Prokaryotic Expression Of Feline Interferon ω2 And Interferon γ And Detection Of Antiviral Effect

Interferon is generally divided into type I,type II and type III interferon according to its difference from binding receptor.Among them,type I interferon has the strongest antiviral effect,while type II interferon mainly plays a role in activating and regulating the immune system.Interferon has been widely used in clinic because of its efficient and broad-spectrum antiviral,bactericidal and antiparasite effects.In recent years,interferon has become the first choice of pet antiviral drugs in the field of veterinary drugs.The aim of this study was to express feline interferonω2（FeIFN-ω2）and feline interferon gamma（FeIFN-γ）with high biological activity,and to verify their antiviral effect.,The specific contents are as follows:1.The signal peptides of FeIFN-ω2 and FeIFN-γwere predicted,and the mature peptide genes were selected for codon optimization and synthesis,and cloned into pET28a-SUMO vector.After the recombinant plasmid was successfully constructed,it was transformed into BL21（DE3）competent cells.The expression was verified by SDS-PAGE and Western Blot,and the expression conditions were optimized.The recombinant protein was purified by nickel column affinity purification.The results showed that the two kinds of cat interferon were soluble in the supernatant of broken bacteria,in which SUMO-FeIFN-ω2 could reach the yield of 90mg recombinant protein per liter of bacterial liquid under the optimized conditions,while SUMO-FeIFN-γcould reach the yield of 27.6mg recombinant protein per liter of bacterial liquid.2.To detect the cytotoxicity of two kinds of feline interferon,F81 cells were incubated with purified recombinant feline interferon after 10-fold dilution.The cell status was recorded at 24 h and 48 h,and the cytotoxicity was determined by ATP method.The results showed that the two kinds of recombinant cat interferon had no significant effect on the morphology of F81 cells,SUMO-FeIFN-ω2 only slightly inhibited the cell viability at low dilution,and SUMO-FeIFN-γhad no significant effect on F81 cell viability.3.To detect the biological activity of two kinds of recombinant cat interferon,the cytopathic effect inhibition experiment based on VSV-GFP was selected to verify the biological activity and antiviral activity of the recombinant protein,and the antiviral titer was compared with that of commercial interferon.At the same time,the ISG molecule in the downstream pathway of type I interferon was detected by relative fluorescence quantitative method,and its protein biological activity and antiviral duration were tested at the transcriptional level.Finally,the expressed product was cleaved with SUMO protease,and the anti-VSV-GFP activity of the fusion protein was compared with that of the recombinant interferon alone to further verify the biological activity of the fusion protein.The results of antiviral activity test showed that the antiviral titer of SUMO-FeIFN-ω2produced by the above method reached 10^10.23U/mg,which was significantly higher than that of cat interferonωproducts sold in the market,and the antiviral titer of SUMO-FeIFN-γreached 10^6.93U/mg,which was similar to that of cat interferonωproducts sold in the market.The two kinds of recombinant cat interferon prepared in this study could significantly stimulate the expression level of downstream genes of interferon for more than72 hours,and the antiviral titer of recombinant cat interferon was not affected after the removal of SUMO tag.4.Three common cat viruses,feline calicivirus（FCV）,feline parvovirus（FPV）and feline herpesvirus（FHV）,were selected for detection.After F81 was incubated with three kinds of SUMO-FeIFN-ω2 and SUMO-FeIFN-γwith different concentration gradients,the above viruses were inoculated respectively,and samples were collected at different time points to determine the titer of the virus.The results showed that the two kinds of recombinant cat interferon had no significant antiviral effect on FPV and FHV in vitro,but had significant antiviral effect on all strains of FCV.In summary,this study successfully expressed cat interferonω2 and cat interferonγwith high biological activity by using prokaryotic expression system,which laid a foundation for the research and development of cat interferon products.