| Chinese suck(Myxocyprinus asiaticus),as the only representative of the family Catostomidae in Asia,holds important research value in taxonomy and animal geography.Meanwhile,Chinese sucker is a freshwater species of high commercial value because of its delicious meat and beautiful body color.However,the resources of Myxocyprinus asiaticus have dramatically declined due to some anthropogenic factors such as overfishing and water pollution,and its low fertility and long development time.Therefore,Myxocyprinus asiaticus was listed as second class national protected animal in China.Artificial propagation and release is an important way to restore its resources,but due to the high density of artificial breeding,the disease of Chinese suck increases and causes higher mortality.The immune system plays an important role in immune response.In this study,research on the structure and function of Chinese suck immune system is helpful to provide theoretical basis for the problems encountered in the artificial breeding of Chinese suck.In immune system,T lymphocytes are mainly responsible for cell-mediated immunity,either by modulating the host’s immune response through the action of T helper cells,or by directly destroying cells infected with an intracellular pathogen through the action of cytotoxic T-cells.Therefore,T cells play an important role in cellular immunity and mechanism against infection of fish.CD3 molecules,as specific surface markers of T lymphocytes,take part in T cell signal transduction by associated with T cell antigen receptors.So CD3 could be used as a molecule marker to detect T cells.The preparation of monoclonal antibodies which can be used for sorting and determination of CD3~+T lymphocyte populations is helpful for understanding the function and status of Chinese sucke cell immunity,and provides a certain theoretical basis for the pathogenesis of Chinese suck.There are five subunits of CD3 molecule,which areε,δ,γ,ε,andδ.Different CD3subunits form multimers and exist on the surface of T lymphocytes.Since CD3molecule contains twoεchains which can provide abundant antigen binding sites.On the other hand,the CD3εchain is relatively conserved,so CD3εwas used for cloningand expression in this paper.In this study,the Chinese sucker CD3ε(designated MaCD3ε)gene was clonedusing RT-PCR and RACE(rapid amplification of cDNA ends)techniques.The MaCD3εis 1317bp in length,including 5’non-coding regions of 157bp and 3’non-coding regions of 653bp.The open reading frame(ORF)of MaCD3εconsists of 507 bases encoding a protein of 168 amino acids.The putative amino acid residues have an estimated molecular mass of 18.52 kDa and an isoelectric point of 7.76.Amino acid sequence alignment results showed that the MaCD3εsequence,like other aligned species,was composed of a signal peptide region,an extracellular region,a transmembrane region,and a cytoplasmic tail region rich in immunoreceptor tyrosine-based activation motif,and has conserved structures such as the CxxCxE motif and negatively charged residues across the membrane.Phylogenetic analysis showed that MaCD3εwas most closelyrelated to CD3εin other cyprinid fishes.Tissue expression profile of MaCD3εdetermined by RT-PCR showed that MaCD3εwas expressed higher in spleen,head kidney,kidney,PBL,heart,gill,intestine,brain,skin,and muscle,and lower expression in liver.Upon induction by A.hydrophila,MaCD3εexpression is significantly down-regulated in spleen、kidney and gill as compared to PBS injected fish at 12h.However,at 24h,the expression of MaCD3εmRNA in spleen was significantly up-regulated,while it was continued to decline in the kidney and gills,and finally recovered to levels similar to control.Meanwhile,MaCD3εexpression in head kidneys and intestine showed a significant up-regulation trend at 12h.The extracellular region except signal peptide of MaCD3ε(designated MasCD3ε)was amplified and inserted into prokaryotic expression vector PET-32a to form a recombinant plasmid.Then transform the recombinant plasmid into E.coli BL21(DE3).After induction expression and purification,the recombinant MasCD3εprotein(rMasCD3ε)was obtained.SDS-PAGE and western blot results showed that the molecular weight of rMasCD3εwas about 27KD,mainly expressed in the form of inclusion bodies.Monoclonal antibody against rMasCD3εnamed 3-E6B8、4-F6C11 and3-G1E9 were prepared by immunizing 6-8 week old Balb/c female mice.The antibody specificity was detected by western blotting and the results indicated that the antibody could react with spleen,head kidney and kidney protein extracts of Chinese sucker.Finally,monoclonal antibodies 4-F6C11 was applied by immunofluorescence and immunohistochemistry.Immunofluorescence results showed that the antibody 4-F6C11can specifically recognize the positive cells in the peripheral blood leukocytes of Chinese sucker.Immunofluorescence results demonstrated that a yellow-brown positive signal was observed in the 1-month-old thymus of Chinese sucker.In this study,to gain further insight into the evolution of CD3εmolecule,wedescribe here the molecular cloning,the bioinformatics analysis,expression pattern andfunctional analysis of CD3εgene from Chinese sucker(Myxocyprinus asiaticus).Ourresults would deepen the understanding on fish immune system and provide a powerful tool for the immunodetection of Chinese sucker.Meanwhile,the preparation of CD3monoclonal antibody also lays a theoretical foundation for the defense of Chinese sucker disease and the development and application of vaccines. |
