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目的构建麻疹病毒血凝素蛋白基因的重组质粒,使其在大肠埃希菌中表达HA蛋白,为麻疹病毒诊断奠定基础。方法从麻疹病毒株MV07-30中提取核糖核酸,用RT-PCR扩增HA基因,用限制性内切酶BamHⅠ和HindⅢ双酶切HA基因片段和pGEM-Teasy载体,连接后获得重组质粒pGEM-Teasy-MVHA并克隆到大肠杆菌表达载体pET28a(+);用IPTG诱导重组质粒在大肠杆菌BL21(DE3)中进行表达;利用SDS-聚丙烯酰胺凝胶电泳(PAGE)技术进行重组蛋白表达的分析;通过Ni-NTA亲和层析获得高纯度目的蛋白。结果 RT-PCR获得的HA基因片段约长1700 bp。转化的大肠杆菌表达产物在SDS-PAGE中出现与目的蛋白分子量相一致的条带,且重组蛋白主要以包涵体形式存在;血凝及血凝抑制实验显示表达产物具有良好的抗原性。结论构建麻疹病毒HA基因的原核表达体系,纯化的重组蛋白可用作麻疹病毒检测的抗原。
Objective To construct the recombinant plasmid of measles virus hemagglutinin gene and make it express HA protein in Escherichia coli, which laid the foundation for the diagnosis of measles virus. Methods Ribonucleic acid was extracted from measles virus strain MV07-30. The HA gene was amplified by RT-PCR. The HA gene fragment and pGEM-Teasy vector were double-digested with restriction endonucleases BamHⅠ and HindⅢ, and the recombinant plasmid pGEM- Teasy-MVHA was cloned into E.coli expression vector pET28a (+). The recombinant plasmid was induced by IPTG in E.coli BL21 (DE3). The recombinant protein was analyzed by SDS-polyacrylamide gel electrophoresis High-purity target protein was obtained by Ni-NTA affinity chromatography. Results The HA gene fragment obtained by RT-PCR was about 1700 bp in length. The expressed product of Escherichia coli transformed by SDS-PAGE showed a band corresponding to the molecular weight of the target protein, and the recombinant protein mainly existed as inclusion body. The hemagglutination and hemagglutination inhibition test showed that the expressed product had good antigenicity. Conclusion The prokaryotic expression system of HA gene of measles virus was constructed, and the purified recombinant protein could be used as antigen detected by measles virus.