The aim of this study is to construct porcine circovirus type 2(PCV2)virus-like particles carrying the CSFV E2 protein(CSFV E2)and to evaluate their immunogenicity in mice.In this study,the PCV2 Cap gene was amplified by PCR,and the decoy epitope(aa169-aa180)of the PCV2 Cap protein was replaced with a fusion gene(PCV2-Cap169-180-E2829-837)encoding two consecutive linear epitopes(aa829-aa837)of the CSFV E2 protein by using overlap-extension PCR,and was cloned into the vector pET-32a(+).The resulting recombinant plasmid p-Cap-E2 was constructed and identified by PCR before transforming into Escherichia coli BL21(DE3),and its expression was induced by IPTG.The recombinant proteins from PCV2-Cap-E2 were purified by using the modified nickel affinity chromatography method,the expression and reactivity of the purified proteins were identified with SDS-PAGE and western blot.Transmission electron microscopy was used to observe whether the purified recombinant proteins could form VLP;mice were immunized with homemade VLP,PCV2,CSFV commercial vaccine and PBS,and the antibody levels and cytokine contents of mice were detected by ELISA at different times after immunization;SDS-PAGE showed that the target band appeared at 49ku,and the recombinant proteins were expressed in the form of soluble proteins;the recombinant protein was mainly expressed as soluble protein,and the purification of the recombinant protein resulted in collecting a single target protein.Western blot results showed that the recombinant protein reacted specifically with porcine PCV2 polyclonal antibody(pAb),porcine CSFV PAb,HRP-labeled 6×His Tag mouse monoclonal antibody(MAb),with specific bands appearing at 49ku;electron microscopy showed that the recombinant protein could form regular VLP;antibody ELISA results showed that the homemade VLP induced higher antibody levels in stimulated mice compared to the PBS control.Cytokine ELISA results showed that the homemade VLP induced higher levels of cytokine production in mice compared with the PBS control.The results of in vitro virus neutralization assay showed there was neutralizing activity of PCV2 in the the serum obtained from mice immunizd with homemade VLP.In summary,PCV2-Cap-E2 recombinant proteins were expressed in E.coli and purified for the first time in this experiment.The resulting proteins can stimulate mice to produce virus-like particles in a self-assembly way in vitro,which can react with specific antibodies against PCV2 Cap protein and CSFV E2 protein.Our study provides the material basis for the development of a new dual virus-like particle vaccine against PCV2 and CSFV.
porcine circovirus type 2virus-like particlesswine fever E2 proteinCap protein
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