【摘要】 目的构建人黑色素浓集激素1型受体(MCHR1)真核表达载体,转染CHO细胞,建立稳定转染的CHO细胞系。方法采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR1基因的全长cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1(+),经酶切和测序鉴定后,用脂质体转染法转染CHO细胞,通过G418筛选,建立稳定转染的CHO细胞系,用RT-PCR、Westernblot及免疫荧光法检测MCHR1的表达。结果成功构建了pcDNA3.1(+)/MCHR1真核表达载体,并建立了稳定转染的CHO细胞系,成功地表达了目的基因。结论真核表达载体的构建和稳定转染CHO细胞系的建立为进一步研究MCHR1功能奠定了良好的实验基础。更多还原

【Abstract】 Objective To construct eukaryotic expressing vector of human melanin-concentrating hormone receptor 1(MCHR1), then to transfect CHO cells with the vector for establishment of stable CHO cell line. Methods The full-length MCHR1 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and then inserted into eukaryotic expression vector pcDNA3.1(+).The recombinant was transfected into CHO cells by lipofectamine TM 2000 after identification of digestion and sequencing on the recombinant eukaryotic expression vector pcDNA3.1(+)/MCHR1. The stable transfected CHO cell line was then established by screening cultures with G418, and the transcription and expression of MCHR1 were identified by RT-PCR, Western blot and immunofluorescence. Results The eukaryotic expression vector pcDNA3.1(+)/MCHR1 was constructed successfully, stable transfected CHO cell line was established, the MCHR1 protein was expressed successfully. Conclusion The construction of eukaryotic expression vector pcDNA3.1(+)/MCHR1 and the establishment of stable transfected CHO cell line provided a solid experimental foundation for further studies on the function of MCHR1.更多还原