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

构建重组真核表达质粒PHLCX Nflag3／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，连接在真核表达栽体PHLCX Nflag3的短肽标签flag的下游，用限制性酶切和泖l序的方法鉴定；将重组质粒以脂质体法转染293T细胞，Western blotting法检测蛋白质的表达．结果显示，双酶切出现两个片段，分别与空栽体和人小窝蛋白-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；Western blotting显示构建的新栽体能够在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栽体PLHCX Nflag3／小窝蛋白-1．

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

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.