人DDR2基因pShuttle-CMV穿梭载体的构建及表达

Construction of pShuttle-CMV vector of human DDR2 gene and its expression

目的:构建带有flag标签的人DDR2 pShuttle-CMV载体,检测其真核表达并观察DDR2分子的亚细胞分布.方法:以含有人全长DDR2cDNA的质粒为模板,用PCR方法扩增DDR2基因的C-端(DDR2-C),并在其C末端带上含24bp的flag标签,NcoI/EcoRV酶切后亚克隆入含有DDR2全长序列的pMD18-T载体,即替换掉原有DDR2序列的C-端,引入flag标签,测序正确后再克隆入pShuttle-CMV表达载体,酶切鉴定正确后采用脂质体法瞬时转染HEK293细胞,West-ern Blot检测DDR2-flag在细胞中的表达.瞬时转染Hela细胞,通过间接免疫荧光法观察DDR2分子在细胞内的分布情况.结果:测序及酶切显示DDR2-flag/pShuttle-CMV载体构建符合预期;脂质体法转染HEK293细胞,24h后用Western Blot方法检测到目的蛋白的表达;对转染了目的载体的细胞应用FITC标记的抗体进行间接免疫荧光实验,激光共聚焦显微镜观察到DDR2分子主要分布于细胞质与细胞膜.结论:成功构建并表达了C-末端带flag标签的DDR2真核表达载体,使其在真核细胞中表达,并观察到其亚细胞分布.

AIM： To construct the pShuttle-CMV vector of human DDR2 with flag tag, to test its expression in human embryonic kidney cells and to observe the subcellular distribution of DDR2. METHODS： DDR2-C gene tagged with 24 bp flag was amplied by PCR using plasmid encoding human DDR2 as tem- plates, and then digested by Nco I/EcoR V, subcloned into the pMD18-T vector which contained the full length DDR2 gene, for replacing the original C-terminal. After splicing and sequencing in T vector, the new full length DDR2 with flag tag was then sub- cloned into pShuttle-CMV. The recombined vector was then trans- fected into HEK293 cells with Lipofeetamine and its expression was detected by Western Blot. The subcellular distribution of DDR2 in cultured Hela cells was studied by confocal microscopy. RESULTS： The eukaryotic expression vector pShutfle-CMV en- coding DDR2 was constructed and identified by the digestion of restriction enzymes. It expressed in HEK293 cells, and could be detected by Western Blot after transfection using Lipofectamine. After the experiment of indirect immunofluorescence, green fluo- rescent signals were detected mainly in the cytoplasm and plasma membrane of Hela cells by confocal microscopy. CONCLU- SION： The eukaryotic expression vector encoding DDR2 has been constructed and it can express DDR2-flag fused protein correctly in HEK-293 cells. DDR2 mainly distributes in the cytoplasm and plasma membrane.