Abstract
Objective VP2 and VP3 surface structural proteins of EV-D68 were expressed in prokaryotic system, and the affinity and binding charactors of two proteins were analyzed to investigate their capacity of binding to memory B cells. Methods The VP2 and VP3 genes of EV-D68 were amplified by PCR and inserted into prokaryotic expression vector pGEX-5X-1.Rosetta(DE3)pLysS, transformed by the constructed recombinant expression plasmids pGEX-5X-1-VP2 and pGEX-5X-1-VP3, were induced by IPTG.The expressed recombinant protein in the form of inclusion bodies were extracted, followed by washing, dialyzing and renaturing.The recombinant protein was then identified by SDS-PAGE electrophoresis and Western blotting.The activity and affinity of VP2 and VP3 were evaluated by competitive ELISA.The binding capacity of VP2 and VP3 to the memory B cells was detected by flow cytometry. Results The recombinant expression plasmids pGEX-5X-1-VP2 and pGEX-5X-1-VP3 were successfully constructed and verified by double restriction enzyme digestion and sequencing.We then obtained both VP2 and VP3 proteins.The purity of VP2 and VP3 proteins were 80% and 75%, respectively.Both proteins showed antigenic specificity.VP2 showed a medium affinity with affinity constant of 2.7 × 107 M-1, and the VP3 showed a low affinity with affinity constant of 3.25 × 106 M-1.Specific memory B cells in spleen after viral challenge to mice can be detected by flow cytometry. Conclusion VP2 and VP3 proteins of EV-D68 were obtained in prokaryotic system with favorable antigen specificity.These proteins can specifically bind to memory B cells with high affinity and binding capacity.These findings offer the targets for sorting EV-68-specific memory B cellsand provide insight for further investigation in the neutralizing epitopes of VP2 and VP3 and their broad-spectrum neutralizing antibodies. Key words: Enterovirus D68; VP2 protein; VP3 protein; Prokaryotic expression
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