hClock-(35-47)的表达、纯化及其功能的检验  

Expression,Purification of hClock(35-47) and Verification of Its Transmembrane Ability in Vitro

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

机构地区：[1]四川大学华西医学中心卫生部时间生物学重点实验室,四川成都610041

出　　处：《航天医学与医学工程》2007年第4期250-253,共4页Space Medicine & Medical Engineering

基　　金：国家自然科学基金资助项目(30070278;39970275)

摘　　要：目的构建hClock-(35-47)的表达质粒,进行诱导表达,纯化,在体外初步鉴定其跨膜功能。方法合成hClock-(35-47)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 bloting鉴定。纯化鉴定后的hClock-(35-47)用FITC标记,以一定的浓度孵育体外培养的血管内皮细胞(ECV-304),荧光显微镜下观察其穿膜活性。结果成功构建了hClock-(35-47)的表达载体,插入片断为51bp,表达产物相对分子质量约为21kD,经Western bloting鉴定,与抗His-Tag抗体有特异性反应,与培养的ECV-304孵化,显示具有穿膜功能。结论原核表达的hClock-(35-47)仍然保留其穿膜功能,为进一步研究提供基础。Objective To clone, express and purify hClock-（35-47） and to verify its transmembrane ability in vitro, Methods cDNA sequence coded full-length hClock-（35-47） was synthesized and cloned into the expression plasmid pET-32a. After sequence analysis, the recombinants were transduced into E. coil Rosetta（DE3）, which was induced with IPTG to express hClock-（35-47）, The products were purified by NTA-Ni affinity chromatography and then verified by means of SDS-PAGE and Western blotting, The vascular endothelial cell （ECV-304） was cultured with hClock-（35-47） which had been labeled with FITC in vitro, and was observed through fluorescence microscope to examine the transmembrane ability of hClock-（ 35-47 ）, Results The hClock-（35-47） expression vector containing the insert of 51 bp was successfully constructed, The product is about 21 kD, which could react immunologically with standard anti-His-Tag antibody, Fluorescence microscopy showed that hClock-（35-47） has the ability to transfer into ECV-304 cells, Conclusion The recombinants hClock- （3547） expressed by E. coli. Rosetta（ DE3）remain its transmembrane ability, Its large-scale preparation will be helpful for further studies.

关 键 词：hClock-(35-47) 纯化 内皮细胞 穿膜功能 

分 类 号：Q786[生物学—分子生物学]