pEYFP-hApelin-R重组真核表达载体的构建及在HEK293细胞中的表达

目的构建与增强型黄色荧光蛋白（EYFP）融合的人Apelin-R（Human Apelin receptor,hApelin-R）真核表达载体,用于Apelin-R介导的细胞内信号转导机制研究。方法以质粒pcDNA3.1-hApelin-R为模板,PCR方法扩增人Apelin-R。扩增的Apelin-R产物与质粒pEYFP-C1按常规方法酶切、连接、转化,并经酶切和测序进行鉴定。将重组质粒用脂质体法转染HEK293细胞,Western blot鉴定人Apelin-R的表达。用Apelin-13诱导后,共聚焦显微镜观察受体在细胞内的位移。结果扩增出了一条1143 bp的基因片段,序列与GenBank（NM_005161）相同。在69 kDa处有一蛋白条带,与预期大小相同。人Apelin-R表达在细胞膜上。诱导30 min后,发生受体向胞浆的位移。结论成功构建了pEYFP-hApelin-R重组表达载体,建立了该质粒转染HEK293的细胞模型。可用于Apelin-R介导的细胞内信号转导机制研究,其结果有助于寻找其作为药物的靶点。

Construction of pEYFP-hApelin-R eukaryotic expression vector and its expression in HEK293 cells

Objective To construct expression vector that enhanced yellow fluorescent protein（EYFP）fused with Apelin receptor（Apelin-R）,to investigate intercellular signal transduction mechanism mediated by Apelin-R.Methods The human Apelin-R gene was amplified by PCR using the plasmid pcDNA3.1-hApelin-R as template.The PCR product was digested,ligased with the plasmid pEYFP-C1 and then be transformed into the competent cell Top10.The construct was identified by DNA sequencing.The recombinant plasmid was transiently transfected into human embryonic kidney 293（HEK293）cells,and the expression of pEYFP-hApelin-R was detected by confocal microscopy and Western blot.Results A fragment of 1143bp was amplified by PCR,and the DNA sequence was identical with the gene in GenBank（NM_005161）.Western blot showed a 69kDa band.Confocal microscopy showed that Apelin-R was expressed on the plasma membrane.However,after induction for 30 min by its ligand Apelin-13,the receptor was endocytosed into cytoplasm.Conclusion The pEYFP-hApelin-R eukaryotic expression vector was successfully constructed,and the expression vector could be used to investigate intercellular signal transduction mechanism mediated by Apelin-R and for drug discovery.