KR101479668B1 - Recombinant adenovirus expressing rabies virus gene and vaccine composition for preventing or treating rabies comprising the same - Google Patents

Abstract

The present invention relates to a recombinant adenovirus expressing a rabies virus gene and a vaccine composition for preventing or treating rabies comprising the recombinant adenovirus. The recombinant adenovirus according to the present invention is a recombinant nucleotide sequence encoding the G protein and the N protein of the rabies virus isolated from the domestic, and is effective for expressing G protein and N protein of rabies virus and is suitable for use as a vaccine strain It is suitable for mass production with its ability to replenish. In addition, it has improved safety compared to existing vaccines and can be used for oral administration and has excellent defense against rabies virus during vaccination.

Description

Technical Field [0001] The present invention relates to a recombinant adenovirus expressing a rabies virus gene and a vaccine composition for preventing or treating rabies comprising the recombinant adenovirus,

The present invention relates to a recombinant adenovirus expressing a rabies virus gene and a vaccine composition for preventing or treating rabies comprising the recombinant adenovirus.

Rabies is the most deadly common infectious disease in humans and animals, with more than 55,000 people infected worldwide annually, with more than 29,000 of them occurring in Asia, including China. Rabies are spread by host animals such as bats, red foxes, skunks, raccoons, dogs, wolves, and mongooses. In Korea, after 1993, the propagation by dogs has disappeared and forest types that are propagated by raccoon or badger have appeared .

A more effective, safe, and inexpensive vaccine is needed to control rabies in animals and prevent rabies in humans. Many new vaccines are being developed and tested, including DNA vaccines and other recombinant vaccines. DNA vectors expressing rabies virus G have been shown to stimulate the production of T helper cells and rabies VNA (Xiang et al., 1994). Methods of inducing mouse immunization with these DNA vectors have been used to protect mice and monkeys against subsequent challenge infections of fatal rabies viruses (Xi et al., 1994; Ray et al., 1997; Lodmell et al., 1998 ). However, the induction of an immune response by a DNA vaccine usually takes a long time and the magnitude of the immune response is lower than that of a conventional vaccine (Xiang et al., 1994; Osorio et al., 1999).

In Korea, since 2000, vaccine-rabies glycoprotein (V-RG) vaccine has been spread in Gyeonggi Province and Gangwon Province to prevent rabies spread by wildlife. However, this V-RG bait vaccine is designed to express the rabies virus G protein in live vaccinia virus, so that the problem of infection with vaccinia when contacted with this bait vaccine is due to the Center for Disease Control control and prevention (CDC), two of which have been reported to be a safety issue for humans.

The rabies live vaccine currently in use in Korea is a purified vaccine strain obtained by cloning the ERA (Evelyn-Rokitnicki-Abelseth) strain using various cells and purifying the virus by limiting dilution method. The live vaccine is allowed to inoculate the dog, but it is not allowed to inoculate other animals such as cats and cows, and it is known that the mice are killed when inoculated into the mouse's brain. These rabies live vaccines have been identified as arginine (R), the 333rd amino acid of the G protein, which is a pathogenic part, and can not be guaranteed to be safe when inoculated for oral administration. Inactivated vaccines have been approved in Korea since the 1980s and are inoculated into various animals such as dogs, cats, and cows. However, these vaccines are known to be ineffective in forming antibodies because they are inactivated when inoculated.

The present rabies vaccine has a problem in safety, and there are limitations in the type of inoculation and in the method of inoculation. Therefore, the development of a vaccine for rabies vaccination using a new recombinant virus which can be safely and effectively inoculated to various animals including wild animals need.

The inventors of the present invention have found that recombinant adenovirus expressing G protein and N protein of rabies virus isolated from domestic can be mass produced with high safety and high content, And the present invention has been completed.

An object of the present invention is to provide a recombinant adenovirus expressing a rabies virus gene capable of effectively preventing or treating rabies and a vaccine composition for preventing or treating rabies comprising the same.

In order to achieve the above object, the present invention provides a recombinant adenovirus expressing a rabies virus gene.

In addition, the present invention provides a vaccine composition for preventing or treating rabies comprising the recombinant adenovirus.

The present invention also provides a diagnostic kit for rabies virus comprising recombinant adenovirus or antigen thereof.

In addition, the present invention provides a method for detecting rabies virus, characterized in that rabies virus is detected in infected or infected cells through an antigen-antibody reaction using recombinant adenovirus or antigen thereof.

The present invention also provides a method for preventing or treating a rabies-infectious disease by administering the vaccine composition to mammals other than humans.

The recombinant adenovirus according to the present invention is a recombinant nucleotide sequence encoding the G protein and the N protein of the rabies virus isolated from the domestic, and is effective for expressing G protein and N protein of rabies virus and is suitable for use as a vaccine strain It is suitable for mass production with its ability to replenish. In addition, it has improved safety compared to existing vaccines and can be used for oral administration and has excellent defense against rabies virus during vaccination.

FIG. 1 is a fluorescence photograph of a rabies virus confirmed by a fluorescent antibody test using a rabies-specific antibody.
Fig. 2 shows RABVN1 gene (A), RABVN2 gene (B), RABVG1 gene (C) and RABVG2 gene (D) cloned using primers.
Fig. 3 shows the nucleotide sequences of N and G full-length genes of the rabies virus isolated.
FIG. 4 is a diagram showing the results of PCR amplification of N full length, G full length and G? TMCD genes.
FIG. 5 is a schematic diagram of a process for preparing a recombinant adenovirus containing the gene of the domestic isolated rabies virus (KRVB0910 strain).
FIG. 6 is a graph showing the results of immunoblot assay to determine whether N alleles, G alleles, and G ΔTMCD proteins, which are rabies virus proteins, are expressed in the recombinant adenovirus.
FIG. 7 is a graph showing the results of Western blot analysis for the release of the G-total length and G? TMCD protein in the recombinant adenovirus.
FIG. 8 shows the results of confirming the content of recombinant adenovirus by the Spear & Kaber method.
FIG. 9 is a graph showing the result of confirming recombinant adenovirus particles in an infected cell through an electron microscope.
FIG. 10 is a graph showing the results of observing changes in the body weight of a mouse after inoculation of mice with recombinant adenovirus (G full: G full gene, G (-TMCD): G △ TMCD gene, N full: gene).
FIG. 11 is a graph showing changes in weight (A) and survival rate (B) of mice after inoculation of recombinant adenovirus into muscles or brains of mice and rabies vaccination (IM: muscle inoculation, IC: G (full): G full-length gene, G (-TMCD): G? TMCD gene, N: N full-

The present invention provides a recombinant adenovirus expressing a rabies virus gene.

Hereinafter, the present invention will be described in more detail.

The recombinant adenovirus according to the present invention is a recombinant adenovirus for producing a rabies vaccine, wherein the recombinant adenovirus has a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a nucleotide sequence having at least 95% homology thereto.

The nucleotide sequence of SEQ ID NO: 1 may encode the N protein of the rabies virus and the nucleotide sequence of SEQ ID NO: 2 may encode the G protein of the rabies virus.

In the present invention, homology refers to the degree of identity shared by a polynucleotide or polypeptide sequence, and comparison can be performed using a visual program or a comparison program that is easy to purchase. Commercially available computer programs can calculate homology between two or more sequences as a percentage, and homology (%) can be calculated for adjacent sequences.

In addition, the present invention provides a vaccine composition for preventing or treating rabies, which comprises a recombinant adenovirus into which at least one of the nucleotide sequences of SEQ ID NO: 1 or SEQ ID NO: 2 is introduced.

The recombinant adenovirus is a recombinant nucleotide sequence encoding the G protein and N protein of the rabies virus isolated from the domestic, effectively expressing the G protein and the N protein of the rabies virus, and has a proper capacity for use as a vaccine strain It is suitable for mass production. In addition, it can be used for oral administration because it has improved safety compared to conventional vaccines, and because it has excellent defense against rabies virus during vaccination, it can be useful as a vaccine for preventing or treating rabies.

The vaccine of the present invention further comprises a pharmacologically acceptable carrier or diluent. Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. Such carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carrier include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic ester such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer ' s dextrose, dextrose and sodium chloride, lactic acid treatment linger or fixed oils. The intravenous carrier includes electrolyte supplements, liquids and nutritional supplements, such as those based on Ringer ' s dextrose. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases and the like may be additionally present. Preferred preservatives include formalin, thimerosal, neomycin, polyamicin B and amphotericin B.

In addition, the vaccine composition may further comprise an adjuvant.

The adjuvant includes any absorption-promoting agent which refers to a compound or mixture that promotes an immune response and / or promotes the rate of absorption after inoculation. Acceptable adjuvants include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oils, hydrocarbon emulsions, But not limited to, dinitrophenol and the like. The preferred adjuvant is the METASTIM adjuvant of Fort Dodge Animal Health.

The vaccine may be a vaccine selected from the group consisting of a live vaccine, an attenuated vaccine, and a sadox vaccine.

The dose of the vaccine composition for the purpose of the present invention is preferably 0.01 ml to 1 ml, preferably 0.2 ml to 0.4 ml per 1 kg of body weight.

The vaccine composition of the present invention may be administered through one or more routes selected from the group consisting of muscles, subcutaneous, intraperitoneal, intravenous, oral, dermal, ocular and intracerebral. In the present invention, oral administration is preferred.

Also, the present invention provides a diagnostic kit for a recombinant adenovirus or a rabies virus comprising the same, wherein the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is introduced.

The diagnostic kit may be prepared using a method commonly used in the art.

Such a diagnostic kit includes not only recombinant adenovirus or its antigen into which the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is introduced, but also tools and reagents commonly used in the art used for immunological analysis do. Such tools / reagents include, but are not limited to, suitable carriers, labeling substances capable of producing a detectable signal, solubilizers, cleaning agents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose and combinations thereof.

The present invention also provides a method for detecting rabies virus in an infected or infected cell through an antigen-antibody reaction using recombinant adenovirus or its antigen into which at least one of the nucleotide sequences of SEQ ID NO: 1 or SEQ ID NO: 2 is introduced And a method for detecting rabies virus.

The antigen-antibody reaction can be assayed by immunohistochemical staining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assays, immunodiffusion assays, (Complement Fixation Assay), Fluorescence-activated cell sorter (FACS) and protein chip analysis.

The present invention also provides a method for preventing or treating a rabies-infectious disease by administering the vaccine composition containing the recombinant rabies virus to a mammal other than a human.

The mammal may be at least one selected from the group consisting of bats, red foxes, skunks, raccoons, badgers, dogs, wolves, mongooses, coyotes, ferrets, dogs and cats.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  1. Recombination with rabies virus Adeno  Production of viruses

1.1 Isolation and N, G Genes of Domestic Rabies Virus Cloning

When the 333rd amino acid of rabies virus G protein has arginine (R) or lysine (K) in representative rabies virus strains (CVS, SAD, HEP and ERA strains) It is widely known to cause our company. Based on this, it was attempted to isolate rabies virus from Korean NG108-15 cells and analyze recombinant G and N protein-encoding genes to isolate recombinant adenovirus.

In order to isolate rabies virus for recombinant adenovirus production, brain tissue from cattle infected with rabies virus in Goseong area was made into 10% brain emulsion in 2009, and inoculated into NG108-15 cells grown 80% Blind passage was carried out, and the presence of rabies virus was confirmed by confirming the intracellular specific fluorescence by fluorescence antibody test using a rabbit-specific antibody.

The confirmed rabies virus was named as KRVB0910 strain, and the virus content was confirmed by decidual dilution method and the growth ability of 10 ^3.0 TCID50 / ml was confirmed. To analyze N and G genes of isolated KRVB0910 strain, primers were prepared as shown in Table 1 and PCR was performed.

After amplifying the gene using the above primer, four genes including N1, N2, G1 and G2 were cloned using PGEM-T easy vector.

The results are shown in Fig.

As shown in FIG. 2, the sizes of the cloned genes were 1047 and 472 bp for N1 and N2, respectively, and the sizes of G1 and G2 were 1052 and 711 bp, respectively. As a result of comparing the nucleotide sequence of this gene with the homology of the rabies live vaccine strain (ERA strain) used in Korea, the nucleotide of N gene showed similarity of 86.9% and amino acid of 92.0%, and the nucleotide of G gene was 89.4 %, And amino acid was 96.2% similarity. As a result, KRVB0910 strain in Korea showed a difference of 14-11% in the vaccine strain and nucleic acid, and 3.8-8.0% in the protein. The full nucleotide sequence of the N and G genes of the isolated rabies virus is shown in Fig.

1.2 Recombination with rabies virus Adenovirus  Produce

In order to prepare recombinant adenovirus containing the rabies virus gene, the N gene was amplified by amplifying all three genes of the GΔTMCD gene in which a portion corresponding to the intracellular part of the full-length gene, the G full- Specific primers were prepared and amplified. The primer sequences used are shown in Table 2.

The gene amplification products amplified using the primers shown in Table 2 are shown in Fig.

As shown in FIG. 4, it was confirmed that the N full length gene, G full length gene and G? TMCD gene of rabies virus were effectively amplified.

The amplified three gene products were electrophoresed to elute the gene. Each gene was ligated to the pENTR / D-TOPO cloning vector, which is an adenovirus introduction vector, and then transformed into TOP10 competent cells to form colonies on LB agar plates containing kanamycin (50 μg / μl) . The generated colonies were selected for propagation in LB broth containing kanamycin, and the plasmid DNA was extracted by alkaline lysis method to confirm the gene. The plasmid DNA with the confirmed gene sequence and the adenoviral vector pAd / CMV / V5-DEST gateway vector were subjected to LR recombination reaction with LR Clonase II enzyme. The recombinant adenovirus plasmid containing the rabies virus gene generated through this LR recombination reaction was transformed into TOP10 transformed cells and colonies were formed in LB agar plates containing ampicillin (50 mu g / mu l). The resulting colonies were selected and amplified in LB broth containing ampicillin. The plasmid DNA was extracted by alkaline lysis method to confirm the gene. Each recombinant adenovirus vector containing the rabies virus gene was treated with Pac I and the DNA was purified and purified. One day before the 293A cells were cultured on a 60 mm diameter culture plate and immediately transfected, the existing culture medium was discarded and 1.5 ml of medium containing no antibiotic was added. 250 μl of the medium containing the purified vector and 250 μl of the medium in which the lipofectamine was diluted were mixed and allowed to stand at room temperature for 20 minutes. The mixed solution, which had been allowed to stand at room temperature, was added to 293A cells and cultured in a CO ₂ incubator for 24 hours. Then, the medium supplemented with fetal bovine serum was added and cultured for 3-5 days. The virus showing the cytopathic effect through the above cultivation was selected as a recombinant adenovirus containing the rabies virus gene. A schematic diagram of a process for preparing a recombinant adenovirus containing the gene of rabies virus (KRVB0910 strain) is shown in Fig.

1.3 Manufactured recombinant Adenovirus  Confirmation of protein expression

1.3.1 Immunofluorescent staining

The selected recombinant adenoviruses were confirmed to contain the rabies virus gene for each of the three major strains. Cells were fixed with 80% cold acetone, 293A cells were fixed at -20 ° C for 30 minutes, and then washed with PBS. Thereafter, the recombinant adenovirus containing the N full length gene of rabies virus was infected with a primary antibody specific for rabies virus N protein, and the rabies G antibody and GΔTMCD-containing recombinant adenovirus were incubated with anti-rabbit G protein specific antibody for 1 hour Lt; 0 > C. Then, the mouse IgG FITC conjugate was reacted and then washed and observed with a fluorescence microscope.

The results are shown in Fig.

As shown in FIG. 6, it was confirmed that the N alleles protein, G battle protein, and G ΔTMCD protein, which are rabies virus proteins, were efficiently expressed in the recombinant adenovirus vector through specific fluorescence emission different from 293A cells.

1.3.2 Western blot

In addition, western blotting using a monoclonal antibody specific for the G protein of the G long arm and G? TMCD recombinant adenovirus and rabies virus was performed to further confirm whether the recombinant protein was effectively released.

The results are shown in Fig.

As shown in FIG. 7, the G battle recombinant adenovirus showed a protein of 67 kDa and the G? TMCD recombinant adenovirus showed a protein of 57 kDa, and the recombinant adenovirus prepared therefrom showed the G electric field of rabies virus and G? TMCD It was confirmed that the protein can be effectively released.

Example  2. Recombination Adenovirus Proliferative ability  Confirm

In order to confirm whether the recombinant adenovirus prepared in Example 1 had adequate growth ability to be used as a vaccine, the three recombinant adenoviruses prepared were inoculated into 293A cells and harvested after 5 days, And fusion were repeated and centrifuged to obtain a supernatant. In order to confirm the content of recombinant adenovirus in the supernatant obtained, the harvested virus was diluted by 10 steps and virus content was confirmed by Spear & Kaber method.

The results are shown in Fig.

As shown in Figure 8, N full-length recombinant adenovirus is 10 ^7.7 TCID ₅₀ /0.1ml of content, G full-length recombinant adenovirus is 10 ^8.0 TCID ₅₀ /0.1ml of content, G △ TMCD recombinant adenovirus is 10 ^7.9 TCID ₅₀ / 0.1 ml, indicating that all three recombinant adenoviruses are suitable for large-scale production with adequate growth capacity for vaccine use. Also, it was confirmed that the recombinant adenovirus of the present invention having a high virus content can be used as an oral vaccine requiring a high virus content in order to be infected with one side.

Example  3. Identification of virus particles in infected cells after recombinant virus infection

When the recombinant adenovirus was infected to the cells, in order to confirm that the recombinant virus was expressed in the infected cells, the virus particles were observed under an electron microscope after infecting the three kinds of adenoviruses recombined in 293A cells. Three recombinant adenoviruses were inoculated on 293A cells for 3 groups and harvested 4 days later. The harvested cells were harvested by centrifugation at 1500 rpm for 10 minutes. The cells were fixed with 2.5% glutaraldehyde at 4 ° C for 2 hours and fixed again with 1% osmium tetroxide. The fixed cells were made into an ultrathin section, stained with uranyl acetate and lead citrate, and observed under an electron microscope.

The results are shown in Fig.

As shown in Fig. 9, the shape of icosahedrons of typical adenoviruses in the cells infected with three kinds of recombinant adenoviruses could be confirmed by an electron microscope.

Example  4. Recombination Adenovirus  safety

To confirm the safety of three recombinant adenoviruses containing the rabies virus gene, changes in body weight before and after virus infection were measured using Balb / C mice at 4 weeks of age. (GΔTMCD, GΔTMCD + N total length, G total length, G total length + N total length) using three kinds of recombinant adenoviruses having a virus content of 10 ^7.50 TCID ₅₀ / ml or more. The safety was confirmed by inoculating 0.2 ml into the muscles of 4-week-old mice.

The results are shown in Fig.

As shown in Fig. 10, after the inoculation of the virus, the body weight of the mice was slightly decreased in the inoculation group of G battle + N battle recombinant adenovirus, but the normal body weight was increased after 7 days. Weight gain was normal. As a result, it was confirmed that the recombinant adenovirus containing the rabies virus gene of the present invention is a virus that is not toxic and considered to be safe. This safety is believed to be due to the fact that the recombinant adenovirus of the invention is infected once in the body and there is no secondary proliferation.

Example  5. Recombination Adenovirus  Check efficacy

(GΔTMCD, GΔTMCD + N total length, G total length, G total length + N total length) using three kinds of recombinant adenoviruses having a virus content of 10 ^7.50 TCID ₅₀ / ml or more. 0.0 > ml < / RTI > On day 21 after inoculation, rabbit virus (25 LD ₅₀ /0.03 ml) was inoculated intramuscularly or intracerebrally to determine the weight change and survival rate of the mice.

The results are shown in Fig.

As shown in Fig. 11, mice inoculated with four types of GΔTMCD, GΔTMCD + N, G, and G total lengths + N were found to have survival rates of 100% in the experimental group inoculated with mouse pathogenic rabies virus by muscle However, mice were 100% dead when they were inoculated into the control group with the river pathogenic rabies virus.

However, when mice were inoculated with four combinations of GΔTMCD, GΔTMCD + N, G, and G battlefields + N battlefields, when inoculation of the intracerebral rabies virus into the brain was inoculated with recombinant adenovirus of G battle + N battle The survival rate of mice with 100% survival rate was 100%, but the survival rate of GΔTMCD, GΔTMCD + N and G battle recombinant adenoviruses was 40%, 20% and 0%, respectively. The survival rate was 0% when the virus was inoculated intracerebrally into the control group. This indicates that the combination of the most effective rabies virus vaccine at the time of inoculation into the brain is a combination of recombinant adenoviruses of G battle + N battle.

GΔTMCD, GΔTMCD + N The mice were inoculated with recombinant adenoviruses of four types, total length G, total length G, and total length G. The mice were inoculated with an intense, pathogenic rabies virus, The result was that they were constantly growing without being affected by pathogenic viruses. However, when inoculated in the brain, the recombinant adenovirus inoculation group of G battle + N battle group increased in weight without dropping from 7th day, unlike the other inoculation groups, while the rest of GΔTMCD and GΔTMCD + The body weight decreased until day and showed a tendency to rise. On the other hand, control mice inoculated with intense, pathogenic rabies virus in the muscle and brain were continuously lost weight until they died.

As a result, the recombinant adenovirus in which the G gene and the N full length gene were introduced among the four recombinant adenoviruses has the highest safety not only in muscle inoculation but also in the brain, Virus combination.

<110> REPUBLIC OF KOREA (Management: Ministry for Food, Agriculture, Forestry and Fisheries.Animal, Plant and Fisheries Quarantine and Inspection Agency (QIA)) <120> Recombinant adenovirus expressing rabies virus gene and vaccine          composition for preventing or treating rabies comprising the same <130> 1-27 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1353 <212> DNA <213> Rabies virus <400> 1 atggatgccg acaagattgt atttaaagtc aataaccagg tggtctcctt gaagcctgag 60 atcatcgtgg atcaatatga atacaagtac cctgcgatca aggacttgaa aaagcccagt 120 attaccctag ggaaagcccc cgatttgaat aaggcatata agtcagttct atcaggtttg 180 aatgctgcca agcttgaccc tgatgatgtg tgttcctact tagcagccgc aatgcagttc 240 tttgagggga catgtcctga agattggacc agctatggaa tcctgattgc aagagaagga 300 gacaagatca ccccggattc tctcgtggag ataaaacgta ctgatgtaga agggaattgg 360 gctttgactg gaggaatgga actgacaagg gaccccactg tccccgagca tgcgtctcta 420 gtcggtcttc tcttgagtct gtataggctg agcaaaatat cggggcaaaa caccggtaac 480 tataagacaa acattgcaga taggatagag caaattttcg agacagcccc ttttattaaa 540 atcgtggaac atcacactct gatgacgact cacaagatgt gtgccaattg gagtactata 600 cccaatttca gattcttggc tgggacctac gacatgtttt tctcccggat tgagcattta 660 tactcagcga tcagagtggg cacggtagtc actgcttatg aggattgctc tgggctggta 720 tcgtttactg ggttcataaa acagataaat ctcaccgcaa gagaagcaat actatatttc 780 ttccacaaaa acttcgagga agagataaga agaatgtttg agccagggca ggagacagct 840 gttcctcatt cttatttcat tcacttccgt tcactgggcc tgagtgggaa gtccccttat 900 tcgtcaaatg cagttggtca tgtgttcaat ctcattcact ttgtcggatg ttatatgggg 960 caagtaaggt ctctgaatgc aacagtcatt gctgcatgtg ctccccatga gatgtctgtt 1020 ctagggggct atctgggaga ggagttcttc gggaaaggaa cgttcgagag aagattcttc 1080 agagatgaga aagaacttca agagtatgag acggctgaat tgacaaagac tgacgtggcg 1140 ctggcagatg acggaacggt caactcggat gacgaggact gtttttctgg tgaaaccaga 1200 agccccgaag ctgtttatgc ccgaatcatg atgaatggag gtcgactaaa gagatcgcac 1260 atacggagat atgtttcagt cagctccaat caccaagctc gtccgaactc atttgccgag 1320 tttctaaaca agacatactc tagtgactca taa 1353 <210> 2 <211> 1575 <212> DNA <213> Rabies virus <400> 2 atggttcctc aggctctcct gtttgtacct cttctggttt tttcaatgtg tttcgggaaa 60 ttccccatct acacgatacc agacaaactt ggtccttgga gcccaattga catacatcat 120 ctcagctgtc caaacaactt ggttgtagag gatgaaggat gcaccaactt gtcaggtttt 180 tcctacatgg aacttaaagt gggatacatt tcggccataa aagtgaacgg gttcacgtgc 240 acaggtgtgg tgacagaggc agaaacttat actaactttg ttggttatgt caccactacg 300 ttcaaaagaa agcatttccg cccgacaccg gatgcatgtc gggccgctta caactggaaa 360 atggccggtg accccagata tgaagaatct ctgcacaatc cttaccctga ctaccactgg 420 cttcgaactg tcaaaaccac gaaggaatcc ctggtcatca tatccccaag tgtagcggac 480 ctggacccat acgataaatc ccttcactct agagtctttc ctaacggaaa gtgctcagga 540 ataacgatat cctctaccta ctgccctacc aaccattatt acactatctg gctccctgag 600 aatccaagac aggagacatc ttgtgacatc cttaccaata gtagaggaa gagagcatcc 660 aaagggagca agacttgcgg gtttgttgat gaaagaggtt tgtataagtc attgaaaggg 720 gcatgcaaac ttaagttgtg tggggtcctt ggacttagac ttatggatgg aacgtgggtc 780 gcaatgcaag cgtcggacga gaccaaatgg tgccccccag atcagctggt aaatctacac 840 gactttcgct cggacgagat cgaacatctc gttgtggagg agttggtcaa gaagagagaa 900 gagtgtctgg atgcactaga gtccatcatg actactaagt cagtgagttt cagacgtctc 960 agccacttga gaaagcttgt ccctgggttc ggaaaagcat acaccatatt caacaaaacc 1020 ttgatggaag ctgatgctca ctacaagtca gtccgaacct ggaatgagat catcccctca 1080 aaagggtgtt tgagagtcgg ggggaggtgt catcctcatg taaacggggt gtttttcaac 1140 gt; cagcaacata tggagttgtt ggagtcctca gttatccccc tgacgcaccc tttagcagac 1260 ccgtctacag ttttcaagga tggtgatgaa gcagagaatt ttgtggaggt tcaccttcct 1320 gatgtacaca aacagatctc gggagttgac ctgggtctcc ctaactgggg gaagtatgtt 1380 ttagtaagtg caggggtcct gatcgccttg atgttgataa tttttctaat gacatgttgc 1440 ggaagagtca atcgaccaaa gtccacacaa cacaatcttg gcgggacagg gagggaggtg 1500 tcagtaactc cccaaagcgg aaaggtgata tcttcatggg agtcatacaa aagcggaggt 1560 gagaccagac tgtga 1575

Claims (13)

A recombinant adenovirus for the manufacture of a rabies vaccine, wherein the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is introduced.
The recombinant adenovirus for rabies vaccine production according to claim 1, wherein the nucleotide sequence of SEQ ID NO: 1 is a nucleotide sequence encoding the N protein of the rabies virus.
2. The recombinant adenovirus for rabies vaccine production according to claim 1, wherein the nucleotide sequence of SEQ ID NO: 2 is a nucleotide sequence encoding G protein of rabies virus.
A recombinant adenovirus into which the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 is introduced.
5. The vaccine composition according to claim 4, wherein the vaccine composition further comprises at least one member selected from the group consisting of a pharmacologically acceptable carrier, diluent, and adjuvant.
6. The composition of claim 5 wherein the adjuvant is selected from the group consisting of Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, mineral gel, surfactant, pluron polyol, polyanion, peptide, oil, hydrocarbon emulsion, And dinitrophenol, wherein the vaccine composition is at least one selected from the group consisting of:
The vaccine composition according to claim 4, wherein the vaccine is a vaccine selected from the group consisting of a live vaccine, an attenuated vaccine, and a sadox vaccine.
5. The vaccine composition according to claim 4, wherein the vaccine composition is administered through one or more routes selected from the group consisting of muscle, subcutaneous, intraperitoneal, intravenous, oral, dermal, Composition.
A diagnostic kit for recombinant adenovirus or rabies virus comprising the same, wherein the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is introduced.
Characterized in that the rabies virus is detected in the infected or infected cells through the antigen-antibody reaction using the recombinant adenovirus or its antigen in which the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is introduced. A method for detecting rabies virus.
11. The method of claim 10, wherein the antigen-antibody reaction is selected from the group consisting of tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assay, immunodiffusion assay Characterized in that the method is carried out using at least one method selected from the group consisting of Assay, Complement Fixation Assay, Fluorescence-activated cell sorter (FACS) and protein chip assay. Detection method.
A method for preventing or treating a rabies-infectious disease by administering the vaccine composition of claim 4 to a mammal other than a human.
13. The rabies virus according to claim 12, wherein the mammal is at least one selected from the group consisting of a bat, a red fox, a skunk, a raccoon, a badger, a dog, a wolf, a mongoose, a coyote, a ferret, A method for preventing or treating an infectious disease.

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