The Study Of A Baculovirus Construction Of The Glycosylated PRRSV Glycoprotein 5 And The Immunogenicity

Porcine reproductive and respiratory syndrome(PRRS)is a reproductive disorder and respiratory disorder in pigs caused by PRRS virus.Clinically,pregnant sows with reproductive disorders appear abortion,piglets with respiratory symptoms,and high mortality after infection,also known as blue ear disease.Porcine reproductive and respiratory disorders were first discovered in the United States,and had a major impact on the global pig industry.At present,this disease has been identified by OIE as a statutory reported disease,belonging to the second class animal disease.In 2006,the disease also spread rapidly in China,and appeared strain variation phenomenon.GP5 protein is a capsule protein encoded by PRRS virus open reading frame ORF5 with high variability.At present,studies have shown that there are two epitopes of GP5 protein:non-neutralizing A epitope and neutralizing B epitope.In the process of PRRSV infection,neutralizing B epitope can induce the body to produce neutralizing antibody.While neutralizing antibody production time is delayed,the body is the first 7d after infection with PRRSV around,the neutralizing antibodies cannot neutralize the virus after infection 28 days around to produce neutralizing antibody and antibody drop degree is relatively low and can not eliminate the virus,which is the main mechanism of PRRSV escape immune surveillance,is also the main characteristics of the infection.At present,some studies have found that there are multiple glycosylation sites on and around the neutralizing epitope B,and it is speculated that the existence of glycosylation sites may cover the B antigen epitope,which hinders the production of neutralizing antibodies,delays the production and reduces the antibody titer.The glycosylation sites reported so far are: amino acids 30,34,35,44,and 51.Our laboratory has conducted site-directed mutations on these five glycosylation sites and successfully constructed the eukaryotic plasmid p VAX1-N30 A,p VAX1-N34 A,p VAX1-N35 A,p VAX1-N44 A,p VAX1-N51A;Western Blot analysis was performed on the proteins transfected into 293 T cells with these plasmids,and the results showed that the protein content of the mutants at positions 35 and 51 was higher than that of the original GP5 At the same time,the results of animal experiments showed that the neutralizing antibody of the 35 th mutant also increased significantly.In view of the high expression level of the baculovirus expression system and the simpler glycosylation modification than mammals,in this study,the 35 th and 51 th mutants and the original sequence plasmids were constructed and recombined through the experimental procedures of restriction digestion,ligation,transformation,and transposition.Bacmid shuttle plasmid.Transfect the constructed recombinant shuttle plasmid into Sf9 cells for proliferation,and collect recombinant baculovirus to infect Sf9 cells.Through SDS-PAGE and Western Blot results,you can see the target protein band imprint;through IFA test,it can be seen that three groups of recombinant baculoviruses are infected The specific fluorescence(green)can be detected in all Sf9 cells,while the wild-type Bacmid-infected Sf9 cells and normal Sf9 cells have no specific fluorescence.It can be seen that the target gene carried by the recombinant baculovirus is in the Sf9 cells.The GP5 protein was successfully expressed and specifically bound to the GP5 mouse monoclonal antibody.The above results indicate that the recombinant baculovirus with glycosylation mutation sites at positions 35 and 51 was successfully constructed and can be translated and expressed in Sf9 cells.The obtained recombinant baculovirus were named:Ac MNPV-ORF5,Ac MNPV-N35 A,Ac MNPV-N51 A.The recombinant baculovirus constructed in the above experiment was immunized to rabbits(combining with the characteristics of baculovirus would not cause disease to mammals),and the rabbits were subjected to regular cardiac blood sampling to determine the titer of the neutralizing antibody.The results of neutralizing antibody showed that the titer of neutralizing antibody in ACMNPV-N35 A group was significantly higher than that in Ac MNPV-ORF5 group on the 14 th day(p < 0.01).On day 28,the ACMNPV-N35 A group was also significantly different from the Ac MNPV-ORF5 group(p < 0.001).In conclusion,the 35 th glycosylated mutant can induce a relatively high level of neutralizing antibody in the body.Above results can preliminary determination recombinant baculovirus build success,at the same time,recombinant baculovirus can induce the body to produce neutralizing antibody,but the best dose of recombinant baculovirus and its influence to the pig is not yet clear,but also can induce the body to produce neutralizing antibody for PRRSV GP5 genetic engineering subunit vaccine development laid a solid foundation.