Study On Anti-RGNNV Effect Of Interferon 1 In Red Spotted Grouper (Epinephelus Akaara)

Red spotted grouper nervous Necrosis virus（RGNNV）has a wide prevalence,strong infectivity and high mortality,which seriously hinders the healthy development of Red spotted grouper breeding industry in China.Interferon（IFN）is a glycoprotein that is produced when an organism is stimulated by viruses or other substances and has antiviral effects.Interferon besides antiviral action,still have a variety of biological activity,if can inhibit tumor cell activity,regulate airbody immunity to wait.This study aims to clone Interferon 1（IFN1）gene in red spotted grouper,induce the expression of the recombinant protein by constructing eukaryotic and prokaryotic expression plasmids of IFN1,explore the antiviral activity of the recombinant protein,and verify the protective effect of the recombinant protein in living grouper.These results lay the foundation for the functional study of IFN1 in red grouper,and provide reference for the prevention and treatment of fish viral diseases.In this study,the full-length fragment of IFN1 gene（519 bp）and the mature peptide gene fragment（459 bp）were amplified,and the eukaryotic expression plasmid pc DNA4.0-IFN1 was successfully constructed,and the experimental results proved that it had significant antiviral function on the cellular level.The prokaryotic expression plasmid p GEX-4T-1-IFN1 was successfully induced to express Recombinant interferon 1（rIFN1）with a size of 43 KDa by the prokaryotic expression system.The concentration of prokaryotic recombinant protein was determined by BCA method.In cell experiments,fluorescence quantitative PCR was used to detect the expression of interferon inducible gene ISGs and the change of virus transcription level,and TCID₅₀was used to detect the change of virus titer level and Western blot was used to detect the change of virus protein level,which verified that rIFN1 had the biological activity of activating interferon signaling pathway and antiviral infection.The results of cell antiviral experiment showed that rIFN1 had significant anti-RGNNV activity in cells pretreated with 400 ng/m L rIFN1for 12 h.The rIFN1 was injected with RGNNV 24 h before infection in vivo.The rIFN1was injected 24 h after RGNNV infection.The rIFN1 was injected into three groups at 0 h after RGNNV infection.The results showed that injection of 1μg rIFN1/g（fish body weight）24 h before RGNNV infection had the most significant protective effect on grouper infected with RGNNV,and the relative protection rate was 64.3%.In addition,under the same experimental conditions,the data of the relative protection rate of 0.1μg rIFN1/g（body weight of fish）and 1μg rIFN1/g（body weight of fish）showed a significant dose dependence of rIFN1.In conclusion,IFN1 gene of red spotted grouper was amplified and identified in this study.The eukaryotic expression plasmid and prokaryotic expression plasmid were constructed.Prokaryotic expression recombinant protein rIFN1 was expressed and purified.It was proved that IFN1 had significant antiviral activity in cells and in vivo of grouper,and its activity was time-dependent and dose-dependent,which provided theoretical reference for the application of interferon in the prevention and treatment of fish viral diseases.