Generation Of The Bovine IFN-λ3and Reseach On Its Antiviral Activity Compared With The Recombinant BoIFN-α, BoIFN-β And BoIFN-γ

IFNs are key cytokines in the establishment of the activities with antiviral, antitumor andimmunomodulatory activities. Three distinct types of IFNs are now recognized (type I, II, and III)based on their structural features, receptor usage and biological activities. The type III interferon isalso known as the IFN-λs and for the amino acid level and protein functions, IFN-λs are morerelated to type I IFNs. However, the more limited tissue expression of IFN-λ receptors suggeststhat type III IFNs do not simply recapitulate the type I IFN antiviral system. It has the antiviraleffects in the respiratory tract, gastrointestinal tract, skin mucosa, epithelial cells and some tumorcells, which can effectively reduce the toxic side effects. In our country, the foot-and-mouthdisease (FMD), bovine viral diarrhea disease (BVD) and infectious bovine rhinotracheitis (IBR)which spread through the digestive tract, respiratory tract is still relatively severe. Therecombinant bovine IFN-λs may play an important role in the control the diseases by its antiviralactivity. For in-depth reseach of antiviral activity of the bovine IFN-λs, revealing its feasibility forthe control of bovine viral disease, the non-redundant amino acid recombinant bovine IFN-λ3wassoluble expressed in Pichia pastoris. The recombinant bovine IFN-α and the recombinant bovineIFN-β was also expressed in Pichia pastoris. On the other hand, the recombinant bovine IFN-β andthe recombinant bovine IFN-γ was espressed from the E.coli expression system. In the direction offurther understand the biological function of bovine IFN-λ, the in-depth reseach of the antiviralactivities of the IFN-λ3was carried out and compared with type I and type II interferons alone orcombination used. The main contents are as follows:1. The recombinant bovine IFN-λ3was soluble expression in Pichia pastoris. Taking into theyeast codon uasage, the free energy and the secondary structure of the bovine IFN-λ3, theoptimized bovine IFN-λ3sequence which named as boIFN-λ3and its allele (named as theboIFN-λ3*that one gene was different which caused the eighteen amina acid of the mature peptideof the bovine IFN-λ3was variability) was designed and the sequences were amplified using thegene splicing by overlap extension (SOE) with the synthesized14oligonucleotide. Then thesecretion expression vector pPICZαA-boIFN-λ3and pPICZaA-BoIFN-λ3*was successfullyconstructed. With the screening and identification of the positive recombinantGS115-pPICZαA-boIFN-λ3, the selection of the high expression stain and the optimization of the induced expression system, we expressed the recombinant boIFN-λ3and boIFN-λ3*with theexpression levels up to1.2g/L. The glycosylated protein (23kDa, about70%of the total protein)and non-glycosylated protein (18kDa, about15%of the total protein) was detected byglycoprotein staining. The protein was purified by the ammonium sulfate precipitation and thecation exchange chromatography. The recombinant protein has good antigenicity with thedeveloped polyclonal antibody. The antiviral activity of the recombinant boIFN-λ3and boIFN-λ3*was2.39±0.15×106U/mg/ml and2.15±0.40×106U/mg/ml in MDBK-VSV detecting system. Therecombinant proteins were inactivatied with the trypsin, not sensitive to heat(63℃) and partlysensitive to pH (pH2activity decreased by2times). With the comprehensive analysis, theexpression, purification, physical and chemical properties, biological activity and inducedcytotoxicity was no difference between the recombinant boIFN-λ3and the boIFN-λ3*.2. The recombinant bovine IFN-α/IFN-β/IFN-γ was expressed, purified and preliminarydetermined their biological activity. For comparison antiviral activity of boIFN-λ3with type I andtype II IFN, The recombinant boIFN-α and the recombinant boIFN-β was expressed in Pichiapastoris and the biological activity was1.50±0.98×107U/mg/ml and1.25±0.38×104U/mg/ml. Thebiological activity of the prokaryotic expressed and purified recombinant boIFN-β and the boIFN-γwas2.44±0.91×103U/mg/ml and0.81±0.21×105U/mg/ml. The antiviral potency was therecombinant boIFN-α>boIFN-λ3>boIFN-γ>boIFN-β in MDBK-VSV system. Cytotoxicitymeasurements by MTT assay in EBK and MDBK cells showed that boIFN-λ3and other IFNsinduced lower cytotoxicity alone or in combination.3. The Mx1protein and the ISRE promoter activity was induced by IFNs. The Mx1proteinwas induced by recombinant boIFN-λ3and other IFNs in epithelial cell-derived cells (EBK, BT,MDBK and BMEC). We used the ISRE (interferon-stimulated response element) luciferasereporter assay to assess activity downstream of the JAK-STAT signaling pathway. The resultsshowed that the every recombinant boIFN induced ISRE luciferase activity in a dose-dependentmanner; and the recombinant boIFN-λ3and boIFN-γ induced reporter gene expression level wastime-dependent and reached a high level at48h, while the recombinant boIFN-α and boIFN-βinduced ISRE luciferase more rapidly that reached the high level at12h and maintained to48h.The combination of the recombinant boIFN-λ3with other IFNs was high than alone except theboIFN-λ3plus the boIFN-γ.4. The anti-FMDV/IBRV/BVDV was comparative research on the recombinant boIFN-λ3andboIFN-α/boIFN-β/boIFN-γ. The methods were about TCID50assay and the plaque reduction assay.The results showed that the recombinant boIFN-λ3and other three recombinant IFNs haveanti-IBRV and anti-FMDV activity, with a certain degree of dose and time-dependent manner. Theactivity of anti-IBRV was weak while anti-FMDV strong. The inhibitory effect of the recombinantboIFN-λ3on cytopathic BVDV (cpBVDV) and noncytopathic BVDV (ncpBVDV) replication wasobvious while treatment of MDBK cells with IFN before and in a dose-dependent manner. While treatment of MDBK cells with IFN after and the BVDV infecton first, any of the IFNsconcentration has no activity to inhibate the BVDV replication.5. The synergistic antiviral research of the recombinant boIFN-λ3with the recombinantboIFN-α/boIFN-β/boIFN-γ. Using the cytopathic inhibition assay (TCID50assay), the virus-cellsuspension harvested from the MDBK or EBK cells treated before with the recombinant boIFN-λ3alone or combination with the boIFN-α/IFN-β/IFN-γ was assessed. In combination, therecombinant boIFN-λ3and the boIFN-γ had a synergistic anti-IBRV effect in MDBK cells, and therecombinant boIFN-λ3with the recombinant boIFN-α or boIFN-β could significantly inhibit theFMDV replication in EBK cells and BT cells. Thus, the synergistic anti-FMDV effect was apparentby combination with the recombinant boIFN-λ3and the boIFN-α/boIFN-β. It was prompted to bewidely used with the combination of the recombinant boIFN-λ3and other IFNs in clinicalapplications in the future. The synergistic enhancement was observed may relate to the ISREactivity or up regulation of the interferon stimulated genes (ISGs).6. The impact on the activity of the recombinant boIFN-λ3when ncpBVDV persisitly infected.To determine whether ncpBVDV can be eliminated from the infected cell monolayer by prolongedinterferons treatment, we passaged ncpBVDV infected in MDBK cells8times in the presence ofvarious amounts of the recombinant boIFN-λ3, which the recombinant boIFN-α as the control.Using the semi-quantitative RT-PCR to detect BVDV nucleic acid, the cell culture passages ofMDBK cells infected with the HLI-11strain in the presence of up to100U/ml recombinantboIFN-λ3were not able to eliminate the virus infection，which was to say the BVDV can toleratethe interferon and the MDBK cells tolerate the BVDV, resulting in the persistent infection of cells.But this type cells was sensitive to VSV virus and the recombinant boIFN-λ3could prevent theinfection of the cell monolayer with VSV. This ncpBVDV pre-infected MDBK cells does not affectboIFN-λ3and other IFNs on to play anti-VSV and anti-IBRV effect.In conclusion, the generation of the bovine IFN-λ3, the research of the antiviral activites ofthe recombinant boIFN-λ3and its synergistic effects with type I or type II interferons will help usto further understand the biological function of bovine IFN-λs. In addition, there is one moreantiviral agents was provided for future use.