人Leptin基因的cDNA的克隆和表达

克隆人Leptin基因的cDNA并获取表达的Leptin蛋白，为进一步研究新的Leptin相关蛋白打下基础。以人基因组DNA为模板，用引物悬挂延伸PCR法，克隆与6个组氨酸（6×His）密码子相连的人Leptin基因的cDNA，并将其克隆到体外表达载体pIVEX23MCS上，通过体外快速翻译系统(Roche公司的RTS500环状模板试剂盒和RTS500 ProteoMaster仪器)在体外表达了带有6×His的Leptin融合蛋白。经SDSPAGE及Western blot方法鉴定，融合蛋白大小正确，有172个氨基酸，分子量为1946kD，具有特异的抗原性，且主要以可溶形式存在于反应液上清中。

cDNA Cloning of Human Leptin and Its Expression

To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.