结核分支杆菌MPT83蛋白基因在大肠杆菌表达载体中的克隆、表达与鉴定

CLONING,EXPRESSION AND IDENTIFICATION OF MYCOBACTERIUM TUBERCULOSIS PROTEIN MPT83 IN EXPRESSION VECTOR OF BACILLUS COLI

[目的]从分支杆菌培养物中提取DNA,扩增结核分支杆菌MPT83蛋白基因,并在大肠杆菌中进行表达、克隆和鉴定。[方法]从分支杆菌培养物中提取总DNA,扩增MPT83因并克隆入T载体,将目的基因插入表达载体pET-32a(+)中,转化BL21宿主菌,IPTG诱导表达构建的表达质粒pET-MPT83经PCR和BamHⅠ+HindⅢ双酶切进行鉴定。[结果]经SDS-PAGE分析,在约42ku处出现新的蛋白条带,5h后表达量即达到高峰;Western blotting分析表明,融合蛋白能够被分支杆菌阳性血清所识别;纯化的表达蛋白经SDS-PAGE电泳,出现清晰的单一条带。表明MPT83基因原核表达载体构建成功。[结论]结核分支杆菌MPT83蛋白基因在大肠杆菌表达载体中的克隆、表达与鉴定,为结核病的诊断以及亚单位疫苗、核酸疫苗的制备和应用奠定基础。

[ Objective] To extract DNA from mycobacterium tuberculosis culture medium, and to amplify the MPT83 proteinum gene of bacillus tuberculosis as well as to express, clone and identify the gene m E. coil [Methods] the total DNA was extracted from the culture of Mycobacteria, and the MPT83 gene was amplified by PCR and then cloned into prokaryotic expression vector pET-32a （＋）. BL21 host germ was transformed. PET-MPT83 induced by IPTG was identified by restriction endonuclease digestion （BamH ＋Hind） and PCR. [Results] SDS-PAGE were performed to analyze. A specific protein showed to be at about 42ku, and the expression quanity reached peak 5 hours later. Western blotting analysis revealed that fusion protein could be identified by positive serum of myeobacteria. The expression of protein purified was performed with electrophoresis of SDS-PAGE, followed by appearance of a clear single strap. The prokaryotie expression vector of MPT83 gene was sucessfully constructed. [Conclusion] Cloning, expression and identification of Myeobacterium tuberculosis protein MPT83 in Expression vector of bacillus coli establish foundation for the preparation of subunit vaccine and nucleic acid vaccine and diagnosis of tuberculosis.