Expression And Application Of Recombinant Goose Interferon-?

China is a big country for raising goose,and our country's breeding industry occupies a major position in the world.Compared with chickens,ducks and other industries,China's goose industry is an emerging industry,and it is also an advantageous industry compared to foreign countries.Therefore,it is not only necessary to improve large-scale feeding and management,but also to increase the prevention and control of goose epidemic diseases,so as to build China's goose industry into a leading industry in the world.It is of great significance for the sustainable development of goose industry in China to research and prepare biological agents which can improve goose immunity.Goose interferon-?is a cytokine secreted in the body,with good functions such as antiviral and immune regulation,and belongs to a broad-spectrum antiviral agent.In this study,the full-length gene fragment(576bp)and mature peptide gene fragment(498bp)of goose interferon-?with high antiviral activity were amplified from goose blood by RT-PCR.Use the pMD18-T vector to link the full-length goose interferon-?gene to construct the recombinant vector pMD18-T-GoIFN-?.Sequencing and bioinformatics analysis.The results show that the homology of GoIFN-?amplified in this research is as high as 99.8%with HQ11583,and the homology with mammals is low at 41.4?45.8%.The successfully amplified goose interferon-?mature peptide gene was double digested with pET32a(+)vector to construct the prokaryotic expression recombinant plasmid pET32a-mGoIFN-?.Transform the recombinant plasmid into competent cells E.coli BL21(DE3),and after IPTG induced expression,a 38KDa recombinant fusion protein was obtained,After the protein was purified by the urea gradient method,the results of SDS-PAGE electrop Horesis showed that the expressed protein was purified as a single target band.And through Western-blot identification,the results were consistent with expectations.Select the pFast Bac-HT A vector,and double digest the vector with the full-length goose IFN-?gene to construct the baculovirus expression recombinant transfer plasmid pFast Bac-HT A-GoIFN-?.Transpose the recombinant plasmid into DH10Bac competent cells to construct the recombinant bacmid plasmid Bacmid-GoIFN-?,and the recombinant bacmid plasmid was transfected into sf9 insect cells to prepare a recombinant baculovirus.A large amount of recombinant baculovirus was amplified and the virus fluid was purified by a nickel column and then identified by SDS-PAGE electrop Horesis and Western-blot,The baculovirus expression recombinant fusion protein was successfully obtained.The antiviral activity of the recombinant fusion protein obtained in two different ways was detected by the cytopathic inhibition method.Determination of the concentration of prokaryotic expression of recombinant proteins is 1.2mg/m L,anti-VSVactivity is1.94×10³U/m L,specific activity of 1.61×10³U/mg,anti-GPV activity of 1.66×10³U/m L;Determination of the concentration of baculovirus expression of recombinant protein is2.01mg/m L,anti-VSV activity was 4.46×10³U/m L,specific activity of 2.21×10³U/mg,anti-GPV activity of 3.8×10³U/m L.The recombinant proteins obtained by the two different expression methods have good antiviral effects.The antiviral activity of the recombinant protein expressed by baculovirus is higher than that of the recombinant protein expressed by prokaryotic expression,which lays the foundation for further research on the anti-GPV proliferation effect of recombinant goose interferon alpha in goslings.The 192 1-day-old healthy goslings were randomly divided into 8 groups,with 24 in each group.1×10³U,3×10³U,5×10³U pET32a-mGoIFN-?prokaryotic interferon-expressing interferon and Bacmid-GoIFN-?baculovirus interferon-expressing interferon were selected as the experimental groups.simultaneously,a infection control group that only injected GPV and The normal control group was injected with normal saline.Group1?6 were the experimental groups.1×10³U,3×10³U,5×10³U prokaryotic interferon and baculovirus-expressing interferon were injected into the leg muscles at the age of 1 day,GPV(TCID₅₀=10^-4.6/0.1m L)0.8m L was injected into the leg muscle at 2 days of age;group 7 was the infection control group;group 8was the normal control group injected with 0.8m L sterile normal saline.Observe for 21 days under the same feeding conditions,record the survival of each group of goslings and 3d,6d,9d,12d,15d,20d after inoculation with GPV virus,randomly select 5 from each group,collect cloaca cotton swabs to test the detoxification status of the goslings.The results showed that different doses of prokaryotic expression of interferon and baculovirus expression of interferon can inhibit the proliferation of GPV,increase the protection rate of goslings and reduce the rate of detoxification.3×10³U Bacmid-GoIFN-?has the strongest inhibitory effect on GPV in goslings.And the same dose of baculovirus expressing interferon inhibits the proliferation of GPV than the same dose of prokaryotic interferon.In summary,In this study,the recombinant goose interferon alpha obtained by the prokaryotic expression system and the baculovirus expression system has high expression and good antiviral activity.Provide certain technical support for the application of goose interferon in the prevention and control of goose viral diseases and the acquisition of new preparations for waterfowl diseases.