Preparation Of Monoclonal Antibody And Identification Of Novel Antigen Sites Against3D Protein Of Foot-and-mouth Disease Virus

To identify the epitope in3D protein of FMDV, the monoclonal antibody against3D was prepared which was screened by FMDV and expressed3D protein. Eventually,The hybridoma cell line(5G2) which secreted stable monoclonal antibody against3Dprotein has been obtained and the epitope was identified, the experiments were asfollow:1. Prokaryotic expression of3D Protein: Recombinant plasmid pET-28a-3Dwas constructed, transformed into the E.coli BL-21(DE3) which was further used inthe induced expression of the recombinant protein with IPTG. SDS-PAGE andWestern-blot results showed that the protein was expressed in E.coli as soluble afterthe induction of IPTG and showed a good reactogenicity with the positive serum ofguinea pigs against FMDV.2. Preparation and identification of the monoclonal antibody against3Dprotein of FMDV: The3-4weeks BABL/c mice were immunized with recombinantserotype A FMDV, and its spleen cells were fused with myeloma cells Sp2/0usingPEG1500. Eventually, one strain of monoclonal antibody to3D named5G2was gotafter screened with indirect ELISA and limited dilution method. After Giemsa stain,the number of chromosome was102to106. Determined by indirect-ELISA, the titerof5G2in supernant was1:1280, while the titer in ascite was1:25600, and the subtypeof the monoclonal antibody was IgM. After purified with saturated ammonium sulfate,the heavy chain (53KD) and the light chain (22KD) were got. The results ofIndirect-ELISA and indirect immunofluorescence assay showed that the antibodycould react with nonstructural protein3D, recombinant serotype A FMDV, serotype AFMDV, serotype Asia1FMDV and serotype O FMDV, but could not react withSVDV and VSV.3. Identification of recognition sietes of monoclonal antibody against3Dprotein: To mapping the epitope, the gene of3D was divided into four overlapfragments:3D1（315bp）,3D2（498bp）,3D3（474bp）, and3D4（288bp）, whichwere amplified with PCR and cloned to PET-28a vector respectively. The fourrecombinant plasmids (pET-28a-3D1, pET-28a-3D2, pET-28a-3D3, and pET-28a-3D4)were expressed in E.coli BL-21with the induction of IPTG. SDS-PAGE showed thatpET-28a-3D2and pET-28a-3D3were successfully expressed. Western-blot showedthat the prepared monoclonal antibody could react with both the products ofpET-28a-3D2and pET-28a-3D3. The recognition sites was initially determinedbetween228~255amino acid residues of3D protein.