[Construction and identification of recombinant shuttle-plasmid with ESAT-6 from Mycobacterium tuberculosis].

Hua xi yi ke da xue xue bao = Journal of West China University of Medical Sciences = Huaxi yike daxue xuebao Pub Date : 2002-01-01

Wei Chen, Lang Bao, Yongen Xie, Changhua Hu, Wanjiang Zhang, Xuemin Li, Huidong Zhang

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Abstract

Objective: To construct a recombinant BCG secretively expressing ESAT-6 of Mycobacterium tuberculosis.

Methods: alpha-antigen(alpha-Ag) signal sequence and esat-6 gene were amplified from the genome of Bacille Calmette-Guerin (BCG) and Mycobacterium tuberculosis by PCR respectively. esat-6 gene was cloned in E. coli-BCG shuttle-plasmid pMV261 to get pME. Then a new recombinant plasmid pSME was constructed by inserting BCG alpha-Ag signal sequence into pME.

Results: The cloned genes alpha-Ag signal sequence and esat-6 were correctly inserted into the vector pMV261, which was confirmed by restriction endonuclease digestion and PCR amplification of pSME.

Conclusion: pSME was expected to secretively express ESAT-6 of Mycobacterium tuberculosis in BCG. This study provides the possibility of further researches on the development of new anti-tuberculosis vaccine.

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结核分枝杆菌ESAT-6重组穿梭质粒的构建与鉴定

目的:构建表达结核分枝杆菌ESAT-6的重组卡介苗。方法:采用PCR法分别从卡介苗和结核分枝杆菌基因组中扩增α抗原(α - ag)信号序列和esat-6基因。将esat-6基因克隆到大肠杆菌-卡介苗穿梭质粒pMV261中获得pME。将BCG α - ag信号序列插入重组质粒pSME中，构建重组质粒pSME。结果:克隆的α - ag信号序列和esat-6基因被正确插入到载体pMV261中，经内切酶切和pSME PCR扩增证实。结论:pSME有望在卡介苗中隐匿表达结核分枝杆菌ESAT-6。本研究为进一步研制新型抗结核疫苗提供了可能。

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Hua xi yi ke da xue xue bao = Journal of West China University of Medical Sciences = Huaxi yike daxue xuebao

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