Fusion Expression And Biological Activity Detection Of Duck Interferon ?/Interferon ??Interferon ?/Interleukin-18

Interferon is a cytokine with a variety of biological functions, including a broad-spectrum antiviral, anti-proliferative and regulation of immune. They are classified into three types according to the different surface receptors by the International Committee on Interferon Nomenclature. IFN-? is type I IFNs, which display markedly antiviral, anti-proliferative and immune regulation activities. Type II IFN, or IFN-?, which could induce the expression of virus antigen by virus infected cells, and increase the ability of the immune system to identify and kill infected cells. IFN-? could participate in immune response as an immunological adjuvant. Different IFNs display distinct antiviral activities and IFN-? exhibits a weaker antiviral effect than type I IFNs. Interleukin-18 is released by cells from mononuclear phagocytic system, which could induce the expression of multiple cytokines such as IFN-?, IL-2, TNF-? and GM-CSF. IL-18 could also play important role in the process of cell immune. While the former research of interferon and Interleukin were still limited in the expression of solo interferon and Interleukin. There are few reports about the antiviral effect of combination of the duck IFN-? and IFN-?, IFN-? and IL-18. Here we combined duck IFN-? and IFN-?, IFN-? and IL-18 fusion gene through SOE-PCR respectively and cloned the fusion genes into expression vectors. We expressed these proteins by Escherichia coli BL21. The antiviral activities of recombinant protein were tested in vivo and vitro. The purpose of this study is to produce antiviral preparation with low-cost, easy production and high-activity product. The content was divided into four parts:1. Three pairs of primers were designed according to duck IFN-?, IFN-? and IL-18 sequences published on the Gen Bank. We extracted DNA templates from duck peripheral blood lymphocytes and based on which, the objective gene without signal peptide were amplified respectively. we combined duck IFN-? and IFN-?, IFN-? and IL-18 fusion genes through SOE-PCR respectively and cloned the fusion genes into expression vectors and then constructed duck IFN?-IFN?, IFN?-IL18, IFN-?, IFN-? and IL-18 prokaryotic expression vectors with p ET-32a(+).2. These proteins were expressed by the induced of IPTG in Escherichia coli BL21. Then the proteins in inclusion bodies were dissolved with urea and purified by Ni-NTA chromatographic column. The renaturation were obtained using gradient dialysis and identification through SDS-PAGE and Western blot analysis.3. The antiviral activity of r Du IFN?-Du IFN?, r Du IFN?-Du IL18, r Du IFN-?, r Du IFN-? and r Du IL-18 against VSV, AIV and DPV were tested in Duck Embryo Fibroblast using Micro cell lesion inhibition method. The result showed that Fusion protein r Du IFN?-Du IFN? and r Du IFN?-Du IL18 exhibit apprent antiviral activity than single protein in vitro.4. The antiviral activity of r Du IFN?-Du IFN?, r Du IFN?-Du IL18, r Du IFN-?, r Du IFN-? and r Du IL-18 against AIV and DPVwere tested in Peking ducks, the result show that Fusion protein r Du IFN?-Du IFN? exhibits a better antiviral activity than other protein in vivo. We tested the immune enhancement effect of r Du IFN?-Du IL18, r Du IFN-? and r Du IL-18 for AIV oil emulsion vaccine in Peking ducks and found that r Du IFN?-Du IL18 and r Du IL-18 could enhance the immune reaction.