KR20030012321A - Expression vectors useful in animal cells and expression vectors for preparing hbv surface antigen - Google Patents

Abstract

PURPOSE: Provided are expression vectors useful in animal cells and expression vectors for preparing HBV surface antigens massively, thereby mass-producing HBV surface antigen by low concentration of DHFR inhibitor. CONSTITUTION: The expression vector useful in dhfr¬-animal cells includes the promotor of Cytomegalovirus where an enhancer region is removed, dhfr linked to the promotor, and a replication origin working in a eukaryote. The expression vector useful in dhfr¬-animal cells for mass-production of HBV surface antigens contains the promotor of Cytomegalovirus where an enhancer region is removed, dhfr linked to the promotor, a replication origin working in a eukaryote, HBV surface antigen coding gene and the promotor linked to the HBV surface antigen coding gene. Wherein the eukaryote is a CHO cell.

Description

Expression Vectors for Animal Cells and Expression Vectors for Preparing HBV Surface Antigen}

The present invention relates to an expression vector for animal cells, and more particularly to an expression vector for producing a large amount of a protein, such as the surface antigen of HBV in animal cells.

HBV (Hepatitis B Virus) is the main pathogen for hepatitis, which is now infected with about 300 million people around the world, representing a serious global health problem. In Southeast Asia and tropical Africa, about 10% of chronic carriers are in Korea. In the mid-1980s, about 10% of them were chronic carriers. Nowadays, they have decreased to 4-5% due to the advancement of public health and vaccination of hepatitis. However, the rate of carriers is higher than that of 1% in developed countries (Choi, BY et al., J. Hanyang Med. Coll. 10: 245-263 (1990)).

HBV consists of 42 nm Dane particles, 22 nm spherical particles and 100 nm tube particles, of which 22 nm HBsAg is used for the preparation of a prophylactic vaccine. HBsAg particles consist of three surface antigen proteins represented by large (L), medium (M) and small (S) produced by a single long open reading frame. The S protein is the main protein encoded by the S region of the HBV genome, and exists in two forms: aglycosylated p22 and monoglycosylated gp26 (Tiollais, P. et al., Science 213: 406-411 ( 1981). The M protein has monoglycosylated gp33 and diglycosylated gp36 by adding 108 amino acids that are pre-S2 domains to the S protein. The L protein has aglycosylated p39 and glycosylated gp42 with the addition of 55 amino acids, the pre-S1 domain, to the M protein. In general, the concentrations of the three peptides in HBsAg derived from chronic carriers or mammalian cells are S >>>M> L.

On the other hand, HBV's mechanism of invading hepatocytes is as follows: HBV invading the human body binds to hepatocytes using specific sites present in the pre-S1 and pre-S2 antigens, and then transfers its DNA into the hepatocytes through fusion. . The pre-S antigen has an amino acid sequence that is very sensitive to proteolysis, which, at the time of fusion, degrades this site, providing a pathway through which HBV DNA can be transported into hepatocytes. Considering the HBV hepatocyte invasion mechanism as described above, it can be seen that the antibody to pre-S should be generated to block the invasion of the virus into hepatocytes and induce complete defense when exposed to HBV infection.

In addition, Thoma et al. ( Progress in Hepatitis B Immunization. 194: 35 (1990)) reported that the protection against hepatitis virus obtained from pre-S antigen was maintained longer than that obtained by S-antigen, and Neurath et al. ( Vaccine 4 : 35-37 (1986)) and Beasly et al. ( Grune & Stratton, New York . Pp 209-224 (1984)) produced pre-S antibodies earlier than S antibodies, resulting in vertical infection of vaccines containing pre-S. Reported the efficiency for. Therefore, it is desirable to develop vaccines containing pre-S in the development of vaccines against HBV, and there is a need in the art as well.

Several vaccines developed to control infection of HBV have been examined. In the early stages, hepatitis B vaccine was purified from HBV-infected carriers with pepsin to purify HBsAg and inactivate it with formalin (Hillman, M). et al., Am. J. Med. Sci. 270: 401-404 (1975). However, this method has been problematic because it is not easy to obtain materials and may be contaminated by bacteria. In order to avoid such a problem, development of recombinant surface antigen proteins has been attempted.

Valenzuela et al . ( Nature 298: 347-350 (1982)) developed a recombinant surface antigen vaccine using yeast, but it consists of the S peptide p24 and is relatively unstable in vivo because it is not glycosylated. It does not contain -S, so there is a problem that the immune induction somewhat falls. In addition, when preparing r-HBsAg (recombinant-HBsAg) particles using yeast as a host, it is impossible to secrete r-HBsAg having a molecular weight of about 2 million daltons out of the cells. Cells must be disrupted to elute the antigen. At this time, physical and chemical treatments are indispensable for breaking down the rigid cell membrane, which affects the immunogenicity and antigenicity of the hepatitis antigen. In addition, host cell disruption has a problem of eluting a large number of host-derived proteins, which makes it difficult to purify the hepatitis antigen.

Meanwhile, Korean Patent Application No. 1989-700312 discloses a novel pre-S1 of HBV having excellent antigenicity, and Patent Application No. 1995-43662 discloses the N-terminal second and third of the surface antigen pre-S of HBV. Discloses a pre-S antigen mutant protein expressed from a transcript in which a mutation is induced in a nucleotide sequence encoding the first amino acid. Patent application 1995-9865 discloses a 3'- of a gene encoding a glutathione-S-transferase. A fusion gene prepared by linking a polynucleotide encoding a pre-S1 peptide consisting of 50 to 58 amino acids from the N-terminus of the pre-S1 protein of HBV to a terminal thereof is disclosed.

In addition, the Republic of Korea Patent Application No. 1991-25893 is a recombinant Dl virus by cutting or removing a portion of the gene encoding the envelope protein of polio virus and inserting the gene frame encoding the surface antigen and fries antigen of hepatitis B virus After the vector is prepared, RNA-transscripts of the reconstituted DI virus produced by the vector itself or in vitro transcription are transfected into primate animal cells, which are then superinfected with the helper virus and enriched. Disclosed is a method for preparing an oral hepatitis B vaccine, characterized in that the virus is passaged, and then purified and isolated DI virus is formulated in an enteric dosage form.

In addition to the above patent documents, Korean Patent Application Nos. 1985-1458 and 1993-29980 also disclose surface antigens of HBV.

Throughout this specification many patent documents and articles are referenced and their citations are indicated. The disclosures of cited patent documents and articles are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

Accordingly, an object of the present invention is to provide an expression vector for dhfr ^- animal cells.

Another object of the present invention is to provide an expression vector for dhfr ^- animal cells for mass production of surface antigens of HBV.

Another object of the present invention to provide a dhfr ^- animal cell line transformed by the expression vector.

It is another object of the present invention to provide a method for preparing an HBV surface antigen.

1 is a schematic diagram showing a manufacturing process of pCMVdE-dhfr which is one embodiment of the vector of the present invention;

Figure 2 is an agarose gel photograph confirming the production of a promoter from which the enhancer site of cytomegalovirus has been removed by the present invention;

Figure 3 is a schematic diagram showing the manufacturing process of the pZeo-dhfr vector produced in the intermediate stage of the method for producing a vector of the present invention;

Figure 4 is a schematic diagram showing the manufacturing process of the pCB2FD vector of one embodiment of the vector of the present invention;

5 is a diagram showing a genetic map of pCMVdE-dhfr which is one embodiment of the vector of the present invention;

Figure 6 shows a genetic map of pCB2FD which is one embodiment of the vector of the present invention;

7 is a phase contrast micrograph showing the cell shape of the cell lines of the present invention;

8 is a SDS-PAGE gel photograph showing the purity and molecular weight of HBsAg produced and purified according to the method of the present invention;

9 is an immunoblotting gel photograph showing the immunological properties of HBsAg produced and purified according to the method of the present invention;

10 is an ELISA assay graph showing the immunological properties of HBsAg produced and purified according to the method of the present invention; And

11 is a gel photograph showing Southern blot analysis to confirm that the HBsAg gene is inserted into the gDNA of the cell line in the cell line of the present invention.

According to an aspect of the present invention, the present invention provides a dhfr ^- expression vector for animal cells comprising a promoter from which an enhancer region of cytomegalovirus has been removed, dhfr operably linked to the promoter, and a replication origin that operates in eukaryotic cells. do.

According to another aspect of the present invention, the present invention provides a promoter in which an enhancer region of cytomegalovirus is removed, a dhfr operably linked to the promoter, a replication origin operating in eukaryotic cells, a gene encoding a surface antigen of HBV and the HBV Provided are expression vectors for dhfr ^- animal cells for mass production of surface antigens of HBV comprising a promoter functionally linked to a gene encoding a surface antigen of.

The present invention relates to an expression vector for mass production of a protein of interest in animal cells transformed with dhfr ⁻ . As used herein, the term "dhfr ^- animal cell" refers to an animal cell that is not normally expressed in dihydrofolate reductase (DHFR), so that there is little or no activity of the enzyme in the cell. The present invention utilizes the principle of gene amplification including a DHFR gene (hereinafter referred to as "dhfr") for the characterization and selection of the host cell. In other words, when dhfr ^- animal cells are transformed with dhfr-containing DNA (e.g., vectors), and the transformed cells are treated with a DHFR inhibitor, cells with a large amount of dhfr-containing vector are amplified in the cell. . As a result, amplification of the vector is achieved.

According to a preferred embodiment of the present invention, a vector was constructed to achieve effective vector amplification even with low concentrations of DHFR inhibitors. For this strategy, the transcriptional activity of a promoter operatively linked to dhfr is artificially reduced. The promoter is preferably a promoter of cytomegalovirus (hereinafter referred to as "CMV") commonly used in animal cells, more preferably the immediate early promoter of CMV. In order to reduce the activity of this promoter, a promoter with the enhancer site removed from CMV is preferred, and triggers the transcription process by interaction with transcription factor and RNA polymerase in the promoter sequence of conventional CMV. The minimum sequence required is more preferred, most preferably the promoter from which 1-428 of the promoter sequences set forth in SEQ ID NO: 1 has been removed.

According to a preferred embodiment of the present invention, the eukaryotic cell is CHO (Chinese Hamster Ovary), because CHO cells deficient in DHFR have been widely used after being approved by the FDA for safety and efficacy.

Replication origins that operate in eukaryotic cells included in the vectors of the present invention include, but are not limited to, f1 origin, SV 40 origin, pMB1 origin, Adeno origin, AAV origin and BBV origin.

In addition, in the vector of the present invention, a promoter linked to a gene other than dhfr is preferably used as a strong promoter, for example, CMV promoter, tk promoter, SV 40 late promoter, SV 40 early promoter, MT promoter. , MMTV LTR promoter, adenovirus late promoter (Ad MLP), herpes simplex virus promoter, promoter of murine metallothionein gene, and the like, may be used. .

According to the most preferred embodiment of the present invention, the expression vector for dhfr ^- animal cells is pCMVdE-dhfr with the genetic map shown in FIG.

In the expression vector for dhfr ^- animal cells of the present invention for producing a large amount of the surface antigen of HBV, the gene encoding the surface antigen of HBV (hereinafter referred to as "HBsAg") is a pre-S1 gene, a pre-S2 gene or It is preferably composed of S antigen genes, or a combination thereof. As can be seen in the known literature, the gene encoding HBsAg more preferably comprises a pre-S gene (ie, a pre-S1 gene and a pre-S2 gene), and antigens comprising such pre-S are premature. Excellent antibody producing ability and antibody persistence.

As the transcription termination sequence, the expression vector of the present invention includes a polyadenylation sequence, and for example, an SV40 polA sequence and a BGH polA sequence may be used.

In a most preferred embodiment of the invention, the expression vector for eukaryotic cells to obtain large amounts of surface antigens of HBV is pCB2FD with the gene map shown in FIG. 6 (KCTC 1025BP).

According to another aspect of the present invention, the present invention provides a dhfr ^- animal cell line transformed with the expression vector for dhfr ^- animal cell of the present invention described above.

According to another aspect of the invention there is provided dhfr for the production of a HBV surface antigen of the ^{bulk-providing} animal ^cell line transformed with an expression vector for animal cell dhfr.

In the most preferred embodiment of the invention, said animal cell line is a CHO / dhfr ⁻ cell line (KCTC 1025BP). HBsAg expressed by such an animal cell line is an antigen that is more similar to the tertiary structure of a naturally-occurring antigenic protein than an antigen derived from microorganisms, and is superior in terms of early antibody production and antibody persistence than currently used yeast-derived HBsAg.

In the present invention, the method of transforming dhfr ^- animal cells with a vector may include micro-injection (Capecchi, MR, Cell , 22: 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology , 52: 456 (1973)), electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982)), liposome-mediated transformation (Wong, TK et al., Gene , 10:87 ( 1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol. , 5: 1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci. , 87: 9568-9572 (1990)).

According to another aspect of the invention there is provided (a) for the production of surface antigen of the above HBV in bulk ^{dhfr-inhibitor} in an animal cell lines dihydro folate ^{reductase-transformed} with an expression vector for animal cells, dhfr Culturing under the addition of; And (b) purifying the HBV surface antigen in culture.

In a preferred embodiment of the present invention, the inhibitor of the dihydrofolate reductase, for example, aminopterin (aminopterin) and methotrexate and the like, but is not limited thereto, most preferably methotrexate.

Since MTX used for gene amplification is expensive, it is not an important consideration in laboratory experiments conducted at the in vitro level, but it is an important consideration when carried out at a high volume level. Thus, the art continues to study ways to reduce the concentration of MTX added for gene amplification. On the other hand, in the art, about 0.5-1 μM of MTX is used for gene amplification. The cell line used in the production method of the present invention is transformed so that gene amplification is performed well even at low concentrations of MTX. Therefore, the concentration of methotrexate added to the present invention is preferably 5-100 nM, more preferably 30-60 nM, and most preferably 40-50 nM. As can be seen in the following examples, when the MTX is 50 nM or more in the present invention, the rate of gene amplification is greatly reduced, considering both the price and the degree of amplification of MTX, the increase in MTX added is It loses meaning.

In the production method of the present invention, the step of culturing may be carried out in a medium commonly used in the present invention, and commercially available medium may be, for example, Hams F10 (Sigma), MEM (minimal essential medium, Gibco BRL), RPMI. -1640 (Sigma), DMEM (Gibco BRL), and the like. Ham and Wallace, Meth. Enz. , 58:44 (1979) and Barnes and Sato, Anal. Biochem. , 102: 255 (1980) can be used.

In the culturing step, HBsAg expressed in the host cell is secreted into the medium, and the HBsAg thus secreted is purified to obtain a large amount of the desired HBsAg. In the present invention, the purification step may be carried out by employing a purification method commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used and are usually purified using a combination of the above methods.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example 1 Isolation of Genes Encoding Pre-S and S Antigens of HBV

HBV gDNA was extracted from the sera of HBV-infected patients using the Genome DNA Separation Kit (Promega), and the gene encoding the pre-S and S antigens of HBV was obtained by amplification by PCR. Pfu polymerase used for PCR was purchased from Solgent, and the temperature conditions were 30 seconds at annealing 58 ° C, 2 minutes 30 seconds at 72 ° C, and 30 seconds at 94 ° C. It was. The forward primer used was 5'-GTGGAAGGCTAGCATTCTATATAA-3 ', the reverse primer was 5'-CGTCCCGCGCAGGATCCAGTT-3', and the primer was designed to generate Nhe I and Bam HI restriction enzyme sites.

Example 2: Vector Construction to Express r-HBsAg Antigen in CHO Cells

I. Fabrication of pCMVdE-dhfr

To obtain a cell line expressing high r-HBsAg in a low concentration of MTX-containing medium, first, pCMVdE-dhfr was prepared as follows (see FIG. 1): First, a pZeo-SV2 (+) vector having an hCMV promoter (Invitogen) HCMV IE promoter portion excluding the enhancer portion was obtained by PCR method. PCR was performed in the same manner as in Example 1, and the forward primer used was 5'-ATCGATTTCCAAAATGTCGTA-3 ', and the reverse primer was 5'-TGCTAGCCGGTGTCTTCT-3'. Subsequently, pCMVdE was prepared by inserting a PCR amplification product into pMOS Blue (Amersham Pharmacia Biotech) and observing the size on the agarose gel to confirm that the size of the promoter was reduced by removing the enhancer portion (see FIG. 2). Sequencing confirmed the removal of the enhancer couple. In FIG. 2, the M lane is a size marker loaded, the CMV lane is a fragment of pCMV (E + P) containing an enhancer cut by restriction enzymes Cla I and Nhe I, and the CMVdE lanes are the pCMVdE restriction enzymes Cla I and Each section was cut with Nhe I. As can be seen in Figure 2, it can be seen that the pCMVdE includes a CMV IE promoter from which the enhancer is removed.

Meanwhile, pZeo-SV2 (+) vector (Invitogen) and pSV2-dhfr vector (ATCC) were treated with Hin dIII and Bam HI, respectively, to insert the dhfr gene included in pSV2-dhfr into pZeo-SV2 (+). dhfr was produced (see FIG. 3).

Subsequently, the vector pCMVdE was digested with restriction enzymes Cla I and Nhe I and inserted into a vector pZeo-dhfr treated with the same enzyme to finally prepare pCMVdE-dhfr.

Ⅱ. pCB2FD Fabrication

First, the product amplified in Example 1 was digested with Nhe I and Bam HI, and then inserted into a pGEM-T Easy vector (Promega). Subsequently, the pGEM-T Easy vector and the animal cell expression vector pcDNA3.1 / Zeo (Invitrogen) were cut with Nhe I and Bam HI, respectively, and the HBsAg gene in the pGEM-T Easy vector was inserted into pcDNA3.1 / Zeo. PCMV-HBsAg was prepared. Then, the vector pCMV-HBsAg was transformed into COS-7 (ATCC), a mammalian cell line, and cultured for 3 days, and the secretion of HBsAg antigen was examined by ELISA (Green Cross, GENEDIA HBsAg ELISA 3.0) method. In Example 1, the functional activity of the PCR amplified product was confirmed, and the ELISA measurement result confirmed that the HBV surface antigen was normally expressed.

Then, in order to obtain a CHO cell line that continuously expresses the HBsAg antigen, the pCMVdE-dhfr vector and pCMV-HBsAg were respectively digested with Eco RI, and the pCMV-HBsAg portion of pCMV-HBsAg was finally inserted into pCMVdE-dhfr. pCB2FD vector was constructed.

4 is a schematic diagram illustrating the construction of the pCB2FD vector, FIG. 5 is a genetic map of the pCMVdE-dhfr, and FIG. 6 shows a final genetic map of the pCB2FD vector of the present invention.

Example 3: Transformation Using Vector of the Present Invention

3 μl FuGENE^TM6 (Roche) was diluted in 100 μl of serum-free IMDM medium (Iscove's Modified Dulbecco's Media, GiBco BRL), mixed with 2 μg of the pCB2FD vector prepared in Example 2, and mixed at room temperature for 40 minutes. CHO / dhfr prepared after leaving^-(ATCC CRL 9096) Evenly put on the cells. Then, the cells obtained by incubation for 72 hours in normal medium (IMDM medium containing 10% FBS and HT (0.1 mM sodium hypoxanthine, 0.016 mM thymidine)) treated with 0.25% trypsin (GiBCO BRL) and centrifuged 1 x 10 per well⁴dog Cells were aliquoted into 96-well plates. Then incubation (37 ° C., 5% CO₂Concentration) and incubated for 2 weeks with changing medium every 4-5 days. Two weeks later, 100 surviving clones were obtained, and HBsAg ELISA (GENEDIA HBsAg ELISA 3.0) was performed to measure r-HBsAg surface antigen production capacity of each clone. Among them, the cell line that produces the most rHBsAg surface antigen is named "CHO D14", and is 1 × 10.⁶Dog cells were harvested and transferred to a 100 mm dish and incubated for 2 weeks with medium exchange at 4-5 days intervals in α-MEM medium containing MTX (Sigma) 5 nM. Colonies generated at the concentration of MTX were treated with trypsin and then transferred to a 96-well plate using a tip, and the expression level of HBsAg was again measured by the same ELISA method. The highest expression cell line was named "CHO D-14-3", and the final r-HBsAg surface antigen high expression cell line obtained by culturing in α-MEM medium containing 50 nM MTX was designated as "CHO D14-3-". 4 "and said cell line" CHO^dhfr-Was deposited with the Korean Collection for Type Cultures on June 1, 2001 and was given accession number KCTC 1025BP.

Example 4 Analysis of Transformed Inventive Cell Lines and rHBsAg

I. Morphological Characterization of Cells

The cell line producing rHBsAg obtained in the above example was observed with a Phase contrast microscope (Nikon), and was observed in a typical epithelial cell shape, which is the same normal shape as the original cell (see FIG. 7). Therefore, there is no problem in using rHBsAg produced in the cell line of the present invention as a vaccine.

Ⅱ. Isolation and Purification of rHBsAg

end. Thickening and dialysis

Natural HBsAg particles have a size of about 22 nm and molecular weights of 1 million to 2 million Daltons (Zaslavsky, V. et al. J. Gen. Virol. , 2: 341-9 (1980)). Purification process was performed as follows: 5 L of the culture medium of the cell line of the present invention producing rHBsAg was concentrated to 500 ml using an MIL of 300,000 cut-off ultrafiltration system (Millipore Pellicon System), followed by PBS ( Phosphate buffered saline) removed material with molecular weight less than 300,000. Subsequently, after treating with 4% PEG under PBS salt conditions, the precipitate was removed by centrifugation, and the obtained supernatant was treated with PEG again to a concentration of 10%, followed by centrifugation at 10,000 rpm for 20 minutes. Collected.

I. KBr concentration gradient ultracentrifugation

In general, the pHBsAg and rHBsAg have a density of about 1.2 g / ml, and the density of lipids or lipoproteins is much lower than this, and most proteins have a higher density. : The PEG precipitate was dissolved in PBS, followed by KBr addition to a density of 1.2 g / ml, followed by centrifugation at 60,000 rpm using a Beckman Ti 70 rotor. After the centrifugation was terminated, the rHBsAg band was separated using a syringe. The rHBsAg thus separated was diafiltered with MW 10,000 cut-off to remove salts and finally purified.

All. SDS-PAGE Analysis

The purified rHBsAg was electrophoresed on a 12% SDS-polyacrylamide gel and then stained using a silver staining plus system (BIO-RAD, 161-0499) (see FIG. 8). Subunit protein pattern analysis using SDS-PAGE revealed bands of S protein and glycosylated S protein at 22 kD and 26 kD, and monoglycosylated pre-S2 and diglyco at 33 kD and 36 kD. Illuminized pre-S2 was identified and the bands of pre-S1 and glycosylated pre-S1 at 39 kD and 42 kD. Therefore, it can be seen that the rHBsAg was purified by the above-described purification process.

III. Immunological analysis

end. Immunoblotting Analysis

The rHBsAg protein purified in the above example was electrophoresed with 12% SDS-polyacrylamide gel and then adsorbed onto NC membrane (Schleicher & Shuell) by electrophoresis. Subsequently, polyclonal rabbit anti-HBsAg (Fitzgerald, 20-HR20) was reacted with the NC membrane, followed by treatment of alkaline phosphatase-conjugated anti-rabbit IgG (KPL, 075-1506) as a secondary antibody, followed by color development. The substrate was added with BCIP / NBT (Sigma) to develop color (see FIG. 9). As a result, it was confirmed that the recombinant protein expressed in the cell line of the present invention is an S surface antigen including pre-S1 and pr-S2 of HBV.

I. ELISA analysis

RHBsAg expressed in the cell lines of the present invention was quantified using GENEDIA HBsAg ELISA 3.0 kit from Green Cross. The transformed cell line CHO D14-3-4 finally obtained by adapting at a concentration of 50 nM MTX showed an expression amount of 2.5 μg / 10 ⁶ cells / 24 hours (see FIG. 10). In addition, CHO D14, the cell line which did not induce gene amplification by MTX, showed the lowest expression level, and the expression level of CHO D14-3-4 was significantly lower than that of CHO D14-3-4 which induced amplification with 5 nM MTX.

All. Southern blot analysis

In order to confirm whether the rHBsAg gene constructed by the present invention was inserted into the gDNA of CHO cells, Southern blot analysis was performed as follows, and a DIG DNA labling & Detection kit (Roche, 1 093 657) was used for the analysis. First, the gDNA of the cell line CHO D14-3-4 transformed with CHO / dhfr- was isolated (Steffen, et al., Proc. Nat. Acad. Sci. , 76: 4554-4558 (1979)). After quantification. Subsequently, 5 μg of each isolated gDNA was digested with restriction enzyme Eco RI and electrophoresed on a 0.7% agarose gel. The DNA is then transferred to a nylon membrane (Amersham Phamacia Biotech) by capillary method, and the formed blot is hybridized with a DIG-labeled probe (probe to identify the HBsAg gene) and then anti-DIG conjugated with alkaline phosphatase The reaction was carried out with the antibody, and color development was performed by adding NBT / BCIP, which is a color substrate (see FIG. 11).

As a result, no signal was detected in CHO / dhfr- cells, but the signals of HBV pre-S and S surface antigen genes were strong in CHO D14-3-4 cell line. As a result, the dhfr gene and the HBV gene were inserted into the gDNA of the CHO D14-3-4 cell line, and the amplification of the HBV surface antigen was confirmed. On the other hand, the band corresponding to the HBV surface antigen was also observed in the CHO D14 cell line, but appeared very weak and is the lowest nonspecific band.

The present invention provides an expression vector for dhfr ^- animal cells. The present invention also provides an expression vector for dhfr ^- animal cells for mass production of surface antigens of HBV. On the other hand, the present invention provides a dhfr ^- animal cell line transformed by the expression vector. The present invention also provides a method for producing HBV surface antigen. According to the present invention, not only a large amount of HBV surface antigen can be obtained by a lower concentration of DHFR inhibitor than the conventional method, but also a large amount of HBV surface antigen excellent in early antibody generating ability and antibody persistence can be obtained.

Claims (16)

A dhfr ^- expression vector for animal cells comprising a promoter from which an enhancer region of cytomegalovirus has been removed, a dhfr operably linked to the promoter, and a replication origin that operates in eukaryotic cells. A promoter from which an enhancer region of cytomegalovirus has been removed, a dhfr operably linked to the promoter, a replication origin that operates in eukaryotic cells, a gene encoding the surface antigen of HBV and a gene encoding the surface antigen of the HBV expression vector for animal cell ^- dhfr for the production of surface antigen of HBV, including associated peuromoteoeul in large quantities. The expression vector for a dhfr ^- animal cell according to claim 1 or 2, wherein the promoter from which the enhancer site is removed is removed from 1-428 of the promoter sequence of SEQ ID NO: 1. The expression vector for dhfr ^- animal cell according to claim 1 or 2, wherein the eukaryotic cell is a CHO cell. The method of claim 1 or 2, wherein the origin of replication in the eukaryotic cell is selected from the group consisting of f1 origin, SV 40 origin, pMB1 origin, Adeno origin, AAV origin and BBV origin Dhfr ^- an expression vector for animal cells. According to claim 1, wherein the expression vector for eukaryotic cells dhfr ^- animal cell expression vector, characterized in that pCMVdE-dhfr having the gene map shown in FIG. The expression vector for dhfr ^- animal cell according to claim 2, wherein the gene encoding the surface antigen of HBV is composed of a pre-S1 gene, a pre-S2 gene, and an S antigen gene. The expression vector for eukaryotic cells for obtaining a large amount of surface antigens of HBV is pCB2FD having a gene map shown in FIG. 6, dhfr ^- expression vector for animal cells (KCTC 1025BP). A dhfr ^- animal cell line transformed with the expression vector of claim 6. A dhfr ^- animal cell line transformed with the expression vector of claim 8. The transformed dhfr ^- animal cell line (KCTC 1025BP) according to claim 10, wherein the animal cell line is a CHO / dhfr ^- cell line. HBV surface antigen production method comprising the following steps: (a) culturing the cell line of claim 11 under the addition of an inhibitor of dihydrofolate reductase; And (b) purifying the HBV surface antigen in culture. The method of claim 12, wherein the inhibitor of dihydrofolate reductase is aminopterin or methotrexate. The method of claim 13, wherein the inhibitor of dihydrofolate reductase is methotrexate. 15. The method of claim 14, wherein the concentration of methotrexate is 5-100 nM. 16. The method of claim 15, wherein the concentration of methotrexate is 30-60 nM.