The Prokaryotic Expression And Antiviral Activity Assay Of Feline Interferon Alpha And Omega

Interferon (IFN) is a kind of cell factors which is generated mononuclear cell and lymphocyte induced by inducer, is a group of glycoprotein controlled by cell genome 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, directly affect 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 steady product quality, high purity, easy to control product quality and being suitable for large-scale production. More importantly, large-scale production of genetically engineered interferon significantly reduced the cost and it is conducive to its widespread use in modern pet industry. This study develop the recombinant feline interferon alpha and omega with technique of genetic engineering and protein engineering and study the antiviral activity of the recombinant protein. All the following clinical research of interferon by produced with the technology of genetic engineering and new drug development of pets can be strongly supported by these results.According to the gene sequences of feline interferon alpha and omega 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 two new gene sequences of feline interferon alpha and omega were 510bp and 525bp. After added BamH Ⅰ and Hind Ⅲ 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-FeIFN-a and pET32a-FeIFN-ω. The two recombinant plasmids were transformed into expressing host E.coli BL21, respectively expressed 33kD protein in the form of inclusion body by inducing of IPTG. Pure recombinant feline interferon alpha and omega were got after the cell disruption, inclusion body washing, protein denaturation and dialysis renaturation.The study use the micro-cytopathic-effect-inhibition method to detect the antiviral activity of the two recombinant fusion protein, the results are as follows:(1) anti-VSV activity on Vero of the recombinant feline IFN-a was about 4.7x103U/mL, protein concentration 0.96 mg/mL, specific activity 4.9x103U/mg; anti-VSV activity on F81 of the recombinant feline IFN-a was about 4.7×107U/mL, protein concentration 0.96 mg/mL, specific activity 4.9×107U/mg. (2) anti-VSV activity on Vero of the recombinant feline IFN-ω was 1.78×104 U/mL, protein concentration 0.84 mg/mL, specific activity 2.12×104 U/mg; anti-VSV activity on F81 of the recombinant feline IFN-ω was about 3.7×105 U/mL, protein concentration 0.84 mg/mL, specific activity 4.4×105U/mg.