Prokaryotic expression of coxsackie virus A6 genes and preparation of polyclonal antibody against the VP1 and VP2 protein

Abstract

Objective To construct the prokaryotic plasmid expressing the recombinant protein Coxsackie virus A6 VP1 or VP2 and prepare the antiserum of rabbit anti-CVA6 VP1 or anti-CVA6 VP2. Methods CVA6 VP1and VP2 gene fragments were amplified by reverse transcription PCR and inserted into the prokaryotic expression vectors. The recombinant plasmids were expressed in E. coli BL21 (DE3). After induction with Isopropyl β-D-Thiogalactoside (IPTG), the fusion proteins were obtained, and then purified by SDS-PAGE electrophoresis and gel extraction. The polyclonal antibodies were prepared by immunizing rabbits with the fusion proteins and analyzed with Western blot(WB) and indirect immunofluorescence assay(IFA). Results The prokaryotic expression vector of CVA6 VP1 or VP2 was confirmed by PCR, double enzyme digestion and sequencing. CVA6 VP1 and VP2 fusion proteins with high purity were obtained. WB and IFA were used to identify polyclonal antibodies. Conclusions The CVA6 VP1 and VP2 prokaryotic expression vectors were successfully constructed, and the recombinant CVA6 VP1 and VP2 proteins and their corresponding polyclonal antibodies were obtained. Key words: Coxsackie virus A6; VP1; VP2; Prokaryotic expression; Polyclonal antibody