人黑色素浓集激素1型受体真核表达载体的构建及稳定转染CHO细胞系的建立

Construction of eukaryotic expressing vector of human melanin-concentrating hormone receptor 1 and establishment of stable transfectant CHO cell line

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。

Objective To construct eukaryotie expressing vector of human melarin-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 eDNA fragment was amplified by PCR from the humann fetal brain eDNA library and then inserted into eukaryotic expression vector pcDNA3. 1 （＋）. The recombinant was transfeeted into CHO cells by lipofectamineTM 2000 after identification of digestion and sequencing on the recombinant eukaryotie expression vector pcDNA3. 1（＋）/MCHR1. The stable transfeeted CHO cell line was then established by screening eultures with G418, and the transcription and expression of MCHRI were identified by RT-PCR, Western blot and immunofluorescene. Results The eukaryotie expression vector peDNA3. 1 （＋）/MCHRI was constructed successfully, stable transfected CHO eell line was established, the MCHRI prote＇m was expressed successfully. Conclusion The construction of eukaryotie expression vector peDNA3. 1（＋）/MCHRI and the establishment of stable transfeeted CHO eell line provided a solid experimental foundation for further studies on the function of MCHR1.