Studies On The Cloning,Expression And Antiviral Activity Of Porcine IFN-λ1 Within Interferon TypeⅢ

Interferons (interferon, IFN) are a class of cytokines that have broad-spectrum anti-viral, anti-tumor and enhancing immune function. According to its biological activity and antigenicity, interferons are divided into three groups:IFN-α, IFN-β, IFN-γ. IFN-αand IFN-P have a common surface receptor and belong to type I interferon; IFN-γhas different surface receptors with IFN-a, IFN-βand belong to type II interferon. Type III interferon, a new interferon family, was first reported in 2003 and different from the type I and II interferon.It was also known asλinterferon, including IFN-λ1, IFN-λ2, IFN-λ3. Becauseλinterferon gene structure is similar with IL-10, so also known as IL-29, IL-28A, IL-28B. This new type of interferon, like other interferons, has anti-virus, anti-tumor and immune-regulatory biological activities. TypeⅠandⅡinterferon have been widely used in the clinical treatment with many diseases, but the research on type III interferon is lagged behind. So the research on typeⅢinterferon will play a role in promoting the control of animal diseases and medical treatment of human disease. In view of this, in this study, we chiefly studied porcine typeⅢinterferon IFN-λ1 and carried out its anti-viral activity to porcine reproductive and respiratory syndrome virus (PRRSV). The main contents include:1. Cloning and sequence analysis of Pig typeⅢinterferon IFN-λ1The total RNA as a template was extracted from Pig peripheral blood mononuclear cells (PBMC), RT-PCR was performed using primers PoIFN-λ1-F2 and PoIFN-λ1-R2, as upstream and downstream primers, to amplify PoIFN-λ1 and gene fragment size is 576bp. Sequence analysis showed that PoIFN-λ1 had a nucleotide sequence homology as high as 76.12% compared with human IFN-λ1 registered in GenBank and the amino acid sequence homology was 69.15%. Analysis of the PoIFN-λ1 amino acid sequence by the SignalP 3.0 Server software predicted that the first 19 amino acids encoded a signal peptide sequence.2. The expression of pig typeⅢinterferon IFN-λ1 in E.coliPrimers were designed to remove the signal peptide-coding sequence by PCR. The PCR product was inserted into prokaryotic expression vector pET-28a(+), after verification by digestion with EcoRⅠ, XhoⅠ, the recombinant plasmid pET-PoIFN-λ1 was obtained, and transformed into host strain BL21. The expression was induced by IPTG, and SDS-PAGE analysis confirmed thatPoIFN-λ1 fused with His tag was expressed, and the fusion protein was essentially present in insoluble inclusion bodies, with the sizeabout 23kDa. The expression was denatured, renatured, dialysed and purified, and then MDBK/VSV (vesicular stomatitis virus) micro-cytopathic effect inhibition method was used for the determination of its antiviral activity. The bioactivity of prokaryotic expression products was 1.8×103U/mg.3. The preparation of poluclonal antibody against pig typeⅢinterferon IFN-λ1The Prokaryotic 1 expression product was used as antigen to immunize a rabbit, and rabbit anti-PoIFN-λ1 polyclonal antibody was produced. ELISA test showed that its antibody titer reachedl:320. This serum and anti-His6 antibody were used as primary antibody to detect PoIFN-λ1 prokaryotic expression product by Western blot, the results showed in the size of about 23kDa both appearred the specific reaction protein band. This shows that the prepared polyclonal antibody has IFN-λ1 antibody, and illustrates the fusion expression of PoIFN-λ1 and His6.4. The eukaryotic expression of pig typeⅢinterferon IFN-X1Eukaryotic expression plasmid pcDNA-PoIFN-λ1 was constructed and transfected into PK-15 cells, then 24h,48h,72h after transfection, the supernatant was collected respectively. Western blot revealed that the highest expression level was reached at 48h after transfection. We used MDBK/VSV CPE inhibition method to detect the anti-virus activity of supernatant collected at 48h, and it was shown that biological activity was 2.3×103U/mL5. Pig typeⅢinterferon IFN-M inhibits PRRSV CH-la, WuH3 and PRV strainsSJPL cells were pretreated with 50U/mL, 100U/mL,150U/mL,200U/mL PoIFN-λ1 prokaryotic expression products or 24h, then infected with PRRSV CH-la and WuH3 strains. Supernatant were collected at 12h,24h,36h,48h respectively and Real-time PCR and TCID50 were performed to detect the proliferation of the virus. The results showed thatPoIFN-λ1 was able to significantly inhibit the mRNA expression and the titre of viral replication of the two strains, and the inhibition effect on CH-1a is better than on WuH3. The anti-viral effect of PoIFN-λ1 presented a dose and time-dependent manner.In order to study the affect of PoIFN-λ1 on PRV proliferation, in this study PoIFN-λ1 prokaryotic expression products were used to pretreat PK-15 cells, which werethen inoculated with PRV. Plaque reduction experiments showed that PRV plaque was gradually decreased with the dose increasing, indicating PoIFN-λ1 inhibited the PRV proliferation in a dose-dependent manner.