ABSTRACT
@#Objective To express the globular head domain HA1 gene of influenza virus(IV)in prokaryotic cells,optimize the induced expression conditions,and purify the protein in order to obtain IV HA1 protein with good immunogenicity.Methods Influenza A virus[A/Saratov/CRIE-250/2017(H3N2)],Influenza A virus[A/Hebei/F076/2018(mixed)]and Influenza A virus[A/USA/LAN＿(P5)＿HA/2018(H1N1)]were truncated,and amino acid sequence of63-286 globular head domain was obtained. The genes were optimized and synthesized according to the codon commonly used in E.coli,and named 17Sa,Hebei and USA. The 17Sa point mutation gene was named as 17Sa change. The four genes were cloned into prokaryotic expression vector pET-28a(+)to construct recombinant plasmids respectively,which were transformed into E.coli BL21(DE3)competent cells and induced by IPTG. The protein expression conditions were optimized,and the protein was purified by His labeled nickel ion protein purification column.Results The 762 bp target gene was successfully inserted,and the recombinant plasmid was confirmed to be constructed correctly by double enzyme digestion(NheⅠ/XhoⅠ). The expressed recombinant proteins USA with a relative molecular mass of about 26 000,17Sa,p17Sa and Hebei with a relative molecular mass of about 28 000,showed specific binding to mouse anti-His antibody. The recombinant proteins were all expressed in the form of inclusion bodies,and the expression was highest after induction at 37 ℃ for 8 h. The purified recombinant proteins 17Sa,17Sa change,Hebei and USA had a purity of 90%,85%,95% and 80%,respectively.Conclusion The target protein was successfully expressed and purified by prokaryotic expression system with good reactivity,which lays a foundation of the detection of influenza antibody and the development of new vaccines.