The antigenic determinants of trichosanthin were predicted by molecular modeling. First, the threedimensional structure model of the antigen-binding fragment of anti-trichosanthin immunoglobulin E was built on the bas...The antigenic determinants of trichosanthin were predicted by molecular modeling. First, the threedimensional structure model of the antigen-binding fragment of anti-trichosanthin immunoglobulin E was built on the basis of its amino-acid sequence and the known three-dimensional structure of an antibody with similar sequence. Secondly, the preferable antigen-antibody interactions were obtained based on the known three-dimensional structure of trichosanthin and of the hypervariable regions of anti-trichosanthin immunoglobulin E. Two regions in the molecular surface of trichosanthin were found to form extensive interactions with the hypervariable regions of the antibody and have been predicted to be the possible antigenic determinants: one is composed of two polypeptide segments, Ile201-Glu210 and Ile225-Asp229, which are close to each other in the three-dimensional structure; and the other is the segment Lys173-Thr178. The former region seems to be the more reasonable antigenic determinant than the latter one.展开更多
Recently we constructed a murine IgE phage surfacedisplay library and screened out two IgE (Fab) cloneswith specific binding activity to Trichosanthin (TCS). Inthis work, the Vε and Vκ genes of the two clones werese...Recently we constructed a murine IgE phage surfacedisplay library and screened out two IgE (Fab) cloneswith specific binding activity to Trichosanthin (TCS). Inthis work, the Vε and Vκ genes of the two clones weresequenced and their putative germline gene usages werestudied. On the basis of the known 3D structure of Trichosanthin and antibody, molecular modeling was carriedout to study the antigen-antibody interaction. The possible antigenic determinant sites on the surface of TCSrecognized by both the clones were analyzed, and the reaction forces between TCS and two Fab fragments werealso analyzed respectively.展开更多
Wheat high molecular weight glutenin subunits(HMW-GS)1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice.Subcutaneous inoculation of the antigen is performed.The intra-peritone...Wheat high molecular weight glutenin subunits(HMW-GS)1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice.Subcutaneous inoculation of the antigen is performed.The intra-peritoneal injection is completed 3 days before fusion with myeloma cell(SP2/0)via PEG-1500.The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay(ELISA).Positive hybrid cells are further verified three times by limit dilution of the culture cells.A hybridoma cell line is successfully obtained.The monoclonal antibody belongs to IgG1 subclass.In immunoblotting,the antibody binds to all HMW-GS of T.aestivum cultivars,but does not bind to other storage proteins in seeds of wheat.This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat.The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T.durum(durum wheat).Furthermore,it also binds to HMW storage proteins in Secale cereale(rye),Hordeum vulgare(barley).However,it never binds seed storage proteins in other cereals such as maize,oat,rice,foxtail millet,sorghum etc.The antigen determinant recognized by the antibody has been located within hexapeptide[PGQGQQ]or/and nonapeptide[GYYPTSPQQ]in the central repetitive region of HMW-GS.展开更多
文摘The antigenic determinants of trichosanthin were predicted by molecular modeling. First, the threedimensional structure model of the antigen-binding fragment of anti-trichosanthin immunoglobulin E was built on the basis of its amino-acid sequence and the known three-dimensional structure of an antibody with similar sequence. Secondly, the preferable antigen-antibody interactions were obtained based on the known three-dimensional structure of trichosanthin and of the hypervariable regions of anti-trichosanthin immunoglobulin E. Two regions in the molecular surface of trichosanthin were found to form extensive interactions with the hypervariable regions of the antibody and have been predicted to be the possible antigenic determinants: one is composed of two polypeptide segments, Ile201-Glu210 and Ile225-Asp229, which are close to each other in the three-dimensional structure; and the other is the segment Lys173-Thr178. The former region seems to be the more reasonable antigenic determinant than the latter one.
文摘Recently we constructed a murine IgE phage surfacedisplay library and screened out two IgE (Fab) cloneswith specific binding activity to Trichosanthin (TCS). Inthis work, the Vε and Vκ genes of the two clones weresequenced and their putative germline gene usages werestudied. On the basis of the known 3D structure of Trichosanthin and antibody, molecular modeling was carriedout to study the antigen-antibody interaction. The possible antigenic determinant sites on the surface of TCSrecognized by both the clones were analyzed, and the reaction forces between TCS and two Fab fragments werealso analyzed respectively.
基金This work was supported by the grants on transgenic plant(J99-A-018)of National 863 Plan(2003AA207090)of lhe Minisury of Science and Technology of China to 2hang Xueyong
文摘Wheat high molecular weight glutenin subunits(HMW-GS)1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice.Subcutaneous inoculation of the antigen is performed.The intra-peritoneal injection is completed 3 days before fusion with myeloma cell(SP2/0)via PEG-1500.The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay(ELISA).Positive hybrid cells are further verified three times by limit dilution of the culture cells.A hybridoma cell line is successfully obtained.The monoclonal antibody belongs to IgG1 subclass.In immunoblotting,the antibody binds to all HMW-GS of T.aestivum cultivars,but does not bind to other storage proteins in seeds of wheat.This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat.The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T.durum(durum wheat).Furthermore,it also binds to HMW storage proteins in Secale cereale(rye),Hordeum vulgare(barley).However,it never binds seed storage proteins in other cereals such as maize,oat,rice,foxtail millet,sorghum etc.The antigen determinant recognized by the antibody has been located within hexapeptide[PGQGQQ]or/and nonapeptide[GYYPTSPQQ]in the central repetitive region of HMW-GS.
