| Chinese sea bass(Lateolabrax maculatus)is an important economic fish in China,with an annual production of more than 160000 tons,ranking second in mariculture fish.With the promotion of intensive and high-density culture model,fish viral diseases seriously affect the development of sea bass aquaculture industry.Basic research is particularly important to solve production problems,and much attention has been paid to the immune mechanism of sea bass.Interferon is a kind of cytokine with antiviral activity,which plays a variety of roles by inducing a large number of IFN stimulating gene(ISGs).In this study,sea bass was used as the research object to clone the sequence of type I interferon IFNd(LmIFNd)and its receptor(LmCRFBs)c DNA,and induce the recombinant proteins;The functional characteristics of LmIFNd-His recombinant protein were analyzed in vivo and in vitro.Pull-down technique was used to analyze the interaction between LmIFNd and LmCRFB1,LmCRFB2 and LmCRFB5 in vitro,and the effects of different ligand-receptor combinations on the expression of downstream ISGs gene were analyzed.The specific research results are as follows:(1)LmIFNd,LmCRFB1,LmCRFB2 and LmCRFB5 c DNA sequences were obtained by RACE technology,and their sequence characteristics and evolutionary status were analyzed.The total length of LmIFNd c DNA is 1190 bp,of which the length of 5’UTR is 354 bp,the length of 3’UTR is 273 bp,and the length of open reading frame is 558 bp,encoding a total of 185 amino acids.The molecular weight of the protein is21.9 KD,and the theoretical isoelectric point is 6.35.The total length of LmCRFB1 c DNA is 2730 bp,5’UTR and the length of 3’UTR is 871 bp.ORF encodes 529 amino acids.The predicted molecular weight of the protein is 58.14 KD,theoretical isoelectric point is 4.35.The length of LmCRFB2 ORF is 1572 bp,which encodes 523 amino acids.The total length of LmCRFB5 c DNA is 1542 bp,5’UTR,the length of 3’UTR is 99 bp,and the length of ORF is 1272 bp.The predicted molecular weight of the protein is47.21 KD,and the theoretical isoelectric point is 5.84.Functional domain prediction found that LmIFNd protein contains an extracellular IFabd domain,which can provide binding sites for cytokine receptors.The three receptor proteins of LmCRFB1,LmCRFB2 and LmCRFB5 can be divided into extracellular domain,transmembrane domain and intracellular domain.The typical FN3 domain of cytokine family can be predicted,and the interferon binding domain can also be predicted in the extracellular domain.(2)The results of real-time fluorescence quantitative PCR(q RT-PCR)showed that the expression of LmIFNd was the highest in the head kidney,followed by gill and spleen;the expression of LmCRFB1 in blood cells was the highest,followed by gill and intestine;the expression of LmCRFB2 was the highest in the head kidney,followed by spleen and gill;the expression of LmCRFB5 was the highest in spleen,followed by head kidney and gill.After being stimulated by polyinosine monophosphate(Poly I:C),frog virus(RGV)and infectious spleen and kidney necrosis virus(ISKNV),the expression of LmIFNd was up-regulated in the immune tissues of Gill,head kidney and spleen.It is speculated that LmIFNd is involved in the immune process of sea bass virus infection and may play an important role.(3)The recombinant LmIFNd-His protein was obtained,and the function of the recombinant protein was analyzed in vivo and in vitro.In the experiment in vitro,the transcription of downstream ISGs genes(IFIT,ISG15,Viperin)was activated after 6hours incubation with recombinant protein LmIFNd-His,and the cells were incubated with different concentrations of recombinant protein LmIFNd-His and inoculated with ISKNV virus 12 hours later,it was found that the cell activity increased gradually with the increase of the concentration of recombinant protein LmIFNd-His,indicating that the recombinant protein LmIFNd-His has a protective effect on ISKNV infected cells.In vivo experiments,recombinant protein LmIFNd-His was intraperitoneal injection juvenile sea bass,the transcription of downstream ISGs genes(IFIT,ISG15,Viperin)was activated 12 h later;Juvenile sea bass were infection with ISKNV,6 hours after injection of PBS and recombinant protein,respectively.It was found that LmIFNd-His recombinant protein could significantly reduce the copy number of ISKNV virus in head kidney tissue.(4)The recombinant extracellular domain proteins(LmCRFB1-GST,LmCRFB2-GST and LmCRFB5-GST)of type I interferon receptor of sea bass with GST tag were induced by prokaryotic expression technique,and the interaction between LmIFNd-His recombinant protein and LmCRFB1-GST,LmCRFB2-GST and LmCRFB5-GST recombinant protein was analyzed by Pull-down in vitro.The results showed that LmIFNd-His recombinant protein could bind to the three receptor extracellular domain proteins in vitro.In order to further determine the relationship between IFNd and different receptors and its induction effect on downstream genes,LmIFNd and different receptor combinations were cotransfected into FHM cells.The results showed that both LmIFNd+LmCRFB1+LmCRFB5 combination and LmIFNd+LmCRFB2+LmCRFB5 combination could induce the transcription of ISGs gene in FHM cells,and it was found that LmIFNd+LmCRFB1+LmCRFB5 combination had a higher induction effect.It is speculated that LmIFNd may give priority to LmCRFB1+LmCRFB5 receptor complex for signal transmission. |
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