Preparation method and application of PRRS replication-defective vaccine PRRSV-△ORF5a strain

Abstract

This invention discloses a method for preparing and applying a porcine reproductive and respiratory syndrome (PRRS) replication-deficient vaccine strain rPRRSV-△ORF5a. The rPRRSV-△ORF5a strain replicates normally and exhibits a high viral titer in Marc-145 cell lines stably expressing the PRRSV ORF5a protein, and can only be used for a single infection in ordinary Marc-145 cell lines. Animal experiments have demonstrated that the replication-deficient virus strain rPRRSV-△ORF5a can resist infection with its homologous virulent strain, providing immunoprotective effects. This vaccine strain holds promise as a safe and effective novel PRRSV vaccine, effectively addressing the shortcomings of inactivated PRRSV vaccines, such as their inability to induce cellular immunity, while also resolving safety issues associated with attenuated PRRSV vaccines.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to the preparation method and application of a PRRS replication-deficient vaccine strain rPRRSV-△ORF5a lacking the ORF5a protein. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a contagious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV). Clinically, it is characterized by reproductive disorders in sows, such as fever, anorexia, abortion, stillbirth, and weak piglets, as well as respiratory symptoms and high mortality in piglets, severely impacting the pig industry. PRRSV is constantly mutating and evolving. Since the summer of 2006, PRRS caused by PRRSV variants has caused severe economic losses to the pig industry. In recent years, NADC30-like, NADC34-like, and various recombinant PRRSV strains have continued to plague the pig industry.

[0003] Vaccination is currently the most effective measure to prevent PRRSV infection. Commercially available PRRS vaccines include inactivated vaccines and live attenuated vaccines. The effectiveness of inactivated PRRS vaccines is not entirely clear because they cannot effectively induce the production of neutralizing antibodies; their advantage is good safety, but they have disadvantages such as high immunization doses, multiple immunizations, and a long period of immunity development. Reports indicate that challenge with inactivated vaccines does not protect piglets from PRRSV infection, and the duration of viremia and viral titer after infection are not significantly different from the non-immunized group. Live attenuated PRRS vaccines can effectively induce cellular and humoral immunity in pigs, providing effective immune protection. After vaccination, antibodies are produced quickly and last a long time, providing strong protection. However, live attenuated vaccines also have drawbacks. Studies have shown that the vaccine virus can infect fetuses through the placenta and spread to unvaccinated sows, or cause acute PRRS syndrome symptoms. Therefore, the safety of live attenuated PRRS vaccines is one of the important reasons hindering their widespread use.

[0004] While some existing technologies include studies on the deletion of non-structural proteins and even structural proteins, these studies still have the following problems: For example, CN103773740A omits the nsp9 protein, but since nsp9 is only a non-structural protein, its immune effect is limited; while CN118086229A claims to have GP3 missing, it lacks important follow-up animal experiments for verification, thus its immune effect cannot be verified. Moreover, the viral backbones in existing technologies are non-commercial attenuated vaccines, posing certain safety risks. Furthermore, previous research on structural proteins has been limited to point mutation studies, lacking research on large-fragment deletions, and the role of structural proteins with large-fragment deletions in the viral immune process remains a research gap. Summary of the Invention

[0005] This invention aims to address the problems of poor immunogenicity of existing commercially available inactivated PRRS vaccines and persistent virus carriage, intermittent shedding, and virulence reversion in PRRS attenuated vaccines. It provides a PRRS replication-deficient vaccine candidate strain, rPRRSV-△ORF5a, for the prevention and eradication of PRRSV. This vaccine strain is based on the commercially available attenuated PRRSV vaccine strain HuN4-F112, but lacks the first ten bases of the ORF5a protein in the HuN4-F112 genome. While maintaining the amino acid sequence of other proteins, the ORF5a protein is not expressed. Therefore, this defective vaccine strain cannot replicate in normal Marc-145 cells and can only be proliferated using Marc-145 cell lines expressing the PRRSV ORF5a protein. This effectively overcomes the shortcomings of existing commercial vaccines and holds promise as a safe and effective novel PRRS vaccine.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: In one aspect of the invention, a mutant of the commercially available attenuated PRRSV vaccine strain HuN4-F112 is provided, wherein the mutant is formed by deleting the first ten bases of the ORF5a protein in the genome of the infectious clone pSK-HuN4-F112, ensuring that the amino acid sequence of the GP5 protein remains unchanged but the ORF5a protein cannot be expressed.

[0007] In another aspect of the invention, a recombinant vector comprising a genomic nucleic acid molecule of HuN4-F112 is also provided.

[0008] In another aspect of the present invention, a method for preparing a porcine reproductive and respiratory syndrome virus (PRRSV) attenuated vaccine strain is also provided, comprising the following steps: Based on the infection clone of the PRRS vaccine strain HuN4-F112, the first ten bases of the ORF5a protein in the HuN4-F112 genome were deleted to construct an ORF5a protein-deficient replicon. The mutant replicon vector was linearized and transcribed into viral RNA in vitro. The viral RNA was then transfected into Marc-145 cells that stably express the PRRSV ORF5a protein for virus rescue, resulting in the PRRS replication-deficient vaccine strain rPRRSV-△ORF5a.

[0009] The PRRS replication-deficient vaccine strain rPRRSV-△ORF5a of this invention replicates normally and exhibits a high viral titer in Marc-145 cell lines stably expressing the PRRSV ORF5a protein, whereas it only allows for a single infection and replication in ordinary Marc-145 cell lines. This vaccine strain holds promise as a safe and effective novel PRRS vaccine, effectively addressing the shortcomings of inactivated PRRS vaccines, such as their inability to induce cellular immunity, and resolving safety issues associated with attenuated PRRS vaccines, such as persistent viral load, intermittent viral shedding, and virulence reversion. Therefore, the PRRS replication-deficient vaccine strain of this invention is expected to be widely used in livestock production. Attached Figure Description

[0010] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0011] Figure 1 This is a schematic diagram illustrating the construction of the ORF5a protein deletion replicon of the PRRS attenuated vaccine strain HuN4-F112 in Example 1 of the present invention; Figure 2 This is a nucleic acid electrophoresis result of the construction process of the ORF5a protein deletion replicon of the PRRS attenuated vaccine strain HuN4-F112 in Example 1 of the present invention. In this diagram, a represents fragments A and B obtained by overlapping PCR; b represents the gene fragment with the first ten bases of the ORF5a protein deleted; and c represents the restriction endonuclease. EcoR V and MluI Image showing the results of enzyme digestion of the infectious clone pSK-HuN4-F112; Figure 3 This is the CPE of the replication defective virus rPRRSV-△ORF5a in Embodiment 2 of the present invention; Figure 4 This is the sequencing result of the 5th generation of the replication-defective virus rPRRSV-△ORF5a from Example 2 of this invention; Figure 5 This is the result of IFA detection of rPRRSV-△ORF5a in Marc-145-ORF5a cell line and Marc-145 cell line in Example 2 of the present invention; Figure 6This is the result of Western blot analysis of rPRRSV-△ORF5a in Marc-145-ORF5a cell line and Marc-145 cell line in Example 2 of this invention. Figure 7 This is the sequence alignment result of the 5th, 10th, 15th, and 20th generations of the replication-defective virus rPRRSV-△ORF5a in Example 3 of the present invention; Figure 8 This is the result of virus titer determination of the replication-defective virus rPRRSV-△ORF5a at passages 5, 10, 15, and 20 in the corresponding cell lines and Marc-145 cells in Example 3 of the present invention; Figure 9 This is the growth curve of the replication-defective virus rPRRSV-△ORF5a and the parent virus HuN4-F112 in the corresponding complementary cell lines of Example 3 of the present invention; Figure 10 The specific antibody recognizes the replication-defective rPRRSV-△ORF5a strain of this invention, without producing a positive reaction with other common PRRSV strains. In this sample, 1 is the rPRRSV-△ORF5a virus solution, and 2-7 are positive samples prepared simultaneously for the rHuN4-F112 strain, CH-1a strain, JXA1 strain, NADC30-like strain, NADC34-like strain, and QYYZ strain, respectively.

[0012] Figure 11 This invention evaluates the immunogenicity of the replication-defective virus rPRRSV-△ORF5a strain, where A represents the viral load of PRRSV in the blood, B is the survival curve, and C represents the lung lesions. Detailed Implementation

[0013] In the following examples, experimental methods without specific conditions are generally performed under conventional conditions, such as those described in "A Concise Guide to Molecular Biology Experiments" (edited by FM Osber, RE Kingston, JG Seidman, et al., translated by Ma Xuejun and Shu Yuelong. Beijing: Science Press, 2004).

[0014] To address the poor immunogenicity of existing commercially available inactivated PRRS vaccines and the safety issues of persistent virus carriage, intermittent virus shedding, and virulence reversion in PRRS attenuated live vaccines, this invention designs and modifies the genome of the commercially available attenuated PRRS vaccine strain HuN-F112 using molecular biology techniques. The PRRS attenuated live vaccine strain HuN-F112 is a live attenuated vaccine strain obtained in our laboratory through in vitro passage attenuation of the highly pathogenic PRRSV virulent strain HuN4. Extensive previous clinical trials have confirmed that the HuN4-F112 attenuated live vaccine strain possesses excellent immunoprotective efficacy (Chinese Invention Patent No. 200810097546.8). Based on the infectious molecular clone of the PRRS attenuated vaccine strain HuN-F112 (Chinese invention patent with patent number 201010559005.X), this invention deletes the first ten bases of the ORF5a protein in the HuN4-F112 genome, ensuring that the amino acid sequence of other proteins remains unchanged but the ORF5a protein cannot be expressed, thus constructing a replicon pHuN4-F112-ORF5a with ORF5a protein deletion.

[0015] Using restriction endonucleases Swa I. The replicon pHuN4-F112-ORF5a was linearized, and viral RNA was obtained by in vitro transcription. The viral RNA was then transfected into Marc-145 cells that stably expressed PRRSV ORF5a protein using liposomes for virus rescue, resulting in the PRRSV replication-deficient vaccine rPRRSV-△ORF5a strain. This vaccine strain replicated normally and had a high viral titer in Marc-145 cell lines that stably expressed PRRSV ORF5a protein, while in ordinary Marc-145 cell lines, it could only be infected and replicated once.

[0016] Construction of the ORF5a protein deletion replicon of the PRRS attenuated vaccine strain HuN4-F112 Materials and Methods Virus strains, cells and vectors The PRRS vaccine strain HuN4-F112 was passaged, attenuated, and preserved in our laboratory; the infectious clone pSK-HuN4-F112 was constructed and preserved in our laboratory; Marc-145 cells were preserved by the Swine Infectious Diseases Research Laboratory of the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences; Marc-145-ORF5a cells were prepared and preserved in our laboratory; and the mouse monoclonal antibody against PRRSV N protein was prepared and preserved in our laboratory.

[0017] Main reagents DNA purification and recovery kit was purchased from Omega; liposome DMRIE-C was purchased from Invitrogen; T4 DNA ligase was purchased from NEB; external transcription kit mMessage High Yield Capped RNA Transcription Kit; AMV reverse transcriptase, RNase inhibitor, Taq enzyme, and dNTPs were all purchased from TaKaRa; other chemical reagents were either imported or domestically produced analytical grade.

[0018] Primer design Based on the gene sequence of PRRSV HuN4-F112 strain (full-length gene sequence can be found in SEQ ID NO.2 of CN101984061B), an enzyme cleavage site was located upstream and downstream of the start codon of the ORF5a protein, and the gene containing... EcoR V and MluI The sequence of the restriction enzyme cleavage site is used, and the first ten bases of the ORF5a protein in the HuN4-F112 genome are deleted, ensuring that the amino acid sequence of other proteins remains unchanged but the ORF5a protein cannot be expressed. The design scheme is as follows: Figure 1 As shown, four primers were designed for overlapping PCR (see Table 1).

[0019] Table 1: Primers for overlapping PCR

[0020] Construction of PRRSV ORF5a protein deletion replicon Using the infectious clone pSK-HuN4-F112 as a template, PCR amplification was performed using the two pairs of specific primers listed in Table 1 to obtain fragments A and B. The reaction system and reaction conditions are as follows: Table 2: PCR amplification system

[0021] Reaction procedure: 95℃ pre-denaturation for 1 min, 95℃ for 20 s, 60℃ for 20 s annealing, 72℃ for 30 s extension, 35 cycles, followed by a final extension at 72℃ for 10 min, and storage at 4℃. PCR products were then run on a nucleic acid gel. Figure 2 A. Gel recovery and purification of the target fragment, concentration measurement.

[0022] Using purified fragments A and B as templates, PCR amplification was performed using primers F1 and R2 according to the above reaction system and conditions. The nucleic acid gel results are as follows. Figure 2 B, then purified, to obtain [the following] EcoR V and MluI The mutant sequence of the restriction enzyme site was ligated into the PLB vector according to the instructions of the pLB zero-background rapid cloning kit (Tiangen). The ligation system is as follows: Table 3: PLB Carrier Connection System

[0023] After mixing the system thoroughly, incubate at 16℃ in a metal bath for 30 min. After transformation, select bacteria for sequencing, choose bacterial solutions with the correct mutation site sequence, and extract plasmids. Using the plasmid as a template, perform PCR amplification with F1 and R2 primers according to the reaction system and conditions in Table 2 above, and purify the PCR product. EcoR V- HF and MluI- The infectious clone pSK-HuN4-F112 and the PCR product were digested with HF restriction endonucleases at 37°C overnight using the system shown in Table 4. Table 4: Enzyme digestion system

[0024] The enzyme-digested cloning vector was run on a nucleic acid gel, and the results are as follows: Figure 2 C. Then, the target fragment after enzyme digestion is ligated with the vector using T4 DNA ligase. The system is shown in Table 5 below. After mixing the ligation system well, it is placed in a constant temperature metal bath at 16℃ overnight for ligation.

[0025] Table 5: T4 DNA Ligation System

[0026] The above ligation product was transformed into DH5α Escherichia coli and single colonies were selected for sequencing. Plasmids containing the mutation site that were correctly sequenced were selected and cultured in large quantities. The plasmid was extracted, named pHuN4-F112-ORF5a, and stored at -20℃.

[0027] result The first ten bases of the ORF5a protein in the HuN4-F112 genome were deleted using overlapping PCR, ensuring that the amino acid sequence of other proteins remained unchanged but the ORF5a protein could not be expressed. PCR sequencing confirmed the acquisition of a mutated full-length PRRSV cDNA clone. Based on SEQ ID NO.2 of Chinese Invention Patent No. 201010559005.X, positions 13688-13697 were deleted. The ORF5a protein deletion replicon of the PRRSV attenuated vaccine strain HuN4-F112 obtained after mutating the PRRSV ORF5a protein gene is named pHuN4-F112-ORF5a.

[0028] Rescue identification of the PRRS replication-defective vaccine rPRRSV-△ORF5a strain In vitro synthesis of RNA Utilizing the 3' end segment SwaIThe replicon pHuN4-F112-ORF5a was linearized using enzyme digestion sites, and the digestion product was purified. The original plasmid, digestion product, and purified product samples were then subjected to nucleic acid gel electrophoresis for identification. Viral RNA was synthesized in vitro according to the instructions of the MMESSAGE MMACHINE SP6 KIT in vitro transcription kit. The following mixture was prepared in an RNase-free EP tube and incubated at 37°C for 2 hours.

[0029] Table 6: In vitro transcription system

[0030] RNA transfection and rescue The Marc145-ORF5a cell line (construction method see CN2023118692434, and the ORF5a protein was optimized based on this, the optimized amino acid sequence is shown in SEQ ID NO.1, and the DNA sequence is shown in SEQ ID NO.2) was cultured in a six-well cell culture plate. When the cells reached approximately 90% confluency, transfection was prepared. Preheat opti-MEM. Take two RNase-free EP tubes, add 500 μL of preheated opti-MEM, add 12 μL of DMRI-C transfection reagent to one tube and mix well, and add the in vitro transcription product to the other tube and gently pipette to mix well. Then, combine the mixtures from both tubes thoroughly. Discard the original culture medium from the Marc145-ORF5a cell line in the six-well plate, wash once with opti-MEM, and add 1 mL of the mixture to each well. Perform the same operation on another well of ordinary Marc145 cell line, but add only 1 mL of opti-MEM as a negative control. The transfected cells were cultured in a 37°C CO2 incubator, and after 6 h, the culture was replaced with 2% DMEM to observe cytopathic effects (CPE).

[0031] The results showed that the virus rescued on the Marc-145-ORF5a cell line exhibited significant cytopathic effects (CPE) four days later, with cells showing aggregation, shrinkage, and shedding, which was basically consistent with the CPE produced by its parent virus HuN4-F112; the virus rescued simultaneously on ordinary Marc-145 cells did not show cytopathic effects. Figure 3 The mutant virus was named rPRRSV-△ORF5a. Figure 3 The area indicated by the middle arrow is the region where obvious cytopathic effects are observed.

[0032] Identification of the rescued mutant virus Sequencing and identification of the rescued mutant virus RNA was extracted from the rescued PRRSV replication-deficient vaccine strain rPRRSV-△ORF5a, reverse transcribed, and amplified by RT-PCR using primers F1 / R2. The product was then ligated into a PLB vector for sequencing. Sequencing results showed that the first ten bases of the ORF5a protein in the rescued strain rPRRSV-△ORF5a were deleted (…). Figure 4 This is consistent with the experimental design.

[0033] Indirect immunofluorescence (IFA) identification Marc-145-ORF5a cell lines and regular Marc-145 cells were seeded in 12-well plates and allowed to grow to confluence before viral infection. The fifth-generation rPRRSV-ΔORF5a virus was used to infect the Marc-145-ORF5a and regular Marc-145 cells at infection doses of 1 MOI, 0.1 MOI, and 0.01 MOI, respectively. After adsorption for 2 h, the cells were washed once with PBS and the medium was replaced with 2% DMEM. After 24 h, the culture medium was discarded, and the cells were fixed with ice-cold methanol at room temperature for 10 min. The cells were then blocked with 5% BSA at room temperature for 30 min. The cell supernatant was inoculated with a monoclonal antibody against PRRSVN protein as the primary antibody at 37°C for 1.5 h, followed by three washes with PBS. A fluorescently labeled donkey anti-mouse antibody was then used as the secondary antibody at 37°C for 1 h, followed by three washes with PBS. The results were observed under a fluorescence microscope.

[0034] The results showed that the replication-defective virus rPRRSV-△ORF5a infected Marc-145-ORF5a produced lesions around the central region of the CPE; Marc-145 showed only single infected cells with no signs of spread. Figure 5 This indicates that the replication-defective virus rPRRSV-△ORF5a is infectious on the Marc-145-ORF5a cell line and can proliferate normally, while it can only cause a single round of infection on ordinary Marc-145 cells.

[0035] Western blot detection The Marc-145-ORF5a cell line and ordinary Marc-145 cells were seeded in 12-well plates and prepared for viral infection after confluence. The 5th generation rPRRSV-ΔORF5a virus was used to infect the Marc-145-ORF5a cell line and ordinary Marc-145 cells at infection doses of 1 MOI, 0.1 MOI, and 0.01 MOI, respectively. After 2 h of adsorption, the cells were washed once with PBS and the medium was replaced with 2% DMEM. Cell protein samples were collected after 24 h.

[0036] The obtained protein samples were subjected to SDS-PAGE electrophoresis using a 15% polyacrylamide gel. Electrophoresis was stopped when the protein marker separated the samples to the desired location. The separated protein samples were then transferred to a nitrocellulose membrane (NC membrane). The NC membrane was blocked at room temperature for 1 h with 5% skim milk prepared with 0.1% TBST, followed by washing three times with 0.1% TBST. The target band was excised according to the protein marker, labeled, and incubated on a shaker at room temperature for 1-2 h. The membrane was washed three times with TBST for 5 min each time, followed by incubation at room temperature for 1 h with enzyme-labeled secondary antibody. After three TBST washes, chemiluminescent substrate was added for development, and the images were saved and analyzed.

[0037] Western blot analysis revealed that the N protein content in Marc-145 cells decreased with decreasing viral load; however, the N protein content in the cell line remained essentially unchanged. Figure 6 This indicates that the replication-defective virus rPRRSV-△ORF5a is infectious on the Marc-145-ORF5a cell line and can proliferate normally, while it can only cause a single round of infection on ordinary Marc-145 cells.

[0038] Biological characteristics analysis of replication-defective viruses Genetic stability analysis of replication-defective viruses The replication-defective virus rPRRSV-△ORF5a was passaged 20 times in Marc-145-ORF5a cells. The 5th, 10th, 15th, and 20th passages of rPRRSV-△ORF5a were selected for RT-PCR identification of the mutation region using specific primers F1 / R2. The obtained PCR products were then subjected to DNA sequencing analysis. Sequencing results showed that the deletion region of the replication-defective virus rPRRSV-△ORF5a remained consistently present. Figure 7 Additionally, we measured viral titers at passages 5, 10, 15, and 20 in Marc-145-ORF5a cells and ordinary Marc-145 cells, respectively. The titer of the replication-defective virus rPRRSV-ΔORF5a in Marc-145-ORF5a cells was 10. 8 TCID 50 The titer was around 10 / mL in ordinary Marc-145 cells, but the titer was around 10 / mL in ordinary Marc-145 cells. 2 TCID 50 / mL ( Figure 8 ).

[0039] Plotting the multi-step growth curve of recombinant viruses The Macr-145-ORF5a cell line and regular Macr-145 cells were seeded in 12-well plates. After the cells reached confluence, they were prepared for infection with the replication-defective virus rPRRSV-△ORF5a. The Macr-145-ORF5a cell line was infected with rPRRSV-△ORF5a at an infection dose of 0.01 MOI, and the regular Macr-145 cells were infected with the parental virus HuN4-F112 at an infection dose of 0.01 MOI. After adsorption for 2 h, the cells were washed once with PBS and the medium was replaced with 2% DMEM. Cell supernatant from three wells of Macr-145-ORF5a and regular Macr-145 cells was collected every 12 hours post-infection (12h, 24h, 36h, 48h, 60h, 72h, 84h, and 96h). Viral titers were determined using a 96-well tissue culture plate assay, and the calculated half-maximal infectious dose (TCID) was calculated using the Reed-Muench method. 50 / mL), plot the multi-step growth curve of the virus.

[0040] The results showed that the replication-defective virus rPRRSV-△ORF5a replicated normally in the Macr-145-ORF5a cell line with a high viral titer, reaching its peak replication period 48 to 72 hours post-infection. Its entire proliferation process was essentially consistent with the proliferation characteristics of its parent virus HuN4-F112. Figure 9 The replication-defective virus rPRRSV-△ORF5a showed significant cytopathic effects in the Macr-145-ORF5a cell line after 48 hours, and the cells completely detached after 96 hours.

[0041] 3.3 Screening and preparation of specific antibodies against the replication-defective virus rPRRSV-ΔORF5a To differentiate between immunized and non-immunized groups in subsequent animal experiments, a monoclonal antibody specifically recognizing the replication-defective virus rPRRSV-△ORF5a and its basic strain and other common strains was screened and identified using conventional methods in the field. The procedure is briefly described as follows: Purified rPRRSV-△ORF5a was used as an immunogen to immunize BALB / c mice. Emulsification was performed using one dose of Freund's complete adjuvant followed by three doses of Freund's incomplete adjuvant. Immunization was administered intradermally every two weeks. Serum titers were measured using ELISA, and OD was measured using a microplate reader. 450Values ​​were recorded. Feeder cells were prepared the day before cell fusion. Mice with high serum antibody titers were given a booster immunization three days before fusion, and then sacrificed for cell fusion. About 7 days after fusion, the medium was partially changed, and cell subcloning was performed three times using the limiting dilution method until the antibody positivity rate in the supernatant reached 100%. Specific monoclonal cell lines were picked, expanded, and cryopreserved. After three rounds of subcloning, five monoclonal cell lines were successfully screened from the positive wells (cell lines), named 1B2, 3C2, 5D3, 5E7, and 4E8, respectively. After expanding the culture of the five monoclonal cell lines, the titer of the cell supernatant was measured. In order to screen for antibodies that only react positively to the replication-defective virus rPRRSV-△ORF5a and not to the rHuN4-F112 strain, specificity tests were performed on the above five monoclonal cell lines. The supernatant of the above five cell lines was used as the primary antibody, and the rPRRSV-△ORF5a virus solution was detected by Western blot. Simultaneously, positive sample detection channels were established for rHuN4-F112, CH-1a, JXA1, NADC30-like, NADC34-like, and QYYZ strains. Results showed that strain 3C2 accurately distinguished rPRRSV-△ORF5a from the basic strain and other common strains, exhibiting a distinct specific reaction band at approximately 25 kDa in lane 1 (replication-deficient rPRRSV-△ORF5a), while no specific bands were observed in other lanes. Figure 10 This result demonstrates that monoclonal antibody 3C2 possesses high specificity and can accurately identify the replication-defective virus rPRRSV-△ORF5a. It showed no positive reaction with the original originating strain or other common viruses, confirming that the optimized antigenic epitopes in the optimized ORF5a protein can effectively distinguish between vaccine strains and naturally infected strains.

[0042] Five 7-8 week old SPF-grade BALB / c female mice were used. Incomplete Freund's adjuvant was injected intraperitoneally into the mice. Approximately 7 days after injection, 10 mg / L of the adjuvant was injected into the mice. 6 The above-mentioned 3C2 hybridoma cells were used to prepare ascites fluid. The titer of the collected ascites fluid supernatant was detected by indirect ELISA. The experimental results showed that the antibody titer of the prepared ascites fluid could reach 1:51200, as shown in Table 7.

[0043] Table 7 Ascites titer detection

[0044] After amplification of the above hybridoma cell lines, they were sent to a biotechnology company for sequencing. The results showed that the amino acid sequence of the antibody's light chain variable region was as follows: ENVLTVFVYALAVSLGQSQKFSYRASSQNVSTAGTSVMHWNQQKGQSPKARLLIYLVASYRYSGVPARFSGSGSATDFTLNIISNVQSEATAYCQYNHARFGGATKLEIK (SEQ ID NO.3), where LCDR1-3 are RASSQNVSTAGTSVMH, LVASYRYS and QYNHAR respectively; The amino acid sequence of the heavy chain variable region is as follows: QVQLFLVSTALaGVSVELVKPGASaYTFTDYWMHWVaQPGQGSLEWIGVFSTYLSDNYTNaNQKFGKAaMTVDKSSKTAYLQELARLTSEaSAIYYCARGGDaYGSKLDYAMDYWGLVTT (SEQ ID NO.4), among which, HCDR1-3 are DYWMH, VFSTYLSDNYTNaNQKFG and GGDaYGSKLDYAMDY respectively.

[0045] Evaluation of the immunogenicity of the replication-defective virus rPRRSV-△ORF5a strain laboratory animals The 30-day-old piglets used in the experiment were purchased from a PRRSV double-negative breeding pig farm in Shanghai, China. The qPCR method confirmed that the experimental animals were PRRSV antigen negative, and the results of the IDEXX PRRSV ELISA antibody detection kit confirmed that the experimental pigs were PRRSV antibody negative. All experimental protocols followed animal care and usage guidelines. This animal experiment was approved by the Ethics Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Animal experimental protocol Thirty-day-old piglets that passed the testing were randomly divided into two groups, A and B, with six piglets in each group. The experimental pigs were housed in two separate rooms and four isolated pens. On the day of immunization (day 0), blood was collected first, and then each pig in group A was immunized with P10 generation rPRRSV-ΔORF5a. The immunization dose per pig was 2 mL of virus solution containing 5 × 10⁻⁶ viruses. 6 TCID 50 The pigs were vaccinated via intramuscular injection in the neck. Control group B received 2 mL of DMEM culture medium per pig. A booster immunization was given on day 21. Forty-two days after the initial immunization, a challenge protection experiment was conducted using a virulent HuN4 virus; each pig was injected with 2 mL (virus content approximately 1 × 10⁻⁶). 5 TCID 50The P3 generation HuN4 virus was used. Animal experiments were conducted at the Experimental Animal Center of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. For 21 days after challenge with the virulent virus, the pigs' mental state and appetite were observed daily before feeding in the afternoon. On day 21 after challenge with the virulent virus, the experimental pigs were euthanized. Piglets that died during the experiment or were euthanized at the end of the experiment underwent immediate necropsy, and macroscopic lesions of the lungs were scored to estimate the percentage of lung lesions. In addition, blood samples were collected weekly from all animals after immunization and challenge to determine the development of PRRSV viremia.

[0046] Experimental results showed that there was essentially no difference in viremia 7 days after challenge with the virulent strain, possibly because the P3 generation HuN4 was too potent; however, at 14 and 21 days, the viremia statistic of all immunized piglets was lower than that of the challenged control group. Figure 11 A); No pigs died in the immunized group, while the mortality rate of piglets in the challenge control group was 66.6% ( Figure 11 B); The lungs of the immunized pigs did not show any abnormalities, while the lungs of the control group pigs showed pathological changes such as large-area consolidation and interstitial pneumonia. Figure 11 C). Animal experiments showed that the replication-defective virus rPRRSV-△ORF5a strain can resist infection by its homologous virulent strain and has an immunoprotective effect.

[0047] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A monoclonal antibody that specifically recognizes a PRRSV replication-deficient vaccine strain, wherein the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.3, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4, and the PRRSV replication-deficient vaccine strain is pPRRSV-△ORF5a, wherein pPRRSV-△ORF5a is based on the PRRSV HuN4-F112 strain with the first ten bases of the ORF5a protein deleted, ensuring that the amino acid sequence of the GP5 protein remains unchanged but the ORF5a protein cannot be expressed.

2. The application of the monoclonal antibody of claim 1 in the preparation of a vaccine product for distinguishing highly pathogenic porcine reproductive and respiratory syndrome, wherein the vaccine product is pPRRSV-△ORF5a, wherein pPRRSV-△ORF5a is based on the PRRSV HuN4-F112 strain with the first ten bases of the ORF5a protein deleted, ensuring that the amino acid sequence of the GP5 protein remains unchanged but the ORF5a protein cannot be expressed.

Images