Cloning, Expression And Antiviral Bioactivity Of Red-crowned Crane Interferon-α

Interferon-α (IFN-α) is a cytokine produced by the body’s white blood cells, which can evoke antiviral, antitumor, immunomodulatory cellular responses and play an important role in the animal’s defence system. Recently, the studies of animal interferon focused on economic animals, and have made great progress, like chickens, dogs, pigs recombinant interferon product were applied on the market. But the information regarding crane IFN-α has not been reported to date. The Red-Crowned Crane is a large wading bird that lives in wetland environments. It is high in the food chain and is the key to wetland biodiversity; it is known as "the god of wetlands." China is the main country for perched cranes throughout the world. The Chinese State Forestry Administration has put red-crowned as the only candidate for the national bird submitted to the State and take it as Country grade one protected animal. In recent years, environmental changes, human interference, Red-crowned Crane bird flu, Newcastle disease, marek’s disease and other infectious diseases have made the number of wild red-crowned crane decline, which is around 2600 in the world, but is only about 1500 in the East Asian mainland.In this study, we cloned Red-Crowned Crane interferon-α (crIFN-α) gene sequence by degenerate primers PCR and gene walking PCR, which is about 1786 bp encoding 246 amino acids, sharing less than 45% identity with mammalian IFN-α and 60 to 80% identity with avian IFN-α, has a closer relationship with the parrot (sharing 80.7% identity with the Ara militaris). The cIFN-a gene encoding the mature peptide was cloned into the expression vector pET32a (+) to construct the recombinant expression plasmid pET32a-IFN-α, which was transformed into E.coli BL21 to express the recombinant protein. The results of SDS-PAGE showed that the protein was mainly expressed in inclusion bodies, approximately 43KDa. To obtain activated proteins, crIFN-α inclusion bodies were purified on a nickel chelated column and refolded by dialysis. The results of cytopathic inhibition assays in vitro indicated that the recombinant crIFN-α could inhibit the replication of vesicular stomatitis virus in chicken fibroblasts (activity:1.87×105 U/mg). These antiviral activities were abrogated by rabbit anti-crIFN-α antibodies in vitro. The immunofluorescence assay indicated that the recombinant crIFN-α could be expressed in chicken fibroblasts cytoplasm. In addition, application results of two pairs of degenerate primers (AIF-TY1a and AIF-TY2b, POAU and AIF-TY2b) show, AIF-TY1a and AIF-TY2b were more specific in cloning avian IFN-α, which provides an important reference in clone avian IFN-α unknown sequence.Through this study, we cloned the red-crowned crane IFN-α gene, expressed the crIFN-α protein in Escherichia coli and chicken embryo fibroblasts respectively, and determined the antiviral effects of crIFN-a using cytopathic inhibition assays for the first time, which leave a good foundation to great scale industrial production.