人MCHR2真核表达载体的构建及稳定转染细胞系的建立.doc

文档介绍：人MCHR2真核表达载体的构建及稳定转染细胞系的建立
作者:杨俊霞石华魏丽丽袁成福陈济易发平马永平宋方洲
【关键词】 MCHR2;真核表达载体;转染;基因表达
【Abstract】 AIM: To construct the eukaryotic expression vector of human melaninconcentrating hormone receptor 2 (MCHR2) and stably transfect HEK293 cells with it. METHODS: The fulllength MCHR2 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and was inserted into eukaryotic expression vector (+). After identification of restriction digestion and PCR, the binant plasmid was transfected into HEK293 cells by lipofectamine. After screening culture by G418, a stablytransfected cell line was established, and the transcription and expression of the MCHR2 gene were identified by RTPCR, Western blot and immunofluorescence assay. RESULTS: The eukaryotic expression vector was essfully constructed and the MCHR2 gene was transfected stably into HEK293 cells. A stablytransfected cell line was established and the MCHR2 gene was expressed essfully. CONCLUSION: The establishment of the stablytransfected cell line and the expression of the target gene provide a solid experimental foundation for further studies on the function of the MCHR2 gene.
【Keywords】 MCHR2;eukaryotic expression vector; transfection;gene expression
【摘要】目的:构建人MCHR2真核表达载体,转染HEK293细胞,建立稳定转染细胞系. 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,(+),经酶切和PCR鉴定后,脂质体转染法转染HEK293细胞,通过G418选择培养,建立稳定转染细胞系,RTPCR,Western Blot及免疫荧光法检测MCHR2的表达. 结果:,建立了稳定转染细胞系,成功地表达目的基因. 结论:稳定转染细胞系的建立和基因表达为进一步研究MCHR2的功能提供了良好的实验基础.
【关键词】 MCHR2;真核表达载体;转染;基因表达
0引言
近年来研究发现肥胖的发生与下丘脑对摄食的神经内分泌调控密切相关[1],继神经肽Y、刺鼠相关蛋白、增食欲素之后,又一个增强食欲的神经肽黑色素浓集激素(melaninconcentrating hormone, MCH)进入人们的视野. 现已发现其有2种受体亚型,即MCHR1和MCHR2均属于G蛋白偶联受体家族. 通过构建转基因和基因敲除动物模型已对MCH及MCHR1与肥胖及能量代谢的关系进行了深入细致的研究,但有关MCHR2的功能至今仍不十分清楚. 为此我们构建了人MCHR2基因的真核表达载体,并转染HEK293细胞,建立了稳定表达MCHR2的细胞系,为深入研究MCHR2的生物学功能奠定实验基础.
1材料和方法
. 菌株E. coli DH5α,(+)和HEK293细胞由本教研室保存.