| 【Objective】The study was carried out to prepare the monoclonal antibodies（MAb）of S10-segmentcoded protein against Tilapia Lake Virus（TiLV）.【Method】A recombinant expression plasmid pET32a -S10 that could efficiently express the TiLV S10 gene segment was constructed and it was converted into BL21（DE3）competent cells, which were induced by IPTG expression and purified by nickel column to obtain recombinant S10 protein with high purity. Female BALB/c mice aged 6-8 weeks were immunized with purified S10 protein to fuse their spleen cells with SP2/0 cells to obtain hybridoma cells. Two hybridoma cell lines that could steadily secrete anti-S10 protein MAb were screened by finite dilution and ELISA method. The hybridoma cells were named 2C3 and 2E3 respectively, and their specificity, subtype and titer were analyzed.【Result】Western blot results show that both 2C3 and 2E3 can recognize S10 recombinant protein and TiLV. Indirect immunofluorescence assay（IFA）results show that 2C3 and 2E3 only react positively with TiB cells infected with TiLV. Subtype detection results show that 2C3 antibodies are IgG1/к and 2E3 antibodies are IgG2a/к. Indirect ELISA shows that the titers of the two MAbs are 1:12 800 and 1:51 200, respectively.【Conclusion】The anti-TiLV-S10 protein MAb is specific and highly effective, which can provide materials and support for the development of TiLV vaccine, the establishment of immunological methods and the study of S10 protein function. |
