MCHR2在CHO细胞中的稳定表达及其信号通路分析  

Stable expression of MCHR2 in CHO cell line and analysis of its signal transduction pathways

作　　者：杨俊霞[1,2] 袁成福[2] 石华[2] 魏丽丽[2] 陈济[2] 易发平[2] 马永平[2] 宋方洲[2] 

机构地区：[1]重庆医科大学药理学教研室,重庆市生物化学与分子药理学重点实验室,重庆400016 [2]重庆医科大学生物化学与分子生物学教研室,临床检验诊断学省部共建教育部重点实验室,重庆400016

出　　处：《中国药理学通报》2008年第3期294-299,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No30671008);重庆市科委自然科学基金资助项目(NoCSTC,2007BA5012)

摘　　要：目的构建人MCHR2基因真核表达载体,在CHO细胞中进行稳定表达,并分析MCHR2介导的信号转导通路。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,定向插入到真核表达载体pcDNA3·1(+),经酶切和测序鉴定后,脂质体转染法转染CHO细胞,通过G418选择培养,建立稳定转染细胞系,经RT-PCR和Western blot鉴定后,通过测定细胞内cAMP、Ca2+浓度的改变分析MCHR2的信号转导通路。结果成功构建了pcDNA3·1-MCHR2真核表达载体并稳定转入CHO细胞,建立了稳定转染细胞系,成功地表达目的基因,MCH作用于MCHR2后不影响细胞内cAMP生成,可促使细胞内钙库释放Ca2+,进而使胞内Ca2+浓度出现明显而短暂的升高,提示MCHR2的信号转导通路主要是与Gq蛋白偶联。结论人MCHR2的稳定表达和信号转导通路分析为进一步体外研究MCHR2的功能提供了良好的实验基础。Aim To construct a eukaryotic expression vector of human MCHR2 and to analyze the stable expression and signal transduction pathways of MCHR2 in CHO cells. Methods The full-length MCHR2 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and inserted into eukaryotic expression vector pcDNA3. 1 （ ＋ ）, resulted in the recombinant expression vector pcDNA3.1-MCHR2. The recombinant plasmid was confirmed by the restriction enzymatic digestion and DNA sequencing analysis, and then transfected into CHO cells by lipofectamine. A stably-transfected cell line was obtained by the dominant G418 selection, and the expression of the MCHR2 gene on transcription and translation levels were identified by RT-PCR and Western blot. Signal transduction pathways mediated by MCHR2 were analyzed by measurement of intracellular cAMP and calcium. Results The eukaryotic expression vector pcDNA3. 1-MCHR2 was successfully constructed and the MCHR2 gene was stably transfected into CHO cells. A stably-transfected cell line was established and the MCHR2 gene was efficiently transcribed and translated. MCH stimulation had no effect on the production of cAMP, however, it could induce a clear and transient increase of intracellular calcium, suggesting that MCHR2 was only coupled to Gq protein. Conclusion The stable expression of MCHR2 and analysis of its signal transduction pathways in CHO cell line provided a solid experimental foundation for further studies on the function of the MCHR2 gene in vitro.

关 键 词：MCHR2 真核表达载体 稳定转染 基因表达 信号转导 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R348.2[医药卫生—基础医学]