Expression Of Chicken Interferon α And Interferon γ In Silkworm And The Antiviral Activity Assay

With the development of chicken industry, the prevention and cure of chicken diseases is becoming a more and more important issue. Interferon is a multifunctional cytokine which plays an important role in antiviral, antineoplastic and antiparasitic activity. Interferon has been proved to be effective in the treatment of commom poultry diseases including Newcastle disease and avian influenza. Thus, developing a highly active interferon product has a favourable prospect in application.In this research, we chose the DNA sequences of Ch IFN-α（Gen Bank:NM₂05427.1）and Ch IFN-γ（Gen Bank:NM₂05149.1）in Genbank database for codon bias optimization. According to the Optimum Gene TM method, we have raised the CAI value of Ch IFN-α and Ch IFN-γ to 0.88 and 0.89 separately and adjusted the GC content in Ch IFN-α and Ch IFN-γ to 49.13% and 42.79%. Meanwhile, we replaced the rare codons and codons which can form hairpin structure in m RNA, removed the restriction enzyme sites in Ch IFN-α and Ch IFN-γ and then obtained the intact sequences through chemical synthesis. Ch IFN-α and Ch IFN-γ were inserted into the genome of baculovirus to construct the recombinant virus and silkworms were infected with the recombinant virus to co-express Ch IFN-α and Ch IFN-γ. The expression of Ch IFN-α and Ch IFN-γ in silkworm has been proved by Western blotting. Antiviral activity of chicken Ch IFN-α and Ch IFN-γ co-expressed in silkworms reached 7.64×106U/m L in CEF/VSV-GFP system. Recombinant baculovirus was purified through plaque screening, and the antiviral activity of chicken interferons expressed by purified baculovirus reached 1.78×107U/m L. The expression level was improved by almost 2.3 fold. At the same time, the antiviral activities of Ch IFN-α and Ch IFN-γ expressed separately by baculovirus expression vector system were detected. The results showed that antiviral activity of Ch IFN-α and Ch IFN-γ was 3.66×106U/m L and 1.73×106U/m L, respectively. Ch IFN-α expressed in silkworm pupa was purified by acid denaturation. The result showed that impurity protein with molecular weight of about 60 k Da was removed after acid denaturation and Ch IFN-α mainly existed in the supernatant gathered after acid denaturation.In this research, we have effectively expressed Ch IFN-α and Ch IFN-γ using baculovirus expression vector system, and obtained high-activity chicken interferons. This provided a foundation to the manufacture of chicken interferon products.