AdEasy重组腺病毒系统对Ad-HIF1α的构建和鉴定

发布时间：2018-04-07 15:54

本文选题：低氧诱导因子α　切入点：重组腺病毒　出处：《中国生物工程杂志》2014年03期

【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。
[Abstract]:Objective: to construct and identify recombinant adenovirus Ad-HIF1 伪 using AdEasy recombinant adenovirus system.Methods: the coding region of HIF1 伪 gene was amplified by high fidelity PCR from EST clone, and the PCR fragment was digested by restriction endonuclease BamH I and Xba I.T4 DNA ligase was used to ligate pAdtrace-HIF1 伪 into the shuttle vector pAdtrace-TO4, which was also digested by BamH I and Xba I restriction enzyme, to construct the shuttle vector pAdtrace-HIF1 伪, and to recombine pAdtrace-HIF1 伪 and pAdEasy-1 in bacterial BJ5183.Recombinant adenovirus plasmid pAd-HIF1 伪 pAd-HIF1 伪 was digested by Pac I and transfected into E1 expression packaging cell line HEK293 for HIF1 伪 gene packaging. The recombinant adenovirus was amplified repeatedly and purified by cesium chloride density gradient centrifugation.High titer Ad-HIF1 伪 was obtained and infected stem cell line C3H10T1 / 2. The infection efficiency of C3H10T1 / 2 was determined by fluorescent protein expression. PCR and Western blot were used to verify the expression of the target gene, and the expression of downstream target gene VEGF was detected.To understand the biological activity of the target protein expressed by the vector.Results: the recombinant adenovirus plasmid pAd-HIF1 伪 was constructed correctly by PCR and restriction endonuclease analysis, and the recombinant adenovirus vector with high titer was obtained by the packaging of HIF1 伪 gene in HEK293.Through the expression of red fluorescence protein (Ad-HIF1), we found that Ad-HIF1 伪 could infect C3H10T1/2 cells highly, and the expression of HIF1 伪 in C3H10T1/2 cells was significantly higher than that in the control group by PCR.In C3H10T1/2 cells infected with Ad-HIF1 伪, the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor (VEGF), the downstream target gene of HIF-1 伪, was significantly increased, which confirmed that the expressed target protein had biological activity.Conclusion: Ad-HIF1 伪 was successfully constructed by using AdEasy recombinant adenovirus system, and its biological activity was confirmed, which laid a foundation for further study.
【作者单位】：重庆医科大学附属第一医院;重庆市中山医院(第十二人民医院);重庆医科大学医学基础医学院;
【基金】：重庆科委基金基础和前沿研究项目(cstc2013jcyjA0108)
【分类号】：R373

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