介导人α-反义寡核苷酸慢病毒载体的构建  

Construct recombinant lentiviral vectors carrying human α-antisense oligonucleotide

作　　者：赖颖晖[1] 赖永榕[1] 杨高晖[1] 

机构地区：[1]广西医科大学第一附属医院血液科,广西南宁530021

出　　处：《中国实验诊断学》2010年第9期1358-1362,共5页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金资助项目(30860307);广西研究生教育创新计划资助项目

摘　　要：目的构建介导人α-反义寡核苷酸的慢病毒载体,为α-反义寡核苷酸对β-地中海贫血基因治疗的体内试验提供稳定的转染细胞载体。方法根据人α-反义寡核苷酸序列设计siRNA、合成DNA片段,通过双限制性内切酶消化和连接的方法构建pGCSIL-vshRNA-GFP载体质粒,接着该质粒转化感受态的大肠杆菌E.coliDH5α,通过PCR及基因测序鉴定阳性克隆,再经Lipofectamine 2000将pGCSIL-vshRNA-GFP,pHelper 1.0和pHelper 2.0三质粒系统共转染293 T细胞包装病毒,通过绿色荧光蛋白的表达测定收集的病毒滴度。结果重组质粒的外源基因PCR鉴定正确,基因测序结果与所需要的α-反义寡核苷酸序列完全一致,浓缩后病毒滴度为5×109TU/ml。结论成功构建介导人α-反义寡核苷酸的慢病毒载体。Objective To construct recombinant lentiviral vectors carrying human α-antisense oligonucleotide.Methods According to the human α-antisense oligonucleotide sequence,pGCSIL-vshRNA-GFP plasmid was constructed by double restriction enzyme digestion and ligation,and then the plasmid was transformed into E.coliDH5α.Purified pGCSIL-vshRNA-GFP plasmids from the positive clones was confirmed by PCR and sequencing.293T cells were cotransfected with lentiviral vector pGCSIL-vshRNA-GFP,pHelper 1.0 and pHelper 2.0 by Lipofectamine 2000 to produce lentivirus.The titer of virus was tested according to the expression level of green fluorescent protein.Results The exogenous gene sequence of the recombinant plasmids was completely in accordance with that of the human α-antisense oligonucleotide.The titer of concentrated virus was 5×109TU/ml.Conclusion The recombinant lentiviral vectors carrying human α-antisense oligonucleotide are successfully constructed.

关 键 词：慢病毒载体 反义寡核苷酸 基因治疗 

分 类 号：R556[医药卫生—血液循环系统疾病]