对硫磷纳米抗体筛选及分子识别机制研究  

Selection of Nanobody and Recognition Mechanism between Nanobody and Parathion

作　　者：张玉琪 张瑾如 王锋[1] 李家冬[1] 司睿 吕丽珊 沈玉栋[1] 王弘[1] HAMMOCK Bruce D 孙远明[1] ZHANG Yu-Qi;ZHANG Jin-Ru;WANG Feng;LI Jia-Dong;SI Rui;LYU Li-Shan;SHEN Yu-Dong;WANG Hong;HAMMOCK Bruce D;SUN Yuan-Ming(Guangdong Provincial Key Laboratory of Food Quality and Safety,National-Local Joint Engineering Research Center for Processing and Safety Control of Livestock and Poultry Products,College of Food Science,SouthChina AgriculturalUniversity,Guangzhou 510642,China;Department of Entomology and UCD Comprehensive Cancer Center,University of California,Davis,California 95616,United States;Guangzhou Annuo Technology Co.,Ltd,Guangzhou 510663,China)

机构地区：[1]华南农业大学食品学院,广东省食品质量与安全重点实验室,畜禽产品精准加工与安全控制技术国家地方联合工程研究中心(广东),广州510642 [2]Department of Entomology and UCD Comprehensive Cancer Center,University of California,Davis,California 95616,United States [3]广州安诺科技股份有限公司,广州510663

出　　处：《分析化学》2019年第9期1419-1426,I0007,I0008,共10页Chinese Journal of Analytical Chemistry

基　　金：十三五重点研发计划项目(Nos.2016YFE0106000,2018YFC1602904);广州市科技计划项目(No.201804020077);广东省重点领域研发计划项目(No.2019B020211002);广东省高等学校珠江学者岗位计划项目(2017)资助~~

摘　　要：对硫磷是一种广谱的有机磷酸酯类杀虫、杀螨剂,毒性极强,可对环境和人类健康造成严重危害。尽管我国已禁止使用对硫磷,但仍存在违禁使用现象,加强对其监测十分必要。纳米抗体是已知最小的与抗原结合的片段(15 kDa),来源于骆驼科动物体内重链抗体的可变区,具有稳定性强、灵敏度高、易表达、水溶性好等特性。本研究基于噬菌体展示技术构建了抗对硫磷纳米抗体库,其库容为2.41×10^7 cfu/mL,经抗原-抗体固相亲和筛选及噬菌体酶联免疫法(Phage ELISA)鉴定,获得5种抗对硫磷纳米抗体VHH1、VHH6、VHH13、VHH21和VHH24。以结合能力最强的VHH1为基础,采用同源建模和分子对接技术探讨VHH1与对硫磷分子的识别机制,发现VHH1与对硫磷存在氢键和疏水间分子作用力,关键氨基酸是Ser60、Lys104、Phe105、Gly106和Arg107。本研究为抗体的进化改造和半抗原的设计提供了新思路。The organophosphorus pesticides are widely used in agriculture and food in China.Parathion,which is highly toxic and persistent,is one of the most widely used organophosphorus pesticides.Variable domain of the H chain of heavy-chain antibodies(VHH)is a fragment derived from camelids antibody.Due to its small size(only 15 kDa),high stability,ease of expression,and intact antigen-binding activity,VHHs have recently emerged as attractive reagents for immunodetection of chemical contaminants in food.In this study,five VHHs namely VHH1,VHH6,VHH13,VHH21 and VHH24 were selected from phage display library.Among these VHHs,VHH1 with the highest binding ability to parathion was selected for further analysis.VHH1 and parathion recognition mechanism predicted by homology modeling and molecule docking revealed the H bond and hydrophobic force,indicating the key amino acids Ser60,Lys104,Phe105,Gly106 and Arg107,which provided insights into antibody modification and antigen design.

关 键 词：纳米抗体 对硫磷 噬菌体展示 分子模拟 

分 类 号：S48[农业科学—农药学]