| 摘要: |
| 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。 |
| 关键词: MCHR2 真核表达载体 稳定转染的SHG-44细胞系 基因表达 |
| DOI：10.3724/SP.J.1008.2008.00896 |
| 投稿时间：2008-03-22修订日期：2008-04-21 |
| 基金项目:国家自然科学基金(30671008);重庆市自然科学基金重点项目(CSTC2007BA5012);国家外专局项目(20075000019). |
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| Establishment of SHG-44 cell line stably and highly expressing MCHR2 |
| ZHANG Qin,BU You-quan,YI Fa-ping,YUAN Cheng-fu,YUAN Fei,SONG Fang-zhou* |
| (Department of Biochemistry and Molecular Biology,Chongqing Medical University,Chongqing 400016,China) |
| Abstract: |
| 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 LipofectamineTM. 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. |
| Key words: MCHR2 eukaryotic expression vector stable transfected SHG-44 cell line gene expression |
