Construction, identification and application of a gI / gE / TK triple gene deletion vaccine strain of pseudorabies virus

Abstract

This study describes the construction, identification, and application of a pseudorabies virus (PRV) triple-gene deletion vaccine strain (gI / gE / TK). Based on the JS-2012-ΔgI / gE double-gene deletion strain, the TK virulence gene was further deleted using CRISPR technology. Its pathogenicity was evaluated in KM mice and piglets, and its immunoprotective efficacy was verified in piglets. This provides theoretical support for the clinical prevention and control of pseudorabies; establishes experimental models in mice and pigs to study the immunoprotective efficacy of the triple-gene deletion strain; and prepares for the subsequent development and evaluation of vaccines using PRV strains as vectors.

Description

Technical Field

[0001] This invention belongs to the field of bioimmunotechnology, specifically relating to a variant strain of porcine pseudorabies virus with the gI / gE / TK / UL21 gene deletion and its applications. Background Technology

[0002] Pseudorabies is a common infectious disease in livestock and wild animals caused by the pseudorabies virus (PRV), characterized by intense itching, fever, and encephalomyelitis. It has a broad host spectrum, but generally does not infect poultry. The PRV belongs to the Varicellavirus genus of the Herpesvirus A subfamily. Its mature virus particles are large, approximately 150–180 nm in diameter, with a four-layered structure and a G+C content as high as 73%. Except for pigs, PRV infection is fatal in all other animals. PRV can infect various pig herds, primarily piglets and pregnant sows. Piglets mainly exhibit persistent high fever, anorexia, diarrhea, and other neurological symptoms; the mortality rate for suckling piglets infected is 100%. Pregnant sows mainly exhibit abortion, the birth of mummified fetuses, or stillbirth.

[0003] PRV encodes 70-100 genes, among which the TK gene, encoded by UL23, is a ubiquitous gene in α / γ herpesviruses. It catalyzes the phosphorylation of deoxythymidine or pyrimidine to dTP to maintain and promote viral replication, participating in the replication and latent infection of pseudorabies virus in the central nervous system. It is a major virulence gene of PRV. Deletion of the TK gene not only significantly reduces the infectivity, transmissibility, and virulence of PRV, but also allows PRV to maintain good immunogenicity.

[0004] In the 1970s, my country imported gene-deleted vaccine strains, represented by Bartha-K61, from Hungary, and their widespread use effectively controlled the outbreak of porcine pseudorabies. However, in 2011, a mutated PRV strain was discovered in my country. This newly emerging PRV variant exhibits enhanced pathogenicity, and the classic pseudorabies virus gene-deleted vaccine, represented by Bartha-K61, cannot provide complete protection against this strain in immunized pigs. Therefore, developing a novel gene-deleted vaccine targeting the PRV variant is essential for controlling this emerging pseudorabies outbreak.

[0005] To develop a PRV variant vaccine with gene deletion, Tong Wu et al. used homologous recombination to delete the gI / gE gene of a pseudorabies virus variant (JS-2012 strain), successfully developing a porcine pseudorabies live vaccine (JS-2012-△gI / gE strain). Results showed that this vaccine had good safety and efficacy in newborn piglets. However, Cao Yanyun et al. inoculated the vaccine strain (JS-2012-△gI / gE strain) into mice, and the results showed that intranasal administration of 10⁴ TCID₅₀ / mouse to KM mice caused a 40% mortality rate. This indicates that although the porcine pseudorabies live vaccine (JS-2012-△gI / gE strain) has good safety in newborn piglets, it still has strong pathogenicity in KM mice. Many existing vaccines contain multiple gene deletions, but these vaccines still face numerous problems that urgently need to be addressed. The main issues are as follows: While the three-gene deletion strains in CN117625561 A and CN113862230 A also utilize CRISPR / Cas9, the deletion of some genes is performed in two steps. Therefore, these gene knockout strategies are cumbersome and inefficient. Furthermore, for pseudorabies virus, the more gene deletions, the higher the safety, but the lower the immunogenicity. Thus, it is difficult to balance safety and immunogenicity. Although CN114045269 A, CN1028883838A, and CN114657151 A use a one-step method to delete the gI / gE genes of pseudorabies virus to obtain a strain with both gI and gE genes deleted, there are potential safety risks because the starting strain is a wild-type strain. CN113373119 A describes a recombinant virus expressing African swine fever virus CD2v and p72 or p54 and p30 using a vector with the deletion of the TK, gI, and gE genes of pseudorabies virus. Its purpose is to highly express foreign proteins, but at the same time, it severely reduces the virulence of the virus. For pseudorabies virus live vector vaccines, the main method is for the recombinant virus to enter the host and replicate extensively. During the replication process, the inserted foreign genes are efficiently expressed, thereby prompting the body to produce antibodies against foreign proteins and achieving the effect of viral immune protection against foreign genes. The level of viral replication in the body is directly related to the virus's virulence. Viruses with high virulence replicate at high levels, while those with low virulence replicate at severely reduced levels, resulting in insufficient antibody production and affecting vaccine efficacy. More seriously, the vectors or originating strains used in these vaccines are products of foreign research institutions or companies, lacking complete intellectual property rights, which severely impacts and restricts the large-scale production and application of subsequent vaccines. Therefore, developing a live pseudorabies virus vaccine strain with complete intellectual property rights in my country, possessing the aforementioned virulence, replication capacity, and safety, has become an urgent challenge that needs to be overcome. Summary of the Invention

[0006] The purpose of this invention is to provide the construction, identification and application of a pseudorabies virus gI / gE / TK triple gene deletion vaccine strain.

[0007] First, this invention seeks protection for a pseudorabies virus vaccine strain with the gI / gE / TK triple gene deletion, wherein the vaccine strain is pseudorabies virus rPRV JS-2012-△gI / gE / TK, which was deposited on January 21, 2025 at the China General Microbiological Culture Collection Center (CGCCC) with accession number CCTCC No. V202518, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, this invention provides a method for constructing a pseudorabies virus gI / gE / TK triple gene deletion vaccine strain, the method comprising the following steps: 1) Based on the genomic sequence of PRV (JS-2012-△gI / gE strain), two sgRNAs were designed, targeting the 5' and 3' ends of the TK gene, respectively. Following the instructions for constructing the lentiCRISPRv2 plasmid, two double-stranded TK-gRNA DNAs were obtained. The pCRISPR / Cas9 vector was digested with BsmBI, and the vector fragments were recovered via gel electrophoresis. The two double-stranded TK-gRNA DNAs were ligated into the BsmBI-digested and recovered vectors using T4 DNA ligase, respectively. The vectors were then transformed into TOP10 competent cells, and plasmids were extracted to obtain donor plasmids Cas9-TK1 and Cas9-TK2. Double digestion with NotI and BamHI was performed for identification, followed by sequencing analysis.

[0009] Among them, sgRNA-TK-1-FCACCGCATCAGCGCGCGGCGCCTTCG; sgRNA-TK-1-RAAACCGAAGGCCGCCGCGCTGATGC; sgRNA-TK-2-FCACCGGTCTGCGCATCGTAAATAC; sgRNA-TK-2-RAAACGTATTTACGATGCGCAGACC; 2) Extraction of the JS-2012-△gI / gE genome; 3) Co-transfect BHK cells with 1 μg of donor plasmids Cas9-TK1 and Cas9-TK2 and 2 μg of JS-2012-ΔgI / gE viral genome. Observe the cytopathic effect daily after transfection. When approximately 80% of the cells show cytopathic effects, freeze-thaw the transfected cells at -80℃, collect the supernatant, and perform selection and purification.

[0010] Furthermore, this invention provides a monoclonal antibody 3G1 that specifically recognizes pseudorabies virus gI / gE / TK triple-gene deletion vaccine strains. The amino acid sequence of the light chain variable region of 3G1 is: ASLAVVAEKVSISC RSSQSIVYSANQGNY LA WYLQKPGQSPKLAIY WLASTRES GVPDGVPDGSGAGTDFTLTASSVKAEAEDLGVYYC QQYYHAPRT FGSATKLEIK; The amino acid sequence of the heavy chain variable region is as follows: DVKLVSGAGLVVKPGGSLKLSCAVAGFATFSDYPAMSWVRQVRQKRLEAVATISSDGGSYTAYPDSVKGRFISRDNAKATNLYLQMSSLKSEDTAMYACTRDYNSMYAWFAYWAQGTSVTVSA.

[0011] Furthermore, the present invention provides an epidemiological survey of the above-mentioned antibody in porcine pseudorabies virus. The monoclonal antibody 3G1 can effectively distinguish between the common domestic vaccine strain PRV (JS-2012-△gI / gE strain), the epidemic strain, and the pseudorabies virus gI / gE / TK three-gene deletion vaccine strain JS-2012-△gI / gE / TK strain.

[0012] Furthermore, this invention seeks to protect the use of the above-mentioned porcine pseudorabies virus variant strain with the gI / gE / TK gene deletion in the preparation of a porcine pseudorabies virus vaccine.

[0013] Beneficial effects: To improve the safety of JS-2012-△gI / gE, this study used CRISPR technology to further delete the TK virulence gene on the JS-2012-△gI / gE double-gene deletion strain. Its pathogenicity was evaluated in KM mice and piglets, and its immunoprotective efficacy was verified in piglets. The aim is to develop a safer vaccine, providing theoretical support for the eradication and control of pseudorabies in clinical practice; to establish mouse and pig experimental models to study the immunoprotective efficacy of the triple-gene deletion strain; and to prepare for the subsequent development and evaluation of vaccines using PRV strains as vectors. Attached Figure Description

[0014] Figure 1. Construction of donor plasmids. A: Enzyme digestion vector (M: Marker; 1-2: Cas9 digestion vector; 3: lentiCRISPRv2 plasmid) B: Enzyme digestion identification (M: Marker; 1-2: gRNA-Cas9).

[0015] Figure 2. PCR screening of three-gene deletion viruses. A: First round of purification (M: Marker; 1-17: JS-2012-△gI / gE / TK; 18: JS-2012-△gI / gE; 19: Negative control) B: Second round of purification (M: Marker; 1-20: JS-2012-△gI / gE / TK; 21: JS-2012-△gI / gE; 22: Negative control) C: Third round of purification (M: Marker; 1-10: JS-2012-△gI / gE / TK; 11: JS-2012-△gI / gE; 12: Negative control).

[0016] Figure 3 shows the one-step growth curve.

[0017] Figure 4. Morphological observation of etch spots.

[0018] Figure 5. Western blot identification results.

[0019] Figure 6. Results of indirect immunofluorescence assay.

[0020] Figure 7. Sequencing analysis of JS-2012-△gI / gE / TK at generations 1, 5, 10, 15, and 20.

[0021] Figure 8. JS-2012-△gI / gE / TK indirect immunofluorescence assays at generations 1, 5, 10, 15, and 20.

[0022] Figure 9. Survival of mice at different time points after immunization.

[0023] Figure 10. Pathological anatomy of mice after immunization.

[0024] Figure 11. Histopathological observation of mice after immunization.

[0025] Figure 12 shows the results of viral load detection in tissues and organs of immunized mice.

[0026] Figure 13. Changes in body temperature at different times after immunization of piglets.

[0027] Figure 14. Pathological anatomy of immunized piglets.

[0028] Figure 15. Histopathological observation of immunized piglets.

[0029] Figure 16. Dynamic changes of gB antibodies after immunizing piglets.

[0030] Figure 17. Dynamic changes of gE antibodies in immunized piglets.

[0031] Figure 18. Changes in body temperature at different times after viral challenge in immunized piglets. Figure 19. Survival of immunized piglets at different time points after viral challenge. Figure 20 Pathological anatomy of immunized piglets after viral challenge. Figure 21. Histopathological observation of immunized piglets after viral challenge. Figure 22 Results of viral load detection in tissues and organs of immunized piglets after challenge with the virus. Detailed Implementation

[0032] The following examples illustrate specific implementation methods to further describe the content of the present invention in detail. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies that can be implemented in the art based on the above-mentioned content of the present invention should be included in the content of the present invention.

[0033] 1.1 Construction of CRISPR / Cas9 donor plasmid Based on the genome sequence of PRV (JS-2012-△gI / gE strain), sgRNAs were designed using an online CRISPR / sgRNA analysis tool. Two sgRNAs were designed, each targeting a specific target. TK The 5' and 3' regions of the gene are shown in Table 1. Following the instructions for constructing the lentiCRISPRv2 plasmid, the complementary TK-gRNA primers TK-sgRNA-F and TK-sgRNA-R were annealed and phosphorylated using phosphorylase. This was followed by high-temperature inactivation (95 °C, 5 min) of T4 PNK and primer denaturation, and then gradual cooling for annealing to allow primer complementarity and double-strand formation, yielding two double-stranded TK-gRNA DNAs. BsmB I. The pCRISPR / Cas9 vector was digested with enzymes, and the vector fragments were recovered by gel electrophoresis. Two double-stranded TK-gRNA DNA molecules were ligated into the BsmBI-digested and recovered vectors using T4 DNA ligase. The vectors were then transformed into TOP10 competent cells, and plasmids were extracted to obtain donor plasmids Cas9-TK1 and Cas9-TK2. The pCRISPR / Cas9 vector was digested with BsmBI, and the vector fragments were recovered by gel electrophoresis. The results are as follows: Figure 1 As shown in Figure 1-A, the vector size is 12984 bp, which matches the target band size. After ligating sgRNA to the Cas9 vector with T4, the vector was identified by double digestion with NotI and BamHI and then sent to the company for sequencing (the results are shown in Figure 1-B), indicating that the Cas9-sgRNA-TK1 and Cas9-sgRNA-TK2 plasmids were successfully constructed.

[0034] Table 1 Primers Table 1 Primer

[0035] 1.2 Extraction of the JS-2012-ΔgI / gE genome JS-2012-ΔgI / gE strain was inoculated onto a monolayer of BHK cells at a 1 MOI dose. When approximately 90% or more of the cells showed cytopathic effects, the culture medium was discarded, cells were scraped off, resuspended in enzyme-free water, and transferred to 1.5 ml centrifuge tubes. 10% SDS and RNase A were added to the centrifuge tubes, and the mixture was incubated at 37°C for 10 min. Proteinase K was added, and the mixture was incubated at 37°C for 4–6 h, inverting the tube once every one hour. Phenol-chloroform was added and the mixture was inverted repeatedly. The mixture was centrifuged at 10000×g for 10 min, and the supernatant was transferred to a new 1.5 ml centrifuge tube. This process was repeated until the supernatant was clear. The supernatant was then transferred to a new 1.5 ml centrifuge tube, and twice the volume of phenol-chloroform in anhydrous ethanol was added. The mixture was inverted several times and incubated overnight at -20°C. Centrifuge at 12000×g for 10 min, discard the supernatant, add 75% ethanol to disperse the precipitate, centrifuge at 10000×g for 10 min, discard the supernatant, air dry, add enzyme-free water, measure the concentration, and store at -20℃.

[0036] 1.3 Co-transfection of donor plasmid with JS-2012-ΔgI / gE genome Using Lipofectamine® 3000 Transfection Reagent, 1 μg of the donor plasmids Cas9-TK1 and Cas9-TK2 were co-transfected with 2 μg of JS-2012-ΔgI / gE viral genome into BHK cells. Pathogenicity was observed daily after transfection. When approximately 80% of the cells showed cytopathic effects, the cells were freeze-thawed at -80°C, and the supernatant was collected and stored at -80°C for later use.

[0037] 1.4 Screening, Purification and Identification of Tri-Gene Deletion Viruses The supernatant collected in step 1.3 was serially diluted 10-fold with DMEM medium, and 10 μL of each solution was taken. -2 ~10 -6One mL of serially diluted virus solution was inoculated into a monolayer of BHK cells in a six-well cell culture plate and incubated at 37°C with 5% CO2 for 1.5 h. After incubation, the supernatant was discarded, and a mixture of MEM containing 2% FBS and 2% low-melting-point agarose was added to each well. After solidification at room temperature, the plate was incubated at 37°C with 5% CO2. After 48 h, cytopathic effects were observed. Once viral plaques had formed, individual plaques were aspirated and added to 1.5 mL centrifuge tubes, resuspended in 500 μL of DMEM, and stored at -80°C for later use. 200 μL of the viral plaque solution was used to extract the viral genome using the TIANGEN Viral Genomic DNA / RNA Extraction Kit. PCR identification was performed using primers (TK identification-F: TCTGTTCGACACGGACA; TK identification-R: AGCCATGTGTATGTCATCC). Nucleic acid electrophoresis results showed that the three-gene deletion virus correctly deleted the target fragment of the TK gene, with a size of 249 bp (as shown in Figure 2). The PCR product was sent to a sequencing company for sequencing. After three rounds of plaque selection, the purified three-gene deletion virus was named JS-2012-△gI / gE / TK and deposited on January 21, 2025, at the China General Microbiological Culture Collection Center (CGCCC), accession number CCTCC No. V202518, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The PCR-positive virus solution underwent the next round of plaque purification. After three rounds of plaque purification, the biological characteristics and genetic stability of the PCR-selected positive strains were analyzed.

[0038] 1.5 Plotting the one-step growth curve of a three-gene deletion virus The triple-gene deletion virus was diluted 10-fold with 2% DMEM medium to obtain a 10-fold solution. -1 ~10 -11 100 μL per well was added to each of 96-well cell culture plates with a cell density of 70-80%, and incubated at 37°C in a 5% CO2 incubator for 3-4 days. Cytopathic effects were then observed, and the viral TCID was calculated using the Reed-Muench method. 50 .

[0039] Calculate TCID 50Subsequently, the triple-gene deletion virus and the parental virus (JS-2012-△gI / gE strain) were inoculated into Vero cells with a cell density of 70-80% at a dose of 1 MOI. The cells were incubated in serum-free DMEM medium at 37°C in a 5% CO2 incubator for 1 h, and then the medium was replaced with 2% DMEM for further culture. Cell supernatants were collected at 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, and 52 h after virus inoculation. The supernatants collected at different times were inoculated into 96-well monolayers of Vero cells with a cell density of 70-80%, and the mean and standard deviation were calculated to plot a one-step growth curve. The results showed that JS-2012-△gI / gE / TK and JS-2012-△gI / gE had similar growth kinetics (as shown in Figure 3).

[0040] The triple-gene deletion virus and the parental virus (JS-2012-△gI / gE strain) were serially diluted 10-fold in DMEM medium, and 10 μL of each was taken. -3 ~10 -5 One mL of the diluted virus solution was inoculated into a monolayer of BHK cells in a six-well cell culture plate. After 1.5 h, the supernatant was discarded, and a MEM mixture containing 2% FBS and 2% low-melting-point agarose was added to each well. After solidification at room temperature, the cells were incubated at 37°C in a 5% CO2 incubator for 3–4 days. After staining with crystal violet, the solid layer was carefully washed off, and the cells were air-dried upside down. Analysis of the viral plaque morphology and size showed that the recombinant virus and the parent virus had similar plaque morphology and clear edges, but the plaque size differed (as shown in Figure 4).

[0041] 1.6 Preparation and screening of monoclonal antibodies that specifically recognize JS-2012-ΔgI / gE / TK Referring to *Molecular Cloning Handbook*, JS-2012-△gI / gE / TK viral fluid was used as the specific antigen to screen for monoclonal antibodies that specifically recognize the viral fluid. The process was briefly described as follows: First, the viral fluid was purified and its titer was determined. After ensuring that the antigen purity and immunogenicity met the standards, Balb / c mice were immunized three times using standard immunization methods. The purified recombinant protein was mixed with an equal mass of Freund's complete adjuvant, thoroughly emulsified, and 0.2 ml / mouse was injected subcutaneously at multiple sites on the back of each mouse, for a total of five mice. Two weeks after the initial immunization, a second immunization was performed, with the recombinant protein mixed with an equal mass of Freund's incomplete adjuvant and injected subcutaneously at multiple sites on the back of the mice. Mice with higher titers received a booster immunization intraperitoneally three days before cell fusion. The antibody titer in the mouse serum was detected by indirect ELISA, and mice with higher titers were selected for booster immunization. Spleen cells from immunized mice were fused with myeloma cells and cultured in HAT selective medium. Successfully fused hybridoma cells were screened. Using JS-2012-△gI / gE / TK virus solution as the coating antigen, hybridoma cell culture supernatant was initially screened by indirect ELISA. Positive wells were then subjected to subclonal culture using limiting dilution. This process was repeated at least three times to obtain monoclonal hybridoma cell lines that stably secreted specific antibodies. Antibody titers were measured in the culture supernatant of successfully fused hybridoma cells. Wells with positive results were selected, resuspended in HT selection medium, and seeded into 96-well cell culture plates. Six replicate groups were set up, each with a 12-fold serial dilution, and cultured continuously for approximately 10 days in an incubator at 50 mL / L CO2 and 37°C. When hybridoma cells aggregated, they were screened again by antibody titer testing. At least four consecutive clonal culture cycles were performed until the percentage of positive hybridoma cell clones in the wells reached 100%, and individual hybridoma cell clusters appeared. The obtained positive individual hybridoma cell clusters were expanded, cryopreserved, and named 2H7 and 3G1.

[0042] BHK cells were seeded in 6-well plates. When the cells reached 80% confluence, they were inoculated with JS-2012-△gI / gE / TK virus solution, with the starting strain JS-2012-△gI / gE virus solution used as a control. After incubation for 2 h, the maintenance medium was replaced. After 48 h, the cell pellet was collected, and the cells were lysed with RIPA lysis buffer for SDS-PAGE. The proteins on the gel were then transferred to a PVDF membrane using a Bio-Rad wet transfer instrument. The membrane was blocked with 5% skim milk dissolved in TBST at room temperature for 1 h, and then incubated overnight at 4 °C with the supernatant of the successfully selected 2H7 and 3G1 hybridoma cells as the primary antibody. The cells were then washed 4 times with TBST and incubated for 1 h at room temperature with a secondary antibody (HRP-labeled goat anti-mouse IgG antibody) diluted 1:10000 with TBST. After washing 4 times with TBST, the cells were developed with ECL chemiluminescence solution from Sanor Biotech. Monoclonal antibody 3G1, which showed positive signals for JS-2012-△gI / gE / TK but no positive signal for JS-2012-△gI / gE, was selected. Its recognition site was analyzed to be a newly formed antigenic epitope after gene deletion, but whether the specific recognition site is a linear epitope or a spatial epitope needs further verification.

[0043] After culturing the selected 3G1 cell line, mRNA was extracted and its reproducible region was determined. The mRNA was then sent to Nanjing Mingyan Biotechnology Co., Ltd. for sequencing. The results showed that: The amino acid sequence of the light chain variable region of 3G1 is: ASLAVVAEKVSISC RSSQSIVYSANQGNYLA WYLQKPGQSPKLAIY WLASTRES GVPDGVPDGSGAGTDFTLTASSVKAEAEDLGVYYC QQYYHAPRT FGSATKLEIK (SEQ ID NO.5); The amino acid sequence of the heavy chain variable region is as follows: DVKLVSGAGLVVKPGGSLKLSCAVAGFATF SDYPAMS WVRQVRQKRLEAVA TISSDGGSYTAYPDSVK G RFISRDNAKATNLYLQMSSLKSEDTAMYACTR DYNSMYAWFAY WAQGTSVTVSA (SEQ ID NO.6), where the underlined portion represents the CDR1-3 of each variable region.

[0044] 1.7 Identification of TK gene activity 1.7.1 Western blot identification of TK gene activity Three-gene deletion virus and parental virus (JS-2012-△gI / gE strain) were inoculated into Vero cells at a dose of 1 MOI, with uninoculated Vero cells serving as a negative control. When 80% cytopathic effect was observed after virus inoculation, the supernatant was discarded, and a mixture of RIPA lysis buffer and a whitening enzyme inhibitor was added and the cells were lysed on ice for 15 min. Cells were scraped off the cell wall, centrifuged, and the supernatant was collected. The supernatant was then added to loading buffer, boiled in water for 10 min, and subjected to SDS-PAGE electrophoresis. After SDS-PAGE electrophoresis, the cells were transferred to an NC membrane using a transfer apparatus. The primary antibody was diluted 1:10,000 with the prepared TK monoclonal antibody, and the secondary antibody was diluted 1:5,000 with HRP-labeled goat anti-mouse antibody. Finally, the cells were mixed with chromogenic buffer at a 1:1 ratio for color development, observed, and photographed using a luminescence imaging system. Western blot results showed that JS-2012-△gI / gE could specifically react with TK monoclonal antibody and a relatively obvious specific band was detected at around 35 kDa. JS-2012-△gI / gE / TK and blank control showed no reaction (as shown in Figure 5).

[0045] 1.7.2 IFA Identification of TK Gene Activity Tri-gene deletion virus and parental virus (JS-2012-△gI / gE strain) were inoculated into Vero cells that had grown to a monolayer at a dose of 1 MOI, with uninoculated Vero cells serving as a negative control. When 40-50% CPE appeared in the cells, the culture medium was discarded, and the cells were washed three times with PBS. Pre-chilled 95% ethanol was added, and the cells were fixed overnight at 4 ℃, followed by three washes with PBS. The cells were incubated with TK monoclonal antibody (1:10,000) as the primary antibody at 37 ℃ for 1 h; after washing with PBS, Alexa Fluor 488-conjugated goat anti-mouse IgG (1:5,000) was added, and the cells were incubated at 37 ℃ in the dark for 1 h, followed by three washes with PBS buffer. The cells were then observed under green light using an inverted fluorescence microscope. IFA results showed that the TK monoclonal antibody exhibited good reactivity with Vero cells infected with JS-2012-△gI / gE, with obvious green fluorescence, while cells infected with JS-2012-△gI / gE / TK and the negative control group showed no reaction (as shown in Figure 6). The experimental results indicate that the triple-gene deletion virus JS-2012-△gI / gE / TK... TK Loss of gene activity.

[0046] 1.8 Genetic stability analysis The triple-gene deletion virus was inoculated into Vero cells at a dose of 1 MOI. When cytopathic effects reached over 90%, the cells were freeze-thawed at -80°C, centrifuged, and the supernatant was labeled P1. This method was used to passage both viruses for 20 consecutive generations. Viral DNA from P1, P5, P10, P15, and P20 was extracted using a commercial kit and identified by PCR using primers (TK-F / TK-R) from section 1.4. The amplified fragments were recovered and ligated into the PMD-18T vector, transformed into TOP10 competent cells, and single clones were picked for colony PCR identification. Positive colonies were sent to a sequencing company for sequencing. Sequencing results showed that the JS-2012-△gI / gE / TK virus, after 20 passages, showed that the virus… TK No base mutations were found in the gene deletion sequence (as shown in Figure 7).

[0047] Simultaneously, viruses P1, P5, P10, P15, and P20 were used to infect Vero cells. JS-2012-ΔgI / gE virus was used as a positive control in Vero cells, and uninoculated Vero cells were used as a blank control. Indirect immunofluorescence assays were performed using the same method as described above (see 1.7.2). The results showed that the TK monoclonal antibody had good reactivity with Vero cells infected with JS-2012-ΔgI / gE, and obvious green fluorescence was observed. However, viruses P1, P5, P10, P15, and P20, as well as the negative control group, showed no reaction (as shown in Figure 8), indicating no significant difference between passages, thus indicating that the virus remained stable during passage.

[0048] 1.9 Safety experiment of triple gene deletion virus in mice Six-week-old KM mice were randomly divided into six groups of ten mice each. Tri-gene deletion virus and JS-2012-ΔgI / gE were administered at doses of 10... 4 10 6 TCID 50 JS-2012 was administered at a dose of 10... 5 TCID 50 Mice were vaccinated with 0.1 mL of JS-2012-ΔgI / gE / TK at the prescribed dose, with a blank control group serving as the control. Clinical symptoms were observed and recorded for 14 days post-vaccination. No PRV-specific clinical reactions were observed in mice immunized with different doses of JS-2012-ΔgI / gE / TK, and no deaths occurred during the observation period, consistent with the blank control group. Mice immunized with different doses of JS-2012-ΔgI / gE began exhibiting severe itching and depression on day 3 post-vaccination, and deaths occurred; by day 4, 10... 6 All members of the high-dose TCID50 group died. 4Two mice in the TCID50 dose group survived after 8 days. Mice inoculated with the JS-2012 virus developed symptoms on day 3 post-inoculation, exhibiting itching, licking of the injection site, and death; all mice died on day 4 (as shown in Figure 9). The experiment demonstrates that JS-2012-ΔgI / gE / TK is not pathogenic in mice.

[0049] The results of mouse tissue anatomy and histopathological changes showed that Pathological changes observed: Mice in the JS-2012-△gI / gE, JS-2012 and three other groups showed typical viral encephalitis with meningeal congestion; the liver was dark red or yellowish-brown, congested and enlarged; the lungs were dark red, enlarged and congested; the kidneys were enlarged; there were no obvious lesions visible to the naked eye in the heart; the spleen was pale, while no obvious lesions were observed in mice injected with JS-2012-△gI / gE / TK and the blank control group (as shown in Figure 10).

[0050] Pathological tissue sections: The JS-2012-△gI / gE and JS-2012 mice showed varying degrees of neuronal degeneration and necrosis in the brain, and glial cell proliferation and aggregation; the alveolar structure was loose and disordered, with a small amount of inflammatory cell infiltration; local punctate necrotic foci were visible in the liver tissue, accompanied by significant inflammatory cell infiltration; a small number of lymphocytes were reduced in the spleen; a small amount of degeneration of renal epithelial cells was observed; local myocardial fibers showed degeneration and necrosis, with a loose and disordered structure; while no obvious pathological changes were observed in the tissues and organs of the JS-2012-△gI / gE / TK and blank control mice (as shown in Figure 11).

[0051] The results of viral load detection in the organs of mice in each group showed that a large amount of viral nucleic acid was present in various tissues and organs of mice immunized with JS-2012-ΔgI / gE and JS-2012, with the highest viral load in the brain, reaching up to 2×10⁻⁶. 4 The viral nucleic acid content in the tissues and organs of mice immunized with JS-2012-△gI / gE / TK was low, only about 2 copies / g, showing a significant difference (as shown in Figure 12). 1.10 Safety experiment of triple gene deletion virus in pigs Fifteen pseudorabies virus double-negative piglets (negative for both gB and gE antibodies) were selected and randomly divided into three groups of five piglets each. Group 1 was intranasally inoculated with 10 doses of the triple-gene deletion virus. 6 TCID 50 / 2mL / head, Group 2 received 10 doses of parental virus (JS-2012-△gI / gE) via intranasal inoculation. 6 TCID 50 / 2mL / head, with group 3 receiving no treatment as a blank control. Patients were observed for 28 days post-vaccination, with daily temperature measurements and clinical symptoms observed and recorded.

[0052] Piglets were inoculated with JS-2012-△gI / gE / TK and the parent virus (JS-2012-△gI / gE) and observed for 28 days. All pigs showed no significant difference from the blank control group during the immunization period. No obvious adverse reactions were observed throughout the observation period. All pigs were healthy and alive, with normal body temperature, appetite, and mental state (as shown in Figure 13).

[0053] Anatomical and histopathological results of pig tissues showed that piglets inoculated with JS-2012-△gI / gE / TK virus all survived, with no significant differences compared to the parent virus (JS-2012-△gI / gE) group and the blank control group. The surviving experimental pigs were then euthanized, and visual and histopathological changes were observed. The results showed that no significant visual or histopathological changes were observed in the brain, lungs, liver, kidneys, inguinal lymph nodes, spleen, and heart of the surviving pigs in the JS-2012-△gI / gE / TK virus inoculation group, and there were no significant differences in any of the tissues and organs compared to the parent virus (JS-2012-△gI / gE) group and the blank control group (as shown in Figures 14 and 15).

[0054] 1.11 Immunoprotective efficacy test of triple gene deletion virus against piglets Ten pseudorabies virus double-negative piglets (negative for both gB and gE antibodies) were randomly divided into two groups of five piglets each. Group 1 piglets were injected intramuscularly into the neck with 10 doses of the triple-gene deletion virus. 5 TCID 50 Group 2 received DMEM via intramuscular injection in the neck, while Group 2 served as the control group. Blood samples were collected every 7 days post-immunization for antibody monitoring. On day 28 post-immunization, all animals were administered 10 ml of virulent pseudorabies virus strain JS-2012 via intranasal instillation. 5 TCID 50 / 2ML / head. After challenge, clinical manifestations were observed daily, body temperature was measured, and nasal and anal swabs were collected for virus shedding tests. Blood was collected every three days for continuous observation and monitoring for 14 days. At the same time, various tissues and organs (including brain, liver, heart, lungs, kidneys, spleen, and inguinal lymph nodes) of diseased and dead pigs and surviving pigs were collected during the observation period for histopathological observation and viral load determination.

[0055] The results showed that gB antibodies gradually turned positive on day 7 after vaccination with JS-2012-△gI / gE / TK virus (as shown in Figure 16), and the antibody level increased over time; while gE antibodies remained negative throughout the entire immunization period (as shown in Figure 17). After immunization of piglets with JS-2012-△gI / gE / TK virus, no adverse reactions were observed at the injection site or throughout the body in either group of experimental piglets. The pigs' feed intake, water consumption, feces, and mental state were all normal, showing no significant difference from the DMEM vaccination group. Following challenge with a virulent variant of porcine pseudorabies virus (JS-2012 strain), the JS-2012-△gI / gE / TK virus inoculation group exhibited a brief period of fever from day 3 to 5, which returned to normal by day 6. Their appetite, water intake, feces, and mental state remained normal, achieving 100% protection (as shown in Figure 19). In contrast, the DMEM inoculation group experienced a rapid increase in temperature to above 41°C on day 2 after inoculation with the virulent JS-2012 strain. By day 6 post-challenge, they began exhibiting typical neurological symptoms such as vomiting, severe depression, ataxia, convulsions, and paddling motions in their limbs. One pig died on days 7, 9, and 10 (as shown in Figure 18). Nasal and anal swabs were collected daily from each group of pigs after challenge for virus shedding testing. The results showed that piglets inoculated with JS-2012-△gI / gE / TK virus shed the virus in nasal or anal swabs during the sporadic fever period after challenge. Piglets in the DMEM-inoculated group developed a rapid increase in body temperature to above 41℃ on days 2-3 after challenge, and subsequently exhibited typical neurological symptoms such as ataxia and circling. Virus shedding was detected in both nasal and anal swabs throughout the entire course of the disease (as shown in Table 2).

[0056] Table 2 Immunoprotective efficacy experiment Tab.2 Immune Protective Efficacy Experiment

[0057] Note: a: Number of pigs detected with viral shedding per day per head; b: Total number of tests, 5 pigs tested once a day, for 0-14 days (5 x 15 = 75), excluding dead pigs. Note: a: Number of detoxification tests detected / day. pig; b: Totalnumber of tests, with 5 pigs tested once per day for 0-14 days (5×15=75), excluding dead pigs After challenge with the JS-2012 strain of porcine pseudorabies virus, all piglets in the JS-2012-△gI / gE / TK virus inoculation group survived. No obvious visual or histopathological changes were observed in the brain, lungs, liver, kidneys, inguinal lymph nodes, and spleen of the surviving pigs. In the DMEM inoculation group, 60% of the piglets died. The deceased pigs showed obvious visual hemorrhage lesions in the brain, lungs, liver, inguinal lymph nodes, and spleen: severe brain hemorrhage with significant vascular dilation; large areas of congestion in the lung lobes with a firm feel; congested liver with a dark red color; hemorrhagic and swollen lymph nodes; congested spleen; and swollen, dark red kidneys. No obvious visual pathological changes were observed in the heart (as shown in Figure 20).

[0058] Histopathology: In the brains of the dead test pigs, there was inflammatory cell infiltration and red blood cells visible outside the blood vessel walls; alveoli were morphologically destroyed and contained large amounts of red blood cells; hepatocytes showed vacuolar degeneration and red blood cell aggregation; renal tubular epithelial cells showed vacuolar degeneration; the number of lymphocytes in the inguinal lymph nodes was significantly reduced and large amounts of red blood cells were visible; the number of lymphocytes in the spleen was reduced, inflammatory cells were observed, and red blood cell aggregation was also visible; large amounts of red blood cells were visible between the myocardial cell fibers (as shown in Figure 21).

[0059] Viral load was measured in relevant organs of pigs that died during the experiment and those euthanized after the experiment. Results showed that piglets in the JS-2012-△gI / gE / TK virus inoculation group achieved good immune protection after challenge, but very low levels of viral DNA were detected in the brain, heart, liver, spleen, lungs, kidneys, and inguinal lymph nodes of the experimental pigs. In the DMEM inoculation group, both morbid and surviving piglets showed viral loads of 10 in their brain, heart, liver, spleen, lungs, kidneys, and inguinal lymph nodes. 8 The viral DNA was present at a rate of 100 copies / g; and the highest viral load was found in the brain, reaching 3.5 × 10⁻⁶. 8 Copies / g (as shown in Figure 22).

[0060] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A pseudorabies virus gI / gE / TK triple gene deletion vaccine strain, characterized in that, The vaccine strain is pseudorabies virus rPRV JS-2012-△gI / gE / TK, which was deposited on January 21, 2025 at the China General Microbiological Culture Collection Center (CCTCC) with accession number CCTCC No. V202518. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The method for constructing a pseudorabies virus gI / gE / TK triple-gene deletion vaccine strain JS-2012-△gI / gE / TK as described in claim 1, the method comprising the following steps: 1) Based on the genomic sequence of PRV (JS-2012-△gI / gE strain), two sgRNAs were designed, targeting the 5' and 3' ends of the TK gene, respectively. Following the instructions for constructing the lentiCRISPRv2 plasmid, two double-stranded TK-gRNA DNAs were obtained. The pCRISPR / Cas9 vector was digested with BsmBI, and the vector fragments were recovered via gel electrophoresis. The two double-stranded TK-gRNA DNAs were ligated into the BsmBI-digested and recovered vectors using T4 DNA ligase. The vectors were then transformed into TOP10 competent cells, and the plasmids were extracted to obtain donor plasmids Cas9-TK1 and Cas9-TK2. Double digestion with NotI and BamHI was performed for identification, followed by sequencing analysis. Among them, sgRNA-TK-1-FCACCGCATCAGCGCGCGGCGCCTTCG; sgRNA-TK-1-RAAACCGAAGGCCGCCGCGCTGATGC; sgRNA-TK-2-FCACCGGTCTGCGCATCGTAAATAC; sgRNA-TK-2-RAAACGTATTTACGATGCGCAGACC; 2) Extraction of the JS-2012-△gI / gE genome; 3) Co-transfect BHK cells with 1 μg of donor plasmids Cas9-TK1 and Cas9-TK2 and 2 μg of JS-2012-ΔgI / gE viral genome. Observe the cytopathic effect daily after transfection. When approximately 80% of the cells show cytopathic effects, freeze-thaw the transfected cells at -80℃, collect the supernatant, and perform selection and purification.

3. A monoclonal antibody 3G1 that distinguishes between vaccine strains and wild-type strains, characterized in that... The amino acid sequence of the light chain variable region of 3G1 is shown in SEQ ID NO.6, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

4. The application of the monoclonal antibody 3G1 as described in claim 3 in the epidemiological investigation of pseudorabies virus.

5. The application of the pseudorabies virus gI / gE / TK triple gene deletion vaccine strain JS-2012-△gI / gE / TK as described in claim 1 in the preparation of porcine pseudorabies virus vaccine.

6. An immune composition, characterized in that... The vaccine includes the pseudorabies virus gI / gE / TK triple gene deletion vaccine strain JS-2012-△gI / gE / TK as described in claim 1 and the monoclonal antibody 3G1 as described in claim 3, wherein the triple gene deletion vaccine strain JS-2012-△gI / gE / TK is used as an effective immunizing component and the monoclonal antibody 3G1 is used as an indicator of immunization efficacy.

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