结核分枝杆菌ESAT-6蛋白的重组表达及膜定位研究

中国生物工程杂志

2011, Vol. 31

Issue (5): 55-59

研究报告

结核分枝杆菌ESAT-6蛋白的重组表达及膜定位研究

李浩¹, 殷瑛¹, 毛亚丽², 董大勇¹, 张军¹, 付玲¹, 郭继红², 徐俊杰¹, 陈薇¹

1. 军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室北京 100071
2. 空军总医院药学部北京 100142

Recombinant Expression of Mycobacterium tuberculosis Protein ESAT-6 and the Study about Its Binding to Cell Membrane

LI Hao¹, YIN Ying¹, MAO Ya-li², DONG Da-yong¹, ZHANG Jun¹, FU Ling¹, GUO Ji-hong², XU Jun-jie¹, CHEN Wei¹

1. State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Beijing 100071,China;
2. Department of Pharmacy, Air Force General Hospital, Beijing 100142,China

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摘要：

从结核分枝杆菌H37Rv基因组中扩增出ESAT-6基因,克隆入pET21a(+)载体。将成功构建的pET21a(+)-ESAT-6重组质粒转化入感受态BL21(DE3)中,经诱导表达后,使用SDS-PAGE和Western blot鉴定。超声破碎后发现目的蛋白以可溶性表达形式存在,经过Ni-NTA柱和DEAE-Sepharose^TMFast Flow柱纯化,获得纯度约为95%的重组ESAT-6蛋白。将目的蛋白和RAW264.7细胞共孵育后,使用免疫荧光技术发现ESAT-6蛋白能够直接和RAW264.7的细胞膜结合。

关键词： 结核分枝杆菌; ESAT-6; 纯化; 免疫荧光

Abstract:

ESAT-6 gene was amplified by PCR from the genome of Mycobacterium tuberculosis H37Rv, and then cloned into pET21a(+) plasmid. The recombinant plasmid that was successfully constructed was transformed into E.coli BL21(DE3). After induced with IPTG, the expressed recombinant protein was confirmed by SDS-PAGE and Western blot. The vector yielded satisfactory levels of recombinant ESAT-6 protein expressed as a soluble protein in E. coli. After ultrasonication, the recombinant ESAT-6 protein was firstly purified by a column packed with Ni-NTA Resin and then a column packed with DEAE-Sepharose^TM Fast Flow matrix. The purity of the purified protein was about 95%. The purified ESAT-6 protein was incubated with RAW264.7 cells, and the result got by Immunofluorescence showed that ESAT-6 could directly bind to the macrophage membrane.

Key words: Mycobacterium tuberculosis ESAT-6 Purification Immunofluorescence

收稿日期: 2010-11-10 出版日期: 2011-05-27

ZTFLH:

Q786

基金资助:

国家自然科学基金资助项目(30972772)

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引用本文:

李浩, 殷瑛, 毛亚丽, 董大勇, 张军, 付玲, 郭继红, 徐俊杰, 陈薇. 结核分枝杆菌ESAT-6蛋白的重组表达及膜定位研究[J]. 中国生物工程杂志, 2011, 31(5): 55-59.

LI Hao, YIN Ying, MAO Ya-li, DONG Da-yong, ZHANG Jun, FU Ling, GUO Ji-hong, XU Jun-jie, CHEN Wei. Recombinant Expression of Mycobacterium tuberculosis Protein ESAT-6 and the Study about Its Binding to Cell Membrane. China Biotechnology, 2011, 31(5): 55-59.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I5/55

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