两种农药单链抗体的亲和力成熟与痕量检测技术研究

Due to its relative lower affinities, engineering antibodies usually have some outstanding problems in practical applications of detecting trace amounts of pesticides. This project intends to use affinity maturation and time-resolved fluoroimmunoassay to resolve these problems.①Two large-capacity affinity maturation libraries are separately constructed for single chain variable fragments(scFv)of Bacillus thuringiensis (Bt) Cry1Ac toxin and ivermectin (IVM) by error PCR and site-directed mutagenesis. ②20-30 positive clones against Cry1Ac and IVM are isolated from affinity maturation libraries by using magnetic beads and free antigen elution. The efficiency of these strategies and the different structural modification requirements of protein or hapten specific antibodies are discussed. The key epitopes of affinity maturation are also predicted. ③The clones which have significantly improvement in affinities or specificities are identified and expressed in soluble formats. Base on these antibodies,3-4 high sensitive or broad specific time-resolved fluoroimmunoassay are established for Cry1Ac, IVM or their analogues. This project will make an exploration in affinity maturation of engineering antibodies and their use in detecting trace amounts of pesticides.

本项目针对基因工程抗体亲和力偏低，在痕量农药残留检测领域实际应用困难的突出问题。拟借鉴亲和力成熟技术的高效抗体重组、改性优势，发挥时间分辨荧光免疫分析高灵敏与多组分分析特点，完成以下工作：①对已筛选到的苏云金杆菌杀虫蛋白Cry1Ac和伊维菌素（IVM）单链抗体基因，采用错配PCR、定点突变等方法，分别构建2种大容量亲和力成熟库。②采用磁珠液相筛选和游离抗原洗脱等优化筛选技术，分离鉴定出20-30种Cry1Ac、IVM成熟抗体株。比较不同成熟策略效率，分析蛋白及小分子农药特异性抗体在成熟过程中结构修饰差异，预测成熟关键表位。③选择亲和力显著提高或具广谱识别能力的抗体株，经可溶性表达后，建立3-4种高灵敏或广谱Cry1Ac、IVM及其家族同系物的时间分辨荧光免疫分析方法。为农药基因工程抗体亲和力成熟技术做出探索，并建立高灵敏度的农药残留检测方法。

本项目以苏云金杆菌Cry1Ac毒素单链抗体和伊维菌素（IVM）单链抗体为研究对象，开展了两种农药单链抗体的亲和力成熟技术研究，具体取得以下结果：（1）采用定点突变和易错PCR技术，分别构建了Cry1Ac单链抗体的2个亲和力成熟库，库容分别为8×106和1×108。（2）以实验室前期筛选到的伊维菌素单链抗体和TomlinsonI库作为模板，采用重、轻链替换方法，分别构建了伊维菌素重、轻链替换库，库容分别为8×105和2×106。（3）采用固相筛选和磁珠液相筛选法，对两种Cry1Ac成熟库进行了3-4轮筛选，初步获得47个阳性克隆，经测序鉴定最终获得12个突变克隆，其中最佳克隆的结合性检测主导参数EC50与模板抗体比值为2.8倍，亲和力获得提高。并采用展示型表达的Cry1Ac单链抗体建立了间接竞争时间分辨荧光免疫学分析方法，方法的检测灵敏度为2.43ng/mL，检测线性范围在10.51ng/mL - 2064.47ng/mL之间。（4）采用多克隆噬菌体免疫分析对IVM重、轻链替换原始抗体库的整体亲和力进行鉴定，结果表明重链替换库的整体亲和力为轻链替换库的4.1倍。采用IVM-BSA对IVM的重、轻链替换库进行了1轮筛选，重轻链替换库的阳性克隆检出率分别为13.5%和57.4%，最佳克隆IVM-C8和模板抗体的EC50比值为1.4倍。采用展示型表达的IVM-C8建立了伊维菌素间接竞争TRFIA分析方法，检测灵敏度（IC10）为76ng/mL，线性范围在160-3840ng/mL。（5）对Cry1Ac单链抗体与毒素进行分子对接，预测的关键氨基酸位点分别是的104位的酪氨酸、105位的苯丙氨酸和107位的酪氨酸。对伊维菌素突变抗体IVM-C8的三维结构进行模拟，并对抗体的CDR区结构进行了分析。项目的开展为为农药基因工程抗体亲和力成熟技术做出探索，并建立高灵敏度的农药残留检测方法。