Preparation And Application Of Monoclonal Antibody Against CD4 Of Chinese Sucker(Myxocyprinus Asiaticus)

Chinese sucker(Myxocyprinus asiaticus)belongs to the family Catostomidae,which is a freshwater fish species in China,mainly distributed in the Yangtze and Minjiang rivers.Meanwhile,it has both ornamental and economic value because of its unique shape,gorgeous body color and delicious meat.In recent years,due to its low reproduction rate,and the impact of human activities such as environmental pollution and overfishing,the number of Chinese sucker has been gradually decreasing.Artificial breeding is an effective way to protect this species,but in the breeding process,the existence of diseases,water quality and other problems will also cause high mortality of Chinese sucker,which is not conducive to the healthy development of aquaculture.This study is based on the understanding of the structure and function of the fish immune system,and attempts to study the surface marker molecules of T lymphocytes of Chinese sucker,hoping to provide theroretical foundation for the prevention of diseases in the aquaculture process of Chinese sucker.The immune system is an important system for the body to carry out immune responses and immune functions.As the earliest lower vertebrates with a complete immune system,fishes occupy an important position in the study of the evolution of the immune system.Therefore,the research of fish immune system can provide a theoretical basis for further understanding of the evolution law of the vertebrate immune system.The immune system is divided into innate immunity and adaptive immunity,and adaptive immunity includes humoral immunity and cellular immunity,in which cellular immunity is an immune response mediated by T lymphocytes.CD4,as an important surface marker molecule of T lymphocytes,can interact with MHC class II molecules to form antigen-MHC class II molecular complexes,thereby participating in humoral immunity and causing inflammation.However,there have been no reports on T cell surface marker molecules in the study of Chinese sucker.In this paper,the transcriptome sequencing data was analyzed to obtain the CD4cDNA sequence of Chinese sucker.The open reading frame(ORF)of MaCD4 consists of 1443bp and encodes 481 amino acids.The predicted amino acid residues mass is approximately 53.89kDa and the isoelectric point is 8.88.Amino acid multiple sequence alignment results show that similar to other species,Ma CD4 consists of an extracellular region containing three Ig domains and an Ig-like domain,a transmembrane region,and a cytoplasmic region containing a conserved CXC motif.At the same time,there are several conserved sites in the extracellular region of MaCD4,such as its WTC motif,N-glycosylation sites and cysteine residues forming disulfide bonds.Phylogenetic tree showed that MaCD4 has close genetic relationship with those of Ctenopharyngodon idella,Cyprinus carpio,Carassius auratus and Danio rerio.SqRT-PCR results during the developmental period of Chinese sucker showed that the transcripts of MaCD4 could be constantly detected from the fertilized egg to 51 days after hatching,and the expression level gradually increased.Tissue distribution analysis showed that MaCD4 was expressed in all tissues examined,and the highest expression level was detected in the gill,the relatively low levels were detected in spleen,heart and PBL,and the lowest expression level was detected in muscle and skin.After stimulating Chinese sucker with inactivated Aeromonas hydrophila,the results showed that the expression levels of MaCD4 were significantly up-regulated in the spleen,head kidney and kidney at 48 h,and the changes in the spleen and head kidney were more significant;while in gills and intestines,the significant up-regulation of MaCD4 was seen at 24h.The highly antigenic CD4 extracellular protein was obtained by prokaryotic expression in E.coli BL21(DE3).The His6-tagged recombinant protein was induced by isopropyl-?-D-thiogalactopyranoside(IPTG)and purified by using Ni-nitrilotriacetic acid resin under optimal conditions,and its molecular weight was approximately36KDa.The CD4 recombinant protein was used as an antigen to immunize Balb/c female mice at 5-6 weeks of age,and the corresponding antiserum was obtained.Finally,hybridoma cells secreting monoclonal antibody against Chinese sucker CD4 was obtained by cell fusion technology and named as 8-G7B12,4-F2C3,8-G6C1 and11-F6A4.Western blotting results showed that these antibodies could specifically react with the head kidney protein extracted from Chinese sucker.The result of monoclonal antibody against Chinese sucker CD4 reacting with the peripheral blood leukocytes of Chinese sucker by indirect immunofluorescence assay(IFA)showed that the presence of CD4~+leukocytes was found in these cells.Immunohistochemical staining results showed that most positive signals were detected in the thymus and few positive cells were detected in head kidney and spleen;no positive signals were observed in the negative control.In this study,we cloned the Chinese sucker CD4 gene,detected the gene expression patterns in different tissues of Chinese sucker,expressed and purified the protein of interest,and prepared and identified anti-CD4 monoclonal antibody.The distribution characteristics of Chinese sucker CD4~+cells in related tissues were initially explored.In view of the role of CD4~+T cells in the adaptive immune system,the preparation of anti-CD4 monoclonal antibody will be expected to lay a foundation for the classification and functional study of T cell subsets in fish.