GalR2基因真核表达质粒的构建及鉴定

Construction and Identification of Eukaryotic Expression Vector for GalR2 Gene

目的构建GalR2基因真核表达质粒,并在HeLa细胞中表达。方法从C57BL∕6J小鼠海马组织中提取总RNA,RT-PCR法扩增GalR2基因,克隆至pEGFP-C1载体中,构建重组真核表达质粒pEGFP-GalR2。将重组表达质粒转染至HeLa细胞,荧光显微镜观察融合蛋白在细胞内的定位,RT-PCR和Western blot分别检测GalR2基因的转录及表达。结果从C57BL∕6J小鼠海马组织扩增出1 116 bp的GalR2基因;重组表达质粒pEGFP-GalR2经PCR、双酶切及测序鉴定证明构建正确;转染细胞融合基因的表达产物定位于细胞核,GalR2基因在mRNA及蛋白水平上均有表达。结论已成功构建了GalR2基因真核表达质粒pEGFP-GalR2,并在HeLa细胞中成功表达了GalR2蛋白,为进一步探讨GalR2的生物学活性奠定了基础,也为寻找抗抑郁药物的靶点提供了新的思路。

Objective To construct a eukaryotic expression vector for GalR2 gene and express in HeLa cells.Methods Total RNA was extracted from the hippocampus of C57BL/6J mice,with which GalR2 gene was amplified by RT-PCR and cloned into pEGFP-C1.The constructed recombinant plasmid pEGFP-GalR2 was transfected to HeLa cells.The intracellular location of fusion protein was observed by fluorescent microscopy.The transcription and expression of GalR2 gene were determined by RT-PCR and Western blot.Results GalR2 gene at a length of 1 116 bp was amplified from the hippocampus of C57BL/6J mice.PCR,restriction analysis and sequencing proved that recombinant plasmid pEGFP-GalR2 was constructed correctly.The expressed fusion protein was located in nuclei of transfected cells.GalR2 gene was expressed at both mRNA and protein levels.Conclusion The eukaryotic expression vector pEGFP-GalR2 was successfully constructed,which laid a foundation of further study on biological activity of GalR2 and provided a novel route for development of drugs for depression.