Nanometer antibody for detecting vomitoxin and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a nano antibody for detecting vomitoxin and application of the nano antibody. The DON nano antibody has the advantages of being small in molecular weight, high in stability, easy to prepare and modify and the like. The nano antibody can be specifically combined with deoxynivalenol (DON) and has high affinity to the deoxynivalenol, IC50 is 1.337 ng / mL, and LOD is 0.38 ng / mL. The DON nano antibody disclosed by the invention can be widely applied to detecting whether vomitoxin exists in food and feed or not, and has important significance on guaranteeing the safety of the food and the feed.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and in particular relates to a nano-antibody for detecting vomitoxin and an application thereof. Background Art

[0002] Deoxynivalenol (DON) is a secondary metabolite produced by the fungus Fusarium and widely found in contaminated grains such as wheat, corn, and barley. DON is a potent mycotoxin that can cause acute poisoning in animals and humans, and long-term exposure may also increase the risk of cancer. Therefore, rapid and sensitive detection of DON is crucial for ensuring food and feed safety.

[0003] Currently, the methods for detecting vomitoxin mainly include the following:

[0004] High-performance liquid chromatography (HPLC): Although highly sensitive, the equipment is expensive, the operation is complex, and professional personnel are required for sample pretreatment and analysis.

[0005] Enzyme-linked immunosorbent assay (ELISA): Although the operation is relatively simple, the detection time is long and the reagent cost is high.

[0006] Immunochromatographic test strips: Although simple and quick to operate, their sensitivity and specificity need to be improved.

[0007] Nanobodies are single-domain antibodies derived from camelids (such as camels and alpacas). Compared to traditional antibodies, nanobodies have higher sensitivity and specificity in toxin detection and are easily integrated into various detection platforms. However, there is currently a lack of high-affinity nanobodies targeting vomitoxin.

[0008] Therefore, there is an urgent need to develop a nanoantibody that can efficiently detect vomitoxin. Summary of the Invention

[0009] The object of the present invention is to provide a nanobody for detecting vomitoxin and its application. The DON nanobody provided by the present invention can specifically bind to vomitoxin and has a high affinity for vomitoxin.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] The present invention provides a nanobody for detecting vomitoxin, the amino acid sequence of the nanobody is shown in SEQ ID NO.1.

[0012] Preferably, the Nanobody can specifically bind to DON.

[0013] More preferably, the affinity of the Nanobody to DON is IC 50 It is 1.337ng / mL.

[0014] The present invention also provides a nucleotide molecule encoding the above-mentioned nanobody, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0015] The present invention also provides a kit for detecting vomitoxin, which comprises the above-mentioned nanoantibody.

[0016] The present invention also provides a recombinant plasmid, which contains the above nucleotide molecule.

[0017] The present invention also provides an expression strain, which comprises the above-mentioned recombinant plasmid.

[0018] The present invention also provides the use of the above-mentioned nanobody, the above-mentioned recombinant plasmid or the above-mentioned expression strain in the preparation of a reagent or kit for binding to vomitoxin.

[0019] The present invention also provides the use of the above-mentioned nanobody in detecting vomitoxin in food or animal feed.

[0020] Beneficial effects of the present invention:

[0021] The nanobody of the present invention has the advantages of small molecular weight, high stability, and easy preparation and modification. The nanobody can specifically bind to vomitoxin and has a high affinity for vomitoxin.

[0022] The nanoantibodies described in the present invention can be used to quickly and sensitively detect vomitoxin, which is of great significance for ensuring the safety of food and animal feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is the result of SDS-PAGE protein electrophoresis detection of DON nanobody;

[0025] Figure 2 3D visualization of the docking results of DON nanobody and DON small molecule;

[0026] Figure 3 This is the result of indirect competitive ELISA assay of DON nanobody sensitivity;

[0027] Figure 4 This is the competitive inhibition curve of DON nanobody and different fungal toxins;

[0028] Figure 5 This is a graph showing the thermal stability test results of DON nanobody;

[0029] Figure 6 This is the test result of DON nanobody's tolerance to different concentrations of methanol. DETAILED DESCRIPTION

[0030] The present invention provides a nanobody for detecting vomitoxin, the amino acid sequence (DON-AA) of the nanobody is shown in SEQ ID NO.1: EVQLQASGGGFVQPGGSLRLSCAASGQNPMRPTMGWFRQAPGKEREFVSAISPCVSSYSYYADSVKGRFTISRDNSKNTVYLQMNSLRAED TATYYCAQTRIRKLQRLLKKRPYWGQGTQVTVSS (SEQ ID NO.1).

[0031] The nucleotide sequence of the DON Nanobody is shown in SEQ ID NO.2: GAAGTTCAGCTGCAGGCAAGTGGCGGCGGCTTTGTGCAGCCGGG TGGCTCACTGCGCCTGAGCTGCGCTGCAAGTGGTCAGAATCCCATGCGACCGACAATGGGCTGGTTTCCGCCAGGCCCCGGGTAAAGAACGTGAATTTGTTAGCGCAATTAGCCCTTGTGTTTCGTCTTATTCTTATTATGCCGATAGTGTTAAAGGTCGTTTTACCAT TAGCCGCGATAATAGTAAAAATACCGTGTATCTGCAGATGAATAGTCTGCGTGCCGAAGATACCGCCACCTATTATTGTGCCCAGACAAGGATCCGTAAACTCCAGAGACTTCTTAAGAAAAGACCCTATTGGGGCCAGGGCACCCAGGTGACCGTGAGTAGCA(SEQ ID NO.2).

[0032] The nanobody of the present invention can specifically bind to vomitoxin. The DON nanobody of the present invention has a high affinity to vomitoxin. The IC 50The results of indirect competitive ELISA showed that the DON nanobody can specifically bind to vomitoxin; the thermal stability test results of the nanobody showed that the nanobody still had 58.6% residual activity after being incubated at 90°C for 75 minutes, indicating that the nanobody of the present invention has a high antigen binding ability and high thermal stability; the methanol tolerance test of the nanobody showed that the nanobody can still maintain a high activity in 80% methanol.

[0033] In some embodiments, the present invention further provides a recombinant plasmid comprising the nucleotide sequence of the Nanobody. The present invention further provides an expression strain, the host cell of which is preferably Escherichia coli, comprising the above-mentioned recombinant plasmid.

[0034] In some embodiments, the present invention also provides the use of the nanobody in the preparation of a reagent or kit for binding to vomitoxin, and the detection technology of the nanobody reagent or kit preferably includes one or more of the following methods: immunohistochemistry, Western Blotting, ELISA, immunofluorescence staining and colloidal gold immunoaffinity chromatography.

[0035] The reagent or kit of the present invention can be used to detect whether vomitoxin is present in food or animal feed, which is of great significance for ensuring the safety of food and feed.

[0036] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Unless otherwise specified, the production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art, and their names and / or abbreviations are conventional names in the field and are very clear and unambiguous in the relevant fields of use. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0038] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources and are all conventional products that can be purchased through regular commercial channels or prepared according to conventional methods well known to those skilled in the art.

[0039] Example 1 Screening and identification of nanobodies

[0040] 1.1 Acquisition and processing of camel blood lymphocytes

[0041] (1) Immunization of camels: Collecting blood lymphocytes from immunized camels;

[0042] (2) Blood lymphocyte separation: Blood lymphocytes were separated using density gradient centrifugation;

[0043] (3) Cell lysis: The isolated blood lymphocytes are lysed to release RNA.

[0044] 1.2 RNA extraction and reverse transcription

[0045] (1) RNA extraction: Total RNA was extracted from B cells using an RNA extraction kit according to the instructions.

[0046] (2) RNA purity and concentration detection: Use a UV spectrophotometer to detect the purity (A260 / A280 ratio between 1.8-2.0) and concentration of RNA.

[0047] (3) Reverse transcription: Use a reverse transcription kit to reverse transcribe RNA into cDNA for subsequent gene amplification.

[0048] 1.3 Amplification of Nanobody Variable Region Genes

[0049] Primer design: Specific primers were designed based on the conserved sequence of the variable region (VHH) of camel nanobodies.

[0050] PCR amplification: Use the designed primers and cDNA as template to perform PCR amplification to obtain VHH gene fragments.

[0051] PCR product detection: The size and purity of PCR products were detected by agarose electrophoresis.

[0052] 1.4 Gene cloning and nanoantibody synthesis library construction Gene insertion: The amplified VHH gene fragment is inserted into the phage display vector, and cloning is usually completed using restriction endonucleases and T4 DNA ligase.

[0053] Transformation into E. coli: The recombinant phage vector is transferred into the E. coli host bacteria for amplification to construct a phage library.

[0054] Library titer determination: The titer of the phage library was determined by plate culture method to ensure the diversity of the library.

[0055] 1.5 Screening of specific nanobodies

[0056] Antigen coating: Deoxynivalenol antigen is coated on a solid phase carrier (such as magnetic beads or ELISA plates).

[0057] Phage library inoculation: The constructed phage library is inoculated onto the antigen-coated carrier for the first round of screening (panning).

[0058] Elution and amplification: Wash away unbound phages, collect bound phages, amplify them and proceed to the next round of screening.

[0059] Multiple rounds of screening: Repeat the screening process to gradually improve the binding specificity and affinity of the nanoantibody to the antigen.

[0060] Monoclonal screening: Through monoclonal picking, nanoantibody monoclones that specifically bind to vomitoxin are obtained.

[0061] 1.6 Sequencing and verification of nanoantibodies

[0062] Gene sequencing: Sequence the nanoantibody genes obtained through screening to obtain their nucleic acid sequences.

[0063] Expression and purification: The sequence-verified gene is introduced into an expression system (such as E. coli cells) to express and purify the nanobody.

[0064] The results of SDS-PAGE protein electrophoresis were as follows: Figure 1 As shown, the molecular weight of the DON nanobody is 12 kDa, indicating that the DON nanobody obtained by the present invention has the characteristics of small molecular weight, high stability, strong penetration and high specificity.

[0065] Example 2 Molecular docking experiment of DON nanobody

[0066] The obtained DON nanobody amino acid sequence was entered into Discovery Studio 2019 software, and matching templates were searched for within the deduplicated database based on a 95% homology criterion. Modeling was performed using E-value, MapView, and bit score data, selecting templates with homology greater than 30%. (Modeling parameters: Number of Models = 20, Optimization Level = High).

[0067] After the model was built, the quality of the model was evaluated with the help of Ramachandran plots, and the model was further analyzed through the UCLA-DOE network platform.

[0068] The screened construction model was input into Discovery Studio 2019 software, and the CDOCKER algorithm was used to perform precise molecular docking between the nanoantibody protein and the DON toxin molecule. After docking, the docking configuration with the highest score was selected and molecular docking visualization was performed using Pymol, and the interaction force was analyzed.

[0069] The molecular docking results are as follows Figure 2 As shown, it shows that DON nanoantibodies have high affinity and can tightly bind to vomitoxin.

[0070] Example 2 Nanobody Sensitivity Detection

[0071] DON-BSA was diluted to 2 μg / mL with coating solution and coated on a 96-well ELISA plate. The plate was incubated at 37°C for 2 h. After washing the plate three times, 100 μL of BSA blocking solution was added for blocking treatment for 1 h. The plate was washed three times, and DON (0.125, 0.250, 0.500, 1.000, 1.125, 1.500, 2.000, 2.125 ng / mL) was diluted with 10% methanol / PBS and added to the ELISA plate. The plate was injected with the diluted solution to 1:5 0 ratio of DON nanoantibody, using a volume of 50 μL per well, the reaction process was carried out at 37 ° C for 1 hour; then, the ELISA plate was washed three times with washing solution, adding 100 μL each time, ensuring that 100 μL of HRP-labeled anti-His mouse monoclonal antibody was added to each well, and incubated at 37 ° C for another 1 hour; then, the ELISA plate was washed six times, and then TMB colorimetric agent was added, and the color reaction was carried out at 37 ° C for 15 minutes, and then 50 μL of stop solution was added to terminate the reaction. By measuring OD 450 The value of , and Graphpad Prism 10.4.0 software was used to draw the curve and then calculate the IC 50 value.

[0072] IC 50 The value represents the drug concentration required to inhibit 50% of biological processes (such as enzyme activity, cell proliferation, etc.). The lower the value, the more effective the drug is at a lower concentration and the stronger the effect.

[0073] The results are as follows Figure 3 As shown, the IC of nanoantibodies against DON 50 It is 1.337, indicating that the nanobody of the present invention is more sensitive to DON targeting.

[0074] Example 3 Nanobody specificity test

[0075] DON-BSA was diluted to 2 μg / mL using the coating solution to coat a 96-well ELISA plate and incubated in a 37°C incubator for 2 h. After washing the plate three times, 100 μL of BSA blocking solution was taken to block the plate for 1 h. The plate was washed three times and AFM1, AFB1, and OTA (0.02, 0.06, 0.18, 0.53, 1.53, 4.375, 12.5, 35, and 100 ng / mL) were diluted with 10% methanol / PBS. ) was added to the ELISA plate, and the DON nanobody diluted to a ratio of 1:50 was dispensed into each well, 50 μL per well, and the plate was incubated at 37°C for 1 hour; the plate was washed again with washing solution 3 times, 100 μL each time, and then 100 μL of HRP-labeled anti-His mouse monoclonal antibody was added to each well and incubated at 37°C for another 1 hour; after washing the plate 6 times, TMB color development solution was added, and after color development at 37°C for 15 minutes, 50 μL of stop solution was added to stop the reaction. OD was measured. 450 Graphpad Prism 10.4.0 was used to plot the curves and calculate the IC 50 , and calculated the cross-reaction rate.

[0076] Competitive inhibition curves of DON nanoantibodies and different fungal toxins Figure 4 shown.

[0077] The cross-reaction rate results are shown in Table 1, which shows that the nanoantibodies have no significant cross-reactions to other mycotoxins such as AFB1 and OTA, which are all <30%, and the cross-reaction rate with vomitoxin is as high as 100%.

[0078] Table 1 Cross-reaction rates of DON nanoantibodies with different mycotoxins

[0079]

[0080]

[0081] Example 4 Thermal stability test of nanobodies

[0082] DON-BSA was diluted to 2 μg / mL using coating solution and coated on a 96-well microtiter plate. The plate was then incubated in a 37°C incubator for 2 hours. After three washes, the plate was blocked with 100 μL of BSA blocking solution for 1 hour and washed again three times. The nanoantibody was then divided into five equal parts and placed in a PCR device. Each part was incubated for 5 minutes at the set temperatures of 25, 50, 65, 80, and 95°C. After the incubation period, the antibody was returned to room temperature. The nanoantibody was diluted to a ratio of 1:50, and its binding efficiency to the antigen was tested using an antibody specificity test. The thermal stability of the nanoantibody after heating at these different temperatures for 5 minutes was evaluated, with the binding rate of the unheated antibody as the benchmark of 100%.

[0083] DON-BSA was diluted to 2 μg / mL and coated on a 96-well microtiter plate. The plate was then incubated at 37°C for 2 hours. The washing step was repeated three times, followed by sealing the plate with 100 μL of BSA blocking solution for 1 hour and washing again three times. The nanoantibody was evenly divided into five portions and heated at 90°C for 5, 15, 30, 45, 60, and 75 minutes in different PCR equipment. After heating, the antibody was returned to room temperature. The nanoantibody was diluted to a concentration of 1:50 and its binding ability to the antigen was evaluated according to the antibody specificity detection procedure. The thermal stability of the nanoantibody after different heating times at 90°C was evaluated, with the binding rate of the unheated antibody as 100%.

[0084] The results are as follows Figure 5 As shown, the activity of the Nanobody was retained at 65.3 ± 2.5% after treatment at 90 °C for 30 min.

[0085] Example 5 Nanobody tolerance test to methanol

[0086] DON-BSA was diluted to 2 μg / mL with coating solution and coated on a 96-well ELISA plate, and incubated in a 37°C incubator for 2 h. After washing the plate three times, 100 μL of BSA blocking solution was injected for 1 h of blocking, and then the plate was washed again three times. Methanol was selected for antibody tolerance test, and methanol was diluted with 0.01 mol / L PBS (pH 7.4) to concentrations of 10%, 20%, 40%, 60%, and 80% to dilute the DON standard step by step. At the same time, the DON nanoantibody was diluted with PBS of the same concentration to a ratio of 1:50. 50 μL of the diluted standard and antibody were taken respectively and added to the pretreated 96-well ELISA plate in sequence. The remaining operations were consistent with the antibody sensitivity test process. The maximum absorbance value of the antibody and antigen binding at a wavelength of 450 nm was compared under different concentrations of organic solvent.

[0087] The results are as follows Figure 6 As shown, the nanobody can maintain high activity in 80% methanol.

[0088] Example 6 Detection of vomitoxin by DON nanobody

[0089] (1) Antibody coating: Dilute the nanobody to 5 μg / mL with coating buffer, add 100 μL to each well, and incubate at 4°C overnight or at 37°C for 2 hours. Discard the coating buffer and wash the plate three times with washing buffer (PBS containing 0.05% Tween-20), each for 5 minutes.

[0090] (2) Blocking: Add 200 μL of blocking solution (5% BSA) to each well and incubate at 37°C for 1 hour. Discard the blocking solution and wash the plate three times.

[0091] (3) Sample addition and incubation: Dilute the DON standard (DON concentration gradient: 0.1 ng / mL to 10 ng / mL) and the sample to be tested to the appropriate concentration, add 100 μL to each well, and incubate at 37°C for 1 hour. Discard the sample solution and wash the plate three times.

[0092] (4) Add enzyme-labeled secondary antibody: Add 100 μL of enzyme-labeled secondary antibody (HRP-labeled) to each well and incubate at 37°C for 1 hour. Discard the secondary antibody and wash the plate three times.

[0093] (5) Color development and termination: 100 μL of substrate solution (TMB) was added to each well. After color development at 37°C in the dark for 10 minutes, 50 μL of stop solution (2 M sulfuric acid) was added to each well to terminate the reaction.

[0094] (6) Reading: Use a microplate reader to measure the optical density (OD value) of each well at a wavelength of 450 nm.

[0095] (7) Standard curve drawing: Use the OD value of the standard to draw a standard curve (the horizontal axis is concentration, the vertical axis is OD value), and calculate the sample concentration based on the standard curve.

[0096] By diluting the standard, the concentration at a signal-to-noise ratio (S / N) of 3 was determined, and the minimum detection limit (LOD) reached 0.38 ng / mL.

[0097] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A nanobody for detecting vomitoxin, characterized in that: The amino acid sequence of the nanobody is shown in SEQ ID NO.

1.

2. The nanobody according to claim 1, characterized in that The nanobody can specifically bind to vomitoxin.

3. The nanobody according to claim 2, characterized in that The affinity IC of the nanobody to vomitoxin 50 It is 1.337ng / mL.

4. A nucleotide molecule encoding the Nanobody according to claim 1, characterized in that The nucleotide sequence of the nucleotide molecule is shown in SEQ ID NO.

2.

5. A kit for detecting vomitoxin, characterized in that: The kit comprises the nanobody according to claim 1.

6. A recombinant plasmid, characterized in that The recombinant plasmid comprises the nucleotide molecule according to claim 4.

7. An expression strain, characterized in that The expression strain comprises the recombinant plasmid according to claim 6.

8. Use of the Nanobody according to claim 1, the recombinant plasmid according to claim 6, or the expression strain according to claim 7 in the preparation of a reagent or kit for binding to vomitoxin.

9. Use of the nanobody according to claim 1 in detecting vomitoxin in food or animal feed.

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