含人谷胱苷肽过氧化物酶基因重组腺病毒的构建

目的构建含人谷胱苷肽过氧化物酶（GSH—Px）基因的重组腺病毒载体，为基因治疗提供一种新的方法。方法从人血中分离出白细胞，提取总RNA，采用RT-PCR法获得人GPx-1和人GPx-2的全长cDNA，克隆到pcD—NA3．14-中，构建pcDNA3．1＋GPx-1和pcDNA3．1＋GPx-2载体，酶切和基因测序鉴定阳性克隆；采用酶切法把GPX-1和GPx-2基因克隆至入门载体pENTRIA中，构建pENTRIA—GPx-1和pENTR1AGPx-2载体。采用LR反应，体外重组pENTR1A-GPx-1或pENTR1A-GPx-2和pAd／CMV／V5-DEST，得重组腺病毒表达载体pAd／C...

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Bibliographic Details
Published in西安交通大学学报（医学版） Vol. 34; no. 1; pp. 7 - 10
Main Author 宁启兰 许楠 王慧莲
Format Journal Article
LanguageChinese
Published 西安交通大学医学院遗传与分子生物学系,陕西西安,710061 2013
Subjects
基因治疗
活性氧
谷胱苷肽过氧化物酶（GSH—Px）
重组腺病毒
基因治疗
重组腺病毒
活性氧
谷胱苷肽过氧化物酶(GSH-Px)
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ISSN1671-8259

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Summary:目的构建含人谷胱苷肽过氧化物酶（GSH—Px）基因的重组腺病毒载体，为基因治疗提供一种新的方法。方法从人血中分离出白细胞，提取总RNA，采用RT-PCR法获得人GPx-1和人GPx-2的全长cDNA，克隆到pcD—NA3．14-中，构建pcDNA3．1＋GPx-1和pcDNA3．1＋GPx-2载体，酶切和基因测序鉴定阳性克隆；采用酶切法把GPX-1和GPx-2基因克隆至入门载体pENTRIA中，构建pENTRIA—GPx-1和pENTR1AGPx-2载体。采用LR反应，体外重组pENTR1A-GPx-1或pENTR1A-GPx-2和pAd／CMV／V5-DEST，得重组腺病毒表达载体pAd／CMV／V5-DEST—GPx-1和pAd／CMV／V5-DESTGPx-2。PCR和序列测定鉴定重组腺病毒载体。293A细胞中包装和扩增GSH—Px-1和GSH—Px-2的重组腺病毒。结果获得GSH—Px-1和GSH—Px-2的重组腺病毒载体和重组腺病毒。结论成功构建了pAd／CMV／V5-DEST—GPx-1和pAd／CMV／V5-DEST—GPx-2重组腺病毒载体，并在293A中获得该重组腺病毒，为进一步研究人GSH—Px基因在基因治疗中的作用奠定了基础。
Bibliography:recombinant adenovirus; glutathione peroxidase （GSH-Px）; reactive oxygen; gene therapy
NING Qi-lan, XU Nan, WANG Hui-lian （Department of Genetics and Molecular Biology, Medical School of Xi＇an Jiaotong University, Xi＇an 710061, China）
Objective To construct recombinant adenovirus vector containing human glutathione peroxidase （GSH-Px） gene so as to provide a new approach for gene therapy. Methods First we isolated the leukocytes from human blood to extract total RNA and obtained full-length human GSH-Px-1 or GSH-Px-2 cDNA by using RT-PCR method. Then GSH-Px-1 or GSH-Px-2 gene was cloned into pcDNA3. 1-F vector and pcDNA3. 1-t-GSH-Px-1 or pcDNA3. I＋GSH-Px-2 was constructed respectively. The positive clones were confirmed by the restriction enzyme digestion and gene sequencing. After that, the GSH-Px-1 or GSH-Px-2 gene was cloned into the entry vector pENTR1A to construct pENTR1A-GSH-Px-1 or pENTR1A-GSH-Px-2 vector. Recombinant adenovirus vector pad/ CMV/VS-DEST-GSH-Px-1 or pAd/CMV/V5-DEST-GSH-Px-2 was obtained b
ISSN:1671-8259