Preparation And Application Of Anti-CD3ε Polyclonal Antibody In Identification Of T Lymphocytes In Paralichthys Olivaceus

T lymphocytes play an important role in the cellular immunity and themechanism against infection of the fish. CD3is the specific surface marker of Tlymphocytes, and it takes part in signal transduction of T lymphocytes by associatedwith T cell antigen receptor (TCR). CD3can be used as a marker for T lymphocytesand thymocytes. The preparation of anti-CD3antibody will be useful for the study ofT lymphocytes because of the specificity conjunction of the antigen and antibody.In this paper, CD3protein of Paralichthys olivaceus was expressed based on thepublished gene sequence of CD3ε, and the anti-CD3polyclonal antibody which canspecifically identify T lymphocytes of P. olivaceus was prepared. In addition, theanti-CD3antibody was applied to detect the tissue distribution of T lymphocytes in P.olivaceus and the amounts change of T lymphocytes after immunization withinactivated Edwardsiella tarda. This paper laid the foundation for the furtherunderstanding the roles of T lymphocytes in the immune system and the immuneresponse mechanisms of fish. The results were as follows.The expression primers were designed based on the reported CD3genesequence of Paralichthys olivaceus and CD3gene was got by PCR. The CD3genewas cloned into pET-30a expression plasmid and the recombinant plasmids weretransformed into Escherichia coli BL21(DE3) strain. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that themolecular weight of recombinant protein was27kDa, including7kDa tag and theresult of protein mass spectrometry analysis showed the recombinant protein wasCD3of Paralichthys olivaceus. The recombinant protein was purified successfully byusing affinity chromatography and then was injected into Balb/c mouse in order toobtain polyclonal antibody. Co-immunoprecipitation of the polyclonal antibody specifically reacting withthe peripheral leukocytes of Paralichthys olivaceus found a20kDa protein could berecognized and it was proven to be CD3of Paralichthys olivaceus by protein massspectrometry analysis. The result of CD3polyclonal antibody and IgM monoclonalantibody reacting with the peripheral leukocytes of Paralichthys olivaceus by indirectimmunofluorescence assay (IFA) showed there were CD3+/IgM-、CD3-/IgM+andCD3-/IgM-leukocytes in the same view, besides CD3+leukocytes and IgM+leukocytes did not cross.Immunohistochemical staining results showed that positive red signals werepresent in the cell membranes of some peripheral blood leukocytes, gill epithelium,epidermis, lamina propria of gastric mucosa and gastric glands, intestinal epithelium,liver, heart, serosa and white pulp surrounding ellipsoids of spleen, and scatteredthroughout the tissue and the surface connective tissue of the head-kidney, and thetubular epithelium in the mid-kidney; no positive signals were observed in the gonadand negative control tissues of P.olivaceus．Flow cytometry results showed that thepercentages of CD3+T cells in the lymphocytes of peripheral blood, spleen and headkidney from P.olivaceus increased and reached the peak (12.6%,9.7%and8.7%respectively) at the fifth day after injected with inactivated E. tarda, and thendecreased gradually to the levels of the controls.