| Porcine circovirus type 2(PCV2)is a major pathogen to induce post-weaning multisystem wasting syndrome(PMWS)in piglets(5-12 weeks),and the main features of the disease includes weight loss,interstitial pneumonia,lymph node enlargement and hepatitis.The disease was first detected in western Canada in 1991,and since then,it has been identified around the world,which caused significant economic losses to the world’s pig industry.At present,vaccine immunization is the main means of prevention and control of PMWS in China.The Cap protein is a structural protein encoded by the PCV2 ORF2 gene,which can mediate the identification and invasion of PCV2,which contains the main neutralizing epitopes of antigens,and can induce effective immune protection.The Cap protein expressed by insect baculovirus system can be self-assembled into virus-like particles(VLP),and can be used as a subunit vaccine or diagnostic antigen of PCV2.Therefore,PCV2 Cap protein has become a hot spot in PCV2 detection and vaccine research and development.The expression of Cap protein using the insect baculovirus expression system is a tool to prepare PCV2 subunit vaccine and diagnostic antigen.In this study,baculovirus GP64 secretory signal gp64sp was used to guide Cap protein Secretory expression in insect cells.Meanwhile,self-cleaving 2A peptide was used to efficiently express Cap protein in insect cells to improve the level of Cap protein expression.A single copy recombinant baculovirus rvAc-gp64sp-capl and two double copy recombinant baculovirus rvAc-gp64sp-capl-2A-cap2 and rvAc-gp64sp-cap1-2A-gp64sp-cap2,were prepared after infecting Sf9 cells.Western Blotting analysis of concentrated supernatant confirmed that Cap protein had been successfully expressed.At the same time,the level of Cap protein was analyzed at different times,and the results showed that the expression level of Cap protein was the highest at 4 d and 5 d after recombinant baculovirus infection.Finally,the secretory expressions of Cap protein in Sf9 cells that were infected with three recombinant baculovirus viruses were compared,and the results showed that the secretory expression of Cap protein of two double copy recombinant baculovirus was similar,and much more than that of single copy recombinant baculovirus(p<0.01).We have built a cap four-copy recombinant baculovirus based on pFastBac-Dual,Western blotting analysis shows that the amount of Cap secreted by four-copy recombinant baculovirus was significantly higher than that secreted by single copy and double copy recombinant baculovirus,and the amount of recombinant baculovirus secreted by four-copy recombinant baculovirus was about 2.7±0.4 times higher than that by single copy recombinant baculovirus(p<0.001).Four-copy recombinant baculovirus were used to optimize the secretory expression of Cap protein by infecting High five(Hi5)cells.After cation exchange chromatography,it was observed by transmission electron microscope that the Cap protein secreted by four copies of recombinant baculovirus could self-assemble into VLP.BALB/c mice were immunized with subcutaneously injected VLP(VLP-Cap),a self-assembled Cap protein enriched by ultrafiltration,spleen lymphocyte proliferation test and indirect ELISA assay showed that Cap protein secreted by single copy recombinant baculovirus and four-copy recombinant baculovirus can induce specific immune response in miceTaken together,in this study,gp64sp secretory signal peptide was used to replace ORF2 localization signal peptide,and then Cap protein expression in insect cells was successfully realized using insect baculovirus expression system.Besides,several PCV2 cap genes were successfully expressed in insect cells using self-clipping 2A peptide.Finally,the specific humoral and cellular immunity were induced by VLP immunization in mice.In this study,The experimental work was carried out to efficiently express PCV2 Cap protein assembly VLP in insect cells by secretion,which laid a foundation for the subsequent practical application. |
