Molecular Cloning Of CD5 In Common Carp(Cyprinus Carpio) And Preliminary Study On Its Immune Response Profile From Lymphocytes

CD5 is a type Ⅰ transmembrane glycoprotein that belongs to the cysteine-rich family of scavenger receptors.In B lymphocytes,CD5 molecule can not only serve as a marker molecule for functional subsets of Bla lymphocytes,but also have an important negative regulation effect on BCR receptor-mediated signaling activation.The lack of signature molecules on the cell surface limits insights into subsets of B lymphocyte function and the mechanisms of differentiation and development,while,as the first vertebrates to produce adaptive immunity,IgM+B cells exhibit cellular characteristics similar to mammalian B1 cells.Therefore,the study of the immune response of CD5 molecules in bony fish lymphocytes and its expression localization in different B lymphocyte subsets will provide new landmark molecules for the subdivision of bony fish B cells,and will also be an important basis for the study of BCR signaling activation and its regulatory mechanisms.Gene expression and functional analysis of carp CD5 molecules were conducted from the following five aspects.First,cloning obtained 984 bp,including the complete ORF region,750 bp long,encoding 249 amino acids,with signal peptide,transmembrane domain and conserved SRCR domain,two N-linked glycosylation sites in the extracellular region,and multiple PKA,PKC and p38MAPK phosphorylation sites in the intracellular region.Second,the prokaryotic expression vector of CD5 of carp was constructed,transformed into E.coli BL21(DE3)and ROSSETA,induced expression of CD5 protein was purified,7 polyclonal antibodies and monoclonal antibodies were prepared and screened,and CD5 polyclonal antibodies C8 and C9 were determined for subsequent experiments.Third,the model of T cell-independent antigen immuno-stimulated carp injected with TNPLPS and T cell-dependent antigen TNP-KLH was constructed,Three derived lymphocytes from spleen,peripheral blood and abdominal cavity were extracted,For pression of 13 genes at 1 d,3 d,7 d and 15 d after immune stimulation,Including:the B cell differentiation-related transcription factors blimp1,pax5,irf-5,The BCR signaling complex,mIgM and CD79a,Immunoglobins sIgM,IgZ3,IgZ2,IgZ1,and the cell surface differentiation antigens CD3,CD4,CD8 and CD5.The results showed that(1)tissue-derived lymphocytes showed significant expression in TI and TD immune response,indicating the difference in cell composition and function.(2)CD5 in spleen and abdominal lymphocytes showed higher expression in TD antigen,1 d and 7 d after stimulation,the expression of peritoneal cavity lymphocytes increased significantly at 3 and 15 days after stimulation.(3)expression of mIgM and CD79a molecules showed significant positive correlation in lymphocytes of three origin,indicating that the activation of BCR signaling complex participates in the immune response process under the immunostimulatory conditions described above.As an important marker of B lymphocyte maturation and differentiation,there is a significant negative correlation between Pax5 and Blimp-1,which is consistent with the characteristics of their expression at specific stages of B cell differentiation.In addition,the expression of Pax5 was positively correlated with the expression of CD79a and mIgM,indicating that there was antigen-dependent activation of B cells in the mature B cell stage marked by Pax5,characterized by the expression of CD79a and mIgM,while in the plasma cell stage marked by Blimp-1,the expression of CD79a and mIgM decreasedFourth,lymphocyte in vitro stimulation experiments to detect the activation of peripheral blood lymphocytes and peritoneal lymphocytes under LPS and BCR crosslinking signal stimulation.Antibodies against 10 molecules associated with BCR signaling were screened and examined,including Syk,p-Syk,ZAP-70,p-ZAP-70,p-BLNK,SHP1(3244),SHP1(2406),p-Akt,MAPK,and p-MAPK,indicating that(1)IgM antibody treatment activated BCR signaling for pBLNK activation in peripheral blood lymphocytes,with elevated expression of the inhibitory molecule SHP1.IgM antibody treatment produced inhibition of MAPK signaling in peritoneal lymphocytes.(2)The above results show that in peripheral blood lymphocytes,the activation of BCR signaling complex can activate the downstream molecule BLNK,and SHP1 is a key molecule of CD5 to inhibit BCR signaling,and its elevated expression provides basic data for further analysis of the inhibitory regulation of BCR signaling in peripheral blood lymphocytes and the determination of upstream signaling.The MAPK signaling pathway is also involved in BCR signaling activation and it’s signaling processes in carp lymphocytes.Fifth,using CD5 and IgM antibodies to label peripheral blood and abdominal cavity-derived lymphocytes,the results showed that CD5 co-localized with IgM in some lymphocytes,and IgM+ CD5+ cells,IgM+ CD5-cells,IgM-CD5+ cells,and IgM-CD5-cells were present in both tissue-derived lymphocytes.Treatment of peripheral blood lymphocytes and peritoneal lymphocytes with LPS and flow test showed that LPS stimulation reduced IgM+CD5+cells from 6.7%to 3.0%and IgM+CD5’ cells from 5.1%to 10.6%;LPS stimulation increased IgM+CD5cells from 8.6%to 11.2%without the proportion of IgM+CD5+cells.In mammals,LPS can activate the proliferation and antibody secretion of B1 cell subsets in vitro.The above experiments show that for carp IgM+B lymphocytes,IgM+B cells without expressing CD5 molecules and IgM+ B cells expressing CD5 molecules have significant differences in LPS stimulation.The results provide an important basis for further investigation of the cell proliferation activity and functional differences of the two cell subsets.In conclusion,the preliminary study of CD5 gene and its immune response to lymphocytes in carp obtained monoclonal antibodies and polyclonal antibodies of CD5,which provided important technical tools for the analysis and functional study of lymphocyte subsets in common carp.The difference of immune response in carp was analyzed by different tissue-derived lymphocytes in TI and TD antigen stimulation,the role of BLNK and MAPK signaling in the BCR signaling complex,IgM+CD5+cells and IgM+ CD5-cells,and the effect of LPS in vitro stimulation on different cell subsets.This experiment laid a foundation for further study of B lymphocyte differentiation and immune function in carp,help to further clarify the molecular mechanism of antibody production in carp,and provide theoretical basis for the selection of immune prevention strategies during breeding.