【摘要】 目的构建人MCHR2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MCHR2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、W estern b lot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,并建立了稳定转染的CHO细胞系,成功地表达目的基因。结论真核表达载体成功构建和稳定转染CHO细胞系的建立为进一步研究MCHR2的功能奠定良好的实验基础。更多还原

【Abstract】 Objective To construct eukaryotic expression vector of human melanin-concentrating hormone receptor 2（MCHR2） and transfect CHO cells to establish stable CHO cell line.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 the identification by digestion and sequencing on the recombinant eukaryotic expression vector pcDNA3.1（+）/MCHR2,the recombinant was transfected into CHO cell by lipofectamine^TM2000.After screening culture by G418,stable transfected CHO cell line was established,and the transcription and expression of MCHR2 were identified by RT-PCR,Western blotting and immunofluorescence.Results The eukaryotic expression vector pcDNA3.1（+）/MCHR2 was constructed successfully.The stable transfected CHO cell line was established.The MCHR2 protein was expressed successfully.Conclusion The construction of the eukaryotic expression vector pcDNA3.1（+）/MCHR2 and the establishment of stable transfected CHO cell line provide solid foundation for further experimental studies on the function of MCHR2.更多还原