Construction of recombinant adenovirns vector containing human beta-interferon gene and its expression in bone mesenchymal stem cells

Abstract

Objective To construct the adenoviruse vector containing human beta-interferon gene (hIFN-β) and investigate the property of the transfeeted bone mesenchymal stem cells (MSCs) in vitro. Methods The recombinant plasmid pAdEasy-hIFN-β DNA was homologously recombinated in bacterial cells. After amplified in HEK293 cells, the obtained adenovirus was transfected into HEK293 cells again, and GFP expression was detected. Ad-hIFN-β was transfected into MSCs cultured in vitro. The expression of hIFN-β in MSCs after transfection was detected by reverse transcription polymerase chain reaction ( RT-PCR). ELISA method was applied to assay the secretion of hIFN-β. MTT method was applied to draw cells growth curve. Results Recombinant adenoviral hIFN-β was constructed successfully,which was confirmed by restriction enzyme digestion, gene sequence and GFP expression. GFP expression could be observed un-der fluorescent microscopy 24 h after transfection, and the expression of hIFN-β was confirmed by RT-PCR. ELISA analysis showed the expression levels of hIFN-β in transfection group were 192,273,436, 957,605,472,279 ng/L respectively after transfection for 1,3,5,7,10,15, and 20 days ,which were higher than in control group (P 0.05). Conclusion The recombinant adenoviral vector carrying hIFN-β was successfully constructed. Exogenous hIFN-β can be expressed in MSCs. which may offer the possibility of a novel approach to local gene therapy of glioma. Key words: Mesenehymal stem cells; IFN-β; Adenovirus vector; Gene therapy