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.
出处
《航天医学与医学工程》
CAS
CSCD
北大核心
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)
