Expression Of Yanbian Breeding Goose Interferon-α Gene In Recombinant Fowlpox And Detection For Its Activity Of GPV

Interferon (interferon, IFN) is a glucoprotein which secreted by the cells with a broad-spectrum anti-viral, anti-cell division, immunomodulatory activity and in different ways affect cell metabolism, growth. Interferon alpha is famous as the antiviral factors and immunoregulation.In order to study Yanbian breeding goose IFN-α gene and its recombinant vector vaccine, the main test as follows:Using phytohemagglutinin (PHA) to stimulate the goose peripheral blood lymphocytes, and extract total RNA.IFN-αgene was amplified by RT-PCR. The fragment was cloned into pMD18-T simple vector and then cloned into pET-30a prokaryotic expression vector. The target protein was expressed in E. coli BL21. Purified recombinant protein was used to immunize mice for the goose IFN-α mature polyclonal anti-serum preparation. Titer was measured by agar diffusion reaction. The results showed that IFN-α gene total length of576bp was amplified, and encoded a polypeptide of192amino acid; The prokaryotic expression vector pET-30a-IFN-α was constructed successfully; The expressed protein about29ku was identified by SDS-PAGE and Western-blot, which showed that the protein has a good reactivity; Successful preparation of polyclonal antibody serum, its agar diffusion reaction antibody titer was1:32.In order to construct the recombinant fowlpox virus (FPV) expression Yanbian breeding goose interferon-α and evaluate its antiviral activity, the test got Yanbian breeding goose interferon-α gene by RT-PCR. Recovery of IFN-α gene fragment from recombinant plasmid pMD18T-IFN-α building pSY681-LP2EP2-IFN-α-LacZ transfer vector by gene recombination technology. The recombinant virus of rFPV-IFN-α was generated by transfection of the transfer vector into the pre-infected CEF with FPV-017and selected the blue virus plaque in present of X-gal. In additionally, the expression of IFN-α from rFPV-IFN-α was identified by indirect immunofluorescent assay (IFA) and Western-blot.Its activity was determined by cytopathic effect inhibition. The results showed that IFN-α gene was amplified; recombinant fowlpox virus rFPV-IFN-α was constructed successfully; the expressed protein was identified by indirect immunofluorescent assay (IFA) and Western-blot, which showed that the protein has a good reactivity; the cytopathic effect inhibition tests showed that Yanbian breeding goose interferon-a possessed antiviral activity evaluated by using gosling plague virus (GPV) in geese embryo fibroblast.Overall, the tests laid the foundation for further study of the recombinant viral vaccines expressing Yanbian breeding goose interferon-a in vivo.