| Classical swine fever virus (CSFV), Bovine viral diarrhea virus (BVDV), and Border disease virus (BDV) are members of the pestivirus genus within the family of Flaviviridae. The viruses are structurally, antigenically, and genetically closely related. Structural and surface/envelope glycoprotein E2 (gp55) of classical swine fever virus (CSFV) is the most antigenic protein being responsible for eliciting neutralizing antibodies and conferring protective immunity. The current structure model of this protein predicts its surface-exposed region at the N terminus with a short stretch of the C-terminal residues spanning the membrane envelope.Eras/EO (gp48) protein has been shown to contain RNase activity, which plays a role in the viral life cycle and is also involved in virus neutralization.E? represents a second determinant for the induction of protective immunity against classical swine fever. Infection of cells with classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E2 and Ems with the cell surface. Previous studies carried out by immunoperoxidase binding assay against 135 pestivirus strains and isolates using the MAbs have revealed that c24\10, a18 , WH303 andMl669 E2 MAbs can detecte all CSFV strains and isolates, but none of the three ErnsMAbs(lB5, b4-22 24/16) detected an epitope common for all CSFV strains, all of the E2 and Erns MAbs have the virus neutralization functions and none of them showed cross-reactivity with ruminant pestiviruses.In this study, the antigenic epitopes or mimotopes of CSFV envelope structural glycoprotein E2 and Ems have been mapped by screening a 12-mer random peptide phage display library using the monoclonal antibodies (MAbs) c24\10, a!8, WH303,M1669 and IBS, b4-22 24/16, which reacted with the E2 and Erns structural proteins respectively. The critical residues in mapped epitopes or mimotopes of E2 and Erns structural proteins have been identified by differential the immunoreactivity using ELISA, Blocking ELISA, Westbern blot, test of neutralizing inhibition of antibodies and test of competitive inhibition of immunofluorecent antibodies, and also the encoding gene of light chain variable region of c24/10, a18 and b4-22, 24/16 MAbs have sequncedand compared.A linear epitope recognized by c24\10, a18, WH303 MAbs which is strongly conserved in CSFV strains but highly divergent among BVDV and BDV strains have been precisely defined and located at ,SPTTLR(aa832-837) of E2 molecule. Among aniino acids of the epiotpe, the residues at the first(S, serine), the second(P, proline), the thrid(T, threonine) and the fifth(L, leucine) sites are critical for binding to c24\10, a18 MAbs, for instance the thrid(T, threonine), if it has been changed to S(serine), lost the binding capability to both MAbs but can still be recognized by WH303 MAbs. Besides the sixth(R, arginine) is also important for strong binding.Two major mimotope motifs WNKPxT,AxWPIATA and three major mimotope motifs DxMRLD, SLMPPF. MLLH only recognized by WH303 and Ml669 respectively in ELISA and western blot analysis have also been defined, and but can not find the consensus or similar sequence in CSFV E2 molecule.Three major mimotope motifs. WxNxxP, DKNR (Q) G and A (T) CxYxKNwere identified respectively and characterized immunologically by the MAbs, Ib5, b4-22 and 24/16. MAbs, b4-22 and 24/16 shared a part of binding motif sequence, KN, and recognized the similar antigenic domain on the Emi protein but showed a distinct pattern of flank sequence and reactivity with the mimotopes by western blot analysis.In virus neutralizing test, the phages bearing the SPTxLR motifs can inhibit the neutralizing capability of c24/10 and WH303 MAbs' but bearing the WNKPxT, AxWPlATA motifs can't in virus neutralizing test.The competitive inhibition of immunofluorecent antibody test by the mimotopes showed the phage carried with the SPTxLR. WxNxxP, DKNR (Q) G or A (T) CxYxKN motifs can block c24/10, Ib5, b4-22 or 24/16 Mabs' reactivity with CSFV antigen respectively.The gen... |
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