【摘要】 为预测并鉴定人凝血因子IX(FIX)的线性B细胞表位.通过IEDB数据库及Discovery Studio软件预测FIX的B表位和白喉毒素T结构域(DTT)的B表位,用所预测的FIX的B表位替换某预测的DTT的B表位形成DTT-表位融合蛋白.通过基因工程技术制备各重组蛋白,并免疫小鼠,通过ELISA和Western-blot检测抗血清效价和特异性,通过等温量热滴定技术(ITC)测定抗体的亲和力.预测到4个FIX的B表位.用重组蛋白FIX-2-DTT免疫的小鼠产生了识别完整FIX的抗体,该抗体具有良好的特异性,其结合常数KD为106 M-1.表明FIX-2(306 NAAINKY312)是FIX的B表位,用重组蛋白FIX-2-DTT免疫小鼠能够产生特异性识别FIX且亲和力温和的抗体.更多还原

【Abstract】 In order to predict and identify linear B cell epitopes of human coagulation factor IX(FIX),the linear B cell epitopes of FIX and diphtheria toxin transmembrane domains structure(DTT)were predicted by IEDB database and Discovery Studio solftware.According to the principle of"similar N-C distance",the predicted B cell epitopes of DTT were replaced by the predicted B cell epitopes of FIX in oder to form DTT-epitope fusion proteins.Fusion proteins were prepared by genetic engineering technology and subsequently used for immunization of Bal b/c mice.The titer and specificity of antiserum was measured by ELISA and Western-blot.The affinity of antibody was determined by isothermal titration calorimetry techniques(ITC).Results showed:Four B cell epitopes of FIX were predicted.The antibody that can recognize the complete FIX was produced in mice immunized with the FIX-2-DTT fusion protein,which has good specificity.The binding constant KD of the antibody is 106 M-1.FIX-2(306 NAAINKY312)is a B epitope of FIX.The anti-FIX antibody with good specificity and gentle affinity could be produced by immunizing mice with FIX-2-DTT fusion protein.更多还原