Replication-restricted recombinant feline herpesvirus and methods of construction and use thereof

Abstract

This invention relates to a replication-restricted recombinant feline herpesvirus, its construction method, and its applications, encompassing the fields of genetic engineering and preventive veterinary medicine. The recombinant feline herpesvirus expresses the exogenous feline parvovirus VP2 protein while simultaneously lacking the gL gene from feline herpesvirus. The construction method is as follows: 1. A QD-1 gL gene stably expressed F81 cell line is constructed using a lentiviral packaging three-plasmid system; 2. A PUC19-gL-LR-eGFP FPV VP2 recombinant plasmid is constructed; 3. The gL gene sgRNA expression vector lentiCRISPRv2-gL-sgRNA is constructed; 4. The recombinant vectors from steps 2 and 3 are transfected into the F81-gL cell line to obtain the QD-1 ΔgL-FPV VP2 recombinant feline herpesvirus. The recombinant feline herpesvirus of this invention can be used as a candidate vaccine strain to resist feline herpesvirus and feline parvovirus infection.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and preventive veterinary medicine, and in particular to a replication-restricted recombinant feline herpesvirus, its construction method, and its applications. Background Technology

[0002] Feline herpesvirus (FHV) is a common respiratory pathogen in cats. It is highly contagious and mainly causes diseases of the upper respiratory tract (nasal cavity, sinuses, pharynx, trachea) and eyes in cats. It can remain latent in the cat's body for life.

[0003] Feline parvovirus is a common gastrointestinal pathogen in cats, possessing extremely strong environmental resistance, which is one of the most frightening characteristics of this virus. It is resistant to most commonly used disinfectants (such as alcohol and quaternary ammonium salts).

[0004] These two antigens severely damage the health of felines. Vaccination is the most crucial and effective means of prevention. Summary of the Invention

[0005] The purpose of this invention is to provide a replication-restricted recombinant feline herpesvirus, its construction method, and its application to resist feline herpesvirus and feline parvovirus infection.

[0006] This invention provides a replication-restricted recombinant feline herpesvirus that expresses exogenous feline parvovirus VP2 protein and lacks the gL gene of feline herpesvirus.

[0007] This invention provides a method for constructing the above-mentioned replication-restricted recombinant feline herpesvirus, comprising the following steps:

[0008] I. Constructing a stable F81 cell line expressing the QD-1 gL gene using a lentiviral packaging three-plasmid system;

[0009] II. The recombinant plasmid PUC19-gL-LR-eGFP FPV VP2 was constructed using homologous recombination and PCR.

[0010] III. Constructing the sgRNA expression vector lentiCRISPRv2-gL-sgRNA for the gL gene using CRISPR gene editing method;

[0011] 4. PUC19-gL-LR-eGFP FPV VP2 and lentiCRISPRv2-gL-sgRNA were transfected into the F81-gL cell line to obtain QD-1 △gL-FPV VP2 recombinant feline herpesvirus.

[0012] Furthermore, the gL gene sequence expressed by the F81 cell line in step one is shown in SEQ ID NO: 1 in the sequence listing.

[0013] Furthermore, the VP2 sequence inserted into the PUC19-gL-LR-eGFP FPV VP2 recombinant plasmid in step two is shown as SEQ ID NO: 2 in the sequence listing.

[0014] Furthermore, the sgRNA primers used in step three to construct lentiCRISPRv2-gL-sgRNA are: sgRNA-F: 5'-CACCGAGCAGATTGTAACCCACCGG-3', sgRNA-R: 5'-AAACCCGGTGGGTTACAATCTGCTC-3'.

[0015] This invention provides the application of the above-mentioned recombinant feline herpesvirus in the preparation of vaccines against feline herpesvirus and feline parvovirus.

[0016] This invention provides a vaccine that simultaneously prevents and treats feline herpesvirus and feline parvovirus, comprising the aforementioned replication-restricted recombinant feline herpesvirus.

[0017] The beneficial effects of this invention are:

[0018] This invention utilizes CRISPR / Cas9 and homologous recombination technology to directionally knock out the gL gene of feline herpesvirus WT QD-1 strain and insert the feline parvovirus (FPV) VP2 gene, constructing a recombinant virus QD-1△gL-FPV VP2 using feline herpesvirus as a vector, capable of simultaneously expressing the feline parvovirus structural protein VP2. In vitro experiments verified that QD-1△gL-FPV VP2 exhibits good genetic stability, stably expressing the VP2 protein even after 20 passages in the defined cell line F81-gL, without a decrease in viral titer. Immunization of cats with QD-1△gL-FPV VP2 did not result in any clinical symptoms, and QD-1△gL-FPV VP2 viral nucleic acid testing was negative. Neutralizing antibodies against FPV reached 2. 10 The above describes the neutralizing antibodies against FHV in 2... 3 -2 5 Between. The recombinant virus QD-1 △gL-FPV VP2 can be used as a candidate strain for vaccine preparation to resist feline herpesvirus and feline parvovirus infection.

[0019] The most significant characteristic of the replication-restricted recombinant feline herpesvirus constructed in this invention is its restricted viral replication. This recombinant feline herpesvirus grows only in F81-gL cells and does not grow or multiply in cells other than F81-gL cells, thus it cannot replicate in animals. Therefore, unlike existing recombinant feline herpesviruses with the gL gene knocked out, even in the presence of exogenous viruses (multiple viral co-infections), the replication-restricted recombinant feline herpesvirus constructed in this invention will not acquire the gL gene of the exogenous virus. The exogenous virus and the recombinant feline herpesvirus of this invention do not share common growth characteristics, therefore, the recombinant feline herpesvirus of this invention cannot acquire regenerative capabilities. Attached Figure Description

[0020] Figure 1 Here is the structure diagram of the pLVX-gL-IRES-mCherry recombinant plasmid;

[0021] Figure 2 Western blot was used to test the stable expression of gL protein in cell lines;

[0022] Figure 3 A schematic diagram illustrating the construction strategy of QD-1 △gL-FPV VP2 recombinant herpesvirus;

[0023] Figure 4 The structure diagram of the pcDNA3.1 eGFP-FPV VP2 plasmid is shown.

[0024] Figure 5 The green fluorescence staining of the fluorescent recombinant virus QD-1 △gL-eGFP FPV VP2 in F81 and F81-gL cells as it was continuously passaged to the P3 generation.

[0025] Figure 6 PCR identification results of gene carrying status of recombinant virus QD-1 △gL-FPV VP2;

[0026] Figure 7 The expression of VP2 protein in recombinant virus QD-1 △gL-FPV VP2 was detected by IFA.

[0027] Figure 8 To test the virus shedding status after QD-1 △gL-FPV VP2 inoculation. Detailed Implementation

[0028] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0029] Example 1: Construction and identification of the F81-gL cell line

[0030] A stable QD-1 gL gene expression F81 cell line was constructed using a lentiviral packaging three-plasmid system. The pLVX-gL-IRES-mCherry recombinant plasmid was synthesized and provided by Nanjing Genscript Biotech Co., Ltd., and its structure is shown below. Figure 1 As shown, the gL gene has an additional 6His codon; the PMD2G and PSPAX2 backbone plasmids are commercially available plasmids. The gL gene sequence inserted into the pLVX-gL-IRES-mCherry recombinant plasmid is shown in SEQ ID NO: 1 in the sequence listing. The gL gene sequence (SEQ ID NO: 1) in this embodiment is an artificial sequence optimized from the original feline herpesvirus gL gene without altering the amino acid sequence of the gL protein encoded by the original feline herpesvirus gL gene.

[0031] 293T cells were cultured in T75 flasks to approximately 80% confluency for transfection. The transfection procedure was as follows: 10 μg of each of pLVX-gL-IRES-mCherry, PMD2G, and PSPAX2 plasmids were added to 100 μL of DMEM and mixed thoroughly to obtain plasmid dilution I; 60 μL of PEI (2 μg / μl) transfection reagent was added to 100 μL of DMEM and mixed thoroughly to obtain PEI dilution I. PEI dilution I and plasmid dilution I were thoroughly mixed and incubated for 25 min to obtain plasmid / PEI complex I. Plasmid / PEI complex I was then uniformly added into the cells. The cells were then transferred to a 37°C, 5% CO2 incubator for culture. 48 h after transfection, the supernatant of the culture medium was collected, and the cells were replaced with fresh complete culture medium. 72 h later, the supernatant of the culture medium was collected again. The two supernatants were mixed and then mixed with fresh complete culture medium at a volume ratio of 1:1. F81 cells with a density of about 50% were infected. 24 h later, the cells were sorted by flow cytometry based on red fluorescence. When the red fluorescence positivity rate reached more than 95% in two consecutive sortings, the expression of the gL gene was detected by Western blot using anti-His antibody.

[0032] Western blot analysis of the stable expression of gL protein in cell lines, such as... Figure 2 As shown in the figure, Western blot analysis revealed the expected protein blot size, between 15 kDa and 25 kDa, indicating that F81-gL cells can stably express gL protein.

[0033] Example 2: Construction of the plasmid with the homologous arm of PUC19-gL-LR-eGFP FPV VP2

[0034] (a) The construction method of pcDNA3.1 eGFP-FPV VP2 plasmid is as follows (plasmid structure as shown in the figure). Figure 4 (as shown)

[0035] PCR amplification was performed using primers listed in Table 1. The PCR system consisted of: 10×PCR Buffer (5 μL), 25 mM MgSO4 (4 μL), 2 mM dNTPs (5 μL), 1.5 μL each of forward and reverse primers, cDNA template (2 μL), KOD high-fidelity enzyme (1 μL), and sterile water (5 μL). The PCR reaction program was: 98℃ denaturation for 10 s, 55℃ annealing for 30 s, and 68℃ extension for 1 min / Kb, for 30 cycles; 68℃ extension for 10 min; and storage at 4℃. The cDNA templates were pEGFP-N1 plasmid and PVAX1-FPV-VP2, respectively. Both PCR amplification products were subjected to agarose gel electrophoresis.

[0036] The commercially available plasmid pcDNA3.1(+) was double-digested with EcoRI and XbaI restriction endonucleases. The digestion system was (50 μL): 10×Buffer (5 μL), 2 μg of plasmid, 2 μL each of EcoRI and XbaI restriction endonucleases, and sterile water was added to 50 μL.

[0037] Two amplified DNA fragments were obtained by agarose gel electrophoresis, gel extraction, and purification. These fragments were then ligated to a linearized pcDNA3.1(+) plasmid after double enzyme digestion. Each fragment had a 20 bp homologous arm. The ligation system consisted of 2×CE mix (5 μL), 50 ng of each DNA fragment, and sterile water to a final volume of 10 μL. After 15 min at 50°C, the ligation product was obtained and transformed into competent JM109 cells to obtain positive clones. The pcDNA3.1 eGFP-FPV VP2 recombinant plasmid was extracted using a plasmid extraction kit and sequenced. The inserted VP2 sequence is shown in SEQ ID NO: 2 in the sequence listing; the VP2 sequence (SEQ ID NO: 2) is an artificially sequenced codon-optimized sequence.

[0038] Table 1 Primers used to prepare pcDNA3.1 eGFP-FPV VP2 plasmid

[0039]

[0040] (II) Construction of the plasmid with homologous arm of PUC19-gL-LR-eGFP FPV VP2

[0041] PCR amplification was performed using primers listed in Table 2. The PCR system consisted of: 10×PCR Buffer (5 μL), 25 mM MgSO4 (4 μL), 2 mM dNTPs (5 μL), 1.5 μL each of forward and reverse primers, cDNA template (2 μL), KOD high-fidelity enzyme (1 μL), and sterile water (5 μL). The PCR reaction program was: 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 68℃ extension for 2 min, 30 cycles; 68℃ extension for 10 min; and storage at 4℃. The corresponding cDNA templates for each primer are shown in Table 2. The four PCR amplification products were subjected to agarose gel electrophoresis.

[0042] Four amplified DNA fragments were obtained by agarose gel electrophoresis, gel extraction, and purification. Each fragment had a 20 bp homologous arm between its ends. The four amplified DNA fragments were ligated using the following ligation system: 2×CE mix (5 μL), 50 ng of each DNA fragment, and sterile water to a final volume of 10 μL. After 15 min at 50°C, the ligation products were obtained and transformed into competent JM109 cells to obtain positive clones. The recombinant plasmid PUC19-gL-LR-eGFP FPV VP2 was extracted using a plasmid extraction kit and sequenced.

[0043] Table 2. Primers used to prepare the PUC19-gL-LR-eGFP FPV VP2 homologous arm plasmid

[0044]

[0045] Example 3: Construction of lentiCRISPRv2-gL-sgRNA

[0046] The gL gene sequence was input into the online sgRNA design website (https: / / zlab.squarespace.com / guide-design-resources). A high-scoring sgRNA sequence (e.g., SEQ ID NO: 19) was selected: AGCAGATTGTAACCCACCGG. Primers sgRNA-F (e.g., SEQ ID NO: 15): 5'-CACCGAGCAGATTGTAACCCACCGG-3' and sgRNA-R (e.g., SEQ ID NO: 16): 5'-AAACCCGGTGGGTTACAATCTGCTC-3' were designed and diluted to 100 mM respectively. The mixtures were then incubated in equal volumes, denatured at 94℃ for 10 min, and annealed at 55℃ for 10 min. The prepared double-stranded DNA was cloned into the plasmid lentiCRISPR v2 digested with BsmBI. The recombinant plasmid was named lentiCRISPRv2-gL-sgRNA.

[0047] Example 4: Construction and identification of QD-1 △gL-FPV VP2 recombinant herpesvirus

[0048] Diagram of QD-1 △gL-FPV VP2 recombinant herpesvirus construction strategy Figure 3 As shown.

[0049] I. Construction and Identification of QD-1 △gL-eGFP FPV VP2 Recombinant Herpesvirus

[0050] PUC19-gL-LR-eGFP FPV VP2 and lentiCRISPRv2-gL-sgRNA were transfected into the F81-gL cell line (the F81-gL cell line prepared in Example 1). The transfection procedure was as follows: F81-gL cells were seeded into 12-well plates. When the cell density reached approximately 90%, 2 μg each of PUC19-gL-LR-eGFP FPV VP2 and lentiCRISPRv2-gL-sgRNA were placed in 100 μL of DMEM and mixed well to obtain plasmid dilution II. 12 μL of PEI transfection reagent was placed in 100 μL of DMEM and mixed well to obtain PEI dilution II. After standing for 5 min, plasmid dilution II and PEI dilution II were thoroughly mixed and then stood for another 25 min to obtain plasmid / PEI complex II. Finally, plasmid / PEI complex II was uniformly dripped into the cells. After culturing cells in a 37℃ 5% CO2 incubator for 12 h, the cells were infected with feline herpesvirus WT QD-1 strain at a volume ratio of 1:1000 to the cell culture medium. After 48 h, the cells were subjected to a freeze-thaw cycle at -80℃. Following the freeze-thaw cycle, the cells were centrifuged at 1000 rpm for 10 min, and the supernatant virus solution was harvested. 2 μL of this supernatant was transferred to 2 mL of fresh 1% FBSDMEM medium and vortexed thoroughly. The cells were then inoculated with F81-gL cell lines at a density of 90%. After 48 h, the cells were observed under an inverted microscope for the appearance of green fluorescent cytokines (CPE). The feline herpesvirus WT QD-1 strain is disclosed in the literature "Construction and Evaluation of Immunogenic Efficacy of Feline Type I Herpesvirus DNA Vaccine" (Chinese Journal of Preventive Veterinary Medicine, July 2025, Vol. 47, No. 7), specifically the FHV-1 QD-1 strain mentioned in the literature.

[0051] QD-1 △gL-eGFP FPV VP2 fluorescent recombinant virus purification steps: Digest the F81-gL cell line and evenly seed it in a 6-well plate. When the cell density reaches 90% or higher, dilute the fluorescent recombinant virus 10-fold, repeating the process 10 times. 1 10 2 10 3Three dilution gradients were used. 0.8 mL of the diluted virus solution was added to each well, and the culture medium was discarded after adsorption at 37°C for 1 hour. The prepared 2% low-melting-point agarose was placed in a 40-50°C water bath until needed, then mixed with 2×DMEM at a 1:1 ratio and added to each well of the culture plate, 2 mL per well, allowing it to cool and solidify into a capping layer. The culture plate was inverted and incubated in a 37°C CO2 incubator for 12-24 h. During this period, the presence of green fluorescent cytokines (CPEs) was observed under an inverted microscope. Green fluorescent CPEs (along with the agarose) were directly aspirated using a 10 μL pipette tip and placed in 1 mL of fresh culture medium. The plate was shaken thoroughly and subjected to a freeze-thaw cycle at -80°C to infect the F81-gL cell line. After three plaque purifications, the fluorescent recombinant virus QD-1 △gL-eGFP FPV VP2 was harvested. F81 and F81-gL cells were infected with an MOI of 0.01. After 48 h, the green fluorescence lesions were observed under an inverted microscope. P1 cells were harvested by freeze-thaw and passaged to P3. The green fluorescence was observed at each passage.

[0052] The fluorescent recombinant virus QD-1 △gL-eGFP FPV VP2 was serially passaged into F81 and F81-gL cells to the P3 generation, and its replication was observed by green fluorescence. The replication restriction of the recombinant virus QD-1 △gL-eGFP FPV VP2 was as follows: Figure 5 As shown, F81: cat kidney cells; F81-gL: F81 cells stably expressing gL; P1-P3: passages of the virus in continuous cell culture. The results indicate that the gL gene-deleted recombinant virus QD-1 △gL-eGFP FPV VP2 can only express eGFP protein in F81 cells and does not produce cytopathic effects. Figure 5 -A), its culture was re-inoculated into F81 cells, and no eGFP protein expression was observed, and cell growth was normal. Figure 5 -B and C). In F81-gL cells, there is abundant eGFP protein expression and CPE is present ( Figure 5 -D), subcultures showed stable traits ( Figure 5 -E and F).

[0053] II. Construction and Identification of QD-1 △gL- FPV VP2 Recombinant Herpesvirus

[0054] pcDNA3.1-Cre was transfected into the F81-gL cell line. The transfection procedure was as follows: F81-gL cells were seeded into 12-well plates. When the cell density reached approximately 90%, 2 μg of pcDNA3.1-Cre was added to 100 μL of DMEM and mixed well to obtain plasmid dilution III. 6 μL of PEI transfection reagent was added to 100 μL of DMEM and mixed well to obtain PEI dilution III. After standing for 5 min, plasmid dilution III and PEI dilution III were thoroughly mixed and allowed to stand for 25 min to obtain plasmid / PEI complex III. Finally, plasmid / PEI complex III was uniformly added to the cells. After culturing the cells in a 37℃, 5% CO2 incubator for 12 h, they were infected with fluorescent recombinant virus at a 1:1000 ratio, and 2 μL of fluorescent recombinant virus QD-1 ΔgL-eGFP FPV VP2 was inoculated. After 48 h, the cells were frozen and thawed at -80℃, and F81-gL cells were inoculated at a 1:1000 ratio. After 48 h, the cells were observed under an inverted microscope to see if CPE without green fluorescence appeared.

[0055] QD-1 △gL-FPV VP2 recombinant virus purification steps: Digest and evenly seed F81-gL cell lines into 6-well culture plates. When the cell density reaches 90% or higher, dilute the fluorescent recombinant virus 10-fold, repeating the process 10 times. 1 10 2 10 3Three dilution gradients were used. 0.8 mL of the diluted virus solution was added to each well, and the culture medium was discarded after adsorption at 37°C for 1 hour. The prepared 2% low-melting-point agarose was placed in a 40-50°C water bath and then mixed with 2×DMEM at a 1:1 ratio. 2 mL of this mixture was added to each well of the culture plate, allowing it to cool and solidify into a capping layer. The culture plate was inverted and incubated in a 37°C CO2 incubator for 12-24 h. During this period, the presence of non-green fluorescent phagocytic plaques (CPEs) was observed under an inverted microscope. A 10 μL pipette tip was used to directly aspirate the non-green fluorescent CPEs (along with the agarose) into 1 mL of fresh culture medium, shaken thoroughly, and then freeze-thawed once at -80°C to infect the F81-gL cell line. After plaque purification three times, the non-fluorescent recombinant virus P1 was harvested. The non-fluorescent recombinant virus was passaged to generation P20, and its gL and VP2 genes were detected by PCR. VP2 protein expression was detected by indirect immunofluorescence assay (IFA). IFA procedure: When the density of F81-gL and normal F81 cells reaches 90%, fix the cells with methanol pre-cooled at -20℃ for 30 min, wash three times with PBST; block with 5% skim milk for 1 h, wash three times with PBST at 50 rpm; incubate with anti-VP2 protein mouse monoclonal antibody (1:5000) for 1 h, wash three times with PBST at 50 rpm; add DyLight 488 labeled goat anti-mouse IgG secondary antibody (1:500) and incubate for 1 h, wash three times with PBST at 50 rpm, and observe for green fluorescence under a fluorescence inverted microscope.

[0056] PCR detection was performed on the gL and VP2 genes of the recombinant viruses QD-1 △gL-FPV VP2 from generations P5, P10, P15, and P20. Figure 6 As shown, M: DL2000 DNA Marker, P5-P20: viral passages, +: recombinant plasmid containing the VP2 gene, QD-1: wild-type virus, -: negative control. Nucleic acid gel imaging results showed that the VP2 gene was detected in the recombinant viruses of passages P5, P10, P15, and P20. Figure 6 A) indicates that the VP2 gene was successfully inserted into the QD-1 genome, and the gL gene was not detected in the P5, P10, P15, and P20 generation recombinant viruses. Figure 6 B) indicates that the gL gene was successfully deleted.

[0057] The expression of VP2 protein was detected by IFA of the P10 and P20 generation recombinant viruses QD-1 ΔgL-FPV VP2. Figure 7 As shown in the figure. The results showed that the VP2 protein was still successfully expressed after the virus was passaged to the P20 generation, indicating that the recombinant virus QD-1 △gL-FPVVP2 has good stability.

[0058] Example 5: Assessment of neutralizing antibody levels following immunization with QD-1△gL-FPV VP2 recombinant feline herpesvirus

[0059] Based on different immunization doses (10gL-FPV VP2) of QD-1 △gL-FPV VP2 7.5 TCID 50 / mL, 10 6.5 TCID 50 / mL, 10 5.5 TCID 50 Three immunization groups and one negative control group were set up. The immunization method was intramuscular injection, and the immunization volume was 1 mL / animal. The second immunization was performed 21 days after the first immunization. On the 7th day after the first immunization, conjunctival swabs were collected, nucleic acid was extracted, and the gD gene was amplified by PCR (primer F (e.g., SEQ ID NO: 17): 5'-ATGATGACACGTCTACATTTTTGG-3', primer R (e.g., SEQ ID NO: 18): 5'-TTAAGGATGGTGAGTTGTATGTAT-3') to detect the replication and shedding of QD-1 ΔgL-FPV VP2 in the body. Blood was collected after each immunization, and serum was separated for neutralization test to detect the level of neutralizing antibodies. Neutralization test procedure: The separated serum sample was heat-inactivated at 56℃ for 30 min, and the treated cat serum was serially diluted 2-fold (2) using serum-free DMEM medium. 3 -2 12 ), and feline herpesvirus WT QD-1 strain or FPV (200 TCID) 50 The cat serum dilution / virus was mixed in equal volumes (100 μL / well), and neutralized at 37°C for 1 h. Then, 100 μL of the cat serum dilution / virus was seeded into each well of a 96-well plate containing 80% confluence of normal F81 cells. Four replicates were set up for each dilution. Virus adsorption was performed at 37°C for 1 h. The culture supernatant was discarded, and the cells were washed twice with PBS. Fresh DMEM medium (1% FBS) was added to each well, and the cells were incubated at 37°C for 48 h. CPE was observed after this incubation period. CPE caused by FPV infection was not significant; therefore, the level of neutralizing antibodies against FPV was detected using indirect immunofluorescence assay (IFA). If CPE appeared in one or more of the four replicates, the serum dilution was deemed incapable of completely neutralizing the virus. If no CPE appeared in any of the four replicates, the serum dilution was deemed capable of completely neutralizing the virus.

[0060] QD-1 △gL-FPV VP2 according to different immunization doses (10 7.5 TCID 50 / mL, 10 6.5 TCID 50 / mL, 10 5.5 TCID50 ( / mL) Immunization of Chinese domestic cats. Results of virus shedding tests after QD-1 △gL-FPV VP2 inoculation are as follows: Figure 8 As shown, M: DNA marker, +: feline herpesvirus WT QD-1 strain, -: negative control. On day 7 after the first immunization, conjunctival swabs were collected, and the gD gene of QD-1 △gL-FPV VP2 was not detected, indicating no viral shedding. Table 3 shows that after two immunizations, the neutralizing antibody level against FHV in cats was 2... 3 -2 5 The results were comparable to those reported in similar studies both domestically and internationally; the level of neutralizing antibodies against FPV was between 2... 5 -2 10 The results above all meet the criteria for positive FHV and FPV antibodies.

[0061] Table 3. Antibody levels in cats after immunization with different groups and doses of the virus.

[0062]

Claims

1. A replication-restricted recombinant feline herpesvirus, characterized in that, The virus expresses the exogenous feline parvovirus VP2 protein and lacks the gL gene from feline herpesvirus. The method for constructing the replication-restricted recombinant feline herpesvirus includes the following steps: I. Constructing a stable F81 cell line expressing the QD-1 gL gene using a lentiviral packaging three-plasmid system; II. The recombinant plasmid PUC19-gL-LR-eGFP FPV VP2 was constructed using homologous recombination and PCR. III. Constructing the sgRNA expression vector lentiCRISPRv2-gL-sgRNA for the gL gene using CRISPR gene editing method; IV. PUC19-gL-LR-eGFP FPV VP2 and lentiCRISPRv2-gL-sgRNA were transfected into the F81-gL cell line to obtain QD-1 ΔgL-FPV VP2 recombinant feline herpesvirus; The gL gene sequence expressed by the F81 cell line in step one is shown in SEQ ID NO: 1 in the sequence listing; In step two, the VP2 sequence inserted into the PUC19-gL-LR-eGFP FPV VP2 recombinant plasmid is shown as SEQ ID NO: 2 in the sequence listing; In step three, the sgRNA primers used to construct lentiCRISPRv2-gL-sgRNA are: sgRNA-F: 5'-CACCGAGCAGATTGTAACCCACCGG-3', sgRNA-R: 5'-AAACCCGGTGGGTTACAATCTGCTC-3'.

2. The use of the replication-restricted recombinant feline herpesvirus as described in claim 1 in the preparation of vaccines against feline herpesvirus and feline parvovirus.

3. A vaccine that simultaneously prevents and controls feline herpesvirus and feline parvovirus, characterized in that, The vaccine comprises the replication-restricted recombinant feline herpesvirus as described in claim 1.

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