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| Ubc9基因重组腺病毒的构建及其在HeLa细胞中的表达 | |
| 引用本文： | 李薇,刘晓萍,徐祥,谭艳,于业军,李艳君,任书亭,贾跃伟.Ubc9基因重组腺病毒的构建及其在HeLa细胞中的表达[J].中国医药生物技术,2011,6(3):173-177. |
| 作者姓名： | 李薇 刘晓萍 徐祥 谭艳 于业军 李艳君 任书亭 贾跃伟 |
| 作者单位： | 1. 青岛大学医学院基础学院组织胚胎学教研室,266000 2. 第三军医大学野战外科研究所,重庆,400042 3. 青岛大学附属医院皮肤科,266000 4. 青岛大学医学院基础学院分子生物学教研室,266000 5. 青岛大学医学院基础学院中西医结合专业,266000 |
| 摘 要： | 目的构建携带Ubc9基因的重组腺病毒质粒pAdEasy-1/Ubc9,制备含Ubc9基因的重组腺病毒Ad-Ubc9,并使Ubc9在HeLa细胞中高效表达。方法 PCR法扩增目的Ubc9基因;用pemI酶切将穿梭质粒pAdTrack-CMV-Ubc9线性化;将线性化的穿梭质粒pAdTrack-CMV-Ubc9与腺病毒骨架质粒pAdeasy-1在感受态BJ5183菌内进行同源重组,筛选阳性重组子pAdEasy-1/Ubc9;再用PacI酶切pAdEasy-1/Ubc9使之线性化,线性化的重组腺病毒质粒经脂质体转染HEK293细胞,进行重组腺病毒的包装和扩增。收集重组腺病毒,感染HeLa细胞,用Westernblot方法检测Ubc9在HeLa细胞中的表达。结果通过PacI酶切证实携带Ubc9基因的腺病毒载体构建成功,包装出携带Ubc9基因的腺病毒能有效感染HeLa细胞。结论利用细菌内同源重组方法成功地构建了携带Ubc9的重组腺病毒载体,并能在HeLa细胞中高效表达。 |
| 关 键 词： | 腺病毒科 HeLa细胞 基因表达 质粒 |
Construction of recombinant adenovirus carrying Ubc9 gene and adenovirus-mediated Ubc9 expression in HeLa cells |
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| LI Wei,LIU Xiao-ping,XU Xiang,TAN Yan,YU Ye-jun,LI Yan-jun,REN Shu-ring,JIA Yue-wei.Construction of recombinant adenovirus carrying Ubc9 gene and adenovirus-mediated Ubc9 expression in HeLa cells[J].Chinese Medicinal Biotechnology,2011,6(3):173-177. | |
| Authors: | LI Wei LIU Xiao-ping XU Xiang TAN Yan YU Ye-jun LI Yan-jun REN Shu-ring JIA Yue-wei |
| Institution: | Department of Histology and Embryology(LI Wei,LIU Xiao-ping,JIA Yue-wei),Department of Biochemistry and Molecular Biology(LI Yan-jun),Department of Clinical medicine combined with Chinese traditional medicine and Western medicine (REN Shu-ting),Medical College of Qingdao University,266000 Qingdao,China,Research Institute of Surgery of Third Military Medical University,400042 Chongqing,China (XU Xiang,TAN Yan),Department of Dermatology,The Affiliated Hospital of Qingdao University Medical College,266000 Qing... |
| Abstract: | Objective To construct the recombinant adenovirus plasmid containing Ubc9 gene pAdEasy-1/ Ubc9,prepare recombinant adenovirus Ad-Ubc9 and efficiently express the Ubc9 gene in HeLa cells.Methods pAdTrack-CMV-Ubc9 was linearized with PmeI and transformed into ultracompletent BJ5183 containing pAdeasy-1 The recombinant adenovirous plasmid pAdEasy-1/Ubc9 was then constructed by homologous recombination in bacteria BJ5183,and was identified by PacI digestion.Linearized pAdEasy-1/Ubc9 was then transfected into HE... |
| Keywords: | Adenoviridae Hela cells Gene expression Plasmids |
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