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。 |
关键词: 牛分枝杆菌,MPB83基因,克隆,原核表达,免疫印迹,蛋白纯化 |
DOI:10.11841/j.issn.1007-4333.2006.06.145 |
投稿时间:2006-05-24 |
基金项目:国家重点基础研究发展计划(973计划);国家自然科学基金;高等学校博士学科点专项科研项目 |
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Cloning and expression of Mycobacterium bovis secreted protein MPB83 in Escherichia coli |
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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. |
Key words: Mycobacterium bovis,MPB83 gene,cloning,prokaryotic expression,western blotting,protein purification |