Cloning And Prokaryotic Expression Of Tibetan Porcine IFN-λ3 Gnen And Studies On Anti-PoRV Activity Of Recombinant Protein

Interferon（Interferon,IFN）as the first line of defense against virus infection,and plays an important role in innate immune response.At present,based on difference in sources,the nucleotide sequences,amino acid sequences,receptors,structurals and biological functions.,IFNs are divided into three categories,respectively,typeⅠ,typeⅡand typeⅢIFNs.TypeⅢIFNs（IFN-λ）were found in 2003,IFN–λbind to a heterodimeric receptor complex formed by the specifc receptor chain IFN-λR1 and the IL-10R2 chain,activates Jak-STAT signaling pathway,induce interferon-stimulated genes（ISGs）expression and induction of an antiviral state.Because of IFN-λR1 more express on the specific tissues such as epithelia,a large number of studies have shown that IFN-λhas centralized antiviral effect at mucosal surfaces,rather than typeⅠIFNs.Porcine Rotavirus（PoRV）is one of the main pathogens causing diarrhea in piglets,which spread widely in the world and has caused great economic losses to the pig industry.Intestinal epithelial cells are main target cells.In this study,IFN-λ3 gene from Tibetan porcine was cloned,prokaryotic expression and studied its antiviral effects against PoRV1.Gene cloning and bioinformatics analysis of Tibetan porcine IFN-λ3 geneAccording to porcine IFN-λ3 gene sequences（registration number:NM₀01166490）in GeneBank,we designd PCR primers.The Tibetan pcrcine IFN-λ3 gene was amplified from lung and intestinal tract by RT-PCR,and the it was cloned into pMD19-T vector,constructed recombinant plasmid pMD19-T-ZPoIFN-λ3.The nucleic acid and amino acid sequence were analyzed by bioinformatics software,The sequence of the Tibetab porcine IFN-λ3 nucleic acid sequence has 100%similarity with porcine.The whole ORF of Tibetab porcine contained 588bp nucleotides,coencoded a polypeptide of 195 amino acids.The signal peptide cleavage site was between the 23th and 24th amino acids.ZPoIFN-λ3amino acid sequence had 13 phosphorylation sites,one potential n-glycosylation site and has four potential o-glycosylation sites.The secondary structure is mainly composed of the alpha-helix and random crimp.2.The prokaryotic expression and antiviral activity detection of the Tibetan porcine IFN-λ3Based on pMD19-T-ZPoIFN-λ3 plasmid,the mature peptide gene of ZPoIFN-λ3 was cloned（pMD19-T-mZPoIFN-λ3）,and the prokaryotic expression vector pET-32a（+）-mZPoIFN-λ3 was constructed and expressed in the E.coli BL21（DE3）,which successfully expressed the protein of 34KDa.The induction time and IPTG concentration were optimized.The protein was purified by Ni-NTA affinity chromatography,SDS-PAGE showed a single band.After renaturation and concentration.The ZPoIFN-λ3fusion protein was used as immunogen to prepared anti-ZPoIFN-λ3 mouse polyclonal antibody.Western Blotting result demonstrated that it had good immunogenicity.The ZPoIFN-λ3 antiviral effect was measured in MDBK/VSV by inhibition of the cytopathic effect,and the results showed that the antiviral effect of ZPoIFN-λ3 in MDBK/VSV was10x2^4.5IU/0.1mL,specific activity was 2x10³IU/mg.3.Research on anti-PoRV activity of ZPoIFN-λ3MA104 cells and IPEC-J2 cells were untreated or pre-treated with ZPoIFN-λ3（10,100,1000ng/mL）for 24 hours.Then the cells were infected with PoRV SC-R strain at 0.1MOI for 36 hours.Infected cells were cultured.IPEC-J2 cells were pre-treated with100ng/mL ZPoIFN-λ3 for 24 hours,and then were infected with PoRV SC-R strain for 12,24,36 hours at 0.1 MOI.The quantification of PoRV VP6 mRNA was performed on total cellular supernatant RNA.The results showed that the replication of PoRV in MA104 cells and the intestinal porcine epithelial cell line J2（IPEC-J2）was significantly reduced after pre-treatment with ZPoIFN-λ3,and such inhibition was dose-and time-dependent.ZPoIFN-λ3 inhibited PoRV infection in IPEC-J2 cells more efficiently than infection in MA104 cells,and the anti-PoRV activity of low concentration of mZPoIFN-λ3（100ng/mL and 10ng/mL）in IPEC-J2 significantly stronger than in MA104 cells,high concentration（1000ng/mL）showed no significant difference.To explore whether co-treatment with mZPoIFN-λ3 and procine IFN-αcould enhance the antiviral efficacy against PoRV in IPEC-J2 cells.IPEC-J2 cells were incubated with increasing amounts of either recombinant IFN-α（10IU/mL,100IU/mL and 1000IU/mL）or ZPoIFN-λ3（0.1ng/mL,1ng/mLand10ng/mL）,incubatedwithmixturesof IFN-α+ZPoIFN-λ3（10IU/mL+0.1ng/mL,100IU/mL+1ng/mL,1000IU/mL+10ng/mL）for24 hours,and then infected with PoRV SC-R strain for 36 hours at an MOI of 0.01.The PoRV titer in the supernatant was titrated by TCID₅₀.The result showed that mixture of IFN-αand ZPoIFN-λ3 could not improve the antiviral activity of ZPoIFN-λ3,activation of ISGs,MxA,OASL and ISG15 also showed same trend.