PHLCX Nflag3/小窝蛋白-1重组质粒的构建及融合基因在293T细胞中的表达  

Construction of Recombinant PHLCX Nflag3/caveolin-1 and Expression of Fusion Gene in 293T Cell

作　　者：杞少华[1] 彭芳芳[1] 张百芳[1] 沈晗[1] 吴少波[1] 武栋成[1] 

机构地区：[1]武汉大学医学院,中国湖北武汉430071

出　　处：《生命科学研究》2005年第4期331-335,共5页Life Science Research

基　　金：湖北省自然科学基金(2002AB00148)

摘　　要：构建重组真核表达质粒PHLCXNflag3/小窝蛋白-1,并在293T细胞中表达.用PCR的方法扩增cDNA文库中的人小窝蛋白-1基因,连接在真核表达载体PHLCXNflag3的短肽标签flag的下游,用限制性酶切和测序的方法鉴定;将重组质粒以脂质体法转染293T细胞,Westernblotting法检测蛋白质的表达.结果显示,双酶切出现两个片段,分别与空载体和人小窝蛋白-1的cDNA分子质量大小相符,测序结果符合人小窝蛋白-1的cDNA序列;Westernblotting显示构建的新载体能够在293T细胞中表达小窝蛋白-1/flag融合蛋白,表明已成功构建了能在293T细胞中高效表达小窝蛋白-1/flag融合蛋白的真核表达载体PLHCXNflag3/小窝蛋白-1.Recombinant PHLCX Nflag3/caveolin-1 plasmid was constructed in which fusion caveolin-1/flag gene can express in mammalian cells. The caveolin-1 fragment was obtained from cDNA bank with PCR technique. PHLCX Nflag3/caveolin-1 was constructed by inserting caveolin-I into PHLCX Nflag3 plasmid vector with routine molecular techniques. Then PHLCX Nflag3/caveolin-1 was transferred into 293T cell using liposomes mediated gene transfer. The expression of caveolin-1 and flag was detected by Western blotting analysis. Restriction analysis showed that the structure of PHLCX Nflag3/caveolin-1 plasmid was the same as anticipated. The Western blotting analysis also showed that fusion caveolin-1/flag gene can express in 293T cell successfully. Recombinant PHLCX Nflag3/caveolin-1 plasmid was constructed as anticipated and fusion caveolin-1/flag gene can express in 293T cell successfully.

关 键 词：小窝蛋白-1 基因表达 293T细胞 

分 类 号：Q782[生物学—分子生物学]