抗苏云金芽孢杆菌Cry1B毒蛋白质单链抗体的筛选与鉴定  

Screening and identification of single-chain antibodies(scFvs) against Bacillus thuringiensis Cry1B toxin

作　　者：徐重新[1,2] 张霄[2,3] 刘媛 王耘[2,3] 张存政 刘贤进 

机构地区：[1]南京师范大学生命科学学院,江苏省微生物与功能基因组学重点实验室,江苏南京210046 [2]江苏省食品质量安全重点实验室--省部共建国家重点实验室培育基地,农业部农产品质量安全控制技术与标准重点实验室,江苏南京210014 [3]南京农业大学植物保护学院,江苏南京210095

出　　处：《江苏农业学报》2012年第4期886-890,共5页Jiangsu Journal of Agricultural Sciences

基　　金：国家“973”计划项目(2012CB722505)

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。A large human synthetic phage displayed human library （Tomlinson J） was employed to generate single-chain antibodies （scFvs） against Bacillus thuringiensis （Bt） CrylB toxin by affinity panning. Specific anti-CrylB toxin an- tibodies were isolated from amplified naive phage-displayed human single-fold scFv Tomlinson J library after four rounds of ＂adsorption-elution-amplification＂ by using Cryl B 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 posi- tive clones with distinct nucleotide sequences and intact scFv gene were confirmed to be specific for the Cryl B recognition. The positive clone, namely 1E2, which showed better binding ability than others, was employed to develop an indirect com-petitive ELISA for the detecting of Cryl B. The results indicated that the IC50 reached 1. 075 μg/ml, and the minimum de-tection limit was 0. 013 4 μg/ml for the determination of Cryl B. The linear range of detection was approximately 0.5-4. 0μg/ml.

关 键 词：噬菌体抗体库 Cry1B毒蛋白质 单链抗体 ELISA 

分 类 号：X836[环境科学与工程—环境工程]