人内皮细胞特异性分子-1表达载体的构建、体外表达及初步纯化

Construction of pET-28b human endothelial cell-specific molecule-1 and its expression in E. coli BL-21

目的 探讨人内皮细胞特异性分子 1(hESM 1)表达载体的构建、体外表达及表达产物的初步纯化。方法 从人脐静脉内皮细胞中提取总RNA ,用反转录 PCR扩增出hESM 1的cDNA。插入质粒 pET 2 8b中构建成表达载体 ,转化入大肠杆菌BL 2 1,经IPTG诱导表达 ,表达产物用电洗脱的方法初步纯化。结果 经限制性内切酶酶切图谱分析和DNA序列测定证明所构建质粒为含hESM 1的重组质粒 ,经SDS PAGE分析证实获得产物为分子量 2 3 80 8u的融合蛋白 ,表达量约占菌体总蛋白的 2 4%。结论 成功构建了重组hESM 1表达载体 ,并在大肠杆菌中获得表达 ,为进一步研究及临床应用奠定了良好基础。

Objective To investigate the construction of the recombinant vector and expression of human endothelial cell-specific molecule-1 (hESM-1) in E.Coli. Methods Total RNA was reconstructed using HUVECs firstly. Then RT-PCR was used to amplify hESM-1 cDNA, which was inserted into pET-28b to construct expression vector. The recombinant vector was transformed into BL21. Expression of ESM-1 was induced by IPTG. SDS-PAGE and electroelution were used to purify the product expressed. Results Restriction endonucleases mapping analysis and DNA sequencing demonstrated that the constructed plasmid was the recombi-nant one. The protein was expressed in E.Coli and purified by electroelution. Conclusion Both the vector construction and hESM-1 expression in E.Coli are successful. This finding lays a good basis for manufacture and clinical application of the enzyme.