麻疹病毒血凝素基因原核表达与抗原性鉴定

Expression and Character of Measles Virus Hemagglutinin Gene

目的利用大肠杆菌表达系统表达麻疹病毒血凝素(Hemagglutinin,H)基因,并将表达产物应用于麻疹病毒抗体的检测。方法从麻疹病毒株MVi/Zhejiang.CHN/7.05/4中提取核糖核酸,用逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR)扩增H基因,并克隆到大肠杆菌表达载体pET28a(+);用异丙基-β-D-硫代半乳糖苷诱导重组质粒在大肠杆菌BL21(DB3)中进行表达;利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis,SDS-PAGE)和蛋白印迹法(Western Blotting)分析重组蛋白表达情况和抗原性:通过Ni-NTA亲和层析获得的高纯度目的蛋白用于酶联免疫吸附试验。结果RT-PCR获得的H基因外段约长1700bp,经测序确证无突变。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;蛋白印迹(Western Blot)显示,表达产物具有良好的抗原性。结论构建麻疹病毒H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 express the hemagglutinin gene of measles virus （MV） in Escherichia coli expression system, and apply the recombinant protein for detection of MV. Method Measles virus genomic RNA was extracted from the strain MVi/Zhejiang. CHN/7.05/4, the interest H gene was amplified by RT-PCR and subcloned into the pET28a （＋） vector, the recombinant plasmid was transformed into E.coli BL21 （DE3） cells and induced with IPTG. After the expression of the recombinant protein, it was analyzed with SDS-PAGE, while Western blotting was performed to determine the specific antigenicity of the target protein. Then, the recombinant protein purified with the Ni-NTA column was applied for the ELISA detection. Results The interest gene amplified by RT-PCR was 1700bp, and there was no mutation by sequencing. The interest protein was expressed in inclusion in Escherichia coli expression system, and showed the high antigenicity in Western blotting. Conclusions the interest protein was successfully expressed in prokaryotic system, and can be used as the antigen in measles virus detection after it was purified.