KR100421945B1 - Recombinant adenovirus(ad/hcv/e3) containing structure protein core gene, and surface glycoproteins e1 and e2 genes of hepatitis c virus - Google Patents

Abstract

PURPOSE: A recombinant adenovirus(Ad/HCV/E3) containing structure protein core gene, and surface glycoproteins E1 and E2 genes of hepatitis C virus is provided, which adenovirus can be useful as an alive vaccine for hepatitis C virus(HCV). CONSTITUTION: The recombinant adenovirus(Ad/HCV/E3)(KCTC 0244BP) is prepared by inserting structure protein core gene of hepatitis C virus(HCV), and surface glycoproteins E1 and E2 genes of hepatitis C virus into the E3 gene region of human adenovirus type 5, wherein the structure protein gene of HCV has a termination codon in the C-terminal. The method for producing HCV E1 and E2 proteins comprises culturing a mammal cell infected with the recombinant adenovirus(Ad/HCV/E3)(KCTC 0244BP) in a medium to express the HCV E1 and E2 protein and isolating them from the cells by using GNA-agarose column and DEAE fractogel column chromatography, wherein the mammal cell is HeLa cell or 293 cell.

Description

Recombinant adenoviruses (Ad / HCV / E3) containing the structural protein core, E1 and E2 envelope glycoprotein genes of hepatitis C virus

The present invention is a recombinant protein prepared by introducing the genes of core protein E1 and E2 of the structural protein core of the hepatitis C virus (hereinafter referred to as HCV) into the human adenovirus type 5 E3 gene region. Adenovirus (Ad / HCV / E3), a vaccine against hepatitis C virus characterized by containing this recombinant virus, and a method for producing HCV core, El and E2 proteins using the recombinant virus.

HCV is a pathogen that is mainly transmitted by blood transfusions and causes chronic hepatitis, cirrhosis and liver cancer when infected with humans (see Choo, QL et al., Science, 244: 359-362, 1989; Kuo, G. et al., Science 244: 362-364, 1989; Saito, L. et al., Proc. Natl. Acad. Sci. USA 87: 6547-6549, 1990). Symptoms when a person is infected with HCV are similar to those of hepatitis B virus (HBV), but are more lethal to humans because HCV induces liver cancer more frequently than HBV. However, there is no effective therapeutic or preventive vaccines for this.

HCV is a single stranded (+) RNA virus with a gene of about 10 kb in size and its structure is similar to flaviviruses or pestiviruses. When the cell is infected by the virus, translation occurs in the HCV RNA, creating a large polypeptide of 3011 amino acids, which is contained in the protease signalase and the virus in the host cell. Proteins such as core, E1 (gp35), E2 (gp70), NS2, NS3, NS4A, NS4B, NS5A and NS5B are prepared by proteolytic enzymes (NS2 and NS3). In particular, the core protein is a nucleocapsid protein that surrounds the RNA of HCV and has high antigenicity and has been reported to have the smallest difference in sequence among several genotypes of HCV. Houghton, M. et al., Molecular Biology of the hepatitis C virus: Implications for diagnosis, developm-ent and control of viral disease, Hepatology 14: 381-388, 1991). In addition, HCV E1 and E2 proteins are the outermost envelope glycoproteins of HCV, and have been reported to bind to each other to form a heterodimer (Balston, R. et al., Journal of Virology 67: 6753-6761). , 1993: Dubuisson, J. et al., Journal of Virology 68: 6147-6160, 1994). Induced immune defense against HCV infection of the same genotype in chimpanzees administered with antigens of purified HCV E1 and E2 proteins together. (Cho, QL et al., Proc. Natl. Acad. Sci., USA 91: 1294-1298, 1994).

On the other hand, adenovirus is a pathogenic virus that induces acute respiratory disease in people living in the group. Therefore, oral adenovirus type 4 and 7 vaccines were developed in 1983 and were approved by the US FDA. Although the vaccine was vaccinated, it had no side effects, and more than 97% had a preventive effect, which is considered to be a highly efficient and safe vaccine. Therefore, several attempts have been made to develop adenovirus type 5 as the in vivo antigen gene transfer vector (vaccine vector) of other pathogenic viruses (Graham, FL and L. Prerec, Adenovirus-based expr-ession vectors and recombinant vaccines. In RW Ellis ed., Vaccines: New approaches to immunological problems.Butternnorth-Heinemann, Boston, 1991).

In particular, adenovirus vectors are inexpensive to produce, exhibit no pyrogen activity, can induce mucosal immunity, and can be used orally.

In this situation, the present inventors have conducted continuous research to develop a vaccine against HCV. As a result, we introduced a recombinant protein gene of HCV into a human adenovirus type 5 vector, thereby replicating the recombinant adenovirus (Ad / HCV). / E3), HCV structural protein cores, E1 and E2 glycoproteins in human cells infected with this recombinant adenovirus are expressed almost identically to those of natural infection, and HCV when inoculated with recombinant adenovirus in mice By confirming that antibodies against core, E1 and E2 proteins are produced, the present inventors have found that the recombinant virus can be usefully used as a live vaccine against HCV, thereby completing the present invention.

Hereinafter, the configuration of the present invention will be described in detail.

An object of the present invention is to provide a recombinant adenovirus (Ad / HCV / E3) prepared by introducing the core protein of HCV, the envelope glycoproteins E1 and E2 gene into the E3 gene region of human adenovirus type 5.

The recombinant adenovirus of the present invention is a gene encoding the HCV type 2 core, E1 and E2 glycoproteins contained in a known C740 plasmid according to conventional methods known in the field of genetic engineering, E3 gene of adenovirus type 5 It was prepared by inserting instead, the process is as shown in Figures 1A and 1B and the specific method can be referred to Example 1. The recombinant adenovirus of the present invention prepared by such a method is a novel one that has not been constructed so far, and the inventors named it human recombinant adenovirus type 5 (Ad / HCV / E3), and as of April 26, 1996, Korea Science and Technology The deposit number of KKTC0244BP was deposited in the Research Institute for Biotechnology Research under the Budapest Treaty on Microbial Deposits.

It is noteworthy that during the production of the recombinant adenovirus of the present invention, a sequence encoding a translational codon and 10 amino acids derived from the pBKS / TAA plasmid used during construction is added to the E2 glycoprotein gene of HCV ( 2, thus, E2 glycoprotein can be expressed in human cells. As such, a sequence of a certain length was added to the C-terminus of the E2 gene so that E2 protein can be expressed in mammalian cells including human cells during the production of recombinant adenoviruses. Proteins, E1 and E2 glycoproteins are produced in one polypeptide and do not interfere with glycosylation, reactivity with the serum of the patient, formation of E1 and E2 heterodimers, and antibody-inducing ability in mice. It was confirmed not to give. Therefore, according to the present invention, there is provided an HCV structural protein gene (see FIG. 2) in which a translation termination codon is introduced at the C-terminus to efficiently express the structural protein gene of HCV in mammalian cells. One purpose.

Meanwhile, the gene of the recombinant adenovirus according to the present invention can be cloned in human cells to express HCV structural proteins, and when the mice are inoculated, antibodies to HCV core, E1 and E2 glycoproteins are generated. (See Examples 2, 3 and 6), the recombinant adenovirus of the present invention can be very usefully used as a live vaccine against HCV, and therefore the present invention aims to provide such a vaccine.

In general, a vaccine can be used as a killed virus, or only an antigenic part of a pathogen can be separately isolated, or a virus whose pathogenicity is lost can be used as a live vaccine, and can also be manufactured as a subunit vaccine using genetic engineering techniques. Can be used. Among these, live vaccine viruses are used as carriers (vectors) for introducing the protective immune genes of other pathogenic viruses in vivo. Vaccine preparations using these live vaccine vectors can allow the production of antigens continuously in vivo. It has the advantage of being able to induce both sexual immune responses and long lasting immune responses.

Core protein of HCV structural protein gene has high antigenicity and epitopes that can induce cytotoxic T cells. E1 and E2 glycoproteins are also thought to have epitopes that can induce viral neutralizing antibodies as components of viral envelopes. Since vaccines using E1 and E2 proteins are glycosylated, it is desirable to develop them to be used as a vaccine by expressing them directly in mammalian cells.However, E1 and E2 glycoproteins are expressed in large quantities in the mammalian expression system. In addition to the development of a technique for the development of the E1 and E2 glycoproteins bind to each other to form a heterodimer, the characteristics of inducing neutralizing antibodies with E1 or E2 protein alone is different from that of E1 and E2 heterodimer Is expected to be. E1 glycoproteins, on the other hand, have been reported to have epitopes capable of inducing cytotoxic T lymphocytes, similar to coa proteins (Koziel, M. J. et al., Journal of Immunology 149: 3339-3344, 1992).

Therefore, a gene encoding the structural protein cores, E1 and E2 glycoproteins of HCV was introduced into an adenovirus vector having the characteristics described above, and then used as a live vaccine to construct the structural proteins of HCV cores, E1 and E2, respectively. Much like in vivo HCV infection, the method of expression in vivo can be very useful for the purpose of inducing protective immunity against HCV. As a result of the experiment, the present inventors have expressed a large amount of core, E1 and E2 proteins in human cells by recombinant adenovirus (Ad / HCV / E3) provided by the present invention, and these expressed proteins are HCV in the serum of the patient. Reacting specifically with antibodies confirmed that almost identical production was made in human cells during the natural infection of HCV. In addition, in human cells infected with recombinant adenovirus, E1 and E2 glycoproteins exist as heterodimers and also as E1 or E2 proteins alone, and thus can induce various antibodies to each protein. It may be expected that each protein of E2 induces the generation of cytotoxic lymphocytes in vivo.

In the case where the recombinant adenovirus according to the present invention is to be used as a live vaccine, a vaccine which is oral preparation is preferably used. In this case, a vaccine consisting of only recombinant adenovirus may be prepared and used, but a pharmaceutically acceptable adjuvant may be added as needed in the vaccine manufacturing process. Preferably, the vaccine formulated for oral use is coated with gelatin. Is to use.

The present invention also provides a method for producing HCV E1 and E2 proteins by culturing mammalian cells infected with the recombinant adenovirus Ad / HCV / E3 according to the present invention to express the protein. Each protein can be isolated and purified in high purity from cell culture media according to conventional methods of the art (Journal of Virology Vol. 67 (11): 6753-6761, 1993). Protein extracts obtained by disrupting and centrifuging mammalian cells, preferably HeLa cells or 293 cell lines, transformed with the recombinant adenovirus according to the invention were subjected to GNA agarose column and DEAE-Fractogel column chromatography. Sequential application to gave a mixture of E1 and E2 glycoproteins with a purity of about 70%. The E1 and E2 glycoproteins thus obtained may be usefully used for the production of subunit vaccines.

Hereinafter, the present invention will be described in more detail based on the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention in any sense is not limited to these examples.

Example 1 Preparation of Recombinant Adenovirus (Ad / HCV / E3)

Known C740 plasmids containing genes encoding cores, E1 and E2 glycoproteins of HCV type 2 (Hijikata, M. et al., Proc. Natl. Acad. Sci. USA, 88: 5547-5551, 1991 ), A 2.3 kb DNA fragment obtained by treatment with EcoR1 was introduced into the EcoRI site of the pBKS / TAA plasmid into which the translation termination codon was introduced as shown in FIG. 1A to prepare a pBKS / A740 plasmid. The pSKS / A740 plasmid was treated with XbaI and XhoI and blunt ends at both ends of the DNA, followed by introduction into the known plasmid pSV2x3 with the SV40 promoter and poly A signal to express the protein pSV / A740 in mammals. Was prepared (see also FIG. 1A). About 3 kb of DNA fragment obtained by treating the pSV / A740 plasmid with XbaI was introduced into the XbaI site of the known recombinant adenovirus production vector pFG144k5 to prepare a pFG144k5 / HCV plasmid, while adenovirus type 5 DNA was infected with adenovirus 293 Isolated from the cell line, treated with EcoRI and electrophoresed on agarose gel to obtain a DNA fragment of about 27.1 kb containing the left inverted terminal repeat (ITR) of the adenovirus. About 2 µg of the obtained DNA fragment and about 6 µg of pFG144k5 / HCV were introduced together into the 293 cell line by the calcium phosphate method to produce a replicable recombinant adenovirus in which the HCV structural protein gene was introduced into the adenovirus E3 gene region. (See also FIG. 1B).

The recombinant adenovirus was designated as human recombinant adenovirus type 5 (Ad / HCV / E3), and as of April 26, 1996, a deposit number of KKTC0244BP was assigned to the Gene Bank of Korea Institute of Science and Technology under the Budapest Treaty on the Microbial Deposit. The deposit was completed.

Example 2: Expression of HCV Core Proteins

Adenovirus Ad / ΔE3 lacking the E3 gene in the monolayer cells (6 cm plate) of HeLa and 293 cell lines exhibiting a confluency of about 80-90% and the recombinant adenovirus Ad / HCV / obtained in Example 1 Each E3 was infected with 20MOI (multiplicity of infection) at room temperature for 1 hour, followed by the addition of dulbecco's modified Eagles' Medium (DMEM) containing 10% fetal bovine serum and incubated in a CO ₂ incubator at 37 ° C. At this time, Hela cells not infected with the virus were cultured simultaneously as a control. After 6, 12, 24, 36 hours post infection, the cultures were removed and 0.5 μg of RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium dioxycholate, 0.1% SDS) ) Was added and the cells were transferred to 1 ml Eppendorf tubes. The cell harvest was sonicated and 120 μl was taken and mixed with 40 μl of 5 × Laemmli sample buffer, boiled in a water bath for 5 minutes and separated by 10% SDS-PAGE. Gels were transferred to nitrocellulose membranes according to the method of Towbin et al. (Proc. Natl. Acad. Sci., USA, 76: 4350, 1979) followed by western blotting. That is, the nitrocellulose membrane was reacted with 10% skim milk suspended in phosphate-buffered saline (PBS) for 2 hours at room temperature, and the serum of HCV positive patients diluted 1: 1000 by PBS was added for 2 hours at room temperature. Then washed three times for 15 minutes with PBST (PBS containing 0.05% Tween 20). Again, the membrane was reacted with chlorine antibody (purchased from Sigma) for 1 hour at room temperature with alkaline phosphatase-linked human IgG diluted 1: 1000 with PBS, washed four times with PBST, and then nitrobluetezozolium And a protein band recognized by the serum of HCV positive patients by reacting in a substrate solution to which 5-bromo-4-chloro-3-indolylphosphate was added (see FIG. 3). As a result, in Hela and 293 cell lines infected with Ad / HCV / E3 virus, a protein having a molecular weight of about 22 kDa specifically reacted with the serum of the patient. Since it was not observed in the cell line infected with the virus, it was found to be HCV core protein.

In addition, even when Western blotting using an antibody specific to the core protein was specifically reacted with the 22kDa protein of the cell line infected with Ad / HCV / E3. The expression of HCV core protein began to be expressed at 6 hours after infection and showed the maximum expression at 12 hours. The expression of HCV core protein was no longer increased even when the culture time was increased to 24 and 36 hours. On the other hand, the Western blotting method did not confirm the expression of the envelope glycoproteins E1 and E2 of HCV, either because the E1 and E2 proteins denatured under SDS-PAGE conditions are not well detected by the antibody or in patients. It is thought that the effect of the antibody on adenovirus present in the serum. Therefore, in Example 3, immunoprecipitation reaction was performed using sera of four HCV positive patients to confirm the expression of E1 and E2 glycoproteins.

Example 3: Expression of HCV El and E2 Proteins

HeLa cells (10 cm dish, about 1x10 ⁷ cells) were incubated for 1 hour in DMEM medium without methionine and cysteine after 12 hours of infection with 20 MOI of Ad / ΔE3 and Ad / HCV / E3 viruses, respectively. Cell proteins were labeled with S ³⁵ by culturing in a CO ₂ incubator at 37 ° C. for 2 hours in DMEM medium containing methionine and cysteine labeled with S ³⁵ and 10% fetal bovine serum. At this time, the protein of HeLa cells not infected with the virus was also labeled with S ³⁵ and used as a control sample. After S ³⁵ labeling, the monolayer cells were washed three times with PBS and 1 μg of lysis buffer (lysis buffer; 100 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5% Triton X-100) was added. Thereafter, the sample was transferred to an eppendorf tube, and the protein was extracted on a nutator at 4 ° C. for 1 hour, followed by centrifugation at 4 ° C. and 12000 rpm for 20 minutes to obtain a supernatant. After washing each supernatant with lysis buffer, added suspension of Protein A suspension (trade name: Pansorbin, Calbiochem, USA) bound with 20 μl of normal human serum, followed by shaking at 4 ° C. for 6 to 8 hours for binding. The supernatant was removed by centrifugation at 4 ℃, 10000rpm for 10 minutes to remove non-specifically bound proteins.

30 μl of patient serum was mixed with 100 μl of Protein A Sepharose Bead Suspension (50:50, v / v), and then the lysis buffer was added so that the total volume was 500 μl. Protein A Sepharose beads were washed twice with Wash Solution A (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.2% NP-40) and then infected with Ad / ΔE3 virus. 1 μg of the extract was added and reacted at 4 ° C. for 6 to 8 hours, followed by washing twice with washing solution B (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% SDS), followed by dissolution buffer. Suspension in 150 μl. 50 μl of protein A Sepharose beads bound to the patient's serum, 500 μl of protein extracted from HeLa cells labeled with S ³⁵ , HeLa cells infected with the Ad / ΔE3 virus, and HeLa cells infected with the Ad / HCV / E3 virus After reacting at 4 ° C. for 12 hours or more, the beads were washed twice with Wash Solution B, twice with Wash Solution C (10 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2 mM EDTA, 0.2% NP-40). Washed once with sterile tertiary distilled water. It was suspended in 50 μl of the Ramoly sample buffer and boiled for 5 minutes, and the gel obtained by performing SDS-PAGE was dried and fluorography was performed. The results are shown in FIG. 4.

As can be seen from the results of FIG. 4, the sera of four HCV-positive patients specifically reacted with proteins of size 70 (E2) and 35 kDa (E1) in HeLa cells infected with Ad / HCV / E3. . More specifically, as apparently visible in the patient's serum 3 (PS3) results, three E2 protein bands were observed when immunoprecipitated with the patient's serum, of which 70 kDa protein reacted most strongly with the patient's antibody. In the case of the E1 protein, three proteins react strongly with the serum of the patient and 35 kDa protein binds the most strongly. The division into three bands is due to the difference in the degree of glycation of the E1 and E2 proteins, which is consistent with previous studies that deglycosylate these proteins into a single band. The antibody possessed by the antibody may specifically react with the E2 and E1 proteins expressed in human cells infected by Ad / HCV / E3, and the expressed E1 and E2 proteins may be larger than those of HCV spontaneous infection. Shows no difference. Therefore, when immunized with Ad / HCV / E3 virus, E1 and E2 antigen proteins are generated in the same manner as in natural infection, and it can be seen that they can induce neutralizing antibodies that can prevent HCV infection.

Example 4 Heterodimer Formation of HCV El and E2 Glycoproteins and Stability of Heterodimers

According to the same method as in Example 3, HeLa cells infected with Ad / ΔE3 virus, HeLa cells infected with Ad / HCV / E3 virus were labeled with S ³⁵ , and the polynucleotides for E1 and E2 proteins prepared in rabbits were labeled. Using the clonal antibody (provided by LG Chem Biotech Research Institute), the immunoprecipitation reaction was performed for each protein extract as follows.

15 μl of antibodies against E1 and E2 were bound to 120 μl of Protein A Sepharose Beads, and then suspended in 150 μl of lysis buffer, 50 μl of each of the protein extracts labeled S ³⁵ and 12 at 4 ° C. It was reacted for over time. The beads were washed twice with Wash Solution B, twice with Wash Solution C, and once with distilled water, followed by addition of Ramolei sample buffer, and boiled for 5 minutes, followed by SDS-PAGE. The gel was also dried and fluorographed and the results are shown in FIG. As can be seen from the results of FIG. 5, when the immunoprecipitation was induced by using an antibody against the E1 protein, the protein extracts of cells infected with the Ad / HCV / E3 virus precipitated E2 proteins together with the three E1 proteins. When the antibody against the E2 protein was used in the immunoprecipitation reaction, only the E2 protein was precipitated, and the E1 and E2 proteins were not observed in the uninfected HeLa cells or HeLa cells infected with the Ad / ΔE3 virus. These results suggest that E1 and E2 proteins expressed in HeLa cells infected with Ad / HCV / E3 virus form heterodimers with each other and E2 proteins alone.

On the other hand, the following immunoprecipitation reaction was performed to investigate the stability of the E1 and E2 heterodimer and whether the antibody against E2 can recognize the heterodimer under conditions different from the above. The HeLa monolayer cells (10 cm dish) labeled S ³⁵ were washed three times with PBS according to the same method as in Example 3. IP buffer containing 0.5% SDS (20 mM Tris-HCl, pH 7.5,100 mM NaCl, 1 mM EDTA) The suspension was passed through a 26-gauge size needle after suspension in 0.5 ml). Samples were treated for 15 minutes at 70 ° C., diluted 4 times with IP buffer containing 0.5% Triton X-100 and bovine serum albumin (0.5 mg / ml) and centrifuged at 4 ° C., 12000 rpm for 20 minutes. Each sample was pretreated with normal human serum and reacted with Protein A Sepharose beads bound to E1 and E2 antibodies for at least 12 hours at 4 ° C., and the proteins bound to the beads were treated as described above. Fluorographies were performed. As a result, it was found that both antibodies against E1 and E2 precipitated the E1 and E2 proteins together (see FIG. 6). This result shows that the E1 and E2 glycoproteins form a stable heterodimer even under 0.5% SDS conditions. It has been shown that antibodies to E2 can recognize heterodimers depending on the conditions of immunoprecipitation.

Example 5 Separation and Purification of HCV E1 and E2 Glycoproteins

About 10 ⁸ (10 cm dish, 10) HeLa cells were infected with 20 MOI of Ad / HCV / E3 per plate and then incubated in DMEM medium containing 10% fetal bovine serum for 12 hours. Infected cells were washed three times with PBS, raked with 3 ml of lysis buffer, and stirred for 1 hour at 4 ° C to disrupt cells and centrifuged at 4 ° C and 12,000xg for 20 minutes to obtain supernatant. This protein extract was purchased from Sigma, USA as lectin from GNA agarose column (Galanthus nivalis cross-linked with 4% beaded agarose; bed volume 2 ml , 1 × 5 cm) and washed with 10-fold column volume of dissolution buffer (20 mM Tris-HOl, pH7.5, 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100), followed by 0.9 M α-D-mannopyrano Proteins bound to the column were eluted with a lysis buffer of the same composition to which side was added. The eluate containing HCV envelope glycoproteins was collected and diluted 10-fold with NaCl-free lysis buffer, and the diluted eluate was added to a DEAE-Fractogel column (0.5x10 cm) to remove the substance bound to the column. (Flowthrough fraction) was obtained. The column flow was concentrated on Amicon YM-10 membrane to give 3 ml and stored at -70 ° C. 50 μl of the concentrate was added to the SDS-sample solution, boiled for 5 minutes in a water bath, separated on SDS-PAGE, and then stained with silver to confirm that the E1 and E2 proteins were separated and purified to about 70% purity (see FIG. 7 A lane). ), E1 and E2 proteins could be identified by Western blotting using E1 and E2 antibodies prepared in rabbits (see FIG. 7 B lane). By isolation and purification as described above, about 100 μg of E1 and E2 proteins were obtained in HeLa cells infected with Ad / HCV / E3 virus.

Example 6 Antibody Induction of Ad / HCV / E3 Virus Against HCV Core, E1 and E2 Proteins

In order to determine whether antibodies to HCV structural proteins are produced in mice infected with Ad / HCV / E3 virus, the following experiments were performed.

10 ⁷ virus particles were firstly inoculated into mice (Balb / C) intraperitoneally, and after 2 weeks, two additional antigens were stimulated at 1 week intervals, and then injected twice at 2 week intervals to obtain mouse serum. At this time, the control mice were injected with 10 ⁷ adenovirus (Ad / ΔE3) particles intraperitoneally under the same conditions to obtain serum. Meanwhile, DH5α was transformed with the respective recombinant plasmids obtained by linking HCV core, E1 and E2 structural protein genes to the E. coli expression vector pGEX-2T. DH5α transformed with pGEX-core (ΔC), pGEX-E1 (ΔC) and pGEX-E2 (ΔC) was inoculated in 5 ml of LB / ampicillin (100 μg / ml) medium and incubated overnight and fresh LB / ampicillin medium Inoculate 500 μl of the preculture solution in 5 ml to incubate at 0.6 to OD ₆₀₀ , and then add IPTG to a final concentration of 0.5 mM and incubate further for 4 hours to fully express the respective fusion proteins. 400 μl of each culture solution was centrifuged at 5000 rpm for 2 minutes, and the pellet obtained by dissolving in 40 μl of the Ramoli protein sample buffer containing 8M urea was boiled for 5 minutes, and 5 μl of the pellet was carried out to perform SDS-PAGE. As a result of staining with Coomassie blue to identify the expressed fusion proteins, GST-core (ΔC), GST-E1 (ΔC) and GST-E2 (ΔC) proteins with the C-terminus of each of the fusion proteins were removed. It was found that an excess of 27 kDa GST (glutathione-S transferase) protein was expressed (see also FIG. 8A). Meanwhile, the same protein samples as those used in FIG. 8B or 8A were transferred to the nitrocellulose membrane, and Western blotting was performed using antibodies diluted 1: 500 of the serum of mice infected with Ad / HCV / E3. It is shown. From this, the serum of this mouse does not react with GST protein (Fig. 8B, lane 2), but GST-core (ΔC) (lane 3), GST-E1 (ΔC) (lane 4) and GST-E2 (ΔC) It can be seen that it reacts specifically with (lane 5) protein and does not have any specific reaction with E. coli proteins (lane 1). That is, the results indicate that HCV core, E1 and E2 proteins were expressed in mice inoculated with Ad / HCV / E3 virus, and these expressed proteins acted as antigens to induce specific antibodies for each.

Antibodies specific to the coa protein and HCV coa protein present in the serum of mice infected with Ad / HCV / E3 were examined by indirect immunoassay (indirect ELISA) as described below. GST-core (ΔC) fusion protein solution (5 μg /) dissolved in coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , 3 mM NaN ₃ , pH 9.6) in each well of a 96-well ELISA plate (Maxisorp, Nunc) 100 ml) was added and coated at room temperature for at least 12 hours. After removing the coating solution, 200μl of 5% skim milk solution dissolved in PBST was added to the plate, which was allowed to stand at room temperature for 2 hours and washed three times with PBST. As a primary antibody, mouse serum was diluted 2-fold stepwise at 100-12800 times, and 100 µl was added to the wells, and then reacted for 2 hours in a humid chamber at 37 ° C., followed by washing three times with PBST. Goat α mouse IgG-HRP (commercially obtained from Sigma) was diluted 1: 1000 with PBS, added to 100 μl in wells, and reacted for 1 hour in a humid chamber at 37 ° C., followed by washing four times with PBST. To 25 ml of PC buffer (48.6 mM citric acid, 102.8 mM Na ₂ HPO ₄ ), 10 µl of 1,2-phenylenediamine dihydrochloride and 10 µl of 30% H ₂ O ₂ were added to the substrate mixture, and 100 µl each was added at room temperature. After reacting for 15 minutes, 50 µl of 2.5MH ₂ SO ₄ was added to stop the reaction, and the absorbance was measured at 492 nm. The result is shown in FIG. As can be seen from Figure 9, the serum of mice infected with Ad / HCV / E3 virus specifically reacted with the GST-core (ΔC) protein more than twice as much as that of Ad / ΔE3 infected mice. It was again confirmed that the antibody specific for was induced in mice infected with Ad / HCV / E3 virus.

Example 7: Propagation and Titer Assay of Ad / HCV / E3 Virus

Ad / HCV / E3 virus solution was inoculated to subconflunt HeLa monolayer cells (10 ⁸ cells, 10 cm dish x 10) so that the MOI was 20, and then infected at room temperature for 1 hour. DMEM medium containing serum (FBS) was added and incubated for 24 to 48 hours in a 5% CO ₂ incubator. At the time when 40-50% of the infected cells fall from the bottom, the cells were harvested, washed twice with PBS, suspended in 10 ml of PBS, and sonicated 30 times at maximum intensity using a microtip. Cells were disrupted (sealed-type sonicator, SONIFIER 450, BRANSON). The cell lysate was centrifuged at 4 ° C. at 10000 rpm for 10 minutes and the virus containing supernatant was dissolved in a discontinuous CsCl gradient layer (10 mM Hepes buffer, (pH7.4) in 10 ml of 4 M CsCl, 2.2 M CsCl 5 ml). Careful dropping was followed by ultracentrifugation at 4 ° C. and 25000 rpm for 2 hours (SW28 rotor, Beckman). The milky virus band formed after centrifugation was separated using a syringe, and the sample mixed with the same amount of saturated CsCl solution was separated at a second CsCl discontinuous phase (4M CsCl 3ml, 2.2M CsCl 4ml) at 4 ° C and 35000rpm. Ultracentrifugation was carried out for 3 hours at (SW41 rotor, Beckman). The band containing the virus particles was extracted and dialyzed for at least 12 hours under PBS containing 1000-2000 times of 10% glycerol to obtain about 2-5 × 10 ¹⁰ virus particles.

Virus titers were assayed using 293 cell lines. The 293 cell line was cultured in a 6 cm dish to be fused, and then the medium was removed, and the virus solution diluted 10-fold sequentially in the presence of 1 × DMEM was evenly distributed on 293 monolayer cells and then infected at room temperature for 1 hour. The virus solution was removed and then carefully covered with agar containing DMEM medium (1% noble agar, 20 mM MgCl ₂ , 5% FBS) and incubated in a 37 ° C., 5% CO ₂ incubator. Virus titers (plaque forming units / ml) were determined by counting the virus plaques formed by adding the agar medium every three days for about 6-9 days. As a result, the viral titer of the recombinant adenovirus Ad / HCV / E3 according to the present invention was found to be 5 × 10 ¹⁰ PFU / ml.

1A and 1B are schematic diagrams showing the manufacturing process of a replicable recombinant adenovirus (Ad / HCV / E3) containing structural proteins of hepatitis C virus (HCV).

Figure 2 shows the nucleotide sequences encoding the structural protein cores, E1 and E2 glycoproteins of HCV used to prepare recombinant adenovirus in the present invention.

3 is a result of Western blotting with serum of HCV-positive patients after electrophoresis of the protein extract obtained by infecting and culturing HeLa cells with the recombinant adenovirus according to the present invention on an SDS-polyacrylamide gel.

4 shows recombinant adenoviruses (Ad / HCV /) by infecting HeLa cells with recombinant adenovirus, labeling viruses and cellular proteins with ³⁵ S-methionine, and performing immunoprecipitation with the sera of four different HCV-positive patients. In E3), HCV envelope glycoproteins E1 and E2 are expressed and specifically bind to the serum of the patient.

FIG. 5 is labeled with ³⁵ S of HeLa cells infected with recombinant adenoviruses as shown in FIG. 4, followed by immunoprecipitation with polyvalent antibodies of HCV E1 and E2 prepared in rabbits, respectively. Analysis on the gel shows that HCV El and E2 proteins bind to each other to form a heterodimer.

FIG. 6 shows that the E1 and E2 glycoproteins stably form heterodimers with each other even in the presence of 0.5% SDS.

FIG. 7 shows the results of silver staining (lane A) of HCV E1 and E2 proteins obtained by passing a HeLa cell protein infected with recombinant adenovirus through a GNA-agarose column and a DEAE-fractogel column. The results of Western blotting (lane B) of the purified E1 and E2 proteins separated as described above with antibodies to E1 and E2, respectively, prepared in rabbits.

FIG. 8A shows SDS-PAGE results of GST-HCV core (ΔC), E1 (ΔC) and E2 (ΔC) proteins expressed in E. coli.

FIG. 8B is a Western blotting result showing the production of antibodies specifically reacting with core, El and E2 proteins of HCV in the serum of mice infected with 10 ⁷ recombinant adenoviruses (Ad / HCV / E3).

9 dilutes serum of mice infected with 10 ⁷ recombinant adenoviruses (Ad / HCV / E3) and adenoviruses (AD / ΔE3), and then expresses the GST-HCV core fusion protein expressed in E. coli as an antigen. Indirect immunoenzyme assay (ELISA) was performed to show the production of antibodies specific to HCV core protein in mice infected with recombinant adenovirus.

Claims (8)

It is prepared by introducing the core of the hepatitis C virus (HCV) structural protein of the nucleotide sequence shown in Figure 2, the E1 and E2 genes of the envelope glycoproteins into the E3 gene region of human adenovirus type 5 Human recombinant adenovirus Ad / HCV / E3 (Accession No .: KCTC 0244BP). HCV structural protein gene of nucleotide sequence shown in FIG. 2 in which a translation termination codon is introduced at the C-terminus. A method of preparing HCV E1 and E2 proteins by culturing mammalian cells infected with the human recombinant adenovirus Ad / HCV / E3 according to claim 1 (Accession No .: KCTC 0244BP) in a medium to express the proteins and purifying them from the cells. . The method of claim 3, wherein the mammalian cells are HeLa cells or 293 cells. The method according to claim 3, wherein the GNA-agarose column and the DEAE Fractogel column chromatography are sequentially applied for separation and purification. A vaccine against hepatitis C viral disease containing the human recombinant adenovirus Ad / HCV / E3 (Accession No .: KCTC0244BP) according to claim 1 as an active ingredient. The vaccine of claim 6 which is an oral formulation. The vaccine of claim 7 coated with gelatin.