JPS6322195A - Viii-factor control method - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to several expression vectors for human factor (2) and a method for preparing this factor (2).

■Recently obtained cloning of factor or antihemophilic factor (references 1-3 and 29 below) has shown that hemophilia A
We can now expect safer treatment for patients than ever before.

(Prior art) Patients with hemophilia A are treated with a concentrated factor preparation (Reference 4) in which the procoagulant activity of the factor is less than 0.1%, but this is a mixture of plasma from many blood donors. It is something that is made.

In this state, hemophiliacs must be at risk of being infected by pathogens such as the hepatitis virus and the LAV/HTL virus, which is the cause of AIDS.

However, by using genetic engineering technology, it has become possible to produce recombinant factors in large quantities without the risk of infection, which has immeasurable advantages from a therapeutic perspective.

However, the efficiency and stability of recombinants depend to some extent on the host system used for their expression.

Special mechanisms are involved in maintaining the efficiency and integrity of expressed proteins. For example, this is the correct modification after translation. There is one powerful test system for evaluating these various cell lines, and especially for checking the suitability of the resulting proteins. In other words, it is a system that relies on vaccinia virus, and this method is an extremely powerful weapon in the sense that wild-type and recombinant viruses can infect an unlimited number of cells and simultaneously produce copies. Furthermore, this system has the advantage of being very simple to use, since it does not require a lengthy selection system such as a host-cell system that exhibits a marker.

Expression of exogenous protein encoding sequences in vaccinia virus (VV) necessarily involves two steps.

1) This needing sequence must be dependent on the vv promoter and must be integrated into a non-essential strand of VV DNA, cloned in a suitable strain plasmid.

2) These sequences located opposite each other must allow in vivo cognate recombination between the plasmid and the viral genome. Mutual double recombination results in the transfer of the plasmid's DNA entry sequence to the viral genome, which allows the DNA to spread and express (References 5: Sm1th&co1..1983;6:
Pantcali&co1. ．． 1983;7:Kien
y&co1. ．． 1984).

(Objects and Structure of the Invention) The present invention relates to a vaccinia virus, particularly a vaccinia virus, which is characterized by having a DNA sequence encoding the factor (1). Preferably, the virus contains all the elements that result in the expression of a complete, mature factor 2 that the cells secrete into the culture medium.

In particular, this virus must carry the promoter of the Voxvirus gene, in particular the vaccinia virus gene located upstream of the factor coding sequence, e.g.
(hereinafter referred to as DNA (F■)) 7.5
This is the promoter of the K gene (denoted as P7°5K).

This promoter/DNA (F■) sequence set is inserted into the non-essential sequences of the vaccine's DNA. The vaccine is equivalent to the gene of vaccinia virus, and T
This also applies to the K (thymidine kinase) gene, which opens up the possibility of selection as will be explained later.

In the present invention, it goes without saying that the term "vaccine virus" refers to the entire virus or a virus from which non-essential parts have been removed.

The present invention further includes within its scope vectors having a sequence involved in encoding at least one polypeptide selected from the high molecular weight portion and the low molecular weight portion of factor ①.

In fact, by purifying the natural molecule of factor (23-28) and cloning its corresponding gene, we were able to understand the structural mechanism of factor (2) and also determined the location of the cleavage site that led to molecular activation. It was also possible to do so.

The first cleavage (by an unrecognized protease) occurs between amino acids 164g and 164g, in the middle of the molecule (B
area) causes protease degradation.

Thrombin then cleaves the terminal NH2 portion at amino acid 740. This produces two polypeptides of approximately 90 Kd and 80 Kd, which are held together by calcium bridges.

■These two polypeptides (the high molecular weight portion is HC) are responsible for the procoagulant activity of factor (F■:C).

(low molecular weight part expressed as LC) was shown to be necessary and sufficient (23-20). In fact, the site of stratification by thrombin is located at amino acid 372 in the macromolecular region.
It is located at amino acid 1689 in the low molecular weight part. This observation suggests that the B region is not required for the procoagulant function of the molecule.

The present invention specifically describes the cloning of sequences encoding the two polypeptides HC and LC separately using vaccinia virus as vectors and the simultaneous production of these two molecules through co-infection of mammalian cells with the two recombinant viruses. It is related to expression.

Therefore, vectors preferably used in the present invention are characterized in that the sequence encoding part of the factor (1) encodes at least one of these polypeptides. H
Either C or LC. If possible, it is desirable that the signal sequence of the factor ① comes before the sequence encoding HC or LC.

Further, the present invention is useful for mammalian cells infected with the above-mentioned vectors, and for the above-mentioned vectors.
It concerns a plasmid that has at least one sequence encoding C or LC.

As cells which may be used in particular, mention must be made of BHK cells, especially BHK-21. This will be described in the examples below.

As described in the main patent, these cells infected after culture, whether transfected with HC or LC coding vectors, or subjected to co-transfection with both HC and LC coding vectors, This results in the secretion of polypeptides that can be therapeutically useful.

In particular, 1nvi with both high and low molecular weight parts synthesized by two independent vectors, one encoding HC and the other encoding LC.
Due to vo recombination, it can be expected that a molecule with the activity of ① factor will be produced from mammalian cells.

Besides the use of the compounds obtained by recombining HC and LC chains, other uses are also conceivable for the polypeptides according to the invention.

In other words, it is known that 8 to 20% of severe hemophilia A patients who require multiple blood transfusions produce factor 2 antibodies in their bodies. It is very difficult to infuse factor concentrates into this type of "inhibited patients."

The epitopes recognized by these antibodies are restricted to certain portions of the molecule. It may also be possible to use the above-mentioned amount of factor ① as an antibody inhibitor for treatment. To facilitate the treatment of inhibited patients, it may be possible to administer this amount prior to the infusion of intact factor ■.

The present invention also relates to a method for preparing factor (2) obtained by culturing the above-mentioned cells in an appropriate medium.

In particular, a suitable medium contains von Willebrand factor (v
This is a ① factor preparation process in which mammalian cells infected with a vector that expresses the ① factor are cultured therein by containing W f ).

In fact, as shown experimentally, the presence of this factor in the culture medium stabilizes the produced factor. Preferably, the von Willebrand factor has a concentration of 0. IU/m
It is desirable to set it to ρ or more.

Finally, the present invention concerns the human factor 2 protein obtained using this method and the mature, glycosylated form of the factor 2 protein obtained in cell culture.

In particular, the present invention also relates to the composition of a therapeutic drug containing factor (2) obtained by the method of the present invention as an active factor.

The present invention also includes polypeptides having HC or LC structures or structures that recombine HC and LC, as well as compositions of therapeutic agents made therefrom.

(Examples) The following examples clarify the characteristics of the various products obtained.

These examples also list the various equipment that was used.

Unless specified to the contrary, enzymes are used under the conditions specified by the manufacturer and techniques of use are known.

Example 1 There are approximately 9000 mRNAs encoding human factor
Since it is known to be a long nucleotide (ntd) (References, 1-3), it is considered impractical to clone it in the form of a complete cDNA molecule by conventional brining methods using oligo(dT). Therefore, based on the cDNA sequence that has already been announced,
We developed a strategy that combines two methods.

a) mRNA for the entire coding sequence
b) Cloning of genomic DNA Synthetic oligonucleotides (18-mer and 21-mer) were used as primers for elongation of cDNA and as probes for searching for sequences cloned at the same time.

It has been revealed that screening of a genome bank in which two 21-mars are sequenced in tandem does not produce any errors after sequence analysis and is an extremely efficient method for clonal selection of factor (2).

As shown in Figure 1, specific brimers or oligos (
From mRNA isolated from human liver using
An aliquot of cDNA was synthesized.

■Since factor mRNA is contained in extremely small amounts in this tissue (total mRNA molecules are 100,000
−200,000 mRNA), λgtlO
(λNM607) type cloning vector was used (Reference, 8). This is a representative bank since it can contain up to 50,000 recombinants per nanogram of double-stranded CDNA.

The conventional methylation and “linker” addition steps are
Generally it is not very effective so avoid it and try rt instead
It was decided to use the a i 1 ing-adaptor J method. (Reference, 9).

The central part of the coding sequence, which has the highest exon value, exon 14, and is 3,106 bp in length (Reference, 10) in the human factor ■ gene, has been found in the human lymphocyte genomic DNA bank. Cloned from. 5″ untranslated portion (UT: untranslated),
Potion 5゛, which has exons 1 and 2゛, is human D
A bank called N A G M 1202A (cell line 4
XY) were isolated in the form of two independent genomic clones. To aggregate the complete coding sequence (see Figure 1), the SUT region and the first exon were concatenated to the second exon by very precise cuts of the intermediary sequence (with one 22.9 kb intron). Exons 1 and 2 are excised in the gene of factor ① (see Figure 1).

Other cDNA or genomic clones were ligated together using control sites placed in a known manner.

The final construction of the sequence encoding the ■ factor introduced into the vector plasmid ptrcg is called pT01080,
■ In addition to the complete coding sequence of the factor, there is a 5-UT portion (
ntd-64 to ntd-1) and 3-UT range (n
td7055 to ntd8235). n
td numbering is Wo.

d according to another method (Reference, 1). The complete 8.3 kb insertion is sandwiched between two 5a11 sites.

More precisely, total NRA was extracted from adult liver using the guanidinium HCl method (ref. 11).

PolyA(+)-RNA obtained by passing oligo(dT)-cellulose twice was used to support cDNA synthesis. Brymer used: cDNAAl
oligo(dT) 12-18 for and 1g- for cDNA2.
mer (ntd6937-6954), and 21-mer (ntd3067-3087) for CDNA3.

Synthesis of the first and second strands, treatment with nuclease S1, and enrichment on a sucrose density gradient for sequences longer than 500 ntd were performed as previously described (Reference, 9
).

However, in the case of specific briming, the reaction of the first strand of CDNA involves at least polyA(+)-
It was found that it was necessary to use 50 gg of RNA and 1 μg/mΩ of specific primer.

cDNA “tailing” oligo (dG).

Phosphorylated oligomer 5′-dAATTCCCCCCCC
Reaction with CCCCC-3-, λN digested with EcoRI
For conjugation with M607 vector, refer to the literature.

This was done using the method described in 9.

The ligated recombinant DNA is injected into phage particles to infect the carpet of E. coli POPlol.
It was packaged in vitro (Reference 913).

Reproduction of filters using plaques (130 mm diameter) containing 2X 104 ufp of lng "tailed" oligo(dG) DNA is typically performed in 6X SSC, 5% dexidran-sulfate, 4X Tenhardt's solution, 10 mM sodium phosphate. , 5DS0.1%, 18/21-mar
Hybridization was performed by soaking overnight in a solution containing phosphorylated synthetic oligonucleotide 32p. The process temperature was 42°C for the 18-mer and 50°C for the 21-mer.

Fitwo is 6XSSC and 0. Washed with 1% SDS solution at appropriate hybridization temperature. As replication expanded, a second round of selection was performed using the 1g-mar and 21-mer placed in close proximity to those used in the first screen. Exon 14 (portion 42 dotted line) is extracted from Genome Bank λEMBL3 (Reference, 14) (2X106
isolated on a unique λ clone made from several recombinant Fache). This genome bank is partially Mb.

The DNA was derived from human lymphocyte DNA that had been digested with I and electrophoresed on an agarose gel. exons 1 and 2
(Portion 51 dotted line) was isolated in λEMBL3 with two clones isolated from Genome Bank 4XY. This bank consists of human lymphoblastoid cell line GM1202
It is derived from A. In either case, the screening was carried out under the same conditions as used for the cDNA bank screening.
Two 1-mer oligonucleotide probes were used.

Two exons were subcloned in M13. These two clones were linearized with restriction enzymes. This enzyme splits 3' in the mediating sequence from exon 1,
It separates exons 2 to 5. The two exons were treated with nuclease Bal 31 and 4d
Repaired by Freneau's polymerase under the intervention of NTPs, 5stI (exon 1 clone) or Ec
by being digested with oRI (exon 2 clone) and inserted into phage M13TG131 (ref.

15), the two exons are placed in the same vector with the same correct orientation, separated by a 1.2 kb intermediate sequence. Single strand support D
A NA was prepared from this clone and a precisely excised intermediate sequence. At this time, 13 bases in the terminal 3' of exon 1,
There are 18 bases at the 5' end of exon 2, and these specific 3
A 6-mar oligonucleotide was used to remove single stranded sequences without aligned complementary sequences.

Correctly sequenced clones are 36-mar 5xS
Hybridization was performed at 60°C in sc.
Recognition was achieved by washing in 2X SSC at 60'C. c, DNA and genomic sequences were ligated one by one using the restriction enzymes indicated above the figure. DNA sequencing of fully assembled molecules can be performed using dideoxynucleotide methods (ref. 16) or chemical degradation techniques (ref. 1).
7) was used.

FIG. 1 schematically shows the construction of the entire code composition of the factor (1). The complete cDNA is shown at the top. The first ATG, stop codon TGA and polyazonylation site AATAAA and restriction sites used for assembly are also illustrated.

UT areas 5' and 3' are shaded. Arrow 1.2.
3 shows the cDNA clones used and their primers. Genome sequences 4 and 5 are marked with dotted lines.

At the bottom of the figure, the final assembly is plasmid pUC
It has been cloned at the 5ai1 site of 8.

Analysis of the DNA sequence of the coding region revealed that cellular AL
It can be seen that there are two differences between the ■ factor synthesized from -7 (Reference, 1) and the ■ factor made from human kidney (Reference, 3). Thus: 1) Position 1035: A→G (silent) 2) Position 3864: A→C (silent) The first alternative to disrupting the unique site ACCI resulted from an error in the reverse transcription process; , 14 independently isolated cDNA clones from the same bank were found to retain this restriction site.

A second alternative exists in the core cloned from genome banks, which may imply genetic polymorphism.

Example 2 In pT01080, the Sma I site of pUC8 is of factor ■! Although located upstream of the IMUT region, the HpaI site is 3-
It is located at position 7434 in the UT area. These two enzymes are ■
The factor sequences were excised and used to insert into the pTG186-poly vector.

This plasmid allows vaccinia virus (VV)
genome allows homologous recombination in vivo.

The DNA of various recombinant v■ is the same as ■ in the viral DNA.
To confirm the integration of the factor sequences, they were prepared and analyzed by Southern method (Fig. 2).

More specifically, the hybridization analysis of factor 2 of v recombinant was carried out by Southern method using HindI [I
(A) or BamHI (B). In both cases, pTG186 Dol
pTG1016 and pTG1015 are pTol by insertion of a ■ sequence placed in the correct or wrong orientation for transcription, respectively.
86-po 1y. 10.1 to 16.
The clones up to
-) represents the DNA of Clones 6.1-6.5 were derived from pTG1015. The products can be either hybridized with (A) to yield a mix of complementary phosphorylated (21-mar or 18-mer) oligonucleotides 32p of HindII [fragment of the factor sequence, or (B) with 71 Embedded,
32p constitutes a complete assembly (Sma I-Hpa I fragment) labeled "nick translation". In part B, only the VV recombinants selected for complementary analysis are shown.

The ■factor sequence located in ptrc 8 (pTo 108 Q) was digested with Sma I + Hpa I, inserted in two directions at the BamHI site, and pTG was generated by treatment with nuclease S1.
186-paly is isolated. This is Polyclinker M
13TG131 (BglII by E coR
It is a derivative of pT0186 containing I).

(Reference, 15). The pTGl 86-po 1 y vector can be obtained, for example, from the vector described in International Publication No. WO 3505376.

The pTG186-poly plasmid can be produced from the pTG188 plasmid that has been digested with PstI and religated with T4 ligase. The pTG393 plasmid is digested with BamHI to create pTG393 after ligation.
"rcIH-TK-P7゜5K plasmid is generated. Next, the polyclinker fiber of bacterial phage M13TG131 (Reference, 15) is conjugated with EcoRI and Bg.
pTGIH-TK-P7.5
K plasmid is inserted between the EcoRI and BamH1 sites to generate pTol 86-poly.

In this construction, 10 restriction sites are available for cloning of foreign genes under P7.5K control.

■Insertion of factor sequence into VV (Copenhagen t
s 26), as already stated (Ref.

7) to produce TK-virus.

After selection with 5-bromodeoxyuridine, recombinant DNA was extracted, cut with HindIII or BamHI, subjected to electrophoresis on a 1% agarose gel, placed on a nitrocellulose fist filter, and hybridized with the probes described above. be done.

After a new screening of TK-clones, 11 recombinants (VV, T
G, F ■ 10.2-1. cl.

A derivative of nelO,2) was selected and used to infect various cells.

After 1 hour of adsorption, the medium supplemented with fetal bovine serum (SFC) was removed, the cells were thoroughly washed, and the factor antibody rate (F■, Ag) was determined by immunoradiometric assay (F). I
Each floating substance was measured using RMA (Reference, 18), and the results are shown in Table 1.

Culture of various cell lines, ratio of ArIF in L (
U/mj) Cell line Bll 21 Vero
He1a 1IepG2W, Ding G, F ■ 4
02-1 0.120 0.049 0.035
0.028WTW --- 2x10 cells are infected for 1 hour at 1 upf/cell. The SFC-containing media is then removed, the cells washed, and they are further soaked in SFC-free media containing 4% live serum albumin (BSΔ) and 1 mM CaCl2. After 24 hours, the content of FVIAg is determined by the 11-way MA method using IgG purified from the serum of an immunized hemophiliac patient as the first antibody and an anti-F■ monoclonal antibody as the second antibody.

Results obtained from suspensions of wild-type infected cells are meaningless.

When the floaters are tested to determine the procoagulant activity of the factor (conventional measurement of activated partial thromboplastin time - APTT (Ref. 19)), values are sometimes so variable that they are completely independent of the corresponding antigen rate. comes out.

BHK2 infected with VV, TG, F■ 10.2-1
One cell secretes factor 2 with an action peak corresponding to about 0.09 units (U/mΩ) in 24 hours. The detection at this time is negative. The activity gradually decreases over 36 to 48 hours, probably due to protein susceptibility at 37°C, a phenomenon already known in the storage of concentrated plasma factor preparations (Ref. 20). ). or
This is due to the elution of cells by VV, the accompanying release of intracellular proteases, and the gradual decomposition of recombinant proteins.

The specific procoagulant activity of factor ■ is expressed in the serum of immune hemophilia patients and the anti-factor monoclonal antibody, CAG-1175A7, which has the ability to inhibit this effect at various concentrations (Reference, 21). It was controlled by using in combination with.

A very loud rumble was heard in the suspension of monkey kidney cells Vero and cells HeLa infected with wild type vaccinia virus. This phenomenon is due to the fact that the elution content from VV is
This may be due to leading to salting out of endogenous proteases which may interfere with the T test.

Human 11TJiaG2 (Vv, T, G, FV
No significant procoagulant effect was found in the suspension of HepG2 infected with I[10,2-1).

This result contrasts with the detection of constant factor ■ antibody rates in the same samples. The question of whether the ■factor is secreted in an inactive form from the beginning or whether it rapidly becomes inactive in these types of cells remains unclear. These results indicate that only active recombinant forms of factor II are found in BHK21 cells.

Example 3 ■ Stabilization of factors Due to the gradual decrease in coagulation activity after 24 hours observed in the case of BHK21 cells, the applicant can increase the stability of the recombinant molecules thus obtained. I decided to do a test to see if that's the case.

Therefore, the von Willebrand factor (v W f )
(This is a fairly large glycoprotein that acts as a carrier molecule for factor 1 in the blood and plays a role in promoting coagulation by platelets after vascular damage.) (Reference, 22 ) BHK21 cells VV, F
After infection with VIII 10.2-1 and washing it several times, vWf preparation (0.5 U/ml) is added to the media.

Figure 3 shows the changes in procoagulant activity.

... In the presence of vWf O-O Intervention of other vWf - ■ After 48 hours in the absence of vWf, the procoagulant effect increases almost linearly, and Maxima is 0.18 U/m in the presence of vWf.
Corresponds to when jJ. In the absence of vWf, the time when the effects normally decline would be after 24 hours. Floating cells infected with wild-type V■ do not exhibit any procoagulant activity under the mediation of vWf, indicating that the infection of the ■ factor in the vWf reagent is not significant. 0
It can be seen that concentrations of vWf up to 1 IU/m,& are effective in stabilizing the recombinant protein.

At lower concentrations, no more molecule is protected after 24 hours.

From these results, two points can be confirmed: (1) Factor-active recombinant is observed, and it is stabilized by natural vWf.

Example 4 ■Construction of a plasmid carrying the coding sequence for the high or low molecular weight portion of the factor ■Plasmid pTG1016 carrying the complete coding sequence for the factor was used for the following construction as shown in Figure 4. Using.

Digestion of plasmid pTG1016 with oxygen 5phIn removes the DNA fragment contained between nucleotides 3935 and 6585 (nucleotide and amino acid numbering according to Wood et al. 1). Therefore, the plasmid that has been reconstituted has a sequence corresponding to amino acids 1 to 1293, that is, the high molecular weight portion of factor (2). In addition, there is a second 5ph1 site and a delay in codon reading based on the position of this site ('Frameshif
t') with the 44 last nucleotides as residues of the sequence located downstream of the 5TOP codon. (This DNA position is indicated by a grid in Figure 4). This plasmid is designated pTG1021.

Digestion of plasmid pTG1016 with the enzyme BclIn removes the DNA fragment contained between nucleotides 402 and 4419. The rearranged plasmid then contains a signal peptide, a high molecular weight amino acid 1 to
116, and then from 1455 to 2232, that is, in the low molecular weight region, there is a sequence corresponding to the natural 5 TOP codon of the Zarani ■ factor gene. This blastid is called pTG1025.

Example 5 Selection of a recombinant vaccinia virus having a sequence encoding the high molecular weight part or low molecular weight part of factor (2) in pTG1021 or pTG1025 construction.

In plasmids pTG1021 and pTG1025K, as in the original pTG1016 plasmid, the coding sequence is
．． Placed under the control of the 5K protein promoter, this entire DNA functional block is inserted into the TK gene of the vaccine.

Therefore, these plasmids can be used to construct DNA functional blocks using now conventional techniques (in particular, French Patent No. 84).
．． 06499) can be used to integrate into the TK gene of wild-type vaccinia virus by homolog recombination in cells infected and transmitted by the plasmid.

The wild type virus used was Copenhagen TS26 virus. Recombination takes place in BHK21 cells. Recombinant viruses are selected by their TK-characteristics.

Recombinant viruses were selected for each of the following constructions.

- Derivatives of plasmid pTG1021 Vv, TG, F■
.

One derivative VV from plasmid pTG1025.

TG, F■.

■VV, which has the sequence of factors and is derived from pT01026, F■10. 2. 1 virus is described in a major patent.

Example 6 Synthesis of factor ■ by cells infected with recombinant virus and antithrombin activity of the synthesized molecule.

In the two constructs introduced in Example 5, the sequences encoding the HC and LC chains are preceded by the natural signal sequence of factor 2, which is responsible for the excretion of the molecule into the medium. In addition, these two sequences contain cleavage sites by thrombin, which effect the proteolytic activity of the molecule.

Therefore, it is possible to measure the amount of the molecule (i.e. factor antigen) and examine its activity in the medium in which the cells are suspended.

Cultures of BHK21 cells (2×10 6 cells) are infected with wild-type vaccinia virus as a control, or with recombinant virus at an infection frequency of 1 u f p/cell.

For recombinants, four independent infections were performed. It is as follows.

-VV, TG, F ■ 10.2.1 - VV, TG, FVlff HC, 1-VV, TG
, FVIII LC, 1-VV, TG, FVI
II HC, 1+VV, TG.

FVIII LC, 1 This is a co-infection.

After 1 hour of adsorption, the cultures were washed and the medium was refreshed. This medium contains 4% ASB (but no calf serum), Ca
C121mM, Fong. Billeplant factor (vWf) 0
．． Contains 5 units/mfl. 24 or 48
After a period of time, the floaters were collected and immediately frozen in liquid nitrogen for subsequent testing.

1) The presence of factor VIII molecules or fragments thereof was determined by immunoradiometry. Antigen (F■:Ag) is made by absorbing immunoglobulin G from the serum of a hemophilia patient into the test tube wall and an anti-factor monoclonal antibody, which has a nibtope in the L chain and is radioactive. (According to the method by Lee et al. 19
).

The results are shown in the table (but only the chains can be detected by the monoclonal antibodies used) - recombinant VV,
Cells infected with TO, F■ LC, 1 were recombinant VV, TG.

F■ Produces an amount of antigen close to that produced by 10.2.1.

-LC,! :Cells co-infected with two recombinants of HC produced about half as few LC antibodies. The reason for this is that at the level of the cellular machinery necessary for the expression of the two proteins, two
This is because two recombinant viruses compete.

2) The activity of factor ■ (F■:C) was measured according to the conventional method.
Partial time activation of thromboplastin was performed (18). The results are listed in Table 2.

2: Factor ■ in the culture suspension of BHK21 cells infected with the recombinant virus - two recombinant viruses LC and HC
In the suspension of cells co-infected with , the coagulation activity was found to be significant, but the value was lower than that of the complete factor 2 recombinant. This activity diminishes after 48 hours, possibly indicating increased instability of the reassembled molecules. This activity is very unique. That's because it's anti-■
This is because the factor can be neutralized by adding monoclonal antibodies or inhibiting hemophilia patient serum.

− Even in cells infected with wild-type virus (negative control),
Even in cells infected with recombinants expressing only H chain or L chain, no procoagulant ability (FVm: C) was found in the suspension. The reason why in vitro mixing of these floaters containing H and L chains did not promote coagulation may be due in part to insufficient production to recombine the two chains, or alternatively, this It is also possible that recombination was unstable or could only occur under specific environmental conditions.

Plasmid pTc 188 is available from Bastoeur Institute, 28
rue-du Docteur-Roux.

75724 PARIS No. at the National Microbial Culture Preservation Center in Cedexl 5.

E, Co11 5KpTG1gg-No, filed on June 20, 1985 under the number 1.458.

The next strain is also from Bastwool Research Institute, 28ru″d
u Docteur-RouX% 75724 P
Submitted to the National Microbial Culture Archives of ARIS Cedexl 5 on June 6, 1986.

φ strain E, coli 5K pTG1016-N
o, 1. 558 ・Strain E, coli 5K pTG1080-N
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(Printed material)

[Brief explanation of the drawing]

Figure 1 is an explanatory diagram illustrating the gene preparation process for factor ■ used in the Examples. Figure 2 is a graph showing the coagulation activity of factor ■ over time in the presence or absence of vWf. The explanatory diagram, Figure 3A, is
One is high molecular weight part (Heavychain) pTG10
21, and the other is a light chai
n) Schematic representation of the construction of two plasmids of the cDNA sequence of factor ■, containing pTG1025 (the first row shows the complete sequence of factor cDNA; untranslated 5
' and 3' sequences are diagonally lined. Initiator codon ATG and 5TOP codon TGA. Elements used in construction, such as restriction sites, are listed. p
In the construction of TG1021, the gridded zones are 5p
Figure 3B shows the products of initial proteolysis (before activation) (numbers indicate the products of the initial proteolysis (before activation)). FIG. 1°: t. Agent Teruo Akimoto “-・1・、?fu and 1 other personFIG-2 <a)2

Claims (23)

[Claims] (1) A method for preparing factor VIII, which comprises culturing mammalian cells infected with a vector that expresses factor VIII in an appropriate medium, and further including the medium containing von Willebrand factor. (2) A method according to claim 1, further comprising adding von Willebrand factor at a concentration of at least 0.1 U/mp. (3) A method according to Claims 1 and 2, further characterized in that the cultured cells are BHK cells. (4) A vector characterized in that the vector for expressing factor VIII in mammalian cells is a box virus containing all or part of the DNA sequence encoding factor VIII. (5) The vector according to claim 4 is a vaccinia virus. (6) The vector according to any one of claims 4 and 5, wherein the DNA sequence encoding factor VIII is dependent on the promoter of the box virus gene. (7) The vector according to claim 6, characterized in that the promoter is a promoter of a vaccine gene. (8) In the vector according to claim 6, the DNA sequence encoding factor VIII is contained in the vaccine.
A vector characterized by being controlled by the promoter of the 5K protein gene. (9) A vector according to any one of claims 4 to 8, characterized in that the residue encoding factor VIII is cloned with the TK gene of the vaccine. (10) In the vector according to any one of claims 4 to 9, the sequence encoding a part of factor VIII is the high molecular weight portion HC of factor VIII: or VII
A vector characterized in that it encodes at least one of the polypeptides of the low molecular weight portion LC of factor I. (11) A vector according to claim 10, wherein the sequence encodes a high molecular weight portion of factor VIII. (12) A vector according to claim 10, characterized in that the sequence encodes a low molecular weight portion of factor VIII. (13) One of the claims 10 to 12 above.
A vector characterized by the fact that the HC or LC coding sequence is preceded by the natural signal sequence of factor VIII. (14) A mammalian cell infected with a vector according to any one of claims 4 to 13. (15) A vector according to one of claim 11 or claim 13 and claim 12.
A mammalian cell co-infected with a vector according to paragraph 1 or one of paragraph 13. (16) A cell characterized in that the mammalian cell according to claim 14 or 15 is a BHK21 cell. (17) A method for preparing factor VIII characterized by culturing cells and collecting the produced protein according to one of claims 14 to 16. (18) culturing mammalian cells infected with the factor VIII expression vector according to one of claims 4 to 13 in a suitable medium; A method for whole or partial preparation of factor VIII, characterized by comprising: (19) A vector in which the vector according to one of claims 10 to 13 is a plasmid having a sequence encoding HC or LC. (20) A polypeptide with a structure corresponding to HC or LC, or HC+LC reconnection. (21) A therapeutic composition comprising at least one polypeptide or one recombination compound according to claim 20 as an active agent. (22) Factor VIII obtained using the method according to claim 17. (23) Factor VIII obtained from cell culture, which is characterized by being mature and glycosylated.