AdEasy重组腺病毒系统对Ad-HIF1α的构建和鉴定

doi:10.13523/j.cb.20140305

中国生物工程杂志

2014, Vol. 34

Issue (3): 34-40 DOI: 10.13523/j.cb.20140305

研究报告

AdEasy重组腺病毒系统对Ad-HIF1α的构建和鉴定

周年¹, 胡宁¹, 龚璇², 汪长东³, 廖军义¹, 梁熙¹, 黄伟¹

1. 重庆医科大学附属第一医院重庆 400016;
2. 重庆市中山医院（第十二人民医院）重庆 400020;
3. 重庆医科大学医学基础医学院重庆 400016

Construction and Identification of Recombinant Adenovirus Ad-HIF1α

ZHOU Nian¹, HU Ning¹, GONG Xuan², WANG Chang-dong³, LIAO Jun-yi¹, LIANG Xi¹, HUANG Wei¹

1. The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China;
2. The Out-patient Department, Chongqing ZhongShan Hospital, Chongqing 400020, China;
3. The Molecular Biochemistry and Molecular Biology Department of School of Basic Medicine, Chongqing Medical University, Chongqing 400016, China

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摘要： 目的：采用AdEasy重组腺病毒系统构建重组腺病毒Ad-HIF1α并进行鉴定。方法：使用高保真PCR从EST克隆中扩增 HIF1α 基因编码区，并用限制性内切酶BamH I和Xba I对PCR片段限制性酶切，用T4 DNA连接酶将其连接至同样经BamH I和Xba I限制性酶切的穿梭载体pAdtrace-TO4，构建穿梭载体pAdtrace-HIF1α；将pAdtrace-HIF1α与腺病毒骨架质粒pAdEasy-1在细菌BJ5183中同源重组，得到重组腺病毒质粒pAd-HIF1α；pAd-HIF1α经Pac I酶切后转染至E1表达包装细胞系HEK293中进行 HIF1α 基因表达腺病毒包装，重复扩增，氯化铯密度梯度离心法纯化，获得高滴度Ad-HIF1α；感染干细胞株C3H10T1/2，通过荧光蛋白的表达了解其感染效率，并应用PCR及Western blot验证目的基因的表达，并通过检测下游靶基因VEGF的表达，了解该载体表达的目的蛋白的生物活性。结果：重组腺病毒质粒pAd-HIF1α，经PCR及酶切分析验证构建正确；在HEK293中进行HIF1α基因表达腺病毒包装，经反复冻融4次使细胞裂解，获得高滴度重组腺病毒载体；通过红色荧光蛋白（Red fluorescence protein，RFP）表达，观察到Ad-HIF1α能够高效感染C3H10T1/2细胞，并通过PCR证实了HIF1α在C3H10T1/2细胞较对照组明显高表达；在感染Ad-HIF1α的C3H10T1/2细胞中，HIF-1α下游靶基因血管内皮生长因子（vascular endothelial growth factor，VEGF）的表达明显增高，证实表达的目的蛋白具有生物活性。结论：应用AdEasy重组腺病毒系统成功构建Ad-HIF1α，并证实其具有生物活性，为后续研究奠定了基础。

关键词： 低氧诱导因子1α; 重组腺病毒; 血管内皮生长因子

Abstract: Objective: To construct and identify the recombinant adenovirus Ad-HIF1α. Methods: HIF1α gene coding region was amplified from EST clones by high fidelity PCR, and the PCR product were digested by restriction enzymes BamH I and Xba I and the fragment was inserted into shuttle plasmid pAdTrace-TO4 by T4 DNA ligase.The pAdtrace-HIF1α and the framework plasmid pAdEasy-1 were transfected into BJ5183 for homogenous recombination, obtaining recombinant adenovirus plasmid pAd-HIF1α. The obtained recombinant adenovirus plasmid pAd-HIF1α was digested with PacⅠ and transfected to HEK293 cells for packaging. Ad-HIF1α was obtained after repeated amplification and purification. The infection efficiency and biological activity of the Ad-HIF1α by the expression of fluorescent protein and downstream gene(VEGF) in C3H10T1/2 wore rested. Results: Recombinant adenovirus plasmid pAd-HIF1α was constructed correctly as proved by PCR and restriction analysis. Packing the HIF1α gene expression adenovirus in HEK-293, we obtained high titer recombinant adenovirus vector Ad-HIF1α after repeated freezing and thawing four times. Ad-HIF1α can infect C3H10T1/2 efficiently and promote the expression of downstream gene (VEGF). Conclusion: The recombinant adenovirus Ad-HIF1α was constructed successfully, laying the foundation for further research.

Key words: Hypoxia inducible factor 1α Recombinant adenovirus Vascular endothelial growth factor

收稿日期: 2013-12-24 出版日期: 2014-03-25

ZTFLH:

R373

基金资助: 重庆科委基金基础和前沿研究项目（cstc2013jcyjA0108）

通讯作者: 周年 E-mail: zhounian_2008@126.com

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引用本文:

周年, 胡宁, 龚璇, 汪长东, 廖军义, 梁熙, 黄伟. AdEasy重组腺病毒系统对Ad-HIF1α的构建和鉴定[J]. 中国生物工程杂志, 2014, 34(3): 34-40.

ZHOU Nian, HU Ning, GONG Xuan, WANG Chang-dong, LIAO Jun-yi, LIANG Xi, HUANG Wei. Construction and Identification of Recombinant Adenovirus Ad-HIF1α. China Biotechnology, 2014, 34(3): 34-40.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140305 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I3/34

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