张薇1 柏银兰2 康健2 王平2 徐志凯2 王丽梅2△.结核分枝杆菌持续感染期抗原Rv1733c 的表达、纯化和鉴定[J].现代生物医学进展英文版,2012,12(10):1868-1871. |
结核分枝杆菌持续感染期抗原Rv1733c 的表达、纯化和鉴定 |
Expression, Purification and Identification of the Mycobacterium tuberculosisProtein Latency Antigen Rv1733c |
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DOI: |
中文关键词: 结核分枝杆菌 持续感染期抗原Rv1733c 原核表达 纯化 |
英文关键词: Mycobacterium tuberculosis (MTB) Latency antigen Rv1733c Prokaryotic expression Purification |
基金项目:国家自然科学基金资助项目(30801055) |
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中文摘要: |
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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