The Gene Synthesis, Prokaryotic Expression And Antiviral Activity Assay Of Chicken, Duck And Canine Interferon Alpha

Interferon (IFN), which is generated by specific cells induced by inducer, is a group of glycoprotein that has antiviral activity, anti-tumor activity and immunoregulation function. IFNs are divided into three types:Ⅰ, Ⅱ,Ⅲ. IFN Type Ⅰ, which has the strongest antiviral activity, is important to the basic research of virus disease of human and animals. It is also has broad clinical application prospect. Genetically engineered interferon not only has familiar biological activity with natural interferon, but also has many benefits such as high purity, being suitable for large-scale production, easy to control product quality and steady product quality. More importantly, large-scale production of genetically engineered interferon significantly reduced the cost and it is conducive to its widespread use in livestock and poultry breeding industry. The aim of the present study is to develop the recombinant chicken, duck and canine interferon alpha with technique of genetic engineering and protein engineering and to study the antiviral activity of the recombinant protein. The study laid the foundation for engineering production and clinical application of the recombinant interferon.According to the gene sequences of chicken, duck and canine interferon alpha which were published in GeneBank, the mature protein coding sequence were reserved after eliminating the signal peptide sequence. The known gene sequences were transformed by replacing the codon with E. coli codon preferences without changing the sequence and composition of amino acid. The three new gene sequences of chicken, duck and canine interferon alpha were489bp,486bp and495bp. After added EcoRⅠ and XhoⅠ restriction sites respectively at the two ends of the sequences, the gene was synthesized by bio-company and cloned into the pET32a vector to construct the recombinant prokaryotic expression vector pET32a-ChIFN-aα pET32a-DuIFN-α and pET32a-CaIFN-α. The three recombinant plasmids were transformed into expressing host E.coli BL21, respectively expressed32kD protein in the form of inclusion body by inducing of IPTG. Pure recombinant chicken, duck and canine interferon alpha were got after the cell disruption,inclusion body extraction, washing, protein denaturation and dialysis renaturation.Using the micro-cytopathic-effect-inhibition method to detect the antiviral activity of the three recombinant fusion protein, the results are as follows:(1)anti-VSV activity of the recombinant chicken IFN-a was about5.6×104U/mL, protein concentration0.53mg/mL, specific activity1.06×105U/mg; anti-NDV activity of recombinant chicken IFN-a was1.35×105U/mL, specific activity2.55×105U/mg.(2) anti-VSV activity of the recombinant duck IFN-a was5.13×103U/mL, protein concentration0.92mg/mL, specific activity5.6×103U/mg.(3)anti-VSV activity of the recombinant canine IFN-a was1.06×107U/mL, protein concentration0.79mg/mL, specific activity1.67×107U/mg.The study on the prevention and treatment effect of chicken interferon-a to Newcastle disease was carried on the chicken. Test chicken were divided into four groups:group1and2were injected with chicken interferon-a respectively24hours before or after the inoculating NDV; group3and4were NDV control group and blank control group. The results showed that the mortality of group1and group2were significantly lower than the NDV control group, and the high-dose interferon was more effective.