JPH022353A - Production of human b cell differentiation factor - Google Patents

Abstract

PURPOSE:To obtain a B cell differentiation factor by treating fused protein of human growth hormone A domain, human growth hormone B domain and B cell differentiation factor prepared by cultivating Escherichia coli containing a specific plasmid with an enzyme. CONSTITUTION:A bacterium belonging to the genus Escherichia coli containing recombinant plasmid which contains human B cell differentiation factor gene with DNA sequence shown by formula I, can be subjected to self-propagation in a bacterium and can manifest human B cell differentiation factor prepared, in the cell is cultivated in a nutritive medium. Human B cell differentiation factor is obtained in the form of protein fused with part of N end side of human growth hormone through base sequence of DNA shown by IleGluGlyArg from N end and is digested with blood coagulation factor Xa.

Description

[Detailed Description of the Invention] (Industrial Application Field) B cell differentiation factor (hereinafter abbreviated as BSr-2) is T
Lymphocyte replacement factor, TRF, is used in the culture supernatant after culture of a mouse lymphocyte mixture, or in the lymphocyte supernatant of mice stimulated with antigens or mitogens, or a lymphocyte cell population from which mouse T cells have been removed. It was discovered as a substance that amplifies the primary immune response of lymphocytes derived from nude mice to sheep red blood cells. Since then, TRF acts on B cells in a non-antigen-specific manner that does not require the matching of the major histocompatibility complex (hereinafter abbreviated as MMC), does not induce the division and proliferation of B cells, It is defined as a humoral factor that induces the differentiation of B cells into antibody-producing cells.

Currently, the factor that differentiates B cells into antibody-producing cells as defined above is called BSF-2. Furthermore, Unauri 5F-2 plays an important role in the antibody production function of B cells in the human body.

As for the clinical application of BSF-2, 1) BSF-2 antibody is made with BSF-2, and BSF-2 and anti-BSF-2
2) Application to the treatment of various diseases using an immunoassay system for BSF-2 using antibodies, such as BSF-2, which is associated with decreased helper function of T cells.
BSF-2 for patients with immunodeficiency due to decreased cellular antibody production ability
By administering alone or together with other lymphokines, the antibody production function is restored to normal. 3) By adding T cell factors including B cell growth factors and other lymphokines to the culture medium, normal B cells or EB virus can be transformed. It is conceivable to produce antibodies in vitro by acting BSF-2 on converted B cells at an appropriate time. Furthermore, monoclonalizing B cells that produce specific antibodies, such as antibodies that recognize specific antigens on the surface of pathogenic bacteria, pathogenic viruses, pathogenic protozoa, cancer cells, etc.
Useful monoclonal antibodies can be produced by culturing cloned normal B cells or cells transformed with EB virus in combination with B S 1;'-2 and other lymphokines. These antibodies can be used to treat and diagnose infectious diseases and cancer.

The present invention relates to a method for producing BSF-2.

(Prior art) As a method for obtaining BSF-2, BSF-2 is obtained by stimulating normal human T cells isolated from human peripheral blood with mitogens.
A method for producing F-2, a method for fusing human T cells with human cancer to obtain human T fusion cells, and producing BSF-2 thereby, and a method for expressing DNA encoding BSF-2 in Escherichia coli. As a method of directly expressing BSF-2 under the control of a tryptophan promoter and human IL
A method of expressing the protein as a fusion protein with a partial N-terminal peptide of -2 is known.

In view of the above circumstances, the present inventors have completed a method for producing BSF-2 in a fused form with human growth hormone. That is, the present invention provides a ribosome binding site, a gene encoding human growth hormone A domain and human growth hormone B domain, a gene encoding BSF-2, and a stop codon downstream of a control gene that regulates the expression of structural genes. BSF-2 is obtained by culturing Escherichia coli transformed with the containing plasmid and treating the expressed fusion protein of human growth hormone A domain, human growth hormone B domain, and BSF-2 with an enzyme. The present invention relates to a method for producing BSF-2. The present invention will be further explained in detail below.

(Structures of the Invention) rBSF-2 gene segment and its preparation" That is, the present invention first provides a BSF-2 gene segment [hereinafter referred to as formula (1), consisting of a base sequence represented by the formula In the middle, A is deoxyadenylic acid, C is deoxycytidylic acid, G is deoxyguanylic acid, and T is deoxythymidylic acid. The encoded amino acid sequence is shown below.

This BSF-2 gene segment preferably has at its upstream end a sequence of the formula % (where X is less than the N-terminus).
The present invention provides a DNA sequence that encodes the amino acid sequence represented by the formula, contains a stop codon downstream thereof, and further ends with a restriction enzyme cleavage end downstream of the stop codon, and has a DNA sequence represented by the formula .

X and X of the DNA sequence added to the upstream side are:
An example can be an array represented by the expression % expression %.

Y and Y of the DNA sequence that is added to the downstream side and includes a stop codon and ends further downstream with a restriction enzyme cleavage end is the sequence of the cleavage end of the restriction enzyme BamHI, a sequence represented by the formula % formula % Examples include:

In formula (2) above, the upstream end ends at the BamHI end. An oligonucleotide having a DNA sequence represented by the formula %, which is added to the upstream side of (1), can be prepared by the method described below.

First, each of the main chain portions of these oligonucleotides may be synthesized and hydrogen bonded. In this case, when ligase is used to join oligonucleotides or double-stranded DNA fragments, the 5' ends of the components that participate in the 5' reactions are phosphorylated in advance. Phosphorylation can be performed using conventional techniques and conditions.

The obtained BSF-2 gene segment can be inserted into an appropriate plasmid, transformed into E. coli, etc., and cloned and amplified using indicators such as drug sensitivity and techniques such as rapid isolation.

"Plasmid and its Preparation" The second aspect of the present invention provides a recombinant plasmid containing a BSF-2 gene segment.

That is, the present invention provides a recombinant plasmid that contains the BSF-2 gene segment having the base sequence represented by formula (1) and is capable of self-replication in microorganisms. And preferably,
It contains a promoter/operator system upstream of the BSF-2 gene that controls the expression of this gene, and a part that enables the plasmid to self-replicate between the upstream of the promoter/operator and the downstream of the BSF-2 gene. It is something that As a promoter/operator,
Conventionally, tryptophan promoter/operator system,
Lactose promoter/operator system, or
The tributophane promoter/lactose operator system may be used. The recombinant plasmid of the present invention is
The BSF-2 gene segment can be prepared by incorporating into a suitable vector plasmid in a conventional manner. In particular, the BSF-2 gene segment having restriction enzyme cleavage ends upstream and downstream thereof can be easily integrated into the corresponding cleavage sites of such plasmids, such as sites such as Bglll.

The recombinant plasmid of the present invention can be transformed into microorganisms such as E. coli to produce the product of the human BSF-2 gene of the present invention in the form of a fusion protein. For example, B.S.
Among the F-2 gene segments, those with an end expressed by formula (2) upstream include those that have a site for the restriction enzyme Bgln in the DNA sequence encoding a protein that can be expressed on the plasmid, and those that have a site for the restriction enzyme Bglll in the DNA sequence encoding a protein that can be expressed on the plasmid. A plasmid that can produce a fusion protein between a part of the N-terminal side of the protein and the BSF-2 protein linked via the amino acid sequence lie Glu Gly Arg is obtained by integrating the protein so that the reading phases match. Can be built. An example of such a protein is human growth hormone, and an example of a DNA sequence containing a Bg111 site is the pGII-L series plasmid disclosed in JP-A-60-234584. I can do it.

The recombinant plasmid thus constructed has a DNA sequence from the Messenger RNA transcription start point of the promoter-operator system to the BSF-2 gene of the formula % (X and X- represent the same meanings as above). Particularly preferred. A plasmid with such a sequence is, for example, a pGH-L9 plasmid digested with BglII, which has a terminal sequence of the formula (2) on the upstream side of the BSF-2 gene segment and a terminal sequence of the formula (3) on the downstream side. It can be prepared by adhesively ring-closing a terminal having the terminal sequence represented by Figure (2) shows this preparation. Plasmid pGBs1m can be constructed in this manner. In addition, E.coli strain carrying plasmid pG■-1,9, E.coli strain
li pGH-L9 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, as Microtechnology Research Institute Deposit No. 7606.

"Transformant" E, col with the recombinant plasmid pGBsl of the present invention
The BSF-2 gene can be efficiently amplified by transforming an i strain, such as the R8791 strain, and culturing it in a nutrient medium. Moreover, the BSF-
Those with a promoter-operator system, especially a tryptophan promoter-operator system, that controls the expression of BSF-2 upstream of the gene express BSF-2 in the form of a fusion protein with a part of the N-terminal side of human growth hormone. , accumulates within the bacterial body.

In this case (tryptophan promoter operator system) BSF-2 production (mRNA synthesis) is strongly induced by the addition of indoleacrylic acid.

The fusion protein of BSF-2 and human growth hormone accumulated in the bacterial cells can be separated and recovered by a normal bioactive protein recovery method after the bacterial cells are lysed. For example, after culturing, the bacterial cells are collected by centrifugation, and Tris-
The protein is suspended in a hydrochloric acid buffer, crushed, and treated with urea, etc., and the resulting supernatant is subjected to gel filtration to obtain the desired protein fraction.

The microorganism-mediated fusion protein of the present invention with human growth hormone has a blood coagulation factor Xa recognition site (l l
e-Glu-Gly-Arg), and by treating with this enzyme, three amino acid residues (G
lu-Phe-Met) can be added to BSF-2.

Example 1 (Preparation of oligonucleotide) (1) S″GATCCTCATCGAAGGTCGTG
3-(2) '-AATTCCAGACCTTC
Preparation of GATGAG 3- The above oligonucleotides (1) and (2) were transferred to a DNA synthesizer model 1-381A (Appl.
It was synthesized using ied Biosys (manufactured by Lem). After the synthesis was completed, the oligonucleotide adsorbed on the carrier in the column was treated with 1 ml of aqueous ammonia for one hour and eluted. This ammonia solution was placed in a container equipped with a screw vial and reacted overnight at 55°C. The solvent was distilled off under reduced pressure, the residue was dissolved in 500 μl of distilled water, and the solution was diluted with diethyl ether 5 times.
After extracting unnecessary substances with 00 μl, the solvent was distilled off under reduced pressure again.

The residue was dissolved in 10mM Tris-HCl buffer solution (pH 7,5)/
They were dissolved in 200 μm of 1 m ethylenediamine tetraacetate, and the absorbance was measured at 260 nm during annealing to determine the concentration, and each solution was prepared at 10 pmol/μl.

The above oligonucleotide (1) obtained by synthesis,
A reaction was carried out using (2) under the following conditions to obtain a synthetic fragment.

Synthetic oligonucleotide (10 pmol) 5μQ5
×Kination buffer IOμeO, 01M
ATP 5μQT4 polynucleotide kinase 2μQ (manufactured by Zenshuzo Co., Ltd., 10U/
μl) The composition of the caination buffer is 50mM) Lis-HCl buffer solution (pH 9', 6), 10mM magnesium chloride,
2mM spermidine, 10mM dithiothretol, 10
It was incubated for 20 minutes at 37"C with 0mM potassium chloride. After incubation, the synthetic oligonucleotide (
The solutions 1) and (2) were mixed and reacted at 65° C. for 10 minutes, and then slowly cooled to room temperature for annealing.

This operation yielded a synthetic fragment at a concentration of 5 pmol/μl.

Example 2 (Preparation of recombinant plasmid and transformed strain) The recombinant plasmid of the present invention is a fusion protein expression type, and will be described in detail in the following example.

The known plasmid pBsF2.38 was digested with the restriction enzyme Alul.
and BamHI to create approximately 1000 base pairs of A I
The u I-BamH1 fragment was extracted.

On the other hand, the known plasmid pGEM4 was converted into Smal and Ba
Digested and cleaved with mHI. Cut out Alul-B
amHI fragment into T, pGEM in the presence of DNA ligase
4 into the Sma L BamH1 cleavage site to prepare plasmid pBSF2.381. The plasmid was then digested with Bco RII and Hindm to remove approximately 1000 base pairs of the translation initiation codon.
The oRII-HindI[I fragment was obtained. Next, the expression containing the translation start codon, '-AATTCATGCCGGTT
The oligonucleotide shown in CCG 3-GTACGGCCAAGGCGGTCC was synthesized, and this synthetic oligonucleotide and Eco from which the above translation start codon was removed
RU-Hi ndII [fragment was converted into a known plasmid pk
k223-3 was digested and cleaved with Eco RI and Hindl II, and then inserted and closed using T and DNA gauze to create pT containing the B cell differentiation factor gene.
AB1 plasmid could be obtained (Fig. 1). The obtained plasmid pTABI was transformed into E. coli and subjected to cloning and amplification. Plasmid pTAB1 was digested with Eco RT and 5ail, and approximately 1000 base pairs of EcoRl-5 were extracted by polyacrylamide electrophoresis.
Ail fragment was obtained. Followed by the formula, “GATCCTCATCGAAGGTCGTG 3
An oligonucleotide represented by GAGTAGCTTCCAGCACTT was synthesized. The above oligonucleotide has a BamHI structure at the 5-end and an Eco structure at the 3-end.
n I structure, lie Glu Gly
It encodes the digestion recognition amino acid sequence of blood coagulation factor Xa, represented by Arg. The above synthetic oligonucleotide and EcoRI-Sail fragment of plasmid pTAB1 were combined with B
The plasmid was inserted into ptJc9, which had been digested and opened with amHl and 5all, and closed to prepare a ptJBs-1 plasmid. Furthermore, the plasmid ptJBs-1 was transformed into Escherichia coli for cloning and amplification.

(Fusion expression type pGBS-1> Plasmid pGH-L9 (E. coli pGH-L9
30 μg of Bgln (manufactured by Zenshuzo Co., Ltd.) was reacted with 100 units of Bgln (manufactured by Zenshuzo Co., Ltd.) at 37° C. for 3 hours. The composition of the reaction solution (ml) is 10mM) Lis-HCl buffer (pH 7.5), 7mM magnesium chloride, 10mM)
0mM sodium chloride, 7mM 2-mercaptoethanol, 100μg/ml bovine serum albumin.

After collecting the DNA by precipitation with ethanol, it was dissolved in 100 μl of Tris-HCl buffer solution and reacted with 0.9 units of alkaline phosphatase (manufactured by Zenshuzo Co., Ltd.) at 65°C for 30 minutes to remove the 5-terminal phosphate group. was removed. Furthermore, phenol treatment and chloroform treatment were performed, and the DNA was recovered by precipitation with ethanol, and the vector DNA pGH-
L9/Bg I II was obtained. Obtained vector DNA
pGH-L9/BglII (0.5μg/ml)lp
1 and ptrBs-1 were digested with BamHI, and an approximately 1000 base pair BamHI fragment (0.1 μg/μl) was obtained by polyacrylamide gel electrophoresis.
5 μl of the mixture was mixed and reacted for 30 minutes at 16° C. using a ligation kit (manufactured by Zenshuzo Co., Ltd.). 10μl of this reaction solution
was transformed into E. coli, and the transformed strain E, coli/pGBS
−1 was obtained (Figure 2).

Example 3 (Expression) E. coli/pGBS-1 obtained in Example 1 was cultured in medium 5.
ml overnight at 37°C. 50 μl of the obtained culture solution
was added to 5 ml of L medium, cultured, and Absorbance 600
When the absorbance reached 0.4 to 0.5 nm, 50 μm of indoleacrylic acid (IOmg/ml) was added and cultured for an additional 4 hours.

After culturing, use a centrifuge for 10. OOOrpm.

The cells were collected by centrifugation for 10 minutes. A portion of it was added to sample buffer (62.5 ml) and Lis-HCl buffer (pH 6).
-8), 2% SDS, 5mM ethylenedeamine chitraacetate, 10% glycerol, 35mM 2-mercaptoethanol, 0.001% bromophenol blue)
After heating in boiling water for 5 minutes, 0-1% 5D
Electrophoresis was performed on S-12, 5% PAGE, and the molecular weight was 3.
6x104 cells were obtained (Fig. 3).

Example 4 (BSF-2 from fusion protein with blood coagulation factor Xa)
E. coli/pGBS-1 was induced with indole acrylic acid in Noshi medium, and after culturing for 4 hours, the bacteria were harvested and approximately IQg (
Wet weight) was obtained. The obtained bacterial cells were mixed with 50mM) Lisu salt?
! l! The suspension was suspended in No. 21 buffer (pH 8,0), crushed, and centrifuged to obtain a precipitate. The obtained precipitate was suspended in 50 mM glycine-sodium hydroxide buffer (pH 9,0), and a buffer solution was prepared so that the final concentration of urea was 8M.

The dialysate containing IM urea (1 hour) was heated at 60°C for 1 hour.
M urea, 50mM glycine-sodium hydroxide buffer (
pH 9,0), 2mM reduced glutathione, 0°2mM
After 12 hours of dialysis with oxidized glutathione, 1mM ethylenediaminetetraacetate), dialysis was performed with 50mM Tris-HCl buffer (pH 8.0) for 6 hours. The dialysate was centrifuged and the supernatant was diluted to a final concentration of 100mM sodium chloride, 1mM
Calcium chloride, 50mM) Lis-HCl buffer (pH 8
-0). To this solution, 10 units of blood coagulation factor Xa (manufactured by Boehringer Mannheim, Inc.) per 50 mg of protein was added and reacted at 37°C for 10 hours. After centrifuging the reaction solution, the supernatant was transferred to Diaflo
Concentrate approximately 10 times using YM-10 (manufactured by Amicon) to give a final solution of 50 mM) Lis-HCl buffer (pH 7.6).
), 2mM dithiothretol, 6M guanidine hydrochloride,
A 0.1M sodium chloride solution was prepared. This solution is
SK Gel G3000SW column (25m
mX 600 mm), and the B cell differentiation factor elution fraction was diluted with 1M guanidine-HCl, 50mM) Lis-HCl buffer solution (
pH 8,0), 1mM ethylenediaminetetraacetate followed by 10mM) Lis-HCl buffer (pH 8,5)
Dialyzed with This dialysate was added to TSKGel DEAE-
Apply to 5FW column (25mm x 200mm) and
It was eluted by a linear concentration gradient method using a 0.8M sodium chloride solution. The eluted fraction was subjected to acrylamide electrophoresis, and molecule 92, which is presumed to be a B cell differentiation factor, was detected.
A fraction of 1°000 was obtained (Figure 3).

(Effects of the Invention) The recombinant plasmid (expression type) of the present invention efficiently amplifies the BSF-2 gene segment, and transforms the BSF-2 protein into a fusion protein with human growth hormone into a microorganism transformed thereby. It can give the production ability in
In addition, the BSF-2 fusion protein of the present invention has blood coagulation factor
BSF-2 with three amino acid residues (Glu-Phe-Met) added from the N-terminus by treatment with a
It can be done.

[Brief explanation of the drawing]

Figures 4 and 2 are diagrams explaining the preparation of the human BSF-2 fusion expression plasmid of the present invention, and Figure 3 is a diagram explaining the acrylamide electrophoresis pattern of the BSF-2 fusion protein obtained by the present invention. Figure 4 is a diagram of BSF-
FIG. 2 is a diagram illustrating an acrylamide electrophoresis pattern of BSF-2 protein obtained by treating and purifying the BSF-2 fusion protein with blood coagulation factor Xa. Figure 4 arker 1 -ν proboscis - # 1 - 15@1.5DS-PAGE ◆-93kD 嬌--68kD 43kD 30kD 噌--14,4kD

Claims (1)

[Claims] (1) A human B cell differentiation factor gene segment consisting of a DNA sequence represented by the formula [There is a gene sequence]. (2) At the upstream end of the human B cell differentiation factor gene, there is a gene sequence with the formula (where X is the DNA sequence encoding the amino acid sequence expressed by IleGluGlyArg from the N terminus, and There is also a DNA sequence represented by the formula [gene sequence], which contains a stop codon at its downstream end and ends with a restriction enzyme cleaved end downstream of it (in the formula, Y represents the first stop). A patent claim having a DNA sequence from the second base of the codon to the 3' end of the restriction enzyme cleaved end, where Y' is a complementary sequence of Y and represents the 5' end of the restriction enzyme cleaved end. Human B according to item 1 within the scope of
Cell differentiation factor gene segment. (3) The DNA sequences X and X' added to the upstream side are
The human B cell differentiation factor gene segment according to claim 2, which has a sequence represented by the formula [There is a gene sequence]. (4) The human B cell differentiation factor gene segment according to claim 2, wherein Y and Y' of the DNA sequence added to the downstream side are sequences containing the second and third base sequences of a stop codon. . (5) A human B cell according to claim 2, in which the restriction enzyme cleavage ends of Y and Y' of the DNA sequence added to the downstream side include a sequence represented by the formula: [There is a gene sequence] Differentiation factor gene segment. (6) [There is a gene sequence] A method for producing a human B cell differentiation factor gene segment consisting of the DNA sequence represented by. (7) At the upstream end of the human B cell differentiation factor gene, there is a gene sequence with the formula (where X is the DNA sequence encoding the amino acid sequence expressed by IleGluGlyArg from the N-terminus, and X' is its complementary sequence. There is also a DNA sequence represented by the formula [gene sequence], which contains a stop codon at its downstream end and ends with a restriction enzyme cleaved end downstream of it (in the formula, Y represents the first stop). A patent claim having a DNA sequence from the second base of the codon to the 3' end of the restriction enzyme cleaved end, where Y' is a complementary sequence of Y and represents the 5' end of the restriction enzyme cleaved end. Human B according to item 6 within the scope of
Method for producing cell differentiation factor gene segments. (8) The DNA sequences X and X' added to the upstream side are
A method for producing a human B cell differentiation factor gene segment according to claim 7, which has a sequence represented by the formula [there is a gene sequence]. (9) The human B cell differentiation factor gene segment according to claim 7, wherein Y and Y' of the DNA sequence added to the downstream side are sequences containing the second and third base sequences of a stop codon. manufacturing method. (10) A human B cell according to claim 7, in which the restriction enzyme cleavage ends of Y and Y' of the DNA sequence added to the downstream side include a sequence represented by the formula: [There is a gene sequence] Method for producing differentiation factor gene segments. (11) [There is a gene sequence] A recombinant plasmid that contains the human B cell differentiation factor gene segment and is capable of self-replication in microorganisms, consisting of the DNA sequence represented by the following. (12) At the upstream end of the human B cell differentiation factor gene, there is a gene sequence with the formula (where X is the DNA sequence encoding the amino acid sequence expressed by IleGluGlyArg from the N-terminus, and X' is its complementary sequence). There is also a DNA sequence represented by the formula [gene sequence], which contains a stop codon at its downstream end and ends with a restriction enzyme cleaved end downstream of it (in the formula, Y represents the first stop). A human B with a DNA sequence represented by the following: from the second base of the codon to the 3' end of the restriction enzyme cleaved end, where Y' is the complementary sequence of Y and represents the 5' end of the restriction enzyme cleaved end. The recombinant plasmid according to claim 11, which contains a cell differentiation factor gene segment. (13) Claim 12, wherein the DNA sequences X and X' added to the upstream side include a human B cell differentiation factor gene segment that is a sequence represented by the formula: [There is a gene sequence] recombinant plasmid. (14) Claim 12, which includes a B cell differentiation factor gene segment in which Y and Y' of the DNA sequence added to the downstream side is a sequence containing the second and third base sequences of a stop codon. Recombinant plasmids as described. (15) A patent claim in which the restriction enzyme cleavage ends of Y and Y' of the DNA sequence added to the downstream side include a human B cell differentiation factor gene segment containing the sequence represented by the formula: [There is a gene sequence] The recombinant plasmid according to item 12. (16) The DNA sequence from the messenger RNA transcription start point of the promoter/operator system to the human B cell differentiation factor gene is expressed as follows: A recombinant plasmid having a DNA sequence represented by (X' indicates its complementary sequence). (17) A microorganism belonging to the genus Esharicha that contains a human B cell differentiation factor gene with the sequence represented by the formula [gene sequence is available] and has a recombinant plasmid capable of self-replication in the microorganism. (18) At the upstream end of the human B cell differentiation factor gene of the recombinant plasmid, there is a gene sequence with the formula indicates the complementary sequence), and contains a stop codon at its downstream end, and ends with the restriction enzyme cleavage end further downstream, and there is a gene sequence with the formula, [in the formula, Y represents the sequence from the second base of the first stop codon to the 3' end of the end cleaved by the restriction enzyme, and Y' is the complementary sequence of Y and represents the 5' end of the end cleaved by the restriction enzyme) The microorganism according to claim 17, which has the following. (19) Claims No. 1 possessing a recombinant plasmid containing a human cell differentiation factor gene in which the DNA sequences X and X' added to the upstream side are the sequences represented by the formula [There is a gene sequence] The microorganism according to item 18. (20) The microorganism according to claim 18, which has a recombinant plasmid in which the DNA sequences Y and Y' added to the downstream side include the second and third base sequences of a stop codon. (21) A recombinant plasmid containing a human B cell differentiation factor gene segment in which the restriction enzyme cleavage ends of Y and Y' of the DNA sequence added to the downstream side are the sequence represented by the formula: [There is a gene sequence] The microorganism according to claim 18, which has the following. (22) The DNA sequence from the messenger RNA transcription start point of the promoter/operator system to the human B cell differentiation factor gene is expressed as follows: A microorganism that possesses a recombinant plasmid having a DNA sequence represented by the following sequence (X' indicates its complementary sequence). (23) A recombinant plasmid containing the human B cell differentiation factor gene with the DNA sequence represented by the formula [gene sequence is available], capable of self-replication in microorganisms, and capable of expressing the human B cell differentiation factor is injected into cells. A microorganism belonging to the genus Esharicia, which is present in the human body, is cultured in a nutrient medium to accumulate human B cell differentiation factor protein, which is collected and digested with blood coagulation factor Xa as necessary. Method for producing B cell differentiation factor. (24) At the upstream end of the human B cell differentiation factor gene, there is a gene sequence with the formula There is also a DNA sequence represented by the formula [gene sequence], which contains a stop codon at its downstream end and ends with a restriction enzyme cleaved end downstream of it (in the formula, Y represents the first stop). A plasmid consisting of a DNA sequence from the second base of the codon to the 3' end of the restriction enzyme cleaved end, where Y' is the complementary sequence of Y and represents the 5' end of the restriction enzyme cleaved end. 24. The production method according to claim 23, using the microorganisms possessed. (25) Using a microorganism carrying a recombinant plasmid containing a human B cell differentiation factor gene segment in which the DNA sequences X and X' added to the upstream side are expressed by the formula [There is a gene sequence] The manufacturing method according to claim 24. (26) A microorganism carrying a recombinant plasmid containing a B cell differentiation factor gene segment in which Y and Y' of the DNA sequence added to the downstream side is a sequence containing the second and third base sequences of the stop codon. The manufacturing method according to claim 24 used. (27) A recombinant plasmid containing a human B cell differentiation factor gene segment whose restriction enzyme cleavage ends of Y and Y' of the DNA sequence added to the downstream side contain a sequence represented by the formula: [There is a gene sequence] 25. The production method according to claim 24, which uses a microorganism having the following. (28) A human B cell differentiation factor is obtained in the form of a protein fused to a part of the N-terminal side of human growth hormone through the DNA base sequence expressed by IleGluGlyArg from the N-terminus, and this is converted into blood coagulation factor Xa. 24. The production method according to claim 23, characterized in that the method is digested with: (29)N
From the terminal side, the cleavage recognition sequence of blood coagulation factor Xa, Ile
29. The production method according to claim 28, wherein the DNA sequence encoding the amino acid sequence represented by Glu Gly Arg has the following formula: [There is a gene sequence]. (30) A claim characterized in that the sequence from the messenger RNA transcription start point of the promoter/operator system to the DNA base sequence encoding the thrombin recognition site is a sequence represented by the formula [There is a gene sequence] The manufacturing method according to item 28. (31) Human B cell differentiation factor fusion protein expressed by the formula [gene sequence available] [gene sequence available].