Expression Of Recombinant Canine IFN-γ Gene In HEK293T Cells And Construction Of Canine IFN-γ Recombinant Adenovirus Vector

Interferon-gamma(IFN-γ),characteristically produced by T cells and NK cells, mitogen or antigen activated T cell clone can also secret IFN-γ.It plays a very critical role in resistance to viruses,anti-tumor proliferation and immunomodulatory activities. Its immune modulation includes activating macrophages,up-regulating the expression of MHC classâ… and classâ…¡molecules,prompting the presentation of antigens,and inducing Th₁ cell growth and differentiation.Due to its enormous regulating function in immune system,IFN-γis a necessary composition in eliminating pathogen by organisim. Accordingly,IFN-γcould be used as therapeutic drug in the treatment of infectious diseases or tumors,or as a vaccine adiuvant to promote vaccination efficacy.To establish the method of canine IFN-γ(CaIFN-γ)expression in HEK293T cells, the canine spleen cells were stimulated by concanavalin A.The cDNA encoding CaIFN-γwas amplified by RT-PCR from the stimulated cells.Then the CaIFN-γgene was inserted into eukaryotic expression vector pcDNA3.1A.The recombinant pcDNA3.1A-CaIFN-γplasmids were transfected into HEK293T cells mediated by calcium phosphate.Results showed that the sequence of CaIFN-γcDNA amplified was identical to that published in GenBank,and the homology is 100%.The expressed products were deteced by Western-blot,which indicated that CaIFN-γgene could be expressed in HEK293T cells and secreted from the cells.The success in construction and expression of canine IFN-γmake it possible to study further on its biological functions.To construct recombinant adenovirus vector containing CaIFN-γgene on the basis of successfully expressing CaIFN-γprotein in HEK293T cells.CaIFN-γcDNA including the end codon was obtained form the expression plasmid pcDNA3.1A-CaIFN-γ-STOP double digestion with Kpnâ… and Apaâ… .then it was cloned into the multiple cloning site(MCS)between the human cytomegalovirus promoter and SV40 ployadenylation sequences of plasmid pShuttle3/gfp.The expression cassette containing CaIFN-γcDNA was obtained from the recombinant pShuttle3/gfp with double digestion of PI-Sceâ… and I-Ceuâ… ,then ligated to Adeno-â…©Viral DNA with in vitro ligation to construct the recombinant adenovirus plasmid pAdeno-CaIFN-γ.The recombinant adenovirus plasmid was digested with Pacâ… to expose the inverted terminal repeats(ITRs)located at either end of the genome.Afeter purification,the linear Pacâ… -digested recombinant adenovirus plasmid was transfected into the low passage HEK293T cells by Lipofectamine2000 mediated gene transfected to generate recombinant adenovirus termed as Adeno- CaIFN-γ.The recombinant adenovirus were amplified and conformed by PCR.Results suggested that the recombinant Adeno-CaIFN-γcan be constructed correctly and be transfected into HEK293 cells,which provide the basis of further research of CaIFN-γmediated gene therapy of viral diseases of canine.