牛结核分枝杆菌MPB70基因的克隆及其在大肠杆菌中的表达

Cloning and expression of Mycobacterium bovis secreted protein MPB70 in Escherichia coli

以牛分枝杆菌DNA为模板,克隆了牛分枝杆菌MPB70基因,构建了克隆载体pGEM-MPB70和表达载体pET30a-MPB70,经IPTG诱导在大肠杆菌BL-21中表达,用SDS-PAGE和免疫印迹分析表达产物并进行蛋白纯化。试验结果表明,牛分枝杆菌MPB70基因体外扩增产物与预期值相符,约582 bp;所构建表达质粒pET30a-MPB70经测序,结果与预期一致;SDS-PAGE分析表明,该融合蛋白以包涵体的形式表达,其分子质量约为29 kD,蛋白表达量约占菌体总蛋白的20%;该蛋白经电洗脱纯化后,纯度达85%以上;免疫印迹分析表明,原核表达的融合蛋白可与兔抗牛分枝杆菌多克隆抗体结合,并且具特异的免疫反应性。

The MPB70 gene of Mycobacterium bovis was cloned from M.bovis genomic DNA.Using the cloned gene,the plasmid pGEM-MPB70 and prokaryotic expression plasmid pET30a-MPB70 were constructed.Recombinant E.coli BL21was induced by IPTG to express the fusion protein.The expressed and purified product was analyzed by SDS-PAGE and Western-Blot.The result showed that PCR product was approximately 582 bp.The results of SDS-PAGE showed that the fusion protein was produced abundantly as inclusion body and the molecular weight of expressed protein was approximately 29 kD.SDS-PAGE analysis also showed that the recombinant protein could reach 20 percent of the whole bacterial proteins.Purified protein was obtained after being eluted from the gel by electrophoresis.The Western-blot analysis showed the fusion protein had the antigenic activity of Mycobacterium bovis.