Ubiquitin真核表达载体构建及在293T细胞中的表达

为构建泛素分子pcDNA3.1-Myc-ubiquitin真核表达载体,实现其在293T细胞中表达。采用人工合成ubiquitin基因序列并加入c-Myc标签序列及EcoRⅠ和BamHⅠ酶切位点的方法,克隆至pcDNA3.1真核表达载体中。重组表达质粒经PCR和测序鉴定正确后,脂质体法转染入293T细胞。Western blot和间接免疫荧光法分析重组质粒在细胞中的蛋白表达及定位情况。菌落PCR及测序等分析结果显示:成功构建了pcDNA3.1-Myc-ubiquitin真核表达载体。免疫荧光和Western-Blot结果显示:Myc-ubiquitin重组表达质粒在293T细胞中获得高效表达且蛋白定位于胞浆。由此可知,成功构建pcDNA3.1-Myc-ubiquitin融合表达载体并在293T细胞中高效表达,为课题组进一步探讨与泛素化修饰相关的neuritin的作用机制及功能奠定基础。

Construction of an Eukaryotic Expression Vector for Ubiquitin Gene and Expression of Ubiquitin in 293T Cells

To construct a new fusion eukaryotic expression vector of ubiquitin and C-Myc tag（pcDNA3.1-Myc-ubiquitin） for the further study of expression in 293T cell,Ubiquitiu gene sequence together with c-Myc tag sequence containing EcoR I and BamH I restriction sites was artificially synthesized and co-cloned to pcDNA3.1（-） eukaryotic expression vector to construct the fusion expressed plasmid of pcDNA3.1 （-）-Myc-ubiquitin.After the identification and certification of the recombinant plasmid and the rightness of the cDNA sequence was done by polymerase chain reaction （PCR）,restriction enzyme digestion and sequencing.The plasmid was transfected into 293T cell by the method of liposome.Then,immunofluorescence and westernblot analysis were used to observe the expression and location of recombinant plasmid protein in cells.The results of colony PCR, sequencing identification and restriction enzyme digestion showed that the pcDNA3.1 （-）-Myc-ubiquitin eukaryotie expression vector was successfully constructed.Immunofluorescence and westernblot suggested that the Myc-ubiquitin fusion protein was effectively expressed in 293T cells and the Myc-ub＇iquitin protein was located in cytoplasm.The conclusion was that pcDNA3.1-Myc-ubiquitin fusion expression plasmid was successfully constructed,and the Myc-ubiquitin protein was expressed in cytoplasm of 293T cells.It provides foundation for further research of ubiquitin function.