牛分枝杆菌外膜蛋白基因ompA的原核表达及免疫活性分析

ANALYSIS OF IMMUNOCOMPETENCE AND EXPRESSION OF OUTER MEMBRANE PROTEIN OMPA GENE OF MYCOBACTERIUM BOVIS

为验证牛分枝杆菌外膜蛋白A是否具有免疫活性,以牛分枝杆菌ValleeⅢ株基因组DNA为模板扩增牛分枝杆菌外膜蛋白基因ompA,采用DNA重组技术将此基因片段连于表达载体pGEX6p-1,构建重组质粒pGEX6p-1-ompA,并将其转化到大肠杆菌中BL21(DE3)中,IPTG诱导(终浓度为0.1mmol/L),表达产物进行SDS-PAGE分析。用glutathione sepharose4B纯化蛋白和Western blot分析该蛋白的免疫学活性。结果表明:pGEX6p-1-ompA以可溶形式表达,蛋白分子量大小为60kDa,与预计大小相符,纯化的蛋白具有良好的免疫学活性。

The outer membrane protein A gene（ompA） of Mycobacterium bovis Vallee strain was amplified by PCR and inserted into the pGEX6p-1 to construct the recombinant plasmid pGEX6p-1-ompA.The recombinant plasmid was then transformed into Escherichia coli BL21（DE3）.Protein expression was induced by IPTG with a final concentration of 0.1 mmol/ L.The expressed protein was purified by glutathione Sepharose 4B.The outer membrane protein A had a molecular mass of 60 kDa and showed good immunological activity in Western blot analysis.Preliminary results have demonstrated that the outer membrane protein A could be developed a promising novel vaccine against tuberculosis.