人ABCB6表达载体的构建及稳定表达细胞株A375的筛选  

Construction of ABCB6 Expression Vector and Screening of Its Stable A375 Cell Line

作　　者：张彩娥[1] 陈岚[2] 任伟萍[2] 梅爱华[2] 邓云华[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院麻醉科,武汉430030 [2]华中科技大学同济医学院附属同济医院皮肤科,武汉430030

出　　处：《中国组织化学与细胞化学杂志》2012年第4期351-354,共4页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金资助(81071357;31171228);教育部博士点新教师基金资助(200804871043)

摘　　要：目的构建表达载体pIRES2-ZsGreen1-ABCB6,在转染的人黑素瘤细胞系株A375中筛选其稳定表达的细胞株。方法抽取健康人外周血,分离外周血单个核细胞,提取总RNA,逆转录获取cDNA序列,加入特异性引物经PCR扩增获得ABCB6cDNA双链,再经过BglII、EcoRI双酶切PCR产物及质粒载体pIRES2-ZsGreen1,酶切产物经回收、T4DNA连接酶连接,产物转化到大肠杆菌DH5α,挑取阳性克隆经菌落PCR鉴定、酶切鉴定和测序分析,以确定构建质粒正确。转染人黑素瘤细胞株A375,G418筛选稳定表达ABCB6的单克隆细胞株,应用荧光显微镜鉴定ABCB6蛋白的表达情况。结果 pIRES2-ZsGreen1-ABCB6质粒经菌落PCR、酶切、测序鉴定正确,经过G418筛选后获得稳定细胞株,在荧光显微镜下可观察到绿色荧光蛋白在A375细胞中的表达。结论表达载体pIRES2-ZsGreen1-ABCB6构建正确,并成功筛选出稳定表达ABCB6的A375细胞株,为进一步研究ABCB6的生物学功能奠定了良好基础。Objective To construct the recombinant plasmid plRES2-ZsGreenI-ABCB6 and detect its stable expression in A375 cells. Methods Total RNA was extracted in peripheral blood mononuclear cells from a health individual for reverse transcription of cDNA. PCR with designed primers was performed to amplify ABCB6,after enzyme digestion by BglII,EcoRI, ligation overnight by T4 DNA ligase which was inserted into plRES2-ZsGreenl vector and the recombinant plasmid pIRES2-ZsGreenl-ABCB6 was trans- formed into E. coli DH5a. The positive clones were screened and their double-stranded DNA sequenced. We got the recombinant plasmid of plRES2-ZsGreenl ABCB6, which was identified by enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into A375 cells cultured in DMEM contai- ning G418. The ABCB6 expression in every stable cell line was detected by fluorescence microscope. Re- sults The recombinant plasmid pIRES2- ZsGreenl ABCB6 was verified by enzyme digestion and DNA se- quencing. The green fluorescence in A375 cells transfected with plRES2-ZsGreenI-ABCB6 vector was ob- served under the fluorescence microscopy. Conclusion The expression vector pIRES2-ZsGreenl-ABCB6 is successfully constructed, which can stably express ABCB6 protein in A375 cells.

关 键 词：ABCB6 表达载体 转染 A375细胞 

分 类 号：R331.1[医药卫生—人体生理学]