hClock-(35-47)_Bax-(55-77)的表达和纯化  

Study on the expressing and purifying of hClock-(35-47)_Bax-(55-77)

作　　者：高梅[1] 刘延友[1] 汪宇辉[1] 赵京卉[1] 叶珊[1] 肖静[1] 王正荣[1] 

机构地区：[1]四川大学基础医学与法医学院生物医学工程研究室,成都610041

出　　处：《四川生理科学杂志》2007年第2期49-52,共4页Sichuan Journal of Physiological Sciences

基　　金：国家自然科学基金资助(N030470623;N030070278)

摘　　要：目的:构建hClock-(35-47)_Bax-(55-77)的表达质粒,进行诱导表达,纯化和鉴定。方法:合成hClock-(35-47)_Bax-(55-77)全长DNA序列,重组入pET-32a表达载体中,测序鉴定后转化入大肠杆菌Rosetta(DE3)菌株,构建好重组体的表达菌株。IPTG诱导并优化表达后,以NTA-Ni亲合层析进行分离纯化,及SDS-PAGE和Western blotting鉴定。结果:成功构建了hClock-(35-47)_Bax-(55-77)的表达载体,插入片断为128bp,表达产物相对分子质量约为23kD,经Western blotting鉴定,与抗His-Tag抗体有特异性反应。结论:hClock-(35-47)_Bax-(55-77)融合蛋白可以用原核表达的方法大量获得,并通过亲和层析纯化达到较好的纯度,为下一步的研究提供基础。Objective： To expressing and purifying of hClock-（35-47）_Bax-（55-77）. Methods： cDNA sequence coding full-length hClock-（35-47）_Bax-（55-77） was synthesized and cloned into the expression plasmid pET-32a. After sequence analysis, the recombinants were transducted into the E. coll. Rosetta（DE3）,which was induced with IPTG to express hClock-（35-47）_Bax-（55- 77）. The products were purified by NTA-Ni affinity chromatography and then verified by means of SDS-PAGE and Western Blotting. Results：The hClock-（35-47）_Bax-（55-77） expression vector containing the insert of 128bp was successfully constructed. The product is about 23kD, which could react immunologically with standard anti-His-Tag antibody. Conclusion： The recombinants hClock-（35-47）_Bax-（55-77） can be expressed by E. coll. Rosetta（DE3）and purified by affinity chromatography. Its large-scale preparation and good purification will profit us to the further study.

关 键 词：hClock BAX 原核表达 纯化 

分 类 号：R914[医药卫生—药物化学]