Study On Antibacterial And Antiviral Function Of IFNf Of Chinese Sturgeon(Acipenser Sinensis)

Type Ⅰ Interferon is an important cytokine in vertebrates,which is mainly involved in antiviral immune response.The function of type I interferon in bony fish is relatively conservative compared with that in mammals,which can activate antiviral signaling pathways and participate in antiviral response.However,studies have shown that type I interferon in bony fish can significantly improve the antibacterial ability of fish,showing direct bactericidal ability.At present,studies on the antibacterial and antiviral function of fish type Ⅰ interferon are relatively lacking.In this study,we conducted a preliminary study on the antibacterial and antiviral function of Chinese sturgeon IFNf.The main results are as follows1.Clone identification and expression pattern analysis of IFNf in Chinese sturgeonThe c DNA sequence of IFNf was screened in the transcriptome of Chinese sturgeon,and specific primers were designed to amplify the IFNf sequence from the c DNA template of spleen cells of Chinese sturgeon for sequencing.After phylogenetic tree analysis and multiple sequence comparison,the IFNf sequence of Chinese sturgeon was found to be clustered into one branch with spotted gar,showing the highest homology.At the same time,we analyzed its gene structure and protein structure and found that,as a member of Group Ⅱ,Chinese sturgeon IFNf only contained a pair of disulfide bonds,which existed in the second and fifth exons,and the structure of disulfide bonds was different from that of Group Ⅰ interferon members.This disulfide bond pattern was different from the interferon of bony fish previously found.The spleen primary cells of Chinese sturgeon were incubated with immune stimulant poly I:C and LPS,and IFNf gene could be upregulated by poly I: C.2.Chinese sturgeon IFNf protein has direct antibacterial effectAlphafold software was used to predict IFNf protein structure of Chinese sturgeon.It was found that the three-dimensional structure of Chinese sturgeon IFNf protein was conservative,mainly composed of 5 α helix domains,and cations were mainly concentrated in α4 domain.It was found to have antibacterial activity.In order to verify the antibacterial effect of IFNf from Chinese sturgeon,we constructed prokaryotic expression vectors p Cold-sumo-IFNf△α4,synthetic IFNfα4 polypeptide(PRLRKYFKTLRTFLRRK),and obtained soluble recombinant protein.The expressed IFNf protein,IFNf△α4 and synthetic IFNfα4 polypeptide were incubated with Escherichia coli and Staphylococcus aureus for 20 min and fixed with 2.5%glutaraldehyde.Scanning electron microscopy(SEM)was performed.It was found that the IFNf protein and IFNfα4 polypeptide had destructive effects on the cell membrane of the two bacteria.The absence of the α4-domain IFNf protein did not cause damage to the cell membrane of the two bacteria.To understand the effects of IFNf and IFNfα4 peptides on clinical bacteria,IFNf recombinant protein and IFNfα4 peptides were incubated with Citrobacter fredii labeled with GFP for 20 min.It was found that IFNf recombinant protein and IFNfα4 polypeptide could reduce the number of Citrobacter fredii.3.The IFNf protein of Chinese sturgeon has a high antiviral abilityThe expression of IFNf gene could be significantly up-regulated by cyprinus spring virus by injecting infected SVCV,which may be involved in the antiviral immune process.And we constructed a SVCV infected Chinese sturgeon fin ray cell model and compared the antiviral ability of IFNf and IFNe2 of Chinese sturgeon.The recombinant expression plasmid p Cold-sumo-IFNe2 was constructed and the recombinant IFNe2 protein was expressed.Fin ray cells were incubated with IFNf and IFNe2 recombinant proteins for 4h,and the downstream viperin and Mx gene expressions were detected.The effects of IFNf and IFNe2 on the resistance of Chinese sturgeon fin ray cells to virus infection were detected by virus infection experiment.By virus infection experiment,IFNf and IFNe2 were tested on the resistance of fin cells to cyprinus spring virus infection.The results showed that both IFNf and IFNe2 proteins could improve the resistance of fin fin cells to cyprinus spring virus infection,and q PCR results showed that IFNf and IFNe2 could induce the up-regulated expression of downstream antiviral genes Mx and viperin...