基于miR30骨架的HPSE-shRNA慢病毒载体的构建及其效应研究

Q343; 目的:模拟miR30的框架结构设计针对HPSE的shRNA,将其克隆至慢病毒载体的CMV启动子调控的表达框内,实现Ⅱ型启动子对RNAi的有效调控.方法:设计3条基于miR30骨架的HPSE-shRNA,通过PCR获得目的片段,回收后与线性化LV PP-GFP载体连接产生重组慢病毒载体LV PP-GFP /miR-HPSE-shRNA,PCR筛选阳性克隆,测序鉴定.用LV PP-GFP/miR-HPSE-shRNA、pHelper 1.0载体和pHelper 2.0载体共转染包装细胞293T细胞,产生慢病毒,感染A375细胞.以实时荧光定量PCR检测HPSE-mRNA的表达情况,以W...

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Published in浙江大学学报（医学版） Vol. 42; no. 1; pp. 67 - 74
Main Authors 刘晓艳, 方红, 朱定仙, 张妤
Format Journal Article
LanguageChinese
Published 浙江大学医学院附属第一医院皮肤科,杭州浙江,310003 2013
Subjects
RNA干扰
RNA聚合酶
转录启动子
葡糖醛酸糖苷酶
慢病毒属
遗传载体
聚合酶链反应
微RNAs
Online AccessGet full text
ISSN1008-9292
DOI10.3785/j.issn.1008-9292.2013.01.011

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Abstract Q343; 目的:模拟miR30的框架结构设计针对HPSE的shRNA,将其克隆至慢病毒载体的CMV启动子调控的表达框内,实现Ⅱ型启动子对RNAi的有效调控.方法:设计3条基于miR30骨架的HPSE-shRNA,通过PCR获得目的片段,回收后与线性化LV PP-GFP载体连接产生重组慢病毒载体LV PP-GFP /miR-HPSE-shRNA,PCR筛选阳性克隆,测序鉴定.用LV PP-GFP/miR-HPSE-shRNA、pHelper 1.0载体和pHelper 2.0载体共转染包装细胞293T细胞,产生慢病毒,感染A375细胞.以实时荧光定量PCR检测HPSE-mRNA的表达情况,以Westen blot检测HPSE蛋白的表达水平.结果:构建出以miR30为骨架的针对HPSE的慢病毒干扰载体,经阳性菌落PCR鉴定与测序,结果正确.实时荧光定量PCR和Westen blot结果表明,LV PP-GFP/miR-HPSE-shRNA重组病毒感染后A375细胞HPSE-mRNA和蛋白的表达明显降低.结论:本研究成功构建了以miR30为骨架的HPSE-shRNA慢病毒载体,实现了Ⅱ型启动子对RNAi的有效调控,为今后实现溶瘤病毒介导RNAi提供了实验依据.
AbstractList Q343; 目的:模拟miR30的框架结构设计针对HPSE的shRNA,将其克隆至慢病毒载体的CMV启动子调控的表达框内,实现Ⅱ型启动子对RNAi的有效调控.方法:设计3条基于miR30骨架的HPSE-shRNA,通过PCR获得目的片段,回收后与线性化LV PP-GFP载体连接产生重组慢病毒载体LV PP-GFP /miR-HPSE-shRNA,PCR筛选阳性克隆,测序鉴定.用LV PP-GFP/miR-HPSE-shRNA、pHelper 1.0载体和pHelper 2.0载体共转染包装细胞293T细胞,产生慢病毒,感染A375细胞.以实时荧光定量PCR检测HPSE-mRNA的表达情况,以Westen blot检测HPSE蛋白的表达水平.结果:构建出以miR30为骨架的针对HPSE的慢病毒干扰载体,经阳性菌落PCR鉴定与测序,结果正确.实时荧光定量PCR和Westen blot结果表明,LV PP-GFP/miR-HPSE-shRNA重组病毒感染后A375细胞HPSE-mRNA和蛋白的表达明显降低.结论:本研究成功构建了以miR30为骨架的HPSE-shRNA慢病毒载体,实现了Ⅱ型启动子对RNAi的有效调控,为今后实现溶瘤病毒介导RNAi提供了实验依据.
Author 刘晓艳
方红
朱定仙
张妤
AuthorAffiliation 浙江大学医学院附属第一医院皮肤科,杭州浙江,310003
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Author_FL ZHU Dingxian
ZHANG Yu
FANG Hong
LIU Xiaoyan
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DocumentTitle_FL Construction of a lentiviral RNA interference system targeting heparanase based on miR30 and its silencing effect
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Keywords RNA干扰
RNA聚合酶
转录启动子
葡糖醛酸糖苷酶
慢病毒属
遗传载体
聚合酶链反应
微RNAs
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Title 基于miR30骨架的HPSE-shRNA慢病毒载体的构建及其效应研究
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