结核分支杆菌分泌蛋白MPT64基因克隆表达

发布时间：2018-05-05 07:20

本文选题：结核分枝杆菌 + MPT64　；参考：《宁夏医科大学》2011年硕士论文

【摘要】：目的:构建结核分枝杆菌MPT64原核表达载体pET32a-MPT64并诱导其表达、纯化及鉴定目的蛋白。方法:采用PCR从MTB H37Rv株DNA基因组中扩增MPT64基因,将目的片段克隆至pET32a载体中进行测序。鉴定阳性重组质粒pET32a-MPT64,经测序证实与Genebank公布的基因序列基本一致。将MPT64基因亚克隆入pET32a原核表达载体,酶切鉴定阳性重组质粒pET32a-MPT64,并用IPTG进行诱导表达。SDS-PAGE检测和Western-blot鉴定表明,在相对分子量为24kDa的位置有特异性目的蛋白表达。采用MagExtractor-His-tag蛋白纯化试剂盒对目的蛋白进行纯化结果:经测序证实,获得的目的基因与GeneBank公布的结核杆菌MPT64基因序列基本一致。构建的原核表达载体pET32a-MPT64在大肠杆菌BL21中经IPTG诱导后表达出相对分子量约为24000的蛋白,MagExtractor-His-tag蛋白纯化试剂盒纯化后经抗组氨酸单克隆抗体进行Westren blotting,在相对分子量约24000处可见特异性着色带。结论:成功构建了原核表达载体pET32a-MPT64,并成功诱导了MPT64蛋白的表达,通过MagExtractor-His-tag蛋白纯化试剂盒纯化获得纯度较高的MPT64蛋白,为MPT64蛋白作为免疫原在下一步研究中提供条件。
[Abstract]:Aim: to construct the prokaryotic expression vector pET32a-MPT64 of Mycobacterium tuberculosis MPT64 and induce its expression, purify and identify the target protein. Methods: the MPT64 gene was amplified by PCR from the DNA genome of MTB H37Rv strain and cloned into pET32a vector for sequencing. The positive recombinant plasmid pET32a-MPT64 was identified by sequencing, and the sequence was basically the same as that published by Genebank. The MPT64 gene was subcloned into the prokaryotic expression vector of pET32a. The recombinant plasmid pET32a-MPT64 was identified by restriction endonuclease digestion, and the expression was induced by IPTG. SDS-PAGE detection and Western-blot identification showed that there was a specific target protein expression in the position where the relative molecular weight was 24kDa. Purification of Target protein by MagExtractor-His-tag protein purification Kit Results: it was confirmed by sequencing that the obtained target gene was consistent with the sequence of MPT64 gene of Mycobacterium tuberculosis published by GeneBank. The constructed prokaryotic expression vector pET32a-MPT64 was induced by IPTG in Escherichia coli BL21 to express a relative molecular weight of about 24000 protein, MagExtractor-His-tag protein purification kit was purified by anti-histidine monoclonal antibody to Westren blotting.The relative molecular weight was about 24000. A specific ribbon can be seen. Conclusion: the prokaryotic expression vector pET32a-MPT64 was successfully constructed, and the expression of MPT64 protein was induced successfully. The purified MPT64 protein was purified by MagExtractor-His-tag protein purification kit, which provided the condition for MPT64 protein to be used as immunogen in the further study.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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