大鼠胰高血糖素样肽-1受体的克隆和原核表达

Cloning and Prokaryotic Expression of Rat Glucagon Like Peptide-1 Receptor

目的从大鼠胰岛细胞INS-1中克隆胰高血糖素样肽-1受体(GLP-1R)的编码序列,构建原核表达载体并在大肠埃希菌中表达。方法培养INS-1细胞,提取细胞总RNA,经RT-PCR技术扩增出GLP-1受体的编码序列。然后将其连接到原核表达载体pGEX-4T-1中,并进行酶切、测序鉴定。将获得的pGEX-4T-1-GLP-1R转化大肠埃希菌感受态细胞BL21(D3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导后收集菌体蛋白,最后通过SDS-PAGE对收获的融合蛋白进行鉴定。结果成功获得了GLP-1R编码序列,构建了原核表达载体pGEX-4T-1-GLP-1R并在大肠埃希菌BL21(D3)中表达。表达的融合蛋白主要存在于上清液中,分子量大小约为83kD。结论本实验为获取GLP-1R大量蛋白样品制备抗体提供了可能;GLP-1R的克隆也为建立2型糖尿病的新药筛选平台提供了条件。

Objective To clone the cDNA of glucagon like peptide-1 receptor （GLP-1R） from the INS-1 ceils and express the protein in E. call Mter constructing the prokaryotic expression vector pGEX-4T-1-GLP-1R. Methods The full length cDNA of GLP-1R was cloned by RT-PCR from the total RNA of INS-1 cells, and then inserted into the vector pGEX-4T-1. After identified by two restriction endonucleases and DNA sequencing, the prokaryotic expression vector pGEX-4T-1-GLP-1R was transSected into BL2I （D3）. The fusion protein GLP-1R/GST was obtained via IPTG inducing, and then confirmed by SDS- PAGE which showed the expected band. Results The coding sequence of GLP-1R has been cloned into pGEX-4T-1 vector and expressed in BL21 （D3） successfully. The fusion protein expressed mainly existed in the supernatant with a molecular weight of 83 kD. Conclusion This study would make it possible to prepare large amounts of protein samples and produce GLP-1R anti- body. The cloning of GLP-1R also makes it possible to create an approach to screen the new drugs of 2-DM.