人脂联素、球状脂联蛋白基因克隆及真核表达载体的构建与表达

Cloning of apM1 and gAdiponectin genes and construction and expression of eukaryotic expression plasmid of gAdiponectin gene

目的构建高效表达球状脂联蛋白的真核表达载体,为研究脂联素、球状脂联蛋白在2型糖尿病及动脉粥样硬化中的作用奠定基础。方法克隆人脂联素基因,构建球状脂联蛋白基因的真核表达载体,转染人脐静脉内皮细胞株(HUVEC),Western blot检测上清中球状脂联蛋白的表达。结果测序结果表明克隆的人脂联素基因序列正确;构建的球状脂联蛋白基因真核表达载体能有效转染HUVEC,并在上清中检测到该基因的表达。结论完成人脂联素基因克隆,成功构建了人球状脂联蛋白基因真核表达载体,并在HUVEC中获得分泌表达,为研究球状脂联蛋白对2型糖尿病的干预作用提供了可能。

Objective To construct eukaryotic expression plasmid of gAdiponectin gene in order to study the function of adiponectin and gAdiponectin in diabetes and atherosclerosis. Methods Human adiponectin gene was cloned, then eukaryotic expression plasmid of gAdiponectin gene was constructed and transfected in HUVEC cells. Results Sequence analysis verified that the fragment cloned in pGEM-T vector was apM1 cDNA and that the fragment in pcDEF/PPT-myc-His（-）A was gAdiponectin gene. In the supernatant of HUVEC cells transfected by the plasmid, 22KDa protein was detected by Western blot. Conclusions Eukaryotic expression plasmid of gAdiponectin gene is successfully constructed and expressed effectively in HUVEC cells.