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目的构建麻疹病毒(measles virus)血凝素蛋白(H蛋白)基因的重组质粒,使其在大肠杆菌中大量表达H蛋白,为制备重组蛋白用于免疫检测奠定基础。方法提取麻疹病毒Leningrad-4株总RNA,RT-PCR法扩增其血凝素基因。克隆入pGEM-Teasy载体,双向测序,测序正确后将Ha基因用BamHⅠ和HindⅢ双酶切后亚克隆至pET28a(+)载体,转化BL21(DE3)。培养后用IPTG诱导表达。SDS-PAGE分离大肠杆菌蛋白,分析H基因表达情况。HisLink树脂纯化目的蛋白用于建立酶联免疫吸附反应,进行血清抗体水平检测。结果扩增了麻疹血凝素基因,与麻疹Leningrad-4株血凝素基因同源性达99.8%,构建了重组血凝素基因的原核表达质粒并进行了诱导表达和SDS-PAGE凝胶电泳,在66 kD处有表达的重组血凝素蛋白条带。结论成功构建了麻疹血凝素基因的表达载体并在大肠杆菌中进行了诱导表达,建立了利用重组血凝素蛋白检测IgG的ELISA方法。
Objective To construct recombinant plasmid of measles virus hemagglutinin (H protein) gene and make it express large amount of H protein in Escherichia coli, which lays the foundation for the preparation of recombinant protein for immunoassay. Methods The total RNA of Leningrad-4 strain of measles virus was extracted and its hemagglutinin gene was amplified by RT-PCR. Cloned into pGEM-Teasy vector and sequenced. After sequencing, the Ha gene was subcloned into pET28a (+) vector and transformed into BL21 (DE3). Induction of expression with IPTG after culturing. The E. coli protein was separated by SDS-PAGE and the expression of H gene was analyzed. HisLink resin purification of the target protein for the establishment of enzyme-linked immunosorbent reaction for serum antibody levels. Results The amplified hemagglutinin gene was 99.8% homologous to the hemagglutinin gene of measles Leningrad-4 strain. The prokaryotic expression plasmid of recombinant hemagglutinin gene was constructed and induced by SDS-PAGE gel electrophoresis , A recombinant hemagglutinin protein band expressed at 66 kD. Conclusion The expression vector of hemagglutinin gene was successfully constructed and expressed in E. coli. The ELISA method for detecting IgG by recombinant hemagglutinin protein was established.