Construction and expression of the prokaryotic expression vector of MTb esat6 gene

CHEN Quan; et al

doi:10.13406/j.cnki.cyxb.2003.01.002

您当前的位置：

首页 >

文章列表页 >

Construction and expression of the prokaryotic expression vector of MTb esat6 gene

更新时间：2024-01-17

- Construction and expression of the prokaryotic expression vector of MTb esat6 gene
- Journal of Chongqing Medical University Issue 1, Pages: 4-7(2003)
- 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：
- DOI：10.13406/j.cnki.cyxb.2003.01.002
  CLC： R346
- Published：2003，
扫描看全文
[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 (1):4-7(2003)
[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 (1):4-7(2003) 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

references

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Construction and expression of the prokaryotic expression vector of MTb lhp-esat6 fusion gene

Construction of the Mycobacterium shuttle expression plasmid with Mycobacterium tuberculosis heat shock protein 70 promoter

Related Author

CHEN Quan

et al

骆旭东

蒋英

江山

et al

骆旭东

Related Institution

Department of Microbiology,College of Basic Medicine,Chongqing Medical University

⁰