人补体衰变加速因子、补体膜辅助调节蛋白及CD59真核表达载体构建及序列分析

目的:构建人补体衰变加速因子(Decayacceleratingfactor,DAF)、补体膜辅助调节蛋白(membranecofactorprotein,MCP)、CD59的真核表达载体pSecTag2/HygroB-DAF、pSecTag2/HygroB-MCP、pSecTag2/HygroB-CD59。方法:应用PCR方法,从DAF-pGEM-TEasyVector、MCP-pGEM-TEasyVector和CD59-pGEM-TEasyVector分别克隆所需的DNA片段,经限制性内切酶BglandXhol消化后,插入到具有相应酶切位点的真核表达载体pSecTag2/HygroB中,分别构建人DAF、MCP、CD59真核表达质粒,并进行酶切鉴定及DNA序列测定分析。结果:DAF基因大小为1049bp,MCP基因大小为1065bp,CD59基因大小为312bp,与genbank中记载的人DAF、MCP和CD59cDNA序列结果基本相同。结论:成功构建DAF、MCP和CD59真核表达质粒,为今后进一步探讨及研究转基因肝脏奠定了基础。

Construction and sequencing of the eukaryotic expression vector of human decay accelerating factor, membrane cofactor protein and CD59

Objective:To construct and sequence eukaryotic expression vector of human decay accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. Methods:The human DAF, MCP and CD59 fragments were obtained by PCR from DAF-pGEM-T Easy Vector, MCP-pGEM-T Easy Vector and CD59-pGEM-T Easy Vector respectively. After digestion by Bgl Ⅱ and Xhol Ⅰ, the fragments were cloned into the eukaryotic expression vector pSecTag2/HygroB and identified by restriction endonuclease's digestion and DNA sequencing. Results: The DAF fragment was 1049bp. The MCP fragment was 1065bp. The CD59 fragment was 312bp. They were approximately the as same the sequence of DAF, MCP and CD59 cDNA in genbank. Conclusion: We have successfully constructed eukaryotic expression vector DAF, MCP and CD59 respectively, which will be very helpful for our further research in transgenic livers.