KR20030054139A - Recombinant expression vector comprising the gD gene of the swine Aujeszky's disease virus and DNA vaccine using the same - Google Patents

Abstract

PURPOSE: A recombinant expression vector comprising the gD gene of swine Aujeszky's disease virus and a DNA vaccine using the same are provided, thereby effectively preventing the infection of swine Aujeszky's disease virus. CONSTITUTION: A recombinant expression vector pMYK/ADV gD(KCCM-10344) comprises the gene encoding a glycoprotein gD isolated from swine Aujeszky's disease virus, wherein the gD gene has the nucleotide sequence of SEQ ID NO: 1. A DNA vaccine for prevention of the infection of swine Aujeszky's disease virus is prepared by infection of bacteria with the recombinant expression vector pMYK/ADV gD(KCCM-10344).

Description

Recombinant expression vector comprising the gD gene of the swine Aujeszky's disease virus and DNA vaccine using the same}

The present invention relates to a recombinant expression vector comprising a gD gene of swine aujeszky's disease virus and a DNA vaccine using the same, and more specifically, to about 1.3 kb isolated from the glycoprotein of swine azesky disease virus. A recombinant expression vector comprising a gD gene and a DNA vaccine capable of increasing the formation of neutralizing antibodies against Ozeski's disease virus and specific / nonspecific cellular immunity.

Ozeski's disease is a type of Herpesviridae alphaherpesvirus (Pseudorabies virus) that infects pigs, cattle, sheep, goats, dogs, cats, mice and most wild animals. It is a contagious disease that affects pigs suffering from stress such as migration and delivery, and has a high mortality rate at a young age, and is economically damaging due to reproductive disorders such as miscarriage and stillbirth. The importance of the prevention of this disease has been highlighted.

Ozeski's disease is a viral disease, so there is no cure at the time of infection and its infectivity is very strong, so it is most desirable to prevent the disease by inoculating a young pig. The most commonly used vaccine is attenuated live virus, which is easy to manufacture and inexpensive to produce. However, in the case of a vaccine using attenuated live virus strains, the attenuated pathogenicity may be revived, or depending on the method of storage and treatment, it may cause a risk of causing fatal damage due to disease.

Therefore, in recent years, many attempts have been made to prepare recombinant plasmid vaccines using peptides or DNA fragments showing immunogenicity rather than vaccines using whole pathogens.

For example, in Korean Patent Application No. 98-25040, a gp50 gene encoding porcine Ozesky's disease important protective antigen was inserted into a recombinant expression vector prepared from human adenovirus type 2, and then the human adenovirus type 2 genomic gene was inserted. Homologous recombination in E. coli and E. coli, which is then transfected into mammalian cells using lipofectin to disclose a gp50 recombinant virus. Thymidine kinase (TK) gene, inserted for expression of porcine interleukin-2 (IL-2) gene under the TK promoter, in addition to deletion of the gl gene and encoding protective antigen against viral pathogens Multivalent vector vaccines with foreign genes inserted were provided.

However, since the vectors and vaccines disclosed in the above patents are not only complicated in manufacturing process but also have low immunity, and also use peptides produced by bacteria as immunogens, they act as foreign antigens and thus are mainly used in the humoral immune system. The disadvantage is that it works locally.

Therefore, in the present invention, the swine Ozesky's disease is inserted into the expression vector of the gD gene of the swine Ozeski's disease virus, which is known to impart a protective ability to the test animal even when the neutralizing antibody suppresses the virus infection and is produced by genetic recombination. Recombinant DNA vaccines for the prevention of viral diseases can be prepared simply without the need to isolate proteins or peptides of interest, and when introduced into cells in the body, the proteins of interest are synthesized in cells to induce humoral immunity as well as cellular immunity. It has been found to increase specific / nonspecific cellular immunity with neutralizing antibody formation, and the present invention has been completed based on this.

Accordingly, an object of the present invention is to provide a recombinant expression vector comprising the gD gene of swine Ozesky's disease virus for disease prevention of swine Ozesky's disease virus.

Another object of the present invention is to provide a DNA vaccine for preventing porcine Ozeski disease produced by introducing the recombinant expression vector into bacteria.

Recombinant expression vector comprising the gD gene of the swine Ozeski disease virus of the present invention for achieving the above object is a recombinant plasmid expression vector comprising a glycoprotein gD gene isolated from swine Aujeszky's disease virus.

The DNA vaccine of the present invention for achieving the above another object is mass produced by infecting the expression vector with bacteria.

1 is a base and amino acid sequence diagram of the swine Ozeski disease virus gD gene.

Figure 2 is a schematic diagram of the cloning process of porcine Ozeski disease virus gD gene.

Figure 3 is a schematic drawing of the pMYK / ADV gD plasmid inserted with the gD gene according to the present invention.

4 is a graph showing serum neutralizing antibody formation in mice immunized with pMYK / ADV gD DNA vaccine according to the present invention.

5 is a photograph showing that the protein derived from the ADV gD gene was expressed on the cell surface when pMYK / ADV gD was introduced into the B6 / wt19 cell line.

6 is a graph showing cell lysis by induction of cytotoxic T lymphocytes of mice immunized with pMYK / ADV gD DNA vaccine according to the present invention.

7 is a graph showing cell lysis by induction of natural killer cells (hereinafter referred to as "NK" cells) of mice immunized with pMYK / ADV gD DNA vaccine according to the present invention.

Looking at the present invention in more detail as follows.

In the present invention, the virus is isolated from a specimen of a pig infected with Ozeski's disease virus, a gD gene, which is one of glycoproteins, is cloned from the DNA of the virus, and a 1.3 kb gD is stored at a multicloning site in the expression vector. Gene was inserted to prepare a recombinant plasmid expression vector.

The genome size of Ozeski's disease virus is about 150 kb of DNA having a double-stranded linear structure, and contains about 20 or more structural proteins, and glycoproteins constitute the envelope. Currently, gI, gII, gIII, gX, gH, gD, gp50, gp53, gp63, and the like are identified as glycoproteins of the Ozeski disease virus genes, and glycoproteins play a very important role when viruses are introduced into target cells. It is also the main point of action for neutralizing antibodies to inhibit the virus's function. In the present invention, even if the neutralizing antibody inhibits virus infection in pig and mouse experiments, the gD gene, which is known to confer defense ability to the test animal even when produced by genetic recombination, was selected as a function point of the DNA vaccine.

In the present invention, the isolated YS strains (Lee Joong-bok, Ahn Soo-hwan, Kim Byung-han, Song Jae-young, Kim Yong-hee, and Seol Dong-seop. Studies on swine Ozeski disease: 1. Isolation and Identification of Causative Agents from Infected Pigs. -103) was isolated from the Ozeski disease virus, referring to the nucleotide sequence of the gD gene such as Petrovsky (Petrovski) from the isolated virus, cloned the gene for the glycoprotein gD of the isolated YS strain in Korea, and sequence Analyzed. Gene cloning and sequencing methods can be performed according to a conventional method, and the nucleotide sequence of the analyzed gD gene is shown in FIG. 1.

The cloned porcine Ozeski disease virus gD gene was inserted between EcoR I and Bam HI of the polycloning site in the pMYK expression vector to prepare a recombinant recombinant plasmid expression vector pMYK / ADV gD of the present invention. It was deposited with the Korea microorganism preservation center with accession number KCCM-10344. Upstream of the polycloning site in the pMYK expression vector is a murine cytomegalovirus early prometer. The pMYK / ADV gD gene recombinant plasmid is shown in FIG. 3. Ori in Figure 3 is E. coli replication origin; MCMV promoters include murine cytomegalovirus early promoter; gD is ADV glycoprotein gD; BGH poly A is a bovine growth hormone polyadenylation signal; Amp ^r is an empicillin resistance gene; Neo ^r is a neomycin resistance gene; And MCS refers to the polycloning site.

The recombinant plasmid pMYK / ADV gD was infected with bacteria, amplified in large quantities and purified, and then inoculated into experimental animals using a gene gun (gene-gun, Bio-Rad), and neutralized against Ozeski disease virus in serum. Formation of the antibody was confirmed. In addition, an increase in specific / nonspecific cellular immunity was confirmed through cell lysis compared to vaccines using conventional peptides.

As such, the DNA vaccine according to the present invention is a vaccine for preventing swine Ozesky's disease viral disease, which is safe because it is not pathogenic, and increases the formation of neutralizing antibody titers and specific / nonspecific cellular immunity. It can be used very effectively as a vaccine for disease viral prevention.

Hereinafter, the present invention will be described in more detail with reference to the following Examples, but the scope of the present invention is not limited to the following Examples.

Example 1 Isolation of Porcine Ozeski Disease Virus DNA

Swine Ozesky's Disease Virus Domestic Isolation Ozesky's disease virus was isolated from YS strain.

DNA extraction of porcine Ozeski disease virus was performed using the Nishmori method (Nishimori T, et al.

Restriction endonuclease analysis of Aujeszky's disease viruses isolated in Japan. Application of Nippon Juigaku Zasshi 1987 Apr; 49 (2): 365-7) in PK-15 cells infected with Ozeski virus YS strain, when 80-90% of cytopathic effect (CPE) was observed The total DNA was extracted from the virus-infected cells and the supernatant at 70,000 × g for 2 hours in ultracentrifuge. They were suspended in TBS (10 mM Tris-HCl, 0.15M NaCl, pH 7.4), and then 0.6% SDS (sodium dodecyl sulfate) and proteinase K (400 μg / ml, BRL.USA) were added at 37 ° C. After 2 hours of incubation, the lysate was first removed with the same amount of phenol / chloroform / isoamylalcohol (25: 24: 1). RNase A (100 μg / ml) and proteinase K (200 μg / ml) were added and treated at 37 ° C. for 1 hour to remove the RNA component, followed by another volume of phenol / chloroform / isoamyl alcohol (25: 24: 1), extracted 1-2 times with 2.5 volume of ethanol and 1/10 volume of sodium acetate for 1 hour at -70 ℃, centrifuged at 12,000 rpm for 10 minutes to precipitate DNA, dried and then 0.1 x TE ( 1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0), stored and used at -20 ° C.

Example 2: Cloning and Sequencing of gD Gene

From the porcine Ozeski disease virus DNA 150 kb isolated in Example 1, the restriction enzyme map for Rice and Ozeski disease virus Rice and Indiana, such as Petrovsky, and the base sequence of the gD gene are referred to. Gene cloning was designed for the glycoprotein gD of the isolated YS strain. The extracted viral DNA was digested with Bam HI (Takara) to clone the seventh largest fragment to make pYSB7. This clone was cut again with Bst XI (Takara) to clone a DNA fragment of about 2 kb to generate pGD (see FIG. 2). Subsequently, pGP50 was treated with KpnI (Takara) to clone 3.1 kb, 1.2 kb, and 0.4 kb segments to generate pK31, pK12, and pK04 subclone, and subjected to sequencing. GP50 shows the molecular weight of 50 kD in gD.

Sequencing of the cloned gD gene was performed using 7-deaza-dGPT sequencing kit (USB) according to the modified dideoxy chain termination method, and the reaction conditions were according to the method proposed by the manufacturer, 8% The sequencing gel was loaded and electrophoresed at 40-50 V / cm for 4-12 hours. After the electrophoresis gel was fixed in 10% methanol, 10% acetic acid for 30-60 minutes, washed with running water for 30 minutes, and then dried at 37 ℃ about 2 hours. The dried gel was adhered to an X-ray film (Kodak) and an intensifying screen, subjected to 18 hours of autoradiography at room temperature, and then developed and the nucleotide sequence was read. DNA sequencing was performed using DNASIS version 7.0 (Hitachi) program.

Example 3: pMYK / ADV gD Expression Vector Preparation

PGP50 in Example 2 was treated with Bam HI (Takara) and EcoR I (Takara), followed by electrophoresis on 0.8% Seakem agarose gel (FMK), and 1.3 kb fragments were cut and purified. This was inserted into pMYK expression vector treated with Bam HI, EcoR I and named as pMYK / ADV gD, which is the DNA vaccine of the present invention. Upstream of the multicloning site, there was an early promoter of the mouse target virus, allowing a large amount of the downstream gD gene to be synthesized intracellularly (see FIG. 3).

Example 4: Confirmation of gD Protein Expression of Recombinant Expression Vector

Whether gD protein is expressed from the pMYK / ADV gD recombinant expression vector was confirmed through the following. In other words, the pMYK / ADV gD recombinant expression vector was ligated into the B6 / wt19 cell line (H- ^2b ) (distributed by Dr. Babiuk of the University of Saskatchewan, Canada) (Lipopctin mediated technique) (Southern, PJ and Berg, P., 1982, J. Mol. Appl. Gen. 1: 327-341). This transformed cell line was lysed with lysis buffer (0.5% Triton X-100, 0.25 M Tris.Cl, pH 7.8), centrifuged at 10,000 xg for 2 minutes, and then the supernatant was 10% SDS-polyacrylamide. After electrophoresis on the gel was transferred to nitrocellulose filter paper (Beoringher Manherim) and Western blot analysis using a monoclonal antibody against ADV gD to detect a protein having a molecular weight of approximately 55 kD.

Example 5: Amplification and Purification of pMYK / ADV gD

In order to secure a large amount of the DNA of the pMYK / ADV gD, it was transformed by introducing into DH5α (Promega) bacteria and purified using a Qiagen maxiprep kit (Qiagen).

Example 6: Confirmation of Neutralizing Antibody Formation of pMYK / ADV gD DNA Vaccine

The vaccine of pMYK / ADV gD was inoculated into experimental animals, and the formation of neutralizing antibodies was confirmed through the following procedure.

As experimental animals, four-week-old female C57BL / 6 mice (Charles Rivers Laboratories), a type of pure chicken, were used and 5 mice per group were donated. The pMYK / ADV gD DNA vaccine was injected into gold-coated microtubes and subcutaneously inoculated at a dose of 1.5 μg per subject using a gene gun (gene-gun, Bio-Rad). Two weeks later, the inoculation was inoculated once more. As the negative control group, only the expression vector containing no Ozeski disease virus gD was used, and as the positive control group, an attenuated vaccine (communication microorganism) currently on the market was used. The attenuated vaccine was inoculated twice into the thigh muscle at a dose of 1/2. At 4 and 6 weeks after the final inoculation, blood was collected from the tail arteries of each animal, and the formation of neutralizing antibodies against the Ozeski disease virus in serum was confirmed by the following method.

Serum was once diluted 10-fold and then further diluted 2-fold on 96 well plates (Falcon). An equal amount of 200 TCID ₅₀ Ozesky's disease virus was added thereto. Incubated at 37 ° C. for 1 hour, 50 μl of Vero cells were added, and then cultured at 37 ° C. for 72 hours. Neutralizing antibody titer was determined as the reciprocal of the maximum dilution ratio that can inhibit the cytopathic effect 100%.

Neutralizing antibody formation in serum of mice immunized with the pMYK / ADV gD vaccine is shown in FIG. In FIG. 4, group A was immunized twice with 1.5 μg of the pMYK plasmid vector and group B was 1.5 μg of the pMYK / ADV gD plasmid vector of the present invention. It is.

Neutralizing antibody titers in serum at 4 weeks after 2 weeks of inoculation of pMYK / ADV gD DNA vaccine were 30 ± 10, which was very similar to 40 ± 10 when attenuated vaccine was inoculated. On the other hand, in the group inoculated with only the expression vector which is a negative control group, neutralizing antibody titers could not be measured at all (see FIG. 4). The neutralizing antibody titers at 6 weeks after the final vaccination of the vaccine were no different than at 4 weeks.

The above results indicate that pMYK / ADV gD stimulates the humoral immune system by injection of DNA without adjuvant with adjuvants to form and increase neutralizing antibodies specific to Ozeski disease virus.

Example 7: Confirmation of Specific Cellular Immune Formation of pMYK / ADV gD DNA Vaccine

In the same manner as in Example 6, two weeks after the final inoculation of the DNA vaccine into the experimental animals, the splenocytes of each individual were isolated and examined to determine whether Ozeski disease virus gD-specific cytotoxic T lymphocytes were induced. Cytotoxicity by cytotoxic T lymphocytes was confirmed by the method of Slezak, S. E. and Horan, P. K., 1989, J. Immunol. Methods 117: 205-214. Target cells were labeled with the fluorescent dye PKH-67, incubated with splenocytes for 6 hours, and then propidium iodide (hereinafter referred to as PI; propidium iodide, Sigma) was added to detect dead cells. The target cell line for cytotoxic T lymphocyte analysis was B6 / wt19 cell line into which pMYK / ADV gD DNA was introduced, and the expression of ADV gD protein on the cell surface was confirmed by immunohistochemistry (see FIG. 5). ). In FIG. 5, A is B6 / wt19 cells before pMYK / ADV gD DNA is introduced and B is a cell into which pMYK / ADV gD DNA is introduced.

This was analyzed using Lysis II program (Beckton Dickinson) of the flow cytometer. Cells that are both positive for PKH-67 and PI are recognized dead among the target cells. The lysis (%) of the target cells by cytotoxic T lymphocytes was calculated by the following analysis by analyzing the PI stained fraction of the target cells labeled with fluorescent dyes.

% Lysis = (P-N) / (100-N)

P: Mortality of Target Cells when Cultured Working Cells and Target Cells Together

N: mortality of target cells when cultured alone

The cell lysis of splenocytes obtained from animals vaccinated with pMYK / ADV gD DNA was 11.6 ± 1.6% when the target cells and the splenocytes were co-cultured at 1:30, and 5.6 ± when only the negative control plasmid was administered. Significant increase over 1.9%. This result indicates that the cytotoxic T lymphocytes specific for gD were induced and increased by inoculation with DNA vaccine (see FIG. 6).

Example 8: Identification of Nonspecific Cellular Immune Formation of pMYK / ADV gD DNA Vaccine

In accordance with recent research reports that nucleic acids increase the immune system nonspecifically, nonspecific cellular immune formation against the pMYK / ADV gD DNA of the present invention has been investigated. The activity of nonspecific cellular immune response was used for NK cells that specifically kill Yac-1 cells as target cells.

Cell lysis against Yac-1 cells was investigated by splenocytes obtained from animals vaccinated with DNA vaccine in the same manner as in Example 6. Cell lysis of splenocytes NK cells in animals that were not administered at all was 7.1 ± 0.3% when the target cells and splenocytes, which are effector cells, were cultured at 1:30, whereas the pMYK / ADV gD DNA vaccine was inoculated. Significantly increased to 21.8 ± 0.8%. On the other hand, even when only plasmids were inoculated, cell lysis was found to be on average 17%, which is presumed to be due to the nucleic acid sequences derived from bacteria contained in the pMYK / ADV gD DNA vaccine of the present invention (see FIG. 7).

The pMYK / ADV gD DNA vaccine according to the present invention was developed for the first time in Korea as a DNA vaccine for preventing swine Ozesky's disease viral disease, and is safe because it is not pathogenic as compared to the conventional attenuated vaccine. It is very easy to manipulate because there is no need to separate peptides. In addition, the DNA vaccine is introduced into cells in the body to synthesize a protein of interest in the cell, thereby inducing humoral immunity as well as cellular immunity. As it increases specific / nonspecific immunity with antibody formation, it can be used very effectively as a vaccine for preventing Ozeki's disease viral when industrialized.

Claims (3)

Recombinant plasmid expression vector pMYK / ADV gD (Accession No. KCCM-10344) comprising a glycoprotein gD gene isolated from Swine aujeszky's disease virus. The recombinant plasmid expression vector pMYK / ADV gD (Accession No. KCCM-10344) according to claim 1, wherein the gD gene has the following nucleotide sequence. A DNA vaccine for the prevention of swine Ozeski disease virus disease, characterized in that the bacterial infection of the expression vector pMYK / ADV gD (Accession Number KCCM-10344) according to claim 1 or 2 in mass production.