Effects Of PRRSV GP5 Glycosylation Sites On Virus-Like Particles Formation And Immunogenicity

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease, which caused by porcine reproductive and respiratory syndrome virus (PRRSV) and characterized by reproductive failure in sows and respiratory problems in piglets. Since the highly pathogenic epidemic PRRSV (HR-PRRSV) emerged in our country in 2006, it becomes one of the most important diseases affecting the swine industry, and causing huge economic losses.As a novel vaccine platform with higher security, virus-like particles (VLPs) structurally resemble the authentic viruses and have a good antibody responses and immunogenicity. To further enhance the immunogenic properties of PRRSV VLPs, in this study, GP5 glycosylation sites had been modified to construct VLPs, and immunogenicity in mince was analased. The details are as follows:1. Polyclonal antibody preparation and development of an indirect ELISA with truncated GP5It was difficult to express the full-length GP5 protein in prokaryotic expression system, so in this study, GP5 C-terminal 130-200 amino acid was selected. The purified tGP5 as immunogens, emulsified to prepare the polyclonal antiserum. The antiserum can react with the tGP5 and display high titers; however, the virus neutralization test had shown that the neutralizing activity was lacking.There are no effective serological methods to assess the relationship between antibody levels and immune protection. To establish a diagnostic method to evaluate the relationship between antibody levels and immune protection, an indirect ELISA was established using recombinant tGP5 protein as coating antigen expressed in E. coli. The optimized reaction conditions were including:coating with 2 μg/mL antigen at 37 ℃ for 2 hours, blocking with 5% skim milk at 37℃ for 2 hours, incubating with sera diluted at 1:100 at 37℃ for 1 hour, then incubating with the secondary antibody diluted at 1:20000 at 37℃ for 45 minutes, and judging with the cutoff value of OD450nm>0.22. Specificity and reproducibility experiments showed that it had no reactions with the positive sera to swine fever virus, pseudorabies virus, porcine circovirus type 2 virus and foot-and-mouth disease virus, and the inter-and intra-batch reproducibility was excellent.30 positive and negative sera, determined by ELISA, were proceeding with neutralization test, the results showed that the neutralization test was inconsistent with the indirect ELISA. It demonstrates that the method of ELISA against tGP5 cannot replace the neutralization test to evaluate the relationship between antibody levels and immune protection.2. Affection of GP5 glycosylation sites on construction of VLPsIn order to study the affection of GP5 glycosylation sites on the formation of VLPs, in this study, compared with the original GP5, the four mutations of single glycosylation sites GN30S, GN35S, GN44S, GN51S were carried out, and an HA tag was inserted between the epitope A and B. Eight pairs of primers were designed, and modified genes were amplified by overlap extension PCR. Recombinant plasmids were constructed by cloned modified PCR products into donor plasmid pFastBac Dual, and then identified by PCR, double enzymes digestion and sequencing analysis. While, construct recombinant plasmid co-expression M and N. The recombinant donor plasmids were transformed into DH10Bac cells, and then the recombinant bacmids were screened and identified by blue-white plaque assay and PCR. The correct recombinant bacmids were transfected into Sf9 cells, and then seven recombinant baculoviurses were obtained. Each of six kinds of GP5 recombinant baculovirus and rBac-MN were co-infected Sf9 cells and confirmed by Western-blot and electron microscope. Experiment results showed that VLPs formed by six types of recombinant baculoviruses can be observed, indicating that the single mutation of GP5 glycosylation sites does not affect the formation of PRRSV VLPs, neither the HA tag inserted between epitopes A and B.3. Immunogenicity analyses of PRRSV VLPs formed by baculovirusesIn order to investigate the immunogenicity of six types of PRRSV VLPs,48 BALB/c mice were divided into eight groups, Sf9 cells lysates and PRRSV vaccine were used as negative and positive controls, respectively. The results showed that the ELISA antibody levels generated by rBac-GP5/MN was higher than rBac-GP5 (HA)/MN and other groups; The neutralizing antibodies and cytokine levels such as IL-4, IFN-y could not detected. Above results demonstrated that the single glvcosylation site mutation and insertion of HA tag between epitope A/B affected the immune properties of PRRSV VLPs, however, neutralizaiton antibody can’t be detected with low amount of VLPs.