Title: Construction of Recombinant Adenovirus pAdxsi-GFP-HIFContaining Hypoxia Inducible Factor Gene and its Expression in Endothelial Cells
Abstract: Objective: To construct a recombinant adenovirus(pAdxsi-GFP-HIF) encoding human hypoxia inducible factor 1αgene(HIF-1α) and human keratinocyte growth factor gene, and investigate its expression in lung epithelium cells. Methods: HIF-1αgene was obtained from human lung cancer cell line A549 which was cultured in hypoxia condition by RT-PCR. The HIF-1αgene was subcloned into shuttle vector pShuttle-CMV-EGFP at KpnI and BamHI sites. And the KGF gene from plasmid pIRES2-EGFP-KGF by PCR was also subcloned into the shuttle vector at NheI and PmeI sites. A shuttle vector pShuttle-GFP-HIF-KGF was obtained. After identified with restriction enzymes, plasmid pShuttle-GFP-HIF-KGF was linearized by digestion with restriction endonuclease I-CeuI and I-SceI, and was subsequently cotransformed into E.coli DH5a with adenoviral backbone plasmid pAdxsi to make homologous recombination. After linearized by PacI, the homologous recombinant adenovirus plasmid was transfected into 293 cells to package and amplify. The recombinant adenovirus was infected A549, and the expression level of HIF-1αprotein and KGF protein were evaluated by ELISA. Results: The recombinant adenovirus vector for HIF-1αgene and KGF gene(pAdxsi-GFP-HIF-KGF)was successfully constructed and amplified with titer of 2.59×1010pfu/mL. The green fluorescence protein could be observed under fluorescent microscope in A549 at 24h after transfection and with a stronger degree after 48h. The amount of HIF-1 protein and KGF protein was 56.36±4.53 ng/ mL and 60.20±2.92 ng/mL in supernatant at 48h after transfection,respectively. Conclusion: A recombinant adenovirus pAdxsi-GFP-HIF-KGF, encoding human hypoxia inducible factor 1αgene and keratinocyte growth factor gene, was constructed in vitro and expressed successfully in A549 cells.It provides the material basis for further studying the biologic function and potential application of the recombinant adenovirus, in order to prepare for gene drug can play a role of KGF and HIF-1 and provide a experimental basis for effective prevention of acute lung injury at high altitudes.
Publication Year: 2013
Publication Date: 2013-01-01
Language: en
Type: article
Access and Citation
AI Researcher Chatbot
Get quick answers to your questions about the article from our AI researcher chatbot