目的:GIP受体激动剂类药物筛选模型的构建.方法:GIP受体激动剂能刺激胰岛素分泌,可作为治疗Ⅱ型糖尿病的潜在药物,因此,GIP受体激动剂的筛选模型十分重要.本研究采用PCR的方法扩增GIPR基因,将扩增产物酶切回收后与表达载体pCMV6-AC-GFP连接,构建pCMV6-AC-GIPR-GFP重组质粒,转化大肠杆菌DH5α经菌落扩增、质粒提取及序列测序重组质粒;利用脂质体lipofectamine2000转染重组质粒到RIN-m5F细胞中,通过抗生素G418筛选,挑单克隆得到稳定的RIN-m5F/GIPR-GFP细胞株.结果:结果表明PCR扩增获得长度为1319 bp的GIPR基因,克隆至pCMV6-AC-GFP真核表达载体,经菌落PCR酶切鉴定及序列分析后,证实质粒pCMV6-AC-GIPR-GFP构建成功;通过荧光显微镜观察到细胞内荧光分布均匀,表明重组质粒成功转到RIN-m5F细胞中.该细胞株经阳性对照(D-Ala2)GIP处理后,与对照组对比,具有荧光斑点聚集.结论:因此,GIP受体激动剂筛选模型RIN-m5 F/GIPR-GFP成功构建,可用于筛选新的GIP受体激动剂,为糖尿病药物开发提供新的筛选模型.%GIP receptor agonists which can stimulate insulin secretion could be potential drugs in the treatment of type II diabetes. Therefore, it is important of the screening model for GIP receptor agonists. In this study, GIPR gene was amplified by PCR, digested by HindIII/XhoI and then connected to the expression vector pCMV6-AC-GFP to construct pCMV6-AC-GIPR-GFP recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli DH5α, subsequently confirmed by colony PCR, restriction endonuclease digestion and sequencing. The recombinant plasmid was transfected into RIN-m5F cells by lipofectamine 2000. After screening with G418, the monoclonal RIN-m5F/GIPR-GFP cell line was selected. The results showed that the GIPR gene with a length of 1319 bp was amplified and cloned into the eukaryotic expression vector of pCMV6-AC-GFP. After analysis of colony PCR, enzyme digestion and sequence, the construction of plasmid pCMV6-AC-GIPR-GFP was proved to be successful. Lots of positive puncta were observed in the treatment of GIPR agonist of ( D-Ala2 ) GIP . The results reveal that GIPR-GFP cell line with stable expression was successfully constructed. This cell line can be used to screen agonists of GIPR for exploring potential new medicines for diabetes.
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