【摘要】 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.更多还原

【Abstract】 AIM: To construct the eukaryotic expression vector of human melanin-concentrating hormone receptor 2 (MCHR2) and stably transfect HEK293 cells with it. METHODS: The full-length MCHR2 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and was inserted into eukaryotic 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-transfected 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 establishedand 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.更多还原