食品中Bt Cry毒素检测用广谱抗体的制备研究

More and more genes of Bt Cry toxins are being employed in transgenic plant breeding. Immunoassay technology based on monoclonal or polyclonal antibodies of the single Cry toxin can't meet the requirements of the huge amount of sample screening, neither in safe food consumption nor in raw material quality control of food processing.Based on the several single chain variable fragments (scFvs) genes of Cry1 toxins obtained in our lab, we are trying to mimic key amino acid recognition sites first by using molecular docking technology and find those genes for further modification. Then we are going to develop three large capacity mutation phage displayed antibody libraries by using site-directed mutagenesis, chain shuffling and other genetic engineering respectively. 20-30 different high affinity antibody mutant strains are going to be isolated from the libraries by similarly domain of Bt toxins coating and toxins alternative panning, combining high throughput Biacore analysis technology. 3-4 Cry broad-spectrum antibodies, which can detect about 10 Cry toxins, could be acquired by different Cry toxin group testing and condition optimizing.Finally we can summarize the experience of broad spectrum antibody developemnt of Bt toxin and provide foundation for establishing high-throughput and high sensitive screening technology.

本研究针对转基因食品中苏云金杆菌（Bt）Cry毒素种类较多，当前食品安全监管急需广谱筛查技术的突出问题。拟发挥基因工程抗体易于定向修饰及Cry毒素具有相似结构的特点，以前期获得的几种Bt Cry1类毒素单链抗体基因为材料，先通过分子对接技术模拟抗体关键氨基酸识别位点，并以此为基础，采用定点突变、链置换等基因操作手段，分别设计、构建3种大容量突变抗体库。再利用Cry毒素共性结构域包被或抗原交替筛选方法，初步获得20-30个能识别Cry毒素的突变抗体株，并通过高通量Biacore分析，采用多种Cry毒素材料验证筛选，最终获得3-4个广谱抗体，检测对象可覆盖10种左右Cry 毒素。探索广谱抗体识别区的分子结构特性，并为Bt毒素基因工程抗体的体外定向修饰和高灵敏、广谱筛选检测方法建立奠定基础。

转基因食品中苏云金芽孢杆菌（Bt）Cry毒素种类较多，进行多种Cry毒素高效广谱抗体筛查技术研究对转基因作物及其产品安全性评估和质量安全监管具有重要意义。基于此，本项目主要开展以下两个方面研究：.（一）依托成熟的单多克隆抗体和基因工程抗体技术，制备Bt Cry1类毒素的单多克隆抗体和基因工程抗体。通过比较7种Cry1类毒素（Cry1Aa，Cry1Ab，Cry1Ac，Cry1B，Cry1C，Cry1E，Cry1F）的三维结构，兼顾一级氨基酸序列和二级抗原性及亲水性分析，选择了三个多肽进行合成（T1，T2和T3），将其与血蓝蛋白（KLH）偶联之后，制备Cry1类毒素的广谱单克隆抗体。筛选到的单克隆抗体对7种Cry1类毒素的平衡解离常数（KD）在1.0-5.0×10−8 M之间。建立的DAS-ELISA检测方法对7种Cry1类毒素的最低检测限（LOD）和最低定量限（LOQ）分别为15和30 ng/mL。进一步采用基因工程抗体技术，将广谱抗体重轻链基因克隆并拼接为scFv，构建了库容约为6.25×107 CFU/mL的鼠源偏向抗体库。经复筛得到的广谱抗体对6种Cry1类毒素的LOD和LOQ分别为3.14-11.07 ng/mL和8.22-39.44 ng/mL。以上得到的广谱抗体灵敏度和精确度均满足实际样品检测要求。.（二）利用噬菌体展示技术从人源化单域抗体库中筛选获得具有抗Cry1Ab毒素活性的单域抗体。采用正负筛选方法，经过4轮富集筛选，成功筛选获得6株具有Cry1Ab毒素蛋白结合活性的噬菌体单域抗体。利用同源建模和分子对接技术对预测得到Cry1Ab毒素单域抗体和5类Cry1类毒素的共同关键氨基酸结合位点为单域抗体重链互补决定簇3区（VH-CDR3）的105位天冬酰胺，106位精氨酸、107位缬氨酸及114位精氨酸。经重叠延伸PCR技术对单域抗体的4个关键氨基酸结合位点进行了协同饱和突变，成功构建了库容为1.2×105的多点饱和突变库。最后利用Cry1类毒素共性结构域蛋白包被和5种Cry1类毒素交替包被策略从单域抗体饱和突变库中筛选获得了5株对Cry1类毒素具有广谱识别活性的突变菌株，与母体相比，活性均提高了2-3倍，并均能用于毒素实际样品的检测。