结核分枝杆菌esat6基因原核表达载体的构建和表达

陈全; 骆旭东; 蒋英; 江山; 朱道银

doi:10.13406/j.cnki.cyxb.2003.01.002

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结核分枝杆菌esat6基因原核表达载体的构建和表达

更新时间：2024-01-17

- 结核分枝杆菌esat6基因原核表达载体的构建和表达
- Construction and expression of the prokaryotic expression vector of MTb esat6 gene
- 重庆医科大学学报 2003年第1期页码：4-7
- 作者机构：
  
  1. 重庆医科大学基础医学院微生物学教研室
  2. 重庆医科大学基础医学院微生物学教研室,重庆,400016
- 作者简介：
- 基金信息：
  
  重庆市卫生局科学基金（00-1006）
- DOI：10.13406/j.cnki.cyxb.2003.01.002
  中图分类号： R346
- 纸质出版日期：2003，
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[1]陈全,骆旭东,蒋英,江山,朱道银.结核分枝杆菌esat6基因原核表达载体的构建和表达[J].重庆医科大学学报,2003(01):4-7.

CHEN Quan, et al. Construction and expression of the prokaryotic expression vector of MTb esat6 gene[J]. Journal of Chongqing Medical University, 2003,(1):4-7.
[1]陈全,骆旭东,蒋英,江山,朱道银.结核分枝杆菌esat6基因原核表达载体的构建和表达[J].重庆医科大学学报,2003(01):4-7. DOI： 10.13406/j.cnki.cyxb.2003.01.002.

CHEN Quan, et al. Construction and expression of the prokaryotic expression vector of MTb esat6 gene[J]. Journal of Chongqing Medical University, 2003,(1):4-7. DOI： 10.13406/j.cnki.cyxb.2003.01.002.

摘要

目的:构建结核分枝杆菌（MTb）esat6基因原核表达载体并进行表达。方法:PCR扩增MTb esat6基因

克隆人pQE30质粒

测序正确后

再亚克隆人pET32a（+）质粒

构建pQE30-ESAT6和pET32a（+）-ESAT6重组体。结果:重组体分别转化DH5a和BL21（DE3）菌后

经IPTG诱导

pQE30-ESAT6未表达目的蛋白

而pET32a（+）-ESAT6表达出19kDa左右重组蛋白。SDS-PAGE分析显示

IPTG诱导4h重组蛋白表达量最高

表达蛋白以可溶性非包涵体形式存在于胞浆中

表达量占全菌蛋白质的42％

用Western blotting证实其有良好的抗原性。经过Ni-NTA柱纯化

获得了纯度为92％的重组蛋白。结论:成功构建原核表达载体pET32a（+）-ESAT6

并获得重组ESAT6蛋白

为MTb重组抗原的应用奠定基础。

Abstract

Objective:To construct prokaryotic expression vector carrying esat6 gene and express in E. coli. Methods: The MTb esat6 gene was amplified by PCR

then cloned into pQE30 plasmid

sequenced and then cloned into pET32a( + ) plasmid. Thus two kinds of prokaryotic expression vectors were constructed. Results: After being transformed into the E. coli and inducted with 1mM IPTG

no protein was expressed in the pQE30 - ESAT6 system

but a recombinant protein

about 19 kDa

was expressed in the pET32a( + ) - ESAT6 system. In the presence of 1mM IPTG for 4h

the protein was expressed to the maximum. The protein existed in cytoplasm in soluble form and represented 42% total protein of E. coli. It’s antigenicity was confirmed by Westem blotting. The protein was purified through the Ni - NTA resin and the purity reached 92 % . Conclusion : The prokaryotic expression vector (pET32a( + ) - ESAT6) was constructed successfully

and the rESAT6 was obtained

providing an experimental basis for application of rESAT6.

关键词

结核分枝杆菌esat6基因重组ESAT6

Keywords

Mycobacterium tuberculosis(MTB)ESAT6 geneRecombinant ESAT6

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