人甲型流感病毒H3N2亚型中和表位串联基因原核表达载体的构建

Construction of Prokaryotic Expression Vector for Multiple Neutralizing Epitopes of Human Influenza A Virus Subtype H3N2

目的构建人甲型流感病毒H3N2亚型中和表位串联基因原核表达载体,并在大肠杆菌中表达目的蛋白。方法合成人甲型流感病毒H3N2亚型中和表位的串联基因,克隆入pET-28a(+)载体中,构建重组表达载体pET-28a-H3,经酶切鉴定和DNA序列分析后,转化大肠杆菌BL21(DE3),IPTG诱导表达,并进行ELISA和Westernblot鉴定。结果重组载体pET-28a-H3酶切片段大小和DNA序列分析与预期结果一致。表达的目的蛋白相对分子质量约9400,与相应的多克隆抗体反应性良好。结论已成功构建人甲型流感病毒H3N2亚型中和表位串联基因原核表达载体pET-28a-H3,并表达了目的蛋白,为制备单克隆和多克隆抗体奠定了基础。

Objective To construct a prokaxyofic expression vector for multiple neutralizing epitopes of human influenza A virus subtype H3N2 and express in E. coli .Methods The cascaded gene of human influenza A virus subtype H3N2 was synthesized and cloned into vector pET-28a（ ＋ ）. The constructed recombinant plasmid pET-28a-H3 was identified by restriction analysis and DNA sequencing,then transformed to E. coli BL21（DE3） for expression under induction of IPFG.The expressed product was identified by ELISA and Western blot.Resuits Both the length and DNA sequence of gene fragment extracted from recombinant plasmid pET-28a-H3 were consistent with those expected.The expressed protein,with a relative molecular mass of about 9 400, showed good reactivity with the corresponding polyclonal antibody. Conclusion The prokaxyotic expression vector pET-28a-H3 for multiple neutralizing epitopes of human influenza A virus subtype H3N2 was successfully constructed and expressed, which laid a foundation of preparation of monoclonal and polyclonal antibodies.