目的 原核表达流感病毒(influenza virus,IV)球状头部结构域HA1基因,优化其诱导表达条件,并进行蛋白纯化,以期获得具有良好免疫原性的IV HA1蛋白.方法 将Influenza A virus[A/Saratov/CRIE-250/2017(H3N2)]、Influenza A virus[A/Hebei/F076/2018(mixed)]和Influenza A virus[A/USA/LAN_(P5)_HA/2018(H1N1)]进行截短,取63-286头状结构域氨基酸序列,按照大肠埃希菌常用密码子进行优化,合成基因命名为17Sa、Hebei和US4,并对基因17Sa进行点突变命名为基因17Sa变.将4个基因分别克隆至原核表达载体pET-28a(+),构建重组质粒,将重组质粒转化至E.coli BL21(DE3)感受态细胞,IPTG诱导表达,优化蛋白表达条件,并利用His标签镍离子蛋白纯化柱纯化蛋白.结果 成功插入长度为762 bp的目的基因,重组质粒经双酶切(NheⅠ/XhoⅠ)鉴定构建正确.表达的重组蛋白USA相对分子质量约为26 000,17Sa、p17Sa和Hebei相对分子质量约为28 000,均可与鼠抗His抗体特异性结合.重组蛋白均以包涵体形式表达,在诱导温度37 ℃,诱导8 h表达量最高.纯化的重组蛋白17Sa、17Sa变、Hebei和USA纯度分别为90％、85％、95％和80％.结论 通过原核表达系统成功表达并纯化了目的蛋白,该蛋白具有良好的反应原性,为流感抗体检测和新型疫苗的开发奠定了基础.
Prokaryotic expression and purification of influenza virus globular head domain HA1
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 of 63-286 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(Nhe Ⅰ/Xho Ⅰ).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.
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