稳定、高效表达人MCHR2的SHG-44细胞系的建立

Establishment of SHG-44 cell line stably and highly expressing MCHR2

目的:构建黑色素浓集激素受体2(MCHR2)真核表达载体pcDNA3.1(+)MCHR2,转染SHG-44细胞,建立稳定、高效表达人MCHR2的SHG-44细胞系。方法:PCR法从人胎脑cDNA文库扩增MCHR2全长cDNA片段。用基因重组方法将其克隆到pcDNA3.1(+),构建真核表达载体pcDNA3.1(+)MCHR2,并用LipofectamineTM转染到SHG-44细胞,通过G418筛选,建立稳定表达MCHR2的SHG-44细胞系,用RT-PCR、Western印迹及免疫荧光法检测MCHR2的表达。结果:扩增出MCHR2的全长cDNA;成功构建pcDNA3.1(+)MCHR2;RT-PCR、Western印迹及免疫荧光法检测到MCHR2的表达,提示成功建立了稳定、高表达MCHR2的SHG-44细胞株。结论:MCHR2-SHG-44细胞株的建立为进一步研究MCHR2的功能奠定了良好的实验基础。

Objective：To construct a eukaryotic expressing vector harboring human melanin-concentrating hormone receptor 2 （MCHR2） and to establish a SHG-44 cell line stably and highly expressing MCHR2, Methods ： The full-length MCHR2 cDNA fragment was amplified from the human fetal brain cDNA library by PCR and was cloned into pcDNA3, 1 （＋） to construct eukaryotic vector pcDNA3. 1 （＋）/MCHR2; the latter was then transduced into SHG-44 cells by I.ipofectamineTM, After screening culture by G418, SHG-44 cells stably expressing MCHR2 were established. The transcription and expression of MCHR2 was identified by RT-PCR,Western blotting and immunofluorescence, Results：The full-length MCHR2 cDNA fragment was amplified and the eukaryotic expression vector pcDNA3, 1 （＋）/MCHR2 was successfully constructed. The expression of MCHR2 was found positive by RT-PCR,Western blotting and immunofluorescence,indicating that the SHG-44 cell line stably and highly expressing MCHR2 was successfully established, Conclusion： The successful establishment of MCHR2-SHG-44 cell line provides a solid foundation for further study on MCHR2 function