A Study On The Soluble Expression Of Porcine Circovirus Type 2 Cap Gene In Escherichia Coli And Its Immunogenicity

Porcine circovirus type 2(PCV2)could cause systemic diseases,clinical symptoms including postweaning multisystem wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS)and reproductive disorders.These diseases are collectively referred to as porcine circovirus associated diseases(PCVAD).In clinical practice,PCV2 could cause secondary or mixed infection of porcine reproductive and respiratory syndrome virus,Mycoplasma pneumoniae,swine flu and other bacterial pathogens,which seriously harm the development of the pig industry in the world.PCV2 is known as the smallest animal virus.Cap protein was encoded by ORF2,which was considered the main structural protein and was the main target for the development of PCV2 gene engineering vaccine.At present,the commercial production of PCV2 inactivated vaccine has been realized and the research of subunit vaccines also have some progress in China,but there are still many limitations.In this study,the soluble exprssion of the Cap protein of PCV2 in E.coli,which laid a foundation for the development of PC V2 subunit vaccine.The main contents of the research are as following:1.The soluble expression of the PCV2 Cap protein in E.coliThe Cap gene condons was optimized according to the preference of E.coli.The Capm gene were cloned into expression vector pRSET B,pET28a,pET32a by PCR and induced for expression in E.coli BL21(DE3)individually.The results of SDS-PAGE showed that the three recombinant proteins were expressed with the expected size and were well secreted.Western-Blot and the sandwich ELISA results confirmed that these recombinant fusion protein had the well antigenic characteristics of PCV2 and the optimized codons were beneficial to the soluble exprssion of the Cap protein in E.coli,which laid an important foundation for the preparation of PCV2 antigen and the development of PCV2 subunit vaccine.2.Prokaryotic expression and immunogenicity of recombinant capsid of PCV2 fused with somatostainIn this study,somatostain gene was fused to the 3'-terminal of the optimized Cap gene of PCV2 SH stain.Then,they were cloned into expression vector pRSET B,pET28a,pET32a by PCR and induced for expression in E.coli BL21(DE3)individually.The results of SDS-PAGE showed that the three recombinant proteins could be expressed with the production of soluble protein and was confirmed by Western-Blot assay with antibodies against PCV2 Cap protein and SS.In the three the pET28a-Capm-SS was selected.Then,the protein was mixed with ISA206 adjuvant.Mice experiment results showed that the immunized groups induced the high levels of antibodies against PCV2 with ELISA antibody assay.Meanwhile,at 14d and 42d post primary vaccination,weight gain in pET28a-Capm-SS group was higher than that in other groups(P<0.05).Following challenge with PCV2,the PCV2 virus loads in spleen in immunized groups were significantly lower than challenge control group(P<0.01).It indicated that the vaccine prepared by the two recombinant proteins and ISA206 could induce immune protection in mice against PCV2 infection,which provided a new idea for the development of PCV2 subunit vaccine.3.Effects of different adjuvant on the immunogenicity of recombinant Cap protein of PCV2In this study,28a-Capm and MrCap espressed in E.coli and TAT-rBCap espressed in baculovirus were mixed with GEL01,ISA206,ISA15A and CPH adjuvant respectively.Mice experiment results showed that the immunized groups induced the high levels of antibodies with ELISA antibody assay at 21d and 42d post primary vaccination.However,different antigens are suitable for different adjuvant.For 28a-Capm and MrCap adjuvant GEL01 was perfect,but for TAT-rBCap the effect of adjuvant CPH was best.The titers of antibody of tne adjuvant ISA15A/three kinds of proteins were lower.The results of neutralization antibody assay showed that TAT-rBCap+GELO1 induced the highest levels,and the titer was up to 1:50.After challenged 21d,the PCV2 virus loads in spleen were detected by Quantitative Real-time PCR.The results showed that PCV2 virus loads in immunized groups with TAT-rBCap were significantly lower than challenge control group(P<0.05),so were immunized groups with 28a-Capm and MrCap with adjuvant GEL01 and ISA206.It indicated that the vaccine prepared by the three recombinant proteins and GEL01 and ISA206 could induce immune protection in mice against PCV2 infection,which laid a foundation for the development of PCV2 subunit vaccine.