An anti-quercetin nanobody and application thereof

Abstract

The application discloses an anit-quinclorac nanobody and application thereof. The amino acid sequence of the nanobody NbFD4 is shown as SEQ ID No. 1. The nanobody NbFD4 still maintains more than 75% activity under the treatment of 30% organic solvent, pH=3.4-9.4 and 45 DEG C, has excellent thermal stability, and has higher resistance to denaturant (acid-base environment, organic solvent). The linear detection range of a detection quinclorac method established by using the nanobody is 3.40-47.38 ng / mL, the LOD value is 1.57 ng / mL, and there is no obvious cross (CR<1.5%) with other structural and functional analogues. The method has the characteristics of high sensitivity, strong specificity and simple operation. Therefore, the nanobody NbFD4 can be used for rapid and effective detection of quinclorac in environment and food samples, and has wide application prospect in detection of quinclorac.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to an antipyretic ester nanobody and its application. Background Technology

[0002] Fludioxonil, a phenylpyrrole fungicide, also known as fludioxonil or flufenoxuron, is widely used to control diseases such as root rot, gray mold, and bakanae disease in crops like soybeans, peanuts, and wheat because it interferes with bacterial biosynthesis and oxidation processes, dissolving the cell walls and causing bacterial death. However, improper use of fludioxonil can lead to its accumulation in the environment, causing ecological pollution of soil and water bodies. Studies have shown that fludioxonil has a slow-growth and slow-lethal effect on algae and fish, and long-term exposure may pose a potential chronic toxicity risk to humans.

[0003] Currently, the main methods for detecting fludioxonil residues include gas chromatography, gas chromatography-mass spectrometry (GC-MS), ultra-high performance liquid chromatography (UHPLC), and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). These methods provide accurate, reliable, and highly reproducible results, but require specialized personnel and sophisticated instruments, are complex to operate, and have long detection cycles, making them unsuitable for rapid on-site screening. In contrast, immunoassay methods based on antigen-antibody specific binding have attracted widespread attention in the field of food safety testing due to their sensitivity, speed, and high throughput, and show promising application prospects. However, research on immunoassay techniques for fludioxonil is limited, mainly focusing on monoclonal and polyclonal antibodies. These antibodies exhibit poor stability under extreme conditions and are prone to inactivation, which imposes significant limitations on sample pretreatment. Therefore, providing a novel, high-performance antibody with high stability and sensitivity is crucial for achieving rapid, sensitive, and accurate detection of fludioxonil. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an antipyretic ester nanobody.

[0005] A second object of the present invention is to provide the application of the aforementioned antipyretic ester nanobody.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention first provides an antipyretic nanobody, the amino acid sequence of which is shown in SEQ ID No. 1. The nanobody is named NbFD4.

[0008] The nanobody NbFD4 is prepared by conjugating a fluocinolone hapten with a carrier protein to obtain an artificial antigen, emulsifying the artificial antigen with an equal amount of Freund's adjuvant, immunizing Bactrian camels, and screening from a camel-derived nanobody library for nanobodies that specifically bind to fluocinolone. This nanobody is then mass-produced through recombinant expression via genetic engineering. The NbFD4 nanobody exhibits good resistance to organic solvents and acids / bases, excellent thermal stability and storage stability, overcoming the shortcomings of monoclonal antibodies such as poor acid / base resistance, organic solvent resistance, and thermal stability. Therefore, it can be widely used in the detection of fluocinolone.

[0009] The present invention provides a gene encoding the nanobody, the nucleotide sequence of which is shown in SEQ ID No. 9.

[0010] The present invention provides a recombinant vector containing a gene encoding the nanobody.

[0011] This invention provides a recombinant cell containing the aforementioned recombinant vector.

[0012] Since the present invention has provided the amino acid sequence of the nanobody NbFD4 and the gene sequence encoding the nanobody, those skilled in the art can obtain the nanobody described in this application using known recombinant DNA techniques. Therefore, any recombinant vectors or recombinant cells that can be used to prepare the nanobody described in this invention should also be within the scope of protection of this invention.

[0013] This invention provides the application of the above-mentioned nanobody in the detection of fludioxonil.

[0014] This invention provides the application of the nanobody, the gene, the recombinant vector, or the recombinant cell in the preparation of fluopyram detection products.

[0015] The present invention provides a kit for detecting fludioxonil, the kit containing the above-mentioned nanobody.

[0016] This invention provides a method for detecting fludioxonil, using an artificial antigen obtained by conjugating fludioxonil hapten with a carrier protein as the coating antigen, and the aforementioned nanobody as the detection antibody for detection.

[0017] Furthermore, the structural formula of the fluopyram hapten is shown in formula (I):

[0018]

[0019] Preferably, the fludioxonil hapten is obtained by hydrolyzing a hapten obtained by replacing the hydrogen atom at the nitrogen atom of the pyrrole ring with a 6-bromoalkane ester. This hapten does not change the skeletal structure of fludioxonil and is highly consistent with the structure of fludioxonil, which is beneficial for the induction of highly specific antibodies. The preparation method of the fludioxonil hapten is to react fludioxonil with ethyl 6-bromohexanoate, DMF and NaH to prepare an intermediate, and then hydrolyze the intermediate in the presence of LiOH to obtain the fludioxonil hapten shown in formula (I).

[0020] Furthermore, the structural formula of the artificial antigen is shown in formula (II):

[0021]

[0022] Furthermore, the artificial antigen is prepared by coupling the hapten FD-H1 with a carrier protein using the active ester method; the carrier protein is concanavalin A or ovalbumin.

[0023] Furthermore, the carrier protein of the coating antigen is ovalbumin.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention provides a nanobody against fluopyram, the amino acid sequence of which is shown in SEQ ID No. 1. The preparation method of the nanobody NbFD4 is simple, and it exhibits good tolerance to organic solvents and pH. It retains high activity in methanol and acetonitrile solutions with concentrations below 30%, and its activity is stable between pH 3.4 and 9.4. Furthermore, it possesses excellent thermal and storage stability, maintaining over 65% activity after treatment at 55°C and retaining over 75% activity after storage at 45°C for 15 days. The linear detection range of the method established using this nanobody for detecting fluopyram is 3.40–47.38 ng / mL, with a half-maximal inhibitory concentration (IC50) of 100%. 50 The concentration of the novel coronavirus was 12.68 ng / mL, the limit of detection (LOD) was 1.57 ng / mL, and there was no significant cross-contamination with other structural and functional analogs (CR < 1.5%). This method is characterized by high sensitivity, strong specificity, and simple operation. Therefore, the described nanobody NbFD4 can be used for rapid and effective detection of fluopyram in environmental and food samples, and has broad application prospects in the detection of fluopyram. Attached Figure Description

[0026] Figure 1 This is the mass spectrum of the fluopyram hapten FD-H1.

[0027] Figure 2 The image shows the UV absorption spectra of the fluopyram immunogen and the coating antigen.

[0028] Figure 3 This is a schematic diagram showing the amino acid numbering and structural domains of the nanobody NbFD4.

[0029] Figure 4 This is a standard curve for an indirect competitive ELISA based on the nanobody NbFD4.

[0030] Figure 5 Activity curves of the nanobody NbFD4 when different ratios of organic solvent / PBS were used as diluents.

[0031] Figure 6 The figure shows the results of the acid-base tolerance analysis of the nanobody NbFD4.

[0032] Figure 7 The figure shows the results of the thermal stability analysis of the nanobody NbFD4.

[0033] Figure 8 The figure shows the results of the storage stability analysis of the nanobody NbFD4. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] Example 1: Construction of a Bactrian camel immune antibody library

[0037] 1. Preparation of fludioxonil hapten FD-H1

[0038] Since fludioxonil is a small molecule and cannot elicit an immune response, it needs to be modified. A hapten is prepared and then coupled to a carrier protein to obtain an artificial antigen, which is then used for animal immunization. Coupling with the carrier protein requires the presence of active groups such as hydroxyl, amino, and carboxyl groups on the small molecule. Since fludioxonil does not possess these groups structurally, they need to be introduced artificially. Starting from the nitrogen atom on the pyridine ring of fludioxonil, a carboxyl group is introduced by reacting with ethyl 6-bromohexanoate, yielding the hapten FD-H1 with six carbon spacer arms. The specific method for preparing the fludioxonil hapten is as follows:

[0039] Weigh 248.18 mg (1 mmol) of fludioxonil, add 5 mL of anhydrous DMF and 96 mg (4 mmol) of NaH, and react at room temperature for 1 h. Add 2 mL of anhydrous DMF and 0.259 mL of ethyl 6-bromohexanoate (1.5 mmol) to a 4 mL centrifuge tube, mix thoroughly, and then add dropwise to the reaction mixture. React overnight at 50 °C. Filter the reaction mixture to remove solid NaH, add an appropriate amount of ice-water mixture, extract four times with ethyl acetate, combine the organic phases, wash four times with saturated brine, dry to anhydrous sodium sulfate, and rotary evaporate to obtain a viscous intermediate. Dissolve this intermediate in 20 mL of tertiary water, add 95.8 mg (4 mmol) of LiOH, and react overnight at 50 °C. After the reaction was complete, an appropriate amount of NaOH aqueous solution was added, and the mixture was extracted four times with ethyl acetate under alkaline conditions. The organic phase was discarded, and the aqueous phase was collected. The pH was adjusted to approximately 3-4 with 6M HCl, resulting in a white precipitate. The reaction solution was filtered, washed twice with ice water, and dried to obtain a white solid, which is the hapten FD-H1. The reaction equation is as follows:

[0040]

[0041] The structural formula of the fluopyram hapten FD-H1 is shown in Formula I:

[0042]

[0043] The mass spectrum of the fluopyram hapten FD-H1 is shown below. Figure 1 As shown in the mass spectrum, 360.8 is the negative ion molecular peak of the hapten FD-H1. Its relative molecular mass was calculated to be 361.8, which is consistent with the actual relative molecular mass (362.11), indicating that the fludioxonil hapten FD-H1 was successfully prepared.

[0044] 2. Preparation of fludioxonil complete antigens FD-H1-ConA and FD-H1-OVA (coating antigens)

[0045] The FD-H1 hapten was conjugated with the carrier proteins ConA and OVA. The specific steps were as follows: 0.03 mmol of the hapten FD-H1, 0.045 mmol of EDC, and 0.045 mmol of NHS were weighed and dissolved in 200 μL of DMF. The mixture was activated at 4°C with stirring in the dark for 12 h, and this solution was designated as solution A. 10 mg of the carrier proteins (ConA and OVA) were weighed and dissolved in 3 mL of 0.01 mol / L PBS buffer solution, and this solution was designated as solution B. Solution A was slowly added dropwise to solution B under magnetic stirring. After stirring and reacting at 4°C for 12 h, the mixture was transferred to a dialysis bag. The product was dialyzed against PBS at 4°C for 3 days, with the dialysate changed twice daily, thus obtaining the artificial antigen.

[0046] Successful conjugation was determined by comparing the changes in the ultraviolet absorption peaks of the hapten molecule, carrier protein, and artificial antigen. Results are as follows: Figure 2 The hapten FD-H1 exhibits characteristic absorption at 270 nm, while the characteristic absorption peaks of the artificial antigens FD-H1-ConA and FD-H1-OVA are at 280 nm and 270 nm, respectively. The peak shapes show a significant shift, indicating successful preparation of the artificial antigen. The structural formula of the fluopyram artificial antigen is shown in formula (II):

[0047]

[0048] 3. Bactrian camel immunization

[0049] Bactrian camels were immunized using the complete antigen FD-H1-ConA as the immunogen. The immunization protocol was as follows: For the first immunization, an equal volume of Freund's complete adjuvant was mixed with the immunogen. Subsequent booster immunizations were performed using Freund's incomplete adjuvant mixed with the immunogen, with each immunization interval of 2–3 weeks and a dose of 2 mg / camel. One week after each booster immunization, 10 mL of antiserum was collected for quality evaluation. Starting from the fourth immunization, 50–100 mL of peripheral blood was collected for each immunization to separate lymphocytes for later use.

[0050] 4. Isolation of lymphocytes

[0051] Peripheral blood from Bactrian camels was diluted with physiological saline at a ratio of 2:1. First, 15 mL of lymphocyte separation solution was added to a 50 mL sterile lymphocyte separation tube. Using a sterile Pasteur pipette, 15 mL of the diluted blood was slowly added dropwise along the tube wall. The tube was centrifuged at 850 g for 25 min at room temperature. The white, misty lymphocyte layer in the center was collected and transferred to a new centrifuge tube. An equal volume of physiological saline was added to wash the lymphocytes, and the tube was centrifuged at 1500 g for 15 min at 4 °C. The supernatant was discarded. 1 mL of cell lysis buffer (Trizol) was added to each lymphocyte sample, and the cells were thoroughly dispersed and collected in a 2 mL centrifuge tube. The samples were then stored at -80 °C for later use.

[0052] 5. Extraction of total RNA

[0053] Total RNA was extracted from the isolated and preserved lymphocytes using the RNA extraction kit (R4105) from Guangzhou Jiebes Biotechnology Co., Ltd. The specific method is as follows: The lysis buffer was removed and thawed. 0.2 mL of chloroform was added to each 1 mL of lysis buffer, and the mixture was gently shaken up and down for 15 seconds. The mixture was then incubated on ice for 5 minutes. After centrifugation at 12000 rpm for 10 minutes at room temperature, no more than 80% of the upper aqueous phase was collected and transferred to a new centrifuge tube. 0.7 volumes of anhydrous ethanol were added, and the mixture was thoroughly mixed. The precipitate and solution were then transferred to a GBC adsorption column. Sample adsorption, impurity washing, ethanol evaporation, and ddH2O elution were performed sequentially. 5 μL of RNA sample was taken for nucleic acid electrophoresis, and the RNA concentration was determined using a Nanodrop 2000C.

[0054] 6. cDNA Synthesis

[0055] Using the extracted RNA as a template, cDNA synthesis was performed according to the instructions of the Takara First-Strand Reverse Transcription Kit. The specific steps are as follows:

[0056] (1) According to the first step reaction system of cDNA synthesis shown in Table 1, mix the reagents in a nuclease-free centrifuge tube and operate under ice bath conditions;

[0057] Table 1. Reaction system for the first step of cDNA synthesis.

[0058] Total RNA 3μg <![CDATA[Oligo(dT) 18 first]]> 1μL <![CDATA[RNase free ddH2O]]> Up to 12μL

[0059] (2) After preparing the reaction system according to the table, incubate at 65℃ for 5 min, then cool in an ice bath;

[0060] (3) Add reagents to the system after the reaction in step (1) according to the second step reaction system of cDNA synthesis shown in Table 2;

[0061] Table 2. Reaction system for the second step of cDNA synthesis.

[0062]

[0063]

[0064] (4) After preparing the reaction system, incubate at 42℃ for 60 min for reverse transcription, and incubate at 70℃ for 5 min to terminate the reaction. Store the obtained cDNA at -80℃ for later use.

[0065] 7. Nanobody target gene amplification

[0066] Nested PCR was used to amplify the target gene, and the primers used are shown in Table 3.

[0067] Table 3. Primers for VHH gene amplification

[0068]

[0069] The first round of PCR amplification used cDNA as a template and CALL001 and CALL002 as primers. The amplification was performed in a PCR tube, and the reaction system and conditions are shown in Table 4. After the reaction, nucleic acid electrophoresis was performed, and the band around 750 bp was excised and recovered from the gel. The second round of amplification used the recovered product from the first round as a template and Fr1-SfiI and Fr4-SfiI as primers, following the reaction conditions in Table 4. After the reaction, nucleic acid electrophoresis was performed, and the 500 bp band was excised and recovered from the gel to obtain the VHH target gene fragment.

[0070] Table 4 Nested PCR amplification system and reaction conditions

[0071]

[0072]

[0073] 8. Document Library Construction

[0074] (1) Enzyme digestion of VHH target gene and vector

[0075] The VHH target gene and pComb3xss vector were digested with SfiI enzyme. Digestion conditions: 50℃ water bath for 16 h.

[0076] (2) Ligation of enzyme digestion products

[0077] The pComb3xss vector and the VHH fragment were mixed (molar ratio 1:3), and the ligation reaction was carried out at 16 °C for 16 h. The fragment was then cleaned and recovered using a kit.

[0078] (3) Electroconversion

[0079] Add 5 μL of the ligation product to 50 μL of electroporation competent E. coli TG1, mix gently, and then transfer to a 0.2 cm electroporation cuvette for electroporation (voltage 2 kV). Immediately add 1000 mL of preheated SOC medium at 37 °C to the electroporation cuvette and incubate at 37 °C and 250 rpm for 1 h. Spread the bacterial culture on LB-Amp plates and incubate upside down at 37 °C overnight.

[0080] 9. The Rescue of the Library

[0081] Inoculate cells at more than 10 times their library volume into 150 mL of LB-Amp medium and culture at 37°C and 250 rpm until the logarithmic growth phase (OD50). 600 nm (Approximately 0.4–0.6); add 1 mL M 13KO7 helper phage (multiplex ratio: 20:1) was used to infect *E. coli* at 37°C for 30 min, followed by incubation at 250 rpm for 1 h. Kanamycin was added to a final concentration of 50 μg / mL, and the mixture was incubated overnight at 37°C and 250 rpm. The mixture was centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected. 1 / 5 volume of PEG / NaCl was added, and the mixture was incubated on ice for 3 h. The mixture was then centrifuged again at 12000 rpm for 15 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in 1 mL of TBS. The precipitate was transferred to a 2 mL centrifuge tube, centrifuged at 12000 rpm for 5 min at 4°C, and filtered through a 0.22 μm polyethersulfone membrane. 10 μL of the solution was used to determine the volume, and the remainder was added to a final concentration of 50% glycerol and stored at -80°C.

[0082] Example 2: Affinity Panning and Identification of Nanobodies

[0083] 1. Affinity screening of nanobodies

[0084] (1) Immobilization of antigens and carrier proteins

[0085] For affinity screening, select microplates with strong adsorption capacity, selecting one column per round of coating, for a total of 4 rounds and 4 columns. The steps are as follows: Dilute the coating agent OVA to 1 mg / mL in wells AB, and dilute the coating agent FD-H1-OVA to 10 μg / mL in coating buffer in wells CDEF. Add 100 μL to each well and incubate overnight at 37°C. In addition, to avoid non-specific adsorption, separately coat one column with the immunogen carrier protein ConA at a concentration of 2 mg / mL. The next day, wash twice with PBST and blot dry. Add 120 μL of 1% fish collagen solution to each well, incubate at 37°C for 3 h, shake off the liquid in the wells, dry at 37°C for 1 h, and store at 4°C for later use.

[0086] (2) Positive phage adsorption

[0087] The phage library from Example 1 was added at 150 μL per well to two wells containing the immunovector protein (this step is only required for the first round; for the second, third, and fourth rounds, start directly from wells AB). The mixture was incubated at 37°C with shaking for 1 h. The free phages were then transferred to wells coated with the immunovector protein (wells AB) and incubated at 37°C with shaking for 1 h. The remaining free phages were transferred to three wells containing immobilized antigen (FD-H1-OVA), 100 μL per well. After incubation at 37°C with shaking for 1 h, the phages in the wells were discarded. The wells were then washed 10 times with 330 μL of PBST (the concentration of Tween-20 in PBST increased sequentially with each washing round, at 0.1%, 0.2%, 0.3%, and 0.4%), followed by 5 washes with 0.01 mol / L PBS.

[0088] (3) Eluting of positive phages

[0089] In the first round of elution, 100 μL of 10 mg / mL trypsin-TBS solution was added to the microwells after washing. The mixture was eluted at 37°C with shaking for 30 min. The eluted phages were collected, and 10 μL of the phages was used to determine the titer. The remaining phages were used to infect 5 mL of E. coli TG1 strain already in the logarithmic growth phase for amplification and subsequent elution. In the second, third, and fourth rounds of elution, the concentration of the original coating agent FD-H1-OVA was successively reduced to 1 μg / mL, 500 ng / mL, and 100 ng / mL. After repeating the adsorption operation and washing the microwells, the drug was added to the microwells for competitive elution. The mixture was incubated at 37°C with shaking for 1 h. The eluent was collected, and 10 μL of the solution was serially diluted and plated on LB-Amp plates for titer calculation. The remaining phages were amplified and cultured for the next round of elution. The concentration of fluopyram decreased sequentially during the screening process, reaching 1000 ng / mL, 500 ng / mL, and 100 ng / mL, respectively.

[0090] 2. Identification of positive clones

[0091] The evaluation of positive phage clones was performed using indirect competitive ELISA. The specific steps were as follows:

[0092] (1) Antigen immobilization

[0093] The antigen FD-H1-OVA was diluted to 1 μg / mL with coating buffer, 100 μL per well, and incubated overnight at 37°C. The next day, the plate was washed twice with PBST (0.01M PBS, 0.05% Tween-20), and then 120 μL of 2% skim milk powder solution was added to each well and incubated at 37°C for 3 hours. The liquid in the wells was poured out, patted dry on absorbent paper, dried at 37°C for 1 hour, and stored at 4°C for later use.

[0094] (2) Small-scale expression of nanobodies

[0095] After each round of titer calculation, 96 single colonies were randomly selected from each plate and inoculated into 96-well deep-well plates containing 0.5 mL of LB-Amp medium per well. A single TG1 colony was also inoculated as a negative control. The plates were sealed and incubated overnight at 37°C and 250 rpm. This plate served as the "mother plate." The next day, 10 μL of bacterial culture was taken from each well of the mother plate and inoculated into another deep-well plate containing 1 mL / well of LB-Amp medium. The plates were incubated at 37°C and 250 rpm for 4 hours until the logarithmic growth phase. IPTG was added to each well to a final concentration of 1 mmol / L, and the plates were incubated overnight with shaking. The next day, the deep-well plates were centrifuged at 4000 rpm for 10 min, and the supernatant was used for positive clone identification.

[0096] (3) Indirect competitive ELISA to identify positive clones

[0097] In pre-coated FD-H1-OVA microplates, add 50 μL / well of bacterial supernatant and 50 μL / well of PBS, respectively, and incubate at 37°C for 40 min. Wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of HRP-labeled anti-VHH secondary antibody diluted 1:5000, incubate at 37°C for 30 min, wash five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop color at 37°C in the dark for 10 min. Stop the reaction by adding 50 μL of stop solution (10% H2SO4, v / v), and measure the absorbance at 450 nm using a microplate reader. Select OD... 450 Phage clones with an OD value greater than 3 times that of the negative control well were considered positive clones. Add 50 μL of PBS or 50 μL of fluopyram standard (1 μg / mL) to the ELISA plate as the titer group and inhibition group, respectively. Then, add 50 μL of the supernatant of the above phage clones sequentially and calculate the inhibition rate. Select clones with an OD value greater than 3 times that of the negative control well in plate 1 and exhibit significant inhibition; record these as strain NbFD4. Transfer the bacterial culture from the corresponding well in the mother plate to sterile centrifuge tubes, add glycerol, and freeze for later use.

[0098] Example 3: Sequencing of the NbFD4 nanobody encoding gene and determination of its amino acid sequence.

[0099] 1. Experimental Methods

[0100] The strain of nanobody NbFD4, which was identified by indirect competitive ELISA, was sent to a sequencing company for sequencing to obtain the nucleotide sequence of nanobody NbFD4; based on the DNA sequencing results and codon table, the amino acid sequence of nanobody NbFD4 was obtained.

[0101] 2. Experimental Results

[0102] The amino acid sequence of VHH in the nanobody NbFD4 is shown below (SEQ ID No. 1):

[0103] EVQLEQSGGGSVQAGGSLRLSCAASGTSTSSFNCVGWFRQAPGKEREGVA AISTSSGSTYYADSVKGRFAISRDYAKRTVYLQMNSLKPEDIAMYYCAAIVGR GCLGSWPQAARYNYWGQGTQVTVSSGQAG.

[0104] The amino acid numbering and structural domain diagram of the nanobody NbFD4 are shown below. Figure 3As shown in the figure, the nanobody NbFD4 includes four framework regions (FRs) and three complementarity-determining regions (CDRs). The order of the four framework regions and the three complementarity-determining regions is FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0105] Among them, amino acid sequences 1-25 are FR1, with the amino acid sequence EVQLEQSGGGSVQAGGSLRLSCAAS (SEQ ID No. 2); amino acid sequences 26-33 are CDR1, with the amino acid sequence GSTSSFNC (SEQ ID No. 6); amino acid sequences 34-50 are FR2, with the amino acid sequence VGWFRQAPGKEREGVAA (SEQ ID No. 3); amino acid sequences 51-58 are CDR2, with the amino acid sequence ISTSSGST (SEQ ID No. 7); amino acid sequences 59-96 are FR3, with the amino acid sequence YYADSVKGRFAISRDYAKRTVYLQMNSLKPEDIAMYYC (SEQ ID No. 4); amino acid sequences 97-116 are CDR3, with the amino acid sequence AAIVGRGCLGSWPQAARYNY (SEQ ID No. 4). No. 8); the amino acid sequence from position 117 to 131 is FR4, and its amino acid sequence is WGQGTQVTVSSGQAG (SEQ ID No. 5).

[0106] The nucleotide sequence of the nanobody NbFD4 is shown below (SEQ ID No. 9):

[0107] gaggtgcagctggagcagtctgggggaggctcggtgcaggctggagggtctctcagactctcctgtgcagcctctggatcgaccagcagtttcaactg cgtgggctggttccgccaggctccagggaaagagcgcgagggggtcgcagctattagtactagtagtggtagcacatactatgccgactccgtgaaggg ccgattcgccatctcccgagactacgccaagagaacggtgtatctgcaaatgaacagcctgaaacctgaggacattgccatgtactactgtgcggcaatagtggggcgcggttgtcttggttcatggccccaagcggctcggtataactactggggccaggggacccaggtcaccgtctcctcaggccaggccggcc.

[0108] Example 4: Large-scale preparation of the nanobody NbFD4

[0109] This invention prepares large quantities of the nanobody NbFD4 in the form of protein expression. The specific method is as follows:

[0110] The plasmid of the nanobody strain NbFD4 was extracted using a kit and transformed into E. coli BL21(DE3) using chemical transformation. A single colony was picked from the transformation plate and inoculated into 10 mL of LB (containing Amp) medium and incubated overnight at 37°C and 250 rpm. The overnight culture was then inoculated at a ratio of 1:100 into 750 mL of LB (Amp) medium and incubated at 37°C and 250 rpm until OD (open-circuit retrieval). 600nm When the bacterial growth rate reaches approximately 0.4–0.6, IPTG (1:1000 ratio, v / v) is added, and the cells are incubated overnight at 37°C and 250 rpm. The next day, the cells are centrifuged at 4°C and 12,000 rpm for 5 min, and the precipitate is collected. The precipitate is then centrifuged at 12,000 rpm for 10 min using the sucrose osmotic pressure freeze-thaw method. The supernatant is collected and purified by affinity chromatography to obtain the expressed specific nanobody NbFD4.

[0111] Example 5: Detection of fluopyram using nanobody NbFD4

[0112] 1. Wrapping and sealing

[0113] Dilute the FD-H1-OVA coating agent to 1 μg / mL with coating buffer and coat overnight at 37°C. The next day, wash the plate twice with PBST (0.01M PBS, 0.05% Tween-20 (v / v)), add 1% fish collagen solution, 120 μL per well, block at 37°C for 3 h, discard the blocking solution, dry at 37°C for 1 h, and store in a sealed bag at 4°C for later use.

[0114] 2. Detection of fludioxonil

[0115] (1) Experimental methods

[0116] The FD-H1-OVA coating agent was diluted to 1 μg / mL with coating buffer and coated overnight at 37°C. The next day, the plate was washed twice with PBST (0.01M PBS, 0.05% Tween-20 (v / v)), and 120 μL / well of 2% skim milk powder solution was added. The plate was blocked at 37°C for 3 h, the blocking buffer was discarded, and the plate was dried at 37°C for 1 h. Add 50 μL of nanobody and a series of different concentrations of 50 μL of fludioxonil to each well, incubate at 37°C for 40 min, wash five times with PBST, blot dry the liquid in the wells, add 100 μL of HRP-labeled anti-VHH secondary antibody diluted 1:5000, incubate at 37°C for 30 min, wash five times with PBST, blot dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop color at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4, v / v) to stop the reaction; read the absorbance at 450 nm using a microplate reader. Plot the OD of the wells with fludioxonil concentration as the x-axis, and the ratio of B / B0 (fludioxonil) as the OD. 450 OD of pores without fluopyram 450 Using the vertical axis as the ordinate, establish an indirect competition standard curve.

[0117] (2) Experimental Results

[0118] The indirect competitive ELISA standard curve based on nanobody NbFD4 is shown in the figure. Figure 4 As shown in the figure, the standard curve exhibits an S-shape, indicating good linear correlation. The linear detection range for fluopyram is 3.40–47.38 ng / mL, and the half-maximal inhibitory concentration (IC50) is [not specified]. 50 The concentration was 12.68 ng / mL, and the limit of detection (LOD) was 1.57 ng / mL.

[0119] Example 6: Organic Tolerance Analysis of the Nanobody NbFD4

[0120] 1. Experimental Methods

[0121] The nanobody NbFD4 was diluted to the same working concentration using mixtures of methanol and PBS at different concentrations (10%, 20%, 30%, 40%, 50%) and mixtures of acetonitrile and PBS at different concentrations (10%, 20%, 30%, 40%, 50%) as diluents to determine the antibody-antigen binding ability. The binding ability of the antibody to the antigen without dilution with organic solvents was taken as 100%. The tolerance of the nanobody to methanol and acetonitrile was evaluated. The specific method is as follows:

[0122] Add 50 μL of diluted NbFD4 nanobody and 50 μL of PBS to the pre-packaged ELISA plate, incubate at 37°C for 40 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of HRP-labeled anti-VHH secondary antibody diluted 1:5000, incubate at 37°C for 30 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of LMB substrate solution, and develop the color at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance at 450 nm using an ELISA reader.

[0123] 2. Experimental Results

[0124] The activity curves of the nanobody NbFD4 with different ratios of organic solvent / PBS as diluents are shown in the figure. Figure 5 As shown in the figure, the activity of the nanobody NbFD4 gradually decreases with increasing organic solvent concentration, but it still has more than 80% activity in 30% methanol / PBS solution and more than 75% activity in 30% acetonitrile / PBS solution. This indicates that the nanobody NbFD4 has good tolerance to methanol and acetonitrile. In the pretreatment process of actual sample detection, methanol / acetonitrile solution with a concentration of less than 30% can be added.

[0125] Example 7: Activity determination of nanobody NbFD4 under different pH conditions

[0126] 1. Experimental Methods

[0127] 0.01M PBS at different pH values ​​(1.4, 3.4, 5.4, 7.4, 9.4, 11.4) was used as the antibody dilution buffer. The NbFD4 nanobody was diluted to the same working concentration to determine the antibody-antigen binding ability. The binding ability of the antibody to the antigen without dilution with organic solvent was taken as 100%. The pH tolerance of the nanobody was evaluated. The specific method is as follows:

[0128] Add 50 μL of diluted NbFD4 nanobody and 50 μL of PBS to the pre-packaged ELISA plate, incubate at 37°C for 40 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of HRP-labeled anti-VHH secondary antibody diluted 1:5000, incubate at 37°C for 30 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of LMB substrate solution, and develop the color at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance at 450 nm using an ELISA reader.

[0129] 2. Experimental Results

[0130] The measurement results are as follows Figure 6 As shown in the figure, the binding activity of the nanobody NbFD4 was above 75% at pH = 3.4, 5.4, and 9.4. At strongly alkaline pH = 11.4 and strongly acidic pH = 1.4, the activity was close to inactivation, indicating that the nanobody NbFD4 has good acid and weak alkali resistance, and its activity is stable between pH 3.4 and 9.4.

[0131] Example 8: Thermal stability analysis of the nanobody NbFD4

[0132] 1. Experimental Methods

[0133] The NbFD4 nanobody was diluted to the same working concentration and then treated at different temperatures (35℃, 45℃, 55℃, 65℃, 75℃, 85℃) for 5 min. The binding ability of the antibody to the antigen was measured. The binding ability of the nanobody to the antigen without high-temperature heat treatment was taken as 100% to evaluate the high-temperature tolerance of the nanobody. The specific method is as follows:

[0134] Add 50 μL of diluted NbFD4 nanobody and 50 μL of PBS to the pre-packaged ELISA plate, incubate at 37°C for 40 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of HRP-labeled anti-VHH secondary antibody diluted 1:5000, incubate at 37°C for 30 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of LMB substrate solution, and develop the color at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance at 450 nm using an ELISA reader.

[0135] 2. Experimental Results

[0136] The activity curves of the nanobody NbFD4 after treatment at different temperatures for 5 minutes are shown in the figure below. Figure 7 As shown in the figure, the activity of the nanobody NbFD4 gradually decreases with increasing treatment temperature. The activity remains above 65% at 55℃ and still has 40% activity at 75℃, indicating that the nanobody NbFD4 has excellent thermal stability.

[0137] Example 9: Storage stability analysis of the nanobody NbFD4

[0138] 1. Experimental Methods

[0139] 0.03% Proclin-300 preservative and 0.01% protease inhibitor were added to the NbFD4 nanobody and stored at 45°C in the dark. The binding ability of the antibody to the antigen was measured at different storage times (1 day, 3 days, 7 days, and 15 days). The binding ability of the antibody to the antigen on day 0 of the nanobody was taken as 100% to evaluate the storage stability of the nanobody. The specific method is as follows:

[0140] Add 50 μL of diluted NbFD4 nanobody and 50 μL of PBS to the pre-packaged ELISA plate, incubate at 37°C for 40 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of HRP-labeled anti-VHH secondary antibody diluted 1:5000, incubate at 37°C for 30 min, wash the plate five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), blot dry the liquid in the wells, add 100 μL of LMB substrate solution, and develop the color at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance at 450 nm using an ELISA reader.

[0141] 2. Experimental Results

[0142] The measurement results are as follows Figure 8 As shown in the figure, when stored at 45°C in the dark, the activity of the nanobody NbFD4 remained at approximately 100% until the third day. The activity gradually decreased from the fifth day onwards, but still maintained more than 75% activity after 15 days of storage. This indicates that the nanobody NbFD4 has excellent storage stability and can save costs during storage and transportation.

[0143] Example 10: Specificity determination of nanobody NbFD4

[0144] 1. Experimental Methods

[0145] The specificity of the method was evaluated by comparing the indirect competitive ELISA detection results of structural analogs of fluopyram (seed dressing fluopyram), and functional analogs of fluopyram (prothioconazole, imazalil, carbendazim, fenpyraclostrobin, dimethomorph, azoxystrobin, pyrimethanil, chlorothalonil, and iprodione). The specific method is as follows:

[0146] Add 50 μL of nanobody and 50 μL of serially diluted drug to each well, incubate at 37°C for 40 min, wash five times with PBST, blot dry the liquid in the wells, add 100 μL of HRP-labeled anti-VHH secondary antibody diluted 1:5000, incubate at 37°C for 30 min, wash five times with PBST, blot dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop color at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4, v / v) to stop the reaction; read the absorbance at 450 nm using a microplate reader. Plot the drug concentration on the x-axis, and calculate the OD of the wells with B / B0 (fludioxonil). 450 OD of pores without fluopyram 450 Using the ordinate, an indirect competition standard curve was established. The cross-reactivity rates of each drug with the NbFD4 nanobody were calculated using the following formula:

[0147]

[0148] 2. Experimental Results

[0149] The results showed that the NbFD4 nanobody had an IC50 value for the structural analogue seed dressing. 50 The concentration was 870.65 ng / mL, with a cross-reactivity of 1.5%; the IC50 for other functional analogues (prothiophanate-methyl, imazalil, carbendazim, fenpyraclostrobin, dimethomorph, azoxystrobin, pyrimethanil, chlorothalonil, and iprodione) was [not specified in the original text]. 50 All values ​​were greater than 2000 ng / mL, and the cross-reactivity rates were all less than 1%. This indicates that the nanobody NbFD4 can specifically recognize fludioxonil, and the detection method established in this invention has high specificity for fludioxonil.

[0150] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nanobody against fluopyram, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID No.

1.

2. A gene encoding the nanobody of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No.

9.

3. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 2.

4. A recombinant cell, characterized in that, The recombinant cells contain the recombinant vector of claim 3.

5. The application of the nanobody according to claim 1 in the detection of fludioxonil.

6. The use of the nanobody of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the recombinant cell of claim 4 in the preparation of fluopyram detection products.

7. A kit for detecting fludioxonil, characterized in that, Contains the nanobody as described in claim 1.

8. A method for detecting fludioxonil, characterized in that, The artificial antigen obtained by conjugating fluopyram hapten with a carrier protein is used as the coating antigen, and the nanobody described in claim 1 is used as the detection antibody for detection.

9. The detection method according to claim 8, characterized in that, The structural formula of the fluopyram hapten is shown in formula (Ⅰ):

10. The detection method according to claim 8, characterized in that, The carrier protein is ovalbumin.

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