JP5280001B2 - Transgenic birds in which foreign gene-derived protein contains Gla residue and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for producing a protein containing a Gla residue. <P>SOLUTION: A transgenic fowl is obtained by a method for infecting the heart or blood vessel formed in an embryo of a fowl in the initial stage of embryo development with a retrovirus vector containing an extraneous gene by micro-injection and hatching the embryo. Thereby, the protein containing the Gla residue is produced. <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

Description

The present invention relates to a method for producing a transgenic bird containing a foreign gene, and relates to the expression of a protein containing a Gla residue, a serine protease, and a protein containing a recognition site for a serine protease by the transgenic bird. More specifically, it relates to the expression of proteins having biological activity for blood coagulation containing Gla residues by transgenic birds.

In recent years, many medicinal proteins have been used. This is because a gene recombination technique for introducing a gene of a target protein into a microorganism or a mammalian cell has been developed and applied, and the protein can be industrially produced by culturing these gene recombinants. In order to exhibit the physiological activity inherent to a medicinal protein, it is necessary to fold the protein in the same manner as it exists in nature and to undergo post-translational modifications such as glycosylation and disulfide bonds.

A method for producing a protein by culturing a microorganism can produce the protein at a low cost because the growth rate of the microorganism is high and the medium composition is simple. However, in many microorganisms, the post-translational modification of the target protein is often not performed correctly. Therefore, it is difficult to obtain a sufficient amount of protein having the same physiological activity as that of the natural type, and there is a long way to put industrial production to practical use.
Until now, a method for producing a target protein by introducing a gene of the target protein into a mammalian cell and culturing the cell has become the mainstream. Pharmaceutical proteins such as blood coagulation factors, thrombolytic agents and antibody drugs are produced, sold and used using genetically modified mammalian animal cells. However, the method using mammalian cells has a problem that a dedicated culture tank or medium is required and the production cost is high.

Animal factories have attracted attention to overcome these problems. This is a technique for producing a target protein using a transgenic animal. Attempts have been made to produce transgenic mammals using goats, sheep, cattle, etc., and to produce the target protein in the milk. Although it depends on the target protein, there is also a report that the antibody was expressed at 10 mg / ml in milk (see Non-Patent Document 1). However, even in this method, the problem of bovine spongiform encephalopathy (BSE) and the available mammals are large in size, difficult to produce, maintain and manage, the period of sexual maturity is 8 months for goats and sheep, There are problems such as being 15 months long.

Therefore, a method for expressing a target protein in eggs using transgenic birds has been studied. This method has high egg productivity, no BSE problem, the sexual maturity period is as short as 5 months in chickens, and it is possible to raise a large number of individuals because of its small size, artificial insemination Advantages include the fact that the law has been established and large-scale transgenic groups can be rapidly grown, and that eggs are generally sterile in nature.
Methods for producing transgenic birds are under investigation, including methods using retroviral vectors, methods using embryonic stem cells, methods using primordial germ cells, and methods for attaching and introducing a target gene into sperm. The method using a retroviral vector is the most common. So far, studies using avian leukemia virus (ALV) replication-defective retrovirus vectors have been reported (see Patent Document 1). Β-lactamase is used as the target protein, and a cytomegalovirus (CMV) promoter gene is used as the promoter gene. A retrovirus vector has been introduced into the stage X blastoderm immediately after egg laying to successfully produce transgenic birds. The expression level is reported to be 0.33 mg / ml (concentration of egg white is 40 ml) in the analysis method using Western blot, and 0.003 to 0.033 mg / ml in terms of β-lactamase activity. . Moreover, the appearance frequency of the G0 transgenic chimera bird at that time is 20%. As a result of examining the efficiency of introduction into germ cells, about 5% of males of G0 transgenic chimeric birds had a transgene in their sperm. As another report, there is a report that the expression of about 1.2 μg / ml in egg white is high in G2 in which genes are introduced into the whole body in the same experiment (see Non-Patent Document 2). . The appearance frequency of G0 to G1 at that time was 3/422 (0.71%). In another report, a high virus titer, infectivity, and expression were obtained using a mouse stem cell virus (MSCV) vector and a VSV-G envelope (see Patent Document 2). Furthermore, in the report, high expression was also achieved by adjusting the timing of introduction of the retroviral vector, and an anti-prion single chain antibody (scFv) was used as the target protein and 0.5% in egg white and egg yolk. Expression of ˜1 mg / ml is realized.

Next, the background of blood coagulation factors will be described. Blood coagulation factor VII is one of the extrinsic blood coagulation factors and is a precursor of serine protease present in plasma at 0.5 μg / μl. In exogenous blood coagulation, factor VII is first between Arg152 and Ile153 by serine proteases such as factor XIIa, Xa, and factor IXa (those with a are active forms and those without a are precursors). The peptide bond is cleaved to become the active form factor VIIa. It is further activated when tissue factor binds to factor VIIa. As a result, factor VIIa has the ability to activate factor X or factor IX, and activation to factor Xa or factor IXa occurs. Thereafter, this action is linked and amplified. After amplification, activated factor Xa activates prothrombin to thrombin, which restricts fibrinogen to fibrin. Fibrin polymerizes to form a fibrin network, completing blood clotting. Among blood coagulation factors, factor II, factor VII, factor IX, factor X and the like are called vitamin K-dependent coagulation factors and require vitamin K to function normally in the blood coagulation process. This is because glutamic acid on the N-terminal side of the vitamin K-dependent coagulation factor is carboxylated to a Gla (γ-carboxyglutamic acid) residue in the presence of vitamin K, and then acquires a normal coagulation function. Specific functions, Gla residues are involved in binding to Ca ^2+, the Ca ²⁺ binds to Gla residues believed Gla domains facilitate the localized chain reaction on a phospholipid membrane Yes. In addition, it is considered that factor VII contributes to the interaction with tissue factor (see Non-Patent Document 3).

So far, factor VII has been produced using mammalian cells. In particular, production using baby hamster kidney (BHK) cells is common. Factor VII has 10 Gla residues on the N-terminal side, but not all of them are completely glazed even in production using BHK cells which are the same mammals (see Non-Patent Document 4). Regarding productivity, the expression level in the medium was 975 ng / ml by the quantitative method using ELISA, and 600 ng / ml by the quantitative method converted from the activity (see Patent Document 3). On the other hand, factor VII is self-activated (see Non-Patent Document 5) or undergoes autolysis that is different from activation during purification (see Patent Document 4). It is a difficult protein.
JP-T-2001-520009 JP 2002-176880 A JP-A-10-117787 Japanese National Patent Publication No. 8-508264 Trends Biotechnol. 1999 Sep; 17 (9): 367-74. Nat Biotechnol. 2002 Apr; 20 (4): 396-9. Hideaki Mizoguchi, Hisamaru Hirai, Yoichi Sakata "Blood Disease" Medical Dentistry Publishing 1st pp. 109-112 Biochemistry. 1988 Oct 4; 27 (20): 7785-93. Biochemistry. 1989 Nov 28; 28 (24): 9331-6.

Some of the blood clotting factors require Gla modification for their activation, such as II, VII, IX, factor X, protein C and the like. These factors have a serine protease recognition site in the sequence and are cleaved and activated at the recognition site to have serine protease activity. In particular, blood coagulation factor VII is a protein that is very easily degraded, and is an unstable protein that undergoes self-activation and cleavage during purification. As a method for producing such a protein, production of blood coagulation factor VII using BHK cells has been performed so far, but human blood coagulation factor VII can also be produced by culturing BHK cells of the same mammal. It is known that Gla conversion is not completely performed. It is also reported that productivity is low.
An object of the present invention is to provide a novel production method for a protein containing a Gla residue, a serine protease, a protein containing a recognition site for a serine protease, and the like.

We aim to obtain proteins containing Gla residues, serine proteases, and proteins containing serine protease recognition sites other than in mammalian cell culture. We considered the use of transgenic birds for the production of the containing protein. Specifically, blood coagulation factor VII having activity only after receiving Gla modification was used as the target protein. Protein modifications vary among tissues within the same individual. Moreover, taxonomically different birds are not normally considered to exhibit human blood coagulation activity due to Gla modification so that mammalian blood coagulation factor VII has activity. In the present invention, transgenic birds that produce active precursors of blood coagulation factor VII can be obtained. Therefore, foreign proteins having a Gla residue, serine proteases, and proteins containing recognition sites for serine proteases by transgenic birds can be obtained. The production method is shown.

That is, the present invention relates to an exogenous gene comprising a coding sequence of at least one protein selected from the group consisting of a protein activated by Gla modification, a serine protease, and a protein containing a recognition site for serine protease, or a precursor thereof. It is a transgenic bird having
Preferably, the foreign gene is a foreign gene containing a coding sequence of a protein activated by Gla modification, or a foreign gene containing a coding sequence of a serine protease and / or precursor protein thereof, An exogenous gene containing a coding sequence of a protein containing a recognition site for serine protease.
The protein activated by the Gla modification is any one of blood coagulation II, VII, IX, factor X, protein C, protein S, and protein Z and / or a protein exhibiting substantially the same biological activity. Preferably, the serine protease and its precursor, and the protein containing the serine protease recognition site are blood coagulation II, VII, IX, X, XI, factor XII, prekallikrein, plasmin, protein C, plasminol It is preferably a protein exhibiting substantially the same biological activity as any one of gen, antiplasmin, tissue plasminogen activator and / or them.
The transgenic birds of the present invention are preferably produced using a retrovirus vector.
More preferably, the transgenic bird of the present invention is produced using a retroviral vector containing a chicken β-actin promoter gene and / or a woodchucks post-translational regulatory element sequence.
More preferably, the transgenic birds of the present invention are prepared using a retroviral vector containing a VSV-G envelope.
More preferably, the transgenic birds of the present invention are produced using a replication-defective mouse stem cell virus as a retroviral vector.
The transgenic bird of the present invention can also be obtained by a method comprising incubating a fertilized avian egg, infecting a retroviral vector containing a foreign gene into an embryo 24 hours after the start of incubation, and allowing the embryo to hatch. Is preferred.
The present invention also provides a protein derived from an exogenous gene comprising any one of the above-mentioned transgenic birds and their progeny extracted from, extracted from, purified from, somatic cells and / or eggs thereof, purified, activated, or a combination thereof. This is the production method.
The present invention is also selected from the group consisting of activated blood coagulation II, VII, IX, factor X, protein C and its precursor containing 0.05 to 0.5 nmol N-acetylneuraminic acid per nmol. A protein selected from the group consisting of activated blood coagulation II, VII, IX, factor X, protein C and a precursor thereof containing 0.2 to 0.4 nmol of N-acetylneuraminic acid per nmol; preferable.
The protein is preferably a protein selected from the group consisting of activated blood coagulation factor VII and its precursor.

The present invention is described in detail below.
The transgenic bird of the present invention is a foreign bird comprising a coding sequence of at least one protein selected from the group consisting of a protein activated by Gla modification, a serine protease, and a protein containing a recognition site for serine protease, or a precursor thereof. It has a sex gene.
A protein is a peptide in which two or more amino acids are peptide-bonded, and includes a protein having a short chain length usually called a peptide or an oligopeptide. In addition, the amino acid may be modified. For protein secretion, it is preferable that a secretory signal sequence is provided in the coding sequence of the protein. The secretory signal sequence is not limited to the self sequence.
Gla refers to 4-carboxyglutamic acid (γ-carboxyglutamic acid), which is obtained by adding a carboxyl group to the γ carbon of glutamic acid. The term “Gla modification” means that at least one glutamic acid residue in a protein becomes Gla (becomes Gla) after the protein is translated.

Examples of proteins activated by Gla modification include, but are not limited to, blood coagulation II, VII, IX, factor X, protein C, protein S, protein Z, osteocalcin, matrix Gla, and the like. Preferably, the protein exhibits any one of blood coagulation II, VII, IX, factor X, protein C, protein S, and protein Z and / or a biological activity substantially the same as them.
“Proteins exhibiting substantially the same biological activity as those” means that one or several amino acid residues in the amino acid sequence of the protein activated by the Gla modification are deleted, added and / or substituted. The amino acid sequences are those that do not change in physiological action. If the physiological actions are the same, they are made substantially the same regardless of the magnitude of the activity.
The same applies to the following “proteins showing substantially the same biological activity”.

Next, the recognition site of serine protease will be described. In general, serine proteases selectively cleave peptide bonds on the carbonyl side of arginine and lysine. Therefore, a protein containing a serine protease recognition site is a protein containing arginine and lysine residues. For cleavage by serine protease, it is more preferable that the peptide chain containing arginine and lysine residues is exposed on the surface of the tertiary structure of the protein. Whether the target protein is exposed on the surface of the tertiary structure is examined by reacting the target protein with serine protease in an appropriate reaction solution in which serine protease can act, and whether the target protein is cleaved by serine protease. . Serine protease and its precursor, and proteins having a recognition site for serine protease, blood coagulation I, II, V, VII, VIII, IX, X, XI, XII, factor XIII, prekallikrein, plasmin, protein C, tissue Examples include, but are not limited to, plasminogen activator and its precursor. Preferably blood coagulation II, VII, IX, X, XI, factor XII, prekallikrein, plasmin, protein C, plasminogen, antiplasmin, tissue plasminogen activator and / or substantially the same It is a protein showing the biological activity of

The transgenic birds of the present invention also contain an exogenous gene containing a coding sequence for a serine protease and / or precursor protein thereof.
Serine protease is a general term for proteases having serine residues in the active site. Serine protease is limitedly decomposed from an inactive or weakly active precursor into an active form. Serine proteases include, but are not limited to, blood coagulation II, VII, IX, X, XI, factor XII, prekallikrein, plasmin, protein C, plasminogen, tissue plasminogen activator, and the like.

The exogenous gene is not particularly limited, and it may include avian genes as well as genes other than birds. Even a sequence possessed by an individual used to produce a transgenic bird is referred to as a foreign gene because the gene is newly introduced into all the genes originally possessed by the individual.
In the present invention, the exogenous gene includes a protein coding sequence, and the boundary of the coding sequence is determined by the 5 ′ terminal initiation codon and the 3 ′ terminal termination codon corresponding to the initiation codon. It is preferable that the vicinity of the initiation codon at the 5 ′ end contains a Kozak consensus sequence. A ribosome binding site is preferably present upstream of the coding sequence.
The foreign gene preferably contains at least part or all of a promoter gene suitable for expression in avian cells. A promoter gene is a region on DNA or RNA that determines the transcription start site of a gene and directly regulates its frequency.
The foreign gene may contain an untranslated region other than the above.

For the production of the transgenic birds of the present invention, a retrovirus vector is preferably used. Retroviral vectors include different forms of plasmids, viral particles, and packaging cells. A packaging cell is a cell into which a gene encoding at least one protein necessary for viral particle replication has been introduced.
The retroviral vector used in the present invention preferably lacks replication ability in consideration of safety. As a method for deleting replication ability, any of protein (group specific antigen: gag), reverse transcriptase (polmerase: pol), and envelope glycoprotein (envelo: env) included in the inner core necessary for virus particle replication It is preferable to delete at least part or all of the coding sequence or a sequence necessary for expression, or to include substitution or insertion mutation so that they cannot be expressed so that any of these or combinations thereof are not expressed. Since retrovirus vectors are limited in the length of genes that can be inserted for each virus, deletion mutations are preferable. From the viewpoint of increasing safety and the length of the inserted fragment, a plurality of gag, pol, and env are deleted. It is preferable to make it.
The retroviral vector preferably contains a viral packaging signal (phi) that functions as a landmark packaged in the viral particle. Since a part of the gag region may function as a virus packaging signal, it is preferable that the viral vector contains at least a part of the gag region that cannot be expressed (J Virol. 1987 May; 61 (5): 1639-46.).

Although it does not specifically limit as a retrovirus, Moloney murine leukemia virus, Moloney murine sarcoma virus, avian leukemia virus (ALV), human immunodeficiency virus (HIV), mouse stem cell virus (MSCV), mouse embryonic stem cell virus (MESV), etc. Is mentioned. Although Moloney murine leukemia virus and / or Moloney murine sarcoma virus are preferable, infecting embryos of birds, mouse stem cell virus (MSCV), mouse embryonic stem cell virus (MESV), etc. are highly infectious to embryonic cells and stem cells. It is preferable to use a virus. More preferred is a replication-defective mouse stem cell virus (MSCV). In the present invention, in order to efficiently infect avian cells with this viral vector, it is preferable to artificially use the coat protein as a VSV-G envelope protein derived from bovine vesicular stomatitis virus, but it is limited to this virus type. It is not something.

The replication-defective retrovirus vector of the present invention preferably contains a tissue-specific promoter gene or a tissue non-specific promoter gene.
A tissue-specific promoter gene is a promoter gene that is particularly strongly active in specific tissues / cells of birds. By using a tissue-specific promoter gene, there is an advantage that the possibility that the expression of the target protein adversely affects the development and survival of birds can be reduced or eliminated.
Although it does not specifically limit as a tissue-specific promoter gene, A part or all of an oviduct-specific promoter gene can be mention | raise | lifted. The fallopian tube tissue is actively induced after sexual maturity because it is active after sexual maturation.
The oviduct-specific promoter genes include avian ovalbumin, ovotransferrin, ovomucoid, ovomucin, lysozyme, G2 globulin, G3 globulin, ovoinhibitor, ovoglycoprotein, ovoflavoprotein, ovomacroglobulin, cystatin, avidin A promoter gene etc. can be mention | raise | lifted and it is preferable that some or all of these are included. When these oviduct-specific promoter genes are used, the target protein can be highly expressed in egg white, which is particularly preferable.

A tissue non-specific promoter gene is a promoter gene that is not a tissue-specific promoter gene. Although it is not specifically limited as a tissue non-specific promoter gene, it means that which is active in almost all somatic cells of birds. In this case, since the target protein is also expressed in blood, there is an advantage that it is possible to detect whether expression is possible at the chick stage.
The tissue non-specific promoter gene is not particularly limited, but β-actin promoter gene, EF1α promoter gene, thymidine kinase promoter gene, simian virus 40 (SV40) promoter gene, cytomegalovirus (CMV) promoter gene, Rous sarcoma virus Examples thereof include virus-derived promoter genes such as (RSV) promoter genes. Alternatively, a tissue non-specific inducible promoter gene such as a tetracycline inducible promoter gene may be used. Examples of the β-actin promoter gene include a chicken β-actin promoter gene, and the tissue non-specific promoter gene preferably includes a chicken β-actin promoter gene.

The replication-defective retrovirus vector of the present invention may contain a transcription enhancer and / or a regulatory element. A transcription enhancer is a sequence on DNA or RNA that is a sequence that promotes transcription from a promoter gene but cannot cause transcription by itself. Since it often functions even when linked to a promoter gene different from the one that originally functions, the combination with the promoter gene is not particularly limited. The transcription enhancer is not particularly limited, and examples thereof include SV40 and CMV-derived thymidine kinase enhancers, steroid response elements and lysozyme enhancers. A regulatory element is a region on DNA or RNA that cannot contribute to transcription by itself, contributing to transcriptional regulation and stabilization of RNA after transcription. Although it does not specifically limit as a regulatory element, Woodchuck hepatitis virus origin regulatory element (Woodchuck post-transcriptional regulatory element: WPRE, US Patent No. 6136597) etc. are preferable.

The retroviral vector used in the present invention may contain a marker gene. A marker gene is a gene that encodes a protein that serves as a marker for identifying and isolating correctly transfected cells. No particular limitation is imposed on the marker gene, green fluorescent protein (GFP) or cyan fluorescent protein (CFP), fluorescent proteins such as luciferase gene, neomycin resistance (Neo ^r), hygromycin resistance ( Hyg ^r ), puromycin resistance (Puro ^r ) and other drug resistance genes; in addition, thymidine kinase, dihydrofolate reductase, aminoglucoside phosphotransferase, chloramphenicol acetyltransferase, β-lactamase, β- Examples include genes such as galactosidase. The marker gene is preferably accompanied by a promoter gene and an element necessary for expression.

Retroviral vectors contain at least a portion of a long terminal repeat (LTR) at the 5 'end and 3' end. Since the LTR has a transcription promoter gene and a polyA addition signal, it can be used as a promoter gene or a terminator gene. In the retroviral vector, the target protein coding sequence, promoter gene, transcription enhancer and / or regulatory element is contained between the 5 'LTR and the 3' LTR. When a promoter other than the LTR is used, it preferably has a structure in which the coding sequence of the target protein is connected downstream of the promoter. In order for a retrovirus vector to be transcribed and a virus particle to be produced, it is preferred that no terminator or polyA signal be included between the 5 'LTR and the 3' LTR.

Next, a preferred embodiment of the method for preparing a replication-defective retrovirus vector suitably used in the present invention will be described.
The replication-defective retrovirus vector used in the present invention preferably lacks the gag, pol and env genes required for replication. A replication-deficient retrovirus plasmid capable of expressing the target protein and a VSV-G expression plasmid are co-introduced into the packaging cells carrying the gag and pol genes, and the culture supernatant is used as a virus solution. Alternatively, desirably, the VSV-G expression plasmid is introduced into the packaging cells infected with the virus solution, and the culture supernatant is used as the virus solution. The virus solution is preferably concentrated as necessary.
The preparation of a replication-defective retrovirus vector is not limited to such a method.

In the above virus solution, the titer of the replication-defective retrovirus vector is preferably 1 × 10 ⁸ to 1 × 10 ¹⁴ cfu / ml, more preferably 1 × 10 ⁹ to 1 × 10 ¹⁴ cfu / ml. preferable.
The titer of the virus solution is defined by the number of cells infected when the virus solution is added to NIH3T3 cells (American Type Culture Collection CRL-1658). Specifically, 1 ml of virus solution diluted at a dilution ratio of 10 ² to 10 ⁶ times is added to 5 × 10 ⁴ NIH3T3 cells present in each well (bottom area of about 9.4 cm ² ) of a 6-well culture plate. The titer of the virus solution is measured by examining the ratio of cells expressing the marker neomycin resistance gene from the resistance to G418 (neomycin).

Next, infection of avian embryos with a replication-defective retrovirus vector will be described.
The transgenic bird of the present invention can be obtained by a method comprising infecting a bird embryo with a replication-defective retrovirus vector containing a foreign gene and hatching the embryo.
Embryos are those in the early ontogeny of multicellular animals and are either encased in egg membranes or eggshells, or in the mother's body and before being eaten independently. Hatching is when the embryo comes out of the egg membrane or eggshell and becomes independent of food.
The embryo infected with the replication-defective retrovirus vector preferably has passed 24 hours or more from the start of incubation. More preferably, the embryo is 32 hours to 72 hours before the start of incubation. More preferably, the embryo is 48 hours to 64 hours before the start of incubation. The place where the retroviral vector is infected, that is, the retroviral vector is introduced is preferably in the heart or blood vessel. In the present invention, in producing a G0 transgenic chimera bird with high gene transfer efficiency, in the initial stage (within 6 hours from the start of the heart beat), the heart beat can be observed. It is preferable to introduce a gene. It is based on the viewpoint that the gene is carried to the whole body in the bloodstream and the number of cells is small.

Microinjection is a method in which a virus solution is directly introduced into a specific location using a micro glass tube having a thin tip under a microscope. In the present invention, microinjection is preferable to other gene introduction methods such as lipofection method and electroporation method, because the virus solution is introduced into a specific place such as heart or blood vessel.
Incubation conditions are not particularly limited. For example, in the case of chickens, the temperature is 37.2 to 37.8 ° C. in a three-dimensional incubator (38.9 to 39.4 ° C. at the top of the egg in a flat incubator), and the humidity is A condition of about 40 to 70% is an optimal environment for occurrence, but is not limited to this environment. In addition, the eggs are turned during incubation. Egg turning is preferably performed twice or more a day at an angle of 30 ° or more, but not limited thereto.

Although it does not specifically limit as birds used by this invention, It is preferable that it is a poultry which can be utilized as livestock. Examples of poultry include chickens, turkeys, ducks, ostriches, quails and ducks. Among these, chickens are particularly preferable because they are easily available, are plentiful as egg-laying species, have large eggs, and have established a large-scale breeding method.

As described above, a G0 transgenic chimeric bird can be obtained by infecting a bird embryo with a replication-defective retrovirus vector containing a foreign gene and hatching the embryo.
A G1 transgenic bird can be obtained by crossing a G0 transgenic chimeric bird carrying a foreign gene in a germ cell with a wild type bird or a G0 transgenic chimeric bird and selecting hatched chicks. G0 transgenic chimera birds have a low probability of introduction of foreign genes into all cells, and in most cases, chimeras in which cells having foreign genes introduced and cells of different genotypes coexist with wild-type cells State. On the other hand, the G1 transgenic birds obtained by the above method have the transgene uniformly in almost all somatic cells. Confirmation of gene introduction into somatic cells and germ cells can be performed by PCR or the like on DNA or RNA derived from blood, somatic cells, sperm, or eggs. It can also be judged from the expression of the target protein. Expression of the target protein can be examined by ELISA, electrophoresis, measurement of target protein activity, or the like.

The G2 and subsequent generations of transgenic birds are produced by crossing G1 transgenic birds. As for the mating type, G1 transgenic male and wild type female, G1 transgenic female and wild type male, G1 transgenic male and female, and the like can be considered, and backcrossing by offspring and their parents is also possible. Among these, the mating type of G1 male and wild type female is preferable from the viewpoint of efficiency because a plurality of wild type females can be mated to one G1 male.
The method for producing a target protein of the present invention is characterized in that the target protein is recovered from the aforementioned transgenic birds. More specifically, the present invention is characterized in that the target protein is collected from the blood, somatic cells and / or eggs of the produced transgenic birds and their progeny by extraction, purification and activation, or a combination thereof. To do. The method used for extraction and purification is not particularly limited. For example, fractional precipitation, centrifugation, two-phase separation, ultrafiltration, membrane separation, chromatography, immunochemical method, crystallization, and / or Examples include a method including the combination. In particular, proteins containing Gla residues may undergo a conformational change due to the presence of a divalent metal cation, and the degree of adsorption to the target protein changes by recognizing the conformational change due to the presence or absence of a divalent metal cation. It is preferable to purify using an antibody. Moreover, you may activate the target protein as needed. In particular, when the target protein is a blood coagulation factor, it may be activated with an appropriate serine protease if necessary.
When the target protein is blood coagulation factor VII, precursor blood coagulation factor VII and / or activity depends on the concentration of a divalent metal cation selected from calcium ion, manganese ion, barium ion, magnesium ion and strontium ion It is preferable to purify using a monoclonal antibody that binds to type II blood coagulation factor VII.
The blood coagulation factor VII has a precursor and an active form, but the blood coagulation factor VII obtained by the above-described production method is mainly composed of the precursor blood coagulation factor VII and may contain the active blood coagulation factor VII. is there.

The protein obtained from the transgenic birds of the present invention is a group consisting of blood coagulation II, VII, IX, factor X, protein C and its precursor containing 0.05 to 0.5 nmol of N-acetylneuraminic acid per nmol. More preferably, the protein contains 0.2 to 0.4 nmol of N-acetylneuraminic acid.
The protein is preferably a protein selected from the group consisting of activated blood coagulation factor VII and its precursor.

According to the present invention, there are provided a protein having a Gla residue, a serine protease, a transgenic bird that produces a protein containing a recognition site for a serine protease, and a method for producing the same. As a result, it is possible to produce a precursor of blood coagulation II, VII, IX, factor X, protein C, etc., and in some cases, a transgenic bird that expresses the precursor and its active protein. At the same time, it was also possible to produce transgenic birds that express unstable proteins that have serine protease recognition sites and serine protease activity.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by these Examples. For those not specifically described for genetic manipulation, representative methods were followed (J. Sambrook, EF Fritsch, t. Maniatis; Molecular Cloning, A Laboratory Manual, 2nd Ed, Cold Spring Harbor Laboratories). For cell cultures not specifically described, typical methods were followed (edited by Hideki Koyama “Cell Culture Lab Manual” Springer Fairlark Tokyo, 1st size). When the product name was described, the instructions in the attached manual were followed unless otherwise specified.

Example 1
Construction of plasmid pMSCVneobactFVII for expression of human blood coagulation factor VII gene pMSCVneobact (SEQ ID NO: 1) is a known document (Gene Ther. 1994 MAR; 1 (2): 136-8.) And information on the Internet (http: // www. ncbi.nlm.NIH.gov/etc.) and inserted between EheI (235) and PvuII (628) of pUC19 (Genbank Accession No. X02514) (manufactured by Toyobo Co., Ltd.). This was cut with HindIII (manufactured by Takara Bio Inc.), treated with Alkaline Phosphatase BAP (manufactured by Takara Bio Inc.), and then purified and collected by MinElute Reaction Cleanup Kit (manufactured by QIAGEN Inc.). This was subjected to 1% agarose gel electrophoresis, and the target fragment was purified and collected by MinElute Gel Extraction Kit (manufactured by QIAGEN) (Example 1 vector fragment).
pCR-BluntII-TOPO-VII is 5'- gtcgac caccATGg ... TAG ctcgac- 3 '(SEQ ID NO: 17, underlined are SalI and XhoI restriction enzyme sites, capital letters are pCR- BluntII -TOPO PCR product insertion site The sequence of blood coagulation factor VII (SEQ ID NO: 2) is encoded in ATGg... TAG by the sequence of start codon and end codon).
Pyrobest DNA Polymerase (manufactured by Takara Bio Inc.) was used as the DNA polymerase, and two chemically synthesized oligonucleotides 5′-agcc aagcttt accatgggtccccccccccctcag-3 ′ (SEQ ID NO: 3) and 5′- A fragment amplified by PCR using cgate aagctt acgcgtctaggggaaatgggggctcgcag-3 ′ (SEQ ID NO: 4) (underlined is a HindIII restriction enzyme site) as a primer was purified and recovered by MinElute PCR Purification Kit (manufactured by QIAGEN) in H The product was cut. This was electrophoresed on a 1% agarose gel, and the target fragment was purified and collected by MinElute Gel Extraction Kit (QIAGEN) (Example 1 insert fragment).
Example 1 Vector fragment and Example 1 insert fragment were ligated to DNA Ligation Kit Ver. 2.1 (manufactured by Takara Bio Inc.). E. coli DH5alpha Competent Cells (Takara Bio Inc.) were transformed. The transformed E. coli was applied to a 2 × YT agar medium containing 50 μg / ml ampicillin and cultured at 37 ° C. for 12 to 15 hours. The grown colonies were cultured in 2 × YT medium containing 5 ml of 50 μg / ml ampicillin at 37 ° C. for 6 to 12 hours, and then the plasmid was extracted. The plasmid was treated with NcoI (Takara Bio) and subjected to 1% agarose gel electrophoresis. A plasmid having the structure of FIG. 1 was selected and designated pMSCVneobactFVII.
In addition, the marker gene beta-lactamase, the virus packaging signal sequence phi +, the marker gene Neomycin resistance gene, the tissue non-specific promoter gene beta-actin promoter, and the long repetitive sequences 5LTR and 3LTR in FIG. 1 are all in pMSCVneobact. It comes from.

(Example 2)
Preparation of retroviral vector using pMSCVneobactFVII and pVSV-G Unless otherwise specified, the medium is Dulbecco's modified Eagle medium containing 10% fetal bovine serum (FBS), 50 units / ml penicillin and streptomycin (Dulbecco's Modified Eagle Medium: DMEM) was used (manufactured by Gibco). Culturing was performed at 37 ° C. and 5% CO ₂ . EndoFree Plasmid Maxi Kit (QIAGEN) was used as the plasmid DNA for the retroviral vector.

In order to prepare a retroviral vector from the plasmid pMSCVneobactFVII constructed in Example 1, 5 × 10 ⁶ cells (70% confluent) were prepared by using packaging cells GP293 having gag and pol genes, and a collagen-coated culture dish having a diameter of 100 mm. ) So that it becomes 90% confluent the next day. On the next day, the medium was removed, and 7.2 ml of medium and 10 μl of 25 mM chloroquine (manufactured by Sigma) were added and further cultured for 1 hour. 56 μl of Lipofectamine. 2000 (manufactured by Invitrogen) was suspended in 1.4 ml of Opti-MEMI medium (manufactured by Gibco) and placed at room temperature for 5 minutes (Lipofectamine. 2000 solution). 12 μg of pMSCVneobactFVII and 12 μg of pVSV-G were suspended in 1.4 ml of Opti-MEMI medium (plasmid DNA solution). Lipofectamine. The 2000 solution and the plasmid DNA solution were mixed and allowed to stand at room temperature for 20 minutes. The whole amount was added to the culture dish and cultured for 6 hours. After 6 hours, the medium was removed, 9 ml of medium and 200 μl of 1M HEPES Buffer Solution (manufactured by Gibco) were added, and further cultured for 24 hours.

The culture supernatant was passed through a 0.45 μm cellulose acetate filter (manufactured by Advantech) and collected in a centrifuge tube. Using an ultracentrifuge CS100GXL (manufactured by Hitachi Koki Co., Ltd.), the mixture was centrifuged at 28000 rpm (50000 g) for 1.5 hours. Add 20 μl of TNE buffer (50 mM Tris-HCl (pH 7.8), 130 mM NaCl, 1 mM EDTA) to the precipitate, leave it overnight at 4 ° C., suspend well, and centrifuge at 12000 rpm for 1 minute in a small high-speed centrifuge. The supernatant was passed through a 0.45 μm Durapore Ultra Free Filter (manufactured by Advantech) to obtain a virus solution.

(Example 3)
Selection of human blood coagulation factor VII stable packaging cells The day before the virus infection, GP293 cells were seeded in a 24-well culture plate at 1.5 × 10 ⁴ cells / well and cultured. On the day of virus infection, the medium was replaced with 1 ml of medium containing 10 μg / ml polybrene. This was infected with the virus solution prepared in Example 2. Thereafter, the cells are cloned by the limiting dilution method. Specifically, the next day, the cells were suspended in a medium containing 800 μg / ml G418 and diluted to 10 cells / ml in the same medium. 100 μl of the diluted cells are seeded in a 96-well culture plate (so that one cell can be contained in the well), and a cell with a high cell growth rate and a shape close to that of GP293 is selected for stable packaging of human blood coagulation factor VII Cell clones were obtained.

Example 4
Preparation of retroviral vector using human blood coagulation factor VII stable packaging cell and pVSV-G 5 × human collagen coagulation factor VII stable packaging cell obtained in Example 3 was added to a collagen-coated culture dish having a diameter of 100 mm. were seeded so that ¹⁰⁶ (70% confluent). Try to be 90% confluent the next day. On the next day, the medium was removed, and 7.2 ml of medium and 10 μl of 25 mM chloroquine were added and further cultured for 1 hour. 56 μl of Lipofectamine. 2000 was suspended in 1.4 ml of Opti-MEMI medium and left at room temperature for 5 minutes (Lipofectamine.2000 solution). 12 μg of pVSV-G was suspended in 1.4 ml of Opti-MEMI medium (plasmid DNA solution). Lipofectamine. The 2000 solution and the plasmid DNA solution were mixed and allowed to stand at room temperature for 20 minutes. This was added to the culture dish and cultured for 6 hours. The medium was removed, 9 ml of medium and 200 μl of 1M HEPES Buffer Solution were added and cultured for 24 hours. The culture supernatant was passed through a 0.45 μm cellulose acetate filter and collected in a centrifuge tube. Centrifugation was performed at 28000 rpm (50000 g) for 1.5 hours using an ultracentrifuge. Add 20 μl of TNE buffer to the precipitate, leave it overnight at 4 ° C., suspend well, centrifuge at 12000 rpm for 1 minute in a small high-speed centrifuge, pass the supernatant through a 0.45 μm Durapore ultrafree filter, and add virus. Liquid. In this manner, a virus solution having a titer of 10 ⁸ cfu / ml or more was obtained.

(Example 5)
Construction of Human Blood Coagulation Factor VII Gene Expression Plasmid pMSCVneobactFVIIwpre Containing WPRE Sequence and Preparation of Retroviral Vector pMSCVneobactFVII obtained in Example 1 was cleaved with ClaI (manufactured by Takara Bio Inc.), and treated with Alkaline Phosphatase BAP. Purified and recovered. The product described in Example 1 was used for purification and recovery. This was electrophoresed on a 1% agarose gel, and the target fragment was purified and collected (Example 5 vector fragment). Two chemically synthesized oligonucleotides 5'-cc atcgaatataccactctggattacaaaaattttga -3 '(SEQ ID NO: 5) and 5'-cc atcgat caggcggggggggg-3' (SEQ ID NO: 6) (underlined ClaI restriction enzyme site) are used as primers, and WP A fragment amplified by PCR from pWHV8 (American Type Culture Collection 45097) containing γ was purified, recovered, and cleaved with ClaI. This was subjected to 1% agarose gel electrophoresis, and the target fragment was purified and recovered (Example 5 insert fragment).
The vector fragment and the insert fragment of Example 5 were ligated. E. coli DH5alpha was transformed. A plasmid having the structure of FIG. 2 was selected from the transformant and designated pMSCVneobactFVIIwpre. Stable packaging cells and retroviral vectors can be prepared in the same manner as in the examples described above. Use of the WPRE sequence facilitates obtaining high titer stable packaging cells. This is probably because transcription activity is enhanced by the WPRE sequence and RNA is stabilized.

(Example 6)
Measurement of virus titer The titer of virus solution was defined by the number of cells infected when the virus solution was added to NIH3T3 cells (American Type Culture Collection CRL-1658). 1 ml of virus solution diluted at a dilution ratio of 10 ² to 10 ⁶ times is added to 5 × 10 ⁴ NIH3T3 cells present in each well (bottom area of about 9.4 cm ² ) of a 6-well culture plate, and the marker neomycin is added. The titer of the virus solution was measured by examining the percentage of cells expressing the resistance gene from the resistance to G418. When 4 colonies appear at 10 ^6- fold dilution, the virus titer is 4 × 10 ⁶ cfu / ml.
Specifically, NIH3T3 cells were seeded and cultured at 5 × 10 ⁴ cells / well in a 6-well culture plate the day before the start of titer measurement. The next day, the cell culture medium was replaced with 900 μg / ml medium containing 9 μg / ml polybrene, the virus solution was diluted 10 ⁻¹ to 10 ⁻⁵ with the medium, and 100 μl was added to each well to infect ( Polybrene final concentration 8 μl / ml). After 4-6 hours of culture, 1 ml of medium was further added. On the next day, the medium was replaced with a medium containing 800 μg / ml G418, and thereafter replaced with a G418-containing medium every 3 to 4 days. About 2 weeks after infection, the plate was stained with a methylene blue solution, and the number of colonies obtained was measured to determine the titer. As a result of titer measurement, the titer of the virus used for injection in Example 7 described later was 4.1 × 10 ^{6 to} 1.6 × 10 ⁹ . The results are shown in Table 1.

(Example 7)
Microinjection and artificial hatching of retroviral vectors into chicken embryos is performed under aseptic conditions. The outside of the chicken fertilized egg (Shiroyama Breeder) is sterilized with disinfectant (Showa Franchi) and ethanol. Set the incubator P-008 (B) (made by Showa Franchi) to an environment of 38 ° C and humidity 50-60%, the time when the power was turned on is the incubation start time (0 hours), and then 15 minutes Incubation was performed while turning 90 ° each time.
After about 55 hours have elapsed from the start of incubation, the egg is removed from the incubator, and the sharp end of the router is a 3.5 cm diameter circle with a diamond blade (blade diameter 20 mm, shaft diameter 2.35 mm) (Proxon) ). The sharp end of a chicken double yellow egg (Shiroyama breeding place) was cut to a diameter of 4.5 cm, the contents of the fertilized egg were transferred to an eggshell where the contents were discarded, and the embryo was moved upward with an inner cylinder of a syringe. Under a stereomicroscope system SZX12 (manufactured by Olympus), a virus solution was injected into a femtochip II (manufactured by Eppendorf), and a femtojet (manufactured by Eppendorf) was used to perform Example 4 (no WPRE sequence) or Example 5 ( About 2 μl of virus solution (with WPRE sequence) was microinjected into the heart.
This hole was closed with Saran wrap (manufactured by Asahi Kasei Co., Ltd.) cut into approximately 8 × 8 cm using egg white as glue, and returned to the incubator to continue incubation. Incubator turning was changed to 30 ° turning every 30 minutes. On the 20th day from the start of incubation, about 20 holes were made in Saran wrap with a 20G injection needle, and oxygen was supplied to the incubator at 60 cc / min for incubation. When the chicks started hammering, they broke the eggshell and hatched. The results regarding this artificial hatching are shown in Table 1.

(Example 8)
Confirmation of blood coagulation factor VII expression in blood and eggs in transgenic chickens expressing blood coagulation factor VII The chicks born in Example 7 were raised and grown. As feed, young SX safety and Neosafety 17 (manufactured by Toyohashi Feed Company) were used. 3.8 g of trisodium citrate dihydrate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved to make 100 ml to obtain a 3.8% sodium citrate solution. Blood from blood coagulation factor VII-expressing transgenic chickens was collected from the subwing vein. Nine volumes of blood were added to 1 volume of 3.8% sodium citrate solution and mixed gently. This was centrifuged at 4 ° C. and 3000 rpm with a small high-speed centrifuge, and the supernatant was used as plasma. When extracting from eggs, egg white and egg yolk were separated. When extracting from egg yolk, a syringe was put in the middle of the yolk and extracted so that no egg white entered. The egg white was prepared so as to be uniform as a whole by ultrasonic and physical techniques. The prepared sample was stored frozen at −80 ° C. until assay. In order to avoid freezing and thawing, it is preferable to perform the lysis quickly at 37 ° C.

Expression level of the blood clotting factor VII ASSRACHROM ^R VII: Ag; was examined by ELISA using (Diagnostica Sutago manufactured Diagnostica Stago, Inc.). A purified product derived from blood was used as a standard. The expression levels (individual number 3-1) were 2.1 μg / ml in plasma, 2.2 μg / ml in egg yolk, and 24 μg / ml in egg white.
In addition, the expression level of blood coagulation factor VII was determined based on the blood coagulation activity. The blood coagulation activity of blood coagulation factor VII was determined by the coagulation time method using deficient plasma. Samples were appropriately diluted with phosphate buffered saline (PBS). Thromborel S (manufactured by Dade Bering) as a reagent, factor VII-deficient plasma (manufactured by George King), and BCS (manufactured by Dade Bering) as a measuring instrument, and the activity of the sample was determined from a calibration curve using normal human plasma. Converted. The reference value for normal human plasma was 100%. When converted to weight, the blood coagulation factor VII concentration in human plasma was converted to 0.5 μg / ml.
The expression result of blood coagulation factor VII in GO transgenic is shown in FIG. After acquiring the injection technique (from the seventh time in Table 1), the appearance frequency of the G0 transgenic in the born chick was 100%. Expression levels were up to 12 μg / ml in plasma and up to 350 μg / ml in egg white. The results are shown in FIG.
The first number of the individual number “3-1” shown in FIG. 3 indicates the number of times of microinjection. For example, the individual number “3-1” is an individual obtained by the third microinjection.
Next, Western blotting of blood coagulation factor VII derived from plasma and eggs of individual numbers 7-9 was performed. Samples were electrophoresed under denaturing conditions using 12.5% e-Pagel (manufactured by Atto), transferred to a PVDF membrane, blocked with PBS containing 10% skim milk and 0.05% Tween 20, and sheep anti-human as an antibody. Using a Factor VII antibody HRP label (Affinity Biologicals), detection was performed with ECL Plus Western Blotting Detection System and Hyperfilm ECL (Amersham). The results of Western blotting are shown in FIG.

Example 9
Confirmation of gene transfer to genomic DNA by PCR Extraction of genomic DNA from chickens can be performed from pellets extracted from plasma, serum-extracted pellets or sperm, but in this example, they were performed from sperm. Extraction was performed using MagExtractor ^R- Genome- and MFX ^R- 2100 (manufactured by Toyobo Co., Ltd.). PCR using Premix Taq (manufactured by Takara Bio Inc.) with two chemically synthesized oligonucleotides 5′-caagaagagaggtgtgggtta-3 ′ (SEQ ID NO: 7) and 5′-ggtgagtgagtgtgggtta-3 ′ (SEQ ID NO: 8) as primers and genomic DNA as a template The presence or absence of gene transfer into genomic DNA was examined by examining whether or not a 97 bp fragment corresponding to the gag region of the retroviral vector could be amplified. Genomic DNA was extracted from the sperm of the transgenic chicken (individual number 9-1), and the amplified fragment by PCR was subjected to 4% agarose gel electrophoresis. As a result, amplification of the target fragment was confirmed. The results are shown in FIG.

(Example 10)
The aforementioned products were used for the construction purification, recovery, etc. of the plasmid pMSCVneobactFIX for human blood coagulation factor IX gene expression. Two chemically synthesized oligonucleotides 5′-atgcagcgcgtgaacatgacatgggcagaa-3 ′ (SEQ ID NO: 9) and 5′-ttaagtgagctttgtttttttccttataccca-3 ′ (SEQ ID NO: 10) were used as primers, and human adult normal liver first-strand cDNA (manufactured by Cosmo Bio) A fragment amplified by PCR as a template was purified and collected, and the ends were blunted and phosphorylated with TaKaRa BKL Kit. After purification and collection, 1% agarose gel electrophoresis was performed, and the target fragment was purified and collected (insert fragment). pUC19 was treated with SmaI (manufactured by Takara Bio Inc.), treated with Alkaline Phosphatase BAP, purified and recovered, and subjected to 1% agarose gel electrophoresis to purify and recover the target fragment (vector fragment). The vector fragment and the insert fragment were ligated, E. coli DH5alpha was transformed. A plasmid having FIX (Genbank Accession No. NM — 000133) inserted therein was selected from the transformant and designated pUCFIX.
Next, two chemically synthesized oligonucleotides 5′-agcc aagctt accatgcagcgcgtgaacatgcatcatgcagaa-3 ′ (SEQ ID NO: 11) and 5′-cgaa agcttt acgcgtttagtgatttttttttttctt The fragment amplified by PCR using pUCFIX as a template was purified, recovered, and cleaved with HindIII. This was subjected to 1% agarose gel electrophoresis, and the target fragment was purified and collected (insert fragment). pMSCVneobact was cleaved with HindIII, purified and recovered after treatment with Alkaline Phosphatase BAP. This was subjected to 1% agarose gel electrophoresis, and the target fragment was purified and recovered (vector fragment). The vector fragment and the insert fragment were ligated, E. coli DH5alpha was transformed. In the same manner as in Example 1, a plasmid was extracted from the transformant, and the plasmid was subjected to restriction enzyme treatment with StuI (Takara Bio) and electrophoresed on a 1% agarose gel. A plasmid having the structure of FIG. 6 was selected and designated as pMSCVneobactFIX.

(Example 11)
The aforementioned product was used for the construction, purification, recovery, etc. of the protein C gene expression plasmid pMSCVneobactprotein C. Two chemically synthesized oligonucleotides 5′-atgtggcagctcacaagcctcctgctgttc-3 ′ (SEQ ID NO: 13) and 5′-ctagtgtccccctctgtggggggggctctc-3 ′ (SEQ ID NO: 14) were used as primers, and human adult normal liver first strand cDNA (Cosmo Bio) A fragment amplified by PCR as a template was purified and collected, and the ends were blunted and phosphorylated with TaKaRa BKL Kit. After purification and collection, 1% agarose gel electrophoresis was performed, and the target fragment was purified and collected (insert fragment). pUC19 was treated with SmaI (manufactured by Takara Bio Inc.), treated with Alkaline Phosphatase BAP, purified and recovered, and subjected to 1% agarose gel electrophoresis to purify and recover the target fragment (vector fragment). The vector fragment and the insert fragment were ligated, E. coli DH5alpha was transformed. A plasmid into which protein C (Genbank accession number NM — 000312) was inserted was selected from the transformed strain, and designated pUCproteinC.
Next, two chemically synthesized oligonucleotides 5'-agcc aagctt accatgtggcagctcacaagcctcctgctgttcctaaggtgcccagctcttctggggggcttc-3 ' ( SEQ ID NO: 15) and 5'-cgat aagctt acgcgtctaaggtgcccagctcttctggggggcttc-3' ( SEQ ID NO: 16) (underlined is HindIII restriction enzyme site) as primers The fragment amplified by PCR using pUCproteinC as a template was purified, recovered, and cleaved with HindIII. This was subjected to 1% agarose gel electrophoresis, and the target fragment was purified and collected (insert fragment). pMSCVneobact was cleaved with HindIII, purified and recovered after treatment with Alkaline Phosphatase BAP. This was subjected to 1% agarose gel electrophoresis, and the target fragment was purified and recovered (vector fragment). The vector fragment and the insert fragment were ligated, E. coli DH5alpha was transformed. In the same manner as in Example 1, a plasmid was extracted from the transformant, and the plasmid was subjected to restriction enzyme treatment with SalI (Takara Bio Inc.) and electrophoresed on a 1% agarose gel. A plasmid having the structure of FIG. 7 was selected and designated pMSCVneobact protein C.

(Example 12)
Purification of blood coagulation factor VII from egg white Blood coagulation factor VII was purified from the egg white of solid numbers 7-9 and 9-6. All purification operations were performed at 4 ° C. The egg white was diluted with 5 volumes of 0.05 M Tris-HCl (pH 6.0) and 0.15 M NaCl and suspended until the viscosity became low. Next, pH was adjusted to 7.4 with 0.2 N NaOH, and CaCl ₂ was added to a final concentration of 5 mM. Centrifugation was performed at 5000 × g, and the supernatant was collected and passed through a 0.45 μm filter to obtain an egg white solution. For purification, an antibody column against blood coagulation factor VII was used (reference document No. 2824430). The antibody column (φ25 cm × 2 cm) was equilibrated with an equilibration buffer of 0.05 M Tris-HCl (pH 7.4), 0.15 M NaCl, 5 mM CaCl ₂ . The egg white solution was passed through the antibody column, washed with an equilibration buffer, and then eluted with an elution buffer of 0.05 M Tris-HCl (pH 7.4), 0.15 M NaCl, 10 mM EDTA. The result of SDS-PAGE of the sample in each step of purification is shown in FIG. SDS-PAGE was performed using 5-20% gradient gel under reducing conditions. From the electrophoretic result of the eluted fraction, three bands around 50 kDa, 30 kDa and 20 kDa derived from blood coagulation factor VII can be confirmed almost purely.
The above purification was performed several times. Table 2 shows the purification yield (%) in each round. The yield was measured by the ELISA method using ASSERACHROM VII: Ag (manufactured by Diagnostica Stago).

(Example 13)
Comparison of physical properties of human plasma-derived and egg white-derived blood coagulation factor VII Using the sample purified in Example 12, the sialic acid content of human plasma-derived and egg white-derived blood coagulation factor VII was measured. The measurement was requested from the Toray Research Center. The results are shown in Table 3. Glycolyl neuraminic acid was below the detection limit.
Next, specific activities were compared. Subsequent experiments were performed only on samples derived from individual number 9-6. The activity was measured by the prothrombin (PT) time method (reference method Methods Enzymol. 1981; 80: 228-37.) Using blood coagulation factor VII-deficient plasma. Mix 0.1 ml of deficient plasma, 0.1 ml of diluted sample, and 0.1 ml of tissue thromboplastin solution, incubate at 37 ° C. for 2 minutes, add 0.1 ml of 0.025 M CaCl ₂ solution, and measure the time until coagulation did. A standard curve was prepared in advance using normal plasma diluted serially instead of the diluted sample, and the blood coagulation factor VII activity in the sample was measured therefrom. Since the degree of activation of human plasma-derived and egg white-derived blood coagulation factor VII varies, each sample was completely activated by Factor Xa (manufactured by Enzyme Research Laboratories), and then the specific activity was measured. Specific activity is 47.4 ± 2.6 KIU / mg for human plasma-derived blood coagulation factor VII, 24.7 ± 1.1 KIU / mg for egg white-derived blood coagulation factor VII, and egg white-derived blood coagulation factor VII is human plasma. The specific activity was about half that of the derived blood coagulation factor VII.
Comparison of the kinetics of blood coagulation factor VII derived from human plasma and egg white in rat blood was performed. Blood coagulation factor VII (500 μg / kg) was administered from the tail vein, blood was collected over time, plasma was separated, and blood coagulation factor VII concentration was measured by the ELISA method described in Example 12. The half-life was about 28.8 minutes for human plasma-derived blood coagulation factor VII and about 7.4 minutes for egg white-derived blood coagulation factor VII. The results are shown in FIG.

The structure of the blood coagulation factor VII expression vector pMSCVneobactFVII obtained in Example 1 is shown. phi + represents the viral packaging signal sequence. A part of gag obtained by changing the start codon (ATG) of gag to TAG is attached to the packaging signal sequence of Moloney murine leukemia virus. FVII indicates a blood coagulation factor VII gene. 5'LTR and 3'LTR indicate the LTR sequence of MSCV. The structure of the blood coagulation factor VII expression vector pMSCVneobactFVIIwpre obtained in Example 5 is shown. phi + represents the viral packaging signal sequence. A part of gag obtained by changing the start codon (ATG) of gag to TAG is attached to the packaging signal sequence of Moloney murine leukemia virus. FVII indicates a blood coagulation factor VII gene. 5'LTR and 3'LTR indicate the LTR sequence of MSCV. WPRE is a post-transcriptional regulator derived from Woodchuck hepatitis virus. Figure 2 shows blood clotting activity in plasma and egg white of transgenic chickens. The activity was converted to a concentration from the standard value of normal human plasma (0.5 μg / ml / 100%). Male and female unknowns were killed before sex determination. (*) Is a transgenic chicken using the pMSCVneobactFVII retroviral vector of Example 4, and the other is a transgenic chicken using the pMSCVneobactFVIIwpre retroviral vector of Example 5. The result of the western blotting using the plasma and egg white anti-coagulation factor VII antibody of a transgenic chicken (individual number 7-9) is shown. The marker is a molecular weight marker. Novo Seven (manufactured by Novo Nordisk) and FVII (purified product of human blood-derived blood coagulation factor VII) are standard samples, and the wild type is a sample derived from a non-transgenic chicken. The molecular weight of blood coagulation factor VII is 50 kDa at the precursor and 30 kDa and 18 kDa when activated by cleavage in the polypeptide chain. Novoseven and FVII are 0.02 mg / lane. Egg white and plasma were electrophoresed at 0.2 μl / lane and 0.4 μl / lane, respectively. The result of the gene transfer confirmation to the sperm genomic DNA of the transgenic chicken (individual number 9-1) by PCR is shown. Lane M is a molecular weight marker, lane 1 is a PCR product of wild-type chicken sperm genomic DNA, lane 2 is a PCR product of transgenic chicken (individual number 9-1) sperm genomic DNA, and lane 3 is a PCR product of pMSCVneobactFVII. The initial template concentration in the reaction solution was 8 ng / ml for genomic DNA and 1 pg / ml for pMSCVneobactFVII. After 40 cycles of reaction, 5 μl was subjected to 4% agarose gel electrophoresis. In lane 1, a band cannot be confirmed at the target position (97 bp), but in lane 2, a band can be confirmed at the target position. The structure of the blood coagulation factor IX expression vector pMSCVneobactFIX obtained in Example 10 is shown. phi + represents the viral packaging signal sequence. A part of gag obtained by changing the start codon (ATG) of gag to TAG is attached to the packaging signal sequence of Moloney murine leukemia virus. FIX indicates a blood coagulation factor IX gene. 5'LTR and 3'LTR indicate the LTR sequence of MSCV. The structure of the protein C expression vector pMSCVneobact protein C obtained in Example 11 is shown. phi + represents the viral packaging signal sequence. A part of gag obtained by changing the start codon (ATG) of gag to TAG is attached to the packaging signal sequence of Moloney murine leukemia virus. proteinC represents the protein C gene. 5'LTR and 3'LTR indicate the LTR sequence of MSCV. The result of SDS-PAGE of the sample in each step of purification in Example 12 is shown. Purification was performed from eggs derived from individual numbers 7-9 and 9-6, respectively. In the electrophoresis results, the egg white is the egg white liquid, the egg white solution is the liquid prepared as in Example 12, the passing fraction is the passing liquid that is not adsorbed through the column, and the washing fraction is the washing liquid passing liquid. The eluate was divided into three fractions. pd FVII is human plasma-derived precursor blood coagulation factor VII, and pdFVIIa is human plasma-derived active blood coagulation factor VII. Human plasma-derived precursor blood coagulation factor VII has a molecular weight of 50 kDa, and human plasma-derived active blood coagulation factor VII has a molecular weight of 30 kDa and 20 kDa. As a result of purification, three bands near 50 KDa, 30 KDa and 20 KDa derived from blood coagulation factor VII can be confirmed almost purely as a result of electrophoresis of the eluted fraction. The results of blood kinetics of human plasma-derived and egg white-derived blood coagulation factor VII of Example 13 are shown. pd represents activated human plasma-derived blood coagulation factor VII, and eg represents activated egg white-derived blood coagulation factor VII. 500 μg / kg was administered from the tail vein, blood was collected over time, and after blood plasma was separated, the blood coagulation factor VII concentration was measured with ASSERACHROM VII: Ag. The horizontal axis of the graph represents time, and the vertical axis represents the converted value of blood coagulation factor VII based on standard human plasma-derived blood coagulation factor VII. The numerical value of 0 minutes after intravenous injection is an estimated value from literature values (reference literature: Thromb Haemost. 1993 Sep 1; 70 (3): 458-64). The number of experiments was 2 times with Pd and 3 times with Eg. The half-life was calculated from the time from 5 μg / ml to 2.5 μg / ml, and the human plasma-derived blood coagulation factor VII was about 28.8 minutes and the egg white-derived blood coagulation factor VII was about 7.4 minutes.

Claims (6)

Activated by Gla modification, blood coagulation II, VII, IX, X factor, protein C, or has a foreign gene including a coding sequence of protein S, from eggs of transgenic birds, as well as their progeny, extraction, purification, activity A method for producing a protein derived from an exogenous gene, comprising any one of the above or a combination thereof. Transgenic birds, a transgenic avian produced using a retroviral vector, method of production according to claim 1. Transgenic birds, a transgenic avian produced using the retroviral vector containing the chicken β-actin promoter gene and / or woodchuck post-transcriptional regulatory element sequence, method of production according to claim 1 or 2. Transgenic birds, a transgenic avian produced using the retroviral vector containing the VSV-G envelope, the method of producing according to any one of claims 1 to 3. The production method according to any one of claims 1 to 4, wherein the retroviral vector is a replication-defective mouse stem cell virus. Transgenic birds, avian fertilized egg incubation, the incubation started after 24 hours after embryos are infected with a retroviral vector containing the foreign gene, in transgenic birds that obtained by a method comprising to hatching the embryo The production method according to any one of claims 1 to 5 .

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