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靶向hTERT基因RNAi慢病毒载体的构建与鉴定 被引量：2

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摘要 目的构建hTERT基因RNAi慢病毒载体,为大肠癌RNAi介导的基因治疗打下基础。方法化学合成3条靶向hTERT基因的siRNA,转染大肠癌细胞SW480,48h后采用RT-PCR方法筛选出一条对hTERT mRNA有明显抑制作用的siRNA。针对已经筛选确定的hTERT基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经XhoⅠ和HpaⅠ酶切后的GP-Supersilencing Vector载体[含U6启动子和绿色荧光蛋白(GFP)]连接产生LV-shhTERT慢病毒载体,PCR筛选阳性克隆,测序鉴定。用LV-shhTERT、pGag/Pol、pRev和pVSV-G4质粒共转染包装细胞293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平测定病毒滴度。结果 PCR和测序证实,成功构建hTERT shRNA的慢病毒载体LV-shhTERT。包装慢病毒,浓缩病毒悬液的滴度为1×108UT/ml。结论成功构建了hTERT基因RNAi慢病毒载体。
作者 刘爱群 葛莲英 罗小玲 蔡艳玲
机构地区 广西医科大学附属肿瘤医院内镜室
出处 《中华临床医师杂志（电子版）》 CAS 2011年第1期253-257,共5页 Chinese Journal of Clinicians(Electronic Edition)
基金 广西自然科学基金(重200971)
关键词 肠肿瘤 RNA干扰 慢病毒属 HTERT
分类号 R735.3 [医药卫生—肿瘤]
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参考文献11

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共引文献4

同被引文献30

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中华临床医师杂志（电子版）
2011年 第1期

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