Structural And Functional Analysis Of Type Ⅰ IFN And Receptor CRFB5 In Barbel Chub(Squaliobarbus Curriculus)

Barbel chub(Squaliobarbus curriculus),commonly known as wild grass carp,possesses strong anti-GCRV ability.Interferon(IFN),as an immune hub in fish,plays an important role in antiviral,antibacterial and immunomodulatory processes of the organism.The structural characteristics,taxonomic status and expression characteristics of IFN in GCRV resistance process are of great significance to the study of the antiviral mechanism of barbel chub.In this study,two type Ⅰ IFNs(named ScIFN1 and ScIFN2)and cytokine receptor 5(named ScCRFB5)were cloned and identified,and their molecular structures,expression patterns,induction pathways and functional properties were explored,with the aim of clarifying the anti-GCRV mechanism of two type Ⅰ IFNs and providing a theoretical basis for future studies on the immune mechanism differences between grass carp and barbel chub.The main findings of this paper are as follows.1.Structural and expression characteristics of ScIFN1,ScIFN2 and ScCRFB5 genesThe full-length c DNAs of ScIFN1,ScIFN2 and ScCRFB5 were 1200,726 and 1542 bp,respectively.And the lengths of ORFs were 543,552 and 1152 bp,encoding 180,183 and383 amino acids.Blast comparison results showed that the similarity between two type ⅠIFNs and the corresponding IFN in grass carp were 100%,while the similarity between ScCRFB5 and CRFB5 of grass carp was as high as 86.95%.The clustering analysis and multiple sequence comparison result showed that ScIFN1 belonged to group Ⅰ of fish typeⅠ IFNs and ScIFN2 belonged to group Ⅱ,while the phylogenetic tree of ScCRFB5 indicated that ScCRFB5 was more closely related to IFNAR1 than IFNAR2,which was presumed to be the short receptor chain of the IFN receptor.Tissue distribution patterns results showed that the three genes were expressed in all 10 tissues examined(liver,spleen,kidney,head kidney,gill,muscle,intestine,skin,heart and brain)and the expression pattern of ScCRFB5 was consistent with that of ScIFN1 in healthy tissues.After GCRV stimulation,the expression levels of ScIFN1 and ScIFN2 were differentially upregulated in all three tissues(gill,liver and head kidney)examined,indicating that both were involved in the anti-GCRV response of the organism.ScIFN2 was down-regulated in gill tissues during the pre-infection period,suggesting that there is also a negative regulatory mechanism of the IFN system in barbel chub and that ScIFN1 and ScIFN2 appear to be complementarily expressed at different stages of viral infection.The expression level of ScCRFB5 were up-regulated to varying degrees in all four tissues examined,suggesting that ScCRFB5 is involved in the anti-GCRV response of the organism.In addition,the trend of expression changes of ScCRFB5 was roughly similar to that of ScIFN1.2.Identification of the induction pathways and exploration of the stimulation function on downstream genes of ScIFN1 and ScIFN2.The induced expression experiments in cell showed that the expression of ScIFN1 and ScIFN2 were positively correlated with TLR7,and the correlation coefficients were 0.936 and 0.943,respectively.But there was no significant correlation between the two and MDA5.The vivo induction experiments showed that in the liver and kidney,the expression of the two ScIFNs did not show a significant correlation with MDA5 and TLR7.It showed that in a single cell line,the expression of ScIFN1 and ScIFN2 was positively regulated by TLR7,but the regulatory relationship in organism was more complicated and requires further exploration.The overexpression challenge results showed that the overexpression of IFN could significantly upregulate its own expression in the cells,but the other IFN in the cells was not upregulated or even inhibited.It is speculated that the active upregulation of IFN may result in intracellular immune overload.To avoid this adverse effect,the cells are forced to suppress the immune response of another IFN.Downstream gene expression level results showed that overexpression of ScIFN1 and ScIFN2 had little effect on the expression of ScCRFB5 and STAT1 in cells,but induction of the Mx showed significant differences.In Sc F overexpressing ScIFN1,Mx was only significantly upregulated at 24 h of GCRV stimulation,while in Sc F overexpressing ScIFN2,Mx was significantly upregulated at the first three time points,indicating hat ScIFN2 has a stronger ability to induce the expression of Mx.The above findings lay the foundation for future studies on the differences in the anti-GCRV mechanisms of IFN systems between grass carp and barbel chub.