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| 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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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.
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Received: 24 December 2013
Published: 25 March 2014
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