Prokaryotic expression, purification and biological properties evaluation of EV-D68 VP1 protein

Objective Prokaryotic expression for the EV-D68 structural protein VP1 and evaluation for its antigenicity character. Methods The EV-D68 VP1 gene was amplified and purified by using the gel extraction kit.Purified DNA was quantified and harboured into pGEX-5X-1 vector according to the manufacturer's instruction.Recombinant plasmid was transformed into the Rosetta (DE3) chemically competent E. coli.The SDS-PAGE, Western-blot was applied to detect the EV-D68 VP1 fusion protein in cellular lysate.The expressed bacterium was destructed by ultrasonication, and subsequently purified by affinity chromatography.The binding ability and affinity between the various kinds of positive serum and EV-D68 VP1 protein is evaluated by Elisa. Results The plasmid pGEX-5X-1-VP1 has been constructed successfully, and is identified by the double-digested method and DNA sequencing.The expression of EV-D68 VP1 protein can be detected on SDS-PAGE with the molecular weight of 61 KD as the predicting.The purified VP1 protein showed special binding to serum from different animals, including rabbit, rhesus monkey and mouse, with affinity constant of 1.6×106、3.9×105、3.1×106, respectively. Conclusion The EV-D68 VP1 fusion protein has been successfully expressed and identified, These results can be the basis for our study on the EV-D68 VP1 protein structure, function, and anti-EV-D68 special neutralization antibodies. Key words: Enterovirus D68; VP1 protein; Prokaryotic expression; Affinity