Plenti/V5-myc-KyoT2-IRES2-EGFP慢病毒载体的构建与鉴定

Construction and Identification of Lentivirus Expression Vector of KyoT2

构建含有myc-KyoT2融合基因片段的病毒载体,并在真核细胞中表达鉴定。方法:由科室保存质粒分别酶切得到Plenti/V5、IRES2-EGFP和myc-KyoT2基因片段,分别将myc-KyoT2基因片段插入质粒pIRES2-EGFP得到myc-KyoT2-IRES2-EGFP,然后将myc-KyoT2-IRES2-EGFP基因片段插入质粒Plenti/V5-GFP,构建病毒载体Plenti/V5-myc-KyoT2-IRES2-EGFP,应用免疫印记和荧光显微镜检测融合蛋白与绿色荧光蛋白的表达。获得了质粒myc-KyoT2-IRES2-EGFP和Plenti/V5-myc-KyoT2-IRES2-EGFP,myc-KyoT2融合蛋白和绿色荧光蛋白均正确表达。说明成功构建了含有myc-KyoT2-IRES2-EGFP融合基因的病毒载体,并在真核细胞中正确表达,为进一步研究急性T淋巴细胞白血病与Notch信号转导通路之间的关系奠定了基础。

To construct lentivirus expression vector of KyoT2 and to identify the expression of the proteins in mammalian cells the fusion gene myc-KyoT2 from vector PSP72-myc-KyoT2 is inserted into pIRES2-EGFP in order to get the eukaryotic expression vector myc-KyoT2-IRES2-EGFP. Then the gene fragment of myc-KyoT2-IRES2-EG- FP was inserted into Plenti/V5-GFP, and the vector Plenti/VS-myc-KyoT2-IRES2-EGFP is got. The enzyme digestion results identified the successful construction of the recombinant plasmids. The fusion protein is successfully expressed in mammalian cells. It is conclysed that the myc-KyoT2 fusion genes have been successfully cloned and expressed, which provide new vectors for further studies on the relationship between T-cell-type acute lymphatic leukemia and Notch signaling pathway.