| Nile tilapia(Oreochromis niloticus)is one of the most important commercial fish cultured in the world.In recent years,with the expansion of aquaculture scale in China,tilapia virus and bacterial diseases occur frequently,causing serious economic losses,which seriously affects the development of the aquaculture industry.Therefore,the study of fish immune mechanism has become an important breakthrough in solving these diseases.CD22 is a member of sialic acid-binding immunoglobulin-like lectin(Sigles)family,which is a type I transprotein.The gene was expressed on mature B cell membrane.The intracellular domain of CD22 includes immune receptor tyrosine inhibitory motif(Immunoreceptor tyrosine-based inhibition motifs(ITIM)and immune receptor tyrosine activated motif(ITAM)).CD22 is an adhesion signal molecule,which is related to the interaction between cells and transduction of B cells.In this study,a CD22 gene(On-CD22)from Oreochromis niloticus(O.niloticus)was cloned,and its expression characteristics and function were investaged.The results are as follows:1.On-CD22 was cloned according to published information on NCBI(denoted as On-CD22)(Gen Bank accession number:LOC102077587;On-CD22).The open reading frame of On-CD22 was 1071 bp,encoding 356 amino acids.The theoretical molecular weight was 39.35 k Da and the isoelectric point was 6.46.On-CD22 contains two IG domains and one IGC2,belonging to the IG domain protein superfamily.Multiple sequence alignment showed that the homology of CD22 protein between tilapia and other species was 31.07 %-96.53 %.It has the highest homology with Oreochromis aureus.Phylogenetic analysis showed that On-CD22 was closely to Oreochromis aureus CD22.2.Real-time fluorescence quantitative PCR analysis showed that On-CD22 was distributed in various tissues of healthy tilapia,with the highest expression in the head kidney and peripheral blood,followed by brain,spleen,intestine,liver,thymus,muscle and gill.The expression level in the skin is the lowest.After challenged with Streptococcus agalactiae,the expression level of On-CD22 is significantly up-regulated in ten tissues including head kidney,peripheral blood,brain,spleen,intestine,liver,thymus,muscle,gill and skin.This results suggest that On-CD22 may be involved in the body’s immune response.3.In order to study the effect of CD22 on the intracellular signaling pathway,a eukaryotic overexpression vector pc DNA3.1-CD22 of this gene was constructed.Double enzyme digestion detection and sequencing analysis showed that the inserted fragment was correct,and pc DNA3.1-CD22 Introduced into HEK293 T cells for expression,Western blot analysis showed that the size was as expected.Subsequently,pc DNA3.1-CD22 and p RL-TK plasmid were co-transfected into HEK293 T cells,and the collected samples were subjected to dual luciferase report detection.It was found that overexpression of CD22 can significantly enhance the transcriptional activity of NF-κB.IFN1 and IFN3 and STAT1 transfect FHM cells,CD22 inhibits IFN1 and IFN3,and activates STAT1.The above results lay a foundation for the subsequent study of On-CD22 function and further exploring the important role of the immunoglobulin-like lectin family in the innate immunity of teleost fish. |
