Cloning, Prokaryotic Expression Of Chicken Interferon-α Gene And Study On Antiviral Effect Of Recombinant Chicken Interferon-α

The protein interferon, produced by animal cells when they are invaded by viruses, is released into the bloodstream or intercellular fluid to induce healthy cells to manufacture enzymes that counters the infection. Interferon has been considered a very exciting group of proteins for many years during which they were initially shown to have antiviral and non-specific effects, and lately, immunomodulatory and immunoregulatory effects. While beneficial aspects of interferon therapy have been shown in a variety of experiments in animals, the great potential for interferon treatment has yet to be achieved, and interferon-alpha is especially well antiviral applied to animals. China is the biggest country of feeding fowl, however, the diseases caused by virus have puzzled the industry for a long time and leaded to huge economic losses. Interferon as biological therapeutic agents is urgently needed to cure the diseases caused by virus. The chicken interferon-alpha (ChIFN-α) was cloned and recombined in this experiment, which mainly targets exploreing the antiviral effect of the recombinant protein both in vitro and vivo. This research project was consisted with several sections:1. The cloning of chicken interferon alpha gene and it's expression in E.coli.The full length of chicken interferon alpha (chicken interferon-α, ChIFN-α)gene was amplified by the polymerase chain reaction(PCR) from total liver genome of Sanhuang meat- chicken and sequenced. The amplified gene was about 582bp, and the similarity was 98% with the gene whose GenBank accession is AB021154. The coding region(489bp) for mature protein was subcloned into pET-28a(+). The recombinant plasmid pET-28a(+)-IFNα was identified by enzyme digestion and DNA sequencing. Data of SDS-PAGE and Western-blot indicated that a 22kD fusion protein was expressed in the form of inclusion bodies with good immunity.2. comparative study on the method of protein depurationThe purity of inclusion bodies was above 70% and that of protein purified by nickel affinity chromatography was 95%. The two experiments in vivo both showed that the bioactivities of recombinant IFNα purified by affinity chromatograph were 20 times higher than that of inclusion bodies.3. the antiviral effect of the recombinant protein both in vitro and vivoThe recombinant protein could inhibit H9N2 avian influenza virus (H9N2 AIV) replication on chick embryo fibroblast. 2μg of recombinant IFNα can completely protect Chick embryo from H9N2 AIV infection. The recombinant IFNα can also delay velogenic strain of Newcastle disease virus (NDV) replication on chick embryo for 12h-24h. Chicken...