【摘要】 以Cry1Ab毒素作为抗原,将扩增后的噬菌体单域抗体库与固相化包被的Cry1Ab毒素特异性结合,利用噬菌体展示技术从人源化噬菌体单域抗体库(DAB)中筛选抗Cry1Ab毒素的单域抗体。采用正负筛选方法,经4轮"吸附-洗脱-扩增"后,富集特异性识别Cry1Ab毒素的噬菌体单域抗体。第4轮筛选得到的单菌落经单克隆ELISA鉴定,获得6株对Cry1Ab毒素具有较强结合活性的阳性单域抗体菌株,经PCR鉴定和DNA测序,均有完整的单域抗体基因片段插入。对6株阳性克隆的氨基酸序列进行比对分析,其序列差异主要存在于单域抗体的CDR区。更多还原

【Abstract】 The amplified phage single domain antibody library was specifically associated with the immobilized cry1Ab protein and human domain antibody library was employed to screen single domain antibodies(sd Abs) against Bacillus thuringiensis(Bt) Cry1Ab toxin by affinity panning using phage display technology.After four rounds of "adsorption-elution-amplification",specificity of phage single domain antibody against Cry1Ab toxin was enriched using posibive and negative screening method.Monoclonal phage enzyme-linked immunosorbent assay(ELISA) was used for the positive clones identification by picking single colonies randomly from the fourth round of panning and six monoclonal phage particles capable of binding with the cry1Ab protein were isolated.The positive clones were confirmed by PCR,DNA electrophoresis and sequencing.Totally 6 positive clones with distinct nucleotide sequences and intact sd Ab gene were confirmed to be specific for the Cry1Ab recognition.The sequence of 6 positive clones was compared and analyzed and the difference of protein sequence was mainly found in the CDR region of single domain antibody.更多还原