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Fusion expression and purification of Mycobacterium tuberculosis difference protein MPT64 and ESAT6
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 20
- DOI:
- 10.3321/j.issn:1000-2790.2005.20.005
- Key Word:
- 分枝杆菌,结核;MPT64;ESAT6;基因表达
Abstract： 目的: 融合表达结核分枝杆菌分泌蛋白MPT64-ESAT6,为结核病的预防提供可供选择的有效亚单位疫苗. 方法: 设计含不同酶切位点的mpt64和esat6引物,采用PCR的方法从结核分枝杆菌毒株H37Rv-DNA中分别扩增出相应大小的DNA片段,将片段分别与T-easy载体连接测序. 将测序正确的mpt64和esat6按照不同的酶切位点克隆入pPro-EX HT表达载体,挑选出阳性克隆,将诱导的表达产物进行SDS-PAGE蛋白电泳分析,同时与6×His的mAb进行Western-blot分析,并用Ni-NTA亲合柱纯化表达产物. 结果: PCR获得的mpt64和esat6序列与GenBank报道的完全一致. 两者在大肠杆菌DH5α株中融合表达的产物Mr为38×103,与预计大小相吻合. Western-blot结果显示,在Mr约38×103处有表达产物与6×His mAb特异性结合带. 表达产物为包涵体,通过Ni-NTA纯化系统,可得到纯化的目的蛋白. 结论: 结核分枝杆菌的分泌蛋白MPT64和ESAT6被成功的融合表达,有望为结核的临床判定提供有效的诊断试剂.
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