Construction and application of PRRSV attenuated vaccine double-gene mutant strain

Abstract

The invention provides construction and application of a PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) attenuated vaccine double-gene mutant strain, the PRRSV attenuated vaccine double-gene mutant strain weakens the inhibition effect of PRRSV coding non-structural protein on an I-type IFN innate immune signal channel, and mutates amino acid sites with potential immunosuppression effect on non-structural proteins Nsp11 and Nsp12 in a PRRSV HuN4-F112 strain, so as to obtain the PRRSV attenuated vaccine double-gene mutant strain. According to the method, mutant genes maNsp11 and maNsp12 are obtained, on the basis, the double-gene mutant strain vmaNsp1112 is successfully rescued and obtained by utilizing a reverse genetic manipulation technology, and the obtained double-gene mutant strain has genetic stability in an in-vitro passage process, and particularly, compared with a parent strain, the double-gene mutant strain has the advantages that the double-gene mutant strain has the advantages that the yield is high; the double-gene mutation strain vmaNsp1112 can significantly weaken the inhibition effect on the activity of an I-type IFN promoter, a novel genetic engineering vaccine candidate strain with the innate immunosuppression reduction effect is developed, and a foundation is laid for improving the immune efficacy of an existing vaccine and effectively preventing and controlling PRRSV.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, and particularly to the construction and application of a double-gene mutant strain of a PRRSV attenuated vaccine. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an important infectious disease that seriously endangers the global pig industry, caused by porcine reproductive and respiratory syndrome virus (PRRSV). The pathogen, porcine reproductive and respiratory syndrome virus (PRRSV), is an enveloped single-stranded positive-sense RNA virus. The main clinical features of PRRSV infection are reproductive disorders such as abortion, premature birth, stillbirth and mummified fetuses in pregnant sows. After piglets and fattening pigs are infected, they will show symptoms such as fever, cough, dyspnea, growth retardation and increased mortality. Since the discovery of PRRSV, it has been seriously threatening the health of the pig industries in various countries around the world. PRRSV belongs to the genus Arterivirus in the family Arteriviridae of the order Nidovirales, and its genome length is about 15 kb, containing at least 11 known open reading frames (ORFs). Among them, the viral replicase genes ORF1a and ORF1b are located at the 5' end of the viral genome, accounting for about three-quarters of the full-length gene, and mainly encode two polyproteins pp1a and pp1ab, which can produce at least 14 non-structural proteins (nsp1α, nsp1β, nsp2-nsp6, nsp7α, nsp7β and nsp8-nsp12) after further processing. The structural protein genes ORF2-7 of the virus are located at the 3' end of the genome and encode 8 structural proteins, mainly including minor envelope proteins (GP2a, E, GP3, GP4 and 5a), major envelope proteins (GP5 and M) and nucleocapsid protein (N). A large number of studies have confirmed that some proteins encoded by PRRSV are antagonists of IFN expression. The viral proteins can target and interfere with a variety of key factors in the host IFN signaling pathway, and inhibit the host innate immune response in various ways, so as to evade the host immune surveillance. Vaccine immunization is an important strategy for the prevention and control of PRRS. Commercial PRRS vaccines induce effective protective immune responses to alleviate the clinical symptoms caused by virus infection, reduce the body's outward virus excretion and reduce secondary infections. However, since PRRSV inhibits the host innate immune response in the early stage of infection, and its immunosuppressive characteristics are related to the viral encoded proteins, the presence of these immunosuppressive genes in the vaccine strain is an important factor affecting the optimal immune effect of the vaccine.

[0003] Currently, there are few reports on strains in which immunosuppressive sites are knocked out or mutated in PRRSV. Some mutate the inhibitory sites existing in one of the genes and then explore the effects after mutation; some cannot rescue the virus after mutating the relevant sites. Therefore, the research on the immunosuppression of the body still stays at the exploratory stage, and there is no suitable platform for systematic research. Summary of the Invention

[0004] To solve the above problems, the present application provides the following technical solutions:

[0005] First, the present application provides a PRRSV-encoded non-structural protein Nsp11 and / or Nsp12 that inhibits the type I IFN innate immune signaling pathway. The nucleotide sequence of the mutated Nsp11 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the nucleotide sequence of the mutated Nsp12 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0006] Furthermore, the present application provides a double-gene mutant strain of a PRRSV attenuated vaccine. The double-gene mutant strain of the PRRSV attenuated vaccine weakens the inhibitory effect of the PRRSV-encoded non-structural protein on the type I IFN innate immune signaling pathway, wherein the Nsp11 and / or Nsp12 of the parental strain are respectively replaced by the PRRSV-encoded non-structural protein Nsp11 and / or Nsp12 that inhibits the type I IFN innate immune signaling pathway.

[0007] Furthermore, the present application provides a recombinant vector carrying the PRRSV-encoded non-structural protein Nsp11 and / or Nsp12 that inhibits the type I IFN innate immune signaling pathway.

[0008] Furthermore, the present application provides a method for preparing a double-gene mutant strain of a PRRSV attenuated vaccine, comprising the following steps:

[0009] Double-digest the above recombinant vector mutant strain, and recover and purify the full-length parental vector after double digestion;

[0010] Perform homologous recombination on the recovered product after the above double-digestion treatment, and screen for positive plasmids;

[0011] Transfect cells, harvest the supernatant of the positive rescued virus, and inoculate it into host cells permitted for virus replication to harvest the infectious double-gene mutant strain of the PRRSV attenuated vaccine.

[0012] Furthermore, the present invention provides a composition containing the above PRRSV-encoded non-structural protein Nsp11 and / or Nsp12 that inhibits the type I IFN innate immune signaling pathway, the recombinant vector, and the double-gene mutant strain of the PRRSV attenuated vaccine.

[0013] Furthermore, the present invention provides an application of the PRRSV-encoded non-structural proteins Nsp11 and / or Nsp12, recombinant vectors, double-gene mutant strains of PRRSV attenuated vaccines, and compositions that inhibit the type I IFN innate immune signaling pathway in the preparation of vaccines for preventing or treating highly pathogenic porcine reproductive and respiratory syndrome.

[0014] Beneficial effects

[0015] Based on the applicant's previous research, the present invention found that several non-structural proteins including Nsp11 in PRRSV can inhibit the production of type I IFN. Therefore, in order to weaken the inhibitory effect of PRRSV-encoded non-structural proteins on the type I IFN innate immune signaling pathway, this study mutated the amino acid sites with potential immunosuppressive effects in the non-structural proteins Nsp11 and Nsp12 of the PRRSV HuN4-F112 strain, obtaining the mutant genes maNsp11 and maNsp12. Moreover, based on the HuN4-F112 vaccine strain, using reverse genetic manipulation techniques, the double-gene mutant strain vmaNsp1112 was successfully rescued. Furthermore, the obtained double-gene mutant strain has genetic stability during in vitro passage. In particular, compared with the parental strain, the double-gene mutant strain vmaNsp1112 can significantly weaken the inhibitory effect on the activity of the type I IFN promoter. This indicates that after the amino acid sites with immunosuppressive effects in the Nsp11 and Nsp12 genes of the PRRSV vaccine strain HuN4-F112 are mutated, the inhibitory effect of the parental strain on innate immunity can be significantly reduced. In summary, this study used reverse genetic manipulation techniques to modify the immunosuppressive amino acid sites in the HuN4-F112 vaccine strain, developing a new genetically engineered vaccine candidate strain with reduced innate immune suppression, laying a foundation for improving the immune efficacy of existing vaccines and the effective prevention and control of PRRSV.

[0016] Through the research of this application, it was further discovered that in the PRRSV genome, Nsp11 has ribonuclease activity and can inhibit the production of type I interferon by downregulating some key molecules (such as MAVS, RIG-I, and IPS-1, etc.) in the interferon signaling pathway. At the same time, it can also antagonize the antiviral activities of interferon-stimulated gene 15 (ISG15), etc., to inhibit the innate immune response of the body. Nsp12 is a non-structural protein with incompletely understood functions. Previous studies by this research team found that Nsp12 can interfere with RIG-I's recognition of viral RNA by targeting and binding to the pattern recognition receptor RIG-I, thereby inhibiting the activation of the type I IFN signaling pathway and further inhibiting the host's innate immune response. PRRSV strains vary greatly, and different strains may have differences in genes or amino acid sites with innate immune inhibitory effects, so it may not be universal. After mutating the genes with the function of inhibiting type I IFN signal transduction in the attenuated vaccine HuN4-F112 strain, it is beneficial to further improve the immune efficacy of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Expression identification of the mutant gene. Among them, A is a schematic diagram of PCR amplification identification of the constructed mutant plasmid, and B is a schematic diagram of Western Blot identification with HA-tag antibody;

[0018] Figure 2 Effects of the mutant genes maNsp11 and maNsp12 on the activity of the IFN-β promoter;

[0019] Figure 3 Construction of the full-length cDNA clone of the mutant strain;

[0020] Figure 4 Observation of the cytopathic effect of the rescued double mutant strain;

[0021] Figure 5 IFA identification of the rescued double mutant strain;

[0022] Figure 6 Plaque morphology of the double mutant strain;

[0023] Figure 7 Proliferation curve of the double mutant strain; [[ID=�2]]

[0024] Figure 8 CPE and IFA identifications of different passages of the double mutant strain;

[0025] Figure 9 The double mutant strain weakens the inhibition of the IFN-β promoter activity. DETAILED DESCRIPTION OF THE INVENTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] HEK293T cells and Marc-145 cells are stored in our laboratory; plasmids containing the full-length cDNA of HuN4-F112 are constructed and stored in our laboratory; PRRSV HuN4 strain and HuN4-F112 strain are stored in our laboratory; monoclonal antibodies against PRRSV N protein are prepared and stored in our laboratory.

[0028] Example 1 Construction and identification of mutant plasmids

[0029] According to the analysis of the research results of relevant literature, amino acid sites with potential inhibitory effects in the nsp11 and nsp12 genes of the HuN4-F112 strain were mutated. Among them, the nucleotide sequence of the mutated Nsp11 is: GGGTCGAGCTCTCCGCTCCCCAAGGTCGCACACAACTTGGGATTTTATTTCTCACCTGATTTAACACAGTTTGCTAAACTCCCAGTAGAACTTGCACCTCACTGGCCCGTGGTGACAACCCAGAACAATGAAAAGTGGCCAGATCGGCTGGTTGCCAGCCTTCGCCCTATCCATAAATACAGCCGCGCGTGCATCGGTGCCGGCTATATGGTGGGCCCTTCGGTGTTTTTAGGCACTCCTGGGGTCGTGTCATACTTTCTCACAAAATTTGTTAAGGGCGAGGCTCAAGTGCTTCCGGAGACGGTTTTCAGCACCGGCCGAATTGAGGTAGACTGCCGGGAATATCTTGATGATCGGGAGCGAGAAGTTGCTGCGTCCCTCCCACACGCTTTCATTGGCGACGTCAAAGGCACTACCGTTGGAGGATGTCATCATGTCACCTCCAGATACCTCCCGCGCGTCCTTCCCAAGGAATCAGTTGCGGTAGTCGGGGTTTCAAGCCCCGGAAAGGCCGCGAAAGCATTGTGCACACTGACAGATGTGTACCTCCCAGATCTTGAAGCCTATCTCCACCCGGAGACCCAGTCCAAGTGCTGGAAAATGATGTTGGACTTCAAAGAAGTTCGACTAATGGTCTGGAAAGACAAAACAGCCTATTTCCAACTTGAA(SEQ ID NO.1);

[0030] The amino acid sequence is: GSSSPLPKVAHNLGFYFSPDLTQFAKLPVELAPHWPVVTTQNNEKWPDRLVASLRPIHKYSRACIGAGYMVGPSVFLGTP GVVSYFLTKFVKGEAQVLPETVFSTGRIEVDCREYLDDREREVAASLPHAFIGDVKGTTVGGCHHVTSRYLPRVLPKESV AVVGVSSPGKAAKALCTLTDVYLPDLEAYLHPETQSKCWKMMLDFKEVRLMVWKDKTAYFQLE (SEQ ID NO.2);

[0031] The nucleotide sequence of the mutated Nsp12 is: GGTCGCTATTTCACCTGGTATCAGCTTGCCAGCTATGCCTCGTACATCCGTGTTCCTGTCAACTCTACGGTATACTTGGACCCCTGCATGGGCCCCGCCCTTTGCAACAGGAGAGTCGTCGGGTCCACCCACTGGGGGGCTGACCTCGCGGTCACCCCTTATGATTACGGCGCTAAAATTATCCTGTCTAGCGCGTACCATGGTGAAATGCCCCCCGGATACAAAATTCTGGCGTGCGCGGAGTTCTCGTTGGATGACCCAGTTAAGTACAAACATACCTGGGGGTTTGAATCGGATACAGCGTATCTGTATGAGTTCACCGGAAACGGTGAGGACTGGGAGGATTACAATGATGCGTTTCGTGCGCGCCAGGAAGGGAAAATTTATAAGGCCACTGCCACCGGCTTGAAGTTTTATTTTCCCCCGGGCCCTGTCATTGAACCAACTTTAGGCCTGAATTGA (SEQ ID NO.3);

[0032] The amino acid sequence is: GRYFTWYQLASYASYIRVPVNSTVYLDPCMGPALCNRRVVGSTHWGADLAVTPYDYGAKIILSSAYHGEMPPGYKILACA EFSLDDPVKYKHTWGFESDTAYLYEFTGNGEDWEDYNDAFRARQEGKIYKATATGLKFYFPPGPVIEPTLGLN (SEQ ID NO.4).

[0033] The mutated gene sequence was synthesized by Shanghai Saiheng Biotechnology Co., Ltd. and named p912 (maNsp11 and maNsp12 connected end to end). Then, using the synthesized p912 gene as a template, PCR amplifications were respectively carried out with primers maNsp11-F / maNsp11-R and maNsp12-F / maNSP12-R. After the obtained PCR products were subjected to gel electrophoresis, the target gene fragments were respectively recovered using a gel extraction kit.

[0034] Among them, maNsp11-F: CATTTTGGCAAAGAATTCATGGGGTCGAGCTCTCCGCTCCCCAAG (SEQ ID NO.5);

[0035] maNsp11-R: AGCATGCATCATGGTACCTTAAGCGTAGTCTGGGACGTCGTATGGGTATTCAAGTTGAAAATAGGCTGTTTTG (SEQ ID NO.6);

[0036] maNsp12-F: CATTTTGGCAAAGAATTCATGGGTCGCTATTTCACCTGGTATCAGCTTGC CAG(SEQID NO.7);

[0037] maNSP12-R: AGCATGCATCATGGTACCTTAAGCGTAGTCTGGGACGTCGTATGGGTAATTCAGGCCTAAAGTTGGTTCAATG (SEQ ID NO.8).

[0038] The eukaryotic expression pCAGGS vector was digested with the restriction enzymes EcoRI and KpnI. The digestion reaction system was configured as follows: 3.5 μL of each of EcoRI and KpnI, 5 μL of 10× buffer, 3 μg of vector, and finally made up to 50 μL with ddH2O. After digestion at 37°C for 3 hours, the vector fragment after double digestion was purified using a gel extraction kit through gel electrophoresis. The gene fragment obtained by the above PCR amplification and the digestion product were ligated at 50°C for 15 minutes using a homologous recombination enzyme (the homologous recombination ligation system was 10 μL: including 5 μL of homologous recombination enzyme, 4 μL of the recovered product of the target fragment, and 1 μL of the recovered product after vector digestion). Subsequently, 10 μL of the ligation product was transformed into TOP10 competent cells, 1 mL of antibiotic-free LB medium was added, and the cells were cultured with shaking at 37°C for 45 min, centrifuged at 4000 rpm for 4 min, the supernatant was discarded, 100 μL of the bacterial solution was spread on an LB plate containing ampicillin resistance, and cultured at 37°C for 12 - 16 h. Single colonies were picked for expansion culture, plasmids were extracted and subjected to PCR identification. The results showed that the size of the Nsp11 mutant gene cloned into the pCAGGS vector was approximately 600 bp, and the size of the Nsp12 mutant gene was approximately 400 bp, which was consistent with the expected size ( Figure 1 A). The eukaryotic expression plasmids with correct sequencing identification were named maNsp11 and maNsp12 respectively. The positive plasmids were transfected into HEK293T cells using lipo2000. After 48 hours of transfection, cell samples were collected, and Western blot identification was performed using an HA-tag antibody to detect the protein expression of the mutant target gene. The results showed that the size of the expressed maNsp11 protein was approximately 25 KDa, and the size of the expressed maNsp12 protein was approximately 17 KDa, indicating that both the mutant genes maNsp11 and maNsp12 could be expressed ( Figure 1 B).

[0039] To explore the effect of the mutant gene on the IFN-β promoter, in HEK-293T cells, transfection was carried out using the transfection reagent lipo2000. In the experimental group, 0.5 μg of the plasmid to be tested, 0.05 μg of the reporter plasmid IFN-β-Luc, and 0.01 μg of the internal reference plasmid PRL-TK were transfected into each well. At the same time, nsp11 and nsp12 of the parental strain HuN4-F112 and the pCAGGS empty vector were set as controls. After 24 h of transfection, the cells were stimulated with Sendai virus (SEV). After 8 h of stimulation, cell samples were collected, and according to the instructions of the dual-luciferase detection kit, the luciferase activity of the samples was detected. The results showed that compared with the parental virus HuN4-F112, both maNsp11 and maNsp12 could significantly weaken the inhibitory effect on the IFN-β promoter activity induced by SEV ( Figure 2) By aligning the amino acid sequence encoded by maNsp11 with the amino acid sequence of Nsp11 of the parental strain HuN4-F112, it was found that there were 12 amino acid mutations between them. By aligning the amino acid sequence encoded by maNsp12 with the amino acid sequence of Nsp12 of the parental strain HuN4-F112, it was found that there were 9 amino acid mutations between them. Therefore, these amino acid differences are the key factors for changing the inhibition of type I IFN expression by Nsp11 and Nsp12.

[0040] Using the synthesized gene p912 as a template, PCR amplification was carried out using primers P912-F and P912-R (P912-F: GGATCCTCGCGGCCCTTGCCTACCATATGAAGGCAAGTAATGTTTCTGAATAC (SEQ ID NO.9); P912-R: GCCAAACAAAATGGCCAAAAATATGATGATATCAACAATGGACACCAGAAATTCCGTGAAAGC (SEQ ID NO.10)). The obtained target fragment was subjected to agarose gel electrophoresis and then gel recovery and purification. Then, the vector pHuN4-F112 containing the full-length gene of HuN4-F112 was digested with restriction enzymes NdeI and EcoRV at 37°C for 3 h, and the vector fragment after double digestion was recovered by a gel recovery kit. The recovered PCR amplification product and the vector after double digestion were added to a 10 μL homologous recombination reaction system, in which 5 μL of homologous recombinase, 4 μL of the target fragment, and 1 μL of the digested vector were added, and ligated at 50°C for 15 min. The 10 μL ligation product was transformed into TOP10 competent cells. After adding antibiotic-free LB medium, it was cultured at 37°C for 45 min. Then, it was centrifuged at 4000 r for 4 min. The bacterial cell pellet was resuspended in 200 μL of LB medium and spread on an LB solid medium with ampicillin resistance, and cultured in a 37°C constant temperature incubator for 12 - 16 h. Single colonies formed were picked for enrichment culture, and then plasmids were extracted, and the bacterial liquid was identified by PCR using identification primers. The plasmids identified as positive by PCR were sequenced and verified, and the plasmids were extracted using a plasmid midiprep kit for subsequent virus rescue.

[0041] Example 2 Analysis of the biological characteristics of the mutant strain

[0042] The successfully identified full-length cDNA clone plasmid of PRRSV was linearized with the restriction enzyme SwaI. The enzyme digestion system was 50 μL, mainly including: 20 μg of full-length cDNA plasmid, 10 μL of restriction enzyme SwaI, 50 μL of 10× NEB buffer, and ddH2O was added to make up to 500 μL. After enzyme digestion at 25 °C for 12 h, the digested linearized product was purified by an agarose gel electrophoresis and a gel extraction kit. Then, using the recovered and purified linearized plasmid as a template, in vitro transcription was carried out using the in vitro transcription kit mMessage High Yield Capped RNA Transcription kit. The in vitro transcription system included: 10 μL of 2× NTP / CAP, 2 μL of Enzyme Mix, 2 μL of 10× Reaction Buffer, 2 μg of linearized plasmid template, and nuclease-free ddH2O was added to make up to 20 μL. It was placed in a 37 °C water bath for transcription for 2 h, and the in vitro transcribed genomic RNA was collected. At the same time, the monolayer Marc-145 cells were washed 3 times with sterile PBS. 12 μL of liposome transfection reagent DMRI-C was added to an EP tube containing Opti-MEM and allowed to act for 2 min. Then the above in vitro transcription product was added to the EP tube, gently mixed, and then added to the washed Marc-145 cells. After culturing in a 37 °C 5% CO2 incubator for 6 h, the supernatant was discarded. After washing with sterile PBS, DMEM cell maintenance medium containing 2% FBS was supplemented and continued to be cultured and observed. When about 80% of the cytopathic effect appeared in the cells, it was stored at -80 °C. At the same time, after three freeze-thaw cycles, the cell debris was removed by centrifugation, and the supernatant was taken for further passage culture and identification.

[0043] The rescued gene mutant strain was passaged to the 3rd generation in Marc-145 cells. 300 μL of the 3rd generation virus was taken to extract RNA and reverse transcribed into cDNA. PCR amplification was carried out using the identification primers maNsp11-JDF / maNsp11-JDR and maNsp12-JDF / maNsp12-JDR. The nucleic acid electrophoresis results showed that the size was about 2700 bp ( Figure 3 A). The cloned positive plasmid was identified by double enzyme digestion with the restriction enzymes NdeI and EcoRV. The results showed that the plasmid band after enzyme digestion was above 15000 bp, and the small fragment was about 2700 bp, which was consistent with the expected size ( Figure 3 B).

[0044] Meanwhile, the PCR amplification products were cloned into the PMD-18T vector and subjected to sequencing analysis (Shanghai Saiheng Biotechnology Co., Ltd.). The results showed that the maNsp1112 gene was successfully introduced into the HuN4-F112 genome without amino acid changes. The correctly identified cDNA clone was named pHuN4-F112-maNsp1112.

[0045] Among them, maNsp11-JDF: GGGTCGAGCTCTCCGCTCCCCAAG (SEQ ID NO.11);

[0046] maNsp11-JDR: TTCAAGTTGAAAATAGGCTGTTTTG (SEQ ID NO.12);

[0047] maNsp12-JDF: GGTCGCTATTTCACCTGGTATCAGC (SEQ ID NO.13);

[0048] maNsp12-JDR: ATTCAGGCCTAAAGTTGGTTCAATG (SEQ ID NO.14).

[0049] Marc-145 cells were seeded into 96-well cell culture plates in advance. After the cells grew to confluence, the rescued third-generation virus strain was inoculated into Marc-145 cells. At the same time, the parental virus HuN4-F112 strain was set as a positive control, and cells without virus inoculation were set as a negative control. The cell morphology was observed. The results showed that cytopathic effects could be observed 48 h after transfection of the in vitro transcribed RNA into Marc-145 cells. As the infection time prolonged, the cytopathic effects became gradually obvious, mainly manifested as cell aggregation, gradual rounding and detachment. The negative control cells without virus infection still had normal cell morphology ( Figure 4 ). The rescued double-gene mutant virus strain was named vmaNsp1112 strain.

[0050] The cells showing cytopathic effects were passaged. The culture medium was discarded, and the cells were washed once with PBS. 100 μL of pre-cooled 80% ethanol was added to each well and fixed at 4°C for 45 min. The fixing solution was discarded, and the cells were washed three times with PBS. Using the PRRSV-specific N protein monoclonal antibody as the primary antibody, 100 μL / well, acting at 37°C for 45 min, and washed three times with PBS. Goat anti-mouse FITC-labeled fluorescent secondary antibody diluted 1:1000 with PBS was added, acting at 37°C in the dark for 45 min. After washing three times with PBS, the fluorescence was observed under an inverted fluorescence microscope. The results showed that the Marc-145 cells infected with vmaNsp1112 produced specific immunofluorescence identical to that of the parental virus HuN4-F112 (Figure 5 ), indicating that the double-gene mutant strain vmaNsp1112 was successfully rescued, and the fluorescence characteristics of the passaged virus were the same as those of the parental strain.

[0051] After Marc-145 cells formed a cell monolayer in a 6-well plate, the rescued virus was serially diluted 10-fold with 2% DMEM (10 -1 to 10 -5 ), inoculated into Marc-145 cells. After 2 h of infection, the cells were washed with sterile PBS. 2% low melting point agarose was mixed with 2×MEM in equal proportion and then added to Marc-145 cells. The mixture was allowed to stand at room temperature for 15 min. After the agarose solidified, it was inverted and cultured under the conditions of 37℃ and 5% CO2 for 4 - 5 days. When typical cytopathic effects were observed, the 6-well cell culture plate was taken out. 4% paraformaldehyde was added to each well to fix for 2 h, then the fixing solution and agarose gel were discarded, and 2 ml of crystal violet solution was added to stain for 5 min. After washing with water, the plaque morphology was observed and recorded. The results showed that the plaque morphology and size formed by vmaNsp1112 in cells were similar to those of the parental virus HuN4-F112 ( Figure 6 ).

[0052] The rescued strain and the parental strain HuN4-F112 were respectively used to infect Marc-145 cells that had formed a cell monolayer in a 6-well plate at a dose of 0.1 MOI. 200 μL of cell supernatant was collected at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after infection respectively for the determination of virus TCID50, and a virus multi-step growth curve was plotted. The results showed that compared with the parental strain HuN4-F112, the replication efficiency of the double-gene mutant strain vmaNsp1112 was significantly lower than that of the parental strain before 48 h of infection, and the replication levels of the two were basically similar after 60 h of infection ( Figure 7 ).

[0053] Example 3 Genetic Stability Analysis of the Mutant Strain

[0054] The rescued virus stored at -80°C was subjected to three cycles of freezing and thawing, followed by centrifugation at 12,000 rpm for 5 min. After taking 500 μL of the virus supernatant and mixing it evenly with 500 μL of cell maintenance medium containing 2% FBS, it was added to Marc-145 cells that had been washed with sterile PBS. The cells were incubated at 37°C in a 5% CO2 incubator for 1 - 2 h, during which they were mixed every 15 minutes. After the incubation ended, the cell supernatant was discarded, and the cells were washed with sterile PBS. Finally, 3 mL of cell maintenance medium containing 2% FBS was added for continued culture. When the cytopathic effect reached 80%, the virus culture fluid was collected as the F1 generation. Using the same method, the virus was passaged continuously on Marc-145 cells up to 20 generations. The cytopathic effects caused by virus strains of different generations were observed, and IFA identification was performed on cells infected with virus strains of different generations using a monoclonal antibody against the specific N protein of PRRSV. The results showed that specific immunofluorescence consistent with the parental virus could be detected in double-gene mutant strains of different generations, indicating that the double-gene mutant strain vmaNsp1112 still maintained the characteristics of PRRSV infection with the increase in the number of passages( Figure 8 ). Gene sequencing was performed on the virus strains of the 5th, 10th, 15th, and 20th generations. The results showed that the Nsp11 and Nsp12 genes replaced in the double-gene mutant strains of each generation were consistent with the introduced gene sequences, and no mutations or deletions occurred, indicating that the double-gene mutant strain vmaNsp1112 had genetic stability.

[0055] Example 4 Analysis of the mutant strain on the IFN-β promoter

[0056] When the culture density of Marc-145 cells in a 24-well plate reached 80%, transfection was carried out using the transfection reagent lip2000 at a rate of 0.05 μg of the reporter plasmid IFN-β-Luc and 0.01 μg of the internal reference plasmid PRL-TK per well. After incubating in a 37°C incubator for 6 h, 10% DMEM was replaced with maintenance medium 2% DMEM, and at the same time, 0.1 MOI of the PRRSV strain HuN4-F112, as well as the rescued 10th and 20th generation viruses, were inoculated. After 24 h of infection, the cells were stimulated with SEV, and cell samples were collected after 8 h. The cell samples were treated with a dual-luciferase detection kit, and their luciferase activities were detected. The results showed that the activities of the IFN-β promoter induced by each virus-inoculated group were lower than those of the non-virus-inoculated control group. However, compared with the parental virus HuN4-F112, the activities of the IFN-β promoter induced by the F10 and F20 generation strains of the gene mutant strain vmaNsp1112 were significantly higher than those of the parental virus( Figure 9 ). This indicates that the double-gene mutant strain weakened the inhibitory effect of the parental strain on the IFN-β promoter.

[0057] To explore the inhibitory effect of the gene-mutated strain vmaNsp1112 on the promoter activity of interferon-stimulated genes, the gene-mutated strain vmaNsp1112 and the parental virus HuN4-F112 were used to infect Marc-145 cells transfected with the reporter gene (ISRE-Luc) containing the interferon-stimulated gene promoter. After 24 h of infection, the cells were stimulated with IFN-α, and samples were collected after 10 h to detect luciferase activity. The results showed that the HuN4-F112 strain could significantly inhibit the ISRE expression induced by IFN-α, while the gene-mutated strain vmaNsp1112 significantly weakened the inhibitory effect of the parental strain on the ISRE promoter activity induced by IFN-α. The above results indicate that the obtained gene-mutated strain vmaNsp1112 can, to a certain extent, antagonize its inhibitory effect on the innate immune response. Through amino acid sequence alignment analysis with the HuN4-F112 strain, 12 and 9 amino acid mutations were found in Nsp11 and Nsp12 of the gene-mutated strain, which are the key factors leading to the attenuation of the immunosuppressive effect of the gene-mutated strain on I-IFN expression. In addition, according to the analysis of the in vitro proliferation characteristics of the gene-mutated strain, it was found that the replication and growth rate of the gene-mutated strain were lower than those of its parental wild-type virus at the early stage of infection. This is because the gene-mutated strain weakened the inhibition of the expression of type I IFN and related interferon-stimulated factors at the early stage of infection, and the result was an increase in the antiviral effect of host cells, which is beneficial to enhancing the immune response induced by the HuN4-F112 vaccine at a higher level.

[0058] The above description of the embodiments is intended to enable those of ordinary skill in the art to understand and use the present invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. All improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A PRRSV-encoded non-structural protein Nsp11 and / or Nsp12 that inhibits the innate immune signaling pathway of type I IFN, characterized in that, The nucleotide sequence of the mutated Nsp11 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the nucleotide sequence of the mutated Nsp12 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.

4.

2. A double-gene mutant strain of PRRSV attenuated vaccine, wherein the double-gene mutant strain of PRRSV attenuated vaccine weakens the inhibitory effect of PRRSV-encoded non-structural proteins on the type I IFN innate immune signaling pathway, and the Nsp11 and / or Nsp12 of the parental strain are respectively replaced by the PRRSV-encoded non-structural proteins Nsp11 and / or Nsp12 having an inhibitory effect on the type I IFN innate immune signaling pathway as claimed in claim 1.

3. A recombinant vector carrying the PRRSV-encoded non-structural proteins Nsp11 and / or Nsp12 having an inhibitory effect on the type I IFN innate immune signaling pathway as claimed in claim 1.

4. A method for preparing a double-gene mutant strain of PRRSV attenuated vaccine, comprising the following steps: Double-digest the recombinant vector mutant strain as claimed in claim 3, and recover and purify the full-length parental vector after double-digestion; Perform homologous recombination on the recovered product after the above double-digestion treatment, and screen for positive plasmids; Transfect cells, harvest the supernatant of positive rescued virus, and inoculate it into host cells permitted for virus replication to harvest the infectious double-gene mutant strain of PRRSV attenuated vaccine.

5. A composition containing one or more of the PRRSV-encoded non-structural proteins Nsp11 and / or Nsp12 having an inhibitory effect on the type I IFN innate immune signaling pathway as claimed in claim 1, the recombinant vector as claimed in claim 3, and the double-gene mutant strain of PRRSV attenuated vaccine as claimed in claim 2.

6. Use of one or more of the PRRSV-encoded non-structural proteins Nsp11 and / or Nsp12 having an inhibitory effect on the type I IFN innate immune signaling pathway as claimed in claim 1, the recombinant vector as claimed in claim 3, and the double-gene mutant strain of PRRSV attenuated vaccine as claimed in claim 2 in the preparation of a vaccine for preventing or treating highly pathogenic porcine reproductive and respiratory syndrome.

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