGGCCT GATATGTGGG CCAACTCACT GGCCTCAGGC     783CCATCCTCTG CCTTCAGCTT CCTCCACAGG GCAGGACCAC AGCTCACAAG GACCCCAGTG     843CCCTCTTCTT GAGCTTGCAA CAACTGCTTC GGGGAAAGGT GCGCTTCCTG CTGCTGGTAG     903AAGGTCCCGC CCTCTGTGTC AGACGGACCC TTCCCACCAC AGCTGTCCCA AGCAGAACCT     953CTCAACTCCT CACACTAAAC AAGTTCCCAA ACAGACTTCT GGATTGTTGG AGACGAACTT    1023CAGTGTTGTA GCCAGAACTG CTGGCCCTGG ACTTCTGAAC AGGCTTCAAG GATTCAGAGC    1083CAAGATTATT CCTGGTCAGC TAAATCAAAC CTCCGGGTCC TTAGACCAAA TCCCTGGATA    1143CCTGAACGGG ACACACGAAC CTGTGAATGG AACTCATGGG CTCTTTGCTG CGACCTCACT    1203ACAGACCCTG GAAGCCCCAG ACGTTGTGCC AGGAGCTTTC AACAAAGGCT CCTTGCCACT    1263CAACCTCCAG AGTGGACTTC CTCCTATCCC AAGCCTTGCT GCTGATGGAT ACACACTTTT    1323CCCTCCTTCA CCTACCTTCC CCACCCCTGG GTCTCCACCC CAGCTCCCCC CCGTTTCCTG    1383ACCCCTCCAC CACCATACCT AACTCTACCA ACCCTCATCC AGGACTTGGT CTCAGTAAGC    1443GTCCCGTGCA CTGGCACGGA GCGCGATCGT CTGCAACATC TCTCAGGGGC AAGCTTCCTC    1503AGGAAGGCTC TGAGGCAGCT CACTAGACAT CCTGCTCTCG CCTAACGGGC CCTGGGAAAG 1563GGATACACAG GCCAGGACAC TGTACAACCT TAGGAGCGAT TTTTTTCTTA ACCTATCAAC 1623AATATTCATC AGAGCAAAAA AAAAAAAAA AAAAAAAAAA 1663(2) Information of SEQ ID NO: 195:

(i) Sequential features:

(A) Length: 861 base pairs

(B) type: nucleic acid

(C) Chain type: double chain

(D) Topology: linear

(ii) Molecular type: cDNA→mRNA

(vi) Source:

(A) Organism: Homo Sapiens

(H) Tissue type: liver

(xi) Sequence description: SEQ ID NO: 195: GGCCAGCCAG ACACCCCGGC CAGA ATG GAG CTG ACT GAA TTG CTC CTC GTG 51

                                                                                     , 

-21 -20 -15GTC ATG CTT CTC CTA ACT GCA AGG CTA ACG CTG CTG CCG GCG GCG GCT 99VAL MET Leu Leu's Ala Ala Leuu Ala Ala Ala Ala Ala Ala Ala's

-10 -5 1CCT GCT GCT GAC CTC CGA GTC CTC AGT AAA CTT CTT CTT GAC TCC Cat 147pro Ala Cys ASP Leu ARG Val Leu Leu Leu ARG ARG AGC AGC AGC CTCA CAGC ’s CAC CCT TTG CCT 195Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro

25 35ACA CCT GTC CTG CTG CCT GCT GCT GAC TTT AGC TTG GAA TGG AAA 243thr Prou Leu Pro Ala Val Ala Val Leu GLY GLU TRP LYSS

40 45 50ACC CAG ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GCA GCA GCA GCA GCA GTG ACC 291thr Gln Met Glu Thr Lys Ala Gln ASLA Val Ala Val Thr Val

55 60 65CTT CTG CTG GAG GGA GGA GCA GCA CGG GGG GGG GGA CAA CTG GGA CCC Act 339leu Leu GLU GLE MET ALA Ala ARG GLN Leu GLY Pro Thr Thr

70                  75                  80TGC CTC TCA TCC CTC CTG GGG CAG CTT TCT GGA CAG GTC CGT CTC CTC       387Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu85                  90                  95                 100CTT GGG GCC CTG CAG AGC CTC CTT GGA ACC CAG CTT CCT CCA CAG GGC 435Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly

105 110 115AGG ACC ACA GCT CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC TTC CAA 483ARG THR Ala His Lys as Ala Ile Phe Leu Seru Leu Gln Gln

120 125 130CAC CTG CTC CGA GGA AAG GTG CGT TTC CTG ATG CTT GGA GGA GGG TCC 531HIS Leu ARG GLY LYS Val ARG PHE Leu Val Gly GLY GLY GLY GLE

135 140 Ar 145ACC CTC TGC GTC AGG CGG GCC CCA CCC ACC ACA GCT GTC CCC AGC AGA 579Thr la Val Leu Cys er Pro TGC Pro TGC Prohr Arg Ala

150                 155                 160ACC TCT CTA GTC CTC ACA CTG AAC GAG CTC CCA AAC AGG ACT TCT GGA       627Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly155                 170                 175                 180TTG TTG GAG ACA AAC TTC ACT GCC TCA GCC AGA ACT ACT GGC TCT GGG 675Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser Gly

185 190 195CTT CTG AAG TGG CAG CAG GGA TTC AGA GCC AAG AAG AAG At GGT CTG 723leu Leu Lys Trn Gln GLN GLN GLN GLN GLN GLN GLN GLN GLN ARLN Leu Leu Leu Leu Leu leu

200 205 20AAC CAA ACC TCC AGG TCC CTG GAC CAA ATC CCC GGA TAC CTG AGG 771asn Gln Thr Serg Serog

215 220 225ATA CAC GAA CTC TTAAAAAAAAA AAAAAAAAA AAAAAAAAAA AAAAAAAAAA 833Ile His Glu Leu

230AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA 861

(2) Information of SEQ ID NO: 196:

(i) Sequence features:

(A) Length: 1267 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D) Topology: Linear

(ii) Molecular type: cDNA→mRNA

(vi) Source:

(A) Organism: Homo Sapiens

(H) Tissue type: Liver

(xi) Sequence description: SEQ ID NO: 196: GGCCACCCAG ACACCCCGGC CAGA ATG GAG CTG ACT GAA TTG CTC CTC GTG 51

                                                                                     , 

-21 -20 -15GTC ATG CTT CTC CTA ACT GCA AGG CTA ACG CTG TCC AGC CCG GCG GCT 99VAL MET Leu Leu THRA Leu Ala Ala Ala Ala Ala Ala's Le Ala's Le Ala's Le

-10 -5 1CCT GCT GCT GAC CTC CGA GTC CTC AGT AAA CTT CTT CTT GAC TCC Cat 147pro Ala Cys ASP Leu ARG Val Leu Leu Leu ARG ARG AGC AGC AGC CTCA CAGC ’s CAC CCT TTG CCT 195Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro

25 35ACA CCT GTC CTG CTG CCT GCT GCT GAC TTT AGC TTG GAA TGG AAA 243thr Prou Leu Pro Ala Val Ala Val Leu GLY GLU TRP LYSS

40 45 50ACC CAG ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GCA GCA GCA GCA GCA GTG ACC 291thr Gln Met Glu Thr Lys Ala Gln ASLA Val Ala Val Thr Val

55 60 65CTT CTG CTG GAG GGA GGA GCA GCA CGG GGG GGG GGA CAA CTG GGA CCC Act 339leu Leu GLU GLE MET ALA Ala ARG GLN Leu GLY Pro Thr Thr

70                  75                  80TGC CTC TCA TCC CTC CTG GGG CAG CTT TCT GGA CAG GTC CGT CTC CTC       387Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu85                  90                  95                 100CTT GGG GCC CTG CAG AGC CTC CTT GGA ACC CAG CTT CCT CCA CAG GGC 435Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly

105 110 115AGG ACC ACA GCT CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC TTC CAA 483ARG THR Ala His Lys as Ala Ile Phe Leu Seru Leu Gln Gln

120 125 130CAC CTG CTC CGA GGA AAG GTG CGT TTC CTG ATG CTT GGA GGA GGG TCC 531HIS Leu ARG GLY LYS Val ARG PHE Leu Val Gly GLY GLY GLY GLE

135 140 Ar 145ACC CTC TGC GTC AGG CGG GCC CCA CCC ACC ACA GCT GTC CCC AGC AGA 579Thr la Val Leu Cys er Pro TGC Pro TGC Prohr Arg Ala

150                 155                 160ACC TCT CTA GTC CTC ACA CTG AAC GAG CTC CCA AAC AGG ACT TCT GGA       627Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly165                 170                 175                 180TTG TTG GAG ACA AAC TTC ACT GCC TCA GCC AGA ACT ACT GGC TCT GGG 675Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser Gly

185 190 195CTT CTG AAG TGG CAG CAG GGA TTC AGA GCC AAG AAG AAG At GGT CTG 723leu Leu Lys Trn Gln GLN GLN GLN GLN GLN GLN GLN GLN GLN ARLN Leu Leu Leu Leu Leu leu

200 205 20AAC CAA ACC TCC AGG TCC CTG GAC CAA ATC CCC GGA TAC CTG AGG 771asn Gln Thr Serg Serog

215 220 225ata Cac GAA CTC TTG AAT GGA ACT CGT GGA CTT CCT GGA CCC TCA 619ile His Glu Leu Asn GLY THR ARG GLY PRE Pro Gly Pro Ser Ser

230                 235                 240CGC AGG ACC CTA GGA GCC CCG GAC ATT TCC TCA GGA ACA TCA GAC ACA       867Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser Gly Thr Ser Asp Thr245                 250                 255                 260GGC TCC CTG CCA CCC AAC CTC CAG CCT GGA TAT TCT CCT TCC CCA ACC 915Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser Pro Ser Pro Thr

265 270 275cat CCT CCT ACT GGA CAG TAG TAG CTC CTC CCA CCC ACC TTG 963HIS Pro Pro ThR Gln Tyr Leu PRO PRO ThR Leu

        280                 285                 290CCC ACC CCT GTG GTC CAG CTC CAC CCC CTG CTT CCT GAC CCT TCT GCT      1011Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro Asp Pro Ser Ala

295 300 305CCA ACC ACC CCT ACC AGC CCT CTT CTA ACA TCC TAC CAC CAC 1059Pro ThR Pro Thr Seru Leu asn Thr Tyr Thr His

310                 315                 320TCC CAG AAT CTG TCT CAG GAA GGG TAAGGTTCTC AGACACTGCC GACATCAGCA     1113Ser Gln Asn Leu Ser Gln Glu Gly325                 330TTGTCTCATG TACAGCTCCC TTCCCTGCAG GGCGCCCCTG GGAGACAACT GGACAAGATT   1173TCCTACTTTC TCCTGAAACC CAAAGCCCTG GTAAAAGGGA TACACAGGAC TGAAAAGGGA   1233ATCATTTTTC ACTGTACATT ATAAACCTTC AGAA                               1267

(2) Information of SEQ ID NO: 197:

(i) Sequence features:

(A) Length: 549 base pairs

(B) type: nucleic acid

(C) chain type: double chain

(D) Topology: Linear

(ii) Molecular type: synthetic DNA

(XI) Sequence description: SEQ ID NO: 197: CTG GTT CCG CGT GGA TCC CCG CCG CCG CCG CCG CCA GCT GAC CTT CGT 48leu Val Pro ARG GLY Serg Gly Serg Val Val-5 10CTG TCT AAA CTG CTT CGC GAC TCT CAC GTG CTG CAC TCT CGT CTG TCC 96Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser

15 20 25CAG TGC CCG GAA GTT CCG CCG CCG CCG ACC CCG GTT CTG CTT CCG GCT 144GLN CYS Pro Glu Val His Pro Leu Pro THR Leu Pro Ala Ala

30 35 40GTC GAC TCC CTG GGT GAA TGG AAA ACC CAG GAA GAG ACC AAA 192VAL ASP PHE Seru GLY GLU TRP LYS THR GLN MET GLU THR LYSS

45                  50                  55GCT CAG GAC ATC CTG GGT GCA GTA ACT CTG CTT CTG GAA GGC GTT ATG       240Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met60                  65                  70                  75GCT GCA CGT GGC CAG CTT GGC CCG ACC TGC CTG TCT TCC CTG CTT GGCAla Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly

80 85 90CAG CTG TCT GGC CAG GTT CGT CGT CTG CTG CTC GGC GCT CTG CAG CTG 336GLN Leu Serg Gln Val ARG Leu Leu GLY Ala Leu Gln Seruu Gln Leu Gln Ser Leu's Le leu Leu Leu Leu Leu Leu Leu's Le's Le's Le's Le's's's

95 1005CTT GGC ACC CAG CCG CCG CCA CAG GGC CGT ACC ACT GCT CAC Gat 384leu GLY ThR Gln Leu Pro GLN GLY ARG THR ALA His Lys Lys ASP

110 115 120CCC AAT GCC ATC TTC CTG AGC TTC CAA CAA CAC CTC CGA GGA AAG GTG 432PRO Asn Ala Ile Phe Leu Leu ARG GLY LY LY LY LY LY LY LY LY LY LY LY -

125                 130                 135CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC       480Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala140                 145                 150                 155CCA CCC ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG 528Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu

                                                                                  

Claims (22)

1. the DNA that forms by one section base sequence, the aminoacid sequence of described base sequence proteins encoded, described albumen is by the 1st to 332 aminoacid sequence among the SEQ ID No:191, or form by the aminoacid sequence that in above-mentioned aminoacid sequence, comprises 1 to 6 amino acid replacement, disappearance, insertion and/or interpolation, described albumen has the TPO activity.

2. the DNA of claim 1 wherein saidly substitutes, disappearance, inserts and/or add and be 1 or 2 and amino acid whosely substitute, disappearance, insert and/or add.

3. the DNA of claim 1 wherein saidly substitutes, disappearance, inserts and/or add and be 1 and amino acid whosely substitute, disappearance, insert and/or add.

4. the DNA that forms by one section base sequence, the aminoacid sequence of described base sequence proteins encoded, described albumen is by the 1st to 231 aminoacid sequence among the SEQ ID No:191, or form by the aminoacid sequence that in above-mentioned aminoacid sequence, comprises 1 to 6 amino acid replacement, disappearance, insertion and/or interpolation, described albumen has the TPO activity.

5. the DNA of claim 4 wherein saidly substitutes, disappearance, inserts and/or add and be 1 or 2 and amino acid whosely substitute, disappearance, insert and/or add.

6. the DNA of claim 4 wherein saidly substitutes, disappearance, inserts and/or add and be 1 and amino acid whosely substitute, disappearance, insert and/or add.

7. the DNA that forms by one section base sequence, the aminoacid sequence of described base sequence proteins encoded, described albumen is by the 1st to 163 aminoacid sequence among the SEQ ID No:191, or form by the aminoacid sequence that in above-mentioned aminoacid sequence, comprises 1 to 6 amino acid replacement, disappearance, insertion and/or interpolation, described albumen has the TPO activity.

8. the DNA of claim 7 wherein saidly substitutes, disappearance, inserts and/or add and be 1 or 2 and amino acid whosely substitute, disappearance, insert and/or add.

9. the DNA of claim 7 wherein saidly substitutes, disappearance, inserts and/or add and be 1 and amino acid whosely substitute, disappearance, insert and/or add.

10. the DNA that forms by one section base sequence, the aminoacid sequence of described base sequence proteins encoded, described albumen is made up of the 1st to 332 aminoacid sequence among the SEQ ID No:191.

11. by the DNA that one section base sequence is formed, the aminoacid sequence of described base sequence proteins encoded, described albumen is made up of the 1st to 231 aminoacid sequence among the SEQ ID No:191.

12. by the DNA that one section base sequence is formed, the aminoacid sequence of described base sequence proteins encoded, described albumen is made up of the 1st to 163 aminoacid sequence among the SEQ ID No:191.

13. the DNA of claim 1, wherein said proteic aminoacid sequence comprise at least following (a) to (at least one amino acid mutation v):

(a) the 1st Ser residue and the 3rd 's Ala residue is substituted by Ala and Val residue respectively;

(b) the 25th Arg residue is substituted by the Asn residue;

(c) the 33rd His residue is substituted by the Thr residue;

(d) the 25th Arg residue and the 231st 's Glu residue is substituted by Asn and Lys residue respectively;

(e) the 33rd His residue disappearance;

(f) the 116th Gly residue disappearance;

(g) the 117th Arg residue disappearance;

(h) between the 33rd His residue and the 34th 's Pro residue, insert the Thr residue;

(i) between the 33rd His residue and the 34th 's Pro residue, insert the Ala residue;

(j) between the 33rd His residue and the 34th 's Pro residue, insert the Gly residue;

(k) between the 33rd His residue and the 34th 's Pro residue, insert the Gly residue, and the 38th Pro residue is substituted by the Ser residue;

(l) between the 116th Gly residue and the 117th 's Arg residue, insert the Asn residue;

(m) between the 116th Gly residue and the 117th 's Arg residue, insert the Ala residue;

(n) between the 116th Gly residue and the 117th 's Arg residue, insert the Gly residue;

(o) the 129th Leu residue is substituted by the Arg residue;

(p) the 133rd His residue is substituted by the Arg residue;

(q) the 143rd Met residue is substituted by the Arg residue;

(r) the 82nd Gly residue is substituted by the Leu residue;

(s) the 146th Gly residue is substituted by the Leu residue;

(t) the 148th Ser residue is substituted by the Pro residue;

(u) the 59th Lys residue is substituted by the Arg residue;

(v) the 115th Gln residue is substituted by the Arg residue.

14. the DNA of arbitrary claim among the claim 1-13, wherein said albumen further adds peptide T hr Ser Ile Gly Thr Pro Tyr Asp Val Pro Asp TyrAla Gly Val His His His His His His. at its C-terminal

15. the DNA of arbitrary claim among the claim 1-13, wherein said albumen its-2 and-1 also add Met and Lys residue respectively.

16. the DNA of arbitrary claim among the claim 1-13, wherein said albumen also adds the Met residue at its-1.

17. the prokaryotic cell prokaryocyte that transforms with the DNA of arbitrary claim among the claim 1-16.

18. the prokaryotic cell prokaryocyte of claim 17, wherein said prokaryotic cell prokaryocyte is a bacterial cell.

19. the prokaryotic cell prokaryocyte of claim 18, wherein said bacterial cell is a Bacillus coli cells.

20. the eukaryotic cell that transforms with the DNA of arbitrary claim among the claim 1-16.

21. the eukaryotic cell of claim 20, wherein said eukaryotic cell is a mammalian cell.

22. the eukaryotic cell of claim 21, wherein said mammalian cell is a Chinese hamster ovary celI.

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