CN110627872A

CN110627872A - Phage display polypeptide specifically bound by imidacloprid antibody and application thereof

Info

Publication number: CN110627872A
Application number: CN201910965913.XA
Authority: CN
Inventors: 王鸣华; 华修德; 杜梅; 丁园; 陈贺
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2019-05-08
Filing date: 2019-10-10
Publication date: 2019-12-31
Also published as: CN110256531A

Abstract

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.

Description

Phage display polypeptide specifically bound by imidacloprid antibody and application thereof

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。

Example 1 panning and preparation of Imidacloprid antibody-specific binding Polypeptides

1. Panning of phage clones specifically binding to Imidacloprid antibodies

The method is carried out according to a cycle of 'adsorption-washing-elution-amplification', and comprises 3 rounds of elutriation, wherein the specific operation is as follows:

(1) adding 100 mu L of imidacloprid antibody purified by 100 mu g/mL protein A column into an enzyme label plate, coating overnight at 4 ℃, and forming three holes in total;

(2) coating a small amount of Escherichia coli ER2738 on LB + Tet plate, and culturing at 37 deg.C overnight;

(3) washing the enzyme label plate in the step (1) for 3 times by using PBST, adding 300 mu L of 5% skimmed milk powder into each hole, incubating for 2 hours at 37 ℃, washing for 3 times by using PBST, and storing at 4 ℃ for later use;

(4) add 100. mu.L of 2X 10 enzyme label plate to each well¹¹The phage (containing 5% skimmed milk powder) was shaken gently at room temperature for 1 hour;

(5) adding 20mL LB culture medium into 250mL triangular flask, adding ER2738 single colony, and culturing at 37 deg.C to OD₆₀₀From 0.01 to 0.05;

(6) washing the micropores in the step (4) for 10 times by using PBST, adding 100 mu L of imidacloprid with the concentration of 10 mu g/mL for elution, and slightly shaking for 1 hour at room temperature;

(7) and (4) collecting the elution buffer in the step (6), adding the elution buffer into a hole coated with 5% skimmed milk powder, and slightly shaking for 1 hour at room temperature to remove non-specific binding. Collecting supernatant, and storing at 4 deg.C;

(8) taking a small amount of eluent to determine the titer of the phage (see titer determination for an operation method);

(9) adding the rest elution buffer solution into the triangular flask in the step (5) for amplification, and culturing for 4 to 4.5 hours at 37 ℃ by using a shaker at 250 rpm;

(10) the amplified phage were transferred to a 50mL centrifuge tube, centrifuged at 12000g at 4 ℃ for 25 minutes, and the supernatant was collected. Centrifuging repeatedly for one time;

(11) taking supernatant of 80% of the upper layer, putting the supernatant into a centrifuge tube, adding 20% PEG-8000/2.5M NaCl in the volume of 1/6% of the supernatant, uniformly mixing, and standing overnight at 4 ℃;

(12) centrifuging the mixed solution obtained in the step (11) at 12000g at 4 ℃ for 15 minutes, removing supernatant, and repeating the steps once;

(13) dissolving the precipitate of step (12) in 300 μ L PBS for the next round of panning, and storing at 4 deg.C for a short period (about three weeks without affecting titer), or adding glycerol and storing at-20 deg.C for a long period;

(14) steps (1) to (13) were one round of panning and amplification, and the second and third rounds of panning and amplification steps were as above, with imidacloprid antibody concentrations used in step (1) of 50. mu.g/mL and 25. mu.g/mL, respectively, and imidacloprid concentrations used in step (6) of 5. mu.g/mL and 2.5. mu.g/mL, respectively.

The phage titer determination procedure was as follows:

(1) 4mL of LB medium was added with 4. mu.L of 20mg/mL tetracycline, and E.coli ER2738 single colony was added and cultured at 37 ℃ to OD₆₀₀Is 0.5;

(2) putting the LB/IPTG/Xgal flat plate into a 37 ℃ incubator to preheat for more than 1 hour;

(3) 5mL of melted top agar (LB +7g/L agarose) was placed in a tube and the temperature of the tube was maintained at 45 ℃;

(4) the phage to be measured is diluted, usually with an elution buffer, from 10 to 10³Fold, phage dilution after amplification 10⁸To 10¹¹Doubling;

(5) adding 10 mu L of diluted phage into 180 mu L of Escherichia coli culture solution obtained in the step (1), and mixing uniformly;

(6) adding the mixed solution obtained in the step (5) into the top agar obtained in the step (3), and uniformly mixing;

(7) uniformly adding the mixed solution obtained in the step (6) onto the flat plate obtained in the step (2), cooling at room temperature, and putting the flat plate into an incubator at 37 ℃ for overnight culture;

(8) the titer of the phage tested was calculated from the number of blue spots on the plate.

The number of eluted phages and amplified phages throughout the panning process is shown in table 1.

TABLE 1 panning of phage display polypeptides conjugated to Imidacloprid antibody (Lindodecapeptide library)

2. Screening of phage clones and determination of displayed polypeptide sequences thereof

After the final panning was completed, the eluates were titered and LB/IPTG/Xgal plates with less than 100 blue spots were selected, from which 30 clones were picked for amplification and identification. The operation procedure is as follows:

(1) the overnight shaken E.coli ER2738 was diluted 1: 100 with LB medium and added to 48 tubes containing 2ml of medium;

(2) selecting 30 clones from LB/IPTG/Xgal plate, placing them into test tube, and shake-culturing at 37 deg.C for 4.5-5 hr;

(3) the culture broth was centrifuged at 12000g at 4 ℃ for 10 minutes, the supernatant was used for phage enzyme-linked immunosorbent assay (P-ELISA) verification (see P-ELISA for procedure), and the pellet was extracted with a plasmid extraction kit and sent to sequencing company for sequencing.

Phage enzyme-linked immunoassay operation steps:

(1) coating: diluting the imidacloprid antibody with PBS buffer solution, adding the diluted imidacloprid antibody into an ELISA plate, incubating at 4 ℃ overnight, wherein each well is 100 mu l;

(2) washing the plate: washing with washing solution PBST (0.05% Tween 20, 0.01mol/L, pH 7.4) for 5 times, and patting dry with absorbent paper;

(3) and (3) sealing: adding 300 mu L of 3% skimmed milk powder into each well, and incubating at 37 ℃ for 2 hours;

(4) washing the plate: the same (2);

(5) addition of analyte and phage: add 50. mu.L PBS or 50. mu.L 10. mu.g/mL imidacloprid standard solution to each well, add 50. mu.L phage polypeptide, shake gently for 1 hour at room temperature, PBST wash 5 times, and set up negative control in parallel.

(6) Washing the plate: the same (2);

(7) adding enzyme-labeled secondary antibody: adding 100 μ L of horseradish peroxidase-labeled anti-M13 monoclonal antibody diluted by PBST at a ratio of 1: 5000 times into each well, and slightly shaking for 1 hour at room temperature;

(8) washing the plate: the same (2);

(9) color development: adding 100 mu L of the prepared color development solution into each hole, and incubating for 15 minutes in an incubator at 37 ℃;

(10) and (4) terminating: adding 50 mu L of 2mol/L H into each hole₂SO₄A solution;

(11) and (3) measuring absorbance: the absorbance of each well at a wavelength of 450nm was measured using a microplate reader.

OD of 13 selected phage clones in P-ELISA for imidacloprid detection₄₅₀The values were significantly reduced (figure 1). The plasmids of the 13 clones are sequenced, the sequencing primer is 5'-CCCTCATAGTTAGCGTAACG-3', 2 sequences are discovered in the sequencing result, and the amino acid sequences are shown as SEQ ID NO 1-2 in the table 2.

TABLE 2 amino acid sequences of phage display polypeptides

3. P-ELISA for imidacloprid detection

3.1 principle of the method

An indirect competitive immunoassay method is used. Coating an imidacloprid antibody on a 96-hole enzyme label plate, adding an object to be detected and phage polypeptide at the same time after closing, adding an anti-M13 monoclonal antibody (only capable of being combined with phage combined on a solid phase antibody) marked by horseradish peroxidase after the immune reaction is finished, then adding an enzyme reaction substrate, and analyzing the content of the object to be detected according to the light absorption value after color development; the color development degree is inversely proportional to the concentration of the pesticide to be detected, and the deeper the color is, the higher the light absorption value is, and the lower the content of the substance to be detected is. Therefore, the concentration of the pesticide to be detected can be calculated according to the standard curve of the known pesticide and the light absorption value of the sample to be detected.

3.2 working concentrations of antibody and phage display polypeptide

The working concentration of the P-ELISA antibody and the phage display polypeptide is determined by a matrix titration method, and the concentration of the antibody with the OD value of 1.0-2.0, namely 20 mu g/mL of the antibody and 3 x 10 of the phage of SEQ ID NO 2 is selected⁹pfu/mL is the mostSuitable for working concentration.

(1) Coating: diluting the imidacloprid antibody to 20 mu g/mL by using PBS buffer solution, adding the diluted imidacloprid antibody into a 96-well enzyme label plate, incubating at 4 ℃ overnight, wherein each well is 100 mu l;

(4) washing the plate: the same (2);

(5) addition of analyte and phage: adding 50 μ L of sample to be tested into each well, and adding 50 μ L of 6 × 10⁹pfu/mL phage polypeptides, room temperature gentle concussion for 1 hours, PBST washing 5 times, and parallel set up positive control and negative control.

(6) Washing the plate: the same (2);

(7) enzyme-labeled antibody was added: adding 100 μ L of horseradish peroxidase-labeled anti-M13 monoclonal antibody diluted by PBST at a ratio of 1: 5000 times into each well, and slightly shaking for 1 hour at room temperature;

(8) washing the plate: the same (2);

(9) color development: adding 100 mu L of freshly prepared color development solution into each hole, and incubating for 15 minutes in an incubator at 37 ℃;

3.4 Standard Curve and sensitivity

According to OD₄₅₀Plotting the values against imidacloprid concentration to obtain a standard curve (FIG. 2), and calculating the Inhibitory Concentration (IC)₅₀) And minimum detection limit (IC)₁₀LOD) of 0.067ng/mL and LOD of 0.024ng/mL, respectively.

3.5 specificity

Under optimal immunoreactive conditions, a gradient standard solution of a structurally similar compound was prepared, a standard curve of the structurally similar compound was established and the cross-reactivity (CR%) was calculated. The lower the cross-reactivity, the stronger the specificity of the reaction and the less interference to the imidacloprid detection. In addition to cross-reacting with imidaclothiz (CR% ═ 102%), there was no significant cross-reaction with other neonicotinoid insecticides in the P-ELISA.

3.6 sample addition detection

3.6.1 sample treatment

Weighing 10g of crushed and uniformly mixed wheat grains, rice, apples, cucumbers and soil samples, putting into a 50mL centrifuge tube, adding 15mL of PBS buffer solution containing 50% methanol, uniformly mixing, whirling for 5min, performing ultrasonic treatment for 15min, whirling for 5min, centrifuging for 5min at 4000rpm, transferring all the supernatant into a 25mL volumetric flask, and fixing the volume to 25mL by using the PBS buffer solution. Then diluted 40-fold with PBS buffer for detection.

3.6.2 sample detection

Sample detection procedure reference 3.3. Analysis shows that the recovery rate of imidacloprid of the P-ELISA is 70.1-102.1%, and the relative standard deviation is 3.3-10.2%.

The detection of the residual quantity of imidacloprid in the actual sample is carried out according to the method 3.6.

The imidacloprid P-ELISA method established by the invention meets the imidacloprid residue analysis standard. The method can be used for detecting imidacloprid residue in environment and agricultural products.

Claims

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.