Construction and expression of prokaryotic expression vector for 3C gene of enterovirus 71 and purification of expressed product

Objective To construct the prokaryotic expression vector for 3C gene of enterovirus 71(EV71),express in E.coli and purify the expressed product.Methods The 3C gene of EV71 was amplified by PCR using the reverse transcription product of the virus as template,and cloned into vector pGEx-4T-1.The constructed recombinant plasmid pGEx-3C was transformed into E.coli BL21(DE3)for expression under induction of IPTG.The expressed recombinant fusion protein GST-3C was purified by GST-Agarose affinity chromatography and molecular sieve chromatography,and determined for reactogenicity by Western blot.Results Restriction analysis and sequencing proved the recombinant plasmid pGEx-3C was constructed correctly.The expressed recombinant fusion protein,with a relative molecular mass of about 46 000,contained about 50% of total somatic protein,reached a purity of more than 90%,and showed specific binding to mouse monoclonal antibody against EV71.Conclusion Recombinant plasmid pGEx-3C was successfully constructed,and recombinant GST-3C fusion protein was highly expressed in secretory form in E.coli,which laid a foundation of study on structure and function of 3C protease of EV71.