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Construction of pShuttle-CMV vector of human DDR2 gene and its expression
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 9
- DOI:
- 10.3321/j.issn:1000-2790.2007.09.001
- Key Word:
- DDR2;聚合酶链式反应;分子;克隆;基因表达;免疫荧光
Abstract： 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DDR2 cDNA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24 bp的flag标签,Nco I/EcoR V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,Western 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,24 h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.
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