Cloning, Prokaryotic Expression And Antiviral Bioactivity Analysis Of Coturnix Interferon-α And Interferon-γ

In China, coturnix is an important economic animal whose breeding amount ranks the first in the world, however some potent viral diseases, such as avian influenza, newcastle disease are potential threats to coturnix scale farming. Interferons are named for their ability to "interfere" with viral replication by protecting cells from virus infections. Interferons have antivirus, immune regulation and other functions, which are proved to be an ideal antiviral biological agent.In this study, coturnix IFN-a fragment was obtained from genome by PCR. The total RNA was isolated from coturnix peripheral blood mononuclear cells stimulated by ConA, and transcribed to cDNA by RT-PCR, the IFN-y fragment was amplified. Then coIFN-α、coIFN-γ were cloned to pEASY-Blunt cloning vectors. The results of sequencing showed that the open reading frame of coIFN-a was consisted of 594bp, encoded a protein of 197 aa, the open reading frame of coIFN-y was consisted of 495bp, encoded a protein of 164 aa. Constructed prokaryotic expression vectors pET-32a-coIFN-a, pET-32a-coIFN-y and induced to express in Escherichia coli. The results of SDS-PAGE showed that the recombiant proteins were 43.0 kDa and 40.0 kDa approximately, mainly expressed in inclusion bodies. Purifyed and refolded coIFN-a and coIFN-y, rabbit anti-coIFN-α、anti-coIFN-γ polyclonal antibodies were obtained by purified proteins. The activities of coIFN-α、coIFN-γ recombinant proteins were detected by anti newcastle disease virus, avian influenza virus, vesicular stomatitis virus in chicken embryo fibroblasts. Calculated the antiviral effect indicated coIFN-a antiviral activity was about 104U/mg; coIFN-γ antiviral activity was about 103U/mg, the maximum value was 104 U/mg. These antiviral activities were abrogated by rabbit anti-coIFN-a, anti-coIFN-y antibodies in vitro, which verify coIFN-a, coIFN-y have biology activities inhibition the multiplication of newcastle disease virus, avian influenza virus, vesicular stomatitis virus.Coturnix IFN-a and IFN-y genes were cloned and expressed in E. coli successfully, its anti-viral effects, physical and chemical properties were studied, which provided physical materials for biological characteristics study and theoretical basis for coturnix antiviral biological agent production.