技术领域technical field
本发明属于生物技术领域，涉及具有与吡虫啉抗体特异性结合的多肽，包括所述多肽在检测吡虫啉中的应用。The invention belongs to the field of biotechnology, and relates to a polypeptide with specific binding to imidacloprid antibody, including the application of the polypeptide in detecting imidacloprid.
背景技术Background technique
吡虫啉是一种烟碱类超高效杀虫剂，并具有触杀、胃毒和内吸等多重作用。由于吡虫啉的广泛、持续和大量的使用导致了许多害虫天敌的死亡，研究发现吡虫啉对稻飞虱的天敌黑肩绿盲蝽和蚜虫的天敌龟纹瓢虫有较大的毒性，且对水生生物、蜜蜂以及土壤中的有益生物均会造成损伤。为预防吡虫啉应用存在的潜在风险，需要一种灵敏、快速、选择性的残留检测方法。Imidacloprid is a super-efficient neonicotinoid insecticide with multiple effects such as contact killing, stomach toxicity and systemic absorption. Due to the extensive, continuous and large-scale use of imidacloprid, the death of many natural enemies of pests has been caused. Studies have found that imidacloprid is highly toxic to the natural enemies of rice planthoppers, Lygus spp. Bees, bees, and beneficial organisms in the soil can all cause damage. In order to prevent the potential risks of imidacloprid application, a sensitive, rapid and selective residue detection method is needed.
目前对吡虫啉的残留检测，主要采用仪器分析方法(如液相色谱、气相色谱-质谱联用仪等)检测吡虫啉在农产品和农业环境中的残留，仪器检测灵敏度高、准确性强，但相对用时较长、操作复杂，不能满足快速检测大量样品的需求。且全新的农药残留检测体系，对农药残留检测分析技术提出更高的标准和更严格的要求：不只推动着农药残留检测技术向着更加快速、简便、灵敏可靠的检测目标发展，而且逐步由原先的在实验室进行少量室内检测演变为如今的现场及时检测以及实验室的大批次、高通量检测。免疫检测技术具有简便、快速、低成本和可靠的特点，极大的推动了农药残留快速检测技术的发展，为保障农产品的质量安全和环境的生态安全提供重要的工具。由于农药等小分子化学品为单抗原决定簇分析物，整个分子只能和一个抗体结合，所以通常选择竞争模式来建立免疫分析方法。在这种形式中，必须存在与分析物竞争结合抗体的竞争物，这个竞争物通常是将半抗原与蛋白、酶或荧光物等连接制备获得。根据竞争物与免疫抗原结构的同异，可以将分析方法分为同源和异源免疫分析。目前，农药异源竞争物通常采用化学合成的方法制备，系列异源半抗原的化学合成及半抗原与蛋白、酶或荧光物的偶联需要很大的工作量，并且不能预知制备的竞争物是否具有活性，所以存在一定的盲目性。At present, for the detection of imidacloprid residues, instrumental analysis methods (such as liquid chromatography, gas chromatography-mass spectrometry, etc.) are mainly used to detect imidacloprid residues in agricultural products and agricultural environments. It is long and complicated to operate, and cannot meet the needs of rapid detection of a large number of samples. Moreover, the brand-new pesticide residue detection system puts forward higher standards and stricter requirements for pesticide residue detection and analysis technology: it not only promotes the development of pesticide residue detection technology to a faster, simpler, more sensitive and reliable detection goal, but also gradually changes from the original A small amount of indoor testing in the laboratory has evolved into today's on-site timely testing and high-volume, high-throughput testing in the laboratory. Immunoassay technology is simple, fast, low-cost and reliable, which greatly promotes the development of rapid pesticide residue detection technology and provides an important tool for ensuring the quality and safety of agricultural products and the ecological safety of the environment. Since small molecule chemicals such as pesticides are single epitope analytes, the entire molecule can only bind to one antibody, so the competition mode is usually chosen to establish an immunoassay method. In this format, there must be a competitor that competes with the analyte for binding to the antibody, and this competitor is usually prepared by linking a hapten to a protein, enzyme, or fluorophore. According to the similarities and differences of competitors and immune antigen structure, analysis methods can be divided into homologous and heterologous immune analysis. At present, pesticide heterologous competitors are usually prepared by chemical synthesis. The chemical synthesis of a series of heterologous haptens and the coupling of haptens with proteins, enzymes or fluorescent substances require a lot of work, and the prepared competitors cannot be predicted. Whether it is active or not, there is a certain blindness.
自噬菌体展示技术报道以来便引起了研究人员的广泛关注，并且成为迄今为止发展最成熟、应用最广泛的抗体筛选技术。该技术已经被广泛应用于各种功能性重组多肽及抗体的高通量筛选。其原理是将编码多肽或外源蛋白的DNA片段与噬菌体表面蛋白的编码基因融合后，以融合蛋白的形式呈现在噬菌体的表面。被展示的多肽或蛋白可保持相对的空间结构和生物活性以利于靶标分子的识别和结合，并使基因型与表型建立直接的联系。导入多种外源基因的一群噬菌体，就构成了一个噬菌体展示库。当噬菌体库与靶标分子即抗体通过3到5轮的“吸附-洗涤-洗脱-扩增”淘选后，与抗体特异性结合的噬菌体就会得到高度的富集。由已经商品化的抗噬菌体的二抗识别，可直接用作竞争物建立异源竞争免疫分析。与化学合成的竞争物相比，具有简单、快捷、安全环保且灵敏度更高的优点。但目前为止，尚未见具有与吡虫啉抗体特异性结合的多肽及其应用的研究和报道。Since the report of phage display technology, it has attracted extensive attention of researchers and has become the most mature and widely used antibody screening technology so far. This technology has been widely used in high-throughput screening of various functional recombinant polypeptides and antibodies. The principle is to fuse a DNA fragment encoding a polypeptide or foreign protein with a gene encoding a phage surface protein, and present it on the surface of the phage in the form of a fusion protein. The displayed polypeptide or protein can maintain relative spatial structure and biological activity to facilitate the recognition and binding of target molecules, and establish a direct relationship between genotype and phenotype. A group of phages introduced with various foreign genes constitutes a phage display library. When the phage library and the target molecule, that is, the antibody, are panned through 3 to 5 rounds of "adsorption-washing-elution-amplification", the phage that specifically binds to the antibody will be highly enriched. Recognized by a commercially available secondary antibody against phage, it can be directly used as a competitor to establish a heterologous competitive immunoassay. Compared with chemically synthesized competitors, it has the advantages of simplicity, quickness, safety, environmental protection and higher sensitivity. But so far, there is no research or report on the polypeptide with specific binding to imidacloprid antibody and its application.
发明内容Contents of the invention
本发明的目的是提供与吡虫啉抗体特异性结合的多肽，及相关多肽在吡虫啉检测中的应用。The purpose of the present invention is to provide a polypeptide that specifically binds to imidacloprid antibody, and the application of related polypeptides in the detection of imidacloprid.
本发明的目的通过下述技术方案予以实现：The purpose of the present invention is achieved through the following technical solutions:
(1)将蛋白A柱纯化的吡虫啉抗体包被在酶标板上，用5％脱脂奶粉封闭酶标板，随后将噬菌体展示随机线十二肽库加入酶标板中进行亲和淘选，淘选过程按照“吸附-洗涤-洗脱-扩增”的步骤进行，经过3轮的富集淘选，从第一轮到第三轮包被抗体的用量和用于竞争洗脱噬菌体的吡虫啉用量依次减少；(1) Coat the imidacloprid antibody purified by the protein A column on the microtiter plate, block the microtiter plate with 5% skimmed milk powder, then add the phage display random line dodecapeptide library to the microtiter plate for affinity panning, The panning process is carried out according to the steps of "adsorption-washing-elution-amplification". After 3 rounds of enrichment panning, the amount of coating antibody from the first round to the third round and the imidacloprid used to compete for the elution of phage The dosage decreases sequentially;
(2)3轮淘选后，挑选30个噬菌体克隆进行ELISA初步鉴定，对于得到的13个阳性克隆进行扩增、质粒提取、测序，共发现2种序列，其序列如SEQ ID NO 1～2所示：His Ser LeuTrp Met Ala Ser Pro Met Pro Gly Tyr和Gln Ile Phe Thr Ser Ser Pro Met Pro AlaMet Val。(2) After 3 rounds of panning, 30 phage clones were selected for preliminary identification by ELISA, and 13 positive clones were amplified, plasmid extracted, and sequenced. A total of 2 sequences were found, such as SEQ ID NO 1-2 Shown: His Ser LeuTrp Met Ala Ser Pro Met Pro Gly Tyr and Gln Ile Phe Thr Ser Ser Pro Met Pro AlaMet Val.
本发明所述的多肽可与吡虫啉抗体特异性结合，建立异源竞争ELISA，用于吡虫啉在环境与农产品中的残留检测。The polypeptide described in the invention can specifically bind to the imidacloprid antibody to establish a heterologous competition ELISA for the detection of imidacloprid residues in the environment and agricultural products.
本发明具有以下有益效果：(1)新颖：为国内外首次报道的与吡虫啉抗体特异性结合的多肽；(2)实用：利用本发明提供的噬菌体展示多肽可以快速建立高敏感性的异源竞争ELISA；(3)特异性强：用本发明提供的噬菌体展示多肽建立的竞争ELISA与吡虫啉类似物的交叉反应，除氯噻啉(102％)外，其余均很低；(4)准确度高：利用本发明提供的噬菌体展示多肽实现的竞争ELISA在环境和农业样品中的添加回收率为70.1-102.1％，变异系数低于10.2％，符合残留分析标准；(5)灵敏度高：利用本发明提供的噬菌体展示多肽实现的竞争ELISA的抑制中浓度(IC50)为0.067ng/mL，检测限(IC10，LOD)为0.024ng/mL。The present invention has the following beneficial effects: (1) Novelty: it is the polypeptide specifically binding to imidacloprid antibody reported for the first time at home and abroad; (2) Practical: Utilizing the phage display polypeptide provided by the present invention can quickly establish a highly sensitive heterologous competition ELISA; (3) strong specificity: the cross-reactivity between the competition ELISA and imidacloprid analogues established by the phage display polypeptide provided by the present invention is very low except for chlorothialine (102%); (4) the accuracy is high : the addition recovery rate of the competition ELISA realized by the phage display polypeptide provided by the invention in environmental and agricultural samples is 70.1-102.1%, and the coefficient of variation is lower than 10.2%, meeting the residue analysis standard; (5) high sensitivity: using the present invention The inhibitory concentration (IC50 ) of the competition ELISA achieved by the provided phage display polypeptide was 0.067ng/mL, and the detection limit (IC10 , LOD) was 0.024ng/mL.
附图说明Description of drawings
图1是挑选的30个噬菌体克隆噬菌体ELISA(P-ELISA)检测的结果；横坐标为噬菌体克隆，纵坐标为OD450值；Fig. 1 is the result that 30 selected phage clones phage ELISA (P-ELISA) detect; Abscissa is phage clone, and ordinate is OD450 value;
图2是P-ELISA检测吡虫啉，OD值与吡虫啉浓度的曲线；横坐标为吡虫啉的浓度，单位为ng/mL；纵坐标为OD450值。Fig. 2 is the curve of P-ELISA detection imidacloprid, OD value and imidacloprid concentration; the abscissa is the concentration of imidacloprid, the unit is ng/mL; the ordinate is OD450 value.
具体实施方式Detailed ways
本发明实施例中所用实验材料、主要试剂及配方如下：Used experimental material, main reagent and formula in the embodiment of the present invention are as follows:
主要实验材料：Main experimental materials:
蛋白A柱纯化吡虫啉单克隆抗体由南京农业大学，植物保护学院，农药残留与环境毒理实验室制备；噬菌体展示随机线十二肽库从NEB公司购买。Protein A column purification of imidacloprid monoclonal antibody was prepared by Nanjing Agricultural University, School of Plant Protection, Pesticide Residues and Environmental Toxicology Laboratory; phage display random line dodecapeptide library was purchased from NEB Company.
主要试剂：Main reagents:
蛋白胨(OXOID)、酵母提取物(OXOID)、琼脂(Amresco)、琼脂糖(Amresco)、四甲基联苯胺(Sigma)、IPTG(Amresco)、Xgal(Amresco)、PEG8000(Sigma)、辣根过氧化物酶标记的抗M13单克隆抗体(GE)Peptone (OXOID), yeast extract (OXOID), agar (Amresco), agarose (Amresco), tetramethylbenzidine (Sigma), IPTG (Amresco), Xgal (Amresco), PEG8000 (Sigma), horseradish Oxidase-conjugated anti-M13 monoclonal antibody (GE)
主要试剂配方：Main reagent formula:
1、LB培养基：每升含10g蛋白胨，5g酵母提取物，5g NaCl，高压灭菌，室温贮存；1. LB medium: each liter contains 10g peptone, 5g yeast extract, 5g NaCl, autoclaved, stored at room temperature;
2、LB/IPTG/Xgal平板：LB培养基+15g/L琼脂粉。高压灭菌，冷却至低于70℃时，加入1mL IPTG/Xgal，混匀倒平板。平板4℃避光贮存；2. LB/IPTG/Xgal plate: LB medium + 15g/L agar powder. Autoclave, cool to below 70°C, add 1mL IPTG/Xgal, mix well and pour plate. Store the plate in the dark at 4°C;
3、顶层琼脂：每升含10g蛋白胨，5g酵母提取物，5g NaCl，7g琼脂粉。高压灭菌，固体培养基室温贮存，用时微波炉融化；3. Top agar: Each liter contains 10g peptone, 5g yeast extract, 5g NaCl, 7g agar powder. Autoclave, store the solid medium at room temperature, and thaw it in a microwave oven when used;
4、四环素贮液：以20mg/mL的浓度溶于50％乙醇中，-20℃避光贮存，用前摇匀；4. Tetracycline stock solution: dissolve in 50% ethanol at a concentration of 20mg/mL, store at -20°C in the dark, and shake well before use;
5、LB-Tet平板：LB培养基+15g/L琼脂粉。高压灭菌，冷却至低于70℃时，加入1mL四环素贮液，混匀倒平板，平板4℃避光贮存；5. LB-Tet plate: LB medium + 15g/L agar powder. Autoclave, when cooled to below 70°C, add 1mL of tetracycline stock solution, mix well and pour plate, and store the plate in the dark at 4°C;
6、封闭液：含有5％脱脂奶粉，0.14M，pH 7.4 PBS，过滤除菌，4℃保存；6. Blocking solution: containing 5% skimmed milk powder, 0.14M, pH 7.4 PBS, filtered and sterilized, stored at 4°C;
7、PEG/NaCl：20％(w/v)PEG-8000，2.5M NaCl，高压灭菌，室温贮存；7. PEG/NaCl: 20% (w/v) PEG-8000, 2.5M NaCl, autoclaved, stored at room temperature;
8、IPTG/Xgal配方：将1.25g IPTG(isopropylβ-D-thiogalactoside)和1g Xgal溶于25mL二甲基甲酰胺中，-20℃避光贮存；8. IPTG/Xgal formula: dissolve 1.25g IPTG (isopropylβ-D-thiogalactoside) and 1g Xgal in 25mL dimethylformamide, and store at -20°C in the dark;
9、显色液(四甲基联苯胺-H2O2底物溶液)：25mL 0.1 M，pH 5.5柠檬酸盐缓冲液中加入0.4mL，6mg/mL四甲基联苯胺，0.1mL 1％H2O2。9. Chromogenic solution (Tetramethylbenzidine-H2 O2 substrate solution): Add 0.4mL, 6mg/mL tetramethylbenzidine, 0.1mL 1% to 25mL 0.1 M, pH 5.5 citrate buffer H2 O2 .
实施例1吡虫啉抗体特异性结合多肽的淘选和制备Example 1 Panning and preparation of imidacloprid antibody specific binding polypeptide
1、与吡虫啉抗体特异性结合的噬菌体克隆的淘选1. Panning of phage clones that specifically bind to imidacloprid antibody
按照“吸附-洗涤-洗脱-扩增”的循环进行，经过3轮的淘选，具体操作如下：According to the cycle of "adsorption-washing-elution-amplification", after three rounds of panning, the specific operation is as follows:
(1)取100μL 100μg/mL蛋白A柱纯化的吡虫啉抗体加入酶标板中，4℃包被过夜，共三个孔；(1) Take 100 μL of imidacloprid antibody purified by a 100 μg/mL protein A column and add it to a microtiter plate, and coat at 4°C overnight, with a total of three wells;
(2)取少量大肠杆菌ER2738涂在LB+Tet平板上，37℃过夜培养；(2) Take a small amount of Escherichia coli ER2738 and spread it on the LB+Tet plate, culture overnight at 37°C;
(3)将步骤(1)的酶标板用PBST洗涤3遍，每孔加入300μL 5％脱脂奶粉，37℃孵育2小时，用PBST洗涤3遍，存放于4℃备用；(3) Wash the ELISA plate in step (1) 3 times with PBST, add 300 μL of 5% skimmed milk powder to each well, incubate at 37°C for 2 hours, wash 3 times with PBST, and store at 4°C for later use;
(4)将酶标板每孔加入100μL 2×1011的噬菌体(含5％脱脂奶粉)，室温轻微震荡1小时；(4) Add 100 μL of 2×1011 phage (containing 5% skimmed milk powder) to each well of the microtiter plate, and shake slightly at room temperature for 1 hour;
(5)取20mL LB培养基加入250mL三角瓶中，加入ER2738单菌落，37℃培养至OD600为0.01至0.05；(5) Take 20mL LB medium and add it to a 250mL Erlenmeyer flask, add a single colony of ER2738, and culture at 37°C until theOD600 is 0.01 to 0.05;
(6)将步骤(4)的微孔用PBST洗涤10遍，加入100μL 10μg/mL的吡虫啉进行洗脱，室温轻微震荡1小时；(6) Wash the microwells of step (4) 10 times with PBST, add 100 μL of 10 μg/mL imidacloprid for elution, and shake slightly at room temperature for 1 hour;
(7)收集步骤(6)中的洗脱缓冲液，加入到5％脱脂奶粉包被的孔，室温轻微震荡1小时，去除非特异性结合。收集上清液，4℃保存；(7) Collect the elution buffer in step (6), add to the wells coated with 5% skimmed milk powder, shake slightly at room temperature for 1 hour, and remove non-specific binding. Collect the supernatant and store at 4°C;
(8)取少量洗脱液测定噬菌体的滴度(操作方法见滴度测定)；(8) Take a small amount of eluate to measure the titer of the phage (see titer determination for the operation method);
(9)剩余的洗脱缓冲液用于扩增，将剩余的洗脱缓冲液加入步骤(5)中的三角瓶中，37℃摇床250rpm培养4至4.5小时；(9) The remaining elution buffer is used for amplification, and the remaining elution buffer is added to the Erlenmeyer flask in step (5), and incubated at 250 rpm at 37 ° C for 4 to 4.5 hours;
(10)将扩增后的噬菌体移入50mL的离心管中，4℃12000g离心25分钟，取上清。重复离心一次；(10) Transfer the amplified phage into a 50 mL centrifuge tube, centrifuge at 12000 g at 4° C. for 25 minutes, and take the supernatant. Repeat the centrifugation once;
(11)取上层80％的上清放入离心管中，加入上清1/6体积的20％PEG-8000/2.5MNaCl，混匀，4℃静置过夜；(11) Take 80% of the supernatant from the upper layer and put it into a centrifuge tube, add 1/6 volume of 20% PEG-8000/2.5MNaCl to the supernatant, mix well, and let stand overnight at 4°C;
(12)将(11)步骤的混合液4℃12000g离心15分钟，去上清，重复一次；(12) Centrifuge the mixture in step (11) at 12,000 g at 4°C for 15 minutes, remove the supernatant, and repeat once;
(13)取300μL PBS溶解步骤(12)的沉淀物用于下一轮的淘选，也可放于4℃短期(大约三周不会影响滴度)保存，或者加入甘油，放于-20℃长期保存；(13) Take 300 μL PBS to dissolve the precipitate from step (12) for the next round of panning, or store it at 4°C for a short period of time (about three weeks will not affect the titer), or add glycerol and store it at -20 ℃ long-term storage;
(14)步骤(1)到(13)为一轮淘选和扩增，第二轮和第三轮的淘选和扩增步骤同上，步骤(1)中使用的吡虫啉抗体浓度分别为50μg/mL和25μg/mL，步骤(6)中使用的吡虫啉浓度分别为5μg/mL和2.5μg/mL。(14) Steps (1) to (13) are one round of panning and amplification, the second and third rounds of panning and amplification steps are the same as above, and the concentration of imidacloprid antibody used in step (1) is 50 μg/ mL and 25 μg/mL, the imidacloprid concentrations used in step (6) were 5 μg/mL and 2.5 μg/mL, respectively.
噬菌体滴度测定操作步骤如下：The steps of phage titer determination are as follows:
(1)取4mL LB培养基，加入4μL 20mg/mL的四环素，取大肠杆菌ER2738单菌落加入其中，37℃培养至OD600为0.5；(1) Take 4 mL of LB medium, add 4 μL of 20 mg/mL tetracycline, add a single colony of Escherichia coli ER2738 into it, and culture at 37°C until theOD600 is 0.5;
(2)将LB/IPTG/Xgal平板放入37℃培养箱中预热1小时以上；(2) Put the LB/IPTG/Xgal plate into a 37°C incubator to preheat for more than 1 hour;
(3)取5mL融化的顶层琼脂(LB+7g/L琼脂糖)放入试管中，并保持试管温度在45℃；(3) Put 5 mL of melted top layer agar (LB+7g/L agarose) into a test tube, and keep the temperature of the test tube at 45°C;
(4)将需要测滴度的噬菌体进行稀释，通常洗脱缓冲液稀释10至103倍，扩增之后的噬菌体稀释108至1011倍；(4) Dilute the phage whose titer needs to be measured, usually the elution buffer is diluted 10 to 103 times, and the amplified phage is diluted 108 to 1011 times;
(5)取10μL稀释后的噬菌体加入到180μL步骤(1)的大肠杆菌培养液中，混匀；(5) Add 10 μL of the diluted phage to 180 μL of the E. coli culture solution in step (1), and mix well;
(6)将步骤(5)的混合液加入步骤(3)中的顶层琼脂中，混匀；(6) Add the mixed solution of step (5) to the top agar in step (3), and mix well;
(7)将步骤(6)的混合液均匀的加入到步骤(2)中的平板上，室温冷却，放入37℃培养箱中过夜培养；(7) Add the mixed solution of step (6) evenly to the plate in step (2), cool at room temperature, and culture overnight in a 37°C incubator;
(8)根据平板上蓝色斑点的数量来计算所测噬菌体的滴度。(8) Calculate the titer of the tested phage according to the number of blue spots on the plate.
整个淘选过程的洗脱噬菌体和扩增后的噬菌体的个数如表1所示。The numbers of eluted phages and amplified phages during the whole panning process are shown in Table 1.
表1与吡虫啉抗体结合的噬菌体展示多肽淘选情况(线十二肽库)Table 1 Panning situation of phage display peptides combined with imidacloprid antibody (line dodecapeptide library)
2、噬菌体克隆的筛选及其展示多肽序列的测定2. Screening of phage clones and determination of displayed polypeptide sequences
在完成最后一次淘选之后，对洗脱液进行滴度测定，选择蓝色斑点少于100个的LB/IPTG/Xgal平板，从中挑选出30个克隆用于扩增和鉴定。操作程序如下：After the last panning, the eluate was titrated, and LB/IPTG/Xgal plates with less than 100 blue spots were selected, and 30 clones were selected for amplification and identification. The operating procedure is as follows:
(1)用LB培养基将过夜摇培的大肠杆菌ER2738以1∶100稀释，加入到含有2ml培养基的48个试管中；(1) Dilute Escherichia coli ER2738 shaken overnight with LB medium at 1:100, and add to 48 test tubes containing 2ml of medium;
(2)从LB/IPTG/Xgal平板挑选出30个克隆放入试管中，37℃摇床培养4.5至5小时；(2) Select 30 clones from the LB/IPTG/Xgal plate and put them into test tubes, and culture them on a shaker at 37°C for 4.5 to 5 hours;
(3)将培养液4℃12000g离心10分钟，上清用于噬菌体酶联免疫分析(P-ELISA)验证(操作方法见P-ELISA)，沉淀用质粒提取试剂盒提取质粒，送测序公司进行序列测定。(3) Centrifuge the culture solution at 12,000g at 4°C for 10 minutes, and use the supernatant for phage enzyme-linked immunoassay (P-ELISA) verification (see P-ELISA for the operation method), extract the plasmid with a plasmid extraction kit for precipitation, and send it to the sequencing company for processing sequence determination.
噬菌体酶联免疫分析操作步骤：Phage ELISA operating steps:
(1)包被：用PBS缓冲液将吡虫啉抗体稀释后加入酶标板，每孔100μl，4℃孵育过夜；(1) Coating: Dilute the imidacloprid antibody with PBS buffer, add to the microtiter plate, 100 μl per well, and incubate overnight at 4°C;
(2)洗板：用洗涤液PBST(0.05％吐温20，0.01mol/L，pH 7.4)洗涤5次，吸水纸拍干；(2) Plate washing: wash 5 times with washing solution PBST (0.05% Tween 20, 0.01mol/L, pH 7.4), and pat dry with absorbent paper;
(3)封闭：每孔加入300μL 3％脱脂奶粉，37℃孵育2小时；(3) Blocking: add 300 μL 3% skimmed milk powder to each well, and incubate at 37°C for 2 hours;
(4)洗板：同(2)；(4) Plate washing: same as (2);
(5)加入分析物和噬菌体：每孔加入50μL PBS或50μL 10μg/mL吡虫啉标准品溶液，再加入50μL的噬菌体多肽，室温轻微震荡1小时，PBST洗涤5次，并平行设置阴性对照。(5) Add analyte and phage: Add 50 μL PBS or 50 μL 10 μg/mL imidacloprid standard solution to each well, then add 50 μL phage polypeptide, shake slightly at room temperature for 1 hour, wash with PBST 5 times, and set up negative controls in parallel.
(6)洗板：同(2)；(6) Plate washing: same as (2);
(7)加入酶标二抗：每孔加入100μL经1∶5000倍PBST稀释的辣根过氧化物酶标记的抗M13单克隆抗体，室温轻微震荡1小时；(7) Add enzyme-labeled secondary antibody: add 100 μL of horseradish peroxidase-labeled anti-M13 monoclonal antibody diluted 1:5000 times in PBST to each well, and shake gently at room temperature for 1 hour;
(8)洗板：同(2)；(8) Plate washing: same as (2);
(9)显色：每孔加入100μL现配制的显色液，37℃温箱孵育15分钟；(9) Color development: Add 100 μL of the newly prepared color development solution to each well, and incubate in a 37°C incubator for 15 minutes;
(10)终止：每孔加50μL 2mol/L的H2SO4溶液；(10) Termination: add 50 μL of 2mol/L H2 SO4 solution to each well;
(11)吸光度测定：用酶标仪测定450nm波长处的各孔吸光值。(11) Determination of absorbance: Measure the absorbance value of each well at a wavelength of 450 nm with a microplate reader.
挑选的30个噬菌体克隆中13个克隆在P-ELISA检测吡虫啉时的OD450值显著降低(图1)。将上述13个克隆的质粒送测序，测序引物为5’-CCCTCATAGTTAGCGTAACG-3’，测序结果共发现2种序列，其氨基酸序列如表2中SEQ ID NO 1～2所示。Among the 30 selected phage clones, the OD450 value of 13 clones was significantly reduced when detecting imidacloprid by P-ELISA (Figure 1). The plasmids of the above 13 clones were sent for sequencing, and the sequencing primer was 5'-CCCTCATAGTTAGCGTAACG-3', and two sequences were found in the sequencing results, and their amino acid sequences are shown as SEQ ID NO 1-2 in Table 2.
表2噬菌体展示多肽的氨基酸序列Table 2 Amino acid sequence of phage display polypeptide
3、P-ELISA对吡虫啉的检测3. Detection of imidacloprid by P-ELISA
3.1方法原理3.1 Method principle
采用间接竞争免疫分析方法。将吡虫啉抗体包被于96孔酶标板上，封闭后同时加入待测物和噬菌体多肽，免疫反应结束后，加入辣根过氧化物酶标记的抗M13单克隆抗体(只能与结合在固相抗体上的噬菌体结合)，随后加入酶反应底物，根据显色后的吸光值大小对待测物的含量进行分析；显色程度深浅与待测农药的浓度成反比，颜色越深，吸光值越高，待测物含量越低。因而可根据已知量农药的标准曲线和待检样品的吸光值，推算出待测农药的浓度。The indirect competitive immunoassay method was used. Coat the imidacloprid antibody on a 96-well ELISA plate, add the analyte and phage polypeptide at the same time after blocking, and add the anti-M13 monoclonal antibody labeled with horseradish peroxidase after the immune reaction Phage binding on the corresponding antibody), then add the enzyme reaction substrate, and analyze the content of the test substance according to the absorbance value after color development; the degree of color development is inversely proportional to the concentration of the pesticide to be tested, the darker the color, the absorbance value The higher the value, the lower the analyte content. Therefore, the concentration of the pesticide to be tested can be calculated according to the standard curve of the known amount of pesticide and the absorbance value of the sample to be tested.
3.2抗体和噬菌体展示多肽的工作浓度3.2 Working concentrations of antibodies and phage-displayed peptides
P-ELISA抗体和噬菌体展示多肽工作浓度的确定用方阵滴定法，选择OD值为1.0～2.0时的浓度，即抗体20μg/mL、SEQ ID NO 2噬菌体3×109pfu/mL为最适工作浓度。The working concentration of P-ELISA antibody and phage-displayed polypeptide is determined by square array titration, and the concentration when the OD value is 1.0-2.0 is selected, that is, the antibody is 20 μg/mL, and the phage of SEQ ID NO 2 is 3×109 pfu/mL. working concentration.
(1)包被：用PBS缓冲液将吡虫啉抗体稀释至20μg/mL加入96孔酶标板，每孔100μl，4℃孵育过夜；(1) Coating: Dilute imidacloprid antibody to 20 μg/mL with PBS buffer, add to 96-well microtiter plate, 100 μl per well, and incubate overnight at 4°C;
(2)洗板：用洗涤液PBST(0.05％吐温20，0.01 mol/L，pH 7.4)洗涤5次，吸水纸拍干；(2) Washing plate: wash 5 times with washing solution PBST (0.05% Tween 20, 0.01 mol/L, pH 7.4), and pat dry with absorbent paper;
(3)封闭：每孔加入300μL 3％脱脂奶粉，37℃孵育2小时；(3) Blocking: add 300 μL 3% skimmed milk powder to each well, and incubate at 37°C for 2 hours;
(4)洗板：同(2)；(4) Plate washing: same as (2);
(5)加入分析物和噬菌体：每孔加入50μL待测样品，再加入50μL，6×109pfu/mL的噬菌体多肽，室温轻微震荡1小时，PBST洗涤5次，并平行设置阳性对照和阴性对照。(5) Add analyte and phage: Add 50 μL of the sample to be tested in each well, then add 50 μL of 6×109 pfu/mL phage polypeptide, shake gently at room temperature for 1 hour, wash with PBST 5 times, and set up positive control and negative in parallel. control.
(6)洗板：同(2)；(6) Plate washing: same as (2);
(7)加入酶标二抗体：每孔加入100μL经1∶5000倍PBST稀释的辣根过氧化物酶标记的抗M13单克隆抗体，室温轻微震荡1小时；(7) Add enzyme-labeled secondary antibody: add 100 μL of horseradish peroxidase-labeled anti-M13 monoclonal antibody diluted 1:5000 times in PBST to each well, and slightly shake at room temperature for 1 hour;
(8)洗板：同(2)；(8) Plate washing: same as (2);
(9)显色：每孔加入100μL新鲜配制的显色液，37℃温箱孵育15分钟；(9) Color development: add 100 μL of freshly prepared color development solution to each well, and incubate in a 37°C incubator for 15 minutes;
(10)终止：每孔加50μL 2mol/L的H2SO4溶液；(10) Termination: add 50 μL of 2mol/L H2 SO4 solution to each well;
(11)吸光度测定：用酶标仪测定450nm波长处的各孔吸光值。(11) Determination of absorbance: Measure the absorbance value of each well at a wavelength of 450 nm with a microplate reader.
3.4标准曲线和灵敏度3.4 Standard curve and sensitivity
根据OD450值与吡虫啉浓度作图即得到标准曲线(图2)，计算抑制中浓度(IC50)及最低检测限(IC10，LOD)分别为0.067ng/mL，LOD为0.024ng/mL。The standard curve was obtained by plotting the OD450 value and the concentration of imidacloprid (Figure 2), and the calculated inhibitory concentration (IC50 ) and the lowest detection limit (IC10 , LOD) were 0.067 ng/mL and the LOD was 0.024 ng/mL.
3.5特异性3.5 Specificity
在最优免疫反应条件下，配制结构类似化合物的梯度标准溶液，建立结构类似化合物的标准曲线并计算交叉反应率(CR％)。交叉反应率越低，反应的特异性越强，对吡虫啉检测的干扰越小。P-ELISA除与氯噻啉有交叉反应外(CR％＝102％)，与其它新烟碱类杀虫剂没有比较明显的交叉反应。Under optimal immune reaction conditions, prepare gradient standard solutions of structurally similar compounds, establish a standard curve of structurally similar compounds and calculate the cross-reactivity rate (CR%). The lower the cross-reaction rate, the stronger the specificity of the reaction, and the less interference to the detection of imidacloprid. Except for cross-reaction with chlorothialine (CR%=102%), P-ELISA has no obvious cross-reaction with other neonicotinoid insecticides.
3.6样品添加检测3.6 Sample addition detection
3.6.1样品处理3.6.1 Sample processing
称取粉碎混匀后的麦粒、稻米、苹果、黄瓜和土壤样品10g，装入50mL离心管中，加入15mL含50％甲醇的PBS缓冲液混匀，涡旋5min，超声15min，再涡旋5min，4000rpm离心5min，将上清全部转移至25mL容量瓶内，用PBS缓冲液定容至25mL。再用PBS缓冲液稀释40倍用于检测。Weigh 10g of crushed and mixed wheat grains, rice, apples, cucumbers and soil samples, put them into a 50mL centrifuge tube, add 15mL of PBS buffer solution containing 50% methanol and mix well, vortex for 5min, sonicate for 15min, and vortex again Centrifuge at 4000rpm for 5min for 5min, transfer all the supernatant to a 25mL volumetric flask, and dilute to 25mL with PBS buffer. Then diluted 40 times with PBS buffer for detection.
3.6.2样品检测3.6.2 Sample testing
样品检测步骤参考3.3。经分析可知，该P-ELISA的吡虫啉回收率为70.1-102.1％，相对标准偏差为3.3-10.2％。Refer to 3.3 for sample detection steps. It can be seen from the analysis that the recovery rate of imidacloprid in the P-ELISA is 70.1-102.1%, and the relative standard deviation is 3.3-10.2%.
实际样品中吡虫啉残留量检测参照3.6方法进行。The detection of imidacloprid residues in actual samples shall be carried out according to the method in 3.6.
本发明建立的吡虫啉P-ELISA方法符合吡虫啉残留分析标准。该方法可用于环境和农产品中吡虫啉的残留检测。The imidacloprid P-ELISA method established by the invention meets the imidacloprid residue analysis standard. This method can be used for the detection of imidacloprid residues in the environment and agricultural products.
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| CN110627872A (en)* | 2019-05-08 | 2019-12-31 | 南京农业大学 | Phage-displayed polypeptides specifically bound by imidacloprid antibodies and uses thereof |
| CN114236113A (en)* | 2021-12-21 | 2022-03-25 | 南京农业大学 | 2, 4-drop instant immunosensor |
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| CN105153280B (en)* | 2015-07-15 | 2018-10-30 | 南京农业大学 | The polypeptide and application thereof of benzothiostrobin immunocomplex specific binding |
| CN104987361B (en)* | 2015-07-15 | 2020-02-14 | 南京农业大学 | Polypeptide specifically bound with benzothiostrobin antibody and application thereof |
| CN106749527A (en)* | 2016-12-28 | 2017-05-31 | 南京农业大学 | Phage-displayed polypeptides that imidaclothiz antibody specificity is combined and application thereof |
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| CN110627872A (en)* | 2019-05-08 | 2019-12-31 | 南京农业大学 | Phage-displayed polypeptides specifically bound by imidacloprid antibodies and uses thereof |
| CN114236113A (en)* | 2021-12-21 | 2022-03-25 | 南京农业大学 | 2, 4-drop instant immunosensor |
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