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Abstract: Objective Construction and identification of recombinant adenovirus vector with human/mouse interferon (IFN-γ) and effectively transfection into human umbilical cord mesenchymal stem cells (HUMSCs). Methods IFN-γ gene of human/mouse were amplified from the plasmid by polymerase chain reaction (PCR) and then inserted into the plasmid pDC316 to generate pDC316-IFN-γ. After being confirmed by restriction enzyme digestion and DNA sequencing, the DNA encoding IFN-γ in the new structure were inserted into the vector of recombinant plasmid adenovirus and confirmed by restricition enzyme digestion. Then the human embryonic kidney cell line 293 were transfected with confirmed Ad-IFN-γ, and the recombinant adenovirus were amplified, and the virus titer were detected using 50% tissue culture infective dose (TCID50) assay. The expression of the green fluorescent protein (GFP) and IFN-γ were detected by fluorescent microsope and Western blot and ELISA after the recombinant adenovirus transfected HUMSCs. Results The recombinant adenovirus Ad-hIFN-γ were constructed successfully, and amplified with titer of 1.6×1010 IU/mL. The titer of Ad-mIFN-γ was 1.0×1010 IU/mL and the titer of Ad-GFP was 1.0×109IU/mL. The green fluorescence proteins could be observed under fluorescent microscope in HUMSCs 24 h after transfection and with a stronger degree after 72 h, and IFN-γ expression in HUMSCs were confirmed by Western blot and ELISA. Conclusion Construction and identification of recombinant adenovirus vector of human/mouse IFN-γ and effectively transfection of HUMSCs were successful.