Study On Preparation And Function Of Canine Albumin Fusion Interferon Gamma

Interferon（IFN）-γis the only member of type II interferon family that is produced primarily by Th1 lymphocytes,CD8+cytotoxic T lymphocytes and natural killer cells.IFN-γexerts antiviral,anti-proliferative,immunomodulatory and is an immunomodulator with pro-inflammatory activities.IFN-γinduces the production of cytokines,increases the expression of class I and II MHC antigens,Fc receptor and leukocyte adhesion molecules.It also inhibits type I hypersensitivity by inhibiting IgE secretion by B cells and relieves the symptoms of rheumatoid arthritis by recovering suppressor T cells function,decreasing immune complex precipitation and inhibiting type III hyper-sensitivity.Although IFN-γprocesses multiple biological functions,its clinical application is limited to a small number of diseases because of its short half-life.Currently,IFN-γis mainly used in the treatment of chronic lymphogranuloma,rheumatic arthritis,atopic dermatitis and renal cancer.Therapeutic blood levels were assured by frequent injections and this leads to increase in side effects and decrease in patient compliance.Current advances in veterinary medicine for dogs has had a positive impact on longevity but cancer,chronic diseases and infectious diseases still threaten their health.Clinical application of veterinary interferon and development of pharmaceutical preparations lag far behind those of human interferon.There are currently few cytokines available for use in the veterinary clinic and the majority is prokaryotic expression products with low-bioactivities.PEG modified IFN-αhas provided human hepatitis patients with great therapeutic benefits without significantly increasing side and toxic effects.However,the high production costs restrict the widespread application of PEGylated interferons for veterinary medicine.A substitute for PEG modification is albumin fusions that have improved cytokine pharmacokinetics compared with their native forms.Baculovirus expression system has the characteristics of good safety,high protein expression,good biological activity and able to co-express multiple gene recombinant proteins.The host cell secrets a small amount of extracellular metabolic protein,the baculovirus system also has the advantages of less clutter and easy purification.In this study,the baculovirus expression system was used to express the canine albumin fusion interferon gamma to find the feasibility of preparation of long-acting drugs by using albumin fusion technique.First,specific primers were used to amplify CSA gene and canine IFN-γgene.CSA gene and canine IFN-γgene were linked by flexible linker GS（GGGGS）₂GS to get fusion gene CSA-IFN-γand IFN-γ-CSA.The fusion genes were inserted into the pFastBac I plasmid to construct the transfer vector pFastBac-CSA-γand pFastBac-γ-CSA.Bacmid-CSA-IFN-γand Bacmid-IFN-γ-CSA were screened to get recombinant baculovirus which could express the fusion protein CSA-IFN-γand IFN-γ-CSA.The fusion proteins were purified and renatured to carry out the further experiments.Based on the biological activity of IFN-γ,the activity of IFN-γcanine albumin fusion IFN-γwas preliminarily determined by in vitro experiments.WB detection result showed that,just like reIFN-γ,the fusion proteins also stimulated Stat1 phosphorylation at 2 h.This indicated that both the N-terminal and C-terminal fusion proteins were functionally active.MDCK-VSV system was used to detect antiviral activity of albumin fusion IFN-γ.The titer of CSA-IFN-γwas 3.3×10⁴ U/mg and IFN-γ-CSA was 9.7×10⁴ U/mg,which was lower than re IFN-γ（1.36×10⁵ U/mg）.We further tested these proteins to evaluate whether they possessed anti-proliferative activity similar to the native canine IFN-γbut both fusion proteins had lower anti-proliferative activities than canine reIFN-γ.IFN-γ-CSA showed slightly higher activity than CSA-IFN-γ.IC50 of CSA-IFN-γwas 106.91 ng/mL,IFN-γ-CSA was 39.840 ng/mL and reIFN-γwas 18.16 ng/mL.The morphology of IFN-γtreated DH82cells were observed.cell refraction change,drawing obvious,individual cells atrophy serious,and there is a lot of dead cells floating in the medium,the preliminary determination of fused interferon may induce the apoptosis of DH82 tumor cells.After 72 h,the refractive index of cells was changed,drawing speed was obvious,and the individual cells were atrophic.A large number of dead cells were suspended in the medium.It was preliminarily determined that IFN might induce the apoptosis of DH82 tumor cells.The effect of albumin IFN-γon apoptosis was verified,the results showed that the apoptotic effect of CSA-IFN-γand IFN-γ-CSA could be detected obviously after 72 h,and it was similar to that of reIFN-γ.The results of cell cycle detection showed that,after treated 48 h,DH82 cells were arrested in G0/G1 phase in IFN-γ-CSA and reIFN-γtreated group,the effect of CSA-IFN-γtreatment group was not obvious.After 72 h,the difference between the groups disappeared,DH82cells were arrested in G0/G1 phase in CSA-IFN-γ,IFN-γ-CSA and reIFN-γtreatment group.We next assessed whether the pharmacokinetics properties of the fusion proteins differed from those of reIFN-γ.The half-lives of reIFN-γand IFN-γ-CSA were 2.22 h and6.51 h respectively.The half-life of CSA-IFN-γwas 21.73 h and statistically different from both IFN-γ-CSA and canine reIFN-γ.To investigate the therapeutic efficacy of the CSA fusion proteins,we tested in vivo antitumor activities of these proteins using a DH82 canine kidney cancer xenograft model.The result showed that,the tumor growth inhibition effect of CSA-IFN-γwas the most obvious.Comprehensive analysis of fused interferon in vivo and in vitro biological activity test results showed that,albumin fusion technology would not lead to IFN-γloss biological activity.These results indicated that fusion of CSA at the N-terminus of canine IFN-γmaximized the therapeutic effects of canine IFN-γby achieving an optimal balance between pharmacokinetics and pharmacodynamics.