PPARγ基因真核表达载体的构建及其在乳腺癌MDA—MB-231细胞中的表达

目的：构建以绿色荧光蛋白（GFP）为报告基因的重组表达质粒pEGFP—C1—PPARγ，观察小鼠PPARγ基因在MDA-MB-231细胞中的表达及定位。方法：采用克隆和亚克隆技术构建小鼠PPARγ基因真核表达载体，脂质体Lip2000介导转染MDA—MB-231细胞，real—time PCR和western—blot验证其mRNA和蛋白的表达，荧光显微镜观察该基因亚细胞定位。结果：酶切和测序结果证实重组质粒含有PPAIh编码区序列且插入方向正确，转染后观察该基因亚细胞定位于胞核，胞质有弥散分布。结论：成功构建了小鼠PPARγ基因真核表达载体，该基因在MDA—MB-231细胞中成功表达，PPARγ基因主要集中表达于胞核。

Construction of Eukaryotic Expression Vector with Mouse PPARγ Gene and Its Expression in Breast Cancer MDA-MB-231 Cells

Objective： To obtain eukaryotic expression vector of mouse PPARγ gene and to detect its expression and subcellular localization in MDA-MB-231 cells. Methods： PPARγ cDNA was amplified by RT-PCR from mouse liver tissues, and fresh PCR products were cloned into pGEM-T vector and sequenced. Then the recombinant ORF was subcloned into pEGFP-C 1 vector and sequenced. The recombinant vector was transfected into MDA-MB-231 cells by lipofectamine 2000. The expression level of PPARγ mRNA and protein were identified by real-time PCR and western-blot, respectively. Immunofluorescence microscope was used to observe the subcellular localization. Results： PPARγ gene was identified to be inserted into pEGFP-C1 vector correctly and the recombinant vector expressed PPARγ mRNA and protein stably. The green fluorescence protein was mainly expressed in the nucleus and dispersed into the whole cytoplasm in MDA-MB-231 cells. Conclusions： The eukaryotic expression vector containing mouse PPARγ gene was successfully constructed and expressed. The PPARγ gene was mainly expressed in the nucleus ofMDA-MB-231 cells.