CD41-42突变型β地中海贫血重组载体pEGFP-C2-CD41-42的构建及其稳定转染HeLa细胞模型的建立  

Construction of recombinant vector pEGFP-C2-CD41-42 of CD41-42 mutation β thalassemia and establishment of its stably transfected HeLa cell model

作　　者：曾雅畅[1] 李慕军[1] 陈萍[1] 陈悦[1] 陈静[1] 庞莉莉[1] 

机构地区：[1]广西医科大学第一附属医院广西地中海贫血防治重点实验室,广西南宁530021

出　　处：《吉林大学学报（医学版）》2014年第2期257-260,I0002,共5页Journal of Jilin University：Medicine Edition

基　　金：广西壮族自治区科技基础条件平台建设项目资助课题(11-031-07,12-071-05,13-051-12);广西壮族自治区医疗卫生重点科研项目资助课题(2012061);广西医科大学优秀博士学位论文培育项目资助课题(YCBZ2012016);广西壮族治自区卫生厅自筹课题(桂卫自Z2013017)

摘　　要：目的:构建CD41-42突变型β地中海贫血重组载体pEGFP-C2-CD41-42和HeLaCD41-42细胞模型,为β地中海贫血CD41-42突变型细胞、小鼠模型建立及进一步基因治疗提供依据。方法:在β地中海贫血CD41-42突变的纯合子患者外周血中提取DNA,PCR法扩增含有βCD41-42的β-珠蛋白基因片段。将PCR产物βCD41-42珠蛋白基因克隆到pEGFP-C2载体中,用脂质体2000将pEGFP-C2-βCD41-42质粒转染HeLa细胞,观察转染后βCD41-42在HeLa细胞中荧光表达情况。RT-PCR法检测βCD41-42在HeLa细胞中的表达。结果:重组质粒pEGFP-C2-βCD41-42转染HeLa细胞24h后细胞发出绿色荧光,G418筛选后,有大量荧光表达。RT-PCR扩增得到227bp的特异性条带,而稳定转染pEGFP-C2空质粒的HeLa细胞中不表达特异性条带。结论:成功构建β地中海贫血pEGFP-C2-βCD41-42突变基因重组载体,并成功构建HeLaCD41-42细胞模型。Objective To construct the recombinant vector pEGFP-C2-CD41-42 of CD41-42 mutation β thalassemia and HeLa cell model, and to provide basis for cell and mouse model establishment of CD41-42 mutation β thalassemia and the further gene therapy. Methods The DNA in peripheral blood of 13CD41-42 homozygote mutation patient was extracted and the DNA flamen of β-globin containing β CD41-42 was amplified by PCR, then the PCR product β CD41-42 globin gene was subcloned into pEGFP-C2 vector. The recombinant plasmid pEGFP-C2-βCD41-42 was transfected into HeLa cells by LipofectamineTM 2000. The fluorescence expression of βCD41-42 in HeLa cells after transfecton was observed. The expression of βCD41-42 in HeLa cells was identified by RT-PCR method. Results The HeLa cells expressed green fluorescence 24 h after transfeeted with the recombinant plasmid pEGFP-C2-βCD41-42 and expressed lots of green fluorescence after G418 screening. The special PCR products （227 bp） was obtained by RT-PCR, but it didn＇t express in the HeLa cells transfected with pEGFP-C2 blank plasmid. Conclusion The recombinant vector pEGFP-C2-βCD41-42 of CD41-42 mutation β thalassemia is successfully estabished and the HeLaCD41-42 cell model is successfully established.

关 键 词：Β地中海贫血 CD41—42突变基因 重组载体 HELA细胞 

分 类 号：R556[医药卫生—血液循环系统疾病]