Expression Of Chicken Interferon λ Using Baculovirus/Insect Cell System And Its Antiviral Effects Evaluation

Interferon (IFN) is essential components of the antiviral defense system of vertebrates. Interferons are divided into three groups:Type Ⅰ, Type Ⅱ and Type Ⅲ. Type Ⅲ interferon is also called λ interferon, which was first reported in 2003. Type Ⅲ interferon has similar antiviral activity with type I interferon, also has similar immune regulating function with type II interferon. The specificity distribution of the IFN-λ receptor causes that IFN-λ plays a relatively independent and specificity of antiviral effect in epithelial cells and immune cells. In this experiment, we studied on ChIFN-λ, used Baculovirus/insect cell expression system to express ChIFN-λ, and carried out its antiviral activity.The total RNA as a template was extracted from chicken kidney tissue, then RT-PCR was performed to amplify ChIFN-λ and gene fragment size was 561 bp. Preliminary analysis showed that the first 23 amino acids of ChIFN-λ amino acids encoded a signal peptide. Primers were designed to remove the signal peptide-coding sequence of ChIFN-λ by PCR. The coding region was subcloned into pET-32a vector, and constructed pET-32a-ChIFN-λ prokaryotic expression plasmid, then transformed the plasmid into competent cells BL21 (DE3). The protein of ChIFN-λ was expressed under IPTG induction. SDS-PAGE and Western-blot indicated that a 36 KD fusion protein was expressed in the form of inclusion bodies. The recombinant protein was purified by High Affinity Ni-NTA Resin. Prokaryotic expression product was used as antigen to immunize BALB/c mice, and mice anti-ChIFN-λ polyclonal antibody was produced. ELISA test showed that its antibody titer reached 1:16000. This serum and anti-6×His antibody were used as primary antibody to detect ChIFN-∵ prokaryotic expression product by Western-blot, and the results showed both of them appeared the specific bands in the size of 36 KD.The synthetic chimaera was obtained by connecting the sequence coding mature ChIFN-λ with gp67 signal peptide by over-lap PCR for improving the secretion expression. This fragment was cloned into the baculovirus transfer vector pFastBacTM1, under the control of pH promoter. Then construct was transformed into DH10Bac E.coli which contained the bacmid with the mini-Tn7 target site and the helper plasmid. The site-specific transposition occurs between the mini-Tn7 element on the pFastBac1 vector and the mini-att Tn7 target site on the bacmid to generate a recombinant bacmid. Subsequently the recombinant bacmid was transfected into the Spodoptera frugiperda 9 (Sf9) cell mediated by lipofectin to produce recombinant baculovirus and express recombinant ChIFN-λ The result showed that the ChIFN-λ was successfully expressed in Sf9 cells by Indirect Immunofluorescence assay and Western Blot analysis.At 96 h after infection, the supernatant and the cell lysis solution of the infected Sf9 cell were collected. The antiviral effects of them were evaluated by their inhibitions to vesicular stomatitis virus (VSV) induced cytotoxicity of chicken embryo kidney cells (CEKC). The antiviral test results showed that the recombinant protein in cell culture supernatant had higher antiviral activity. The antiviral potency of supernatant was 1.44 x 106 U/mL, the antiviral potency of insect cell lysis solution was 4.76×105 U/mL. Avian influenza virus (AIV) and Newcastle disease virus (NDV) could not grow in CEKC pre-treated with recombinant ChIFN-λ. Cell pathogenic efficient (CPE) in the CEKC infected with AIV and NDV was apparently inhibited by the recombinant ChIFN-λ, and the antiviral potency of them were 1.82×106U/mL and 1.94×106 U/mL respectively.In summary, in this experiment, ChIFN-∵ was cloned and expressed in baculovirus/insect cell expression system, and we carried out its antiviral effect to VSV, AIV and NDV. This study showed that recombinant ChIFN-λ could have good application prospect and lay a foundation to mass-produce recombinant ChIFN-λ cheaply.