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目的：克隆结核分枝杆菌持续感染期抗原Rv1733c 基因，构建其原核表达载体，并在大肠杆菌中进行表达和纯化。方法：采用聚合酶链反应（PCR）方法从结核分枝杆菌H37Rv 基因组中扩增出Rv1733c 基因片段，克隆入pMD18-T 载体，序列测定正确后将其亚克隆入原核表达载体pPro-EXHTb，并在大肠杆菌DH5α 中进行表达，表达蛋白经SDS-PAGE 及Western-blot 分析后，以 Ni-NTA 亲和层析纯化蛋白。结果：成功克隆了Rv1733c 基因片段并构建了其原核表达载体pPro- EXHTb-1733c，转化E.Coli DH5α后能表达大小约30 KD 的蛋白，Western-blot 分析表明表达产物正确。通过亲和层析获得纯化蛋白。结论：成功构建结核分枝杆菌持续感染期抗原Rv1733c 原核表达载体pPro- EXHTb-1733c，并获得纯化蛋白，为研究新型结核疫苗的靶抗原奠定了基础。

英文摘要:

Objective：To clone the Rv1733c gene of Mycobacterium tuberculosis and express and purify the fusion protein. Methods：The Rv1733c gene was firstly amplified by PCR from genome of M. tuberculosis H37Rv strain and cloned into pMD18-T vector. After sequencing, the gene fragment was subcloned into the prokaryotic expression vector pPro-EXHTb and expressed in E.coli DH5a. The expressed protein was identified by SDS-PAGE analysis and Western-blot by using monoclonal antibody against 6×His, and the recombinant 6×His fused protein was purified by Ni-NTA purification system. Results：The gene of Rv1733c was cloned and expressed in E.coli DH5a. The relative molecular mass of this protein was consistent with the predicted. In Western-blot analysis, a specific binding band of this protein with 6×His mAb could be detected at the corresponding site with relative molecular mass of 30 kDa. Conclusion: The Rv1733c was successfully expressed and purified, which may be used as the target antigen for the novel vaccine against TB.

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