pDsRed-hAPJ质粒载体构建及在人HEK293细胞中的表达

背景:Apelin/APJ系统具有广泛的生理作用,但其在细胞内信号转导,特别是apelin受体的脱敏,内化、复敏降解等方面仍无一致性见解.目的:构建人apelin受体APJ与红色荧光蛋白pDsRED-express-C1融合的真核表达载体并检测其在人胚胎肾293细胞中的表达.方法:以质粒pcDNA3.1-hAPJ为模板,扩增人APJ,用EcoR I和BamH I分别酶切扩增的产物和pDsRED-express-C1载体,常规连接、转化感受态大肠杆菌TOP10.提取质粒,进行酶切鉴定,最后进行测序.将测序正确的重组载体用脂质体法转染,PI染色,激光共聚焦显微镜观察.结果与结论:PCR扩增出约1.2 kb的片段,与预期的人APJ大小序列相符,酶切结果显示重组质粒被切成2条片段,其中一条为pDsRED载体大小,另一条为目的片段大小.共聚焦显微观察显示APJ主要表达在细胞膜上.说明实验成功构建了pDsRed-hAPJ真核重组质粒,此表达载体为研究人APJ的亚细胞区域的位移、内吞定位提供了重要的工具.

Construction and expression of pDsRed-human apelin receptor recombinant plasmid in human embryo kidney 293 cells

BACKGROUND: Apelin/APJ system has a wide range of physiological functions,but its intracellular signal transduction,in particular,apelin receptor desensitization,internalization,resensitization degradation,have still no consistent opinion.OBJECTIVE: To construct eukaryotic expression vector expressing human apelin receptor(APJ)tagged to red fluorescent protein(pDsRED-express-C1),and to determine the expression in human embryo kidney 293 cells.METHODS: The plasmid pcDNA3.1-hAPJ was used as a template for PCR amplification of human APJ.Following PCR amplification the PCR product were removed and enzymatic digestion with EcoR I and BamH I.Same enzymes were used to cut vector pDsRED-express-C1.The digestive product was ligated by conventional methods of connection,then transfected into Competent E.coli TOP10.Single clones were picked plasmid extraction,followed by restriction enzyme digestion and finally DNA sequencing.The recombinant plasmid with correct sequencing was transfected into human embryonic kidney cells,PI staining,followed by the observation under a confocal microscope.RESULTS AND CONCLUSION: PCR amplified a 1.2-kb fragment,which was consistent with the expected size of the human APJ.The pDsRed-hAPJ recombinant plasmid was cut into two fragments,one corresponded to the pDsRED-express-C1 vector size,and the other fragment corresponded to APJ target fragment.Confocal microscopy analysis showed that,APJ was expressed mainly in the membrane of human embryo kidney 293 cells.The pDeRed-hAPJ eukaryotic plasmid expression vector was successfully constructed and effective expression of this fusion protein is achieved,which might be instrumental in the study of displacement and intracellular localization of human APJ.