结核分支杆菌CFP10/pET32a重组质粒的构建、原核表达及纯化  

CFP10 gene of mycobacterium tuberculosis construction of recombinant plasmid,prokaryotic expression and purification

作　　者：丁淑琴[1] 杨园园[2] 杨风琴[1] 朱佳佳[1] 

机构地区：[1]宁夏医科大学,宁夏银川750004 [2]宁夏银川市第一人民医院,宁夏银川750001

出　　处：《宁夏医学杂志》2013年第5期390-392,I0001,共4页Ningxia Medical Journal

基　　金：宁夏自然基金资助项目(NZ21196)

摘　　要：目的构建结核分支杆菌重组CFP10/pET32a表达质粒,原核表达及纯化重组蛋白。方法将目的基因CFP10亚克隆到pET32a表达载体,转化入大肠杆菌BL21(DE3)plysS,鉴定后以IPTG诱导表达,通过SDS-PAGE检测其表达水平,经含Ni2+的His-bind树脂柱纯化CFP10。结果菌落PCR鉴定及测序分析表明,成功构建CFP10/pET32a重组质粒;IPTG诱导表达后,融合蛋白占菌体总蛋白的40%,占裂解物上清总蛋白的72.4%,表明重组的CFP10基因能在BL21中高效表达,表达的蛋白大部分以可溶性状态存在。结论成功构建结核分支杆菌CFP10/pET32a重组质粒,CFP10获得高效表达。Objective To construct a recombinant plasmid carrying CFP10 from mycobacterium tuberculosis, to express prokaryotically,and to purify the recombinant protein. Methods CFP10 gene was subcloned into expression vector pET32a,then expressed and purified by His - bind affinity chromatography with Ni^2 ＋ of the induced expression level were detected by SDS - PAGE. The recombinant protein was purified by affinity chromatography. Results The recombinant plasmid CFP10/ pET32a of Mycobacterium tuberculosis had been constructed successfully and confirmed by restriction endonuclease analysis and DNA sequencing analysis. The expressed protein de- tected by SDS- PAGE was up to 40% in total bacterial protein and 72.4% in the total protein of cell lysate supernatants, respectively, which demonstrated the recombinant CFP10 gene could be expressed in BL21 with high efficiency and the expressed proteins were mainly soluble. The recombinant protein was purified by affinity chromatography. Conclusion The recombinant plasmid CFP10/pET32a of Mycobacterium tuberculosis is successfully constructed and CFP10 gene could be expressed in BIS21 with high efficiency.

关 键 词：结核分支杆菌 CFP10 重组质粒 表达 纯化 

分 类 号：R378[医药卫生—病原生物学]