人脂联素基因真核表达载体的构建及在HEK293细胞中的表达

Construction of eukaryotic expression plasmid of APM1 gene and its expression In HEK 293 cells

目的构建人脂联素(APMl)基因真核表达载体,并检测其在HEK293细胞中的表达水平。方法用PCR法从测序正确的pGEM-T-APM1质粒上,扩增出两端带有SfiⅠ酶切位点的人脂联素基因,再将扩增出的片段亚克隆到T载体上,用SfiⅠ酶切T载体和pCDEF空载体,将酶切后的片段进行连接、转化、筛选、鉴定、测序。将成功构建的pCDEF-APM1质粒转染HEK293细胞,ELISA检测培养上清中脂联素的水平。结果构建的pCDEF-APM1质粒能有效转染HEK293细胞,并在培养上清中检测到较高水平的脂联素蛋白。结论成功地构建了人脂联素基因的真核表达载体,并在HEK293细胞中获得高效表达。

Objective To construct an eukaryotic expression vector with human adipose most abundant gene transcript 1 （ APM1 ） gene, and to investigate the transfection and expression of pCDEF-APM1 eukaryotic expression plasmid in HEK293 cells. Methods pCDEF-APM1 eukaryotic expression plasmid was constructed by DNA recombinant method. Expression vector pCDEF-APM1 was transfected into HEK293 cells with Effectene reagent. The level of human adiponectin protein in the superuatant of cell culture media was detected with double antibody sandwich ELISA. Results The sequence of DNA fragment from constructed pCDEF-APM1 plasmid was identical to that published in GenBank. There was raised human adiponectin protein level in culture supernatant of HEK293 cells transfected with pCDEF-APM1. Conclusion The pCDEF-APM1, an eukaryotic expression plasmid for APM1 gene is successfully constructed. High protein expression of adiponectin can be obtained in HEK293 cells transfected with pCDEF-APM1 eukaryotic expression plasmid.