Vomitoxin anti-idiotype nano antibody as well as preparation method and application thereof

Abstract

The invention provides a vomitoxin anti-idiotype nano antibody as well as a preparation method and application thereof. The amino acid sequence of the vomitoxin anti-idiotype nano antibody is selected from any one of the following items: (a) an amino acid sequence as shown in SEQ ID NO: 1; and (b) an amino acid sequence formed by adding a tag sequence at one end or two ends of the SEQ ID NO: 1. The vomitoxin anti-idiotype nano antibody can be used as a substitute of vomitoxin, is applied to immunoassay detection of vomitoxin, is high in sensitivity and good in specificity, can be massively produced in a genetic engineering recombinant expression manner, and is simple to prepare.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a deoxynivalenol anti-idiotypic nano antibody and a preparation method and application thereof. Background Art

[0002] Deoxynivalenol (DON), also known as vomitoxin, belongs to the B-type trichothecenes, mainly produced by the Fusarium graminearum species complex, and is an important pathogen causing wheat fusarium head blight and corn ear rot. DON is toxic to animals and humans, including acute and chronic toxicity, cytotoxicity, skin toxicity, immunotoxicity, etc.; it has obvious embryotoxicity and teratogenic effects, and is classified as a Class III carcinogen by IARC. my country has clearly stipulated that the DON content in wheat, barley, corn and their products shall not exceed 1000μg / kg.

[0003] The detection methods of DON mainly include physical and chemical analysis methods and immunoassay methods. The physical and chemical analysis methods mainly include thin layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC), and near infrared spectroscopy (NIR). The above physical and chemical analysis methods all rely on expensive large-scale precision instruments, professional and skilled experimental personnel, and complex sample pretreatment, which limits the wide application of this method. Immunoassay methods include enzyme-linked immunosorbent assay (ELISA), fluorescent polar immunoassay, (fluorescent) immunochromatography technology, etc., which are based on the specific binding of antigen and antibody, and have the advantages of sensitivity, rapidity, specificity, and simplicity. In recent years, they have been widely used in the detection of vomitoxin. However, in some immunoassays, vomitoxin needs to be used as a coating antigen or competitive antigen, and some need to use vomitoxin as a calibrant and standard for calibration and preparation of standard curves, while the more toxic vomitoxin poses a potential threat to operators and the environment. Therefore, high-quality non-toxic substitutes to replace vomitoxin are a way to solve this problem, and also provide core reagent raw materials for the development of a green method for detecting vomitoxin (DON). Summary of the invention

[0004] In order to solve the problems existing in the background technology, the present invention provides an anti-idiotypic nanoantibody of vomitoxin and its preparation method and application. The anti-idiotypic nanoantibody of vomitoxin can replace vomitoxin as a competing antigen, coating antigen, calibrator or standard, etc., and is applied in the immunoassay of vomitoxin with high sensitivity and specificity.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] In a first aspect, the present invention provides a deoxynivalenol anti-idiotypic nanobody, the amino acid sequence of which is selected from any one of the following:

[0007] (a) the amino acid sequence shown in SEQ ID NO: 1;

[0008] (b) An amino acid sequence formed by adding a tag sequence, a restriction site sequence or a signal sequence to one or both ends of SEQ ID NO: 1.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned DON anti-idiotypic Nanobody.

[0010] In a third aspect, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecule.

[0011] In a fourth aspect, the present invention provides a host bacteria for transforming or transfecting the above-mentioned expression vector.

[0012] In a fifth aspect, the present invention provides a kit for detecting vomitoxin, comprising the above-mentioned vomitoxin anti-idiotypic nanoantibody.

[0013] In a sixth aspect, the present invention provides a method for preparing the above-mentioned DON anti-idiotypic nanoantibody, using the above-mentioned host strain to produce the DON anti-idiotypic nanoantibody, or using phage amplification to obtain phage particles displaying the DON anti-idiotypic nanoantibody.

[0014] In a seventh aspect, the present invention provides the use of the above-mentioned DON anti-idiotypic nanobody in immunoassay detection of DON.

[0015] According to the above scheme, the immunoassay detection includes enzyme-linked immunosorbent assay, fluorescent polar immunoassay, colloidal gold immunochromatography, time-resolved fluorescent immunochromatography, electrochemical immunosensor, biological immunosensor, nanoenzyme immunoassay, nanoimmunosensor immunospot hybridization and latex immunoturbidimetry.

[0016] According to the above scheme, the deoxynivalenol anti-idiotypic nanobody is used as a substitute for deoxynivalenol in the form of a competing antigen, a coating antigen, a calibrator, a quality control product or a standard product in immunoassay detection.

[0017] The beneficial effects of the present invention are:

[0018] 1) The present invention uses phage display technology to construct an alpaca-derived phage-displayed nanoantibody library, and through optimized affinity panning conditions, screens anti-idiotypic nanoantibodies that can specifically bind to vomitoxin monoclonal antibodies. The obtained vomitoxin anti-idiotypic nanoantibodies have good specificity and high sensitivity, and can replace vomitoxin as a competitive antigen, coating antigen, calibrator, quality control product or standard product in the field of immunoassay detection of vomitoxin. This method avoids the use of toxic pure toxins, is green, non-toxic, non-polluting, and is friendly to operators and the environment;

[0019] 2) The expression vector containing the deoxynivalenol anti-idiotypic nanobody encoding gene is transformed or transfected into the host bacteria, and after induced expression, separation, purification, and identification, a nanobody in the form of a soluble protein with good performance can be obtained. The deoxynivalenol anti-idiotypic nanobody of the present invention can be prepared in large quantities in the host bacteria, effectively reducing the production cost of the nanobody. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the test result for determining the diversity of the phage-displayed nanobody immune library in Example 1 of the present invention;

[0021] Figure 2 It is a positive clone identified by phage-ELISA in Example 2 of the present invention;

[0022] Figure 3 The binding of VHH DO-7 in Example 4 of the present invention to monoclonal antibodies against vomitoxin (DON), ochratoxin A (OTA), T-2 toxin (T-2), dihydrotoxin (DAS), fumonisin B1 (FB1) and aflatoxin B1 (AFB1);

[0023] Figure 4 The checkerboard method is used to optimize the coating concentration of VHH DO-7 and the working concentration of the DON monoclonal antibody in Example 5 of the present invention;

[0024] Figure 5 The cross-reaction of the VHH-ELISA based on VHH DO-7 in Example 5 of the present invention to deoxynivalenol (DON), ochratoxin A (OTA), T-2 toxin (T-2), dihydrotoxin (DAS), fumonisin B1 (FB1) and aflatoxin B1 (AFB1);

[0025] Figure 6 This is a schematic diagram of the structure of the colloidal gold immunochromatographic test strip prepared in Example 6;

[0026] Among them, the components represented by each number are as follows: 1 bottom plate; 2 water absorbent pad; 3 detection pad; 4 quality control line; 5 detection line; 6 gold label pad; 7 sample pad. DETAILED DESCRIPTION

[0027] The principles and features of the present invention are described below in conjunction with the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Anti-idiotype antibodies are specific antibodies produced against the antigenic determinants (idiotypes) in the variable region of antibodies. They are all antigenic, so anti-idiotype antibodies have the molecular basis to simulate the binding of natural antigens to corresponding antibodies, can block the binding of natural antigens to antibodies, and can be used as a substitute for natural antigens in immunoassays.

[0033] Heavy chain antibodies (HCAbs) are antibodies with only heavy chain dimers that exist in the camelid family (camels, dromedaries and llamas). Their antigen binding site consists of a domain, called the VHH region, so this type of antibody is also called a single domain antibody (sdAb). Since this type of antibody is the variable region sequence after the constant region is removed, the molecular weight is only 12 to 15 kD and the diameter is about 10 nanometers, it is also called a nanobody (Nbs). Compared with the scFv of conventional four-chain antibodies, nanobodies have advantages in solubility, stability, resistance to aggregation, foldability, expression yield, and ease of DNA manipulation, library construction and 3-D structure determination.

[0034] In order to obtain a substitute for vomitoxin used in immunoassay detection, the inventors constructed an alpaca-derived phage-displayed nanoantibody library through phage display technology, and screened out a unique nanoantibody VHH DO-7 that can specifically bind to the vomitoxin monoclonal antibody through optimized affinity panning conditions. Its amino acid sequence is as follows:

[0035] MKKTAIAIAVALAGFATVAQAAQVQLVESGGDLVQPGGSLRVSCAAS

[0036] GGNLDIDDMGWYRQPPGKQRELVATISRYGNTNYADYVKGRFTISRDNA

[0037] NNTVYLHMNSLKPEDTAVYYCNVGLYLNERSDRTEIWGQGTQVTVSSAH

[0038] HSEDPHGQAGQ (SEQ ID NO: 1)

[0039] The obtained DON anti-idiotypic nanoantibody VHH DO-7 has high sensitivity, and its IC 50 The value was 49.2645 ng / mL, and there was no cross reaction with ochratoxin A (OTA), T-2 toxin (T-2), diatoxin (DAS), fumonisin B1 toxin (FB1) and aflatoxin B1 (AFB1), with good specificity.

[0040] In the art, adding one or more amino acids at the C-terminus and / or N-terminus, such as adding a tag sequence, an enzyme cleavage site sequence, a signal sequence or a secretion signal sequence, generally does not change the function of the resulting protein. The sequence formed by adding a tag sequence, an enzyme cleavage site sequence or a signal sequence at one or both ends of the sequence shown in SEQ ID NO: 1 is also within the scope of protection of the deoxynivalenol anti-idiotypic nanobody of the present invention.

[0041] In a specific embodiment of the present invention, in order to facilitate the purification of the DON anti-idiotypic Nanobody, a histidine tag was added to the C-terminus of the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence of the DON anti-idiotypic Nanobody after adding the tag is shown in SEQ ID NO: 2:

[0042] MKKTAIAIAVALAGFATVAQAAQVQLVESGGDLVQPGGSLRVSCAASGGNLDIDDMGWYRQPPGKQRELVATISRYGNTNYADYVKGRFTISRDNANNTVYLHMNSLKPEDTAVYYCNVGLYLNERSDRTEIWGQGTQVTVSSAHHSEDPHGQAGQHHHHHHGAYPYDVPDYAS(SEQ ID NO:2)

[0043] Those skilled in the art may also select other tags according to actual needs.

[0044] The nucleic acid molecule encoding the above-mentioned deoxynivalenol anti-idiotypic nanobody VHH DO-7 is also within the scope of protection of this patent. The nucleic acid molecule of the present invention can be in the form of DNA or RNA, the DNA form includes genomic DNA, cDNA or artificially synthesized DNA, and the DNA can be a coding strand or a non-coding strand.

[0045] The expression vector containing the above nucleic acid molecule, and the host bacteria produced by genetic engineering using the vector of the present invention are all within the protection scope of the present invention.

[0046] The vomitoxin anti-idiotypic nanobody VHH DO-7 of the present invention can be prepared in large quantities by phage amplification or genetic engineering recombinant expression. Phage amplification refers to the production of phage particles displaying vomitoxin anti-idiotypic nanobody by biological amplification of phages that display anti-idiotypic nanobody. The genetic engineering recombinant expression method refers to the transformation or transduction of a suitable host bacterium with a recombinant expression vector containing the nucleic acid molecule, the host bacterium is cultured in a suitable culture medium and induced to express, and the nanobody in the form of soluble protein is separated and purified from the culture medium or bacteria.

[0047] In the present invention, the host bacteria can be any microorganism suitable for expressing the deoxynivalenol anti-idiotypic nanobody VHH DO-7, such as bacteria or fungi. In the following examples, Escherichia coli Top10F' is preferably used.

[0048] The present invention also provides the use of the above-mentioned vomitoxin anti-idiotypic nanoantibody in immunological detection and analysis. The immunological detection and analysis methods include enzyme-linked immunosorbent assay (ELISA), immunochromatography (colloidal gold immunochromatography, time-resolved fluorescence immunochromatography), fluorescence polarity immunoassay, immune dot hybridization, latex immunoturbidimetry, nanoimmunosensor, electrochemical immunosensor, biological immunosensor, nanoenzyme immunoassay and other immunological analysis detection types based on antigen-antibody specific reactions.

[0049] When used, the anti-idiotypic nanoantibodies of the present invention can be amplified by phage to obtain phage particles displaying the anti-idiotypic nanoantibodies of the vomitoxin, which can be directly used for analysis and detection. The anti-idiotypic nanoantibodies of the vomitoxin can also be expressed in prokaryotes or eukaryotes and then used as nanoantibodies in the form of soluble proteins for immunoassay detection.

[0050] Specifically, the anti-idiotypic nanoantibody of vomitoxin is used as a substitute for vomitoxin in the form of a competing antigen, a coating antigen, a calibrator, a quality control product or a standard product in immunoassay detection.

[0051] The amino acid sequence of the vomitoxin anti-idiotypic nanobody of the present invention can be used as a precursor and modified by random mutation or site-directed mutagenesis technology to obtain mutants with better properties (affinity, stability, etc.).

[0052] The deoxynivalenol anti-idiotypic nanobody of the present invention can be connected to various signal materials (such as fluorescent protein, fluorescein, enzyme, etc.) by chemical coupling or biological coupling, and used as a detection antigen in immunoassay.

[0053] The molecular biology experiments in the following examples include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., mainly with reference to "Molecular Cloning Laboratory Guide" (3rd Edition), edited by J. Sambrook et al., Science Press, and the specific experimental conditions can be determined by simple experiments when necessary. PCR amplification experiments are carried out according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier, and can be adjusted by simple experiments when necessary.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0055] Monoclonal antibodies against vomitoxin can be made at home or purchased commercially.

[0056] Example 1 Construction of phage-displayed nanoantibody immune library

[0057] 1. Alpaca Immunity

[0058] Weigh 200 μg of anti-vomitoxin monoclonal antibody (dissolved in PBS 7.4), emulsify with an equal volume of Freund's incomplete adjuvant, and inject subcutaneously into three-year-old male alpacas (Alpaca) at multiple points. After that, immunize once every two weeks for a total of 8 times. Blood collection and testing began after the fourth immunization. 7 to 10 days after the fourth immunization, 10 mL of blood was collected from the jugular vein using an EDTA vacuum blood collection tube. At the same time, the blood collection tube was gently inverted to avoid blood coagulation. The blood was then processed using the filter in the LeukoLOCK kit, and then the filter was rinsed with 3 mL of PBS buffer and 3 mL of RNAlater buffer in turn. The required white blood cells were retained in the filter. Finally, the filter was sealed and stored at -80°C for RNA extraction.

[0059] 2. Total RNA Extraction

[0060] Total RNA was isolated from alpaca blood according to the operating manual of the LeukoLOCK total RNA extraction kit from Life Technology. The specific steps are as follows:

[0061] (1) Add 70 μL of pH adjustment buffer to 2.5 mL of lysis / binding solution.

[0062] (2) Allow the sealed filter to return to room temperature, open the filter cap, and flush the remaining RNAlater buffer in the filter with a 2 mL syringe;

[0063] (3) Use a 2.5 mL syringe to draw 2.5 mL of freshly prepared lysis / binding buffer to rinse the filter once, and collect the effluent in a 15 mL RNAse-free centrifuge tube;

[0064] (4) Add 2.5 mL of nuclease-free ddH2O, vortex to mix, add 25 μL of proteinase K, and shake the centrifuge tube at room temperature at 250 g for 5 min;

[0065] (5) Take out the RNA-binding magnetic beads stored at 4°C, vortex to mix, and then pipette 50 μL into the above centrifuge tube, vortex the centrifuge tube, add 2.5 mL of isopropanol to the centrifuge tube, and shake at room temperature for 5 min;

[0066] (6) Centrifuge the above centrifuge tube at 3200g for 3 min, carefully aspirate and discard the supernatant, taking care not to aspirate the precipitated magnetic beads;

[0067] (7) Add 600 μL of washing solution I, repeatedly blow and beat the precipitated magnetic beads to make them evenly dispersed, transfer the suspension to a 1.5 mL centrifuge tube, rinse the 15 mL centrifuge tube once with 600 μL of washing solution I, and transfer it to the same 1.5 mL centrifuge tube;

[0068] (8) Centrifuge the 1.5 mL centrifuge tube at 16,000 g for 30 seconds and carefully discard the supernatant;

[0069] (9) Add 750 μL 2 / 3 of the washing solution, vortex vigorously for 30 seconds to disperse the aggregated and precipitated magnetic beads, centrifuge at 16000g for 30 seconds to collect the magnetic beads, and carefully discard the supernatant;

[0070] (10) Place the centrifuge tube with the lid open in a clean bench for 2 min to evaporate the alcohol remaining in 2 / 3 of the washing solution. During this time, prepare a TURBO DNase master solution, take 4 μL of TURBO DNase (20 U / μL) and add it to 296 μL of LeukoLOCK DNase buffer. After mixing evenly, transfer the mixture to the above centrifuge tube. Use a pipette to repeatedly blow the precipitated magnetic beads to disperse them evenly. Shake at 1000g for 10 min at room temperature, gently inverting several times during the process.

[0071] (11) Add 300 μL of lysis / binding solution (without pH adjustment buffer) and isopropanol to the above centrifuge tube, mix well, centrifuge for 2 seconds, and then incubate at room temperature for 3 minutes;

[0072] (12) Centrifuge at 16000g for 30s, discard the supernatant, add 750μL 2 / 3 of the washing solution, vortex vigorously for 30s, centrifuge at 16000g for 30s, discard the supernatant, add 750μL 2 / 3 of the washing solution again, vortex vigorously for 30s, centrifuge at 16000g for 1min, discard the supernatant as much as possible, and let it stand at room temperature for 3min to allow the residual washing solution to evaporate. Be careful not to leave it for too long to avoid excessive drying of the magnetic beads;

[0073] (13) Add 60 μL of elution buffer, vortex for 30 seconds, and centrifuge at 16,000 g for 2 minutes. Transfer the eluted RNA solution to another 1.5 mL centrifuge tube that does not contain nucleases. Take 1 μL of RNA solution and measure the concentration using nanodrop. Take about 3 μL of RNA solution for agarose gel electrophoresis analysis. Immediately invert the remaining RNA solution into cDNA to prevent degradation.

[0074] 3. Synthesis of cDNA

[0075] Synthesize the first strand of cDNA according to the instructions of the reverse transcription kit. The steps are as follows:

[0076] (1) Take two 200 μL nuclease-free PCR tubes, add 3 μL 10 mM dNTPmix, 3 μL 50 μM oligo(dT)20, and 24 μL RNA to each tube, and mix gently;

[0077] (2) Heating at 65°C for 5 min to denature the template and open up the secondary structure;

[0078] (3) Immediately cool on ice for at least 1 min;

[0079] (4) Prepare the premix for cDNA amplification:

[0080]

[0081] (5) Add 30 μL of premix to each of the two PCR tubes and mix thoroughly by pipetting;

[0082] (6) heating the reaction mixture: annealing at 50°C for 50 min, and then heating at 85°C for 5 min to terminate the reaction;

[0083] (7) Add 3 μL of RNAse H to each reaction mixture and mix well. Heat at 37°C for 20 min to decompose the incompletely reacted RNA.

[0084] (8) After the synthesis of the first-strand cDNA is completed, it is packaged and stored at -20°C for future use.

[0085] 4. Amplification of VHH gene of heavy chain antibody variable region

[0086] Degenerate primer pairs of IgG2 and IgG3 variable region VHH genes were used for polymerase chain reaction (PCR) amplification using cDNA as template. Primer pair F and R2 were used for cloning IgG2 subtype, and primer pair F and R1 were used for cloning IgG3 subtype. The reaction system was as follows:

[0087]

[0088] The PCR program is:

[0089]

[0090] The upstream primer corresponding to amplification of IgG2 and IgG3 is VHH-F, and the downstream primers are VHH-R2 and VHH-R1, respectively. The primer sequences are shown in Table 1.

[0091] Table 1 VHH antibody gene amplification and sequencing primer sequence list

[0092]

[0093] 5. Construction of phage display nanoantibody library

[0094] The vector pComb3X and VHH were digested with SfiI, and the VHH fragments after digestion were ligated with the vector pComb3X using T4 ligase. 3 μL of the ligation product was added to 25 μL of E. coli ER2738 competent cells. After gentle mixing, all the products were aspirated and transferred to a pre-cooled electrotransfer cup (with an inner diameter of 1 mm), and quickly placed in an electrotransfer instrument for electrotransformation. After electroporation, 1 mL of 37°C preheated SOC medium was immediately added to the electrotransfer cup, and the mixture was gently aspirated and mixed with a pipette, and the mixture was transferred to a shaking tube, and cultured at 250 g in a 37°C shaker for 1 h. The electroporation was repeated 10 times, with 3 μL of the ligation product each time. The bacterial liquid from 10 transformations was pooled and 1 μL was taken and diluted 10 times with sterile water, and then spread on an LB-Amp plate, and cultured in a 37°C incubator overnight for estimating the library capacity. Transfer all transformed bacteria to 200 mL SB medium, add carbenicillin to 50 μg / mL and tetracycline to 20 μg / mL; culture at 250 rpm and 37°C until OD 600 is 0.6; add 1mL of helper phage (1×10 13 pfu / mL), infect at 37°C for 30 min; 250g, culture at 37°C for 2h, add kanamycin to a concentration of 70μg / mL, and continue to culture overnight; the next day, centrifuge the bacterial solution, centrifuge at 4°C and 10000g for 15min; take the supernatant into a sterile centrifuge tube, add 1 / 4 volume of PEG / NaCl solution, ice bath for 2h, centrifuge again, discard the supernatant, and resuspend the precipitate with 10mL resuspension solution (PBS buffer containing 1× protease inhibitor, 0.02% NaN3, and 0.5% BSA); filter the phage solution with a 0.22μm filter to remove residual bacteria, etc.; the obtained phage solution is the phage display nanoantibody library, which is packaged, marked, and stored at -70°C.

[0095] 6. Identification of phage-displayed nanoantibody immune libraries

[0096] Pick 30 single clones from the plate and add them to 1 mL of SB medium. Cultivate at 37°C until the bacterial solution OD is 600 The bacterial solution was taken out and sent to the company for sequencing using gback as primer (sequence as shown in SEQ ID NO:6). The clone sequence was analyzed to determine the diversity of the library. Figure 1 As shown, the amino acid sequences of the inserted fragments of the selected 30 clones are all different, indicating that the constructed phage-displayed nanoantibody library has good diversity and can be used for subsequent screening work.

[0097] SEQ ID NO:6: 5'-GCCCCCTTATTAGCGTTTTGCCATC-3'

[0098] Example 2

[0099] 1. Selection of anti-idiotypic nanobodies against vomitoxin

[0100] Using the DON monoclonal antibody as the target, the concentration of the coating source and the concentration of the DON standard in competitive elution were reduced in succession, and the blocking reagent was used alternately to perform affinity enrichment panning to obtain antibodies against the DON monoclonal antibody, namely, anti-antibodies, also known as anti-idiotypic antibodies. The panning steps are as follows:

[0101] (1) Coating: Coating with DON monoclonal antibody, 50 μg / mL, 100 μL / well, 6 wells, diluting with coating buffer; de-adsorption wells, coating with 3% BSA / PBS, 300 μL / well, 6 wells; overnight coating at 4°C is better than incubation at 37°C for 2 hours;

[0102] (2) Blocking: 3% PBSTM, 300 μL / well, incubate at 37°C for 2 h, and wash the plate three times;

[0103] (3) Add the constructed phage display nanoantibody library to the coated antigen wells: 100 μL / well, react at 37°C for 1 h, then react on a shaker at room temperature for 1 h, then wash the 6 wells by hand with a filter tip, washing the plate 10 times;

[0104] (4) Elution: Prepare 100 ng / mL DON standard and react on a shaking table at room temperature for 30 min;

[0105] (5) Desorption: transfer the eluate to the desorption well, 100 μL / well, and react at room temperature for 1 h;

[0106] (6) 600 μL of the eluate is pooled and called the “1st output”, and the first round of panning is completed; the 1st output needs to be amplified before it can be used in the second round of panning;

[0107] (7) Determine the titer of "1st output", take 10 μL of "1st output", and dilute it to 10 3 , 10 4 , 10 5 , take 10 μL of each of the three dilutions to infect 90 μL ER2738 (OD is 0.8), let it stand at 37°C for 30 minutes, spread it on LB-Amp (ampicillin) plates, and culture it at 37°C overnight. The next day, count the number of single colonies on the plates and estimate the titer.

[0108] In the subsequent panning, the coating antibody concentration was gradually reduced, and the DON concentration in the eluate was gradually reduced. Five rounds of panning were performed. The panning process is shown in Table 2 , and the panning enrichment results are shown in Table 3 .

[0109] Table 2 Panning of phage nanobody library

[0110]

[0111] Table 3 Phage enrichment results in each round of panning

[0112]

[0113] 2. Screening of positive phage clones

[0114] (1) Randomly pick 30 single clones from the output titer plate of the last round of panning, incubate on LB-Amp plates, and inoculate with 3 mL of SB medium;

[0115] (2) Cultivate at 37°C in a shaking incubator for 4-5 h until the OD value reaches 0.8, add 30 μL of M13KO7 helper phage, and incubate at 37°C for 30 min;

[0116] (3) Incubate at 37°C in a shaking incubator for 1 h, add Kana at a final concentration of 70 μg / mL, and incubate at 37°C;

[0117] (4) The next day, 500 μL of bacterial solution was centrifuged at 5000 g for 10 min, and the supernatant was directly used for Phage-ELISA;

[0118] (5) ELISA plate blocking, 3% PBSTM, 300 μL / well, incubated at 37°C for 1 h, and machine washed 3 times;

[0119] (6) Add the phage supernatant of each clone to the corresponding three wells, 50 μL per well. Add 50 μL of standard to columns 1, 4, 7, and 10, and add 50 μL of 10% methanol / PBS buffer to columns 2, 3, 5, 6, 8, 9, 11, and 12. After adding, shake and mix well with a microplate reader.

[0120] (7) Incubate at 37°C for 1 h and wash the plate 10 times;

[0121] (8) Secondary antibody, Anti-M13 horseradish peroxidase, diluted 1:5000, incubated at 37°C for 1 h;

[0122] (9) Color development: incubate at 37°C for 15 min and measure the absorbance at 450 nm.

[0123] Phage-ELISA was performed on 30 clones, and it was found that their binding reactions with the DON monoclonal antibody were quite different. 450 The value is higher, and the OD after adding DON 450 The phage clones with significantly reduced values ​​were judged as positive. Figure 2As shown, 12 positive clones (clones 2, 7, 8, 17, 18, 19, 21, 22, 26, 28, 29, and 30, respectively) were obtained through five rounds of panning. According to the above ELISA results, the positive clones were picked from the bacterial plate, activated and cultured, and sent to Shanghai Bioengineering Company for sequence analysis. The sequencing primer was gback. The 12 positive clones were sequenced and the phage-displayed vomitoxin nanobody VHH DO-7 with the highest OD value was selected. It has a purification tag and the amino acid sequence is shown in SEQ ID NO: 2.

[0124] Example 3 Expression and purification of DON anti-idiotypic nanobodies

[0125] 1. Preparation of Top10F' competent cells

[0126] (1) Pick an appropriate amount of Top10F' and streak it on LB-tetracycline plate and culture it at 37℃ overnight;

[0127] (2) Pick out the Top10F' single clone and place it in 5 mL LB medium. Cultivate at 37°C and 250 rpm until OD 600 0.6~0.8;

[0128] (3) Place the bacterial solution on ice for 30 min;

[0129] (4) Centrifuge at 5000 g for 5 min at 4°C and quickly place back on ice. Discard the supernatant and add 1 mL of pre-cooled 0.1 M CaCl2 solution to resuspend the cells. Pipet and mix well and place on ice for 7 min.

[0130] (5) Repeat the above steps once;

[0131] (6) Centrifuge at 5000 g for 5 min at 4°C and quickly return to ice. Aspirate all the liquid and resuspend the pellet in 100 μL of pre-cooled 0.1 M CaCl2 solution to obtain Top10F' competent cells.

[0132] 2. Extraction and transformation of positive phage plasmid

[0133] (1) From the glycerol stock of VHH DO-7 / ER2738, streak an appropriate amount onto LB-ampicillin plates and culture at 37°C overnight;

[0134] (2) Pick a single colony of VHH DO-7 / ER2738 and place it in 2 mL of SB medium and culture at 37°C until OD 600 was 0.8, and the plasmid of VHH DO-7 was extracted;

[0135] (3) On ice, add 1.5 μL of VHH DO-7 plasmid to 100 μL of Top10F' competent cells prepared above, mix gently, and place on ice for 30 min;

[0136] (4) Heat shock at 42°C for 90 seconds, then quickly return to ice for 5 minutes;

[0137] (5) In a clean bench, add 700 μL of LB medium to each centrifuge tube and incubate at 37°C and 200 rpm for 45 min.

[0138] (6) Centrifuge at 12,000 g for 4 min at 4°C, discard the supernatant, add 100 μL LB medium to the centrifuge tube to resuspend the precipitate, mix well, spread on LB-ampicillin plates, and culture at 37°C overnight.

[0139] 3. Induced expression and purification of nanobodies

[0140] On the VHH DO-7 / Top10F' plate, pick a single colony into 3 mL SB medium, culture overnight at 37°C, 250 rpm in a shaking incubator, transfer 200 μL of overnight bacteria into 200 mL SB medium, and continue to culture until OD 600 The concentration of 1 μg / mL of 1 M IPTG was 0.6, and 200 μL of 1 M IPTG was added. The mixture was induced at 37°C overnight. The bacteria were collected by centrifugation at 4°C, 8000 g, and 15 min. According to the mass of bacterial precipitate, 20 mL / g of lysis buffer B-PER was added. After the precipitate was fully dispersed, it was slowly shaken at room temperature for 10 min, and then centrifuged at 12000 g for 20 min. The supernatant was the crude extract of the nanoantibody. The crude extract was dialyzed with 0.01 M PBS 7.4, and then filtered through a 0.22 μm aqueous filter membrane for nickel column purification.

[0141] Use Ni-NTA His·Bind Resin kit to purify the crude extract of nanoantibodies, the steps are as follows:

[0142] (1) Equilibrate the column with 10 column volumes of sterile ddH2O and 0.01 mol / L PBS 7.4;

[0143] (2) Add the filtered and sterilized crude extract to the chromatography column, mix with the filler, and shake at room temperature for 1 h to allow the nanoantibody to fully bind to the filler;

[0144] (3) Load the mixture into the column and equilibrate the column with 10 column volumes of 0.01 mol / L PBS 7.4;

[0145] (4) Prepare 20 mM, 40 mM, and 300 mM imidazole-PBS 7.4 (10 mL each) from 1 M imidazole, filter through a 0.22 μm aqueous filter membrane, use as the eluent for gradient elution, and collect the effluent in a 2 mL centrifuge tube;

[0146] (5) Pass 10 mL of 20 mM imidazole-PBS 7.4 through the column without collecting the effluent;

[0147] (6) Pass 10 mL of 40 mM imidazole-PBS 7.4 through the column and collect the first 3 mL of the effluent;

[0148] (7) Pass 10 mL of 300 mM imidazole-PBS 7.4 through the column and collect all the effluent;

[0149] (8) Wash the column with 10 column volumes of 1 M imidazole-PBS 7.4 without collecting the effluent to allow all nonspecifically bound proteins to be eluted;

[0150] (9) Wash the column with 10 column volumes of PBS, 10 column volumes of ddH2O, and 10 column volumes of 20% ethanol in sequence, and finally seal the column with an equal volume of 20% ethanol for storage.

[0151] The effluent from each tube was subjected to SDS-PAGE electrophoresis analysis, and the effluents with obvious nano-antibody bands were mixed, dialyzed in PBS buffer at 4°C overnight, and then concentrated using an ultrafiltration tube to obtain VHH DO-7 nano-antibody solution, which was packaged and stored at -20°C.

[0152] Example 4 Specificity analysis of anti-idiotypic nanobody VHH DO-7

[0153] (1) Prepare 0.2 μg / mL of DON-BSA, ochratoxin A antigen OTA-BSA, T-2 toxin antigen T-2-BSA, serpentine toxin antigen DAS-BSA, fumonisin B1 toxin antigen FB1-BSA and aflatoxin antigen AFB1-BSA with coating buffer, and coat the ELISA plate overnight at 4°C; the next day, block with 3% skim milk powder / regular phosphate buffer / regular phosphate Tween buffer solution, 300 μL / well, incubate at 37°C for 1 h, dilute six monoclonal antibodies against DON, OTA, T-2, DAS, FB1 and AFB1 starting from 10 μg / mL with regular phosphate buffer, and incubate at 37°C for 1 h; add goat anti-mouse monoclonal antibody labeled with horseradish peroxidase, and incubate at 37°C for 1 h; add color developing solution, develop at 37°C for 15 min, add stop solution, and measure OD 450 .

[0154] After coating and blocking according to the above method, the anti-idiotypic nanoantibody VHH DO-7 of DON was diluted three times with conventional phosphate buffer, and six monoclonal antibodies against DON, OTA, T-2, DAS, FB1 and AFB1 were prepared according to the above optimized concentrations. 50 μL of VHH DO-7 dilution and 50 μL of the corresponding monoclonal antibody were added to each well, and the microplate reader was shaken and mixed, and incubated at 37°C for 1 hour; then horseradish peroxidase-labeled sheep anti-mouse antibody was added at a dilution of 1:5000, and reacted at 37°C for 1 hour; color development was carried out in the same way as above, and OD was measured 450 value.

[0155] Test results such as Figure 3 As shown, Figure 3 The results showed that VHH DO-7 could inhibit the binding of DON-BSA monoclonal antibody to VON. The inhibitory effect became more and more obvious with the increasing concentration of VHH DO-7. However, it had no inhibitory effect on the binding of anti-OTA, T-2, DAS, FB1 and AFB1 monoclonal antibodies to the corresponding antigens, indicating that VHH DO-7 had good selectivity and specifically bound only to the variable region of DON-BSA monoclonal antibody.

[0156] Example 5 Establishment of ELISA (VHH-ELISA) method using anti-idiotypic nanoantibody VHH DO-7 of DON as a surrogate antigen for DON

[0157] The operation process of the competitive inhibition ELISA reaction of the anti-idiotypic nanoantibody VHH DO-7 against vomitoxin as a surrogate antigen for vomitoxin is as follows:

[0158] 1. VHH-ELISA steps

[0159] (1) The anti-idiotypic nanoantibody VHH DO-7 of DON was diluted and coated on the ELISA plate instead of the DON antigen at 4°C overnight; the next day, the plate was blocked with blocking solution and incubated at 37°C for 1 hour;

[0160] (2) Add methanol / PBS 7.4 buffer to the ELISA wells, dilute the DON monoclonal antibody and competitor with PBS 7.4 buffer and add them to the ELISA wells, and incubate at 37°C for 1 h;

[0161] (3) Add horseradish peroxidase-labeled goat anti-mouse antibody and incubate at 37°C for 1 h;

[0162] (4) Add colorimetric solution and react at 37°C for 15 min. Measure OD 450 .

[0163] 2. VHH-ELISA Condition Optimization

[0164] Optimal VHH coating concentration and DON monoclonal antibody working concentration: VHH DO-7 was diluted to concentrations of 20, 10, 5, and 1 μg / mL, respectively, and coated on the ELISA plate. The chessboard method was used to optimize the coating concentration of the nanoantibody VHH DO-7 and the DON monoclonal antibody working concentration, and the OD 450 The nanobody concentration and DON monoclonal antibody concentration with a value of about 1.0 were the optimal working concentrations. The DON monoclonal antibody was diluted 3 times to concentrations of 100.00, 33.33, 11.11, 3.70, 1.23, 0.41, and 0.14 μg / mL, respectively. Indirect ELISA analysis was performed at these concentrations to establish a standard curve ( Figure 4 ). According to the different color values ​​of each combination, the final working concentration of VHH DO-7 was selected as 5 μg / mL, and the corresponding concentration of DON monoclonal antibody was 8.39 μg / mL.

[0165] 3. Cross-reactivity rate of the anti-idiotypic nanoantibody VHH DO-7 against vomitoxin as a coating antigen in VHH-ELISA

[0166] Using VHH DO-7 as a surrogate antigen, six fungal toxins, DON, OTA, T-2, DAS, FB1 and AFB1, were selected as competitors according to the optimized conditions to determine the cross-reaction rate of VHH-ELISA. Figure 5 As shown. Figure 5 It can be seen that the ELISA has a high sensitivity to DON, and the IC 50 The value reached 49.2645 ng / mL, and there was no cross-reaction with OTA, T-2, DAS, FB1 and AFB1. VHH-ELISA had good specificity and could specifically detect vomitoxin.

[0167] Example 6 Establishment of colloidal gold immunochromatography (VHH-GICA) method using anti-idiotypic nanoantibody VHH DO-7 of vomitoxin as a surrogate antigen for vomitoxin

[0168] The establishment of the colloidal gold immunochromatography (VHH-GICA) method using the anti-idiotypic nanobody VHH DO-7 of DON as a surrogate antigen for DON includes the following steps:

[0169] 1. Establishment of colloidal gold immunochromatography (VHH-GICA) test strips using VHH DO-7 as a surrogate antigen for vomitoxin

[0170] The structure of the test strip is as follows Figure 6As shown, it includes a bottom plate 1 and a sample pad 7, a gold label pad 6, a detection pad 3 and a water absorbent pad 2 which are sequentially overlapped on the bottom plate 1. The detection pad 3 is provided with a detection line 5 and a quality control line 4. The detection line 5 is arranged on one end of the detection pad 3 close to the gold label pad 6, and the quality control line 4 is arranged on one end of the detection pad 3 close to the water absorbent pad 2. The detection line 5 is coated with VHH DO-7 prepared in Example 3, the quality control line is coated with rabbit anti-mouse polyclonal antibody, and the gold label pad 6 is combined with anti-vomitoxin monoclonal antibody labeled with nanogold. The specific preparation method is as follows:

[0171] (1) Preparation of water-absorbent pad 2

[0172] Cut the absorbent paper to obtain an absorbent pad 2;

[0173] (2) Preparation of detection pad 3

[0174] Coating of test line 5:

[0175] VHH DO-7 is prepared into 0.25-0.5 mg / mL coating solutions with coating buffer, which are coated on nitrocellulose membranes to obtain detection lines 5, and then dried at 37-40° C. for 30-60 minutes; the coating amount of VHH DO-7 required per centimeter on the detection line 5 of VHH DO-7 is 100-300 ng; the distance between the detection line 5 and the upper edge of the nitrocellulose membrane is 15-20 mm; and each 10 mL of the coating buffer contains: 0.08 g of sodium chloride, 0.002 g of potassium chloride, 0.002 g of potassium dihydrogen phosphate, 0.1-0.2 g of ovalbumin, and 0.029 g of disodium hydrogen phosphate dodecahydrate.

[0176] Coating of quality control line 4:

[0177] The rabbit anti-mouse polyclonal antibody was prepared into a coating solution of 0.2-0.4 mg / mL using coating buffer, and the solution was coated horizontally on the nitrocellulose membrane to obtain the quality control line 4. The coating amount of the rabbit anti-mouse polyclonal antibody required per centimeter of the quality control line 4 was 50-200 ng, and then dried at 37-40 for 1-2 hours; the coating buffer was the same as above.

[0178] (3) Preparation of sample pad 7

[0179] The glass fiber membrane is soaked in the blocking solution, taken out, and dried at 37-40°C for 6-10 hours to obtain a sample pad 7, which is then stored in a desiccator at room temperature; the blocking solution used contains 2-5 g sucrose, 0.8 g sodium chloride, 0.02 g potassium chloride, 0.02 g sodium azide, 0.02 g potassium dihydrogen phosphate, 1-2 g bovine serum albumin, and 0.29 g disodium hydrogen phosphate dodecahydrate per 100 mL;

[0180] (4) Preparation of gold label pad 6

[0181] The glass fiber membrane is soaked in a blocking solution, taken out, and dried at 37-40° C. for 6-10 hours, and the anti-DON monoclonal antibody solution labeled with nano-gold is sprayed horizontally on the dried glass fiber membrane in a spot spraying manner, wherein the amount of the anti-DON monoclonal antibody labeled with nano-gold required for each centimeter of spraying length is 100-200 ng, and then vacuum freeze-dried for 2-4 hours, and stored in a desiccator at room temperature;

[0182] (5) Assembly of test strips

[0183] The absorbent pad 2, the detection pad 3, the gold label pad 6 and the sample pad 7 are pasted on the bottom plate 1 from top to bottom, overlapping each other by 1 to 2 mm, so as to obtain an immunochromatographic test strip for detecting DON.

[0184] 2. Application of VHH DO-7 as a colloidal gold immunochromatography (VHH-GICA) test strip for the detection of vomitoxin (DON) content

[0185] It was determined that the detection limit of the test strip prepared above for vomitoxin was 5 ng / mL, which is far lower than the national standard of 500 ng / mL.

[0186] The specific detection method is as follows: after the sample extract is diluted with the detection liquid sample sustained-release solution, it is added dropwise to the sample pad of the immunochromatographic test strip for rapid detection of DON for detection, which is used as the detection test strip; another equal volume of the detection liquid sample sustained-release solution is taken as the negative control solution, and is added dropwise to the sample pad of another immunochromatographic test strip for rapid detection of DON, which is used as the control test strip. After 15 to 20 minutes, the detection test strip and the control test strip are subjected to color comparison. When the color of the test line on the test strip is close to the color of the corresponding test line on the control test strip, it indicates that the DON content in the test sample solution is lower than 5ng / mL; when the color is lighter than the corresponding test line on the control test strip, it indicates that the DON content in the test sample solution is equal to or higher than 5ng / mL and lower than 50ng / mL; when no color is shown, it indicates that the DON content in the test sample solution is equal to or higher than 50ng / mL; when the quality control line does not show color, regardless of whether the test line of the test strip is colored, the test strip is judged to be invalid; finally, the DON content in the test sample is obtained by conversion.

[0187] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A deoxynivalenol anti-idiotypic nanobody, characterized in that: The amino acid sequence of the Nanobody is selected from any one of the following: (a) the amino acid sequence shown in SEQ ID NO: 1; (b) An amino acid sequence formed by adding a tag sequence, a restriction site sequence or a signal sequence to one or both ends of the sequence shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the deoxynivalenol anti-idiotypic Nanobody according to claim 1.

3. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 2.

4. Transform or transfect the host bacteria with the expression vector as claimed in claim 3.

5. A kit for detecting vomitoxin, characterized in that: Comprising the vomitoxin anti-idiotypic nanobody of claim 1.

6. The method for preparing the anti-idiotypic nanobody of vomitoxin according to claim 1, characterized in that: The host bacteria of claim 4 are used to produce the anti-idiotypic nanobody of vomitoxin, or phage particles displaying the anti-idiotypic nanobody of vomitoxin are obtained by phage amplification.

7. Use of the anti-idiotypic nanobody of vomitoxin as claimed in claim 1 in immunoassay detection of vomitoxin.

8. The use according to claim 7, characterized in that: The immunoassay detection includes enzyme-linked immunosorbent assay, fluorescent polar immunoassay, colloidal gold immunochromatography, time-resolved fluorescent immunochromatography, electrochemical immunosensor, biological immunosensor, nanoenzyme immunoassay, nanoimmunosensor, immunospot hybridization and latex immunoturbidimetry.

9. The use according to claim 7 or 8, characterized in that: The deoxynivalenol anti-idiotypic nanobody is used as a substitute for deoxynivalenol in the form of a competitive antigen, a coating antigen, a calibrator, a quality control product or a standard product in immunoassay detection.

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