A replication-defective recombinant rabies virus chimerically expressing feline herpesvirus type I proteins and uses thereof

Abstract

This invention discloses a replication-deficient recombinant rabies virus chimerically expressing feline herpesvirus type I protein and its applications. It belongs to the field of biomedical technology. The purpose of this invention is to provide a genetically engineered vaccine for feline infectious rhinotracheitis. This invention provides a replication-deficient recombinant rabies virus chimerically expressing feline herpesvirus type I protein, using plasmid combination 1 or plasmid combination 2 to rescue rabies virus strains; plasmid combination 1 is shown below: pD-N, pD-P, pD-G, pD-L, and pD-SRV9-△G-gB; plasmid combination 2 is shown below: pD-N, pD-P, pD-G, pD-L, and pD-SRV9-△G-gD. The goal is to develop a safe and effective novel vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a replication-deficient recombinant rabies virus that chimerically expresses feline herpesvirus type I protein and its applications. Background Technology

[0002] Feline viral rhinotracheitis (FVR) is an acute upper respiratory tract infection caused by feline herpesvirus type 1 (FHV-1). After infection, the virus forms a latent infection in the trigeminal ganglion and carries the virus for a long period. Approximately 50-75% of confirmed feline viral upper respiratory tract infections are caused by FHV-1 infection. Vaccination is the most effective way to prevent feline herpesvirus infection. Currently available inactivated FHV-1 vaccines have no risk of viral transmission or virulence reversion and offer higher safety, but require multiple doses and have a short duration of immunity. Live attenuated vaccines, on the other hand, pose safety risks such as virulence reversion. Therefore, developing a safe and effective new vaccine is crucial for the prevention and control of feline viral rhinotracheitis. Summary of the Invention

[0003] The purpose of this invention is to provide a genetically engineered vaccine for feline infectious rhinotracheitis.

[0004] This invention provides a replication-deficient recombinant rabies virus chimerically expressing feline herpesvirus type I protein, and a rabies virus strain rescued using plasmid combination 1 or plasmid combination 2; plasmid combination 1 is shown below: pD-N, pD-P, pD-G, pD-L, and pD-SRV9-△G-gB; plasmid combination 2 is shown below: pD-N, pD-P, pD-G, pD-L, and pD-SRV9-△G-gD.

[0005] Further, the plasmid combination was transfected into BSR-G cells, and the cell supernatant was used to rescue the replication-deficient recombinant rabies virus.

[0006] Further specifying, the N gene, P gene, G gene, and L gene are derived from SRV9 strain; the N gene is shown in SEQ ID NO.5, the P gene in SEQ ID NO.7, the G gene in SEQ ID NO.9, and the L gene in SEQ ID NO.11.

[0007] Further specifying, the gB gene is as shown in SEQ ID NO.1.

[0008] Further specifying, the gD gene is as shown in SEQ ID NO.3.

[0009] To further define it, pD-SRV9-△G-gB is a sequence in which the P and M genes in pD-SRV9-△G-eGFP are inserted, with the sequences PE, PS, Kozak, and gB linked together in sequence.

[0010] To further define it, pD-SRV9-△G-gD is a sequence in which the P and M genes in pD-SRV9-△G-eGFP are inserted, with the sequences PE, PS, Kozak, and gD linked together in sequence.

[0011] Further specified, the PE nucleotide sequence is as shown in SEQ ID NO.13, the PS nucleotide sequence is as shown in SEQ ID NO.14, and the Kozak nucleotide sequence is as shown in SEQ ID NO.15.

[0012] This invention provides the application of the above-mentioned replication-defective recombinant rabies virus in the preparation of vaccines or drugs for the prevention or treatment of feline viral rhinotracheitis.

[0013] This invention provides the application of the above-mentioned replication-defective recombinant rabies virus in the preparation of rabies vaccines or drugs for prevention or treatment.

[0014] Beneficial effects: This invention utilizes a rabies virus reverse genetics system to construct a replication-deficient recombinant RABV vector vaccine expressing FHV-1 gB / gD protein. The overall titers of the recombinant viruses rSRV-ΔG-gB and rSRV-ΔG-gD are lower than those of the parent virus SRV9, but their growth trends are similar. The recombinant viruses rSRV9-ΔG-gB and rSRV9-ΔG-gD can successfully express FHV-1 gB / gD protein in BSR cells and localize it on the cell membrane. The FHV-1 gene can still be detected in the recombinant viruses rSRV9-ΔG-gB and rSRV9-ΔG-gD after 14 generations of passage, and they can express FHV-1 gB / gD protein normally after cell infection, exhibiting good genetic stability. One week after booster immunization with the recombinant viruses rSRV9-ΔG-gB and rSRV9-ΔG-gD, FHV-1 neutralizing antibodies can be detected in mouse serum, and the highest neutralizing antibody titer can reach 1:32 three weeks after booster immunization. Attached Figure Description

[0015] Figure 1 The recombinant plasmid in Example 1 pcDNA3.1-SRV9-△G-gB , pcDNA3.1-SRV9-△G-gD Double enzyme digestion identification; M: DL15000 Marker; 1: pcDNA3.1-SRV9-△G-gB 3: pcDNA3.1-SRV9-△G-gD ; Figure 2For the direct immunofluorescence identification of the recombinant virus in Example 2, A: rSRV9-△G-gB; B: rSRV9-△G-gD; C: cell control; Figure 3 Indirect immunofluorescence identification of recombinant virus in Example 2, where A: rSRV9-△G-gB; B: rSRV9-△G-gD; C: cell control; Figure 4 Laser confocal microscopy identification of the recombinant virus in Example 2; Figure 5 For the identification of the growth characteristics of the recombinant virus in Example 2, where A: rSRV9-△G-gB; B: rSRV9-△G-gD; Figure 6 The growth kinetics curve of the recombinant virus in Example 2; Figure 7 For the genetic stability determination of the recombinant virus in Example 2, A: rSRV9-△G-gB genome stability determination, M: DL5000 Marker; 1: P3; 2: P6; 3: P10; 4: P14; B: rSRV9-△G-gD genome stability determination, M: DL5000 Marker; 1: P3; 2: P6; 3: P10; 4: P14; Figure 8 The detection of FHV-1 neutralizing antibodies in mouse serum in Example 3; Figure 9 This is a schematic diagram of gene insertion in a recombinant plasmid. Detailed Implementation

[0016] pD-SRV9-△G-eGFP is described in my master's thesis, "Construction and Experimental Immunological Study of Recombinant Rabies Virus with Replication-Defective G Gene Deletion".

[0017] BSR-G cells were documented in my master's thesis, "Construction and Experimental Immunological Study of Recombinant Rabies Virus with Replication-Defective G Gene Deletion".

[0018] Implementation Case 1. Expressing FHV-1 gB / gD Construction and identification of full-length recombinant RABV plasmids with replication-defective proteins 1. Primer design and synthesis Primers were designed based on the published FHV-1 genome sequence in GenBank (Genebank No. MT813047.1) for amplification. gB , gD Genes. Primers were synthesized by Changchun Sangon Biotech Co., Ltd., and primer sequence information is shown in Table 1.

[0019] Table 1 Primer sequence information

[0020] Note: Underlined areas indicate restriction enzyme sites. 2. Construction and identification of a full-length recombinant replication-deficient RABV plasmid expressing FHV-1 gB / gD protein. Using the primer pairs gB-F / gB-R and gD-F / gD-R in Table 1, respectively, with the contents of gB Recombinant plasmids containing genes gD Using a recombinant plasmid as a template, the gene was amplified by PCR. gB、gD The gene and PCR product were identified and purified by 1% agarose gel electrophoresis and stored at -20℃ for later use.

[0021] The target fragment amplified by PCR gB , gD Genes and recombinant plasmids pD-SRV9-△G-eGFP The products of double enzyme digestion were ligated to obtain a recombinant plasmid, which was named pD-SRV9-ΔG-gB, pD-SRV9-ΔG-gD . ( Figure 1 The specific operating steps are as follows: Use seamless cloning technology to amplify the PCR-obtained... gB、gD Gene fragments were ligated into recombinant plasmids. pcDNA3.1-SRV9- △G The ligation product was transformed into DH5α competent cells, and the plasmid was extracted from the harvested bacterial culture. The plasmid was then identified by BsiwI / PmeI double enzyme digestion, and the results are as follows: Figure 2 As shown, the two recombinant plasmids obtained exhibited target bands at 2844 bp and 1122 bp, respectively. Sequencing analysis confirmed that the results were consistent with the target gene sequence, and the correctly sequenced recombinant plasmid was named... pD-SRV9-ΔG-gB, pD-SRV9-ΔG-gD.

[0022] Implementation Case 2: Rescue and Identification of Recombinant Viruses Expressing FHV-1 gB / gD Proteins 1. Rescue of Recombinant Viruses Two recombinant full-length plasmids were respectively pD-SRV9-ΔG-gB, pDNA-SRV9-ΔG-gD With helper plasmids expressing RABV structural proteins pD-N , pD-P , pD-G , pD-L Co-transfect BSR-G cells.

[0023] helper plasmids pD-N , pD-P , pD-G , pD-L The backbone is pcDNA3.1, which connects to the N gene, P gene, G gene and L gene respectively.

[0024] The specific method is as follows: Approximately 12 hours before transfection, BSR-G cells were passaged into 6-well plates. Transfection was performed when the cells reached approximately 80% confluence. The recombinant full-length plasmid (2.5 μg) was added... pD-SRV9-△G-gB or pDNA-SRV9-△G-gD )and pD-N (0.625μg) pD-P (0.3125μg) pDNA3.1-G (0.1875μg) pD-L Add 3.75 μg (0.125 μg) of the mixture to 250 μL of opti-MEM and mix thoroughly. Add 7.5 μL of Lipofectamine 2000 to another 250 μL opti-MEM. Let both solutions stand at room temperature for 5 min, then mix and incubate at room temperature for another 15 min. Add 500 μL of the plasmid-liposome mixture to the cell culture medium and incubate at 37°C and 5% CO2 for 6 h. Replace the cell supernatant with DMEM medium containing 4% FBS. Collect the cell supernatant on days 3, 5, 7, and 9 post-transfection, and use direct immunofluorescence to assess virus rescue.

[0025] 2. Direct immunofluorescence assay (IFA) BSR-G cells were passaged into 96-well plates at a rate of 100 μL / well, with 100 μL of the viral sample (supernatant from transfected cells) added to each well simultaneously. After incubation at 37°C and 5% CO2 for 48 h, the supernatant was discarded, and the cells were fixed with 80% cold acetone. After washing with PBST, 40 μL of FITC-labeled anti-RABV N protein monoclonal antibody (1:500) was added to each well, and the plates were incubated at 37°C for 1 h. The plates were washed three times with PBST, and the fluorescence results were observed using a fluorescence microscope. Results are as follows: Figure 3 As shown, the expression of RABV N protein (green fluorescence) was detected in BSR-G cells infected with both recombinant RABV strains, indicating that the two recombinant RABV strains were successfully rescued and named rSRV9-△G-gB and rSRV9-△G-gD, respectively.

[0026] 3. Expression and identification of exogenous genes from recombinant viruses Indirect immunofluorescence assay (IFA): BSR-G cells were passaged into 96-well plates at a rate of 100 μL / well. 50 μL of the viral sample to be tested was added to each well simultaneously. The plates were incubated at 37°C and 5% CO2 for 48 h, after which the supernatant was discarded. Cells were fixed with 80% cold acetone. After washing with PBST, 40 μL of PBST-diluted primary antibody (rabbit anti-FHV-gB protein monoclonal antibody, cat anti-FHV-gD protein polyclonal antibody) was added to each well, and the plates were incubated at 37°C for 1 h. After washing three times with PBST, 40 μL of PBST-diluted secondary antibody (FITC goat anti-rabbit IgG antibody, FITC goat anti-cat IgG antibody) was added to each well, and the plates were incubated at 37°C in the dark for 1 h. After washing three times with PBST, the fluorescence results were observed using a fluorescence microscope. Results are as follows: Figure 4 As shown, the expression of gB and gD proteins (green fluorescence) could be detected in BSR-G cells infected with the four recombinant RABV strains, indicating that the exogenous proteins of the two recombinant RABV strains could be normally expressed in BSR-G cells.

[0027] Laser scanning confocal microscopy (LSCM): LSCM was used to observe the expression and localization of exogenous genes in BSR cells after infection with recombinant virus. BSR cells were divided into groups of 2 × 10⁻⁶ cells. 4 Pass the recombinant viruses rSRV9-ΔG-gB and rSRV9-ΔG-gD, and the parent virus SRV9, into 24-well plates at a density of 500 μL / well. Dilute the recombinant viruses rSRV9-ΔG-gB and rSRV9 and the parent virus SRV9 to a concentration of 10⁻⁶. 4 TCID 50 / 50μL(50%Tissue culture infectious dose, TCID 50100 μL / well was simultaneously seeded into 24-well plates (Multiplicity of infection (MOI) = 1) and incubated at 37°C and 5% CO2 for 48 h. The supernatant was discarded, and 500 μL of 4% paraformaldehyde was added to each well for fixation at room temperature for 30 min. The plates were then washed once with phosphate-buffered solution (PBS), and 300 μL of 2% Triton-X-100-PBS was added to each well, incubating for 5 min at room temperature. The plates were then washed three times with PBS, 5 min each time. 500 μL of PBS containing 1% BSA was added to each well for blocking, and the plates were blocked for 30 min at room temperature. The supernatant was carefully discarded, and 50 μL of primary antibody diluted in blocking buffer (mouse anti-RABV G protein monoclonal antibody, rabbit anti-FHV-gB protein monoclonal antibody; mouse anti-RABV) was added to each well. G protein monoclonal antibody, feline anti-FHV-gD protein polyclonal antibody), incubated at 37℃ for 1 h; discard the supernatant, wash 3 times with PBS, 5 min each time; discard the supernatant, add 50 μL of secondary antibody diluted with blocking buffer (FITC goat anti-rabbit IgG antibody, TRITC goat anti-mouse IgG antibody; FITC goat anti-cat IgG antibody, TRITC goat anti-mouse IgG antibody) to each well, incubate at 37℃ in the dark for 1 h. Discard the supernatant, wash 3 times with PBS, 5 min each time; discard the supernatant, add 1 drop of anti-quenching mounting medium containing DAPI to the center of the slide, and observe the results using a laser confocal microscope. Results are as follows. Figure 5 As shown, after infection with recombinant RABV, only the expression of exogenous proteins was detected in the cell membrane and cytoplasm of BSR cells, while only the expression of RABV G protein was detected in the cell membrane and cytoplasm of BSR cells infected with the parental RABV9. This confirms that after infection of BSR cells with the two recombinant RABV strains, exogenous proteins can be successfully expressed and localized on the cell membrane.

[0028] 4. Growth characteristics of recombinant viruses To identify the in vitro growth kinetics of the recombinant virus, BSR cells and BSR-G cells were infected with the recombinant virus at an MOI of 0.1, and the proliferation of the recombinant virus in different cell types was observed using DFA. The results are as follows: Figure 6 The results show that the recombinant virus rSRV9-△G-gB 、 Following infection of BSR-G cells with rSRV9-ΔG-gD, the green fluorescence gradually increases over time. However, after infection of BSR cells with the recombinant virus, the green fluorescence does not increase over time.

[0029] 5. Growth curve of recombinant virus To detect RABV GThe effects of gene deletion and exogenous gene expression on the growth kinetics of recombinant viruses were investigated. BSR-G cells were infected with rSRV9-ΔG-gB, rSRV9-ΔG-gD, and the parental SRV9 virus at an MOI of 0.1. Cell culture supernatant was collected every 24 hours for viral titer determination. Results are as follows: Figure 7 The results showed that, compared to the parent virus SRV9, the recombinant virus had an overall lower viral titer, but its growth trend was basically the same as that of the parent virus SRV9, indicating that RABV... G Gene deletion and foreign gene insertion have no significant effect on viral replication.

[0030] 6. Genetic stability of recombinant viruses Using the correctly identified recombinant virus as the seed, it was passaged continuously for 14 generations in BSR-G cells. Genomic RNA of the recombinant virus was extracted from generations F3, F6, F10, and F14 and identified by exogenous gene PCR. The results are as follows: Figure 8 The results showed that the PCR identification of the exogenous gene was positive, and the sequencing results were consistent with the expectations, indicating that the genomes of the recombinant viruses rSRV9-△G-gB and rSRV9-△G-gD are stable and the exogenous gene will not be lost during viral passage.

[0031] Implementation Case 3: Immunogenicity Evaluation of Recombinant Virus Expressing FHV-1 gB / gD Protein (1) Mouse grouping To evaluate the immunogenicity of recombinant viruses rSRV9-△G-gB and rSRV9-△G-gD, 15 female BALB / c mice aged 6-8 weeks were randomly divided into 3 groups of 5 mice each. The mice in the three groups were immunized by intramuscular injection (IM) on day 0 and day 21, respectively. The specific grouping of the mice is shown in Table 2.

[0032] Table 2. Mouse immunization grouping and protocol

[0033] (2) Detection of neutralizing antibodies after mouse immunization Blood samples were collected from the orbital rims of mice at weeks 2, 4, 6, 8, and 10 post-immunization, and serum was separated for the detection of FHV-1 neutralizing antibodies. The specific method was as follows: the serum samples were inactivated at 56°C for 30 min and then serially diluted 2-fold with serum-free MEM medium, with two replicates for each sample; FHV-1 was diluted to 10 TCID using serum-free MEM medium. 5050 μL. Add the diluted virus to a 96-well cell plate containing serum (50 μL / well). Incubate the 96-well cell plate in a 5% CO2, 37°C incubator for 1 h. Include virus control, positive serum control, and cell control wells. Add F81 cell suspension to the 96-well cell plate (100 μL / well) and incubate in a 5% CO2, 37°C incubator for 3 days. After 3 days, observe cytopathic effects under a microscope and calculate the FHV-1 neutralizing antibody titer in the serum.

[0034] like Figure 9 As shown, no FHV-1 neutralizing antibodies were detected in any of the three groups of mice after the initial immunization. Three weeks after the booster immunization, the highest levels of FHV-1 neutralizing antibodies were observed in the recombinant virus rSRV9-△G-gB and recombinant virus rSRV9-△G-gD groups, both with a level of 2. 4.5 No FHV-1 neutralizing antibody was detected in the PBS group.

[0035] 1. FHV-1 gB protein gene sequence, 1605bp (SEQ ID NO.1): 2. FHV-1 gB protein amino acids, 948 aa (SEQ ID NO.2): MSTRGDLGKRRRGSRWQGHSGYFRQRCFFPSLLGIAATGSRHGNGSSGLTRLARYVSFIWIVLFLVGPRPVEGQSGSTSEQPRRTVATPEVGGTPPKPTTDPTDMSDMREALRASQIEANGPSTFYMCPPPSGSTVVRLEPPRACPDYKLGKNFTEGIAVIFKENIAPYKFKANIYYKNIIMTTVWSGSSYAVTTNRYTDRVPVKVQEITDLIDRRGMCLSKADYVRNNYQFTAFDRDEDPRELPLKPSKFNTPESRGWHTTNETYTKIGAAGFHHSGTSVNCIVEEVDARSVYPYDSFAISTGDVIHMSPFFGLRDGAHVEHTSYSSDRFQQIEGYYPIDLDTRLQLGAPVSRNFLETPHVTVAWNWTPKSGRVCTLAKWREIDEMLRDEYQGSYRFTAKTISATFISNTSQFEINRIRLGDCATKEAAEAIDRIYKSKYSKTHIQTGTLETYLARGGFLIAFRPMISNELAKLYINELARSNRTVDLSALLNPSGETVQRTRRSVPSNQHHRSRRSTIEGGIETVNNASLLKTTSSVEFAMLQFAYDYIQAHVNEMLSRIATAWCTLQNREHVLWTETLKLNPGGVVSMALERRVSARLLGDAVAVTQCVNISSGHVYIQNSMRVTGSSTTCYSRPLVSFRALNDSEYIEGQLGENNELLVERKLIEPCTVNNKRYFKFGADYVYFEDYAYVRKVPLSEIELISAYVDLNLTLLEDREFLPLEVYTRAELEDTGLLDYSEIQRRNQLHALKFYDIDSIVRVDNNLVIMRGMANFFQGLGDVGAGFGKVVLGAASAVISTVSGVSSFLNNPFGALAVGLLILAGIVAAFLAYRYISRLRANPMKALYPVTTRNLKQTAKSPASTAGGDSDPGVDDFDEEKLMQAREMIKYMSLVSAMEQQEHKAMKKNKGPAILTSHLTNMALRRRGPKYQRLNNLDSGDDTETNLV; 3. FHV-1 gD protein gene sequence, 1122 bp (SEQ ID NO.3): 4. Amino acids of FHV-1 gD protein, 374 aa (SEQ ID NO.4): MMTRLHFWWCGIFAVLKYLVCTSSLTTTPKTTTVYVKGFNIPPLRYNYTQARIVPKIPQAMDPKITAEVRYVTSMDSCGMVALISEPDIDATIRTIQLSQKKTYNATISWFKVTQGCEYPMFLMDMRLCDPKREFGICALRSPSYWLEPLTKYMFLTDDELGLIMMAPAQFNQGQYRRVITIDGSMFYTDFMVQLSPTPCWFAKPDRYEEILHEWCRNVKTIGLDGARDYHYYWVPYNPQPHHKAVLLYWYRTHGREPPVRFQEAIRYDRPAIPSGSEDSKRSNDSRGESSGPNWIDIENYTPKNNVPIIISDDDVPTAPPKGMNNQSVVIPAIVLSCLIIALILGVIYYILRVKRSRSTAYQQLPIIHTTHHP; 5. Gene sequence of RABV N protein, 1341 bp (SEQ ID NO.5): 6. Amino acid sequence of RABV N protein, 402 aa (SEQ ID NO.6): MDADKIVFKVNNQVVSLKPEIIVDQYEYKYPAIKDLKKPCITLGKAPDLNKAYKSVLSGMSAAKLNPDDVCSYLAAAMQFFEGTCPEDWTSYGIVIARKGDKITPGSLVEIKRTDVEGNWALTGGMELTRDPLSLSMRPMTTHKMCANWSTIPNFRFLAGTYDMFFSRIEHLYSAIRVGTVVTAYEDCSGLVSFTGFIKQINLTAREAILYFFHKNFEEEIRRMFEPGQETAVPHSYFIHFRSLGLSGKSPYSSNAVGHVFNLIHFVGCYMGQVRSLNATVIAACAPHEMSVLGGYLGEEFFGKGTFERRFFRDEKELQEYEAAELTKTDVALADDGTVNSDDEDYFSGETRSPEAVYTRIMMNGGRLKRSHIRRYVSVSSNHQARPNSFAEFLNKTYSSDS; 7. RABV P protein gene sequence, 895 bp (SEQ ID NO.7): CATGAGCAAGATCTTTGTCAATCCTAGTGCTATTAGAGCCGGTCTGGCCGATCTTGAGATGGCTGAAGAAACTGTTGATCTGATCAATAGAAATATCGAAGACAATCAGGCTCATCTCCAAGGGGAACCCATAGAGGTGGACAATCTCCCTGAGGATATGGGGCGACTTCACCTGGATGATGGAAAATCGCCCAACCATGGTGAGATAGCCAAGGTGGGAGAAGGCAAGTATCGAGAGGACTTTCAGATGGATGAAGGAGAGGATCCTAGCTTCCTGTTCCAGTCATACCTGGAAAATGTTGGAGTCCAAATAGTCAGACAAATGAGGTCAGGAGAGAGATTTCTCAAGATATGGTCACAGACCGTAGAAGAGATTATATCCTATGTCGCGGTCAACTTTCCCAACCCTCCAGGAAAGTCTTCAGAGGATAAATCAACCCAGACTACTGGCCGAGAGCTCAAGAAGGAGACAACACCCACTCCTTCTCAGAGAGAAAGCCAATCATCGAAAGCCAGGATGGCGGCTCAAATTGCTTCTGGCCCTCCAGCCCTTGAATGGTCGGCTACCAATGAAGAGGATGATCTATCAGTGGAGGCTGAGATCGCTCACCAGATTGCAGAAAGTTTCTCCAAAAAATATAAGTTTCCCTCTCGATCCTCAGGGATACTCTTGTATAATTTTGAGCAATTGAAAATGAACCTTGATGATATAGTTAAAGAGGCAAAAAATGTACCAGGTGTGACCCGTTTAGCCCATGACGGGTCCAAACTCCCCCTAAGATGTGTACTGGGATGGGTCGCTTTGGCCAACTCTAAGAAATTCCAGTTGTTAGTCGAATCCGACAAGCTGAGTAAAATCATGCAAGATGACTTGAATCGCTATACATCTTGCTAA; 8. Amino acid sequence of RABV P protein, 297 aa (SEQ ID NO. 8): MSKIFVNPSAIRAGLADLEMAEETVDLINRNIEDNQAHLQGEPIEVDNLPEDMGRLHLDDGKSPNHGEIAKVGEGKYREDFQMDEGEDPSFLFQSYLENVGVQIVRQMRSGERFLKIWSQTVEEIISYVAVNFPNPPGKSSEDKSTQTTGRELKKETTPTPSQRESQSSKARMAAQIASGPPALEWSATNEEDDLSVEAEIAHQIAESFSKKYKFPSRSSGILLYNFEQLKMNLDDIVKEAKNVPGVTRLAHDGSKLPLRCVLGWVALANSKKFQLLVESDKLSKIMQDDLNRYTSC; 9. RABV G protein gene sequence, 1573 bp (SEQ ID NO. 9): 10. Amino acid sequence of RABV G protein, 428 aa (SEQ ID NO.10): MSQPRSKESISAQHQMHVEPRTTGRWPVTPDMKSLHNPYPDYRWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPSGKCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVSMQTSNETKWCPPDKLVNLHDFRSDEIEHLVVEELVRKREECLDALESIMATKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVSTWNEVLPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLESSVIPLVHPLADPSTVFKDGDEAEDFVEVHLPDVHNQVSGVDLGLPNWGKYVLLSAGALTALMLIIFLMTCCRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGETRL; 11. RABV L protein gene sequence, 6384 bp (SEQ ID NO.11): 12. RABV L protein amino acid sequence, 2127aa (SEQ ID NO.12): 13.PE:tctcccctcag(SEQ ID NO.13); 14.PS: TGAAAAAAACAGGCAACACCCCT(SEQ ID NO.14); 15. fairy tale: GCCGCCACC.

Claims

1. A replication-deficient recombinant rabies virus chimeric expressing feline herpesvirus type I protein, characterized in that, Rabies virus strains rescued using plasmid combination 1 or plasmid combination 2; plasmid combination 1 is shown below: pD-N, pD-P, pD-G, pD-L and pD-SRV9-△G-gB; plasmid combination 2 is shown below: pD-N, pD-P, pD-G, pD-L and pD-SRV9-△G-gD.

2. The replication-defective recombinant rabies virus according to claim 1, characterized in that, The plasmid combination was transfected into BSR-G cells, and the cell supernatant was used to rescue the replication-deficient recombinant rabies virus.

3. The replication-defective recombinant rabies virus according to claim 1, characterized in that, The N, P, G, and L genes were derived from SRV9 strain; the N gene is shown in SEQ ID NO.5, the P gene in SEQ ID NO.7, the G gene in SEQ ID NO.9, and the L gene in SEQ ID NO.

11.

4. The replication-defective recombinant rabies virus according to claim 1, characterized in that, The gB gene is shown in SEQ ID NO.

1.

5. The replication-defective recombinant rabies virus according to claim 1, characterized in that, The gD gene is shown in SEQ ID NO.

3.

6. The replication-defective recombinant rabies virus according to claim 1, characterized in that, pD-SRV9-△G-gB is in pD-SRV9-△G-eGFP The P and M genes were inserted with sequences PE, PS, Kozak, and gB linked together in sequence.

7. The replication-defective recombinant rabies virus according to claim 1, characterized in that, pD-SRV9-△G-gD is in pD-SRV9-△G-eGFP The P and M genes in the sequence are PE, PS, Kozak, and gD linked together in sequence.

8. The replication-defective recombinant rabies virus according to claim 1, characterized in that, The PE nucleotide sequence is shown in SEQ ID NO.13, the PS nucleotide sequence is shown in SEQ ID NO.14, and the Kozak nucleotide sequence is shown in SEQ ID NO.

15.

9. The use of the replication-defective recombinant rabies virus according to any one of claims 1-8 in the preparation of a vaccine or medicament for the prevention or treatment of feline viral rhinotracheitis.

10. The use of the replication-defective recombinant rabies virus according to any one of claims 1-8 in the preparation of a vaccine or drug for the prevention or treatment of rabies.

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