hLMO1编码基因重组质粒的构建及蛋白的表达和定位

Construction of the Recombinant Plasmid of Human LMO1 Gene and the Expression and Localization of Fusion Protein

目的构建hLMO1真核表达载体并证实融合蛋白在细胞内的表达及定位。方法以人胎脑文库cDNA为模板,PCR扩增hLMO1基因cDNA全长,亚克隆至pEGFP表达载体中。将构建的重组质粒进行酶切测序鉴定,并转染到人上皮细胞——HEK293细胞中,提取细胞蛋白进行Western blot检测。利用激光扫描共聚焦显微镜观察pEGFP-hLMO1在HEK293细胞内的定位。结果hLMO1基因cDNA全长克隆到了真核表达载体pEGFP中,酶切鉴定片段为471bp。Western blot检测到了融合蛋白表达,分子量约为45kDa。pEGFR-LMO1在人HEK293细胞核与细胞质均有表达。结论成功构建了hLMO1基因cDNA全长的真核表达载体,pEGFP-LMO1蛋白定位于HEK293细胞的细胞质和细胞核内。

Objective To construct the expression plasmid of human LIM domain only 1(hLMO1)gene and identify the expression and localization of its fusion protein.Methods The hLMO1 coding sequence was amplified by polymerase chain reaction by using cDNA library derived from human fetal brain as the template and subcloned into pEGFR vector.After the target region was identified by enzyme digestion and sequencing,the plasmid was transfected into HEK293 cells.The expression of the recombinant plasmid in HEK293 cells was d...