Preparation,Lyophilization Of Recombinant Porcine Interferon Alpha And Efficacy Of Anti PRRSV Replication In Vivo And In Vitro

Interferon alpha(IFN-α)is a cytokines with antiviral activity,immunomodulatory activity and antitumor activity,mainly derived from plasmacytoid dendritic cells(pDCs).Studies have confirmed that porcine interferon alpha(pIFN-α)can inhibit the replication of a variety of viruses in vivo and in vitro,and has a good therapeutic effects.Artificial preparation of IFN-α from leukocytes has many disadvantages,such as high cost,safety,poor quality control,unstable curative effect.Developing efficient methods for producing recombinant porcine interferon a(rPoIFN-α)with high activity,low cost and easy batch-scale production is of great significance for large scale and practical use of the reagent.In this study,rPoIFN-α expressed by Pichia Pastoris was purified and lyophilized,whose inhibitory effect on replication of porcine reproductive and respiratory syndrome virus(PRRSV)was confirmed by in vivo and in vitro experiments.These experiment results reveal rPoIFN-α has important application prospects in the prevention and control of porcine viral diseases.1.High level expression and purification of recombinant porcine interferon-a from Pichia pastorisPichia pastoris KM71 was cultivated and induced based on DO-Stat fed-batch strategy during high density cultivation in 7 L fermenter.The technology of secretory expressing rPoIFN-a in Pichia pastoris has been established.Affinity chromatography,ion exchange chromatography and gel filtration were sequentially used to purify rPoIFN-α.The results showed that the highest concentration of rPoIFN-a expressed by Pichia pastoris was 650.3μg/mL at 84 h of induction,and the antiviral activity of rPoIFN-α was 1.09×106 IU/mg.Afere purification,the purity of rPoIFN-α was detected of 95%,and the inhibitory activity of rPoIFN-α against VSV was about 5.63×106 IU/mg.2.Screening of freeze-drying stabilization additives for recombinant porcine interferon-a preparationFreeze drying technology was applied to prepare rPoIFN-a products.14 cryoprotectants were screened by judging three indexes of physical appearance,residue moisture and bioactivity of the preparations after freeze-drying with conservative method.Three protocols,which had mannitol concentrations of 2%,4%and 5%,could retain preparation bioactivity at 102.8±5.6%,95.1±2.5%and 108.3±2.3%,respectively.Freeze-dried preparations of rPoIFN-α with the three protocols were employed for accelerating stability test at 40℃.The test result showed that the bioactivity of all preparations did not change significantly during the test period of 3 months,which proved that the product had good stability and the expiration time could be two years.These expetiments laid the foundation for preparation of rPoIFN-α products.3.Studies on the inhibitory effect of rPoIFN-α on replication of PRRSV in vitroThe inhibitory effects of rPoIFN-a on PRRSV infection were investigated using cytopathogeuic effect inhibition assay.The results demonstrated that the replication of PRRSV was completely inhibited in Marc 145 cells pretreated 24h with 341 IU/mL rPoIFN-α.After 24 hours post-infection with PRRSV in Marc 145 cells,1367 IU/mL rPoIFN-α could completely inhibit the proliferation of PRRSV.To observe the effect of rPoIFN-α on the titers of PRRSV,Marc 145 cells were incubated with twofold serial dilutions of rPoIFN-α for 24 h,and then inoculated with tenfold serial dilutions of PRRSV.Inhibitory effect on PRRSV propagation gradually diminished with increasing dilution of the rPoIFN-α reagent.The results indicated that rPoIFN-α has inhibition effects on PRRSV infection in vitro,which provided reference data for animal experiments of rPoIFN-α.4.Studies of rPoIFN-α against PRRSV in vivoThe efficiency of rPoIFN-α on prevention and treatment of PRRSV in vivo was evaluated.Pigs were challenged with HP-PRRSV at first,then given rPoIFN-α for three consecutive days at the time as follows:the same time as challenge,1d after challenge,the day of appearing of clinical symptoms.The results showed that seroconversion of pigs against PRRSV and virus propagation were delayed by treatment of rPoIFN-α on the same day of challenge and 1d after challenge.Pigs were given rPoIFN-α at first day appeared symptom.After treatment of rPoIFN-a,virus load in the blood and the body temperature decreased comparing with the positive control group without treatment.After 3 days of injection,the virus load in the blood was significantly lower than that in the positive control group.Therefore,the results proved that rPoIFN-α treatment could inhibit HP-PRRSV infection in pigs and had important application prospect.