Cloning And Expression Of α - Interferon From Yangzhou Goose And Its Biological Activity

Interferon is a kind of important cytokines, which has broad-spectrum antiviral activity, excellent inhibition of cell division and antitumor activity, efficient immune regulation and other functions. Recently, a series of the studies of poultry interferon have been reported, especially about which of chicken and duck, but rare reports of goose interferon have been published.This experiment chose to study type Ⅰ interferon——the interferon-alpha because its antiviral activity is much stronger, and according to the published in the GenBank of goose interferon alpha gene sequences(HQ115583) designed two pairs of primers, and chose the local varieties of Yangzhou goose as experimental animals. By the method of RT-PCR successfully amplified the Yangzhou goose interferon-alpha’s both whole-gene and mature-gene, their sizes are576bp and486bp respectively. The IFN-α whole gene was cloned into pGEM-T carrier and then the recombinant plasmid pGEM-T-GoIFN-α was sent to the sequencing company. Yangzhou GoIFN-α and HQ115583two sequences’ homology at the nucleotide level was revealed99.6%.pET32a-mGoIFN-α were constructed for IFN-α mature gene expression. The recombinant plasmid was transformed into E.coli DH5-α(DE3), and was induced with IPTG for mGoIFN-α gene expression. The expressed product was identified by SDS-PAGE, a fusion protein about36.3kDa was found. After the purification of this inclusion body, can almost get a single specific band, and using Western Blotting method to detect the protein, the result was the same as the expected.Used the recombinant protein pET32a-mGoIFN-α to immune BALB/c mice, then obtained the rat anti mGoIFN-α polyclonal antibody, measured the antibody titer by ELISA method, the result was1:16000. The hyper-immune serum’s specificity was tested by Western Blotting, the results was in the location of the36.3kDa appeared a specific band, this suggesting that the serum has good specificity and reactionogenicity.Yangzhou goose IFN-α whole-gene cloning to pcDNA3.1(-) carrier, thus successfully constructed the eukaryotic expression system pcDNA3.1-GoIFN-α, and adopt the method of indirect immunofluorescence to confirm the GoIFN-a expression in the embryonic fibroblasts. Through the cytopathic inhibition method to detect the antiviral activity of GoIFN-a, the results proved the GoIFN-a can effectively help embryonic fibroblasts against VSV infection.