抗杀螟硫磷纳米抗体表达、纯化策略研究及检测方法建立  

Expression, Purification Strategy and Detection Method Establishment of Anti-Fenitrothion Nanobody

作　　者：郭鹏燕 徐振林[1] 陈子键 沈兴 GUO Pengyan;XU Zhenlin;CHEN Zijian;SHEN Xing(College of Food Science,South China Agricultural University,Guangzhou 510642,China;School of Food Pharmaceutical Engineering,Zhaoqing University,Zhaoqing 526061,China)

机构地区：[1]华南农业大学食品学院,广东广州510642 [2]肇庆学院食品与制药工程学院,广东肇庆526061

出　　处：《食品工业科技》2023年第23期298-305,共8页Science and Technology of Food Industry

基　　金：广东省基础与应用基础研究基金项目(2021A1515110513)。

摘　　要：为了高效制备高纯度的抗杀螟硫磷纳米抗体,本研究对一株低表达水平的重组抗杀螟硫磷纳米抗体在大肠杆菌中的表达条件进行优化,以IPTG浓度、温度为自变量,Nb表达量为因变量进行单因素实验,同时对抗体纯化方法进行研究。结果表明,在37℃、无IPTG条件下抗杀螟硫磷纳米抗体表达量最高,达到6 mg/L,并且发现IPTG用量与诱导温度存在交互效应,通过Ni亲和层析与凝胶过滤层析两步纯化法,最终得到了纯度98%以上的抗杀螟硫磷抗体,表明该表达与纯化策略可以高效制备抗杀螟硫磷纳米抗体。利用所获得的纳米抗体,建立了icELISA检测方法,其IC50为5.81 ng/mL,检出限为0.25 ng/mL,检测范围为0.78~43.07 ng/mL。选择生菜和白菜为样品进行添加回收实验,添加回收率在93.3%~111.7%之间,变异系数(CV)值在2.3%~18.2%之间。基于杀螟硫磷纳米抗体所建立的方法灵敏度高,能够满足国标规定的杀螟硫磷监测需求,可用于杀螟硫磷的快速筛查。本研究为开发高效快速的杀螟硫磷免疫检测方法奠定了基础。For the purpose of preparing anti-fenitrothion nanobodies with high-purity efficiently,the expression conditions of a recombinant anti-fenitrothion nanobody with low expression level in E.coli was optimized.The IPTG concentration and inducing temperature were selected as the independent variables and the nanobody expression level was used as the dependent variable for single-factor experiments,and the purification strategy was also investigated.The results showed that the highest expression level of 6 mg/L anti-fenitrothion nanobody was achieved at 37℃without IPTG.Moreover,an interactive effect of IPTG dosage and inducing temperature was found.After a two-step purification of Ni affinity chromatography and gel filtration chromatography,the anti-fenitrothion nanobodies was finally yielded with more than 98%purity,indicating that the expression and purification strategies in this study can obtain anti-fenitrothion nanobodies efficiently.An ic-ELISA assay was then established based on the obtained nanobody,with an IC50 of 5.81 ng/mL and an LOD of 0.25 ng/mL,and a detection range of 0.78~43.07 ng/mL.The assay was applied to determine fenitrothion in Chinese cabbage and lettuce samples.The recovery rate of addition was between 93.3%~111.7%,and the coefficient of variation(CV)was between 2.3%~18.2%.The proposed assay based on anti-fenitrothion nanobody has high sensitivity,which can meet the requirements for monitoring of fenitrothion under the national standard,thus can be used for rapid screening of fenitrothion.This study laid a foundation for the development of efficient and rapid immunoassays for fenitrothion.

关 键 词：杀螟硫磷 纳米抗体 诱导表达 纯化策略 活性鉴定 

分 类 号：TS201.3[轻工技术与工程—食品科学]