牛蛙生长激素基因在COS_7细胞中的转染表达  

Expression of Bullfrog Growth Hormone Gene in Transfected COS_7 Cells

作　　者：尉研[1] 喻麟[1] 崔佳[1] 杨土凤[1] 刘燕飞[1] 刘世贵[1] 龙章富[1] 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610064

出　　处：《应用与环境生物学报》2007年第3期341-344,共4页Chinese Journal of Applied and Environmental Biology

基　　金：教育部重点项目(国教00104);四川省应用基础研究项目(02NY029-079)~~

摘　　要：应用RT-PCR技术克隆牛蛙生长激素(BfGH)的cDNA,T-A克隆法构建反向插入的pMD18-T/BfGH重组质粒,回收BamHⅠ酶切的BfGH cDNA片段,并与去磷酸化处理的真核表达载体VR1020/BamHⅠ,酶切鉴定方法筛选BfGH正向插入的重组真核表达质粒VBfGH.脂质体法介导质粒VBfGH转染哺乳动物细胞COS7,对转染后的COS7细胞进行RT-PCR、ELISA和免疫荧光检测,分别在转录和翻译水平证实BfGH基因在COS7细胞中得到了正确的转染表达.The cDNA of bullfrog growth hormone （BfGH） was amplified by RT - PCR and it was inserted into pMD18-T vector by T- A cloning method. Then, a recombinant plasmid pMD18-T/BfGH was obtained by screening the plasmids of which BfGH cDNA fragments were oppositely inserted into the pMD18-T vector. The fragments recovered from plasmid pMD18-T/BfGH that was digested by BamH Ⅰ were linked with eukaryotic expression vector VR1020 digested by BamH I and then treated by dephosphorylation. The recombinant expression vector VBfGH was constructed after qualified by different restriction digestions. The recombinant expression plasmids VBfGH were mediated by liposome and were transfected into one kind of mammalian cells called COS7 cells. RT - PCR, ELISA and immunofluorescence assay confirmed that the BfGH gene had been correctly transcripted and translated in COS7 cells, and the expression of BfGH was durative. Fig 4, Tab 1, Ref 17

关 键 词：牛蛙生长激素基因 真核表达质粒 转染 COS7细胞 

分 类 号：Q78[生物学—分子生物学]