摘要
克隆结核分枝杆菌培养滤液蛋白CFP10基因,并在大肠杆菌中进行表达和纯化。用PCR方法从结核分枝杆菌H37Rv基因组扩增出CFP10基因片段,克隆至pMD18-T载体中,序列测定正确后,将其亚克隆到表达载体pGEX-4T-1并在大肠杆菌BL21中表达。表达蛋白经SDS-PAG及Western-blot分析后,亲和层析法纯化蛋白。成功克隆了CFP10基因,并对其在E.coli中进行了表达,SDS-PAGE及Western blot分析表明表达产物正确。通过GST纯化系统获得36 kD纯化蛋白,与文献报道相符,该蛋白可与CFP10 mAb特异结合,并且同时与活动期结核病人血清发生反应。成功获得了纯化的CFP10蛋白,为进一步研究CFP10蛋白的致病机理提供了实验依据。
In order to clone the gene of Mycobacterium Tuberculosis fosters filtrate protein CFP10,then express the culture filter protein of CFP10 in Escherichia.Coli BL21（E.coli） and purify the expressed protein,the CFP10 gene was amplified from the genome of M.tuberculosis H37RV by PCR and cloned into plasmid pMD18-T.The fragment sequenced correctly was subcloned into the expression vector pGEX-4T-1 and expressed in E.coli BL21.The expressed protein was identified by SDS-PAGE analysis and Western blot method,and subsequently purified it by GST-tag purification system.The gene of CFP10 was successfully cloned and expressed in E.coli,and the expressed protein was identified correctly by SDS-PAGE analysis and Western blot analysis.The expressed protein had both the binding site of anti-CFP10 mAb and TB patient＇ serum.The CFP10 was successfully expressed and purified,which provided material foundation for its pathogenic mechanism study.
出处
《科学技术与工程》
北大核心
2012年第5期1017-1019,1029,共4页
Science Technology and Engineering
基金
国家自然科学基金(30972767)
陕西省自然科学基金(2010JM4012)资助
关键词
结核分枝杆菌
CFP10
表达
纯化
Mycobacterium tuberculosis CFP10 expression purification
