CN116004551B - Construction and application of porcine reproductive and respiratory syndrome virus live vaccine for regulating MHC-II transactivator CIITA gene - Google Patents
Construction and application of porcine reproductive and respiratory syndrome virus live vaccine for regulating MHC-II transactivator CIITA gene Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to a site-directed mutagenesis porcine reproductive and respiratory syndrome virus, which is characterized in that the expression of a key gene CIITA for regulating and controlling an MHC-II molecular antigen presenting path can be recovered. The mutant porcine reproductive and respiratory syndrome virus mutates the key amino acid site 199 of Nsp4 regulatory CIITA to aspartic acid. Nsp4 can degrade CIITA through ubiquitin proteasome pathway while cutting CIITA,199 sites are key sites for inhibiting CIITA, and V199D mutant virus can reduce inhibition of CIITA after vaccine strain infects bone marrow-derived dendritic cells to a certain extent, so that MHC-II molecule mediated antigen presenting pathway is restored. Compared with the existing commercial vaccine, the vaccine strain can reduce the immunosuppressive property, ensure the immunogenicity and improve the safety.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a mutant plasmid and a genetic engineering vaccine of viruses, and more particularly relates to a mutant plasmid and a genetic engineering vaccine which can enhance early adaptive immune response and have effective immune protection on porcine reproductive and respiratory syndrome viruses.
Background
Porcine Reproductive and Respiratory Syndrome (PRRS), commonly called as "blue ear disease", clinically causes respiratory system disorder of piglets, and sow abortion and stillbirth reproduction disorder, and causes huge economic loss for pig industry in China since PRRS bursts in China in 1996. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) belongs to the order of the mantle viridae, the genus arterivirus. The nucleic acid line of PRRSV is a non-segmented, single-stranded, positive-stranded RNA of about 15 kb in size, containing 8 Open Reading Frames (ORFs). ORF1a and ORF1b account for about two-thirds of the entire genome, and after expression of 2 multimeric proteins pp1a and pp1ab, 12 viral nonstructural proteins (Nsps) are cleaved off, which play a vital role in viral replication and gene expression. Among them, NSP4 has 3C-like serine protease activity (3 CLSP), which plays an important role in viral replication and immune evasion.
At present, attenuated live vaccine (MLV) immunization is a means which accords with the national conditions of China and effectively prevents PRRS, but the PRRS attenuated live vaccine still has some problems in clinical prevention and control, and especially, early-stage and low-efficiency humoral (neutralizing antibodies) and cellular immune response of PRRS vaccine immunization are a great problem which restricts PRRS vaccine use and interferes with prevention and control strategy formulation. After single immunization, PRRS attenuated live vaccine can induce body to produce high level antibody reaction, mainly specific antibody against N protein, GP5 protein and partial non-structural proteins (Nsp1α, nsp1β, nsp2 and Nsp7), but these antibodies in early stage have no neutralizing activity. The neutralizing antibody is produced at a late time and low level, the neutralizing antibody at the low level is insufficient to protect pigs against PRRSV attack, and cell immunity caused by immunization is not active, and immunosuppression caused by PRRSV can cause secondary infection or co-infection of other pathogenic microorganisms such as Swine Influenza Virus (SIV), porcine Respiratory Coronavirus (PRCV), porcine circovirus (PCV 2) and the like, and the co-infected pigs only show clinical symptoms and growth retardation which are more serious than that of single pathogen infection. It was found that although the neutralizing antibodies were produced at a later time after a single vaccine immunization and at a low level, repeated immunization multiple times could produce high levels of neutralizing antibodies, which could provide effective immunoprotection. Although PRRS vaccines have been put into use for nearly 20 years, the acquired immune response after immunization of PRRS vaccines has been largely studied, the immune protection mechanism of PRRS vaccines still has no accurate answer, and the research lag still restricts the development of vaccines and the formulation of epidemic prevention and control strategies.
Antigen presentation as the initiation of an acquired immune response plays a key role in virus recognition, antigen modification processing, effector cell activation, and the like, and Dendritic Cells (DCs) as the primary antigen presenting cells are the fundamental vectors for a series of processes to be realized. The research on the DC cell anti-presentation function after vaccine immunization is a key for revealing whether the PRRS vaccine is immune and whether the immune suppression is generated or not, is hopeful to reveal the reasons of low humoral (neutralizing antibody) and cell immune response levels after vaccine immunization, and provides theoretical support for comprehensively revealing vaccine immune protection mechanisms and improving novel vaccines. The laboratory early-stage test result shows that the quantity of CD4+ T cells in peripheral blood is obviously lower than that of a control group after the HuN-F112 vaccine strain is immunized on the pig for 14 days; huN4-F112 after infection of bone marrow derived differentiated dendritic cells (BMDC), the transcriptional level of MHC-class II transactivators (CIITA) was significantly down-regulated, suggesting that PRRSV vaccine strains might interfere with MHC-class II molecule mediated antigen presentation by modulating CIITA genes, thereby suppressing the immune response in the body.
CIITA is the most predominant protein regulating MHC class II molecule mediated antigen presentation. CIITA participates in transcription by a variety of mechanisms, such as recruitment of transcription factors TFIID, TFIIB to target gene promoter regions, forming transcription initiation complexes; inducing phosphorylation of RNA polymerase II; interact with P-TEFb; recruiting activators that induce histone acetylation or methylation to alter chromatin; pigment reconstitution factor BRG1 was recruited. CIITA regulation varies between immature and mature DC cells, being regulated by PU.1, IRF8, NF-. Kappa.B and SP1 in immature DC cells and by PRDM1 in mature DC cells. The transcription of CIITA is also regulated by the upstream factors CDCA3, RMND B, CNOT1, MAPK1 and PLEKHA4, requiring both phosphorylation and ubiquitination to perform its function. Phosphorylation is an important modification that directs the localization of the CIITA nucleus and increases trans-activity, and a variety of residues are identified on the CIITA's translation initiation factor IF3 as phosphorylation sites, ser280 being the center of the phosphorylated active site. Once phosphorylated, monoubiquitination follows, resulting in an increase in CIITA transactivation, with a consequent increase in transcription of MHC class II molecules, lysine residues Lys-315, lys-330 and Lys-333 of CIITA, and phosphorylation sites Ser-280, which regulate CIITA ubiquitination, stability and MHC class II expression, this ubiquitination being K63-linked ubiquitination, which is capable of tandem phosphorylation enhancing CIITA transactivation and is important for CIITA movement from the cytoplasm to the nucleus. While polyubiquitination results in degradation of CIITA by the ubiquitin-proteasome pathway, lysine residues Lys-141 and Lys-145 of CIITA regulate the K48-linked ubiquitination modification of CIITA, resulting in recognition and degradation of CIITA by the proteasome. A variety of microorganisms can evade the immune response of the body by interfering with the CIITA gene, for example, the transcriptional activator of HIV interferes with the function of CIITA by binding to cyclin T1, thereby preventing the expression of MHC class II molecules during HIV infection; helicobacter pylori can inhibit MHC-II expression by inhibiting CIITA expression, thereby inhibiting antigen presentation; mycobacterium tuberculosis inhibits the function of CIITA by producing 19kDa lipoprotein so as to achieve the aim of evading the immune response of the organism; CIITA inhibits viral entry by activating constant chain CD74 p41 subtype expression, preventing cathepsin-mediated ebola glycoprotein processing, and also blocks the endosomal entry pathway of coronaviruses including SARS-CoV-2.
Since PRRS first appeared, scientists worldwide have studied PRRS vaccines and numerous data show: the PRRS inactivated vaccine and the subunit vaccine can not provide complete immune protection for pigs, only the PRRS attenuated live vaccine can effectively protect pigs against the attack of homologous PRRSV, but NSP4 is used as a conserved nonstructural protein, viruses are easy to kill after mutation, and NSP4 is not a neutralizing antigen, so that the study on the NSP4 in the aspect of vaccines is less. Aiming at the practical problems of low-level humoral (neutralizing antibodies) and cellular immune responses caused after PRRS vaccine immunization, the patent finds that the CD4+ T cell number in peripheral blood is obviously reduced 14 days after HuN-F112 immunization, and then finds that the transcription level of a key gene CIITA for regulating and controlling an MHC-II molecular antigen presenting path is obviously down-regulated, so that PRRSV vaccine strains possibly interfere with antigen presentation by regulating CIITA genes, thereby inhibiting the immune responses of organisms. Further research shows that Nsp4 can degrade CIITA through ubiquitin proteasome pathway while cutting CIITA,199 sites are key sites for inhibiting CIITA, and V199D mutant virus can reduce the inhibition of CIITA after vaccine strain infects bone marrow-derived dendritic cells to a certain extent, thereby restoring MHC-II molecule mediated antigen presenting pathway. Compared with other known commercial vaccines, the improved vaccine strain reduces the immunosuppressive property of PRRSV in early stage of immunization, ensures the immunogenicity and improves the safety of PRRSV, and is beneficial to the establishment of immunization programs in production.
Disclosure of Invention
The invention aims to provide a construction method of a virus mutant plasmid capable of effectively improving the immunity early humoral immunity level and the cellular immunity level of a porcine reproductive and respiratory syndrome vaccine based on mechanism research of PRRSV (porcine reproductive and respiratory syndrome virus) regulation CIITA.
The invention establishes a method for constructing a mutant plasmid of Nsp4 mutation on the whole genome of PRRSV. According to the single enzyme cutting site position containing Nsp4 fragment in HuN-F112 genome and the key amino acid site position of Nsp4 regulation CIITA, huN-F112 whole genome segment PCR amplification primer and key amino acid site-directed mutagenesis primer on Nsp4 are respectively designed. After obtaining the genome mutant sequence including Nsp4 of HuN-F112 whole genome, T4 ligation was performed with the segmented sequence of pBluescript SK (+) vector whole genome to construct a mutant plasmid pBlue-V199D of HuN-F112 whole genome transcription regulatory sequence.
The construction method of the mutant PRRSV plasmid of the key amino acid site of the Nsp4 regulatory CIITA comprises the following steps: firstly, amplifying by using designed whole genome segment amplification primers to obtain HuN-F112 sequence segments of whole genome including Nsp 4; then, after the fragment is connected with an intermediate vector, the Val199 site in Nsp4 is subjected to site-directed mutagenesis by using a site-directed mutagenesis primer; finally, the genomic fragment of Nsp4 that had been successfully mutated was ligated via T4 to another full-length genome that did not contain Nsp4, which was SpeI and PmeI double digested. The plasmid which is successfully connected in full length is identified and screened, top10 competent cells are transformed, full length sequencing is carried out after plasmid extraction, so that the full length infectious clone plasmid pBlue-V199D with Nsp4 key site mutation which has no mutation at other positions only with Nsp4 target site mutation is obtained, and a corresponding method is established.
After the constructed NSp4 mutant plasmid pBlue-V199D is subjected to linearization, in vitro transcription and transfection of MARC-145 cells, the packaged virus is continuously passaged on Marc-145 cells, and the successfully rescued NSp 4V 199D mutant virus can be detected by three generations of blind transmission.
The full-length sequence of the porcine reproductive and respiratory syndrome virus NSp 4V 199D for regulating CIITA gene is shown as SEQ ID NO. 1;
Further, the present invention provides an application of the porcine reproductive and respiratory syndrome virus Nsp 4V 199D for regulating CIITA gene, which is an application for preparing a kit for treating or preventing porcine reproductive and respiratory syndrome virus, which is used for preparing a medicament, preferably a vaccine;
Further, the present invention provides an immunogenic composition or combination vaccine or combination comprising porcine reproductive and respiratory syndrome virus Nsp 4V 199D which modulates the CIITA gene, optionally with the addition of a pharmaceutically acceptable carrier.
Drawings
FIG. 1 is a graph showing the effect of different PRRSV nonstructural proteins on CIITA at the transcriptional and protein level.
FIG. 2 shows the effect of F112-framed point mutations Nsp4 on CIITA at the transcriptional and protein levels, his39, asp64, ser118 are key enzyme active sites of NSP4 protease, the enzyme active site mutated attenuated NSP4 is no longer functional, the 199 site is the key site for NSP4 to inhibit CIITA, V199L and V199P are similar in effect to V199D, and V199G and V199F appear to lose the ability to cleave CIITA while weakening the ability to inhibit CIITA.
FIG. 3 is a graph showing the results of inhibitor method analysis of the pathway of modification of CIITA by NSP4, where NSP4 is capable of degrading CIITA via the proteasome pathway.
FIG. 4 is a graph showing the results of detection of NSP4 interaction and co-localization with CIITA, where all mutant NSP4 interact directly with CIITA, and CIITA co-localizes with NSP4 and its mutant plasmids in 293T cell nuclei.
FIG. 5 is a graph showing the results of the identification and electrophoresis of the plasmid of the full-length genome of the final screening and construction of the successfully constructed Nsp4 point mutation F112 infectious clone.
FIG. 6 shows the detection result of the number of copies of the rescue virus obtained by extracting viral RNA from the cell supernatant after the blind transfer of the rescue virus to the third generation, reverse transcribing the viral RNA into cDNA, and detecting the N protein by absolute fluorescent quantitative PCR.
FIG. 7 is the results of indirect immunofluorescence of Marc-145 cells from a rescue toxin infection for 48 hours.
FIG. 8 is a multi-step growth curve plotted against the titer change of the rescued virus versus the parental virus.
FIG. 9 shows the results of transcription level detection of bone marrow-derived dendritic cells infected with V199D mutant virus, wherein CIITA transcription level of the V199D group can be significantly increased at 12h and 24h, and CIITA upstream regulatory factor SP1 can be significantly increased at 24 h.
Detailed Description
In the invention, the Nsp4 point mutation PRRSV plasmid refers to a method for mutating a key site of Nsp4 in a high-pathogenicity PRRSV passage attenuated strain HuN-F112 genome by utilizing a reverse genetic technology, wherein the mutation point can not seriously inhibit CIITA under the condition of maintaining normal functions of the Nsp4, and the mutant plasmid is pBlue-V199D.
In the present invention, the NSP4 point mutant virus refers to a live virus V199D which is rescued after Marc-145 cells are transfected by a full-length NSP4 mutant plasmid pBlue-V199D obtained by utilizing a genome site-directed mutagenesis technology.
In the present invention, the reverse genetic manipulation means that on the infectious clone backbone of the obtained highly pathogenic PRRSV attenuated vaccine strain HuN-F112, site-directed mutagenesis is performed on the bases of Nsp4 after the full-length genome is segmented, and then the mutated HuN-F112 full-length infectious clone is reconstructed, so that the infectious clone is assembled into virus particles with bioactivity, and the change of the mutant virus and the parent virus in the biological characteristics of the virus, and the influence of the PRRSV attenuated strain mutated by Nsp4 on CIITA, and the influence on humoral (neutralizing antibody) and cellular immune response after vaccine immunization are studied.
In the present invention, the Genbank accession number of the highly pathogenic porcine reproductive and respiratory syndrome virus HuN is EF635006.
In the present invention, the infectious clone HuN-F112 of the attenuated vaccine strain of highly pathogenic porcine reproductive and respiratory syndrome virus refers to an infectious clone constructed by the method of reference ShanruZhang, YanjunZhou, Yifeng Jiang, Guoxin Li, LipingYan, Hai Yu, Guangzhi Tong. Generation of an infectious clone of HuN4-F112, an attenuated live vaccinestrain of porcine reproductive and respiratory syndrome virus.
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The experimental procedure, which does not specify specific conditions in the following examples, is generally followed by conventional conditions, such as "molecular cloning: the conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989).
In an embodiment of the invention, the cell line used is Marc-145 cells (African green monkey kidney cell line); the vector used was the pBluescript SK (+) vector (available from Invitrogen); the competent cells used were TOP10 competent cells (purchased from TIANGENE company); the enzymes used were SpeI-HF, pmeI-HF, T4 DNA LIGASE (available from NEB), PRIMESTAR HS DNA Polymerase (available from TAKARA), gold-brand Mix (green) (available from full gold); kits QIAquick PCR Purification Kit, QIAPREP SPIN MINIPREP KIT, RNEASYKITS, QIAAMP VIRAL RNA MINI KIT (available from QIAGENE), MMESSAGE MMACHINETM SP6 (available from Invitrogen); transfection reagent DMRIE-C was purchased (from Thermo Fisher Co.); culture medium Opti-MEM, DMEM (available from Gibco).
In the embodiment of the invention, the construction of the HuN4-F112 mutant plasmid with the Nsp4 point mutation is to design HuN-F112 whole genome segment PCR amplification primers and key amino acid site-directed mutation primers on Nsp4 according to the single enzyme cutting site position of the Nsp4 fragment in the HuN-F112 genome and the key amino acid site position of the Nsp4 regulatory CIITA obtained in the earlier study. After obtaining the genome mutant sequence including Nsp4 of HuN-F112 whole genome, T4 ligation was performed with the segmented sequence of pBluescript SK (+) vector whole genome to construct a mutant plasmid pBlue-V199D of HuN-F112 whole genome transcription regulatory sequence. The specific process is as follows:
1.1 primer design
PCR amplification primers as well as site-directed mutagenesis primers were designed based on the position of NSP4 on HuN-F112 full-length infectious clone and the position of the single cleavage site in the genome. The sequences are as follows:
pBlue-F：GGTCGACGGTATCGATAACTAGTGGAAACCTGAACTTTCAACAAAG（SEQ ID NO.2）
pBlue-R：TCTAGAACTAGTGGATCGCTAGCAGTTTAAACACTGCTCCTTAGTC（SEQ ID NO.3）
V199D-F：CCCTGCTTGCTGACAAACCCGAACTGGAA（SEQ ID NO.4）
V199D-R：AGTTCGGGTTTGTCAGCAAGCAGGGCACAAAGATCTGAAG（SEQ ID NO.5）
The specific construction steps are as follows:
1.2 amplification of PRRSV genome segment products
Using pHuN-F112 as a template and the primers pBlue-F, pBlue-R as an upstream primer and a downstream primer, the segmented sequences containing Nsp4 in the genome were amplified by PCR as follows:
The PCR reaction system is as follows: pHuN4-F112 plasmid (1:1000 dilution) was used as a template 1. Mu.L, 1. Mu.L each of the upstream and downstream primers (10. Mu.M), 5X PRIMESTAR BUFFER. Mu.L, 4. Mu.L of dNTP mix, PRIMESTAR HS DNA Polymerase (2.5U/. Mu.L) and water was added to 50. Mu.L.
The PCR reaction parameters are as follows: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 2min, followed by 40 cycles and then extension at 72℃for 5min.
The PCR reaction products are taken, detected by 1% agarose gel electrophoresis, and the products are recovered by gel and then connected to a vector pBluescript SK (+) by a homologous recombination method.
1.3 Site-directed mutagenesis of the Nsp4 Critical site
On the PRRSV genome segment plasmid constructed in 1.2, the 199-locus amino acid of Nsp4 was mutated to aspartic acid by site-directed mutagenesis PCR using the primer V199D-F, V199D-R, as follows:
The PCR reaction system is as follows: PRRSV genome segment plasmid was used as a template 1. Mu.L, each of the upstream and downstream primer pairs (10. Mu.M) 1. Mu.L, 5X PRIMESTAR BUFFER. Mu.L, dNTP mix 4. Mu.L, PRIMESTAR HS DNA Polymerase (2.5U/. Mu.L) 0.5. Mu.L, and water was added to 50. Mu.L (two for each sample system).
The PCR reaction parameters are as follows: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 2min, followed by 40 cycles and then extension at 72℃for 5min.
A total of 100. Mu.L of the PCR reaction product was taken, added with 200. Mu.L of absolute ethanol, mixed uniformly, left to stand for 10min, centrifuged at 12000rpm at 4℃for 10min, and the supernatant was discarded. The plasmid was amplified in Top10 competent cells by suspending with 20. Mu.L of DpnI in ddH 2 O17. Mu.L, cutsmart Buffer. Mu.L and 1. Mu.L of DpnI enzyme in a 37℃water bath for 3 hours, and transferred to Shanghai qing Biotechnology Co., ltd for sequencing and verification, and successfully mutated plasmids were selected and kept for use.
1.4 Construction of Nsp4 Point mutant infectious clone
The properly sequenced mutant plasmid of 1.3 and pHuN-F112 full length infectious clone plasmid were digested with SpeI and PmeI to give a genomic fragment containing the Nsp4 mutation and another fragment containing no Nsp4 over its length. After gel recovery, the fragment concentration was measured, the two sections were ligated together in a 1:1 manner using T4 ligation, the reaction conditions for T4 ligation were overnight ligation at 16℃and the ligation products were competent to transform with Top10, plated with ampicillin LB plates, and positive colonies were selected by picking after 12 hours. When colony screening is carried out, gold-plate Mix (green) is used as bacterial liquid PCR, positive colonies are screened out, plasmids are extracted after the positive colonies are subjected to 1% agarose gel electrophoresis, the positive colonies are compared with positive pHuN-F112 plasmids, the comparison strips are sequenced after the comparison strips are consistent in size (shown in figure 1), and Nsp4 point mutation PRRSV positive cloning plasmids are obtained through screening.
1.5 SwaI linearization of NSP4 Point mutation PRRSV positive cloning plasmid
The Nsp4 point mutant plasmid pV199D and the parent plasmid pHuN-F112 were digested with SwaI in a linear manner, and the 500. Mu.L reaction system was: 20 mu LSwaI enzyme, 50 mu L NEB Buffer 3.1, 20 mu g-30 mu g plasmid, the remainder supplemented with ddH2O to 500 mu L. The reaction conditions are enzyme digestion at 25 ℃ for 12 hours. Referring to the methods of the specification, the cleaved products were purified using QIAquick PCR Purification Kit to obtain purified linearized products, respectively.
1.6 In vitro transcription
Referring to the method of the specification, the linearized product after in vitro transcription purification using MMESSAGE MMACHINETM SP kit was 20 μl:2 x NTP/CAP 10. Mu.L, 10 x Reaction Buffer 2. Mu.L, enzyme Mix 2. Mu. L, GTP. Mu.L, linearization template 4. Mu.L. The reaction conditions were 37℃for 2 hours.
1.7 Transfection of RNA
The in vitro transcribed product is immediately transfected. MARC-145 cells were seeded and cultured in six well plates and transfected when cell densities were 80-90%. The transfection steps are: 1 mL Opti-MEM was added to each EP tube, 5. Mu.L of transfection reagent DMRIE-C was added, one tube was left as negative control, the other tube was added with 20. Mu.L of corresponding in vitro transcribed RNA, vortexed, shaken, washed once with PBS, the whole liquid in the corresponding EP tube was added to each well, the incubator was incubated for 8 hours, the culture was continued with medium changed to 2% FBS, and cytopathy was observed daily.
After cytopathy occurs, collecting cell supernatant and then carrying out passage, wherein the specific process is as follows: MARC-145 cells were grown as a monolayer on six well plates with DMEM medium containing 10% FBS, the medium was discarded, PBS was washed twice, the supernatant five days after transfection was aspirated, 200. Mu.L of the supernatant was inoculated with a maintenance solution (DMEM containing 2% FBS) at a ratio of 1:10, and the culture was continued at 37℃and passaged as described above, and the third generation virus supernatant was collected for storage.
1.8 Fluorescent quantitative PCR and Indirect immunofluorescence detection
Infecting Marc-145 cells with a dose of 20 μl/well of the third-generation virus supernatant of 1.7, extracting viral RNA from the cell supernatant 24h, 48h, 72h, 96h after infection, reverse transcribing into cDNA, detecting with fluorescent quantitative PCR, and the PCR reaction system is: premix Ex TaqTM (Probe qPCR) 10. Mu. L, PRRSV-N-F and PRRSV-N-F each 0.4. Mu.L, probe 0.6. Mu. L, ddH2O 6.6. Mu. L, cDNA. Mu.L. The results of the fluorescent quantitative PCR are shown in FIG. 2. MARC-145 cells infected for 48h were detected by indirect immunofluorescence, fixed with ice methanol for 10min, blocked with 1% BSA at room temperature for 30min, incubated with PRRSV nucleocapsid protein specific mab (1:800 dilution) for 2h at room temperature, then incubated with FITC-labeled goat anti-rabbit secondary antibody for 1h at room temperature, washed with PBS for five times, and observed under a fluorescence microscope, the results are shown in FIG. 3. According to the above results, the obtained mutant virus pV199D was infectious, was able to successfully transform from a single genomic sequence into active virions, and was virus-infectious.
1.9 Drawing of virus multistep growth curve
The third-generation virus supernatant of 1.7 and the parental strain F112 were subjected to TCID 50/mL assay, marc-145 cells were infected at an infectious dose of 1 MOI based on the assay result, and cell supernatants were collected at time points of 12h, 24h, 36h, 48h, 60h, 72h after infection. And determining TCID 50/mL of virus supernatant at different time points, and finally drawing a multi-step growth curve of the virus by taking virus infection time as an abscissa and taking logarithmic value of TCID 50/mL at different time points as an ordinate. As shown in the graph, the V199D mutant strain has no difference from the growth curve of F112, the virus titer of the rescue strain reaches a maximum value of 1.58×10 8TCID50/mL basically at 72h after the inoculation, and the parent strain F112 reaches 6.31×10 7TCID50/mL.
The above embodiments are only for describing the technical solution of the present invention, and not for limiting the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the design of the present invention.
Claims (4)
1. A porcine reproductive and respiratory syndrome virus NSp 4V 199D for regulating expression of an MHC-II transactivator CIITA is characterized in that the porcine reproductive and respiratory syndrome virus NSp 4V 199D mutates a key amino acid site 199 of NSp4 for regulating CIITA into aspartic acid, a wild type HuN4 is GCTGCCAAA, a vaccine strain HuN-F112 is GCTGTCAAA and GCTGACAAA after mutation, and the porcine reproductive and respiratory syndrome virus NSp 4V 199D can reduce the inhibition effect of the vaccine strain on CIITA after infection of bone marrow-derived dendritic cells, thereby restoring an MHC-II molecule-mediated antigen presentation pathway, and the full-length sequence of the porcine reproductive and respiratory syndrome virus NSp 4V 199D is shown as SEQ ID NO. 1.
2. The method for preparing porcine reproductive and respiratory syndrome virus for regulating expression of MHC-II transactivator CIITA according to claim 1, wherein the method comprises obtaining a genome sequence containing Nsp4 on the whole genome of HuN-F112 according to full-length sequencing of HuN-F112 infectious clone and key amino acid sites for regulating CIITA by nonstructural protein Nsp4, obtaining a sequence containing Nsp4 according to genome sequence design primer PCR amplification, connecting the sequence to an intermediate vector, carrying out site-directed mutation on key amino acid site 199 of Nsp4, carrying out T4 connection with a segmented sequence containing Nsp4 of the whole genome of pBluescript SK (+) vector after obtaining a genome mutant sequence containing Nsp4 of the whole genome of HuN-F112, and constructing a mutant plasmid pBlue-V199D of HuN-F112 whole genome transcription regulating sequence, and then carrying out in vitro transcription; the in vitro transcribed product is subjected to RNA transfection and subculture to obtain porcine reproductive and respiratory syndrome virus NSp 4V 199D, the full-length sequence of the porcine reproductive and respiratory syndrome virus NSp 4V 199D is shown as SEQ ID NO.1, and the regulation of MHC-II transactivator CIITA expression is to improve the transcription level of CIITA genes.
3. Use of a porcine reproductive and respiratory syndrome virus Nsp4V199D which modulates the expression of MHC class II transactivator CIITA according to claim 1 in the preparation of a vaccine for the treatment or prevention of porcine reproductive and respiratory syndrome.
4. An immunogenic composition or combination vaccine comprising the porcine reproductive and respiratory syndrome virus Nsp 4V 199D of claim 1 that modulates expression of MHC class II transactivator CIITA.
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