阅读说明：本技术 吡虫啉抗体特异性结合的噬菌体展示多肽及其用途 (Phage display polypeptide specifically bound by imidacloprid antibody and application thereof ) 是由 王鸣华 华修德 杜梅 丁园 陈贺 于 2019-10-10 设计创作，主要内容包括：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,以及该多肽在检测吡虫啉中的应用。本发明采用噬菌体展示技术,用蛋白A柱纯化的吡虫啉抗体对噬菌体展示随机线十二肽库进行3轮淘选,淘选出与吡虫啉抗体结合的噬菌体克隆；随机挑取若干噬菌体克隆进行扩增和质粒提取,通过噬菌体ELISA方法选择阳性噬菌体克隆,并将阳性克隆进行测序获得多肽序列。本发明还涉及该噬菌体展示多肽在吡虫啉检测中的应用。用该噬菌体展示多肽建立的噬菌体酶联免疫分析方法可用于快速、灵敏、简便和廉价检测环境和农产品中吡虫啉残留。(The invention belongs to the technical field of biology, and relates to a polypeptide specifically bound with an imidacloprid antibody, and application of the polypeptide in detection of imidacloprid. The invention adopts a phage display technology, and performs 3 rounds of panning on a phage display random linear dodecapeptide library by using an imidacloprid antibody purified by a protein A column, and panning phage clones combined with the imidacloprid antibody; randomly picking a plurality of phage clones for amplification and plasmid extraction, selecting positive phage clones by a phage ELISA method, and sequencing the positive clones to obtain a polypeptide sequence. The invention also relates to application of the phage display polypeptide in imidacloprid detection. The phage enzyme-linked immunoassay method established by the phage display polypeptide can be used for quickly, sensitively, conveniently and cheaply detecting imidacloprid residue in environment and agricultural products.)

1. A polypeptide having specific binding to an imidacloprid antibody, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 2.

2. The polypeptide having specific binding to imidacloprid antibody according to claim 1, which is linked to the M13 phage coat protein via GGGS.

3. The use of a polypeptide according to claim 1 having specific binding to an imidacloprid antibody for the detection of imidacloprid.

Technical Field

The invention belongs to the technical field of biology, and relates to a polypeptide specifically bound with an imidacloprid antibody, including application of the polypeptide in detection of imidacloprid.

Background

Imidacloprid is a nicotine ultra-high-efficiency pesticide and has multiple effects of contact poisoning, stomach toxicity, systemic absorption and the like. Since the wide, continuous and large-scale use of imidacloprid leads to the death of a plurality of natural enemies of pests, researches show that the imidacloprid has greater toxicity to the natural enemies of rice planthopper, namely lygus lucorum and aphid, namely propylaea japonica, and can cause damage to aquatic organisms, bees and beneficial organisms in soil. To prevent the potential risks associated with imidacloprid application, a sensitive, rapid, selective residual detection method is needed.

At present, the residue of imidacloprid is detected by mainly adopting an instrumental analysis method (such as liquid chromatography, gas chromatography-mass spectrometer and the like) to detect the residue of imidacloprid in agricultural products and agricultural environments, and the instrument has high detection sensitivity and strong accuracy, but has long relative time consumption and complex operation and can not meet the requirement of rapidly detecting a large number of samples. And a brand-new pesticide residue detection system provides higher standards and stricter requirements for pesticide residue detection and analysis technologies: the pesticide residue detection technology is not only promoted to develop towards a faster, simpler, more convenient, more sensitive and more reliable detection target, but also gradually changes the prior small amount of indoor detection in a laboratory into the current on-site timely detection and the large-batch and high-throughput detection in the laboratory. The immunoassay technology has the characteristics of simplicity, convenience, rapidness, low cost and reliability, greatly promotes the development of the rapid detection technology of pesticide residues, and provides an important tool for ensuring the quality safety of agricultural products and the ecological safety of the environment. Since small molecule chemicals such as pesticides are single epitope analytes and the whole molecule can only bind to one antibody, a competition mode is usually selected to establish an immunoassay method. In this format, a competitor must be present which competes with the analyte for binding to the antibody, and this competitor is usually prepared by linking the hapten to a protein, enzyme or fluorochrome, etc. The assay methods can be divided into homologous and heterologous immunoassays based on the structural homology of the competitor to the immunizing antigen. At present, pesticide heterologous competitors are generally prepared by adopting a chemical synthesis method, the chemical synthesis of a series of heterologous haptens and the coupling of the haptens and proteins, enzymes or fluorescent substances require a great deal of workload, and whether the prepared competitors have activity cannot be predicted, so that certain blindness exists.

The report of the autophagosome display technology has attracted the extensive attention of researchers, and becomes the most mature and widely applied antibody screening technology so far. The technology is widely applied to high-throughput screening of various functional recombinant polypeptides and antibodies. The principle is that DNA fragments of coding polypeptide or foreign protein are fused with coding genes of phage surface protein, and then are displayed on the surface of phage in the form of 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 direct connection between genotype and phenotype. A phage display library is formed by introducing a plurality of exogenous genes into a phage. When the phage library is panned with the target molecule, i.e., antibody, by 3 to 5 rounds of "adsorption-washing-elution-amplification", the phage specifically bound to the antibody is highly enriched. Recognition by a secondary antibody against phage, which is already commercially available, can be used directly as a competitor to establish a heterogeneous competitive immunoassay. Compared with a chemical synthesis competitor, the method has the advantages of simplicity, rapidness, safety, environmental protection and higher sensitivity. However, no research and report on the polypeptide specifically binding to the imidacloprid antibody and the application thereof has been found so far.

Disclosure of Invention

The invention aims to provide a polypeptide specifically bound with an imidacloprid antibody and application of the related polypeptide in imidacloprid detection.

The purpose of the invention is realized by the following technical scheme:

(1) coating an imidacloprid antibody purified by a protein A column on an enzyme label plate, sealing the enzyme label plate by using 5% skimmed milk powder, then adding a phage display random line dodecapeptide library into the enzyme label plate for affinity panning, wherein the panning process is carried out according to the steps of 'adsorption-washing-elution-amplification', and the dosage of the coated antibody and the imidacloprid dosage for competitively eluting the phage are sequentially reduced from the first round to the third round through 3 rounds of enrichment panning;

(2) after 3 rounds of panning, 30 phage clones are selected for ELISA preliminary identification, 13 obtained positive clones are amplified, extracted by plasmids and sequenced, 2 sequences are found in total, and the sequences are shown as SEQ ID NO 1-2: his Ser LeuTrp Met Ala Ser Pro Met Pro Gly Tyr and Gln Ile Phe Thr Ser Ser Pro Met Pro AlaMet Val.

The polypeptide can be specifically combined with an imidacloprid antibody to establish heterologous competitive ELISA (enzyme-linked immuno sorbent assay) for detecting imidacloprid residues in the environment and agricultural products.

The invention has the following beneficial effects: (1) the method is novel: is polypeptide which is firstly reported at home and abroad and is specifically combined with an imidacloprid antibody; (2) the method is practical: the phage display polypeptide provided by the invention can be used for quickly establishing high-sensitivity heterologous competitive ELISA; (3) the specificity is strong: the cross reaction between competitive ELISA established by the phage display polypeptide and the imidacloprid analogue is very low except for imidaclothiz (102 percent); (4) the accuracy is high: the addition recovery rate of competitive ELISA realized by using the phage display polypeptide in environment and agricultural samples is 70.1-102.1%, the variation coefficient is lower than 10.2%, and the competitive ELISA accords with the residue analysis standard; (5) the sensitivity is high: inhibition of competitive ELISA by phage display polypeptides provided herein₅₀) 0.067ng/mL, detection limit (IC)₁₀LOD) was 0.024 ng/mL.

Drawings

FIG. 1 shows the results of ELISA (P-ELISA) detection of 30 selected phage clones; the abscissa is the phage clone and the ordinate is OD₄₅₀A value;

FIG. 2 is a P-ELISA curve of OD value and imidacloprid concentration for detecting imidacloprid; the abscissa is the concentration of imidacloprid in ng/mL; ordinate is OD₄₅₀The value is obtained.

Detailed Description

The experimental materials, main reagents and formula used in the embodiment of the invention are as follows:

the main experimental materials:

the protein A column purified imidacloprid monoclonal antibody is prepared by Nanjing university of agriculture, plant protection academy, and pesticide residue and environmental toxicology laboratory; phage display random linear dodecapeptide library was purchased from NEB corporation.

The main reagents are as follows:

peptone (OXOID), yeast extract (OXOID), agar (Amresco), agarose (Amresco), tetramethylbenzidine (Sigma), IPTG (Amresco), Xgal (Amresco), PEG8000(Sigma), horseradish peroxidase-labeled anti-M13 monoclonal antibody (GE)

The main reagent formula is as follows:

1. LB culture medium: 10g peptone, 5g yeast extract, 5g NaCl per liter, autoclaved, stored at room temperature;

2. LB/IPTG/Xgal plates: LB medium +15g/L agar powder. Autoclaving, cooling to below 70 deg.C, adding 1mL IPTG/Xgal, mixing and pouring onto a plate. Storing the plate at 4 ℃ in the dark;

3. top agar layer: each liter contains 10g of peptone, 5g of yeast extract, 5g of NaCl and 7g of agar powder. Autoclaving, storing the solid culture medium at room temperature, and thawing with microwave oven;

4. tetracycline stock solution: dissolving in 50% ethanol at a concentration of 20mg/mL, storing at-20 deg.C in dark, and shaking before use;

5. LB-Tet plates: LB medium +15g/L agar powder. Autoclaving, cooling to below 70 deg.C, adding 1mL tetracycline liquid, mixing, pouring into flat plate, and storing at 4 deg.C in dark place;

6. sealing liquid: contains 5% skimmed milk powder, 0.14M PBS (pH 7.4), filtering for sterilization, and storing at 4 deg.C;

7. PEG/NaCl: 20% (w/v) PEG-8000, 2.5M NaCl, autoclaving, storing at room temperature;

8. IPTG/Xgal formulation: 1.25g of IPTG (isoproyl. beta. -D-thiogalactoside) and 1g of Xgal were dissolved in 25mL of dimethylformamide and stored at-20 ℃ in the dark;

9. color development liquid (tetramethyl benzidine-H)₂O₂Substrate solution): 25mL of 0.1M, pH 5.5 citrate buffer was added with 0.4mL of 6mg/mL tetramethylbenzidine, 0.1mL1％H₂O₂。