SET-EGFP重组表达质粒的构建及表达

目的构建癌蛋白SET与增强型绿色荧光表达载体（EGFP）的融合表达质粒pEGFP—N2-SET并获得表达。方法根据人SET基因序列。设计了一对含有Kozak序列、终止密码子、HindⅢ和KpnI酶切位点的引物。从人L-02肝细胞提取总RNA，以逆转录后cDNA为模板，PCR扩增获得SET基因片段，经双酶切后回收得到有以上两个酶切位点的SET基因片段，将此基因片段克隆至相同酶切回收后的pEGFP—N2载体中，获得重组质粒pEGFP—N2-SET。以Lipofectamine^TM2000为载体，将构建好的真核表达质粒转染到L-02肝细胞中进行瞬时表达。结果。经DNA测序鉴定证实，pEGFP—N2-SET重组质粒构建成功，经荧光倒置显微镜观察，证实能在细胞内进行蛋白表达。结论SET表达质粒的成功构建及表达为其进一步结构和功能研究奠定了基础。

Construction of the eukaryotic expression vector of human SET gene and its expression in L-02 liver cells

Objective To construct a recombinant plasmid carrying enhanced green fluorescent protein and the inhibitor of protein phosphatase 2A （SET） and detect its expression in L - 02 liver cells. Methods The SET eDNA of human L - 02 liver cells was cloned by RT - PCR and the eukaryotic expression vector of SET gene was constructed by gene recombination technique. The recombinant plasmid pEGFP - N2 - SET was verified by restriction enzyme digestion analysis and sequenceing, then transfected into human L - 02 liver cells using LipofectamineTM 2000. Then the expression level of the fusion protein was detected by fluorescence microscope. Results The eukaryotic expression plasmid pEGFP - N2 - SET was successfully constructed. The SET gene was expressed successfully in transfected L -02 liver cells. Conclusion The successful construction of and expression of SET recombinant palsmid has set foundation for further investiagtion of its structure and function.