JPS63503275A - Factor 8: Manufacturing method of C - Google Patents

Abstract

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

Description

[Detailed description of the invention] □ Activation factor ■: C and activation factor ■: encoding a C-like polypeptide. DNA sequences and methods for high-yield production of these polypeptides.

Technical field of the invention The present invention provides activation factor ①: a DNA sequence encoding a C-like polypeptide; The present invention relates to a method for producing the polypeptide using the DNA sequence. More particularly, the present invention Activated factor ■: production of C, and factor ■: biological activity of C The present invention relates to the production of an activated factor 2C-like polypeptide shown in FIG. Furthermore, the present invention The polypeptide has a higher yield than the previously produced Factor ■: C-like polypeptide. A biochemically pure maturation factor that can be produced in large quantities and more easily: C Refined.

Background of the invention Factor ■: C1, a type of high molecular weight plasma glycoprotein, is involved in blood coagulation that progresses in multiple stages. Functions as a coagulation-promoting component of factor ■, which plays a central role in the reaction. (Generally W, J, Williams et al, Hematol ogy, pp.

1085-90. ) lcGraw-Old II, New York (t972 )reference). Factor ■: C is a high molecular weight protein that is involved in platelet aggregation and adhesion. A certain factor ■R: Heg (also as a von Willebrand factor protein) known) and circulates in the blood.

Factor ■: C is synthesized as a single-stranded high molecular weight precursor and is then cleaved to form “ 'Mature' factor ■: Generates fragments constituting C.

Maturation factor ■: C is a double strand cross-linked by calcium ions, i.e. 740 An amino terminal H chain consisting of 684 amino acids and a carboxyl chain consisting of 684 amino acids. It consists of a xyl-terminated long chain.

Factor ■: The primary translation product of C is single-stranded, and the maturation factor ■ :The H chain of C is a “matured polypeptide” consisting of 908 amino acids from the L chain. (maturation polypeptide) tail. Excision of the t-(D matured polypeptide is Arg1648-Glu1 by an unknown, i.e., as yet unidentified, protease acting on the 649 bond. The primary translation product begins with proteolytic cleavage. H produced by the first cut The chain is shortened from its C-terminus by subsequent proteolytic cleavage. the result, A mature heavy chain of 740 amino acids is produced, which is then expanded to a trailing chain of 684 amino acids. together with yield the maturity factor ■:C [L, -0, Andersonet al, “l5olation and Character erization of HumanFactor VIII: Mo1e cular Forms In Commercial Factor VIII Concentrate, Cryoprecipitate, and P lasma, “PNAS (USA), 83.1)l), 2979-83 (1986)]. The resulting complex is then treated with thrombin as Arg 1689-3e. r1690 bond is cleaved and activated. [D. Eaton et al. , Biochemistry.

25, pp. 505-12 (1986)].

Hemophilia A is a linked bleeding disorder characterized by factor ■: either the amount or biological activity of C. Disease occurs when there is a defect.

Acute hemorrhagic symptoms in hemophilia patients can be detected using a factor obtained from normal serum and purified using conventional methods. ■It is being treated with. Various methods for purification are described in the literature [Z immermanetat, USP 4,361,509; 5 ound rey et at, USP 4,578,218; E, G, D, Dingudden hem et al,,”The Properties of Factor VIII Coagulant Activity Prepared By Immunoad-sorbent Chromatography, Jour nalof Laboratory Clinical Medicine, 9 3. pp. 40-53 (1979); D, E, G.

Au5ten, “The Chromatographic 5eparati on of Factor VIII on Am1nohexyl 5epha rose,” Br1tish Journal of Hematology , 43. pp, 669-74 (1979); H, Weinstein etat,,”Analysis of Factor VIII Coa gulant Antigen Inall“purificatiort A ndCharacterization of H+(2hlyPurifi ed Human Factor VIII Consisting of S ingleType of Polypeptide Chain,” PNA S (USA), 79. pp, 7200-04 (1982); C, A, Fulcher and T.S. Zimmerman.

“Characterization of Human Fact r　VlllProcoagulant　Protein　with　A　He terologous Precipi-tating Antibody, "PNAS (USA), 79. Dp, 1648-52 (1982); F. Rotblat et at, Thromb, Haemostasis , 50゜p, 108 (1983); C, A, Fulcher et. al., Blood, 61°pp, 807-11 (1983)].

However, its purification is difficult due to the relatively low concentration of Factor ■ in serum. Big factor ■R: Strong association with Ag, resistance to serum proteases I've come to realize that it's difficult because I'm so sensitive. For that reason, the factor -■: When C is purified from plasma, various proteolytic reactions occur, and the chain length increases to 16 A heterogeneous mixture of day chains ranging from 48 amino acids to 740 amino acids is mixed. Coming in [^nderson et al, 5upra, p, 2983 ). Factor ■ purified from plasma: Heterogeneous chain length mixture of peptides found in C Therefore, it is almost impossible to recover a substantially pure maturation factor ■:C. It was. Furthermore, the conventional method of treating hemophilia using factor■ purified from plasma has a fatal flaw. In particular, this treatment method is effective against hepatitis pathogens or AIDS (AIDS). Acquired Immune Deficiency Syndrome) The troublesome situation of virus metastasis can occur.

In view of the importance of hemophilia treatment, a large amount of factor Many attempts have been made to produce [e.g. Genetics In5 titute, PCT Application WO35101961; G enentech European Patent Application t60,457; Chiron European Patent Appli cation 150,735; J, J, Toole et at.

“Mo1ecular Cloning of a cDNA Encodin g Human Antihaemophilic Factor” Nature e, 312. pp, 342-47 (1984); and W, 1. W ood et at, Nature, 312. pp.

330-37 (1984)].

However, these attempts proved not to be as successful as hoped. the One of the causes is a factor of 2332 amino acids obtained through recombinant methods: C can be susceptible to proteolysis at many positions on the chain. By recombinant method, Another reason for the failure is that it is difficult to produce C at a sufficiently high yield. It is.

Book R Sarabe Kanhyou The present invention solves the above-mentioned problems by using activation factor ■:C and activated factor ■. :The problem can be solved by providing a DNA sequence encoding a C-like polypeptide. That is. With the DNA sequences encoding these polypeptides, Factor ■: Approximately 20 times the amount of Factor ■ that has been recombinantly manufactured in C. :C is produced and more easily purified into biologically pure maturation factor ■2C. Ru.

According to the present invention, a DNA sequence encoding activation factor It is expressed in high yield. As is clear from the specification and subsequent examples, the present invention Activation factor ■2C and activation factor ■: C-like polypeptide of the invention is Factor ■: The main part of the mature polypeptide of C is removed and deleted. It is a characteristic.

In our preferred embodiment, the DNA sequence encodes a mature polypeptide. Substantially all of the nucleotides responsible for this are deleted. Our most preferred embodiment In this case, the DNA sequence encoding the mature polypeptide is completely deleted. our When a DNA sequence is expressed, the maturation factor ■: The heavy chain of C binds directly to the downstream chain. ing. When one proteolytic cleavage occurs, the mature form of Factor ■:C is formed.

Finally, the present invention provides an activation factor produced by the DNA sequence of the present invention: C and activation factor ■: Various anti-hemophilia compositions containing C-like polypeptides are provided. provide and these compositions for the treatment of acute or chronic bleeding in hemophilia A. Provide various uses of objects.

Yahada Dansara Single Oniho” FIG. 1 depicts a restriction enzyme cleavage map of Factor ■:C cDNA.

Figure 2 schematically depicts the construction of a recombinant DNA molecule with a QD deletion. .

Figures 3A and 38G, Schematic construction of a recombinant DNA molecule with a tRE deletion. This is expressed in

Figure 4 shows the replication/enhancer start position of SV40, adenovirus major late promoter. data, RE deletion factor ■: CcDNA, factor ■: 3° non-removal of CmRNA. Mammalian cell expression vector pBG showing location of translated regions and polyadenylation sites A restriction enzyme cleavage map of the RE deletion inserted at 312 is drawn.

Figure 5 shows factor ■:C mRNA isolated from transfected B) ITIO cells. This figure depicts the results of analysis using Sl.

Figure 6 shows 5 out of plasmid DNA isolated from transfected B) ITIO cells. This figure shows the results of analysis using the Hern method.

Figure 7 shows the published sequence of the DNA and amino acids of Factor ■:C. (EPO 160, 457).

Oidan q anal emperor play blue shell In order to provide a better understanding of the present invention, the present invention will be described in detail below.

The following terms will be used in the explanation.

Nucleotides: from sugar moieties (pentose), phosphate esters and nitrogen-containing heterocyclic bases A monomeric unit of DNA or RNA. The base is the glycosidic carbon (1" of the pentose The combination of base and sugar is called a nucleoside. Ru. Bases characterize nucleotides.

There are four types of DNA bases: adenine (A) and guanine (G).

They are cytosine (C) and thymine (1). The four types of RNA bases are A, G, C and and uracil (U).

DNA sequence: A phosphodiester bond is formed between the 3° carbon and 5° carbon of the adjacent pentose. A single chain of linked nucleotides.

Codon: Amino acid, II translation start signal or translation stop signal throughout the mRNA A DNA sequence consisting of three nucleotides that encodes null. For example, TTA, T TG, C1-cho, CTC.

The nucleotide triplet of CTA and CTG encodes leucine (LeLl) However, TAG, TAA, and TGA are translation stop signals, and ATG is a translation initiation signal. It's a signal.

Amino acid: A monomeric unit of a peptide, polypeptide or protein. 20 kinds of amino acids, Namely, phenylalanine (Phe or F), leucine (teu or [), isolo Ile.

■), methionine () let, ) l), valine (Val, V), serine (Ser, S), Proline (Pro, P), Threonine ('rhr.

Shun, alanine (^Ia, A), tyrosine (Tyr, y), histidine ( Old S, H), glutamine (Gln, Q), asparagine (Asn, N ), lysine (Lys, K), aspartic acid (Asp.

D), glutamine M (Glu, E), cysteine (Cys, C), tri Ptophan (Trp, W), arginine (Arq, R), and glycine ( There is Gry, G).

Reading frame (Rreading Frame): conversion to the amino acid sequence of mRNA Grouping when translating.

Correct reading frame must be maintained during translation. For example, GCTGG TTGTAAG can be expressed in three open reading frames or phases, each with a different gives the amino acid sequence.

GCT CCT TGT AAG-Ala-Gly-Cys-Lys GCTG GTT GTA AG-Leu-Val-Val-GCTGG TAA G-Trp-Leu-(STOP) polypeptide: α-amino acids of adjacent amino acids A single chain of amino acids linked by a peptide bond between a group and a carboxyl group.

Genome: The entire DNA of a cell or virus, which contains, among other things, the polypeptides of a substance. Encoding structural gene, operator gene, promoter gene, 5hin e-Dal(learn.

Included are liposome binding and interaction sequences, including various sequences such as liposome binding and interaction sequences.

Gene: Generates a specific polypeptide through template or metsenger RN A (mRN A) A DNA sequence that encodes the unique amino acid sequence of a peptide.

Interpretation: The process of producing mRNA from a gene or DNA sequence.

Cultivation: The process of producing polypeptides from mRNA.

Shari: The process of producing polypeptides from genes or DNA sequences. Transcription and translation become a pair.

Plasmid: a non-chromosomal double-stranded DNA sequence consisting of a complete replicon (replication unit) The plasmid is then replicated in the host cell. Place the plasmid inside a unicellular organism Then, the characteristics of the organism change or transform due to the plasmid's DNA. . For example, a plasmid containing the gene responsible for tetracycline resistance (TETR) Therefore, when cells that were previously sensitive to tetracycline are transformed, they become Gain resistance. Cells transformed by a plasmid are called transformants.

Phage or bacteriophage: Many bacterial viruses have a protein sac or outer shell ( CAPSID).

Cloning vehicle: Plasmid, phage DNA, cosmid capable of replicating in host cells DNA, or any other form of DNA, whose DNA sequence can only be determined in a certain and verifiable manner. It also performs essential biological functions of DNA, such as copying.

Loss of outer shell protein production or loss of promoter genes or binding sites, etc. Labels that can be cleaved independently and are suitable for use in identifying transformed cells, e.g. endonucleases, including resistance to tetracytalin or ampicillin It is characteristic that it has one to several recognition sites. The cloning medium is often Also called a vector.

Cloning: By asexual reproduction, from one organism or DNA sequence, create multiple identical The process of obtaining an organism or DNA sequence.

Recombinant DNA molecule or HybridDNA: A method that combines different genomes into living cells. D obtained by joining the ends together on the outside and able to maintain them in adult cells. A molecule consisting of an NA moiety.

Expression control sequence: When linked to the above gene by manipulation, it controls and regulates gene expression. nucleotide sequence. These include ring systems, β-lactamase systems, υ■-systems, linked systems, υ engineering system, main operator gene and promoter gene region of phage λ, outside fd Shell protein control region, SV40 early and late promoter genes, polyoma Metallothionein, a promoter gene derived from viruses and adenoviruses Glycolysis of promoter gene, 3-phosphoglycerol ester kinase or its counterpart Enzyme promoter genes, acid phosphatase, e.g. Ph05 promoter genes; genes, yeast α-mating factor promoter genes and other prokaryotic or eukaryotic cells and known sequences that control gene expression of those viruses or their combinations. There is.

Factor ■: C: Polypeptide having the amino acid sequence shown in Figure 7, mature and activated Then, due to the multi-step mechanism of blood agglutination reaction, factor IXa-dependent factor It can function as a cofactor for maturation. Files used in this invention Factor ■: C Hata, Factor ■ Aggregation precursor activation protein, Factor ■ Coagulation activation protein White, antihemophilic globulin (AHG>, antihemophilic factor (AHF>) and antihemophilic factor Contains glycoproteins, also known as A [W, J, Williams et al, Hematology, pp,’1056.1074 and 1 0811° Maturation polypeptide: Factor ■: The maturation polypeptide of C is the 741st cell. Consists of 908 amino acids from phosphorus to arginine at position 1648 ( (See Figure 7).

Factor ■: Maturation of C is cleaved between amino acids 1648 and 1649 occurs (at this time, a C-terminal chain is generated). A series of cuts then occur, resulting in A mature N-terminal heavy chain is produced.

Maturation factor ■: C: Maturation factor ■ used in this application: C is C-terminus chain (Glu 1649-Tyr 2332) and alkali metals, e.g. from the N-terminal H chain (^la 1-Ara 740) bonded via a mu bridge. Ru.

Activation factor ■:C: ``Activation factor ■:C'' used in this application is Factor ■:C which lacks the main part of the mature polypeptide. example For example, activation factor ■: C is a factor in which the entire mature polypeptide is missing. ■: A protein in which the N-terminal mature H chain and the C-terminal mature chain of C are linked together in a single chain. Including white.

Activation factor ■: C-like polypeptide: "activation factor used in this application" Tar■:C-like polypeptide'' has the biological activity of activation factor■:C Contains protein. The same polypeptide is a protein containing an amine-terminated methionine, e.g. f-Net factor ■: Deletion, addition or substitution of C and other amino acids Activation factor ■: A protein that has the characteristics of retaining the biological activity of C. Contains.

Paraactivation factor ``C-like polypeptide'' within the scope of the above definition also includes each It also includes the natural allelic variations that exist and can occur in polypeptides. more sugar The degree of chain formation and position of glycans or other post-translational modifications may vary depending on the production host or tissue cell. Activation factor that differs depending on the environment ■: Contains C-like polypeptide I can do it.

The present invention relates to the production of activation factor ■:C and activation factor ■-like polypeptides. Regarding the law. More particularly, the present invention provides activation factor ■:C in a suitable host. and activation factor ■: DNA sequence that can produce C-like polypeptide in high yield. provide. Polypeptides produced by the DNA sequences of the invention can be used for the treatment of hemophilia A. It is useful for medical treatment.

Factor ■: Activation factor produced by the DNA sequence of the present invention compared to C. Factor ■:C has a deletion of the main part of the factor ■:C maturation polypeptide. . The DNA sequence of the present invention surprisingly activates activation factor ■:C, factor ■: Expressed in much higher yield than the DNA sequence encoding C itself.

While not wishing to be bound by theory, the inventors believe that the present invention The DNA sequence provides high yields due to the absence of most or all of the mature polypeptide I believe that activation factor ■:C will be produced. For example, mR of an activated gene NA does not require long RNA encoding the mature polypeptide to be translated. So it will be translated more efficiently. Furthermore, factor ■: C is abundant in cells. However, in activation factor ■:C, the mature polypeptide This type of attack is not possible because the site in the putid that undergoes proteolysis is no longer present. It becomes difficult to accept. Furthermore, in the absence of a maturing polypeptide, the natural factor ■: N- of C Of the 25 binding glycosylation sites, 19 are missing, and only a few are present on the activated polypeptide. Only 6 N-linked glycosylation sites remain (3 on the H mirror and 3 on the L mirror). location). Because there are clearly fewer glycosylation sites, the production and purification of activation factor ■:C is difficult. It becomes easier to manufacture.

The main part of the DNA sequence was removed. Find the DNA sequence containing the desired deletion site. The inventors used the same sequence in various expression vectors and hosts, and Activation factor ■:C encoded by

For example, a wide variety of expression vectors encode the activation factor ■:C of the invention. used to express the sequence. Activation factor ■: C-like polypeptide It will also be understood that encoding DNA sequences may be similarly produced by the present invention. .

Useful expression vectors include, for example, chromosomal, non-chromosomal, and synthetic RNA sequence segments. vectors, such as various known derivatives of SV40, known bacterial plasmids, For example, colEl, pcRl, pBR322,0839 and derivatives thereof plasmids from L. coli, including broader host range plasmids, e.g. RP4, a number of inducers of phage DNA, e.g. phage λ, e.g. NM989. Conductors and other DNA phages, such as old 3, and filamentous single-stranded DNA phages, yeast plasmids such as the 2μ plasmid or derivatives thereof, and plus Vectors derived from a combination of mid and phage DNA, e.g. or plasmids modified using other expression control sequences. of the present invention In a preferred embodiment, the pBR327-related vector pBG312 is used. [R, Cate et al, Ce1l.

45, pi), 685-98 (1986)].

Furthermore, a wide range of expression control sequences, i.e., control the expression of manipulated linked DNA sequences. sequences can be used in these vectors to express the DNA sequences of the invention. I can do it. Useful expression control sequences include, for example, the early phase of SV40. and the late promoter gene, lac system.

Two systems, TAC or science, main operator and promoter gene of phage λ, f control region of d% shell protein, 3-phosphoglycerol ester kinase, or other Promoter gene of glycolytic enzyme, acid phosphatase, e.g. Pho 5 promoter gene Motor genes, yeast α-mating factor promoter genes and other prokaryotic or eukaryotic Controlling gene expression in biological cells and their viruses or their combinations Includes known sequences. In a preferred embodiment of the invention, adenovirus -2 major late promoter gene expression control sequences are used.

A wide variety of host cells are also useful for producing Activated Factor ■:C of the present invention. this host cells include well-known nucleated and prokaryotic cell hosts, such as E, col i.

Pseudomonas, Bacillus, Streptomyces Bacteria, fungi such as yeast, and animal cells such as CHO cells, African green monkey cells. cells such as CO31, CO37, B5Cl, B5C40 and B) IT-10 and Includes human cells and plant cells in tissue culture.

In a preferred embodiment of the invention, B) ITIO African green monkey cells use.

Of course, all vectors and expression control sequences function equally well and exhibit the activity of the present invention. Activation factor ■:C of the present invention is produced. It should be understood that this is not the case. Also, all hosts are equally good with the same expression system. It doesn't mean it works. However, if one skilled in the art However, if the expression control sequence and host are appropriately selected, unreasonable experimental results may be obtained, and the present invention may There is no deviation from the range. For example, when selecting a vector, the vector should be The host must be taken into consideration. Number of vector copies, Production control capacity and expression of other proteins encoded by the vector, e.g. Antibiotic resistance markers should also be considered.

Various factors must also be considered in the selection of expression control sequences. Among them are For example, the relative strength of the system, its controllability, and the activation factor of the present invention ■: This includes compatibility with the encoded DNA sequence, particularly possible secondary structure. with host selection is the compatibility with the selected vector, our activation factor ■: against the host of C. toxicity, host secretion properties, ability to fold proteins accurately, fermentation conditions9. Activation factor■: The ease and safety of purifying C from the host must be considered. Must be.

Within these parameters, those skilled in the art will be able to determine the activation of the present invention by fermentation. Factor ■: The combination of vector/expression control system/host that produces a useful amount of C. You have to make various choices. For example, in a preferred embodiment of the invention, adeno B) The pBG312 vector of the virus 2 main late promoter expression system was Use with Rica Green Monkey Cells.

Activation factor ■20 and activation factor ■2C produced by the present invention Similar polypeptides can be purified by a variety of conventional methods and procedures. useful spirit As for the manufacturing method, natural and recombinant factors are used to purify C. There are methods (for example, -0, Anderssonet al, PNAS (USA), 83. pp. 2979-83 (198B)).

After purification, activation factor ■: C and activation factor ■: C-like polype of the present invention Putide is useful as a composition and as a therapeutic method for the treatment of hemophilia A, and It is useful as a therapeutic agent for various types of uncontrolled bleeding.

Activation factor ■: C and activation factor ■ = C-like polypeptide of the present invention are , prepared as a composition in the form of a single chain polypeptide, or administered in a variety of ways. However, within the scope of the present invention, activation factor ■:C can be proteolyzed. Of course, it is understandable that it is also possible to administer the drug after the treatment. For example activation Factor ■: C is cleaved in vitro and matures Factor ■: C H chain and and L chains, and before, during, or after purification, calcium or other Can be linked by an alkali metal bridge.

Activation factor ■: C and activation factor ■: C-like polypeptide of the present invention are can be formulated by known methods to produce pharmaceutically useful compositions. The same composition is preferred or a known pharmaceutically acceptable carrier, and may also contain other drugs, carriers, agents, etc. Also includes adjuvants, excipients, and other e.g. human serum albumin or plasma preparations. . For example, Remington's Pharmaceutical 5science See (E, W, Hartin). The resulting formulation may cause uncontrollable discharge in the recipient. Contains an amount of activation factor ■:C that is effective enough to treat blood. Ru. Administration of these polypeptides or their pharmaceutically acceptable derivatives has traditionally been Any method that has been accepted as a vector administration method may be used.

They include parenteral, subcutaneous, and intravenous administration.

The compositions of the present invention used in the treatment of hemophilia can be formulated in a variety of dosage forms. preferred agent The form will vary depending on the intended mode of administration and treatment. Administration methods and dosages vary. Depending on factors, e.g. whether to treat acute bleeding patients or to be used prophylactically. I'm coming. However, Factor ■:C levels prevent bleeding and promote epithelial formation. (Williams, Hemato Logy.

see the discussion in Pi), 1335-43).

In order to better understand the present invention, the present invention will be explained below with reference to Examples. Main implementation The examples are for illustrative purposes only and do not limit the scope of the invention in any way. It's not something you can do.

Example The present inventor has proposed an activation factor in which a major part or all of the mature polypeptide is missing. -■: A CDNA sequence encoding the C molecule was constructed. To test the limits of the invention , the inventors also found that Factor ■: is missing more than the mature polypeptide of C. A CDNA sequence encoding the polypeptide was also constructed.

A. Complete factor ■: Construction of CCDNA In Figure 1, complete factor Factor ■: Shows the restriction enzyme map of CCDNA [W, 1.　Wood　 al.

Nl Kyo 4, 312. [l]l), 330-37 (1984); Figure 7].

Double lines (bars) represent coding sequences. Below the restriction enzyme map, Calcium Maturation factor ■: Maturation factor attached to the L chain at the C carboxyl terminal Factor ■: The H chain at the C amino terminal is drawn. Chains matching restriction map Screening of human placenta, liver and kidney cDNA libraries under the protein model The oligonucleotide probes (indicated by *) used for the determination are shown. child These libraries used oligo(d) as the first chain primer and was constructed using λgt10 as a vector.

On this secondary chain, the inventor used human kidney mRNA as Tenbrei 1~ The oligonucleotide primers used to initiate the first cascade of CDNA synthesis ( There is an arrow pointing left. The inventors converted these single-stranded CDNA chains into GLIbler and H It was made into a double strand by offman's technique [υ.

Gubler, and B. J., Hoffman, Gene 25.　 pp, 263-69 (1983)], Kot'L La D N A 1) B It was cloned into the EcoRV site in R322 with dC at the end. instructor The kidney cDNA plasmid library obtained by The oligonucleotide probe was sieved 5° above the primer.

Below the primer/probe chains in Figure 1, the inventors can simply Separated partial factor ■: Indicates CCDNA and genomic subclones.

Together, they seize the complete length CDNA gene and update these clones. The detailed points are shown in Table 1.

Table 1 Factor ■: Summary of C Genomic and CDNA clones Cosmid 1) Isolated from Genomitsuta library constructed with TCP [Grosveld, F, G, et at, Nucleic Ac1 ds Re5search, 10, t) I), 6715-6732 (198 2) ] Subclone length tissue 1) UC19, 28742874bp 48. XXXX human lymphoblasts Amount from oligo (dT) prime λgtlO cDNA library Clone length probe hybridization 1.7977 (placenta > 1728 79+, 77+2.73 (liver) ~70 0 73+ 4.73 (kidney PM) ~220 73÷85.86-Prime Gubler-H Isolated from offman kidney l1iFf cDNA library Clone length probe hybridization 1.82 ~ 1200 82+, 79-. 77-2.82 ~ 1200 82+ , 79-. 77-3, 7573 ~ 2700 74-. 75+, 73+4, 75 73 ~ 2700 74-. 75+, 7346, 7573 ~ 2700 74- ．． 75+, 73+10.797783 > 1263 82-. 79+, 77÷, 83+11.797783 >1263 82-. 79+, 77+, 83+12 ．． 797783 >1263 82-. 79+, 77+, 83+13.7977 83>1263 82-. 79+, 77÷, 83+75.7 Who Prime Gu Isolated from bl er-Hof fman kidney cDNA library Clone length probe hybridization Before assembling the complete CDNA gene The inventor constructed two intermediate plasmids. This is a factor ■: CCD This is necessary because the NA is too long.

In the first pre-assembly, the inventor started from the 5163rd PSt1 site to the 5755th PSt1 site. A fragment from clone 7, 7475, which is continuous to the PSt1 site of the eye, was isolated. I let go. This fragment was cloned into 4.75 at the 5755th PstI site. 73, thereby extending clone 4.7573. This b month part is shared by the inserts of clones 7, 7475 and 4.7573. is possessed. By extending clone 4.7573 in this manner, the present invention The author has created a unique NdeI site in the insert of this clone 4.75731 conductor. Ta. Because we needed a specific site to extend this chain of inserts at the 5° end, we developed this invention. The enlightened person had to create this Nde part.

As a second preassembly, the inventors added the polynucleotide linker to clone 2. 82, Factor ■: 5 immediately adjacent to the translation initiation codon of the C signal sequence. It was introduced in the position of ゛. Clone 2.82 inserts into the EcoRV site of pBR322. This direction is opposite to that of tetracycline resistance. clone 2.8 The 5° end of the insert in 2 is at position −133 in the 5° untranslated leader sequence. Book The inventor cloned clone 2.82 into the 5alI site in the tetracytarif resistance gene. , Sac in the sequence encoding the signal peptide in the insert of clone 2.82. I and introduced the synthetic duplex.

SAI N HBBHNSS ACN RCAPISAS CCU ONIAPCT GTCGACTCGCGACCATGGATGCAAATAGAGCTCi - −−1−−−−−1−−−−−−−−+−−−−−−−−−+−−−−33CA GCTGAGCGCTGGTACCTACGTTTATCTCGAGHetGl nI　IeGIuLeu As a result of this binding, the 5alI-Nrul-NCOI polylinker becomes :Introduced at 5° immediately adjacent to the translation start codon of the C signal sequence. these Three types of restriction enzymes are full chain length factor■: They do not cut the CcDNA gene. stomach.

These two intermediate constructs were used in a 6-fragment ligation reaction to generate the complete factor. ■: C was assembled (Figure 1R bottom row). The present inventors obtained a complete D in a single-stranded clone state. Since I have never isolated DNA, I have to use this method to construct DNA of the complete chain length. did not become. The inventor identified fragment 1 as the above-mentioned derivative of clone 2.82. isolated from Fragment 1 is AVa from position 5alI to position 731 in the polylinker. It's transparent. Fragment 2 is λ(]t10 recombinant gene 1.797 isolated from the insert in 7. Fragment 2 is from AVal at position 731 to 2289 This continues up to EcoRI, which ranks second. Fragment 3 is dienomic cosmid recombination It was isolated from the prey subclone pUC19,2874. The same fragment is 22 From EcoRI at 89th place to BamHI at 4743rd place, it is clear. fragment 4 was isolated from clone 7, 74575, and originated from the BamHI site at position 4743. It continues up to Dan 1el, which is ranked 5522nd.

Fragment 5 was isolated from clone 4.7573 described above.

Fragment 5 extends from NdeI at position 5522 to Ncol at position 7991 . Fragment 6 was used as an origin of replication and selection marker in E. coli. This is an assembled vector containing a phosphorus resistance gene. Fragment 6 is pAT, SV2. Separated from tPA. 1) AT, SV2. tPA is Richard d Presented by Mr. Fisher. This suggests that the transcription of the tpAz gene occurs during the SV40 early stage. It is a plasmid under the control of a promoter. The inventor pAT, SV2゜t 5alI and SV40 before cleaving PA within the tetracycline resistance marker gene Enzymatic degradation was performed using Ncal, which cuts within the phase region.

Analysis of 96 recombinants revealed that 32@ was the control factor for all 591 factors ■: C. Contains limited fragments. The inventor has determined the DNA sequence of one of these clones. It was confirmed that there were two differences from the published sequence.

One is Leu242, where CTG becomes CTA, and the other is amino acid residue 1. At 880, TTC becomes CTC (t'he becomes Leu) (see FIG. 7).

B, Transfer of full-length CDNΔ into mammalian cell expression vector The present inventor has developed a complete factor ■: C cDNA gene from a recombinant vector. It was cut out using l. The resulting NC0I restriction fragment was digested with nuclease S1. to form blunt ends. pBG in which this fragment was digested with SmaI Combined with 312. pBG312, whose structure has been described by other researchers It is an animal cell expression vector [R.

Cate et at, Ce1l, 45. pp, 685-98 (1 986) l. The pBG312 sequence from EcoRI to the old one is (clockwise) SV40 origin of replication, adenovirus-2 main light promoter and complete trypa -ticle leader sequence [S, Zain et at,.

Ce1l, 16. pp, 851-61 (1979)]: Adenowi Rousse 5 splicing donor and immunoglobulin variable region 10. gene splicing A hybrid splice consisting of an acceptor and a bio1. ．． f59. pp, 601-21 (1982; HindIII, XhoI, Majesty RI, Sm Polylinker containing sites for aI, NdeI, DanStI and Machie II: Splice SV40 with single donor and acceptor attached to the side is an antigen intron , and SV40, which has the above-mentioned polyazonylation site.

The present inventors discovered 1) a factor containing a signal sequence in a part of the polylinker of BG312; : prove two independent loans 8.1 and 8.2 CDNA encoding C. Ta. Clone 8.1 differs from clone 8.2 in the 3' untranslated region; clone 8. 17806 in 1 is fused to the SmaI site of pBG312, whereas In loan 8.2, C at the NcoI site at position 7990 is fused.

Furthermore, the present inventor has determined that within the sequence encoding the signal peptide of Factor ■:C, Factor ■: Another product in which the cDNA gene encoding C is fused to pBG312. Roan was also isolated. This clone is called Signal Minus by the inventor. is a transient expression test (described later). ays) was used as a negative control.

Construction of C1GLN744-ASP1563 (abbreviated as QO) DNA encoding expression of the mature polypeptide (amino acids 741-1648) A method for creating QD deletions by removing part of the sequence is shown. Lack of QD is a mature polype The approximately 90 amino acids of the polypeptide (the 4 amino acids at the N-terminus of the mature polypeptide) 84 amino acids at the carboxyl terminus remain.

Figure 2 shows a configuration lacking QD. The inventor creates a certain amount of complete factor ■:C The gene expression plasmid was partially digested with EcoRI. This endonuclea cut between the Gln744 codon and the Asn745 codon. The inventor is 5 'AATT overhang was removed with nuclease S1 and the resulting plasmid was Pvu It was completely degraded within the ampicillin resistance gene by I. The inventor also separately provides a certain amount of Take and cut between the Leu1562 codon and Asp1563 codon...In II Partially disassembled (see Figure 7). 5'GATC overhang is Klenow fragment and once again the resulting plasmid was digested in-house with Pvu1.

The two mixtures of fragments were put together and both were ligated with T4 DNA ligase. . Activated polypeptide produced upon expression as a result of Gln744 QD deficiency Tide is the 4@ C-terminal nucleotide attached to the carboxy-terminal Arg740 of the mature H chain. amino acids, and the 86 N-terminal amino acids attached to the amino terminal Glu1649 of Lm. Of the mature polypeptide consisting of 908 amino acids, excluding amino acids, 818 The @ amino acid is missing (Figure 7). Consists of 908 amino acids like this The mature polypeptide - the first maturation of the translated polypeptide and then unchanged 90 by both protease substrates used in the subsequent maturation of the remaining heavy chain. It is replaced by a mature polypeptide consisting of 5 amino acids.

D, ARG740-GLU1649 (abbreviated as RE) missing construction In this chapter How to create a lack of RE by removing the entire DNA sequence encoding the mature polypeptide Let's talk about.

Figures 3A and B show how this RE-deficient fusion was obtained in two steps. 1st stage In the second step, the four fragments are ligated to obtain one intermediate plasmid. 4 frames What is Ragment? (1) 462bl) fragment: Full length gene expression plasmid, Hind Decompose between Arg740 and 5er741D tons using III and 5°^GC T was removed using nuclease S1 and specifically 1yr586 and Leu5 (decomposed with KpnI, which cuts between codons 87) (2) Synthetic oligonucleotide doublet fragment 5°pGAA ATA AC T CGT ACT ACT C T CAG TCACTT Ding AT Ding GA GCA TGA TGA GAA GTCAGT CTA Gp 5゜Glu Ile Thr Arg Thr Thr Leu Gin Ser 85p (3) The full-length gene expression plasmid was first digested with 5au3A and 135b p fragment is obtained. The inventor has developed 38M657 and Asp1658D and between the GIU1794 and Asp1795 codons using bag 3A. An 11 bp fragment was isolated. The 411bl) fragment was then converted to Ala Digested with PStI, which cuts between the 1702 and Val1703 codons, resulting in a 135bp A 5° fragment was obtained.

(4) pucis decomposed in Hayabusa I and Fun 4I.

From this intermediate plasmid, the inventors obtained a fragment encoding RE@ . For this purpose, an intermediate plasmid generated by ligating the four fragments was used as ASp7. 18 and PstI. QD using fragments encoding RE fusions Replaced the corresponding fragment in the fusion expression plasmid. The obtained RE melt A 624 bp fragment encoding the sequence was added to a plasmid for expressing the QD internal deletion. Different! First cut completely at the J718 site, then cut the 5” GTAC overhang into the calf. phosphorylated with intestinal phosphatase, and the resulting plasmid was partially excised with Pstl. The mixture was then ligated.

A map of the RE-deficient insert introduced into pBG312 is shown in FIG.

The activated polypeptide produced by gene expression consists of 908 amino acids. The mature polypeptide is completely removed. Containing such recombinant molecular species The superior polypeptides produced by these cells are secreted, and the H chain is directly attached to the L chain. It may be purified as a single strand that appears to be linked.

In this absence, during the first cleavage of the full-length primary translation product, the cleaved Is the peptide bond between Arg at position 1648 and Glu at position 1649 conserved? This internally missing primary translation product then initiates the cleavage of the full chain length primary translation product. It is cleaved by the same proteolytic enzyme that In this way, factor ■: Maturation of the H chain of C. Produce molecules directly.

In Western analysis (data not shown), we tested 90 KH and 80 KH in culture medium. The single-stranded molecule that is processed into the K l chain is the RE activation factor ■: coded by C. I confirmed that it was.

The resulting L chain contains 1649 where the double-chain complex binds to the Huonbillbrand protein. There is a peptide consisting of Glu at position 1689 and Arc at position 1689.

Therefore, when this recombinant product is secreted from animal cells, it is not present in the cell culture medium. It will bind to the 7-onvirbrand protein present in the protein.

Similarly, when injected, it forms a complex with the 7-onvirbrand protein in the serum and It will circulate within. Thrombi between Arg at position 1689 and Ser at position 1690 The double-stranded maturation factor ■:C is activated by cleavage by Dissociates from Rand protein and forms three components, Factor IXa and Factor Xa, on the platelet surface. will form a complex consisting of minutes.

E, construction of ARG740-3ER1690 (abbreviated as R8) lacking outside these missing In order to test the limitations of the mature polypeptide and other encodes a polypeptide (i.e., the inventor has determined that the maturation factor ■: the N-terminus of the light chain of C. The DNA sequence encoding the expression of the 41 amino acids at the end has been deleted) plus Made up Mido.

The inventor created this R3 fusion in the two-step manner described for the RE fusion. in the first stage An intermediate plasmid was constructed by ligating the three fragments. 3 flags used Ment is (1) 462bp fragment: Hind the full-length gene expression plasmid Cut between Arg 740 and Ser 741 with III to remove 5°AGCT. Removal with Rease S1 followed by specific codons of 1yr586 and Leu587 It can be obtained by cutting the gap with the blade ■.

(2) Synthetic double-stranded oligonucleotide fragment: 5' pAGCTTTC AA AAG AAA ACA CGA CACTAT TTT AT GC T GCATCG AAA GTT T Ding C Ding TT TGT GCT GTG ATA AAA TAA CGp 5゜Ser Phe Gln Lys Ly s Thr Arg His Dyr Phe Ile Ala Ala (3) With this fusion of PstIr and pUC18, Ar! 11740 and 5er 741 (now Ser 1690) Recreated HindIII site between D tons .

A fragment encoding the R3 fusion was isolated from this intermediate plasmid, and this flag was used in the second step to extract the corresponding fragment in the QD at Godagawa expression plasmid. replaced. In this second step, we identified the 501b encoding R3 fusion. The p fragment was isolated. Cut the intermediate plasmid with layer 718 and PStI, R A fragment encoding the 3 fusion was isolated. Next, the method used in RE fusion is The relevant fragment in the expression plasmid for QD fusion was Replaced with fragment.

The entire matured polypeptide was removed and further Gl (J1649-Arg1689 peptide DNA encoding the petido, i.e. the putative von Willebrand binding domain. A deletion was performed in the absence of R3. Therefore, this recombinant molecule is transferred from animal cells to the culture medium. circulating vonWi l when secreted into the body or injected into the receptor. Does not bind to 1ebrand protein.

F. Transfection of African Green Monkey Kidney Cells The inventors transfected BHTIO cells [ R, D, Gerard and Y.

Gluzman, Hot, Ce11. Biol, 5. pp, 32 31-40 (1985) was transfected with the supercoiled expression plasmid.

Also DEAE-dextran method [L,)1. Sompayvac and K.

J, Danna, PNAS, 78. pp, 7575-78 (198 1)] and Kuro.

Kin [H, Luthman and G, Manusson, Nuclei c　Ac1dsResearch,　11. pp, 1295-1308 ( (1983)] were used to transfect the cells. The transfectants were infected with the 5V40T antigen. Inducibly supplied in trans by BHTIO cells, expression plasmid Activation factor ■: Binds to the replication origin of SV40, which is bound to the C gene. It is known that the introduced expression plasmid can be replicated with high replication yield. Ru. However, this method shows that only a few percent of transfected cells actually contain DNA. It is inefficient because it is not connected.

The resulting transfectants secrete activation factor ■:C for up to 120 hours.

In most experiments, the cm2 lrd ratio was approximately 5.5, i.e., for a 100 mm diameter Petri [ [Culture of 10 ml of confluent monolayer of group 110 transfectants in L (55 cm2) covered with liquid.

G, factor ■: C activity measurement After double transfection into 10 cells of 88 cells, we obtained a signal minus 8.1. , QD, RE, and R3 expression construct factor ■20 production activity was analyzed. Kab i　Coatest■ manufactured by Vitrum, Factor■: 96 C kits Used with well plate. RN for 81 analyzes using one Petri dish Prepare A and extract the old rtDNA used in the 5outhern analysis from the other one. Prepared. After 120 hours of culture, factor ■:C activity in the cell culture medium was analyzed. Ta. The results are factor ■: C plasma concentration, approximately 20 On! Based on 11/d and expressed as a percentage of that level.

Repeated transfections yielded both a signal-minus construct (negative control) and an R8-deficient construct. Factor ■: C activity was undetectable.

This is due to the lack of VOn Willebrand protein binding region in the R3-deficient body. It may be explained as follows.

As shown below in a 120-hour experiment, cells transfected with the full-length gene were The lack of RE showed approximately 5% of the activity of the lack of RE. The activity observed in the absence of QDs is 1. 46% plasma level and 1.30% plasma level in absence of RE.

Thus, Bl(T10 cells) transfected in the absence of QDs and REs are fully chained. Producing factor ■:C that is 20 times more than cells transfected with a long gene. do.

H, Factor ■: mRNA level in the CmRNA nuclease 81 analysis platform construct. Nuclease 81 analysis was performed to analyze the cell line. This analysis This helps determine why the expression levels were improved in the absence of QD and RE.

BHTIOrim cells were transfected and cultured in iomm diameter Petri Tsukuda for 120 hours. , and then the RNA, which has not yet been published.

Isolated using the method of Schleuning and J. Bertoni S.

Briefly describing this method, 100μ (containing 1/d of proteinase K) 50mM Tris-HCffl (ptR,5)-5m)l EDTA-1% SDS solution 3InI! BHTIO cells were lysed for 20 minutes at 37°C using

Transfer the lysate into a 50d conical tube containing 3d phenol and Centrifugation was performed at high speed for 15 seconds in a tron (Brinkmann). water phase Extracted with ether and adjusted to 0.25 NaG. Improper tool of the same volume The nucleic acid portion was precipitated at 4°C, the precipitate was collected by centrifugation, and the 3d Redissolved in water. Adjust the solution to 2.8) 1 ti, keep it at 4℃ overnight, and select RNA was precipitated.

The amount of activation factor ■:C mRNA for each construct was quantified. QD expression The lasmid was cleaved with P-de-I and Pro-1 was isolated for 81 analysis. …■Fragmen Label the 5′ end of the protein with [γ-32p]ATP and 4 polynucleotide kinase. It rang. 10 μq of RNA was isolated on a 5% strand separation gel with 477 nucleotides. 5000 cpm 32P-antisense strike of cleotide EspI fragment 10 μl of 80% deionized formamide annealed to the land-400m HNaG-40mM PIPES (pH 6,4)-11r+HEDTA overnight Cultivate. The obtained hybrid molecules were nucleated at a concentration of 1oo units/mj'. Crease S1 0.2 & HNaG-50mM NaOAc (pH 4,6)-4 , 5+++H2nSO, add 170 ul of solution and cut. EDTA at 10 mH Add until the time is up to stop the elimination, and extract with phenol. Denature the protected fragment and electrophores it on a 5% strand separation gel. The dried gel is immersed in Li. (]htinjJ Plus brightness enhanced screen (manufactured by DuPOnt) Exposure was carried out overnight at -70° C. to hardened Kodak XAR-5 X-ray film.

The 477 nucleotide EspI fragment is a 5° non-factor of Factor ■:C mRNA. One end is within a hybrid intron excised from the translated region [R, J, Kaufman and P, A.

5har, J,) lot, Biol, 159. pp, 601-2 1 (January 1981, and the other end is within the Ala 62 codon (Fig. 4 ).

Activation factor■: CmRNA is a single-stranded 300 nucleotide DNA fragment was detected by protecting it from cutting. Experiments with excess single-stranded probe In order to prove this, we conducted several experiments using 1 μg of RNA.

Activation factor for each construct■: Results of nucleotide 31 analysis of CmRNA The results are shown in Figure 5. According to the results of the present inventor, activation factor ■: CmR NA levels are the same for the three deletion and complete factor ■:C genes. figure 5A is the analysis result using RNA1μ9, and Figure 58 is the analysis result using RNA1μ9. This is the analysis result. Lane 1 in the drawing shows activation factor ■: CmRNA. Labeled 477 nucleotide single-stranded DNA used to protect from S1 cleavage Contains 500 cpm of fragments as markers. It is for each hybrid This corresponds to 10% of the amount used for lysis. Lane 2 is a signal minus configuration Contains RNA isolated from in vitro transfected BMTIO cells. Lane 3 is Full length factor ■: BHTIO transfected with C cDNA (construct 8.1) Cells; lane 4 is transfected with activation factor ■: CCDNA (lacking QD) 8MT10 cells; lane 5 is activation factor ■: C cDNA (RE lacking ); lane 6 is transfected with cDNA from R3 deletion Transfected BMTIO cells; Lane 7 shows the 3′ Label the ends using [α-32P]dATP and KlenOW enzyme. (a gift from Mr. Tizard). equal length The protected fragment with the expected length of 300 bases was clearly 8.1, QD, RE, Seen in both figures showing the R3 construct. Protected fragment approximately 220 bases long The signal peptide is clearly visible in both figures for the signal minus construct, and the signal peptide There is no part of the DNA sequence that encodes this.

Comparing Figures 5A and 5B, it can be seen that the 477 probe used It can be seen that there is a molar excess of each constituent during the process. Activation factor■: The C activity level is lower in the QD and RE deficient forms compared to the R3 deficient form and the complete construct. The amount of mRNA in these four constructs is almost exactly the same, although at least 20 times higher. It is the same. Therefore, the reason for the increased expression in QD and RE-deficient cells is due to post-translational properties. It depends.

■ Southern analysis of plasmid DNA from transfected B) ITIO cells. Newly replicated activation factor ■: C plus Mido DNA levels were quantitatively analyzed and compared with those of the full-length gene. Matako The test is based on the reason why the QD and RE-deficient activation factor of the present invention ■:C has a high yield. It will also help clarify the reason.

In BHT10 cells, each trait was Southern analysis of non-chromosomal DNA isolated from the transfectants was performed. For those of old r’t Law [B, Hil't.

J, ) lol, Biol, 2B, pp, 365-69 (1967 )] and cultured for 120 hours in 100 mm diameter Petri dishes after transfection. DNA was isolated from TIO cells. Methylated bacterial DNA input for each construct To differentiate newly replicated (valve 1 resistant) DNA from A (Sir I susceptible) 0.5 to 260 units were cut with DpnI. DNA fragments were added to 0.7% a Electrophoresed on a galose gel and plotted on Gene 5clean Plus. The DNA was analyzed. The filter is 1) INaα-50m) lTrisH ( ffl　(DH7,5)-0,1% sodium pyrophosphate-0,2% polyvinyl Pyrrolidone-○, 2% Ficoll-0, 2% BSA-1% SOS solution, 6 Hybridization was performed at 5°C using a denatured probe of 105 cpm/rrdl.

Filters were washed at 65°C using the same buffer solution and Lightin KO backed with g-Plus brightness enhancement screen (manufactured by DuPont) Exposure to dakXAR-5 X-ray film overnight at -70°C.

Factor ■: The C probe was isolated from the RE expression plasmid (see Figure 4). 924 bp Espl fragment, re inberg and voge + random hexadeoxynucleotide primer method [A, P, Feinberg and Vogelstein.

Anal, Biochem, 132. pp. 6-13 (1983)] The specific radioactivity of 1109C1)/μg was 32. marked with.

As shown in Figure 6, the newly replicated activation factor ■:C plasmid DN The A level is the same for all three deletions and the full length gene. Lane 1 is Labeled with T4 DNA polymerase according to the manufacturer's experimental method obtained from BRL. Chain length size marker with 1 kb as a constituent unit, lane 2 is 1 ng of supercoil Coiled RE DNA, lane 3 contains 100 g of supercoiled RE DNA, lane 3 Lane 4 is 10ngRE DNA cut with DpnI, lane 5 is signal minus. 0,5 A260 units of old rt region obtained from BHTIO cells transfected with the construct Minute Dpnl ice-melting product ice-melting 6 is the complete chain length factor ■: CcDNA (construct 8 ．． 1), lane 7 is transfected with QD deficient, lane 8 is RE deficient. Transfection, lane 9 is transfected with R8 deletion. Figure 6 shows DpnI off. After disconnection (lanes 5-9>, each construct has almost the same amount of supercoiled form. It has been shown that differences in DNA replication enhance the expression of QD and RE deficiency. The possibility of doing so is excluded. Lane 2 contains 108 molecules of the RE construct; Contain 3 contains 109 molecules and has a replication number of approximately 105 successfully transfected cells. This shows that there are approximately 103 of them.

J, ARG 740-ASP 1658 (hereinafter abbreviated as RD) Construction of lack The chapter describes the DNA sequence encoding the expression between Ser 741 and Ser 1657. A method of manufacturing a RD-deficient structure excluding columns will be shown.

The inventor performed this RD-deficient fusion in three steps. In the first step, plasmid QD ( Figure 2) between Ser 1657 and Asp1658 codons and GltJ 17 It was cut with 5aU3A which cuts between 94 and Asp 1795+ton. thus A 411 bp fragment is generated. Also, plasmid tsa p) IL [1, Dailey et al.

J. Virol, 54. pp. 739-49 (1985)] It was made into a line at the c11 site. The 411 base pair frame derived from plasmid QD was then ligase with T4 DNA ligase (the ligase used for this purpose and in the following examples). , connects to the linearized tsa pML at the unique dazzling site and creates a 411 bp surplus ■ site. Contains on the Asp1658 side of the insert (i.e., ASI) a sequence encoding 1658. 5° to the column @) Plasmid 4118CII was generated. Plasmid 41 1. ``fall'' I can be specifically linearized in 无■, and is connected to the GAT codon of Asp 1658. 5° GATC overhang consisting of the first base of the CAA codon of Gln 1659 give.

In addition, plasmid QD was cut with HindlII, and the same plasmid was transformed into Arc740. 1258bp separated between Ser　741 and within the codon of Glu　321 fragment was generated. Next, attach the 5°AGCT protrusion to the mung bean nuc. Excise it with lyase and then add KIenOWl and 4 deoxynucleosides. 411Bcll fragment with blunt ends treated with total phosphoric acid ester connected to the The thus obtained plasmid RD411 is a 1258 bp carrier I The 5' end of the fusion site within the II fragment contains 718. R.D., 41 1 is a 411 bp...3A fragment located 3' to the fusion site. Contains parts.

Next, plasmid RE (Figure 3B) was cut at gon718, and the Trp585 codon It was cleaved inside.

The 5° GTAC protrusion was then dephosphorylated with calf intestinal phosphatase and further inhibited. Partially severed. By this partial cleavage, the linearized RE plasmid becomes Ala1 628 was cleaved between the 702 and Vat1703 codons and incorporated the RE fusion region. bp fragments are retrieved.

Furthermore, plasmid RD, 411 was cleaved with Asp718 and PStl, and RD fused A 601 bp fragment incorporating the junction was generated.

This plasmid fragment was cleaved into Oki718 and PSt1 partially cleaved RE plasmid. plasmid RD was generated. As shown below, plasmid R D is Ar (fusion factor between 1740 and Asp 1658) Controls expression. By cleavage of the RD polypeptide after Ar(1740), The mature H chain has 69L chains, i.e., the amine-terminated first nine amino acids are missing or the chain is calcified. A double-stranded Factor ■ molecule that is cross-linked is obtained.

K, Construction of ARG 740-SER1657 (R8D missing and abbreviated) In this chapter describes the expression of the mature polypeptide from Ser 741 to G1n1656. A method for creating R3D3D deletion by removing coding DNA is shown.

First, plasmid 411. Construct αJ−■ and write it as in Example J. Then, it was made linear with Bcll. Subsequently, plasmid QD was cut with HindIII, AI’ (between codons 1740 and Ser 741 and for Glu 321 The same plasmid was cut within the codon, creating a 1258 bp fragment.

AGC codon is converted into 5 using KIenOW enzyme and dATP, dGTA and dCTP. ゜Secure it within the AGCT protrusion and place the remaining 5°T protrusion into the mungbean nucleus. Removed using Aze. This modified anal ndlII fragment was previously converted into Kl treated with enOW enzyme and all four deoxynucleoside triphosphates. BclI linearized 411.8cII NiL/, 1258bpLinIII fraction 1718 at a position 5° to the fusion site within the ment and 411 bp 5 au A plus containing a PstI site at 3° to the fusion site within the 3A fragment. Mido R3D, 411 is produced.

Then k-sweat 718-cleavage as described in the Examples.

(Second) A partially cut RE plasmid DNA was prepared. Next, plasmid R3D, 411 was cut with Asp718 and PstI, resulting in 604 containing the R3D fusion region. bp fragment cut with Asp718, PStI partially cut RE plasmid It was ligated to DNA to create plasmid R3D. During expression, R8D plasmid is a factor ■ polypeptide fused between Arg 740 and Ser 1657 code. When the R3D polypeptide is cleaved after Arj;l 740, the resulting It has a mature H chain and a Δ8-L chain, that is, an L chain without the first eight amino acids at the amino terminus. Generates a double-stranded factor ■ molecule. Furthermore, in the primary translation product, Ser is also 7 Since it is also in the 41 position, R8O is also between Ser 741 and Asp 165B. It can also be seen as a fusion of Disconnecting after Ser 741 ends with Ser 741. It is possible to generate a double-stranded Factor 1 molecule with a H chain and a Δ9-L chain at the ends.

Transfection of African Green Monkey Kidney Cells. B5Cl African Green Monkey Kidney Cells (African Green Monkey Kidney Cells) 1 11 cell strain B5C40 [W, Brackman and D, Nathan, Proc, Natl.

^cad, Sci, USA, 71. pp, 942-46 (1974) , p[Ding RtsA58 and hygromycin B phosphotransferase [ K.

Blocklinger, and A, Dtggelmann, Mo1. Ce1l Biol.

4, pp. 2929-31 (1984)]. The African green monkey kidney cell line 6L was created by transfection with both of 3 and 3. P Lasmid LTRtsA5B contains a temperature sensitive SV40 T-antigen allele. There is. The tsA58 mutant virus does not produce progeny at 39°C, the temperature of SV40. It is a susceptible mutant strain. T-antigen specified by TtsA58 mutant strain The protein is much more unstable at intolerable temperatures than the wild-type antigenic protein [H , Tegtmeyer et al., J.

Virol, 16. DI), 168-78 (1975)], obtained Cell line 6L inducibly expresses the 5V40T antigen at 33°C.

6L cells were then transfected with supercoiled expression plasmids RD or R2O. Ta. This transfection was performed using the DEAE-dextran method and as described in Example F. It was performed using chloroquine. Transfected cells were cultured at 33°C. culture During this time, the transfected cells synthesized and secreted activation factor ■:C into the culture medium.

Transfectants secrete activation factor ■:C for 120 hours. in most of the tests , the Cm2/7 ratio was about 5.5. That is, a 100mm diameter beetle dish (55c A monolayer of 6 transfected cells in m2) was covered with 10 d of culture medium.

M, factor ■: C activity measurement After transfection and incubation at 33°C for 3 days, KabiVitrum coates RE (Example D), RD and R in 96 wells using t■Factor■ kit. Factor ■ of 3DRD construct: C production status was inspected. Transfection with plasmid RE The transfected cells produced a factor of 0.48% of the plasma concentration in the culture medium (generally Normal factor ■Plasma concentration is approximately 150 nG/snake). Transfected with plasmid RD The cells produced 0.41% of the plasma concentration of Factor 2 in the culture medium. Plasmi Cells transfected with R5D produced a factor of 0.71% of the plasma concentration. .

In the same test, cells transfected with plasmid RE or R8O were incubated at 33°C for 3 days. After culturing, after culturing for two more days, the concentration in the cell doubling solution was as follows: was produced.

Culture medium factor ■: C concentration expressed as plasma concentration % after 3 days after 5 days RE transfected cells 0.30% 0.77% R8D transfected cells 1.50% 1.16% Microorganism of the present invention, recombinant DNA molecule, activation factor ■: CDN The A-code sequence was acquired on July 22, 1986, at the Rockville, Maryland, USA AmeriCan Culture Co. 11ection) as E. coli, 88101 (RE). Ta. The ATCC accession number of the same culture was 53517. The other two cultures July 27, 1981 American Microbial Strain Library, Rockville, Maryland, USA Ad, RD, 2[E, coli, HBIOI (R[))] in the existing institution ATCC accession number: 67475 and Ad, R2O, 1,2 [E, coli, HBIOI (R2O)] AT CC acceptance number: 67476 Deposited as.

Although several embodiments of the present invention have been described above, the basic configuration of the present invention is partially explained. With modifications, the method of the invention.

It is clear that other embodiments using the compositions are also applicable. Therefore , the scope of the invention extends beyond the limited embodiments of the embodiments described above) to: It is intended to be limited by the scope of the claims appended hereto.

F/G4 rza, s　a ala thr arg arg tyr tyr leu gly ala val glu leu 5erGCCACCAGA AGA TACTACC TG GGT GCA GM; GAA CTG TCAtrp asp ty r met gin ser asp leu gly glu leu pr o valTGG GACTAT AT CAA AC; T CHAT CT CGGT GAG CTC; CCT GTGasp ala arg phe pro pro arg val pro lys ser phe pr.

GAG C; CA AC; A TTr CCT CCT AGA GT CC A AAA TCT TrT CCAphe asn thr ser val val tyr lys lys thr leu phe valTTCA ACACCTCA GTCGT TACAAA AAG ACT CTG T TI’ GTAecoRI 60 g1u phe thr asp his leu phe asn ile ala lys pro argGAA TTCAC to AT CACCTT TTCAACATCCCT AAG CCA AGGpro pro trp met gly leu leu gly pro thr ile gln alaCCA CCCTGG ATC; GGT CTC; CTA GGT CCT ACCATCCAG GCTg1u val tyr asp th r val val ile thr leu lys asn metGAG GTr TAT GAT ACA to TC; GTCATr ACA CTI’ MG AACAT ala ser his pro val ser le u his ala val gly val 5erGCTTCCCAT C CT GTCAC;T CTT CAT GCT TTα;’r GTA T CCbindHI 110 tyr trp lys ala ser glu gly ala glu tyr asp asp ginTACTGt:; AAA, GCT TCT GAG (4A GCT cAATAT GAT (1; AT CA thr ser gin arg glu lys glu asp asp, Ly s　vaL　phe　pr.

ACCAGT CAA AGG GAG AAA GAA CAT (?Jlt T-AAA CTCTTCCCCTgly gly ser his thr t yr val trp gin val leu 1ys qluGGT GG Ａ　AGcCAT　ACA　TAT　GTCTGG　CAGσr　CTC”　A AA GAGasn gly pro met ala ser asp pr o leu cys leu thr tyrMT GOT CCA ATG GCCTCT ACCCA CTG TGCCTr AcCTAC160ec oRr sar tyr lau ser his val asp, 1eu val lys asp leu asnTCA TAT CTT TCT CAT GTG GACCTG GTA AAA CACTTG AATser gly leu ile gly ala leu leu val ays arg glu glyTCA GCiCCTCATT GCA GCCCTA CT A GTA TGT AGA GM GGGsar leu ala lys glu lys thr gin a leu his lys pheA(i To CT GCCAAG GAA AAG ACA CACACCTTG CA CAAA T1’Ti1e leu leu ph@ala val phe asp glu gly lys ser trpATA CTA CTr T TT GCT GTA TTT GAT GAA GGCAA AGT TG Ghis war glu thr lys asn ser leu met gin asp arq aspCACTCA GAA ACA AAG A ACTCCTTGATG CAG GAT AGG to Aτala ala se r　ala　arg　ala　trp　pro　lys　met　his廿w　 valGCT GCA TCT GCT CGG GCCTGGCCT AAA ATGCACACA GTCasn gly try val agn ar q ser leu pro gly leu ila glyAAT GGT TAT CTA AACAGG TCT CTG CCA GGT CTG ATTGGAcys his arg 1ys ser val tyr tr p his val ila qIY matTGCCACAGOAAA TC A GTCTAT TGGCAT GTG ATT GA ATGgly t hr thr pro glu val his ser ile ph@le u glu glyGGCACCACT CCT GAA GTG CACTC A ATA TTCCTCGAA GGτhis thr phe leu v al arq asrr his arg gin ala ser 1euC ACACA TrT CTT GTG AGGAACCAT COCCAG G CG TOCTTGglu ila ser pro ile thr pha leu thr ala gin thr 1euGAA ATCTCG C CA ATA ACT TrCCTT ACT GCT CAA ACA CT C1eu met asp leu gly gin pha leu leu pha ays his 1leTTG ATCGACCTT GCA CA G TrT CTA CTG Tri”T CAT ATC320hin dlII tar ser his gin his asp gly met glu ala tyr val 1ysTCT TCCCACCAA CAT GAT GGCATG GAAαπτAτGTc AAAval asp ser C Y! ! pro glu glu pro gin leu arg met 1ysGTA ACAGCTOτCCA GAG GAA CCCCAA C TA CGA AT AMasn asn glu glu ala glu asp tyr asp asp asp lauυ "AAT AAT GA A GAA GCG GM C; ACTAT pAT CAT GAT CTT AC? asp ser glu met asp val val arg phe asp asp asp asnGAT TCT GAA ATCGA T GTG G1 (: AGG TrT CATCAT (uACAAC3) O ger pro ser ph@ile gin ile arg ser v al ala lys 1ysTCT CCT TCCTTT ATCCM A TT CGCTCA GTT Gee AAGAAGhis pro lys thr trp val his tyr ila ala ala glu gluCAT CCT AAA ACT TGG GTA CAT TACAT TαGo αte G Crystal GAGglu asp trp asp, tyr ala p ro leu val leu ala pro aspGAG GACTC; G GACTAT GCT CCCTTA CTCCTCG (CCCCGATa sp arg ser tyr lys ser gin tyr leu a sn asn gly pr.

GACACA AGT TAT AAA AGT CM TAT TTG MC AAT GGCCC cho-cho 20 gln arg ile gly arg lys tyr lys lys val arg phe metCAG CGGATT GGTAGOAAGτ ACAAA AAA to τCCGA ffr AT Gala tyr thr asp glu thr phe lys the: arq glu ala 1leGCA TACACA GAT Gkk ACCT! 'T AAG A CT C to T GAA GCT ATTgln his glu ser gl y ile leu gly pro leu leu tyr gly CAG CAT GAA TCA GCA ATCTrG GGA CCT TrA CTT TAT GGGglu val gly asp thr leu l eu ile ile phe lys asn ginGAA CTT GC A GAG ACA CTG TTG ATT ATA TTT AAG AA T CAAala ser arg pro tyr asn ile tyr pro his gly ile thrGCA AC, CAGA CCA TAT Me ATCTACCCT CACGGA ATCAC? asp va l arg pro leu tyr ser arg arg leu pr o lys glyCHAT CTCCOT CCT TTG TAT TCA AGGAGA TrA CCA AAA GGTval lys his l eu lys asp pha pro ile leu pro gly g lucTA AAA cAT TTG AAG GAT TrT CCA AT T CTG CCA GA GAAi1a phe lys tyr lys trp thr val thr val glu asp gayATA TTCAAA TAT AAA TGG ACA GT to ACT, CTA GAA GAT GGG pro pi v lys ser asp pro arq ays leu thr arg tyr tyrCCA ACT AAA TCA CAT CCT CGG TC; CCTG ACCC (1; CTAττ ACser ser phe van asn met glu arg as p leu ala sar gly TCT AGT TTC TT MT A TG GAG AGA GAT CTA GCTTCA GGAasp gin arg gly asn gin ile met ser asp lys arq asnCAT CAA AGA GCA AACGAG ATA A TG TCA GACAA AGOAAT580k val ila leu phe ser val phe asp glu asn arg ser trpGTCATCCTG TTT TCT GTA TTT CHAT GAG AACC to A GCTGGpnZ 590 tyr leu thr glu asn ile gin arg phe leu pro asn pr.

TACCTCACA GAG AAT ATA CAA CGCTTT CTC CCCMT CCA600 b amHI 610 ala gly val gin leu glu asp pro glu phe gin ala 5erGCT GGA GTG CAG CTr G AG GAT CCA GAG TTCCAA GCCTCCasnile me sehis ser ile asn gly tyr val phe a sp 5erAACATCATG CACAGCATCAAT GGCTAT GTT TTT GAT AGT1eu gin leu ser val a ys leu his glu val ala tyr trpτTG CA to TTG TCA (TT TGT TTG CAT GAG GTG GC A TACTGGtyr ile leu ser ile gay ala gin thr asp phe leu 5erTACATT CTA AG CATT GGA GCA CAG ACT GACTTCCTr TCTva l　phe　pha　ser　gly　tyr　thr　phe　3. ys　h is lys met valC; TCTTCTrCTCT GA TAT ACCTTCAAA CACAM ATC TOtyr glu asp th r leu thr leu phe pro phe ser gly gl uTAT GM GACACA CTC Ace CTA TrCCCA TTC TCA GCA GMthr val phe met gl u asn pro g], y leu trp 1leACT CTCTTC ATOTCG ATG GAA AACCCA Gcr CTA TGGATT tau gay ays his asn sar asp phe arg asn arg gay metCTG GGG TGCCACAACTCA GACT1+r, CGG AACAGA GGCATG pi v ala leu leu lys val ser sex ays asp lys asn 廿 "ACCGCCTTA CTC; AAG GTT TCT AGT TGT GACAAG AACACTgly asp tyr tyr glu asp sar tyr glu asp ile ser ala GGT GAT TAT TAG GAG GACAGT TAT GAA GAT A Ding ττC A CCA730 hindX tyr leu leu ser lys asn asn ala ile glu pro arq serτACTTG CTCAGT AAA AAC AAT GCCATT GAA CCA AGA AGClr ecoRI 7 50 phe ser gin asn ser arq his pro ser 廿 a　arq　gin　1ysTTCTCCAG　MT　TCA　AGA　CA CCCT AGCACT AGG CAA AAGgln phe asn a la thr thr ile pro glu asn asp il@gL uCAA TTT AAT ccCAce ACA ATT CCA cAA AAT (1; ACATA CRAG1ys thr asp pro trp phe ala his argυtr pro met pr.

AAG ACT GACCCT TGG TTr GCA CACAGA AC A CCT ATG CCT1ys ile gin asn val ser ser ser asp leu leu mat 1euAAA ATA CAA AAT GTCTCCTCT AG”l” GAT TTG TTG ATI:i CTClau arg gin ser pro thr pro his gly leu ser lau 5arTTG CCA CAG A to T CCT ACT CCA CAT GGG CTA TCCTTA T CTasp leu gin glu ala lys tyr glue th r　phe　ser　asp　aspCAT　CTCCAA　GAA　GCCA AA　TAT　GAG　ACT　TTT　TCT　GAT　CATprose r pro gly ala ile asp ser asn asn se r leu 5erCCA TCA CCT GGA C, CA ATA CA CACT MT MCAGCCTCTCTglu met thr his p he arg pro gin leu his his ser glyGA A ATG ACA CACTTCAGG CCA CAG CTCCAT C ACAGT GGGasp mat val phe thr pro glu ser gly leu gin leu argGAG ATG GTA 'I71'' AcCCCT GAG TCA GCCCTCCAA TTA A GA1eu asn glu lys leu glyυhr thr ala ala thr glu 1euTTA AAT GAG AAA CTCG GG ACA ACT GCA GCA ACA GAC’ITG1ys ly s leu asp phe lys val ser ser thr se r asn asnAAG AAA CTT GAT TTCAAA GTT TCT AGT ACA TCA AAT AAτ1au ila ser t hr　ile　pro　sar　asp　asn　lau　ala　ala　g lyCTG ATT TCA ACA ATT CCA TCA GACAAT TTG CC 8 to CA GGTυa asp asn thr ser se r leu gly pro pro ser met pr.

ACT CAT AAT ACA ACT TCCTTA GGA CCCCC A ACT AT CCAval his tyr asp ser gln leu asp thr thr leu phe glyGTT CAT TAT CAT AGT CAA TTA CAT ACCACT CTA T TT GGC1ys lys ser ser pro leu thr gl u ser gly gly pro 1euAAA AAG TCA TCT CCCCTr ACT GAG TCT GOT GGA CC”l’CT Gsac engineering 980 1ys’arg ala his gly pro ala lau leu thr lys asp asnMA AGA GCT CAT GGA C CT GCT TTG TrG ACT AM GAT AATala leu phe lys val ser ile ser leu leu lys 　thr　asnGCCTTA　TTCAAA　GTT　AGCATCTCT　 TTG TTA AAG ACA AAC1ys thr ser asn a sn ser ala 廿a asn axq lys 廿w hisA enter A AC r　TCCAAT　AAT　TCA　(,CA　ACT　AAT　ACA　AA G ACT CACile asp gly pro ser leu leu ile glu asn sar pro 5arATT GAT GGCC CA　TCA　’! ? A TTA ATT to AG AAT AGT CCA TO8 030 1040 val trp gln asn ile leu glu ser asp thr glu phe 1ysCTc TGG CAA AAT ATA T TA GAA AGT CACACT GAG TrT AAA1ys val thr pro leu ile his asp arq met leu met aspAAA GTG ACA CCT TTG ATT CAT GACAG7k ATG CTT ATCGACo60 1ys asn ala thr ala leu arg leu asn his met ser asnAAA AAT GCT ACA GCT T TG AGOCTA AAT CAT ATCTCA AATo70 1ys thr thr ser lys asn met glu met val gln glnAAA ACT ACT TCA TCA A AA AACATG to AA ATG GTCCAA CAG1ys lyg glu gly pro ila pro pro asp ala gin asn pr.

AM AAA GAG GGCCCCATT CCA CCA CAT (,C A CAA AAT CCA1oO asp met ser phe phe lys met leu phe leu pro glu 5erGAT ATG TCG TTCTIT AA G kTG CTA TTCTTG CCA to AA TCAala arg trp　il@gin　arg　thr　his　gly　lys　asn　s er 1euGCA AGG TGG ATA CAA AGG ACT CA T GGA AAG AACTCT CTCasn ser gly gin gly pro sar pro lys gin leu val 5erA ACTCT　n　CAA　GGCCCCAGT　CCA　AAG　CAA　’I TA (iA TCCleu qly pro gLu 1ys-ser va l glu gay gin asn phe 1auTTA GGA CCA GAA AAA TCT TG GAA GGT GAG AAT T’1 ℃TTGsar glu lys asn lys val val val gly lys gly glu pheTCT C;A(: AAA AAC AAA CTG C: rA to TA CCA kAG GG? ' CAA TT Tthr lys asp val gLy leu lys glu met val phe pro 5erACA AAG GACCTA GGA C TCAAA GAG kTG GTTTrT CCA AGCser arg asn leu phe leu thr asn leu asp asn leu　hisAC;CAGA　AACCTA　TTT　CTr　ACT　Me TTC; GAT AAT TTA CATglu asn asn thr his asn gin glu lys ile gin glu GMAAT AAT ACA CACAAT CAA GAA AM AAA ATT　cAG　GAAglu　ile　glu　lys　lys　glu廿a leu ile gin glu asn vaLGAA ATA GAA AAG AAG GAA ACA TTA ATCCAA GAG AAT G Tkval leu pro gin ile his thr val th r gly thr lys asnGTT TTG CCT CAG ATA CAT A (A GTG ACT GGCACT AAG AATphe m et lys asn leu phe leu lau ser thr a rg gin asnTrCATG AAG AACCTT TTCTTA C TG AC; CACT AGG CAA AAT1240 5caX val glu gly ser tyr asp guy ala tyr ala pro val 1auCTA GAA GCTTCA TAT A CGGG GCA TAT GCT CCA GTA CTTg1n asp pha ar:q ser leu asn asp ser thr asn arg “CM GAT Trr AGGTCA TTA AAT GAT TCA ACA AAT AGAACA127゜ lys lys his thr ala his phe ser lys lys gly glu gluAAG AAA CACACA GCT CA TTTCTCA AAA AAA GGOGAG GAAglu asn l eu glu qIY 1eu VIY asn gln thr lys g in　1leC, AA　AACTTG　GAA　GGCTTG　G[l;A　A AT CAA ACCkAG CAA ATT1290 sph engineering 1300 val glu lys tyr ala cys thr thr arg ile ser pro asnGTA GAG AAA TAT GCA T GCACCACA ACC; ATA TCT CCT AATthr sar gin gin asn phe val thr gin arg ser lys arqACA AGCCAG CAG AAT TTT GTCAC G CAA CCT AGT AAG AGA132゜ ala leu lys gin pha arg leu pro leu glu glu thr gluGCT TTG AAA CAA TTCAG A CTCCCA CTA C; AA GAA ACA GfiA1eu gl u lys arg ile ila val asp asp thr se r thr ginCTT GAA AAA AGOATA ATT GTG CAT GACACCTCA ACCCAGtrp ser lys JISn met IYs his leu thr proser thX 1eu TGG TCCAAA AACATG AAA CAT TTG Ace CO G AGCACCCTCthr gin ile asp tyr asn g lu　lye　glu　lys　gly　ala　1laACA　CAG　AT A GACTACAAT GAG AAG GAG AAA GGG GCCA Tτ廿a gin ser pro leu ser asp Cys, 1e u thr arg ser hisACT CAG TCT CCCTTA TCA GAT TCOCTT ACCAGGAGT CATser ile pro gin ala asn xrg ser pro leu pro ile alaAGCATCCCT CAA GCA AAT AGA TCT CCA TTA CCCATT GCA1ys val ser ser p 'rie pro ser ile arg pro ile tyr 1eu AAG to TA TCA TCA TTT CCA TCT ATT %GA CCT ATA TAT CTGthr arg val leu phe g in asp asn ser ser his leu pr.

ACCAGG GTCCTA TTCCAA GACAACTCT TCT C AT CTT CCAala ala serseyr arg lys lys asp ser gly val gin gluGCA GCA TCT TAT AGA AAG AAA GAT TCT GGG GTCCAA G AAser ser his phe leu gin gly aha ly s lys asn asn 1euAGCAGT CAT Trc TrA CAA GGA GCCAAA AAA AAT AJ CTarg glu val gly ser leu gly thr sar ala thr asn 5erAGA GAG GTT GGCTCCCTG GGG AC A AGT GCCACA MT TCAval thr tyr lys l ys val glu asn thr val leu pro 1ysGT CACA TACAAG MA GTT GAG AACACT GTT CT CCCG AAApro asp leu pro lys thr ser gly lys val glu leu 1euCCA GACTTG CC CAAA ACA TCT GGCAA GTr GM TTG CTrpr o lys val his ile tyr gin lys asp le u pha pro thrCCA AM GTT CACATT TAT C AG AAG CRAG CTA TTCCCT ACGg1u thr se r asn gly ser pro gly his leu asp le u valGAA ACT AGCAAT GGG TCT CCT GGCC AT CTG GAT CTC to TG153゜ glu gly ser leu leu gin gly thr glu gly ala ile 1ysGAA GGG AGOCTT CTT CA G GCA ACA GAG GC, AC, CG ATT AAGtrp a sn glu ala asn arg pro gly lys val p rophe 1euTcGAAT GAA GCA AACAGA CCT CAA AAA GTT CCCTTT CT Garg val ala th r　glu　ser　ser　ala　lys　thr　pro　sar　1y sAC:A GTA GCA ACA GAA AGCTCT GCA AAG ACT CCCTCCAAGb amHI 1570 1eu leu asp pro leu ala trp asp asn his tyr gly thrCTA TTG GAT CCT CTT G CT TGG CAT AACCACTATαπM=gln ile pro lys glu glu trp lys set gin glu lys 5erCAG ATA CCA AAA GAA GAG TGG AAA T CCCAA GAG AAG TCApro glu lys thr ala phe lys lys lys asp thr ile 1euCCA GAA AAA ACA GCT TTT AAG AAA AAG GAT Ace ATT TrGser lau asn ala cys glu s er　asn　his　ala　ila　ala　ala　alaTCCCTG　AAC cCTT GAA AGOAAT CAT GCA ATAαmA GCA ila asn glu gly gln asn lys pro glu ile glu val “ATA AAT CAG (14A CAA AA T AAG CCCGkk ATA GAA TCACCtrp ala l ys gin gly ar: q thr glu arg leu cys ser ginTGG GCA AAG CM GGT AGG ACT GA A AGOCTG TGCTCT CAAasn pro pro val l eu lys arg his gin arg glu ala “AACC CA CCA CTCTTG AAA CGCCAT CAA COG GAA ATA ACTarg thr thr leu gin ser asp gin glu glu ile asp tyrCGT ACT ACT C TI’ CAG TCA GAT CAA GAG GAA ATr GAG TAτasp asp thr ile ser val glu met l ys lys glu asp pheGAT GAT ACCATA TCA GTT GAA ATG AAG M GAA GAT TTT1680 , 1690 asp ile tyr asp glu asp glu asn gin ser pro arg 5arGACATT TAT GAT GAG GA 'r GAA AAT CAG AGOCCCCC1CAGCphe gin lys lys thr arg his tyr phe ile ala ala valTTT CAA AAG AAA ACA CGA CACTA T TTT ATT GCT GCA GτGglu arg lau trp asp tyr gly met ser ser ser pro his To GA AGG CTCTGG GkT TAT GGG ATG AGT A GCTCCCCA CATval leu arq asn arg ala gin ser gly ser val pro ginGTT CTA A GA MCAGGα: T CA AGTαX: AGT CTCCCT CA Gphe lys lys val val phe gin glu phe Thr asp gly ser? TCAA (1; AAA GTT C-T T TTCCAG GAA TTr ACT CAT GGCTCCphe t hr gin pro leu tyr arg gly glu lsu a sn glu hisTTT ACT CAG CCCTTA TACC to τG GA　GAA　CTA　AAT　AACAT1eu　gly　leu　leu gly pro tyr ila arg ala glu val glu TTG GCA CTCC'J℃αX; CCA TAT ATA AGA G CA GM GTT GAAasp asn ile met val thr phe arq asn gin ala ser argGAT MT A TCATCGTA ACT TTCAGA AAT CAG GCCTCT C Oτpro tyr ser phe tyr ser ser leu il e sar tyr glu gluCCCTAT TCCTTCTAT TC T AGCCTT AT! 'TC'! ’　TAT　GAG　G入Aasp　g in arg gin gly ala glu pro arg lys a sn phe valGAT CAG AGG CAA GCA GCA GM CCT AGA AAA AACTTT GTC1ys pro asn g lu thr lys thr tyr phe trp lys val g inAAG CCT AAT GAA Ace AM ACT TACTTT TGG AAA GTG CAAhis his mat ala pro t hr lys asp glu phe asp ays 1ysC8T CA T ATG GCA CCCACT AAA CAT GAG TTT GAC TGCAAAala trp ala tyr phe ser asp va l asp lau glu lys aspGCCTGG GCT TAT TTCTCT CAT GTr GACCTG AA AM GATval his ser gly leu ile gly pro leu leu val ays his TG CACTCA GCCCTG ATT GGA CCCCTr CTC TOTGCCACthr asn thr leu asn pro ala his gly arq gin val thrA CT AACACA CTG AACCCT GCT CAT (, CI4 A GA CAA GTG ACAval gin glu phe ala le u phe phe thr ila phe asp gluGTA CAG GAA TrT GCT CTC; ffr TTCACCATCTTT C AT GAGthr lys ser trp tyr phe thr gl u asn met glu arg asnAcCAAA AGCTGGTA CTTCACT GAA AAT ATG GAA AGA AACays a rg ala pro ays asn ile gin met glu a sp　pro　thrTGCAGG　GCT　CCCTGCAAT　ATCCA GATG GAA GAT CCCACTpha lys glu asn t yr　arg　phe　his　ala　ile　asn　gly　tyrτT r AAA GAG MT TAT CGCTTCCAT GCA ATCAA T　GGCTAC193゜ ile met asp thr leu pro gly leu val met ala gin aspATA A’M; CAT ACA CTA CCT GC; CTTA GTA ATCGCT CAG GATg1n ar g ile arg trp tyr leu leu ser met gl y ser asnCAA A (1; G ATT C to A TC; G TAT CTCCTCACCATG CJ:, CAGCAATglu asn ile his ser ile his phe ser gly his val pheGAA AACATCCAT TCT ATT CAT TrCAGT GGA CAT GTG’ITC197゜ 廿a val arg lys lys glu glu tyr lys m et ala leu tyrACT GTA CCA AAA AAA GA to GAG TAT AAA ATG GCA CTG TACasn leu tyr pro gly val pha glu thr val glu met 1euAAT CTCTAT CCA GGT GTT TTr G AGλCA GTG GAA ATG TTApro ser lys ala gly ile trp arg val glu cys leu 1le CCA TCCAAA GCT GGA ATT TGC, CC1G GTG GAA TGCCTT AτTgly glu his leu his al a gly met ser thr leu phe leu to GC(1;A G CAT CTA CAT GCT GGG ATC; AGCACA CT T TrT CTCval tyr ser asn lys ays gin thr pro leu gly met ala GTG TACAGCAA T AAG TGT CACAC’l” CCCCTG GGA ATG CC T203α 2o40 ser gly his ile arg asp pha gin ila thr ala ser glyTCT GCA CACATT AGA GA T m CAG ATT ACA GCT TCA GGAgln tyr g ly gin trp ala pro lys leu ala arg l eu hisCAA TAT GGA CAG TGG GCCCcA AAG CTG ccc AGAcrr cp, τ206゜ tyr ser gly sar ila asn ala txp mar thr lys glue pr.

TAττCCGGATCA ATCAAT GCCT G AGCAcCMG To GA CCCpha ser trp ila lys val asp l eu leu ala pro met 1leTrT TCT TGGATC AAG GTG (1; AT CTG T’l’G GCA CCA ATG ATT engineering le his gly ile lys thr gin gly a la arg gin lys phaATT CACGGCATCAAG A ce CAG GG’r (1; CCC to T CAG kAG TrC21oO Ser　Ser　leu　leu　tyr　ile　ser　gin　phe 工le　i le met tyr 5erTCCAGCCTCTACATCTCT CAG TTT Arc ATCATCTAT AGτ1eu asp gly ly s lys trp gin thr tyr arg gly asn 5e rCTr (1; AT GGG AAG kAG TOCCAG ACT TA T CGA GGA JltAT TCCthr gly thr leu m et val phe phe gly asn val asp 5erAC T GGA AcCTrA ATG C1TCTTCTTT GGCAAT G TG CAT TCAser gly ile lys his asn il a　phe　asn　pro　pro　ile　1leTCT　GC;G　AT A AAA CACMT ATT TTT AACCCT CCA ATT A TTala arq tyr ile arg lsu his pro pi lol his tyr ser 1leGCT CCA TACATCCGT TTG CACCCA ACT CAT TAT AGCATrarq ser th r leu arg met glu leu met gly Cys as p　euCGCAGCACT　CTT　CGCATG　GAG　TTG　AM ; GGCT(T GAT TTAsph!2180 asn ser ays ser met pro leu gly met glu ser lys alaAAT A to T TGCAGC ATG CCA TTG GGA ATCGAG AGT AAA CCAile ser a sp ala gin ile thr ala ser ser tyr p he Phi ATA TCA GAT (1; CA CAG ATT ACT GC T　TCA　TCCTACTTrT　ACCasn　phe　ala　t hr trp ser pro ser lys ala axq 1euAA T ATG TrT GCCACCTOG TCT CCT TCA AAA GCT CCA CTrhis leu gin gly arg ser a sn ala trp arg pro gin vanCACCTCCAA GGC, AGG ACT AAT GCCTGGAC; A CCTCkG CT Casn　asn　pro　lys　q1＊　trp　leu　gin　val asp phe gin 1ysAAT MT CCA AAA GAG T GCi CTG CAA to TGCACTTCCAG AAGthr met lys val thr qLYval thX thr gin 91Yva l 1ysACA ATG AM GTCACA GGA CTA ACT A CT CAG GGA GTA AAAser leu leu tJzr er met tyr val lys glu pha leu ileτC T CTG CTT ACCAGCATG TAT GTG MG GAG T r(CTCATCgln asn gly lys val lys val pha gin gay asn gin aspCAG AAT to GCAA A GTA AAG GTT TTr CAG GA AAT CAA GA Csar pha thr pro val val asn sar leu asp pro pro 1euTCCTTCACA CCT to TG GT G AACTCT CTA GACCCA CCG TTAecoRI 231 0 1eu thr arg tyr l@u &! :q ila his pro gin ser trp valCTG ACT CGCTACCTT CG AATT CACCCCAG ACT TGG GTG232゜ his gin ile ala leu arg met glu val leu gly cys gluCACCAG ATT Gee CTG AG G ATG GAG TT CTG GGCTGCCRAG international investigation report

Claims (19)

[Claims] 1. DN encoding for expression of the mature polypeptide of Factor-VIII:C A major part of the A sequence is missing and activation factor-VIII:C-like poly A recombinant DNA molecule characterized by a DNA sequence encoding a peptide. 2. of the DNA sequence encoding the mature polypeptide expression of Factor-VIII:C. The recombinant DN according to claim 1, characterized in that it is completely absent. A molecule. 3. Activation factor-VIII: DN encoding C-like polypeptide expression Claims characterized in that the A sequence is selected from the group consisting of the following sequences: Recombinant DNA molecule according to item 1. [There is an array]; [There is an array]; [There is an array]. 4. Activation factor-VIII: D encoding for expression of C-like polypeptide The claimed invention is characterized in that the NA sequence is functionally linked to an expression control sequence. A recombinant DNA molecule according to any one of Items 1 to 3. 5. The current control sequences are lac system, trp system, trc system, phage λ main Operator and promoter region, control region of fd coat protein, early stage of SV40 and late promoter, polyoma, adenovirus, and simian virus. induced promoter, 3-phosphoglycerate kinase or other carbohydrate degrading enzyme promoter, yeast acid phosphatase promoter, yeast α-gametophyte Child promoters and other known promoters of prokaryotic and eukaryotic cells and their viruses. characterized by being selected from the group consisting of sequences that control gene expression or combinations thereof; A recombinant DNA molecule according to claim 4. 6. Activation factor-VII characterized in that it has a formula selected from the following group: I: C-like polypeptide. [There is an array]; [There is an array]; [There is an array]; and [There is an array]. 7. Maturation factor-VIII: Is the N-terminal H chain directly linked to the C-terminal L chain of C? an activation factor characterized by being essentially free of other serum proteins. -VIII: C-like polypeptide. 8. Activation factor-VIII:C-like polypeptide of claim 7 The N-terminal H chain of maturation factor-VIII:C and the maturation factor -VIII: A polypeptide characterized by comprising a step of converting C into a C-terminal L chain. manufacturing method. 9. Maturation factor-VIII:C's N-terminal H chain and maturation factor-VIII:C's A special method characterized by comprising a step of linking the C-terminal L chain with an alkali metal bridge. The method according to claim 8. 10. A recombinant DNA molecule as defined in claims 1 to 5. Activation factor-VI characterized by comprising a step of culturing a transformed host II: Method for producing C-like polypeptide. 11. Host: BMT10, BSC1, BSC40, COS1, COS7, CHO A patent claim characterized in that it is selected from cells, and other animal and human cultured cells. The method according to any one of item 8, 9, or 10. 12. Polype produced by the method according to claim 8, 9 or 10 contains an amount effective as a blood clotting agent and a pharmaceutically acceptable carrier. A pharmaceutical composition characterized by: 13. Activation factor defined in any one of claims 6 to 9 -VIII: The C-like polypeptide is effective as a blood clotting agent and a pharmaceutically acceptable carrier. 1. A pharmaceutical composition comprising a pharmaceutically effective amount. 14. Pharmaceutical compositions defined in claims 12 and 13 for use in humans. A hemophilia treatment method characterized by consisting of therapeutic steps. 15. Transformed host, E. coli. HB101(RE),E. coli. H.B. 101 (RD) or E. Recombinant D contained in E. coli HB101 (RSD) The recombinant DNA according to claim 4, selected from the group consisting of NA molecules. molecule. 16. Transformed host, E. coli. HB101(RE),E. coli. H.B. 101 (RD) and E. coli. Recombination contained in HB101 (RSD) produced by a host transformed with a recombinant DNA molecule selected from Activation factor-VIII:C-like polypeptide. 17. Transformed host, E. coli. HB101(RE),E. coli. H.B. 101 (RD) and E. coli. Recombination contained in HB101 (RSD) culturing a host transformed with a recombinant DNA molecule selected from DNA molecules; Production of activation factor-VIII:C-like polypeptide characterized by consisting of steps Construction method. 18. The polypeptide produced by the method described in claim 17 is treated with a coagulant and and a pharmaceutically acceptable carrier, in an effective amount. composition. 19. From the step of treating a human with the pharmaceutical composition according to claim 18 A new hemophilia treatment.