| 牛分枝杆菌MPB83基因的克隆及其在大肠杆菌中的表达 |
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| 引用本文： | 李晶晶,赵德明,徐广贤,周向梅,尹晓敏.牛分枝杆菌MPB83基因的克隆及其在大肠杆菌中的表达[J].中国农业大学学报,2006,11(6):19-22. |
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| 作者姓名： | 李晶晶 赵德明 徐广贤 周向梅 尹晓敏 |
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| 作者单位： | 中国农业大学,动物医学院/国家动物海绵状脑病实验室,北京,100094 |
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| 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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| 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。
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| 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 |
| 文章编号： | 1007-4333（2006）06-0019-04 |
| 收稿时间： | 2006-05-24 |
| 修稿时间： | 2006年5月24日 |
Cloning and expression of Mycobacterium bovis secreted protein MPB83 in Escherichia coli |
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| Li Jingjing,Zhao Deming,Xu Guangxian,Zhou Xiangmei,Yin Xiaomin.Cloning and expression of Mycobacterium bovis secreted protein MPB83 in Escherichia coli[J].Journal of China Agricultural University,2006,11(6):19-22. |
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| Authors: | Li Jingjing Zhao Deming Xu Guangxian Zhou Xiangmei Yin Xiaomin |
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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. |
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| Keywords: | Mycobacterium bovis MPB83 gene cloning prokaryotic expression western blotting protein purification |
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