学术
人MCHR2真核表达载体的构建及稳定转染细胞系的建立
杨俊霞[1,2] 石华[1] 魏丽丽[1] 袁成福[1] 陈济[1] 易发平[1] 马永平[1] 宋方洲[1]
[1]重庆医科大学生物化学与分子生物学教研室,重庆市生物化学与分子药理学重点实验室,临床检验诊断学省部共建教育部重点实验室,重庆400016 [2]重庆医科大学药理学教研室,重庆400016
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，DNA重组技术将其定向插入到真核表达载体pcDNA3．1（＋），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，RT-PCR，Western Blot及免疫荧光法检测MCHR2的表达．结果：成功构建了pcDNA3．1-MCHR2真核表达载体并已稳定转入HEK293细胞，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．[著者文摘]
文章出处:
《第四军医大学学报》-2007年28卷3期 -210-213页
栏目信息:
分 类 号:
文献标识码:
A
文章编号:
1000-2790（2007）03-0210-04
Construction of eukaryotic expression vector of human MCHR2 and estabiishment of stably transfected cell line
YANG Jun-Xia, Sill Hua, WEI Li-Li, YUAN Cheng-Fu, CHEN Ji , YI Fa-Ping , MA Yong-Ping , SONG Fang-Zhou（ 1.Department of Biochemistry and Molecular Biology, Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education, 2Department of Pharmacology, Chongqing Medical University, Chongqing 400016, China）
Abstract:
AIM： To construct the eukaryotic expression vector of human melanin-concentrating hormone receptor 2 （MCHR2） and stably transfeet HEK293 cells with it. METHODS： The fulllength MCHR2 eDNA fragment was amplified hy PCR from the human fetal brain eDNA library and was inserted into eukaryotie expression vector pcDNA3.1 （ ＋ ）. After identification of restriction digestion and PCR, the recombinant plasmid was transfected into HEK293 cells by lipofectamine. After screening culture by G418, a stably-transfeeted cell line was established, and the transcription and expression of the MCHR2 gene were identified by RT-PCR, Western blot and immunofluorescence assay. RESULTS： The eukaryotic expression vector pcDNA3.1-MCHR2 was successfully constructed and the MCHR2 gene was transfected stably into HEK293 cells. A stably-transfected cell line was established and the MCHR2 gene was expressed successfully. CONCLUSION： The establishment of the stably-transfected cell line and the expression of the target gene provide a solid experimental foundation for further studies on the function of the MCHR2 gene.[著者文摘]
Key words:
MCHR2 ;eukaryotic expression vector; transfection; gene expression
基金资助:
国家自然科学基金（30671008）
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