人MCHR2真核表达载体的构建及稳定转染细胞系的建立

Construction of eukaryotic expression vector of human MCHR2 and establishment of stably transfected cell line

目的:构建人MCHR2真核表达载体,转染HEK293细胞,建立稳定转染细胞系.方法:采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR2基因的全长cDNA编码区序列,DNA重组技术将其定向插入到真核表达载体pcDNA3.1(+),经酶切和PCR鉴定后,脂质体转染法转染HEK293细胞,通过G418选择培养,建立稳定转染细胞系,RT-PCR,WesternBlot及免疫荧光法检测MCHR2的表达.结果:成功构建了pcDNA3.1-MCHR2真核表达载体并已稳定转入HEK293细胞,建立了稳定转染细胞系,成功地表达目的基因.结论:稳定转染细胞系的建立和基因表达为进一步研究MCHR2的功能提供了良好的实验基础.

AIM： To construct the eukaryotic expression vector of human melanin-concentrating hormone receptor 2 （MCHR2） and stably transfeet HEK293 cells with it. METHODS： The fulllength MCHR2 eDNA fragment was amplified hy PCR from the human fetal brain eDNA library and was inserted into eukaryotie expression vector pcDNA3.1 （ ＋ ）. After identification of restriction digestion and PCR, the recombinant plasmid was transfected into HEK293 cells by lipofectamine. After screening culture by G418, a stably-transfeeted cell line was established, and the transcription and expression of the MCHR2 gene were identified by RT-PCR, Western blot and immunofluorescence assay. RESULTS： The eukaryotic expression vector pcDNA3.1-MCHR2 was successfully constructed and the MCHR2 gene was transfected stably into HEK293 cells. A stably-transfected cell line was established and the MCHR2 gene was expressed successfully. CONCLUSION： The establishment of the stably-transfected cell line and the expression of the target gene provide a solid experimental foundation for further studies on the function of the MCHR2 gene.