广谱性磺胺类药物纳米抗体筛选及分子识别机制研究

Studies have indicated that light chain is an important cause of nonuniform affinity between traditional antibodies and different sulfonamides. Nanobody consists only of variable region of heavy chain antibody and lacks light chain naturally. Compared with the traditional antibody, whether it has broad-specific and uniform affinity is still unknown. This project aims to prepare anti-sulfonamides nanobodies and clarify the recognition mechanism. The main research contents include: (1) Isolate the heavy chain antibody according to the diverse surface charge between different IgG subtypes, and evaluate the property of the heavy chain antibody accurately; (2) Construct phage display libraries by DNA terminal repair, joint connection, purification, amplification and other techniques; (3) Screen anti-sulfonamides nanobodies with high affinity and broad-specificity based on multiple antigens and the gradient screening strategy; (4) Compared with traditional antibodies, elucidate the molecular recognition mechanism and the structural basis of nanobodies, confirm whether it has broad-specific and uniform affinity by circular dichroism, isothermal titration calorimetry, homology modeling, molecular docking, molecular dynamics and other experimental and simulate means; (5) Establish immunoassay for the detection of multi-residues of sulfonamides in food sample. The results of this project can provide new ideas, technical means and theoretical basis for the development of broad-specific biological identification materials against chemical hazards in food.

研究表明轻链是造成磺胺类药物传统抗体亲和力不均一的重要原因。纳米抗体仅由重链抗体的可变区组成，天然缺失轻链，与传统抗体相比，其是否具有广谱均一识别性尚未可知。本项目拟制备磺胺类药物的纳米抗体并明确其识别机制。主要研究内容包括:（1）根据IgG亚型表面电荷的差异分离重链抗体，精准评价重链抗体的免疫效果；（2）采用DNA末端修复、接头连接、纯化扩增等技术构建噬菌体展示文库；（3）选择组合抗原、梯度筛选策略，筛选出高亲和力、广谱性的磺胺类药物的纳米抗体；（4）采用圆二色光谱、等温滴定量热分析、同源建模、分子对接、分子动力学等实验和模拟手段，阐明纳米抗体分子识别机制和结构基础，明确纳米抗体是否具有广谱均一识别性；（5）建立食品样本中磺胺类药物多残留检测的免疫分析方法。本项目的研究结果可为开发食品中化学性危害物广谱性生物识别材料提供新的研究思路、技术手段和理论依据。

根据羊驼血清中不同亚型IgG在不同pH 条件下，所带电荷不同，采用离子交换层析精准分离重链抗体，通过ELISA测定特异性重链抗体的免疫效果。结果确定重链抗体更能反应纳米抗体的免疫水平。共筛到两株广谱识别性纳米抗体。SA6可识别24种SAs，其中IC50＜100 ng/mL的有13种；SA7可识别27种SAs，其中IC50＜100 ng/mL的有19种。SA7与SAs之间产生的作用力主要包括氢键、范德华力及疏水相互作用，关键氨基酸残基为VAL33、CYS50、ARG57、ASN58、ASN107、PHE104和ARG101。其具有广谱识别性。SA6可耐受0-80%的甲醇，0-35%的乙腈以及0-55%的丙酮。SA7可耐受0-80%的甲醇，0-20%的乙腈以及0-45%的丙酮。建立了3种磺胺类药物多残留检测的免疫分析方法：ELISA免疫分析方法、侧流层析免疫检测方法以及荧光偏振免疫分析方法，可用于原奶、鸡蛋、鸡肉和猪肉样本的检测。培养博士研究生2名，2023年06月拟毕业博士研究生1名，发表SCI文章2篇，均为第一标注。项目负责人于雪芝于2021年获得中国商业联合会科学技术奖-全国商业科技进步奖一等奖（第三完成人）。