Expression Of Porcine Circovirus Type 2 Cap Protein In E.coli

Porcine circovirus type 2 is the major pathogen responsible for diseases associated with multiple system failure syndromes in weaned piglets.The Cap protein encoded by PCV2 ORF2 is the only structural protein of the virus and contains specific antigenic epitopes that can induce immune response.The N-terminal nuclear localization signal(NLS)severely affectsexpression ofthe Cap protein in E.coli.Based on the gene sequence of PCV2 ORF2 published in NCBI,according to the codon usage bias in E.coli,the entire ORF2 gene sequence was optimized and modified.The 702 bp ORF2 gene sequence was amplified by PCR and inserted into the PshA?restriction site of the expression vector pET41 to constract a recombination vector.Plasmid pET41-Cap was identified by PCR and sequencing.The recombinant plasmid was transformed into E.coli BL21(DE3).The induced expression was analyzed by SDS-PAGE gel electropHoresis.The Cap protein existed in the form of inclusion bodies in E.coli.The optimal expression conditions of Cap protein were determined by optimization of IPTG concentration,timeand temperature.At 37?,the final concentration of IPTG was 1 mmol/L,and the Cap protein reached maximum after 4 hours of IPTG inducing.The inclusion body protein was washed and dissolved in 8M urea,and the denatured protein was diluted and renatured in a dialysate whose urea concentration was gradually reduced,and the renatured protein was purified by Ni-sepHarose column affinity chromatograpHy.The fused protein tag,GST tag was removel by adding a suitable ratio of recombinant enterokinase.The size of the 29-kDa and relatively pure Cap protein was obtained.The transmission electron microscopy showed that the target protein could self-assemble into virus-like particles(VLPs)with a size of 17-20 nm.