大鼠大麻素Ⅰ型受体绿色荧光融合蛋白真核表达载体的构建与鉴定

目的:构建大鼠大麻素型Ⅰ受体绿色荧光融合蛋白真核表达载体并观察其在细胞中的表达。方法:大鼠CB1基因序列设计引物,以大鼠脑组织为模板扩增CB1基因编码区片段,克隆至增强型绿色荧光蛋白表达载体pEGFP-N3中,构建重组融合蛋白表达载体pCB1-EGFP。将pCB1-EGFP质粒转染HeLa细胞,通过观察EGFP报告基因的表达以及免疫荧光,Western Blot方法鉴定CB1可在真核细胞中过表达情况。结果:构建重组融合蛋白表达载体pCB1-EGFP,单双酶切和测序验证正确。将pCB1-EGFP质粒转染HeLa细胞,荧光显微镜下观察到融合表达的绿色荧光蛋白,且呈胞膜表达。免疫荧光试验也证明重组载体转染后,CB1基因和GFP共同定位于胞膜部分。Western Blot实验证明表达CB1蛋白。结论:成功构建了高表达的CB1-EGFP融合蛋白真核表达载体。

Construction of Eukaryotic Expression Vector pCB1-EGFP and its Expression in HeLa Cells

Objective:To construct the pCB1-EGFP plasmid and investigate its expression in HeLa cells.Methods:Rat CB1 coding sequence (CDS) was obtain, and primers were designed according to the mRNA sequence published in NCBI databas. With rat brain RNA as template, CB1 CDS was amplified by RT-PCR and cloned into pEGFP-N3 vector. Proved by enzyme digestion and sequencing the recombinant plasmid was constructed named pCB1-EGFP. The vector with pCB1-EGFP was transfected into Hela cells, then it was analyzed by immunofluorescence staining and western blot.Results:The specificity of PCR products by DNA scquencing and restriction endonuclease reactions demonstrated that recombinant pCB1-EGFP plasmid was successfully constructed. Green fluorescence expression of the HeLa cells can be observed under fluorescence microscope and CB1-GFP fusion protein was expressed predominantly in cytomembrane while GFP was alone located in cells and expression of CB1 protein was detected in 1HeLa cells by western blot.Conclusion:pCB1-EGFP has been successfully cloned, which provided a basis for further investigation of CB1''s physiological and physiopathological functions and regulatory mechanism.