KR101119079B1 - Method for producing human coagulation factor VIII - Google Patents

Abstract

The present invention relates to a cell line which simultaneously produces a synthetic protein comprising a heavy chain of human coagulation factor 8, a part of B domain and a light chain, and von Willibrand factor, and a method of producing human coagulation factor 8 using the same.

The production cell line according to the present invention and a method for producing human coagulation factor 8 using the same significantly increase the productivity of human blood coagulation factor 8 and produce it by expressing the stabilized human blood coagulation factor 8 in a serum-free medium with high efficiency. The cost can be greatly reduced.

Human Blood Coagulation Factor 8, Recombinant Protein

Description

Method for producing human coagulation factor 8 {Method for producing human coagulation factor VIII}

The present invention relates to a cell line which simultaneously produces a synthetic protein comprising a heavy chain of human coagulation factor 8, a part of B domain and a light chain, and von Willibrand factor, and a method of producing human coagulation factor 8 using the same.

Hemophilia A and B are caused by a deficiency of the gene encoding the protein of the human X-chromosome. Hemophilia is divided into hemophilia A, a deficiency of the coagulation factor 8, and hemophilia B, a deficiency of the coagulation factor nin. Because of its involvement with the X-chromosome, one deficiency of the coagulation factor 8 and 9 factor genes causes hemophilia in men, while factors involved in other coagulation are linked to autosomal or generally two Deficiency alleles cause blood clotting disease. Therefore, hemophilia A and hemophilia B are the most common blood clotting deficiency, and is a genetic disease that occurs only in men, and occurs in about 1 in 50,000 to 100,000 people. The cause of gene binding is known to be due to mutant proteins that interrupt protein expression or inhibit activity.Severe hemophilia when about 50% of the deficient patients show less than 5% of normal 8 activity Treated as patients, these patients should continue to receive coagulation factor 8 protein throughout their lives.

Among the 13 coagulation factors involved in coagulation, the eighth factor is a protein that plays an important role in accelerating the coagulation process by being involved in the intermediate stage of the coagulation process, a proenzyme that activates prothrombin to thrombin. Once thrombin is formed, it activates fibrinogen and causes blood to coagulate, which is a very important coagulation regulator in and outside the body. Therefore, the eighth factor for coagulation is provided with various regulatory devices for gene transcription and protein expression. As a result, blood coagulation factor 8 is linked to A1, A2, B, A3, C1 and C2 domains in order. Among them, the B domain plays an important role in gene structure and protein expression.

In Korea, patients with hemophilia factor 8 deficiency were used whole plasma from 1950 to the early 1970s, and since then, the protein factor 8 has been concentrated. A large amount of human plasma is needed to obtain human coagulation factor 8, and the coagulation factor 8 is inactivated during a simple purification process for concentration due to the instability of the activity. The product is very low, and the product is very low in purity and has an inertness of about 0.5 to 2 units per mg protein, and the purity of the concentrate is known to be about 0.04%. In addition, there is a problem that increases the chance of infection, such as very high levels of other proteins and denatured proteins, and other hepatitis viruses and AIDS viruses. In order to solve this problem, the introduction of high-purity purification methods and the implementation of double inactivation methods have reduced the chance of infecting a deadly external virus, but the manufacturing cost has increased. The production and supply of recombinant clotting factor 8 using genetic engineering techniques has resulted in a very low infection of foreign viruses. However, the medical expenses of the eighth factor used per year for hemophilia patients have been so high that the burden of medical expenses for the whole nation has increased. As of 2007, there were 1,875 hemophilia patients registered among them, 1,423 hemophilia A patients, and the insurance price paid by the National Health Insurance Corporation was over 40 billion won, about twice that of the late 1990s. Increased over. In addition, as the number 8 gene for coagulation is supplied to patients under 18 years of age from 2006, more insurance drug costs are expected to increase. Therefore, low-cost product supply is very necessary due to the development of higher productivity cell lines.

Genetic engineering methods are needed to produce recombinant hemagglutinin factor 8 in a biologically active and stable state. The conventional method uses a whole gene (J. Biol. Chem., 263: 6352 (1988)) containing all of the A, B and C domains of the hemagglutination factor 8 to produce recombinant hemagglutination factor 8. In the case of making recombinant cells by injecting the host cells to be expressed, the amount of the coagulation factor 8 expression is relatively low when the whole gene is injected. Therefore, the drug cost to the hemophilia patients is high. When the linked gene (Eur. J. Biochem. 232 19-27 (1995); Blood, Vol. 81, No. 11, 2925 (1993)) is used, it is possible to stabilize the expressed coagulation factor 8 There is a need to increase production by introducing methods.

When recombinantly producing the coagulation factor 8, it is essential to reduce the production cost, and for this purpose, it is essential to use a serum-free medium without adding plasma in the medium. However, coagulation factor 8 is greatly dependent on the activity and expression method of various proteins present in the right plasma, the removal of cow albumin and the like causes a problem that the activity of the coagulation factor 8 expressed is greatly reduced . The von Willibrand factor, which is mainly known to stabilize the coagulation factor 8 in the right plasma, has been reported to significantly increase the amount and activity of reduced coagulation factor 8 by adding it to the medium without serum. Mol. Cell Biol., 9: 1233 (1989). However, the addition of human von Willibrand, etc. to the serum-free medium should be examined for the exclusion of the virus-derived infectious virus, and there was a limitation in that it is expensive to add in the medium.

Recombinate (Baxter), Advate ™ (Baxter), Kogenate ™ (Bayer), Refacto ™ (Wyeth), etc. are the 8th factor for gene coagulation clotting supplied to the world. Preparing for registration (Green-Gene ™). There is a difference between the vector composition of genes produced by each company and the method of producing blood coagulation factor 8. Baxter's Chinese hamster Ovary (CHO) cells expressing the whole coagulation factor gene (A, B, C domain) and stabilizing protein von Willibrand Factor (vWF) simultaneously, and Bayer's coagulation factor 8 Baby Hamster Kidney (BHK) cells expressing the entire factor gene (A, B, C domains), CHO cells expressing genes (A, C domains) that removed most of the B domain of the coagulation factor 8 Eur. J. Biochem. 232, 19-27), Green Cross Co., Ltd. expressed the heavy chain (A1, A2 domain) expression vector, the light chain (C domain) expression vector of the coagulation factor 8 and the stabilizing protein von Willibrand factor (vWF). A method of producing coagulation factor 8 using vector-transfected CHO cells (Korean Patent No. 025286) is used. Looking at the expression of the coagulation factor 8 protein as described above, there is a need for a method of lowering the production cost and insurance cost by improving the invention of gene composition, vector composition, and method of making recombinant cells.

In this regard, the present inventors have improved the conventional method for establishing cells, injecting a gene vector expressing the stabilizing protein von Willibrand factor (vWF) to complete recombinant CHO cells first, and then the protein expression of blood coagulation factor 8 is B. The present invention was completed by finding that the use of a genetic vector to remove most domains and linking heavy and light chains resulted in higher protein expression and less recombinant protein degradation.

Therefore, the main object of the present invention is to provide a cell line which simultaneously produces human coagulation factor 8 protein and von Willibrand factor.

Another object of the present invention to provide a method for producing a human coagulation factor 8 using the cell line.

According to an aspect of the present invention, the present invention provides an expression vector of von Willibrand factor (vWF), a stabilizing protein, and a signal amino acid (19 amino acids), heavy chains (1 to 740), and B domain of human coagulation factor 8 (741-745, 1640-1648) and the human blood coagulation factor 8, sequentially transfected with an expression vector of a synthetic protein having the amino acid sequence of SEQ ID NO: 2 connecting the light chain (1649-2332) Provided are cell lines that simultaneously produce synthetic proteins and von Willibrand factors.

In the present invention, "sequential transfection" refers to transfection with an expression vector of von Willibrand factor (vWF) and later transfection with an expression vector of a synthetic protein of human coagulation factor 8 (Factor VII). do. In the present invention, the amino acid sequence of von Willibrand factor (vWF) is well known in the art and has been deposited as Gene Bank P04275 of NCBI, and the amino acid sequence of the original non-recombinant human coagulation factor (Factor VII) It is well known in the art and deposited as NCBI's Gene Bank NP_000123.

In the present invention, a method for producing a recombinant cell line was modified for the purpose of expression of coagulation factor 8 protein having higher productivity than the conventional method. Cells capable of expressing von Willibrand factor were first made, followed by the expression of von Willibrand factor, and the gene of blood coagulation factor 8 from which most of the B-domain was removed. 745, 1640-1648) and the vector containing the gene prepared by the direct linkage of the C-domain (light chain), and by the method of gene amplification and expression of the blood coagulation factor 8 produced by the blood It was found that the productivity of the protein of coagulation factor 8 was significantly increased.

In the present invention, the expression vector of von Willibrand factor (vWF) may be any animal cell expression vector into which a gene encoding a known von Willibrand factor (vWF) is introduced, preferably, the cleavage map of FIG. It is characterized in that the pCMV-vWF having. The pCMV-vWF is prepared by inserting a gene encoding von Willibrand factor (vWF) between Hind III and Not I recognition sites of pCMV-Zeo (Invitrogen, USA), an animal cell expression vector.

In the present invention, the expression vector of the synthetic protein of the human coagulation factor 8 is a signal amino acid of the human coagulation factor 8 (19 amino acids), heavy chain (1 ~ 740), part of the B domain (741-745, 1640) 1648) and any animal cell expression vector into which a gene encoding a synthetic protein connecting the light chain (1649-2332) is possible, but is preferably pCMV-BDDFVIII having a cleavage map of FIG. 2. The pCMV-BDDFVIII is the human blood coagulation factor 8 factor between the Nhe I and Xho I recognition sites among the polyclonal sites located between the cmv promoter and the terminator of the pRC-CMV vector (Invitrogen, USA). It is prepared by inserting a gene encoding a synthetic protein.

In the present invention, the gene encoding the synthetic protein of the human coagulation factor 8 is a signal amino acid of the human coagulation factor 8 (19 amino acids), heavy chain (1 ~ 740), part of the B domain (741-745) , 1640-1648) and any gene sequence capable of encoding the synthetic protein connecting the light chain (1649-2332), but are preferably characterized by having the nucleotide sequence of SEQ ID NO: 1. The nucleotide sequence of SEQ ID NO: 1 is a gene of the signal amino acid (19 amino acids), heavy chain (1 ~ 740) and part of the B domain (741-745) and the B domain of the eighth factor through multi-step PCR in the embodiment of the present invention CDNA sequence of the hFVIII gene synthesized by linking the genes of some (1640-1648) and light chain (1649-2332). The synthetic gene encodes a truncated recombinant human hemagglutinin factor 8 precursor of 1457 amino acid residues comprising a 19 amino acid signal peptide, which is a previously reported native human hemagglutinin factor 8 precursor (GenBank Accession No. It is shorter than NP_000123).

In the present invention, preferably, the animal cell line is deposited with the accession number KCLRF-BP-172 in the Bank of Korea cell line. In Example 4 of the present invention, a G4 cell line having high productivity of human coagulation factor 8 and excellent adaptability in serum-free medium was selected and deposited with the Korean Cell Line Bank on accession number KCLRF-BP-172 on October 27, 2007. .

According to another aspect of the present invention, the present invention comprises the steps of (i) transfecting the expression vector of von Willibrand factor (vWF) stabilizing protein to Chinese Hamster Ovary (CHO) cells; (ii) the amino acid (19 amino acids), heavy chains (1 to 740), part of the B domain (741 to 745, 1640 to 1648) and the light chain (1649 to 2332) of the human coagulation factor 8 in the transfected CHO cell line. Transfecting again the expression vector of the synthetic protein linking); (iii) amplifying a target gene by adapting the CHO cell line transfected with the expression vectors to methotrexate (MTX); And (iv) culturing the CHO cell line adapted with methotrexate in a serum-free medium to obtain an eighth factor for human coagulation.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Preparation of Von Willy Brand Factor Expression Vector

The gene of von Willi Blant factor was obtained by cutting the pMT2-vWF vector (ATCC 67122) with restriction enzyme EcoR I as a vWF gene of about 8 kb, and it was confirmed that the gene was identical to Gene Bank P04275. This was introduced into the EcoR I position of the vector pCDNA1 (Invitrogen, USA) to prepare a pCDNA-vWF vector. The vector (pCDNA-vWF) was then digested with restriction enzymes Hind III and Not I to obtain the gene of von Willibrand's factor, which was placed between the Hind III and Not I recognition sites of the vector pCMV-Zeo (Invitrogen, USA). The expression vector pCMV-vWF was prepared by insertion. Then, E. coli DH5a cells were transformed with this recombinant plasmid, and then plasmids were isolated from the transgenic strain to prepare pCMV-vWF, which is a desired expression vector (FIG. 1).

Example 2 Von Willy Brand Factor Cell Line Selection

One day prior to transfection , CHO (DG44) cells dhfr- deleted were placed in a T-25 flask in medium containing 10% fetal calf serum and HT (hypoxanthine & thymidine) (Gibco 11900) at a concentration of 1 x 10 ⁵ cells / mL. Passage was incubated with. After 24 hours of culture, the cell confluency was confirmed to be about 50% and homogeneously distributed, and 5 μg of von Willibrand factor pCMV-vWF was introduced into the cells using a lipofectin kit (Gibco, USA). To screen for the expression vector pCMV-Zeo-vWF cell line, antibiotic Zeocin was added to the final 250ug / mL in alpha MEM (Gibco 11900) medium containing 10% fetal bovine serum and HT, and per well. Cells were inoculated into 96 well plates to accommodate 3 x 10 ³ cells, and then cultured with 96-well plates at 2-3 days intervals. Between 7 and 14 days after incubation, colonies resistant to zeocin were formed in each well of the 96 well plate and started to grow. After subcultured and divided into two 96-well plates, one prepared the culture supernatant and examined the expression level of von Willibrand factor expressed in the culture supernatant by EIA method, the other MTT (3- [ 4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide: Thiazolyl blue) analysis was performed to select excellent cell lines. The final selected cell line was found to express about 500ng / mL von Willibrand factor.

Example 3 Preparation of Blood Coagulation Factor 8 Expression Vector

Amplify the signal amino acids (19 amino acids) of the coagulation factor 8, heavy chain gene base pairs (1 to 740 as amino acids), part of the B domain (741 to 745, 1640-1648), and light chain gene base pairs (1649 to 2332 as amino acids) To this end, RT-PCR (reverse transcriptase-polymerase chain reaction) method was used in which cDNA was synthesized from total RNA of human liver and then used as template DNA.

The priming method for synthesizing cDNA was oligo (dT) priming method, and cDNA synthesis was performed using a primary chain cDNA synthesis kit (Promega, USA). In other words, 1ug of human liver RNA was added to a micro tube, DEPC (diethyl pyrocarbonate) -treated distilled water was added to adjust the volume to 12.5ul, and then added 1.0ul (20 pmol) oligo (dT) primer Heated at 70 ° C. for 2 minutes and left on ice. 4ul of buffer solution, 1ul of dNTP, 1ul of RNase inhibitor, and 1ul of reverse transcriptase were added to the reaction solution and reacted at 42 ° C for 1 hour.

This reaction solution was used as template DNA for the next PCR reaction. In order to obtain a heavy chain gene and a portion of the B domain (1 ~ 745) including the linking site, PCR was performed using the following primers.

(Primer1: 5'-CTA GCTAGC CACCATGCAAATAGAGCTCTCCACC-3 ', Nhe I site underlined; Primer2: deleted after 5'-ATTCTGGGAGAAGCTTCTTGGTTC-3')) Also, obtain a light chain gene (1640-2332) comprising a terminal linking site of the B domain. In order to perform the PCR reaction using the following primers.

(Primer 3: 5'-GAACCAAGAAGCTTCTCCCAGAATCCACCAGTCTTGAAACGC-3 '; Primer 4: 5'-CCG CTCGAG TCAGTAGAGGTCCTGTGCC-3', Xho I site underlined) A primer was prepared to include an antisense. To amplify each gene fragment, a PCR system (expand long template PCR system, Invitrogen, USA) was used, the annealing temperature during the PCR reaction was carried out at 65 ℃ to reduce the non-specific product. After the PCR reaction, the reaction solution was electrophoresed to confirm whether the desired genes were amplified. As a result, PCR products of about 2.3 Kb for heavy chains and 2.1 Kb for heavy chains were identified.

The gene product was purified by electrophoresis of the PCR product, and the heavy and light chain genes were separated, and then the amount of the genes was adjusted to 50 ng or more. Add NTP, buffer, and pfu Tubo DNA polymerase to this solution, heat at 95 ℃ for 5 minutes, and repeat 40 times of 95 ℃ 30 seconds, 60 ℃ 45 seconds, 72 ℃ 4 minutes so that heavy and light chains can be connected. do. As a PCR reaction, 1 uL of a solution in which the linkage of the heavy and light chains was completed was carried out, and the second PCR amplification reaction was performed under the same conditions. The primers required for the second PCR nucleic acid amplification were used as the heavy primer sense primer: 5'-CTA GCTAGC CACCATGCAAATAGAGCTCTCCACC-3 'and the light chain antisense primer: 5'-CCG CTCGAG TCAGTAGAGGTCCTGTGCC-3'. After completion of the reaction, agarose gel analysis showed a 4.4 Kb size PCR product.

The identified synthetic gene was digested with restriction enzymes and inserted between the Nhe I and Xho I recognition sites among the polyclonal sites located between the cmv promoter and terminator of the plasmid pRC-CMV vector (Invitrogen, USA). Then, E. coli DH5a cells were transformed with this recombinant plasmid, and then plasmids were isolated from the transgenic strain to prepare pCMV-BDDFVIII, which is a desired expression vector. The nucleotide sequence of the synthetic FVIII cDNA inserted into the expression vector confirmed that 14 amino acids were inserted into the B domain sequence by automatic DNA sequencing. The construction of the expression vector of the coagulation factor 8 is shown in FIG. 2.

Example 4 Preparation and Selection of Blood Coagulation Factor 8 Cell Lines

One day before the gene was introduced, CHO cells (the cell line obtained in Example 2), in which the dhfr gene was deleted and von Willibrand factor were expressed, were placed in a T-25 flask with 10% fetal bovine serum, HT (hypoxanthine & Thymidine), and Zeocine 250ug. Subcultured at a concentration of 1 x 10 ⁵ cells / mL in a medium containing / mL (Gibco 11900). After 24 hours of incubation, the cell confluency was about 50% and homogeneously distributed, and the expression vector pCMV-BDDFVIII linked to the heavy and light chains of the coagulation factor 8 using a lipofectin kit (Gibco, USA). 5 ug and 25 ng of DHFR plasmid (pDCH1P) were coat transformed into cells. After gene introduction, the cells were incubated for 6 hours at 37 ° C. and 5% CO ₂ , and then cultured for 48 hours by removing the medium and replacing 5 mL of fresh culture medium. Cultured cells were passaged to contain 3 × 10 ³ cells per well of a 96 well plate. In order to select cell lines into which the expression vector was introduced, zeocin was 250 ug / mL and the antibiotic geneticin (G418) was 500 ug / mL in alpha MEM (Gibco 11900) medium containing 10% fetal bovine serum. It was added, and cultured with medium replacement at 2-3 days intervals. Colonies were formed in each well of the 96 well plate after 7 to 14 days of incubation. Subcultured and divided into two 96 well plates, one prepared the culture supernatant and expressed in the culture supernatant by the EIA method. The expression level of the coagulation factor 8 was investigated, and the other one was subjected to MTT analysis to select excellent cell lines.

As a method for amplifying the heavy and light chain genes of the coagulation factor 8 in a cell line having a relatively high productivity, the method based on the amplification of the dhfr gene using methotrexate (MTX) was used. First, MTX was added to the alpha MEM medium containing 10% fetal bovine serum to the final 20 nM, and colony formation was induced by changing the medium every three days. The productivity of cell lines adapted to this concentration to recover the growth rate was examined by the above-described method, and cell lines with increased productivity of coagulation factor 8 were selected, and the MTX concentrations were 200nM, 1uM, and 5uM. The cells were selected to show an increase in productivity with increasing concentration in the same manner as in the case of 20 nM while gradually increasing. Finally, cell production at the single cell level was examined by passage dilution using a limiting dilution method to select cell lines D3, G1, and G4 producing coagulation factor 8 (FIG. 3).

<Comparative Example 1> Preparation of blood coagulation factor 8 cell line expressing no expression of von Willibrand factor

In order to compare the effect with the cell line of the present invention, an animal cell line of blood factor 8 without von Willibrand factor expression was established. One day prior to transfection , CHO (DG44) cells lacking the dhfr gene were passaged at a concentration of 1 × 10 ⁵ cells / mL in a medium containing 10% fetal bovine serum (Gibco 11900) in a T-25 flask. After 24 hours of incubation, the cell confluency was about 50% and homogeneously distributed. Using the lipofectin kit (Gibco, USA), the heavy and light chain-linked expression vectors pCMV-BDDFVIII 5ug of the 8th coagulation factor were used. And 25ng of DHFR plasmid (pDCH1P) were coat-transfected into cells. After gene introduction, the cells were incubated for 6 hours at 37 ° C. and 5% CO ₂ , and then cultured for 48 hours by removing the medium and replacing 5 mL of fresh culture medium. Cultured cells were passaged to contain 3 × 10 ³ cells per well of 96 well plates. In order to select the cell line into which the expression vector was introduced, antibiotic Geneticin (G418) was added to the final 500 ug / mL in alpha MEM (Gibco 11900) medium containing 10% fetal bovine serum, and 2 The cultures were carried out with medium replacement at intervals of 3 days. Colonies were formed in each well of the 96-well plate after 7 to 14 days of incubation. Subcultured and divided into two 96-well plates, one prepared the culture supernatant and expressed in the culture supernatant by the EIA method. The expression level of coagulation factor 8 was investigated, and the other one was subjected to MTT analysis to select excellent cell lines.

As a method for amplifying the heavy and light chain genes of the coagulation factor 8 in a cell line having a relatively high productivity, a method based on amplification of the dhfr gene using MTX was used. First, MTX was added to the alpha MEM medium containing 10% fetal bovine serum to the final 20 nM, and colony formation was induced by changing the medium every three days. The productivity of cell lines adapted to this concentration of MTX to recover growth rate was examined by the method described above, and cell lines with increased productivity of coagulation factor 8 were selected, and the MTX concentrations were 200nM, 1uM, and 5uM. Cells showing increasing productivity with increasing concentrations were selected in the same manner as 20 nM with increasing gradually. Finally, cell line A23-4 producing coagulation factor 8 was selected by investigating cell productivity at the level of single cells while subcultured using limiting dilution.

Experimental Example 1 Blood Coagulation Factor 8 Production Cell Line Productivity Measurement

The coagulation factor 8 expressed by the cell line selected by the method of Example 4 was confirmed using a COAMATIC Factor VIII activity assay kit (CHROMOGENIX). The productivity of the D3, G1, and G4 cell lines is shown in Table 1 below. The heavy chain expression vector and the light chain expression vector were separately prepared, and it was confirmed that the productivity was increased by 150% to 200% compared with the conventional V88 cell line (Korea Patent No.0251286) transfected with the von Willibrand factor expression vector. . In other words, if too many genes are amplified during MTX amplification, MTX concentration is limited because it inhibits cell growth. In the case of V88, the maximum productivity is only 4.97 units / mL because the concentration of MTX concentration is 1uM. However, the cell lines of the present invention are amplified in cell growth up to 5uM MTX concentration because the highest productivity is from 6.90 units / mL to 9.05 units / mL.

Table 1 Comparison of blood coagulation factor 8 cell line production

MTX concentration (uM) Productivity (units / mL) D3 G1 G4 V88 0.0 0.0 0.0 0.0 0.0 0.02 0.12 0.10 0.09 0.30 0.2 1.35 1.05 0.82 1.20 One 4.80 3.20 3.50 4.97 5 9.05 7.05 6.90 -

< Experimental Example 2> Double doubling time measurement in cell-free medium

Using 10% bovine serum medium, the cell lines D3, G1, and G4 selected were identified as follows. Selected cryopreserved cell lines D3, G1, and G4 are thawed in a 37 ° C. incubator, and then grown in T-175 flasks in medium containing alpha MEM and 5 uM MTX with 10% bovine serum. Once the cells have grown completely in the T-175 flask, remove the medium from the cell container and add 5 mL of 0.25% trypsin solution to separate adherent cells and centrifuge the cells. The collected cells were passaged in T-175 flasks after releasing medium containing alpha MEM and 5 uM MTX containing 10% bovine serum. Three passaged cells are removed from the flask with trypsin solution and released in 20 mL of serum-free medium (HyQ SFM4CHO, Hyclone). The released cells are collected by centrifugation. The cells collected by centrifugation are removed three times in the same manner to remove the remaining alpha MEM and MTX. The collected cells are suspended in a serum-free medium solution, made into a solution containing 40 mL of cells adjusted to an initial cell culture concentration of 5 × 10 ⁵ cells / mL and placed in a 125 mL suspension culture flask. Suspension cultures are grown in 5% CO ₂ , 37 ° C. incubator with stirring at 90-100 rpm. Serum-free medium was changed every 4 days. D3 and G1 cell lines grow very well in 10% serum medium, but attempted adaptation through serum-free medium exchange more than 40 times, but the doubling time of the cell number was as shown in Table 2.

Table 2. Doubling time of cell number of selected cell lines

division In 10% bovine serum medium
2x increase in cell count In serum-free medium
2x increase in cell count D3 cell line To 62 hrs 240 hr or more G1 cell line To 36 hrs 200 hr or more G4 cell line To 35 hrs ~ 28 hrs

Highly productive media are important for commercially cultivating cell lines in serum-free medium to mass-produce coagulation factor 8, but cell proliferation in serum-free medium is more important. The G4 cell line adapted to serum-free medium and mass-producible rather than cell line was finally selected and named as NXT-G4 (CHO-FVIII) and deposited on October 27, 2007 with Bank No. KCLRF-BP-172. .

Experimental Example 3 Identification of Proteins Produced by Cell Lines

The selected NXT-G4 cell line was cultured in serum-free medium for 2 days, and then the protein of blood coagulation factor 8 and von Willibrand factor expressed in the culture medium was determined by Western blot. First, 40uL of the culture solution was subjected to electrophoresis using 10% SDS-PAGE, which was transferred to nitrocellulose membrane, and then treated with 5% skim milk solution. In order to identify the coagulation factor 8 adsorbed on the membrane, polyclonal antibody bound to horse radish peroxidase (HRP) and light chain-specific monoclonal antibody bound to alkaline phosphatase were used. Substrate solution was added to determine the coagulation factor 8 by the chemiluminescence method. As a result of the Western blotting, when the polyclonal antibody was used, a heavy chain of 90,000 daltons and a light chain of 80,000 daltons could be confirmed (FIG. 4).

Experimental Example 4 Measurement of Productivity in Serum-free Medium of Blood Coagulation Factor 8 Cell Lines

1. Adaptation method of suspension culture of serum-free medium of blood coagulation factor 8

Selected cryopreserved cell lines NXT-G4 and A23-4 were thawed in a 37 ° C. thermostat and then grown in T-175 flasks in medium containing alpha MEM and 5 uM MTX in 10% bovine serum to prepare cells. Once fully grown in a T-175 flask, remove the medium from the cell container, add 5 mL of 0.25% trypsin solution to detach adherent cells, and then collect the cells by centrifugation. Collected cells are released in medium containing alpha MEM and 5 uM MTX containing 10% bovine serum and passaged in a T-175 flask. Three passaged cells are removed from the flask with trypsin solution and released in 20 mL of serum-free medium (HyQ SFM4CHO, Hyclone). The released cells are collected by centrifugation. The cells collected by centrifugation are removed three times in the same manner to remove the remaining alpha MEM and MTX. The collected cells are suspended in a serum-free medium solution, made into a solution containing 40 mL of cells adjusted to an initial cell culture concentration of 5 × 10 ⁵ cells / mL and placed in a 125 mL suspension culture flask. Suspension cultures are grown in 5% CO ₂ , 37 ° C. incubator with stirring at 90-100 rpm. Change serum-free medium every 4 days, dilute when cell concentration reaches ~ 1.5 X 10 ⁶ cells / mL and adjust cell concentration to 5 × 10 ⁵ cells / mL again. If more than 30 passages are performed in such a manner, more than 90% of the cells grow normally (FIG. 5). Cells are collected by centrifugation, adjusted to 1 x 10 ⁷ cells / vial, and adjusted with Cell Freezing Medium (DMSO serum free, Sigma) and frozen.

2. Productivity Measurement

Thaw frozen cells, transfer to 50 mL conical tubes and dilute with 10 mL serum-free medium (HyQ SFM4CHO, Hyclone) solution. The cells are collected by centrifugation, released with 10 mL of serum-free medium, and then transferred to a 125 mL suspension culture flask container with the addition of 30 mL of serum-free medium. This flask vessel is grown in a 5% CO ₂ , 37 ° C. incubator with stirring at 90-100 rpm. When the cell concentration reaches ~ 1.5 X 10 ⁶ cells / mL, dilute and adjust the cell concentration again to 5 x 10 ⁵ cells / mL. If more than 90% of cells are lived normally over 3 times, adjust the cell concentration to 5 × 10 ⁵ cells / mL and then stir at 90-100 rpm for 10 days without changing medium in 5% CO ₂ , 32 ℃ incubator. Raise From the 5th day of culture, the blood coagulation factor 8 concentration was measured using the COAMATIC Factor VIII activity assay kit (CHROMOGENIX). On the 9th day, the blood coagulation factor 8 was produced at 27.4 unit / mL. (FIG. 6). Table 3 shows a comparison of titers in serum-free medium of factor 8 of blood coagulation.

TABLE 3 Comparison of titers in serum-free medium of coagulation factor 8

division NXT-G4 A23-4 V88 Station
(Biological activity) 27.4 units / mL 12.6 units / mL 9.5 units / mL

From this, the cell line NXT-G4 of the present invention exhibited better blood coagulation factor 8 in serum-free medium than animal cell line A23-4 of blood factor 8 without expression of von Willibrand factor or V88 cell line of the prior art Patent No. It can be seen that it is expressed.

As described above, according to the present invention, the productivity of human coagulation factor 8 is achieved by using an animal cell line expressing the von Willibrand factor and the synthetic protein including the heavy chain of the human blood coagulation factor 8 part of the B domain and the light chain simultaneously. Significantly increase the production efficiency of the human blood coagulation factor 8 stabilized in serum-free medium can be significantly reduced.

1 is a view showing the manufacturing process of the expression vector of human von Willi Blant factor.

Figure 2 is a diagram showing the manufacturing process of the expression vector of the human coagulation factor eight.

3 is a view showing the manufacturing process of von Willibrand and Factor 8 coexpression cell line.

4 is a photograph confirming the expression level of the coagulation factor 8 by Western blot. Lane 1; Sample with serum, lane 2: no serum on day 5, lane 3: no serum on day 6, lane 4: no serum on day 7, lane 5: no serum on day 8

Figure 5 shows the adaptability (viability and cell density) to serum-free suspension culture of blood coagulation factor 8 expression cell line.

Fig. 6 shows the productivity of the blood coagulation factor 8 in the serum-free suspension culture of the blood coagulation factor 8 expression cell line.

<110> Nexentech Co. Ltd <120> Method for producing human coagulation factor VIII <130> PN080531 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 4374 <212> DNA <213> Human <400> 1 atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60 accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120 ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180 acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240 gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300 gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360 ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420 gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480 aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540 gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600 gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660 tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720 gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780 ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840 accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900 cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960 gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020 gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080 gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140 gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200 tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260 cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320 aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380 attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440 ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500 gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560 ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620 actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680 gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740 agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800 aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860 cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920 tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980 attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040 atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100 atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160 atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220 agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280 ttctcccaga atccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340 cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400 gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460 cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520 catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580 caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640 ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700 agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760 cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820 ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880 gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940 ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000 tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060 gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120 tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180 caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240 attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300 tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360 tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420 gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480 cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540 tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600 ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660 ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720 cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780 aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840 tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900 atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960 tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020 agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080 aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140 tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200 cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260 tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320 cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta ctga 4374 <210> 2 <211> 1457 <212> PRT <213> Human <400> 2 Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe   1 5 10 15 Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser              20 25 30 Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg          35 40 45 Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val      50 55 60 Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile  65 70 75 80 Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln                  85 90 95 Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser             100 105 110 His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser         115 120 125 Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp     130 135 140 Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu 145 150 155 160 Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser                 165 170 175 Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile             180 185 190 Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr         195 200 205 Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly     210 215 220 Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 225 230 235 240 Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr                 245 250 255 Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val             260 265 270 Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile         275 280 285 Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser     290 295 300 Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met 305 310 315 320 Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His                 325 330 335 Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro             340 345 350 Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp         355 360 365 Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser     370 375 380 Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro                 405 410 415 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn             420 425 430 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met         435 440 445 Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu     450 455 460 Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro                 485 490 495 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys             500 505 510 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe         515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp     530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu                 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val             580 585 590 Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu         595 600 605 Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp     610 615 620 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp                 645 650 655 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe             660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr         675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro     690 695 700 Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp                 725 730 735 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys             740 745 750 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu         755 760 765 Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln     770 775 780 Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu 785 790 795 800 Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe                 805 810 815 Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp             820 825 830 Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln         835 840 845 Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr     850 855 860 Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His 865 870 875 880 Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile                 885 890 895 Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser             900 905 910 Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg         915 920 925 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val     930 935 940 Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp 945 950 955 960 Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu                 965 970 975 Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His             980 985 990 Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe         995 1000 1005 Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys    1010 1015 1020 Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn 1025 1030 1035 1040 Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly                1045 1050 1055 Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met            1060 1065 1070 Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe        1075 1080 1085 Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr    1090 1095 1100 Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile 1105 1110 1115 1120 Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser                1125 1130 1135 Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met            1140 1145 1150 Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr        1155 1160 1165 Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile    1170 1175 1180 Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu 1185 1190 1195 1200 Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln                1205 1210 1215 Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu            1220 1225 1230 Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu        1235 1240 1245 Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile    1250 1255 1260 Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His 1265 1270 1275 1280 Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu                1285 1290 1295 Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp            1300 1305 1310 Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp        1315 1320 1325 Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp    1330 1335 1340 Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln 1345 1350 1355 1360 Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu                1365 1370 1375 Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp            1380 1385 1390 Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe        1395 1400 1405 Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro    1410 1415 1420 Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His 1425 1430 1435 1440 Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu                1445 1450 1455 Tyr      

Claims (6)

Human blood, which is sequentially transfected with the expression vector of the stabilizing protein von Willibrand factor (vWF) and the expression vector of the synthetic protein having the amino acid sequence of SEQ ID NO: 2 of the factor 8 of human blood coagulation (Factor Ⅷ) A CHO animal cell line expressing simultaneously said synthetic protein of coagulation factor 8 and von Willibrand factor. The CHO animal cell line according to claim 1, wherein the expression vector of von Willibrand factor (vWF) is pCMV-vWF having a cleavage map of FIG. The CHO animal cell line according to claim 1, wherein the expression vector of the synthetic protein of the human coagulation factor 8 is pCMV-BDDFVIII having a cleavage map of FIG. 4. The CHO animal cell line according to claim 3, wherein the gene encoding the synthetic protein of the human coagulation factor 8 has a nucleotide sequence of SEQ ID NO: 1. The CHO animal cell line according to claim 1, wherein said animal cell line has been deposited with the accession number KCLRF-BP-172 at the Bank of Korea Cell Line. (i) transfecting an expression vector of von Willibrand factor (vWF), a stabilizing protein, into a Chinese Hamster Ovary (CHO) cell line; (ii) transfecting the expression vector of a synthetic protein (SEQ ID NO: 2) of human coagulation factor 8 (Factor VII) into the transfected CHO cell line; (iii) amplifying a target gene by adapting the CHO cell line transfected with the expression vectors sequentially with methotrexate; And (iv) culturing the CHO cell line adapted with methotrexate in a serum-free medium to obtain an eighth factor for human coagulation.

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