ISOLATION AND IDENTIFICATION OF T-LYMPHOCYTES FROM PERIPHERAL BLOOD AND IMMUNE RESPONSE ANALYSIS OF TCRβ CHAIN GENE IN CYNOGLOSSUS SEMILAEVIS

The density gradient method was used to isolate lymphocytes from peripheral blood of Cynoglossus semilaevis, and the isolated lymphocytes were cultured in complete DMEM medium (with 0.3 μg/mL PHA) for 3—4 days. Peripheral blood lymphocytes were separated to non-adherent lymphocytes and adherent lymphocytes using self-made nylon hair column. Non-adherent lymphocytes specifically bind with mouse anti-human FTIC-CD3 monoclonal antibody, while adherent lymphocytes specifically bind to anti-human FTIC-CD19 monoclonal antibody.TCRβ specifically expressed on non-adherent lymphocytes. Taken together, non-adherent lymphocytes were confirmed as T lymphocytes. The results of qRT-PCR showed that TCRβ was expressed in all detected 13 tissues with high expression level in the intestine, stomach, spleen, head kidney and the lowest level in muscle. The expression of TCRβ gene was significantly induced in the liver, spleen and gill with peak level at 72—96h afterV. Anguillarum infection. These results indicate that TCRβ gene plays an important role in adaptive cellular immune response.