Study On The Assembly Of Porcine Reproductive And Respiratory Syndrome Virus Virus-like Particles And DNA Vaccine

Porcine respiratory and reproductive syndrome (PRRS) was an acute and highly lethal infectious disease, which leads to sows’reproductive failure and piglets’ respiratory tract symptoms. PRRS has been a serious problem in swine industry after it was first reported in United States in1987. And the emergence of the highly virulent PRRSV mutant as well as its occasional epidemic outbreaks since2006have caused great economic lost in China.The major envelope protein GP5, coded by ORF5, is the main protective immunogen of PRRSV, and it was also the focus of current research of vaccine against PRRSV. GP5and M could form heterodimers when they were co-expressed, which may facilitate transportation of GP5from Endoplasmic reticulum to Golgi apparatus, and increase the immune response induced by GP5. The structural protein N is the most abundant viral protein expressed in infected cells, and it is also the most immunogenic viral protein. Although anti-N antibodies are non-neutralizing and non-protective, N protein could induce strong cellular immune response.Virus-like particles structurally resemble authentic viruses, and have strong immunogenicity, but are non-infectious and safe because they do not contain any viral genetic material. They have a promising future for application in vaccine research. And baculovirus expression system have been broadly applied in VLPs assembly research for its safe and abundant production of recombinant protein.For more safe and rapid production of genetically engineered vaccine against PRRSV, in this study, we used baculovirus expression system to investigate the structural proteins of PRRSV that participate in the process of VLPs assembly. And recombinant eukaryotic expression piasmids that co-expressed GP5, M and N were also constructed and evaluated in mice for their immunogenicity. The main research works were as following:1. Construction of recombinant eukaryotic expression piasmidsPCR replicate the ORF5, ORF6and ORF7of PRRSV WUH3, and then inserted these genes into eukaryotic expression plasmid pcDNA3.1. Three recombinant eukaryotic expression piasmids were constructed to express various combination of PRRSV protein, with the plasmid pcDNA3.1-ORF5/6/7expressing M, GP5and N, pcDNA3.1-ORF5expressing M alone, and pcDNA3.1-ORF5/6expressing M and GP5. The ability of the piasmids to express the correspondent virus proteins have being verified by Western blot.2. The establishment of ELISA of PRRSV GP5proteinPlasmid pKG-ORF5, which have already been constructed in our lab to express GP5in the prokaryotic expression system, was transformed into Escherichia coli. Following IPTG induction and separation of inclusion body, the GST labeled recombinant GST-GP5was specifically purified. SDS-PAGE analysis showed its molecular weight was38KD. GP5specific ELISA analysis was established using this recombinant protein as antigen.3. Humoral immune response induced by immunizition of mice with recombinant eukaryotic expression plasmidsTo investigate whether N could increase the immunogenicity of M and GP5, BALB/c mice were separately immunized with pcDNA3.1-ORF5, pcDNA3.1-ORF5/6, pcDNA3.1-ORF5/6/7and control plasmid pcDNA3.1. The results indicated that co-expression of GP5and M by immunizition with pcDNA3.1-ORF5/6, could visually add to the immunogenicity of GP5, and increase the level neutralizing antibodies to PRRSV (P<0.05). However, co-expression of GP5, M and N together could not further increase the immunogenicity (P>0.05)4. Cellular immune response induced by immunizition of mice with recombinant eukaryotic expression plasmids5weeks after immunizition with DNA vaccine, mice were killed and the spleen lymphocytes were then separated and treated with inactivated PRRSV. The expression level of IFN-y was evaluated by real-time RT-PCR and ELISA analysis. The results showed that co-expression of GP5and M as well as co-expression of GP5, M and N induced higher level of IFN-y than expression of GP5alone (P<0.05). Moreover, co-expression of GP5, M and N produced the higher IFN-y level than co-expression of GP5and M, and the difference was significant (F<0.05). This result suggest that, when N protein was co-expressed with GPS and M, it could further enhance the cellular immune response induced by these two protein.5. Study on the assembly of PRRSV virus-like particles by recombinant baculovirusPlasmids pFastBac-ORF5/6、pFastBac-ORF5/6/7and pFastBac-ORF2/5/6/7were separately transferred into DH10BacTM E. coli for transposition, and then the recombinant Bacmid-ORF5/6、Bacmid-ORF5/6/7and Bacmid-ORF2/5/6/7were identified by PCR. The corresponding recombinant baculoviruses BV-ORF5/6、BV-ORF5/6/7and BV-ORF2/5/6/7were collected after transfecting Sf9cells with Bacmid DNA. Expression of foreign proteins was checked by western blot, and then the supernatant were harvested for ultracentrifugation. The final pellets were analyzed by electron microscopy assays. The PRRSV virus-like particles were observed in all supernatants, but there are much more VLPs by co-expressing GP5、M、N and GP2b proteins, in comparison with other groups. The result indicated the VLPs can be produced by co-expression of GP5and M proteins and GP2protein will be beneficial for release of VLPs.