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In this study, the antigenic site B/C of S gene of transmissible gastroenteritis virus (TGEV) was cloned into prokaryotic expression vector pET-28a and the recombinant plasmid was transferred into E.coli BL21 (DE3). The fusion protein was expressed after induction with IPTG. The SDS PAGE purified protein was used as immunogen. Six-week-old BALB/c mice were immunized three times with recombinant protein. The hybridoma cell strain, designated 5G, steadily secreting monoclonal antibodies (McAbs) against the protein of site B/C, was obtained by hybridoma technique. It belonged to IgM subclass and could recognize the site B/C recombinant protein of S gene of TGEV. It was confirmed that the McAbs only could react with TGEV using indirect immunofluorescence assay (IFA) and indirect ELISA, indicating the McAbs had good specificity.