【摘要】 利用人源化噬菌体抗体库筛选抗Bt Cry1B毒蛋白质的单链抗体(Single-chain antibodies,scFv)。将扩增后的噬菌体抗体库与固相化包被的Cry1B毒蛋白质特异性结合,经4轮"吸附-洗脱-扩增"后,富集特异性识别Cry1B毒蛋白质的噬菌体单链抗体。从最后一轮筛选中随机挑取单菌落进行单克隆ELISA鉴定,对阳性克隆进行PCR扩增、DNA电泳鉴定及测序,成功筛选获得8个阳性噬菌体scFvs,经鉴定均有完整外源基因片段插入。挑取阳性值最高的scFv(1E2)建立了基于单链抗体的Cry1B毒蛋白质间接竞争ELISA检测方法。结果表明,Cry1B毒蛋白质对噬菌体scFv(1E2)的抑制中浓度(IC50)为1.075μg/ml,最低检测限(IC10)为0.013 4μg/ml,线性检测范围在0.5μg/ml至4.0μg/ml之间。更多还原

【Abstract】 A large human synthetic phage displayed human library(Tomlinson J) was employed to generate single-chain antibodies(scFvs) against Bacillus thuringiensis(Bt) Cry1B toxin by affinity panning.Specific anti-Cry1B toxin antibodies were isolated from amplified naive phage-displayed human single-fold scFv Tomlinson J library after four rounds of "adsorption-elution-amplification" by using Cry1B toxin protein as immobilized antigen.Monoclonal phage enzyme-linked immunosorbent assay(ELISA) was used for the positive clones identification by picking single colonies randomly from the final round of panning.The positive clones were confirmed by PCR,DNA electrophoresis and sequencing.Totally 8 positive clones with distinct nucleotide sequences and intact scFv gene were confirmed to be specific for the Cry1B recognition.The positive clone,namely 1E2,which showed better binding ability than others,was employed to develop an indirect competitive ELISA for the detecting of Cry1B.The results indicated that the IC50 reached 1.075 μg/ml,and the minimum detection limit was 0.013 4 μg/ml for the determination of Cry1B.The linear range of detection was approximately 0.5-4.0 μg/ml.更多还原