稳定表达人MCHR2的SH-SY5Y细胞系的建立  

Establishment of SH-SY5Y cell line expressing human MCHR2 stably

作　　者：张琴[1] 卜友泉[1] 易发平[1] 袁成福[1] 秦琴[1] 宋方洲[1] 

机构地区：[1]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：《吉林大学学报（医学版）》2009年第2期258-262,F0002,共6页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30671008);重庆市自然科学基金重点项目资助课题(CSTC2007BA5012);国家外专局项目资助课题(20075000019)

摘　　要：目的:构建黑色素浓集激素受体2(MCHR2)/增强型绿色荧光蛋白(EGFP)融合蛋白真核表达载体pEGFPN1-MCHR2,转染人神经母细胞瘤细胞株SH-SY5Y,建立稳定表达人MCHR2的SH-SY5Y细胞系MCHR2-SH-SY5Y。方法:采用聚合酶链反应(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质粒pEGFPN1,构建真核表达载体pEGFPN1-MCHR2,并用LipofectamineTM转染到SH-SY5Y细胞,通过G418筛选,建立稳定表达MCHR2的MCHR2-SH-SY5Y细胞系,采用逆转录聚合酶链反应(RT-PCR)、Western blotting检测MCHR2的表达并用激光共聚焦显微镜观察融合蛋白亚细胞定位。结果:扩增出人MCHR2全长cDNA;成功构建真核表达载体pEGFPN1-MCHR2;RT-PCR、Western blotting检测到MCHR2-SH-SY5Y细胞MCHR2的表达,激光共聚焦显微镜观察转染pEGFPN1-MCHR2的MCHR2-SH-SY5Y细胞融合蛋白定位于细胞膜,而转染空质粒pEGFPN1的SH-SY5Y细胞EGFP定位于细胞质。结论:成功建立稳定表达人MCHR2的MCHR2-SH-SY5Y细胞株。Objective To construct eukaryotic expressing vector of human melanin-concentrating hormone receptor 2 （MCHR2） / enhancer green fluorescent protein （EGFP） and establish a SH-SY5Y cell line expressing MCHR2 stably. Methods Human MCHR2 cDNA was amplified from the human fetal brain cDNA library by PCR. It was cloned into pEGFPN1 and the eukaryotic expressing vector pEGFPN1-MCHR2 was constructed. Then the vector was transfected into human neuroblastoma cells SH-SY5Y by LipofectamineTM. After screening culture by G418, the SH-SY5Y cells expressing MCHR2 stably were established. The transcription and expression of MCHR2 were identified by RT-PCR and Western blotting. The location of MCHR2 was observed by laser confocal microscope. Results Human MCHR2 cDNA was amplified and the eukaryotic expression vector pEGFPN1 MCHR2 was constructed successfully. The expression of MCHR2 was detected by RT-PCR and Western blotting successfully. The fusion protein MCHR2/EGFP was located in the cytomembrane of the MCHR2-SH-SY5Y cells transfected with pEGFPN1-MCHR2 while EGFP was located in the cytoplasm of the SH-SYSY cells transfected with pEGFPN1. Conclusion The cell line MCHR2-SH-SY5Y expressing MCHR2 stably has been established successfully.

关 键 词：MCHR2 真核表达载体 稳定转染的SH-SY5Y细胞系 基因表达 

分 类 号：Q786[生物学—分子生物学]