Expression And Function Of Zebrafish Type ? Interferon And Tumor Necrosis Factor(TNF?)

Interferon(IFN)is a high-performance,low-molecular-weight secretable protein consists of 2 subgroups.Numerous studies have shown that fish type I interferons play an important role in protecting fish from viral infection.However,the two subgroups of type I interferon have temporal and spatial differences in antiviral effects.The Group I can rapidly induce antiviral response but has a weak effect,but the Group II induces delayed but a highly effective antiviral response.There are two members in the Group I of type I interferon in zebrafish: IFN?1 and IFN?4.There are many studies on IFN?1,including the expression of IFN?1 and the function of its protein.There are little studies on IFN?4,especially in terms of its functional aspects.Up till now there are many doubts about the role of IFN?4 in protecting fish from viral infection.Tumour necrosis factor(TNF),as an important inflammatory factor,plays an important role in resisting bacterial and viral infections and clearing infected cells.In this study,zebrafish were intraperitoneally injected with an immunostimulant,bacterial and viral pathogens,and the expression of IFN?1 and IFN?4,as well as TNF? and TNFRSF1 a in kidney and spleen were analyzed by real-time PCR.At the same time,IFN?1 was used as a control to further study the biological function of IFN?4.In this experiment,IFN?1 was used as a control to assess the biological function of IFN?4.The zebrafish were intraperitoneally injected with immunostimulatory,bacterial and viral pathogens,and the expression levels of IFN?1 and IFN?4 in the kidney and spleen were analyzed by real-time PCR.The results showed that IFN?1 and IFN?4 were relatively high in the kidney,spleen and heart of zebrafish,and the expression level in the liver was extremely low.LPS and PolyI:C can regulate the expression of IFN?1 and IFN?4 genes in kidney and spleen.Different bacterial and viral pathogens were injected to zebrafish,respectively.1)After infection with A hydrophila,IFN?1 transcript in the spleen(6 h And 24 h)and kidney(24 h)was significantly elevated;IFN?4 was also significantly up-regulated in the spleen and kidney(6 h and 48 h).2)when the SVCV was infected,the expression IFN?1 was significantly increased in the 1-7 d infection;IFN?4 was only found a slight up-regulation in the spleen(7 d)and the kidney(3 d).3)After the E.tarda infection,the expression of IFN?1 and IFN?4 genes in the kidney and spleen has been down-regulated.The results showed that the zebrafish IFN?1 and IFN?4 genes play an important role in the resistance to viral and bacterial infections,and the IFN?4 antiviral effect is weaker than the IFN?1 gene.To further investigate the function of IFN?4,polyclonal antibodies to the IFN?1 and IFN?4 genes were prepared.Immunohistochemical analysis was performed on the kidney,spleen,gill and intestine of zebrafish infected with delayed E tarda and wild SVCV by injection of zebrafish with equal volume of PBS.The results showed that SVCV can induce zebrafish IFN?1 and IFN?4 protein synthesis;delayed Edwardella infection can inhibit zebrafish IFN?1 and IFN?4 protein synthesis.In order to further study the related signalling pathways involved in IFN?1 and IFN?4.IFN?1 and IFN?4 recombinant proteins were prepared by a or the prokaryotic expression,and ZF4 cells were incubated with 0.1 ng/mL,1 ng/mL,10 ng/mL,100 ng/mL,and 300 ng/mL protein concentrations,respectively.At 6 h,the activity and function of the recombinant proteins were identified by measuring the transcription level of the Mx gene.The experimental results showed that different concentrations of IFN?1 and IFN?4 recombinant proteins could significantly up-regulate the expression level of Mx antiviral gene.Based on the actual situation,ZF4 cells were incubated with 20 ng/mL IFN?1 and IFN?4 recombinant protein for 6 h to analyze the transcriptome of mRNA and miRNA.The results showed that IFN?1 and IFN?4 proteins up-regulated 966 mRNA genes and 21 miRNA genes together,down-regulating 548 mRNA genes and 24 miRNA genes,and the up-regulated and down-regulated signal pathway genes were very similar,especially on the signal pathways that were up-regulated together.After data analysis,the genes on the antiviral,interferon pathway,apoptosis,cytokines and other pathways were selected for fluorescence quantitative verification.The results were consistent with the results of the transcriptome assay.Delayed E.tarda infection in zebrafish and related immunohistochemistry experiments showed that the expression of IFN?1 and IFN?4 genes was significantly down-regulated,and the expression of IFN?1 and IFN?4 genes was significantly downregulated in order to further validate both transcript and immunohistochemistry experiments.To further verify the effects of IFN?1 and IFN?4 on E.tarda,the expression of Mx,IFN?1 and IFN?4 genes was measured by E.tarda infection of ZF4 cells for 6 h and 24 h.The protein was extracted and the expression of IFN?1 and IFN?4 protein was verified by Western blot with a polyclonal antibody.The experimental results show that E.tarda can significantly up-regulate the expression of Mx,IFN?1 and IFN?4 genes in ZF4 cells.The same batch of zebrafish samples injected intraperitoneally with an immunostimulant,bacterial and viral pathogen samples,and the transcripts of Tumor necrosis factor(TNF)and TNF receptor superfamily member1a(TNFRSF1a)were quantified.TNF? and TNFRSF1 a play important roles in fish resistance to bacterial and viral infections.