人SYNOVIOLIN基因真核表达载体的构建及表达  

Construction and expression of eukaryotic expression vector of human SYNOVIOLIN gene

作　　者：陈钢[1] 张正治[1] 

机构地区：[1]第三军医大学基础医学部中心实验室,重庆400038

出　　处：《中国临床解剖学杂志》2009年第6期704-707,共4页Chinese Journal of Clinical Anatomy

基　　金：国家自然科学基金(30672122)

摘　　要：目的:构建携EGFP的人SYNOVIOLIN基因真核表达载体。方法:应用基因重组技术,根据人SYNOVIOLIN基因序列和表达载体pIRES2-EGFP质粒上的多克隆位点设计引物,对含有SYNOVIOLIN基因的质粒pCDNA3-syno扩增,得到约1900bp的目的片段,进行T-A克隆。SalⅠ/BamHⅠ双酶切测序正确的重组质粒,回收SYNOVIOLINcDNA片段,将其亚克隆于pIRES2-EGFP载体的多克隆位点内得到质粒pIRES2-EGFP-syno。脂质体法转染HEK293细胞,用激光共聚焦显微镜和Western blot检测EGFP和SYNOVIOLIN在HEK293细胞的表达。结果:PCR,酶切及测序结果表明pIRES2-EGFP-syno真核表达载体构建成功,激光共聚焦显微镜和Western blot显示EGFP和SYNOVIOLIN蛋白在HEK293细胞中成功表达。结论:成功构建pIRES2-EGFP-syno真核表达载体并在HEK293细胞表达,为抗肌腱粘连的SYNOVIOLIN基因治疗研究奠定基础。Objective： To construct eukaryotic expression vector containing the EGFP-SYNOVIOLIN gene. Methods： According to the sequence of SYNOVIOL1N gene and the multiple clone sites of the expression vector plRES2-EGFP plasmid, a specific pair of primers were designed and synthesize. PCR amplification of SYNOVIOL1N gene from the pCDNA3-syno plasmid was performed, and an approximate 1900bp objective fragment was achieved. The fragment was then cloned into T-A vector. The recombined vector confirmed by sequencing was digested by Sal I/BamH I to obtain SYNOVIOLIN cDNA fragment, then the fragment was subcloned into the multiple sites of plRES2-EGFP vector. The recombinant plasmid plRES2-EGFP-syno was transfected into HEK293 cell by lipofectamine 2000. The result was examined using confocal microscopy. The expression of SYNOVIOLIN was detected by Western blotting. Results： PCR, DNA sequencing and restriction enzyme digestion analysis indicated that the eukaryotic expression vector plRES2-EGFP-syno was constructed successfully. The expression of EGFP can be seen under confocal microscopy. Western blotting proved the protein expression of SYNOVIOLIN in HEK 293 cells. Conclusions： The SYNOVIOL1N cDNA is acquired and the eukaryotic expression vector, plRES2-EGFP-syno is successfully constructed with efficient expression in HEK293 cells, which provide a good experimental basis for further study on the gene therapy of anti-tendon adhesion.

关 键 词：SYNOVIOLIN 绿色荧光蛋白 真核表达载体 基因治疗 

分 类 号：Q813[生物学—生物工程]