Mink Interferon-α,ε Gene Engineering Expression And Its Preliminary Application In The Treatment Of Mink Viral Enteritis

Interferons(IFNs)are glycoprotein secreted by vertebrate cells.IFN-a as one family member of type I IFNs was the earliest and the most extensive cytokine used in clinical treatment.IFN-ε was found much later than IFN-a.It is constitutively expressed in mucosal epithelial cells,has antiviral and mucosal immunity functions,and maintains the stability of the microbial environment.Mink(Mustela vison)viral enteritis is an acute viral disease caused by mink enteritis virus(MEV),with intestinal diarrhea as the main clinical symptom.The mortality rate is 30%～80%,causing huge economic losses to the breeding industry.With the continuous progress in veterinary clinical antiviral drug research technology,IFNs have already been used in clinical antiviral treatment in the domestic market.The effects of MEV infection on the expression of mink interferon-a(MiIFN-α)and interferon-ε(MiIFN-ε)was analyzed in this study.The therapeutic effect of mink viral enteritis was observed,which laid the foundation for mink genetic engineering antiviral reagent development.1 The effects of MEV infection on the transcription levels of IFN-α,ε mRNA.To investigate different MiIFN-α,ε transcription levels of mink tissues.Specific primers were designed to amplify the coding region of MiIFN-α,ε gene.The transcription levels of MiIFN-α,ε mRNA before and after virus infection were detected by using S YBR Green real-time PCR method.The results showed that IFN-ε was highly expressed in the brain,lung,trachea,kidney,intestine,bladder,ovary,and testis but not in the muscle and spleen.MEV infection had no effect on the expression of MiIFN-ε in tissues.The expression of IFN-α was significantly higher than that in normal tissues(p<0.001).The expression of MiIFN-α in spleen was increased by more than 2.86×103 times.The results showed that MiIFN-ε is constitutively expressed in mink tissues and is not induced by virus.2 Soluble expression and identification of recombinant rMiIFN-α,εIn order to obtain soluble rMiIFN-α,ε with biological activity,The prokaryotic expression vector pET28a-SUMO-rMiIFN-a and pET28a-SUMO-rMiIFN-ε were constructed separately by fusing the N-terminus of mature peptide gene with a SUMOsolubilizing tag.SUMO-rMiIFN-α and SUMO-rMiIFN-ε was co-expressed with the Tf16 chaperone plasmid in E.coli.BL21 strain.Soluble 32 kDa combined SUMOrMiIFN-ε protein was obtained.It was 20 kDa after SUMO protease digestion,which was consistent with the expected size.SUMO-rMiIFN-α was a 31 kDa inclusion body protein.The mature peptide gene encoding MiIFN-α was cloned into the yeast expression vector pPICZaA,containing the AOX1 promoter and α-factor secretion signal,after codon optimization.pPICZaA-rMiIFN-α plasmid was linearized by Sac I and transformed into the P.pastoris X33 strain to screen for anti-Zeocin transformants.Secretory expression was induced by methanol.SDS-PAGE and Western blot analyses showed that a 25 kDa fusion protein was expressed in the culture supernatant.rMiIFNα has cross reaction with canine IFN-α monoclonal antibody.3 The antiviral activity analysis of rMiIFN-α,ε in vitrorMiIFN-α of yeast secreted expression and rMiIFN-ε of E.coli.soluble expression were selected for antiviral assay by cytopathic effect inhibition(CPEI)in vitro.Purified rMiIFN-α and rMiIFN-ε significantly inhibited the cytopathic effect of vesicular stomatitis virus with a green fluorescent protein(VSV-GFP)in F81 feline kidney cells,with an antiviral activity of 6.4×107 IU/mL and 1.3×104 IU/mL respectively,and also significantly inhibited MEV replication in F81 cells.The inhibition efficiency of rMiIFN-α was higher than that of rMiIFN-ε at the same dose.rMiIFN-α and rMiIFN-εwere both sensitive to pH changes.Compared with pH7.4 control group,the activity of rMiIFN-α in pH2,pH6,pH10 and pH12 treated groups decreased significantly(p<0.05).There was no significant difference between pH6 treated groups and control group.Significant difference were found in other PH treated groups(p<0.05).The results showed the antiviral activity of rMiIFN-α secreted by recombinant yeast was 4.92×103 times higher than soluble rMiIFN-ε expressed in E.coli.4 The evaluation of therapeutic effect of lyophilized rMiIFN-α to mink viral enteritisAntiviral experiments in vitro shows that rMiIFN-α is an ideal choice for antiviral therapeutic reagents development.We prepare freeze-dried rMiIFN-α.The blood concentration of rMiIFN-α was determined in rats.The safety experiment was carried out in mink,and the therapeutic effect of anti-MEV virus was observed.The results showed that the blood concentration of rMiIFN-α peaked at 36 h after intramuscular injection in rats.rMiIFN-α with l×107 IU/kg was safe to use and did not cause adverse reactions and lesions in mink tissues and organs.Treatment results for mink artificially infected MEV showed that the survival rate of diseased mink was 80%after injected with 5×105 IU/kg rMiIFN-α.In 5×105 IU/kg rMiIFN-α and 5×106 IU/kg rMiIFN-αgroups,the survival rates of the two groups were the same,reaching 60%,significantly higher than the adjuvant treatment group(p<0.05).The above results indicate that rMiIFN-α has a significant preventive and therapeutic effect on mink viral enteritis.In summary,rMiIFN-α showed obvious preventive and therapeutic effect on artificial infected and natural infected samples.It can reduce clinical symptoms、virus loads,mink morbidity and mortality.It has potential application prospect.This study laid a foundation for mink genetic engineering antiviral agents development.