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

CHEN Quan; et al

doi:10.13406/j.cnki.cyxb.2003.03.009

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

更新时间：2024-01-17

- Construction and expression of the prokaryotic expression vector of MTb lhp-esat6 fusion gene
- Journal of Chongqing Medical University Issue 3, Pages: 284-287(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.2003.03.009
  CLC： R346
- Published：2003，
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[1]陈全,朱道银,骆旭东,蒋英,江山.结核分枝杆菌lhp-esat6融合基因原核表达载体的构建和表达[J].重庆医科大学学报,2003(03):284-287+333.

CHEN Quan, et al. Construction and expression of the prokaryotic expression vector of MTb lhp-esat6 fusion gene. [J]. Journal of Chongqing Medical University (3):284-287(2003)
[1]陈全,朱道银,骆旭东,蒋英,江山.结核分枝杆菌lhp-esat6融合基因原核表达载体的构建和表达[J].重庆医科大学学报,2003(03):284-287+333. DOI： 10.13406/j.cnki.cyxb.2003.03.009.

CHEN Quan, et al. Construction and expression of the prokaryotic expression vector of MTb lhp-esat6 fusion gene. [J]. Journal of Chongqing Medical University (3):284-287(2003) DOI： 10.13406/j.cnki.cyxb.2003.03.009.

摘要

目的 :构建结核分枝杆菌（MTb） 1hp -esat6融合基因及其原核表达载体并进行表达。方法 :用GeneSOEing法

将1hp基因和esat6基因体外重组

构建融合基因

克隆入pQE30质粒中进行表达。结果 :构建的融合基因经测序证实完全正确

重组体转化表达菌DH5α后

经过IPTG诱导

表达出 2 6kDa左右的CFP10 -ESAT6融合蛋白。SDS -PAGE分析显示

IPTG诱导 4h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 4 0 %

Westernblotting证实其有良好的抗原性。表达蛋白经过Ni-NTA柱纯化后

获得了纯度为 98%的重组蛋白。结论 :成功构建MTblhp -esat6融合基因

成功构建原核表达载体pQE30 -CFP10 -ESAT6

并获得重组CFP10 -ESAT6融合蛋白

为MTb重组抗原的应用奠定了基础。

Abstract

Objective:To construct lhp-esat6 fusion gene and its prokaryotic expression vector and express it in E.coli.Methods:By Gene SOEing techniques

a fusion gene was constructed by splicing lhp gene and esat6 gene

then cloned into pQE30 plasmid and expressed in DH5a.Results:The fusion gene was identified by DNA sequencing.A fusion protein about 26kDa was expressed in the E.coli.In presence of 1mM IPTG for 4h

the fusion protein was expressed to the maximum.The fusion protein existed in cytoplasm in soluble form and represented about 40% total bacterial protein of E.coli.Its antigenicity was confirmed by Western blotting.The fusion protein was purified through the Ni-NTA resin and the purity reached 98%.Conclusion:The lhp-esat6 fusion gene and the prokaryotic expression vector (pQE30-CFP10-ESAT6) were constructed successfully

and the fusion protein CFP10-ESAT6 was obtained

so that it can provide an experimental basis for application of the recombinant CFP10-ESAT6.

关键词

结核分枝杆菌lhp-esat6融合基因重组CFP10-ESAT6原核表达

Keywords

Mycobacterium tuberculosis(MTB)Lhp-esat6 fusion geneRecombinant CFP10-ESAT6prokaryotic expression

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