Prepartion And Application Of Monoclonal Antibodies Against Goose CD3ε Chain Extracellular Domain

CD3is one of the most important cell surface markers of T lymphocytes. CD3complex play an important role in signal transmission of antigen recognition. CD3is an important indicator of detection in the study of many related immune diseases. Specific monoclonal antibody anti-goose CD3molecule can be used for measuring and sorting goose CD3+T cells in the body, thus understand the function of cellular immune function in the goose body, for the further study of cellular immune mechanism.Firstly, in this study, the primers for PCR amplication were designed according to public Mallard duck reference sequence in GenBank. Then we obtained goose CD3ε gene by RT-PCR successfully. Then the character of sequence cloned here was analyzed, such as the similarity of amino acid sequence, predication of signal peptide, glycosylation sites, construction of phylogenetic tree, which indicates the sequence cloned here was belong goose CD3s gene.Secondly, according to goose CD3ε ORF sequence, specific primers were designed to amplify the goose CD3ε extracellular region gene, and cloned it into the eukaryotic expression vector pcDNA3.1(+). Transfected the eukaryotic expression plasmid into chicken embryo fibroblasts, and indirect immunofluorescence test confirmed that goose CD3ε extracellular protein was produced by eukaryotic expression system successfully. Simultaneously, the extracellular region gene was cloned into prokaryotic expression vector pET-28a (+), and we produced recombinant goose CD3ε extracellular region protein in E. coli Rosetta (DE3) pLysS. The recombinant protein was purified by Ni-NTA column affinity chromatography. And the PAb against goose CD3ε extracellular domain was prepared with rGoCD3s.At last, to prepare monoclonal antibodies against goose CD3ε extracellular domain, the recombinant plasmid pcDNA3.1-CD3was used as immunogen to immunize intramuscularly BALB/c mice. The immune dose was100μg per mouse, and the interval between two immunizations was2-3weeks.5days after the last immunization, splenocytes from immunized mice were fused with SP2/0myeloma cells. Using recombinant protein as detecting antigen, the supernatant of hybridoma clones were screened by indirect ELISA. Simultaneously, using the whole-cell ELISA mothod assisted selection. Seven hybridoma cell lines secreting McAbs against GoCD3e were obtained, and named as1G1,2C1,3C5,3C11,5G2,5A3and9F9. The immunoglobulin subclasses of1G1and5G2were IgGl,5A3was IgG2b,9F9was IgG3,2C1and3C5and3C11were IgM. The ELISA titers of these McAbs ascites were1:3200and1:1:3267800respectively. Western-blot analysis of the seven McAbs against rGoCD3s further confirmed that they only recognized the14kDa protein of rGoCD3ε. The results of IFA demonstrated that7McAbs could react with pcDNA3.1-CD3transfected CEF cells. All these results suggested that these McAbs were specific to goose CD3ε extracellular domain.Flow cytometric analysis showed that the ascites of3C11and5A3strain could identify peripheral blood T lymphocytes and the experimental group showed a more pronounced positive peak. The fluorescence signal was detected on the cell membrane.McAbs developed in this study provide a foundation for the study of avian immune cell function, immune regulations, and mechanisms of preventing and controlling poultry infectious diseases.