【摘要】 为观察重组MPB83蛋白的免疫活性,揭示该蛋白在牛结核病的诊断和防治中的作用,克隆了牛分枝杆菌MPB83基因,构建了克隆载体pGEM-MPB83和表达载体pET30a-MPB83,经IPTG诱导在大肠杆菌BL21(DE3)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MPB83基因体外扩增产物与预期值相符,约600 bp;所构建表达质粒pET30a-MPB83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26 ku,蛋白表达量占菌体总蛋白的20%;该蛋白经电洗脱纯化后,纯度达95%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。更多还原

【Abstract】 In order to determine the function of the MPB83 in the prevention and diagnosis of tuberculosis in cattle,the MPB83 gene of Mycobacterium bovis was cloned.Then vector pGEM-MPB83 and prokaryotic expression vector pET30a-MPB83 were constructed.Recombinant E.coli BL21(DE3) was 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 about 600?bp as expected.Expression vector pET30aMPB83 was conformed confirmed by sequencing.The results of SDS-PAGE showed that the fusion protein was produced abundantly as inclusion body and the molecular weight of expressed protein was about 26?ku.SDS-PAGE analysis also showed that the recombinant protein expressed could reach 20 percent of the whole bacterial protein.Purified protein was obtained after being eluted from the gel by electrophoresis.The Western-blotting analysis showed the fusion protein had the antigenic activity of Mycobacterium bovis.更多还原