A recombinant pseudorabies triple vaccine and a preparation method and application thereof

Abstract

The application discloses a recombinant pseudorabies triple vaccine and a preparation method and application thereof, and belongs to the technical field of multiple vaccines. The recombinant pseudorabies triple vaccine provided by the application is rPRV-ΔTK-ApxII-eGFP-ΔgI / gE-E2-mCherry, takes PRV as a parent strain, simultaneously carries two protective antigens of CSFV E2 and APP ApxII, can simultaneously induce three kinds of antibodies against PRV, CSFV and APP through one-time immunization, can effectively reduce the number of immunization times, labor intensity, immunization cost and pig group stress, is high in safety, is high in humoral immune effect, and the humoral immune effect is significantly improved after secondary immunization; the PRV neutralizing antibody titer reaches 1:39.89, the CSFV neutralizing antibody titer reaches 1:21.46; the attack protection effect is exact; and the genetic stability and the proliferation stability are both excellent.

Description

Technical Field

[0001] This invention relates to the field of multivalent vaccine technology, and in particular to a recombinant pseudorabies trivalent vaccine, its preparation method, and its application. Background Technology

[0002] Intensive pig farming faces the synergistic threat of several major diseases, among which porcine pseudorabies (PR), classical swine fever (CSF), and porcine contagious pleuropneumonia (PCP) are three highly dangerous and difficult-to-control diseases. The causative agent of PCP is Actinobacillus pleuropneumoniae (APP), which produces four different RTX toxins. ApxI, ApxII, and ApxIII are expressed both in vitro and in vivo, exhibiting varying degrees of cytotoxicity against lung macrophages, PMNs, lung epithelial cells, and endothelial cells. ApxI and ApxII also possess hemolytic activity.

[0003] Porcine pseudorabies is caused by pseudorabies virus (PRV). Studies have shown that the PRV genome contains some non-essential genes, such as gE, gC, and thymidine kinase (TK) genes. These genes are not indispensable in the basic replication process of the virus. After knocking them out, the virus can still complete normal replication in the host cell.

[0004] Classical swine fever (CSF) is a Class I notifiable animal disease in my country, a highly contagious and deadly disease caused by the classical swine fever virus (CSFV). The CSFV virus is highly resistant and can survive for months in pork, feces, and under low-temperature conditions. It can be transmitted over long distances through pork products, personnel, vehicles, equipment, and insect vectors. There is no effective treatment; prevention relies solely on vaccination.

[0005] Most existing swine vaccines are single-pathogen vaccines, requiring multiple separate immunizations to control PR, CSF, and APP. This immunization procedure is cumbersome, labor-intensive, and costly, and can easily cause stress in pig herds. There is also a risk of immune interference, affecting the overall immunization effect, making it difficult to achieve simultaneous protection and address the high incidence of mixed infections and secondary infections of the three pathogens in clinical practice. Furthermore, most multivalent vaccines suffer from problems such as easy loss of exogenous genes, poor genetic stability, and impaired viral replication, making them unsuitable for industrial application. Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant pseudorabies trivalent vaccine and its preparation method and application, so as to provide a multivalent vaccine that can induce three antibodies against PRV, CSFV and APP with a single immunization, and has excellent genetic stability and proliferation stability, high safety and definite protection against challenge.

[0007] To achieve the above objectives, the present invention provides a recombinant pseudorabies trivalent vaccine, wherein the recombinant pseudorabies trivalent vaccine is rPRV-ΔTK-ApxII-eGFP-ΔgI / gE-E2-mCherry.

[0008] Preferably, the recombinant pseudorabies trivalent vaccine uses pseudorabies virus PRV SX-10-2015 as the parent strain, and is prepared by deleting three virulence genes, TK, gI, and gE, and inserting optimized APP ApxII and CSFVE2 gene coding sequences at the deletion sites, respectively.

[0009] Preferably, the optimized APP ApxII gene coding sequence is shown in SEQ ID NO.5; the optimized CSFV E2 gene coding sequence is shown in SEQ ID NO.2.

[0010] A method for preparing the recombinant pseudorabies trivalent vaccine as described above, comprising the following steps: S1 and BHK-21 cells were co-transfected with pX335-TK-sgRNA plasmid and TK site donor plasmid, and inoculated with parental strain PRVSX-10-2015. The TK gene was knocked out and inserted into the ApxII-eGFP expression cassette. After multiple rounds of purification with green fluorescent plaques, rPRV-ΔTK-ApxII-eGFP was obtained. S2. Using rPRV-ΔTK-ApxII-eGFP as the parent, gI / gE targeting sgRNA plasmid and gI / gE site donor plasmid were co-transfected. The gI / gE gene was knocked out and inserted into E2-mCherry at a specific site. After screening for plaques with red and green fluorescence, the final strain rPRV-ΔTK-ApxII-eGFP-ΔgI / gE-E2-mCherry was obtained after 5-7 generations of continuous purification.

[0011] Preferably, the TK site donor plasmid in S1 is pUC19-TK-CMV-HA-APXII-eGFP-polyA, and the construction method is as follows: The optimized APP ApxII gene coding sequence was sent to the company for synthesis to obtain the recombinant pET-45b(+) vector with the target sequence inserted. Using the recombinant pET-45b(+) vector as a template, two rounds of PCR amplification were performed using APXII-HA-F and APXII-HA-R, CMV-HA-F and GSG-HA-R as primers, respectively. The 2967bp target fragment HA-APXII was recovered by gel extraction. use Nhe I and Sph The pUC19-TK-CMV-EGFP-polyA fragment was double-digested with enzyme I, and the 7156 bp fragment was recovered by gel extraction. It was then seamlessly cloned and ligated with the HA-APXII fragment.

[0012] Preferably, the APXII-HA-F sequence is shown in SEQ ID NO.17; the APXII-HA-R sequence is shown in SEQ ID NO.18; the CMV-HA-F sequence is shown in SEQ ID NO.19; and the GSG-HA-R sequence is shown in SEQ ID NO.20.

[0013] Preferably, the gI / gE site donor plasmid in S2 is pUC19-gI / gE-CMV-SPZJ-E2-TM-mCherry-polyA; the construction method is as follows: The optimized CSFV E2 gene coding sequence was sent to the company for synthesis to obtain the recombinant pET-30a(+) vector containing the target sequence. Using the recombinant pET-30a(+) vector as a template, two rounds of PCR amplification were performed using E2-Flag-F and E2-TM-R, and CMV-SPZJ-F and GSG-TM-R as primers, respectively. The 1235bp target fragment SPZJ-E2-TM was recovered by gel extraction. Using pUC19-gI / gE-CMV-mCherry-polyA as a template, PCR amplification was performed using AMP-2283-F and CMV-R as primers. The amplification product was then recovered by gel to obtain the AMP-CMV-3447 fragment. Using pUC19-gI / gE-CMV-mCherry-polyA as a template, PCR amplification was performed using GSG-F and AMP-2000-R as primers. The amplification product was then recovered by gel to obtain the GSG-AMP-3548 fragment. The SPZJ-E2-TM, GSG-AMP-3548, and AMP-CMV-3447 fragments can be seamlessly cloned and joined.

[0014] Preferably, the E2-Flag-F sequence is shown in SEQ ID NO.7; the E2-TM-R sequence is shown in SEQ ID NO.8; the CMV-SPZJ-F sequence is shown in SEQ ID NO.9; the GSG-TM-R sequence is shown in SEQ ID NO.10; the GSG-F sequence is shown in SEQ ID NO.11; the CMV-R sequence is shown in SEQ ID NO.12; the AMP-2283-F sequence is shown in SEQ ID NO.13; and the AMP-2000-R sequence is shown in SEQ ID NO.14.

[0015] The application of the preparation method of the recombinant pseudorabies trivalent vaccine described above in the prevention and control of diseases, wherein the disease is one or more of porcine pseudorabies, classical swine fever, and porcine infectious pleuropneumonia.

[0016] The application of the recombinant pseudorabies trivalent vaccine as described above in the prevention and control of diseases, wherein the disease is one or more of porcine pseudorabies, classical swine fever, and porcine infectious pleuropneumonia.

[0017] Therefore, the recombinant pseudorabies trivalent vaccine, its preparation method, and its application provided by this invention have the following specific technical effects: (1) The recombinant pseudorabies trivalent vaccine provided by the present invention uses PRV as the parent strain and carries two protective antigens, CSFV E2 and APP ApxII. One immunization can induce three antibodies against PRV, CSFV and APP at the same time, which can effectively reduce the number of immunizations, labor intensity, immunization costs and pig stress. (2) The recombinant pseudorabies trivalent vaccine provided by the present invention has high safety. The immunized mice showed no death, no neurological symptoms, no fever, and a survival rate of 100%; while all the parent virulent strains died within 4 days. (3) The recombinant pseudorabies trivalent vaccine provided by the present invention has significant humoral immunization effect, and can induce a continuous increase in PRV gB antibody, CSFV E2 antibody and APP ApxII antibody, which significantly increases after the second immunization; the PRV neutralizing antibody titer reaches 1:39.89 and the CSFV neutralizing antibody titer reaches 1:21.46. (4) The recombinant pseudorabies trivalent vaccine provided by the present invention has a definite protective effect against viral challenge: PRV virulent challenge, 100% survival rate in the immunized group, which is significantly better than Bartha-K61 vaccine (only 20% protection); APP virulent challenge, 60% survival rate in the immunized group, which is not significantly different from the commercial trivalent inactivated vaccine (80%), significantly reduces the bacterial load in the lungs, and significantly reduces pathological damage. (5) The recombinant pseudorabies trivalent vaccine provided by the present invention has good genetic stability. After 20 consecutive passages, ApxII and E2 are stably expressed, TK and gI / gE are stably deleted, and the viral titer is not significantly different from the triple-deficient strain. (6) The recombinant pseudorabies trivalent vaccine provided by the present invention has good proliferation stability. The one-step growth curve shows that the replication kinetics of the recombinant pseudorabies trivalent vaccine is highly consistent with the parent strain and the three-deficient strain.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a diagram of the carrier of pUC19-gI / gE-CMV-mCherry-polyA in Embodiment 3 of the present invention; Figure 2 This is a diagram showing the PCR identification results of the donor plasmids constructed in embodiments 3 and 4 of this invention; lane 1 is the donor plasmid pUC19-TK-CMV-HA-APXII-eGFP-polyA; lane 2 is the donor plasmid pUC19-gI / gE-CMV-SPZJ-E2-TM-mCherry-polyA; Figure 3 These are the sequencing results of the donor plasmids in embodiments 3 and 4 of this invention; where A is the donor plasmid pUC19-TK-CMV-HA-APXII-eGFP-polyA; and B is the donor plasmid pUC19-gI / gE-CMV-SPZJ-E2-TM-mCherry-polyA. Figure 4 This is a vector diagram of pUC19-TK-CMV-EGFP-polyA in Embodiment 4 of the present invention; Figure 5 This is a vector diagram of pX335-TK-sgRNA-32-955 in Embodiment 5 of the present invention; Figure 6 This is a fluorescence microscope image of the recombinant virus rPRV-ΔTK-APXII-eGFP in Embodiment 5 of this invention; Figure 7 This is the PCR result of the recombinant virus rPRV-ΔTK-APXII-eGFP in Embodiment 5 of this invention; Figure 8 This is the Western Blot identification result of the recombinant virus rPRV-ΔTK-APXII-eGFP in Embodiment 5 of this invention; Figure 9 This is a fluorescence microscope image of the recombinant virus rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry in Embodiment 5 of this invention; Figure 10 This is the PCR identification result of the recombinant virus rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry in Embodiment 6 of this invention; Figure 11 This is the Western Blot identification result of the recombinant virus rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry in Embodiment 6 of this invention; Figure 12This is the stability test result of the recombinant virus rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry in Embodiment 7 of this invention; where A is the APXII gene; B is the E2 gene; C is the TK gene; D is the gI / gE gene; lane 1 is the 5th generation of the recombinant virus, lane 2 is the 10th generation of the recombinant virus, lane 3 is the 20th generation of the recombinant virus, + represents the constructed donor plasmids pUC19-TK-CMV-HA-APXII-eGFP-polyA and pUC19-gI / gE-CMV-SPZJ-E2-TM-mCherry-polyA, and - represents the genome of PRVSX-10-2015; Figure 13 This is a one-step growth curve of the recombinant virus rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry in Embodiment 7 of the present invention; Figure 14 These are photographs of the lungs, spleen, and liver of mice that died after APP inoculation in Embodiment 8 of this invention; where A is the lung; B is the liver; and C is the spleen. Figure 15 These are the survival curves of mice in each group after immunization in Embodiment 8 of this invention; Figure 16 These are the results of the detection of PRV, CSFV, and APP-specific antibodies in mice after immunization in Embodiment 8 of this invention; where A is CSFV E2; B is PRV gB; and C is APP APXII. Figure 17 These are the results of neutralizing antibody detection in mouse serum after immunization in Embodiment 8 of this invention; where A represents PRV; and B represents CSFV. Figure 18 This refers to the survival rate of mice after being challenged with the APP in the 8th embodiment of this invention; Figure 19 This refers to the survival rate of mice after challenge with PRV SX-10-2015 in Embodiment 8 of this invention; Figure 20 This is the distribution of viral load in mouse tissues after challenge with PRV SX-10-2015 in Embodiment 8 of this invention; Figure 21 This refers to the bacterial load in the lungs of mice in each group after being challenged with the APP in 8 different embodiments of the present invention. Figure 22 These are photographs (200×) of lung tissue sections from mice after APP challenge in Embodiment 8 of this invention. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0024] Example 1 The coding sequence of the CSFV E2 gene was optimized as follows: The CDS sequence of the CSFV E2 gene is shown in SEQ ID NO.1. The transmembrane region of the CSFV E2 gene was predicted using the protein transmembrane region prediction website (DeepTMHMM 1.0 - DTU Health Tech - Bioinformatic Services). The results showed that the transmembrane region and the intracellular region are located at 342-373 aa. Linear antigenic epitopes in the CSFV E2 sequence were predicted using the linear epitope prediction tool (SVMTriP). The results showed that no linear B-cell antigenic epitopes were predicted in the intracellular region.

[0025] SEQ ID NO.1: The transmembrane and intracellular regions of the CSFV E2 gene (342-373aa) were removed, retaining the antigen core fragment. The remaining CSFV E2 gene core fragment sequence was then optimized based on PRV codon usage preferences, resulting in the codon-optimized sequence shown in SEQ ID NO.2. A natural signal peptide SPZJ was added to the 5' end, and a PEDV S2-derived transmembrane TM sequence was inserted to the 3' end. The optimized CSFV E2 gene encoding nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.3. The optimized nucleotide sequence information was sent to a biotechnology company for sequence synthesis. The received sequence was the target sequence linked to the backbone vector pET-30a(+), named pET-30a(+)-E2.

[0026] SEQ ID NO.2: SEQ ID NO.3: RLACKEDYRYAISSTNEIGPLGAGGLTTTWKEYSHDLQLYDGTVKAICVAGSFKVTALNVVSRRYLASLHKGALLTSVTFELLFDGTNPSTEEMGDDFGFGLCPFDTSPVVKGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFRREKPFPHRMDCVTT TVENEDLFYCKLGGNWTCVKGEPVVYTGGQVKQCKWCGFDFNEPDGLPHYPIGKCILANETGYRIVDSTDCNRDGVVISAEGSHECLIGNTTVKVHASDERLGPMPCRPKEIVSSAGPVRKTSCTFNYAKTLKNKYYEPRDSYFQQYMLKGEYQYWFDLDVTDRHSDYFAE The codon fitness index (CAI) was increased from 0.22 in the CSFV E2 gene to 0.99 using the company's synthetic assay, and the GC content was optimized from 48% in the CSFV E2 gene to 65%, effectively eliminating the risk of rare codons and significantly improving the sequence's fit in the PRV expression system.

[0027] Example 2 The coding sequence of the APP ApxII gene was optimized as follows: The coding sequence of the APP ApxII gene (as shown in SEQ ID NO.4) was codon optimized according to PRV codon usage preference, and HA tag sequences were added to both ends of the gene. The optimized coding nucleotide sequence of the APP ApxII gene is shown in SEQ ID NO.5, and the encoded amino acid sequence is shown in SEQ ID NO.6.

[0028] The optimized nucleotide sequence information was sent to a biotechnology company to synthesize the sequence. The received sequence was the target sequence linked to the backbone vector pET-45b(+), named pET-45b(+)-APXII.

[0029] Using the company's synthetic assay CAI, the optimized APP ApxII gene changed from 0.10 to 0.98, and the GC content increased from 36% to 65%, improving the adaptability and expression efficiency of the APXII gene in the PRV expression system.

[0030] SEQ ID NO.4: SEQ ID NO.5: SEQ ID NO.6: * Example 3 The donor plasmid pUC19-gI / gE-CMV-SPZJ-E2-TM-mCherry-polyA was constructed as follows: (1) Using the pET-30a(+)-E2 recombinant plasmid synthesized by the biotechnology company as a template, PCR amplification was performed using primers E2-Flag-F (sequence shown in SEQ ID NO.7) and E2-TM-R (sequence shown in SEQ ID NO.8). The amplification system was prepared according to the instructions attached to the high-fidelity enzyme, and the amplification program was 98℃ for 30s; 98℃ for 15s, 59℃ for 15s, 72℃ for 1min, 35 cycles; 72℃ for 5min. Using the first amplification product as a template, PCR amplification was performed using primers CMV-SPZJ-F (sequence shown in SEQ ID NO.9) and GSG-TM-R (sequence shown in SEQ ID NO.10). The amplification system was prepared according to the instructions attached to the high-fidelity enzyme, and the amplification program was the same as the first amplification, except that Tm was 54℃. The target fragment SPZJ-E2-TM was obtained by gel recovery of the second amplification product using a kit. Then, the signal peptide sequence was spliced ​​onto the fragment SPZJ-E2-TM.

[0031] SEQ ID NO.7: GGTGTGATATGGCTGCTACTAGTAACTGGGGCACAAGGCGATTACAAGGATGACGACGATAAGCGCCTGGCCTGCAAGGAG SEQ ID NO.8: GAAGACCAGCAGCGAGACGACGAAGATCAGCACGATGAAGATGATCAGCCACACCCACCACTCGGCGAAGTAGTCCGA SEQ ID NO.9: GTCTATATAAGCAGAGCTGCTAGCGCCACCATGAAAGTATTAAGAGGACAGATCGTGCAAGGTGTGATATGGCTGCTA SEQ ID NO.10: CTTCCTCTGCCCTCTCCACTGCCACGCGTGAAGACCAGCAGCGAGAC (2) Using pUC19-gI / gE-CMV-mCherry-polyA (carrier diagram as shown) Figure 1Using pUC19-gI / gE-CMV-mCherry-polyA as a template, PCR amplification was performed using AMP-2283-F (sequence shown in SEQ ID NO.13) and CMV-R (sequence shown in SEQ ID NO.12) as primers. The amplification products were then recovered from the gel to obtain the GSG-AMP-3548 fragment. Using pUC19-gI / gE-CMV-mCherry-polyA as a template, PCR amplification was performed using AMP-2283-F (sequence shown in SEQ ID NO.13) and CMV-R (sequence shown in SEQ ID NO.12) as primers. The amplification products were then recovered from the gel to obtain the AMP-CMV-3447 fragment. The amplification system was prepared according to the instructions accompanying the high-fidelity enzyme. The amplification program was 98℃ for 30s; 98℃ for 15s, 59℃ for 15s, 72℃ for 1.5min, for 35 cycles; 72℃ for 5min.

[0032] SEQ ID NO.11: GGCAGTGGAGAGGGCAGAG SEQ ID NO.12: AGCTCTGCTTATATAGACCTCCC SEQ ID NO.13: CAACGTTGCGCAAACTATTAACTGGCG SEQ ID NO.14: TTGCCGGGAAGCTAGAGTAAGTAGTTC (3) The SPZJ-E2-TM, GSG-AMP-3548, and AMP-CMV-3447 fragments with homologous arms were seamlessly cloned and ligated to construct a plasmid containing the CSFV E2 gene and an mCherry fluorescent tag. PCR was performed using the sequences shown in SEQ ID NO.15 and SEQ ID NO.16 as primers and the constructed plasmid as a template. The electrophoresis results are shown below. Figure 2 As shown, the PCR amplification products all exhibited clear characteristic bands at the expected molecular weight positions, and the band sizes were consistent with theoretical values, preliminarily confirming the successful construction of the donor plasmid. Some sequencing results are shown below. Figure 3 As shown in Figure B, the target gene sequence is complete, with no base deletions, insertions, or mutations, further confirming that the donor plasmid construction was completely correct. After verification, it was named pUC19-gI / gE-CMV-SPZJ-E2-TM-mCherry-polyA.

[0033] SEQ ID NO.15: TATCATATGCCAAGTCCGCCC SEQ ID NO.16: CTCTGCCCTCTCCACTGCC Example 4 The donor plasmid pUC19-TK-CMV-HA-APXII-eGFP-polyA was constructed as follows: (1) Using pET-45b(+)-APXII synthesized by the biotechnology company as a template, PCR amplification was performed using APXII-HA-F (sequence shown in SEQ ID NO. 17) and APXII-HA-R (sequence shown in SEQ ID NO. 18) as primers. Using the amplification product as a template, PCR amplification was performed using CMV-HA-F (sequence shown in SEQ ID NO. 19) and GSG-HA-R (sequence shown in SEQ ID NO. 20) as primers. The amplification system was prepared according to the instructions attached to the high-fidelity enzyme, and the amplification procedure was the same as the procedure for the first amplification in (1) of Example 3. The target fragment HA-APXII was obtained by gel recovery of the second amplification product using a kit.

[0034] SEQ ID NO.17: TACCCATACGATGTTCCAGATTACGCTTCGAAGATCACGCTGAGC SEQ ID NO.18: AGCGTAATCTGGAACATCGTATGGGTAGGCGGCGCGGCCAGCTGG SEQ ID NO.19: GTCTATATAAGCAGAGCTGCTAGCGCCACCATGTACCCATACGATGTTCCAGA SEQ ID NO: 20: TCCACTGCCGCATGCAGCGTAATCTGGAACATCGTATGGGTA (2) Utilization Nhe I and Sph I. Enzyme interaction with pUC19-TK-CMV-EGFP-polyA (vector diagram shown) Figure 4 (As shown) Double digestion was performed, following the enzyme digestion system and procedure specified in the enzyme's instruction manual. The 7156 bp fragment was recovered by gel extraction.

[0035] (3) The gel recovery product from step (2) was seamlessly cloned and ligated with the HA-APXII fragment to construct a plasmid containing the APXII gene and an EGFP fluorescent tag. The electrophoresis results are as follows: Figure 2As shown, PCR was performed using the sequences shown in SEQ ID NO.15 and SEQ ID NO.16 as primers and the constructed plasmid as a template. The PCR amplification products all showed clear characteristic bands at the expected molecular weight positions, and the band sizes were consistent with the theoretical values, preliminarily confirming the successful construction of the donor plasmid. Some sequencing results are shown below. Figure 3 As shown in Figure A, the target gene sequence is complete, with no base deletions, insertions, or mutations, further confirming that the donor plasmid construction was completely correct. It was correctly identified and named PUC19-TK-CMV-HA-APXII-eGFP-polyA.

[0036] Example 5 The recombinant virus rPRV-ΔTK-ApxII-eGFP-ΔgI / gE-E2-mCherry was constructed as follows: (1) Construction and purification of recombinant virus rPRV-ΔTK-APXII-eGFP.

[0037] Healthy and viable BHK-21 cells were evenly seeded in 6-well plates and cultured in DMEM containing 10% FBS at 37°C and 5% CO2. Cell growth was observed periodically using an inverted microscope. When cell growth stabilized at 60%–70% and the cells were regular in morphology and firmly adhered to the plate, a mixture of transfection reagent and plasmid (2 µg of both plasmids, totaling 4 µg of plasmid, mixed with 8 µL of Turbo Fect transfection reagent in 200 µL of Opti MEM per well) was added, mixed well, and cultured for a longer period.

[0038] The mixture of transfection reagent and plasmid was prepared by cutting plasmid pX335-TK-sgRNA-32-955 (vector diagram shown). Figure 5 As shown in Example 4, the donor plasmid PUC19-TK-CMV-HA-APXII-eGFP-polyA was constructed and transfected with Turbo Fect reagent. The mass-volume ratio of the cleavage plasmid to the donor plasmid to the Turbo Fect transfection reagent was 1g:1g:4L.

[0039] Once the transfection efficiency reached its peak, 0.01 MOI of PRV-SX-10-2015 virus solution was inoculated into each well. After incubation at 37°C with 5% CO2 for 2 hours, the medium was discarded and replaced with DMEM maintenance medium containing 2% FBS for continuous culture. As viral replication and homologous recombination occurred, the TK gene of the parental PRV-SX-10-2015 was knocked out, and a foreign gene expression cassette, ApxII-eGFP, carrying APXII was inserted at that site, ultimately generating the recombinant virus rPRV-ΔTK-APXII-eGFP intracellularly. To fully release the intracellular recombinant virus, the cells in the 6-well plate were subjected to a freeze-thaw cycle at -80°C and then at room temperature three times to lyse the cells. After the freeze-thaw cycle, the cells were centrifuged at 12000 rpm for 3 minutes to remove cell debris. The clear viral supernatant (virus solution) was aliquoted and stored at 80°C for long-term use.

[0040] BHK-21 cells were cultured in 6-well plates using standard methods. Once the cell density exceeded 80%, the harvested virus solution was serially diluted for inoculation. Each well was serially diluted 10-fold before infecting BHK-21 cells. Pre-prepared 1.8% low-melting-point agarose was thawed in a 72°C water bath. After thawing, it was mixed with 2×DMEM containing 4% serum and incubated in a 42°C water bath. Static adsorption was allowed for approximately 2 hours to ensure sufficient viral invasion of the cells, avoiding premature washing that could lead to virus loss. The cells were then gently washed with sterile PBS. Low-melting-point agarose and 2×DMEM were mixed at a 1:1 volume ratio and spread onto each well. After solidification in a clean bench for 5 minutes, the wells were incubated at 37°C with 5% CO2. Cytopathic effects and plaque formation were observed under a fluorescence inverted microscope after 24 hours.

[0041] Daily observations were then conducted until specific green fluorescent patches were clearly observed under a fluorescence microscope, indicating successful expression of the eGFP-labeled recombinant virus. Individual fluorescent positive spots were precisely selected under fluorescence microscopy, and the virus was enriched through freeze-thaw lysis. The spot-picking and purification steps were repeated multiple times until uniform green fluorescence was observed across the entire field of view, indicating successful purification of the recombinant virus plaques. Finally, dominant single spots were selected again and inoculated into BHK-21 cells for large-scale amplification. After harvesting the virus, PCR molecular identification (using the sequences shown in SEQ ID NO.15 and SEQ ID NO.16 as primers and the harvested virus as a template) verified the correct gene insertion. The identified recombinant virus was aliquoted and cryopreserved at 80°C, and formally named rPRV-ΔTK-APXII-eGFP.

[0042] Fluorescence microscope images as follows Figure 6As shown, only a small amount of recombinant virus expressing green fluorescence was observed in the F1 generation, with most cells infected by the parental PRV SX-10-2015; a large number of recombinant viruses expressing eGFP were observed in the F3 generation; and almost all cells in the F5 generation culture plate contained recombinant virus, with all fields of view showing green fluorescence, indicating that the recombinant virus rPRV-ΔTK-APXII-eGFP had been purified.

[0043] The purified recombinant virus rPRV-ΔTK-APXII-eGFP was identified by PCR and Western blot. The PCR results showed no characteristic band of the TK gene. Figure 7 The results indicated that the TK gene had been completely knocked out, and the presence of a characteristic band of the APXII gene suggested stable expression of the target gene. Western blot results showed a specific band at the expected location. All of these results confirmed the successful construction of the recombinant virus rPRV-ΔTK-APXII-eGFP. Figure 8 ).

[0044] (2) Construction and purification of recombinant virus rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry.

[0045] The procedure is the same as in (1) above, except that the vectors and inoculated strains are replaced. The cutting vector in (1) is replaced with pX335-gIgE-sgRNA-18-1686, and the donor plasmid is replaced with PUC19-gI / gE-CMV-SPZJ-E2-TM-mCherry-polyA. PRV-SX-10-2015 is no longer used for inoculation; instead, the recombinant virus rPRV-ΔTK-APXII-eGFP purified in (1) is used. The subsequent virus picking and purification procedures are performed according to the picking and purification method in (1). Viral plaques are observed under green and red light fields of view, and images of the plaques under two different fluorescence fields are taken using a microscope.

[0046] After imaging, the two fluorescence images were precisely superimposed using computer image processing software to locate the area where the two fluorescence beams overlapped. Viral plaques in this area were then aspirated using a sterile pipette. This plaque-picking and purification process was repeated 5-7 times until the entire field of view under the fluorescence microscope contained only the target virus plaques, with no other plaques or negative areas. The final purified target virus plaques were then seeded into BHK-21 cells for large-scale amplification. After amplification, the recombinant virus was identified by PCR (using the sequences shown in SEQ ID NO.15 and SEQ ID NO.16 as primers and the extracted recombinant virus genome as a template). After successful identification, the virus was aliquoted, labeled, and stored at -80°C for later use. The final recombinant virus was named rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry.

[0047] In the F1 generation, the red fluorescence appeared only as tiny clumps in the field of view, indicating that few viruses expressed mCherry, and most viruses expressed only eGFP; in the F3 generation, the cell community morphology observed in the red fluorescence and green fluorescence fields was similar, suggesting a significant increase in viral purity; in the F5 generation, all viruses simultaneously expressed both eGFP and mCherry, and the observations in the red fluorescence and green fluorescence fields were consistent and almost completely overlapped. Figure 9 ).

[0048] Example 6 Molecular identification of the recombinant virus rPRV-ΔTK-ApxII-eGFP-ΔgI / gE-E2-mCherry is as follows: (1) PCR identification.

[0049] The genomes of recombinant viruses rPRV-ΔTK-APXII-eGFP and rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry were extracted using kits, and the CSFV E2 gene was amplified by PCR using E2-Flag-F (sequence shown in SEQ ID NO.7) and E2-TM-R (sequence shown in SEQ ID NO.8) as primers.

[0050] The APP APXII gene was identified by PCR using APXII-HA-F (sequence shown in SEQ ID NO.17) and APXII-HA-R (sequence shown in SEQ ID NO.18) as primers. Furthermore, the recombinant virus was identified by PCR using designed specific primers for the TK and gI / gE genes (TK specific primers are shown in SEQ ID NO.21 and SEQ ID NO.22; gI / gE gene specific primers are shown in SEQ ID NO.23 and SEQ ID NO.24).

[0051] SEQ ID NO.21: CGCACTCTGTTCGACACGGA SEQ ID NO.22: GCTGATGTCCCCGACGATGA SEQ ID NO.23: TTTGGATCCATGCGGCCCTTTCTG SEQ ID NO.24: TTTGAATTCTTACGACACGGCGTCGCA PCR identification results showed that the recombinant virus rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry did not show the characteristic bands of the gI / gE gene, indicating that the gI / gE gene has been completely knocked out. Figure 10 The presence of a characteristic band of the E2 gene at the same time indicates that the target gene can be stably expressed. (2) Western Blot identification.

[0052] The recombinant virus was inoculated into PK-15 cells in 6-well cell culture plates. A PRV SX-10-2015 infection group and a PK-15 cell blank control group were simultaneously set up. When 60-70% of the PK-15 cells showed cytopathic effects, the culture medium was slowly aspirated, and the cells were washed twice with sterile PBS. 200 µL of cell lysis buffer and 5 µL of protease inhibitor were added to each well, and the mixture was gently mixed for 1-2 min. The lysis buffer was then aspirated. Protein loading buffer was added to the aspirated lysis buffer and mixed thoroughly. The mixture was then boiled at 100°C for 10 min and cooled on ice for 2 min before use.

[0053] When identifying the expression of the target protein using Western blot, anti-Flag monoclonal antibody and anti-HA monoclonal antibody were used as primary antibodies (dilution ratio 1:1000), and horseradish peroxidase-labeled goat anti-mouse IgG was used as secondary antibody (dilution ratio 1:5000).

[0054] Western blot results showed specific bands at the expected locations. All of the above results indicate that the recombinant virus rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry was successfully constructed. Figure 11 ).

[0055] Example 7 The biological characteristics of the recombinant virus rPRV-ΔTK-ApxII-eGFP-ΔgI / gE-E2-mCherry were determined as follows: (1) Determination of the titer of recombinant virus.

[0056] PK-15 cells were uniformly seeded into 96-well cell culture plates and incubated statically under standard cell culture conditions until the cells covered the bottom of the wells and formed a complete and dense monolayer. The recombinant viral strain rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry was then serially diluted 10-fold to prepare 10 different concentrations of viral dilution. Each dilution was seeded into eight parallel replicates in the same row, with 100 µL of cell maintenance medium added to uninoculated wells as a negative control. The cells were gently mixed and returned to an incubator for further incubation. Cell status in each well was observed under a microscope at fixed time intervals for three consecutive days, and the number of positive wells showing typical cytopathic effects (CPE) in each row was precisely recorded. After the observation period, all data were compiled and statistically analyzed, and the median tissue infectious dose (TCID) of the recombinant virus was accurately determined and calculated strictly according to the classic Reed-Muench statistical method. 50 ).

[0057] On the first day, 10 were observed to have been vaccinated. 1 10 2 10 3 CPE was observed in all 8 wells of the diluted virus solution; on the second day, cells inoculated at 10... 4 10 5 CPE was observed in all 8 wells of the cell culture with the diluted virus solution. 6 Five wells showed obvious CPE at the dilution factor; 10 was observed on the third day. 7 At dilution factor 10, obvious CPE was observed in 3 wells. 6 Despite the dilution factor, CPE was observed in 5 wells. Following strict adherence to the classic Reed-Muench statistical method, the median tissue infectious dose (TCID) of this recombinant virus was accurately determined and calculated. 50 ) is 10 6.5 .

[0058] (2) Stability determination of recombinant virus.

[0059] Monolayers of PK-15 cells were prepared, and the constructed recombinant virus was inoculated into these cells and cultured continuously at 37°C and 5% CO2 for a total of 20 passages. During passage, the recombinant virus solutions from passages 5, 10, and 20 were collected, and total viral genomic DNA was extracted. Using the obtained total viral genomic DNA as a template, PCR identification of CSFV E2 was performed using primers E2-Flag-F (sequence shown in SEQ ID NO. 7) and E2-TM-R (sequence shown in SEQ ID NO. 8); PCR identification of APP APXII was performed using primers APXII-HA-F (sequence shown in SEQ ID NO. 17) and APXII-HA-R (sequence shown in SEQ ID NO. 18). PCR identification verified the presence and integrity of the target gene in each generation of recombinant virus, thereby determining the genetic stability of the recombinant virus during continuous passage.

[0060] The results are as follows Figure 12 As shown, no bands were observed in the TK and gI / gE genes by PCR electrophoresis, indicating that the two genes were stably deleted; the APXII and E2 genes both showed specific bands at the expected positions, indicating that the two genes were stably inserted.

[0061] (3) Determination of the one-step growth curve of recombinant virus.

[0062] Recombinant virus rPRV-ΔTK-eGFP-ΔgI / gE-mCherry, parental virus PRV SX-10-2015, and recombinant virus rPRV-ΔTK-APXII-eGFP-ΔgI / gE-E2-mCherry were selected, all at a ratio of 10... 5 TCID 50 The appropriate dose was inoculated into monolayers of PK-15 cells. After inoculation, the cells were incubated under suitable conditions for approximately 2 hours until the virus was fully adsorbed. The cells were then washed three times with sterile PBS to remove unadsorbed virus and the original culture medium. Subsequently, 1 mL of fresh 2% DMEM medium was added to each well for continued culture. Starting from the start of inoculation, the supernatant was collected from the culture plate every 12 hours for a total of 72 hours. The collected viral supernatant at each time point was repeatedly freeze-thawed three times to fully release the virus, and then inoculated into monolayers of PK-15 cells in 96-well plates. The TCID of the virus at each time point was calculated using the Reed-Muench method. 50 The values ​​were used to plot the one-step growth curve of the recombinant virus.

[0063] The results are as follows Figure 13As shown, the three strains exhibited the same proliferation trend, all rapidly proliferating and reaching peak titers within 48 hours post-infection, followed by a slow decline. Compared to PRV SX-10-2015, the three-gene deletion strain and the recombinant virus showed slightly lower cell replication rates, but there was no significant difference between them, suggesting that the insertion of exogenous genes does not significantly affect viral replication.

[0064] Example 8 The biosafety assay for the recombinant virus rPRV-ΔTK-ApxII-eGFP-ΔgI / gE-E2-mCherry is as follows: (1) APP pathogenicity test.

[0065] APP S15 strain was purified by streaking on TSA-YE plates. After incubation at 37°C for 24 h, single colonies with uniform morphology and neat edges were picked and inoculated onto TSB-YE liquid medium. After shaking incubation at 37°C for 24 h, a uniformly turbid bacterial suspension was obtained. The supernatant was discarded by centrifugation, and the bacterial cells were resuspended in physiological saline to prepare a bacterial suspension. After serial dilutions of 10-fold, the suspension was plated onto TSA-YE plates and incubated upside down at 37°C for 24 h. Plates with single colony counts in the range of 30-300 were selected for counting, and the final concentration of the original bacterial suspension was determined to be 3 × 10⁻⁶. 10 CFU / mL, the bacterial concentrations after dilution in each experimental group were 1.5 × 10⁻⁶. 11 CFU / mL, 3×10 10 CFU / mL, 3×10 9 CFU / mL, 3×10 8 The CFU / mL concentration meets the appropriate concentration requirements set in the preliminary experiment.

[0066] Five groups of healthy, 6-week-old SPF-grade female Kunming mice (weighing 18-22g) were intraperitoneally injected with 0.2 mL of the APP solution. Four mice in each group served as a blank control and were intraperitoneally injected with an equal volume of TSB-YE medium (containing 5% newborn calf serum and 2% NAD). Morbidity and mortality were continuously observed and recorded in each group. The median lethal dose (LD50) of APP in SPF-grade mice was calculated using Formula I. 50 ).

[0067] logLD 50 =X k -i(∑P-0.5) (Formula I), Where X k ∑P represents the logarithmic dose of the highest dose group, i represents the logarithm of the dilution factor, and ∑P represents the sum of the mortality probabilities of each group.

[0068] The calculation results are shown in Table 1. The LD50 of this APP strain in SPF mice... 50 It is 1.685×10 9The CFU / mL level indicates that this strain is highly pathogenic to mice.

[0069] Table 1 Results of APP toxicity test in mice

[0070] There were significant differences in the incidence and mortality of mice in each group. The mice in the blank control group showed no symptoms of disease throughout the experiment, were in good mental condition, had a normal diet, and were active. There were no deaths during the experiment, and no abnormal lesions were found in any organs after necropsy.

[0071] In the experimental group of mice, the onset time after inoculation was positively correlated with the bacterial concentration; the high concentration group (1.5×10⁻⁶) showed a higher incidence. 11 Mice with a CFU / mL concentration showed obvious symptoms 12-24 hours after inoculation, including lethargy, immobility, loss of appetite, and ruffled fur, accompanied by respiratory distress and abdominal distension. All mice died within 24 hours of inoculation, resulting in a 100% mortality rate. (3×10⁻⁶) 10 Mice in the CFU / mL group developed symptoms 12-24 hours after inoculation, consistent with those in the high-concentration group. All mice died within 24 hours of inoculation, resulting in a 100% mortality rate. (3×10) 9 The onset of illness in mice in the CFU / mL group was delayed to 24-36 hours post-inoculation, and the symptoms were relatively mild. Some mice exhibited lethargy and decreased appetite. Three mice died within 7 days post-inoculation, resulting in a mortality rate of 75%. (3×10) 8 Mice in the CFU / mL group only showed transient lethargy 48-72 hours after inoculation, with no other obvious symptoms. There were no deaths during the experiment, with a mortality rate of 0%. Autopsy of the deceased mice in each group yielded the following results: Figure 14 As shown, the lungs are enlarged, dark red, and have become solid and inelastic, with diffuse hemorrhages on the lung surface; the spleen is significantly enlarged, soft, and dark red; the liver is enlarged, pale yellow or dark red in color, and brittle in texture, with scattered grayish-white necrotic foci and ecchymoses on the surface, and subcapsular hemorrhage.

[0072] (2) Evaluation of mouse immune safety.

[0073] Fifty healthy 6-week-old female Kunming mice were randomly divided into 10 groups of 5 mice each. One group of mice received an intramuscular injection of 100 µL of 1×10⁻⁶ titer in their hind limbs. 5 TCID 50 Mice in groups 2 and 3 were administered 0.1 mL of PRV SX-10-2015 via intramuscular injection in the hind limbs; 100 µL of a 1×10⁻⁶ titer was administered to mice in these groups. 5 TCID 50 / 0.1mL of rPRV-ΔTK-eGFP-ΔgI / gE-mCherry; mice in groups 4 and 5 were injected intramuscularly with 100µL of a 1×10⁻⁶ titer in their hind limbs. 5 TCID50 Mice were injected with 0.1 mL of recombinant virus rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry; groups 6 and 7 were injected with 100 µL of DMEM as negative controls; group 8 was injected with 100 µL of purchased trivalent inactivated APP vaccine as an APP vaccine control; group 9 was injected with an equal volume of PRV Bartha-K61 as a PRV vaccine control; group 10 received no treatment and served as a blank control. Mice in each group were isolated and housed separately. Clinical symptoms and survival status were observed and recorded daily, and survival curves were plotted. Dead mice were promptly dissected, and brain, lung, liver, and spleen tissues were collected for subsequent viral load detection. Simultaneously, brain, lung, and liver tissues were fixed in 4% paraformaldehyde and sent to a biotechnology company for histopathological section preparation.

[0074] Survival rates of mice in each group after immunization were as follows: Figure 15 As shown, mice in group 1 of the PRV SX-10-15 variant test began to die on day 3 after injection, and all died within 4 days; while the survival rate of groups 4-5, 9, and 10 (recombinant virus group, PRV Bartha-K61 vaccine group, and DMEM control group) was 100% during the 7-day observation period, and no obvious clinical symptoms were observed. These results indicate that the recombinant virus strain rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry prepared in this invention has high safety.

[0075] (3) Detection of specific antibody levels in mouse serum.

[0076] Following the initial immunization, blood was collected weekly from the orbital venous plexus of mice. Serum was separated by centrifugation at 1700×g for 5 min and properly stored. gB antibody detection was performed using the Yisenbao PRV gB antibody detection kit. The procedure was identical to the instructions, except that the secondary antibody was replaced with a 1:5000 diluted goat anti-mouse IgG. Simultaneously, E2-specific antibodies were detected using the CSFV E2 ELISA method, and the OD was set. 450nm A value ≥0.2198 was used as the positive criterion; APXII-specific antibodies were detected using the APP APXII ELISA method, and the OD value was set. 450nm A value ≥0.2788 is the criterion for a positive result.

[0077] PRV gB antibody test results are as follows Figure 16 As shown, both the experimental group and the PRV Bartha-K61 vaccine immunization group showed antibody seroconversion on day 7 after the first immunization, and the antibody levels were relatively low at this time; after the second immunization, the antibody levels in both groups increased rapidly. There was no significant difference in gB antibody levels between the recombinant virus strain immunization group and the three-gene deletion strain immunization group, while the antibody level in the PRV Bartha-K61 vaccine control group was significantly lower than that in the first two groups.P <0.05%. APP APX II antibody detection results showed that both the experimental group and the vaccine control group showed antibody seroconversion 14 days after the first immunization, with relatively low antibody levels. Antibody levels increased significantly after the second immunization. There was no significant difference in APX II antibody levels induced by the recombinant virus strain and the commercial vaccine. P >0.05). CSFV E2 antibody test results showed that the experimental group showed antibody seroconversion 14 days after the first immunization, with low antibody levels; after the second immunization, antibody levels rose rapidly, entered a stable rising phase after 28 days, and reached a peak at 35 days.

[0078] (4) Detection of neutralizing antibody titer in mouse serum.

[0079] ① Detection of anti-CSFV neutralizing antibody levels in mouse serum.

[0080] PK-15 cells were seeded into 96-well cell culture plates and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2 until the cell monolayer confluence was ≥95%. The cells were washed twice with sterile PBS before use.

[0081] The serum samples collected in (3) were placed in a 56°C water bath for 30 min to inactivate the virus. After inactivation, the serum was serially diluted 2-fold in a 96-well plate, with a dilution range of 1:2 to 1:256. Each dilution was used in 8 replicates, with a final volume of 50 μL per well. The experiment also included the following controls: serum toxicity control (only serum samples collected in (3) were added, without virus), negative control (a mixture of DMEM and virus), and cell blank control (only DMEM was added).

[0082] Dilute the CSFV virus to 200 TCID. 50 Add 50 μL of serum to each well containing diluted serum and incubate at 37°C and 5% CO2 for 1 hour. After incubation, transfer the serum-virus mixture to the prepared PK-15 cell monolayer, allow it to adsorb for 1 hour, discard the supernatant, wash the cells three times with PBS, and then continue culturing at 37°C and 5% CO2 for 48-72 hours.

[0083] Using the SYBR Green I RT-qPCR method for classical swine fever virus (CSFV) as the criterion, the Reed-Muench method was used to calculate the neutralizing antibody titer; the half-maximal protective titer (PD) was calculated based on the cumulative protection rate and the non-protection rate. 50 The reciprocal of this is the final titer of the neutralizing antibody in the sample.

[0084] ② Detection of anti-PRV neutralizing antibody levels in mouse serum.

[0085] BHK-21 cells were seeded into 96-well cell culture plates and cultured until the cell monolayer confluence was ≥95%. The cells were washed twice with sterile PBS before use.

[0086] The serum samples collected in (3) were placed in a 56°C water bath for 30 min to inactivate the virus. After inactivation, the serum was serially diluted 2-fold in a 96-well plate, with a dilution range of 1:2 to 1:256. Each dilution was used in 8 replicates, with a final volume of 50 μL per well. The experiment also included the following controls: serum toxicity control (only serum samples collected in (3) were added, without virus), negative control (a mixture of DMEM and virus), and cell blank control (only DMEM was added).

[0087] Dilute PRV SX-10-2015 virus to 200 TCID. 50 Add 50 μL of serum to each well containing diluted serum and incubate at 37°C and 5% CO2 for 1 hour. After incubation, transfer the serum-virus mixture to a BHK-21 cell monolayer washed twice with sterile PBS. After adsorption for 1 hour, discard the supernatant, wash the cells three times with PBS, and then continue culturing at 37°C and 5% CO2 for 48-72 hours.

[0088] The neutralizing antibody titer was calculated using the Reed-Muench method, with complete inhibition of CPE as the criterion; the half-maximal protective titer (PD) was calculated based on the cumulative protection rate and the non-protection rate. 50 The reciprocal of this is the final titer of the neutralizing antibody in the sample.

[0089] ③ To evaluate the efficacy of neutralizing antibodies induced by the recombinant virus strain rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry, serum samples were collected from mice on days 0, 14, and 28 after the first immunization. The levels of PRV and CSFV-specific neutralizing antibodies in mouse serum were detected by neutralization experiments. The results showed that both the recombinant viral strains rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry and rPRV-ΔTK-eGFP-ΔgI / gE-mCherry (a triple-gene deletion strain) produced high levels of PRV neutralizing antibodies after immunization of mice, and these levels increased significantly over time, showing a significant increase at 14 days compared to day 0, reaching peak values ​​of 1:39.89 and 1:38.34 at 28 days, respectively. According to the CSFV neutralizing antibody detection results, the recombinant viral strain rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry began to produce neutralizing antibodies 14 days after immunization, reaching a peak value of 1:21.46 at 28 days. However, the triple-gene deletion strain and the DMEM control group did not produce neutralizing antibodies against CSFV during immunization. Figure 17As shown, the recombinant viral strain rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry can efficiently induce neutralizing antibodies against CSFV and PRV.

[0090] (5) Protection test of mice after immunization against viral infection.

[0091] Two weeks after the initial immunization, mice in groups with a higher safety profile were given a second immunization, with the same method and dosage as the first immunization. Two weeks after the second immunization, mice in groups 2, 4, 6, and 8 were intraperitoneally injected with 5 times the median lethal dose of APP. Mice in groups 3, 5, 7, and 9 were intramuscularly injected with 100µL of 1×10⁻⁶ APP in their hind legs. 5 TCID 50 / 0.1mL of PRV SX-10-2015; similarly, each experimental group was kept in separate isolation and the clinical symptoms and survival status of mice were observed daily.

[0092] Fourteen days after the second immunization, mice in groups 2, 4, 6, and 8 were challenged with APP. All mice in the negative control group and the group with the three-gene deletion strain rPRV-ΔTK-eGFP-ΔgI / gE-mCherry died within one day. Two mice in the group with the recombinant virus strain rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry died within 2 days. One mouse in the commercial inactivated vaccine group died on day 2. These results indicate that the protective effect of the recombinant virus rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry was not significantly different from that of the commercial inactivated vaccine. The survival rates of mice in each group were as follows: Figure 18 .

[0093] Fourteen days after the second immunization, mice in groups 3, 5, 7, and 9 were challenged with the PRV SX-10-2015 strain. Following challenge, all mice in the negative control group died within 4 days; mice in the PRV Bartha-K61 vaccine group began dying on day 3, with one death on days 4 and 5, and only one surviving one week after challenge, indicating a 20% protective efficacy of the vaccine in mice. Mice immunized with the recombinant virus strain and the three-gene deletion strain only showed symptoms of ruffled fur 3-4 days after challenge, gradually recovering from day 5 onwards, with no deaths during the challenge period. The results indicate that both the recombinant virus strain rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry and the three-gene deletion strain rPRV-ΔTK-eGFP-ΔgI / gE-mCherry can provide 100% immune protection in mice, and the survival rates of each group are as follows: Figure 19 .

[0094] (6) Distribution of load in each tissue.

[0095] After PRV challenge, 0.1g each of brain, lung, and liver tissues from mice were placed in 2mL grinding tubes, 1mL of PBS was added, and the tissues were thoroughly ground. After three freeze-thaw cycles, the tissues were centrifuged at 12000r / min for 5 minutes. 300µL of the supernatant was collected to extract DNA. Using the extracted DNA as a template, the viral load in each tissue was detected using laboratory-preserved primers gD-F (sequence shown in SEQ ID NO.25) and gD-R (sequence shown in SEQ ID NO.26), as detailed below: ① Distribution of Actinobacillus pleuropneumoniae load in lung tissue.

[0096] After APP infection, 0.1g of mouse lung tissue was placed in a 2mL grinding tube, and 1mL of PBS was added to thoroughly homogenize the tissue. The homogenate was then divided into 10... 1 -10 7 A 10-fold serial dilution was performed, and 100 μL of bacterial culture from each dilution was inoculated onto TSA-YE plates. Each dilution was replicated in triplicate and incubated upside down at 37°C for 24 h. After incubation, single colony counts were performed, and the bacterial load in the lung tissue of mice in each group was calculated.

[0097] SEQ ID NO.25: TTATCGAGTACGCCGACTGC SEQ ID NO.26: CCTCCGTGGGGAACATGTAG ② To investigate the immunoprotective efficacy of the recombinant virus rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry vaccine against PRV SX-10-2015 strain, mice were challenged 14 days after secondary immunization, and PRV load in brain, lung, and liver tissues was measured 7 days post-challenge (all mice in the DMEM group died 4 days post-challenge, and samples were collected post-mortem). Results are as follows: Figure 20 As shown: It can be seen that the viral load in various tissues of mice in the DMEM group was significantly higher than that in other immunized groups after challenge. P <0.0001); the viral load in the tissues of mice in the PRV Bartha-K61 group was significantly higher than that in the groups immunized with recombinant virus strains and triple gene deletion strains ( P <0.001. There was no significant difference between the recombinant viral strain and the triple-gene deletion strain. P >0.05). In summary, the recombinant virus rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry vaccine has a significant effect on inhibiting viral replication in brain, lung, and liver tissues, and its protective effect is superior to that of the traditional Bartha-K61 vaccine.

[0098] ③ To investigate the immunoprotective efficacy of the recombinant virus rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry vaccine against APP, mice were challenged 14 days after secondary immunization, and the bacterial load in the lungs was measured 7 days post-challenge (all mice in the DMEM group and the three-gene deletion strain immunization group died 1 day post-challenge; samples were collected post-mortem). Results are as follows: Figure 21 As shown: There was no significant difference in the bacterial load in the lungs between the recombinant virus immunization group and the commercial vaccine group. P >0.05), and both were significantly lower than the DMEM group and the three-gene deletion strain immunization group ( P <0.001). In summary, the recombinant virus rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry vaccine can effectively inhibit the proliferation of APP in the lungs of mice, and its protective efficacy is comparable to that of traditional trivalent inactivated vaccines.

[0099] (7) Observation of pathological tissue sections of the attacking APP.

[0100] Fourteen days after the second immunization, mice in groups 2, 4, 6, and 8 were intraperitoneally injected with five times the median lethal dose of APP. Each group was kept in isolated isolation, and clinical symptoms and survival rates were recorded daily. Sample collection: One week after challenge, surviving mice were euthanized, and lung tissue was collected after dissection. The tissue was immediately fixed in 4% paraformaldehyde at room temperature for 24 hours, and then sent to a biotechnology company for paraffin embedding and H&E staining.

[0101] Pathological tissue section observation of PRV challenge.

[0102] Fourteen days after the second immunization, mice in groups 3, 5, 7, and 9 were injected intramuscularly with 1×10⁻⁶ mg / L of the drug via the hind limb. 5 TCID 50 / 0.1mL of the PRVSX-10-2015 mutant strain (0.1mL / mouse) was administered to each group in independent isolation. Clinical symptoms and survival rates were recorded daily. Sample collection: One week after challenge, surviving mice were euthanized, and liver tissue was collected after dissection. The liver tissue was immediately fixed in 4% paraformaldehyde at room temperature for 24 hours, and then sent to Wuhan Saiwei Biotechnology Co., Ltd. for paraffin embedding, sectioning, and H&E staining.

[0103] To evaluate the protective effect of the recombinant viral rPRV-ΔTK-APXⅡ-eGFP-ΔgI / gE-E2-mCherry vaccine against histopathological damage in mice challenged with the APP strain, lung tissues were collected from all mice in the DMEM control group and the three-gene deletion strain rPRV-ΔTK-eGFP-ΔgI / gE-mCherry immunization group after all mice had died. Histopathological sections were prepared, and the pathological damage in each tissue was observed. Figure 22The results showed that the lungs of mice in the DMEM control group and the mice in the three-gene deletion strain immunization group showed different degrees of pathological damage: the lungs were characterized by destruction of alveolar structure, consolidation and diffuse hemorrhage in lung tissue, inflammatory cell infiltration in alveolar cavities and thickening of alveolar walls.

[0104] Therefore, the recombinant pseudorabies trivalent vaccine provided by this invention is rPRV-ΔTK-ApxII-eGFP-ΔgI / gE-E2-mCherry, using PRV as the parent strain and carrying two protective antigens, CSFV E2 and APP ApxII. A single immunization can induce three antibodies against PRV, CSFV, and APP simultaneously, effectively reducing the number of immunizations, labor intensity, immunization costs, and stress on the pig herd; it has high safety; significant humoral immunity, with a significant increase after a second immunization; PRV neutralizing antibody titer reaches 1:39.89, and CSFV neutralizing antibody titer reaches 1:21.46; it has definite protective effect against challenge; and it has excellent genetic and proliferative stability.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A recombinant pseudorabies trivalent vaccine, characterized in that: The recombinant pseudorabies trivalent vaccine is rPRV-ΔTK-ApxII-eGFP-ΔgI / gE-E2-mCherry.

2. The recombinant pseudorabies trivalent vaccine according to claim 1, characterized in that: The recombinant pseudorabies trivalent vaccine was prepared using pseudorabies virus PRV SX-10-2015 as the parent strain. It was made by deleting three virulence genes, TK, gI, and gE, and inserting optimized APP ApxII and CSFV E2 gene coding sequences into the deletion sites, respectively.

3. The recombinant pseudorabies trivalent vaccine according to claim 1, characterized in that: The optimized APPApxII gene coding sequence is shown in SEQ ID NO.5; the optimized CSFV E2 gene coding sequence is shown in SEQ ID NO.

2.

4. A method for preparing a recombinant pseudorabies trivalent vaccine as described in any one of claims 1-3, characterized in that, The steps are as follows: S1 and BHK-21 cells were co-transfected with pX335-TK-sgRNA plasmid and TK site donor plasmid, and inoculated with parental strain PRV SX-10-2015. The TK gene was knocked out and inserted into the ApxII-eGFP expression cassette. After multiple rounds of purification with green fluorescent plaques, rPRV-ΔTK-ApxII-eGFP was obtained. S2. Using rPRV-ΔTK-ApxII-eGFP as the parent, gI / gE targeting sgRNA plasmid and gI / gE site donor plasmid were co-transfected. The gI / gE gene was knocked out and inserted into E2-mCherry at a specific site. After screening for plaques with red and green fluorescence, the final strain rPRV-ΔTK-ApxII-eGFP-ΔgI / gE-E2-mCherry was obtained after 5-7 generations of continuous purification.

5. The method for preparing a recombinant pseudorabies trivalent vaccine according to claim 4, characterized in that, The donor plasmid for the TK site in S1 is pUC19-TK-CMV-HA-APXII-eGFP-polyA, and its construction method is as follows: The optimized APP ApxII gene coding sequence was sent to the company for synthesis to obtain the recombinant pET-45b(+) vector with the target sequence inserted. Using the recombinant pET-45b(+) vector as a template, two rounds of PCR amplification were performed using APXII-HA-F and APXII-HA-R, CMV-HA-F and GSG-HA-R as primers, respectively. The 2967bp target fragment HA-APXII was recovered by gel extraction. use Nhe I and Sph Double digestion of pUC19-TK-CMV-EGFP-polyA with enzyme I, gel recovery of the 7156bp fragment, and seamless cloning and ligation with the HA-APXII fragment are performed.

6. The method for preparing a recombinant pseudorabies trivalent vaccine according to claim 5, characterized in that: The APXII-HA-F sequence is shown in SEQ ID NO.17; the APXII-HA-R sequence is shown in SEQ ID NO.18; the CMV-HA-F sequence is shown in SEQ ID NO.19; and the GSG-HA-R sequence is shown in SEQ ID NO.

20.

7. The method for preparing a recombinant pseudorabies trivalent vaccine according to claim 4, characterized in that, The donor plasmid for the gI / gE site in S2 is pUC19-gI / gE-CMV-SPZJ-E2-TM-mCherry-polyA; the construction method is as follows: The optimized CSFV E2 gene coding sequence was sent to the company for synthesis to obtain the recombinant pET-30a(+) vector containing the target sequence. Using the recombinant pET-30a(+) vector as a template, two rounds of PCR amplification were performed using E2-Flag-F and E2-TM-R, and CMV-SPZJ-F and GSG-TM-R as primers, respectively. The 1235bp target fragment SPZJ-E2-TM was recovered by gel extraction. Using pUC19-gI / gE-CMV-mCherry-polyA as a template, PCR amplification was performed using AMP-2283-F and CMV-R as primers. The amplification product was then recovered by gel to obtain the AMP-CMV-3447 fragment. Using pUC19-gI / gE-CMV-mCherry-polyA as a template, PCR amplification was performed using GSG-F and AMP-2000-R as primers. The amplification product was then recovered by gel to obtain the GSG-AMP-3548 fragment. The SPZJ-E2-TM, GSG-AMP-3548, and AMP-CMV-3447 fragments can be seamlessly cloned and joined.

8. The method for preparing a recombinant pseudorabies trivalent vaccine according to claim 7, characterized in that: The E2-Flag-F sequence is shown in SEQ ID NO.7; the E2-TM-R sequence is shown in SEQ ID NO.8; the CMV-SPZJ-F sequence is shown in SEQ ID NO.9; the GSG-TM-R sequence is shown in SEQ ID NO.10; the GSG-F sequence is shown in SEQ ID NO.11; the CMV-R sequence is shown in SEQ ID NO.12; the AMP-2283-F sequence is shown in SEQ ID NO.13; and the AMP-2000-R sequence is shown in SEQ ID NO.

14.

9. The application of the preparation method of the recombinant pseudorabies trivalent vaccine according to any one of claims 5-8 in the prevention and control of infectious diseases, characterized in that: The disease is one or more of the following: swine pseudorabies, classical swine fever, and swine infectious pleuropneumonia.

10. The application of the recombinant pseudorabies trivalent vaccine as described in any one of claims 1-3 in the prevention and control of infectious diseases, characterized in that: The disease is one or more of the following: swine pseudorabies, classical swine fever, and swine infectious pleuropneumonia.

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