Preparation Of Three Kinds Of Bovine Interferon Fusion Proteins And Identification Of Biological Activity

Interferon(IFN)as a glycoprotein with antiviral and immunomodulatory effects,commonly used to treat and prevent viral diseases.Interferon has a short half-life,requires repeated injections during treatment of the disease,and may be accompanied by side effects of cytotoxicity.These disadvantages greatly limit its clinical application.Therefore,exploring and developing low-toxic,long-acting interferon is of great significance.In this study,three interferon recombinant proteins were constructed to extend the half-life of bovine interferon-?(BoIFN?)and reduce its cytotoxicity.According to immunoglobulin G(IgG),which has the advantage of long half-life,the recombinant fusion protein BoIFN?-IgC? composed of BoIFN? and bovine IgG heavy chain constant region(BoIgC?).According to the low cytotoxicity of bovine interferon tau,the recombinant hybrid protein BoIFN?/? composed of BoIFN?(163 amino acids of amino terminus of mature peptide)and BoIFN?(10 amino acids of carboxy terminus of mature peptide).At the same time,the recombinant fusion protein BoIFN?/?-IgC? composed of BoIFN?/? and BoIgC? was constructed,which is expected to have a long-acting and low-toxicity effect.The expression of three interferon fusion proteins was achieved using E.coli prokaryotic expression system,and the physicochemical properties and biological activities were studied.The study of toxicity of the three interferon fusion proteins on MDBK cell,and the half-life was studied in 50% plasma of BALB/c mice,SD rats vitro,and Holstein cattle,and the half-life was studied in SD rats vivo.Studies have shown that the three interferon fusion proteins in the MDBK/BT/PK-15/F81/RK-13-VSV system have antiviral activity from high to low is BoIFN?,BoIFN?/?,BoIFN?-IgC?,and BoIFN?/?-IgC?,without antiviral activity on BHK-21/MDCK-VSV systems.Three interferon fusion proteins have against BEV,BPIV3 activity on MDBK cells.Three interferon fusion proteins are highly sensitive to trypsin and are resistant to high temperature and pH condition.BoIFN?-IgC? and BoIFN?/?-IgC? are more sensitive than BoIFN? in high temperature and acidic environment.Toxicity studies showed that three interferon fusion proteins could inhibit the growth of MDBK cells in a dose-dependent manner.The order of cytotoxicity from weak to strong is BoIFN?/?,BoIFN?/?-IgC?,BoIFN?,and BoIFN?-IgC?.In vitro half-life studies have shown that in 50% plasma of BALB/c mice,SD rats and Holstein cattle,the half-life of BoIFN?-IgC?(about 24h)is significantly longer than the half-life of BoIFN?(about 12h).The half-life experimental results in SD rats showed that the injection of high and low doses of BoIFN?-IgC? exerted antiviral activity in plasma 1.4 times and twice as long as BoIFN?,and BoIFN?-IgC? and BoIFN?/?-IgC?exert longer antiviral activity than BoIFN? and BoIFN?/? in plasma.In conclusion,this study has made a meaningful exploration for the development and clinical application of long-acting and low-toxic interferon.