A nanobody nb3-p-3 specifically recognizing fludioxonil and application thereof

Abstract

The application discloses a nanobody Nb3-P-3 capable of specifically recognizing fludioxonil and an application thereof. An amino acid sequence of the nanobody Nb3-P-3 is shown as SEQ ID No. 1. An AI-guided affinity maturation strategy is adopted to perform multi-point synergistic mutation and computer-aided screening on a key binding site of an initial nanobody Nb3, so as to obtain the nanobody Nb3-P-3. The nanobody can specifically recognize fludioxonil, a half-inhibitory concentration (IC 50 ) of the nanobody for fludioxonil is 1.95 ng / mL, a lowest detection limit (LOD) is 0.09 ng / mL, a linear range (IC 20 -IC 80 ) is 0.29-19.30 ng / mL, and the nanobody has excellent organic solvent resistance. The nanobody Nb3-P-3 has an excellent application prospect in rapid detection of fludioxonil residues.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nanobody Nb3-P-3 that specifically recognizes fludioxonil and its applications. Background Technology

[0002] Fludioxonil (FD) belongs to the phenylpyrrole class of fungicides. Due to its high efficiency, low toxicity, and broad spectrum, it is widely used in the control of gray mold in fruits, vegetables, and other crops. However, improper use can lead to fludioxonil residues in agricultural products.

[0003] Currently, the main methods for detecting fludioxonil include instrumental analysis methods such as gas chromatography and high-performance liquid chromatography-tandem mass spectrometry. While these methods provide accurate and reliable results, they require expensive equipment and specialized personnel, and the sample pretreatment is complex, making them unsuitable for rapid screening of large numbers of samples on-site. Immunoassay methods based on the specific binding of antigens and antibodies, due to their high sensitivity, strong specificity, ease of operation, and low cost, are gradually becoming the mainstream technology for the rapid detection of harmful substances in food.

[0004] Antibodies are the core components of immunoassay methods. Nanobodies (Nb) are variable region fragments of heavy chain antibodies derived from animals such as camels. They possess unique advantages such as small molecular weight (approximately 15 kDa), structural stability, strong tissue permeability, and ease of genetic engineering modification, showing great application potential in the detection of small molecule pollutants. Chinese patent application CN118459601A discloses an anti-cyfluthrin nanobody, NbFD4. This antibody was obtained through initial screening using animal immunization and phage display. The linear detection range of the method established using this antibody for detecting cyfluthrin is 3.40–47.38 ng / mL, with a half-maximal inhibitory concentration (IC50) of 100%. 50 The limit of concentration (LOD) was 12.68 ng / mL, and the limit of detection (LOD) was 1.57 ng / mL. However, its affinity is often limited, making it difficult to meet the practical needs of trace detection; therefore, affinity maturation modification is required.

[0005] Traditional affinity maturation strategies mainly fall into two categories: one is in vitro evolution techniques based on random mutation (such as error-prone PCR and DNA shuffling), which has high throughput but is prone to blindness, requires a large screening workload, and struggles to balance affinity with other key properties (such as stability); the other is rational design based on structure, which relies on high-resolution antigen-antibody complex crystal structure information. However, for small molecule targets, high-resolution complex structures are often difficult to obtain, severely limiting its application. In recent years, the rapid development of artificial intelligence (AI) technology has provided new ideas for protein engineering. Structure prediction tools, such as AlphaFold2, can predict antibody three-dimensional structures with high accuracy, and generative AI models, such as PocketGen, can design sequences based on a given structural framework. Applying AI technology to the affinity maturation of nanobodies is expected to overcome the blindness and dependence on crystal structures of traditional methods, achieving efficient and precise targeted optimization, significantly reducing screening costs, and shortening the R&D cycle. There are currently no reports of using AI-guided affinity maturation strategies to prepare high-affinity antifluopyrrolizidine nanobodies. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a nanobody Nb3-P-3 that specifically recognizes fludioxonil.

[0007] A second objective of this invention is to provide a gene encoding the aforementioned nanobody Nb3-P-3.

[0008] A third objective of this invention is to provide a recombinant vector / recombinant cell.

[0009] A fourth object of the present invention is to provide the use of the above-mentioned nanobody Nb3-P-3, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant cells in the detection of fludioxonil or in the preparation of fludioxonil detection reagents / kits.

[0010] The fifth object of the present invention is to provide a kit for detecting fludioxonil.

[0011] The sixth object of the present invention is to provide a method for detecting fludioxonil.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution: The present invention provides a nanobody Nb3-P-3 that specifically recognizes fludioxonil, the amino acid sequence of which is shown in SEQ ID No. 1.

[0013] In previous studies, phage display technology was used to screen for an initial nanobody Nb3 that specifically binds to fluticasone from a camel immune gene library. Further, an AI-guided / driven affinity maturation strategy was employed to perform multi-point synergistic mutations and computer-aided screening on key binding sites of the initial nanobody Nb3. This identified six residues contributing to binding: Thr28, Pro30, Phe31, Arg72, Tyr74, and Arg77. Arg72 was identified as the most critical, and it was retained. Synergistic optimization was performed on five sites: Thr28, Pro30, Phe31, Tyr74, and Arg77, ultimately yielding a nanobody Nb3-P-3 with significantly enhanced affinity. The VHH amino acid sequence of the nanobody Nb3-P-3 is shown in SEQ ID No. 1; the nucleotide sequence encoding the nanobody Nb3-P-3 is shown in SEQ ID No. 9.

[0014] Furthermore, the screening method for the nanobody Nb3-P-3 includes the following steps: S1. The three-dimensional structure of fludioxonil nanobody was predicted to obtain a high-confidence model; S2. The fluopyram molecules are docked to the high-confidence model of step S1, and amino acid scanning mutations are used to identify the amino acid residues that contribute to the binding. S3. Perform synergistic optimization on the amino acid residues obtained in step S2 to obtain the final product.

[0015] The affinity maturation strategy provided by this invention integrates the efficient sequence search capabilities of artificial intelligence with drug design concepts, overcoming the blindness of traditional random mutation methods and the dependence on high-resolution complex crystal structures. It achieves rapid and precise targeted optimization of nanobody affinity maturation, significantly reducing screening costs and shortening the R&D cycle, and providing a referable technical solution for affinity maturation of other small molecule target nanobodies.

[0016] Furthermore, the prediction is a three-dimensional structure prediction of the initial fludioxonil nanobody using AlphaFold3.

[0017] Furthermore, the collaborative optimization involves using PocketGen to construct a library of virtual mutants based on binding contributing amino acid residues, and then screening out the optimal mutant sequence based on the predicted binding affinity score.

[0018] The present invention also provides a gene encoding the above-mentioned nanobody Nb3-P-3, the nucleotide sequence of which is shown in SEQ ID No. 9.

[0019] Further, the nanobody Nb3-P-3 comprises four framework regions FR1, FR2, FR3, and FR4, and three complementarity-determining regions CDR1, CDR2, and CDR3; the arrangement order of the four framework regions and the three complementarity-determining regions is FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; wherein the amino acid sequence of FR1 is shown in SEQ ID No. 2, the amino acid sequence of FR2 is shown in SEQ ID No. 3, the amino acid sequence of FR3 is shown in SEQ ID No. 4, the amino acid sequence of FR4 is shown in SEQ ID No. 5, the amino acid sequence of CDR1 is shown in SEQ ID No. 6, the amino acid sequence of CDR2 is shown in SEQ ID No. 7, and the amino acid sequence of CDR3 is shown in SEQ ID No. 8.

[0020] The present invention also provides a recombinant vector containing the above-mentioned gene.

[0021] The present invention also provides a recombinant cell containing the above-mentioned gene or the above-mentioned recombinant vector. The recombinant cell can express the above-mentioned nanobody Nb3-P-3.

[0022] The nanobody Nb3-P-3 provided by this invention can specifically recognize fludioxonil, exhibiting a 90% inhibition rate against 100 ng / mL fludioxonil. Based on an indirect competitive ELISA standard curve established using Nb3-P-3, the half-maximal inhibitory concentration (IC50) of the nanobody Nb3-P-3 against fludioxonil was determined. 50 The limit of detection (LOD) was 1.95 ng / mL, the limit of detection (LOD) was 0.09 ng / mL, and the linear range (IC50) was [not specified]. 20 -IC 80 The detection sensitivity ranged from 0.29 to 19.30 ng / mL, showing a significant improvement compared to the initial nanobody Nb3. Furthermore, no cross-reactivity was observed with various structural and functional analogs (CR < 1.0%). Further analysis of the organic solvent tolerance of the Nb3-P-3 nanobody revealed that it retained over 75% activity in 10% methanol, acetonitrile, and acetone solutions, and maintained approximately 50% bioactivity in 50% methanol, 50% acetonitrile, and 40% acetone solutions. Therefore, this nanobody not only possesses high sensitivity and specificity but also excellent organic solvent tolerance (methanol, acetonitrile, acetone), making it suitable for rapid detection of fludioxonil residues under complex conditions and demonstrating significant application value.

[0023] Therefore, the present invention also provides the application of the above-mentioned nanobody Nb3-P-3, the above-mentioned gene, the above-mentioned recombinant vector or the above-mentioned recombinant cell in the detection of fludioxonil.

[0024] The present invention also provides the application of the above-mentioned nanobody Nb3-P-3, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant cells in the preparation of fludioxonil detection reagents / kits.

[0025] The present invention also provides a kit for detecting fludioxonil, the kit comprising the above-mentioned nanobody Nb3-P-3.

[0026] The present invention also provides a method for detecting fludioxonil, wherein the method uses a complete antigen obtained by conjugating fludioxonil hapten with a carrier protein as a coating antigen, and uses the above-mentioned nanobody Nb3-P-3 as a detection antibody for detection.

[0027] This method is simple to operate, takes little time, and produces highly sensitive and accurate results, making it well-suited for detecting fludioxonil residues in actual samples.

[0028] Furthermore, the structural formula of the fludioxonil hapten is shown in formula (I): .

[0029] Furthermore, the carrier protein is ovalbumin.

[0030] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a nanobody Nb3-P-3 that specifically recognizes fludioxonil, the amino acid sequence of which is shown in SEQ ID No. 1. This invention employs an AI-guided affinity maturation strategy, performing multi-point co-mutation and computer-aided screening on the key binding sites of the initial nanobody Nb3, ultimately obtaining a nanobody Nb3-P-3 with significantly enhanced affinity. This nanobody Nb3-P-3 can specifically recognize fludioxonil, exhibits no cross-reactivity with various structural and functional analogs, and maintains a half-maximum inhibitory concentration (IC50) for fludioxonil. 50 The limit of detection (LOD) was 1.95 ng / mL, the limit of detection (LOD) was 0.09 ng / mL, and the linear range (IC50) was [missing value]. 20 -IC 80 The concentration ranges from 0.29 to 19.30 ng / mL, exhibiting high sensitivity and excellent resistance to organic solvents. The nanobody Nb3-P-3 provided by this invention, capable of specifically recognizing fludioxonil, shows excellent application prospects in the rapid detection of fludioxonil residues. Attached Figure Description

[0031] Figure 1 This is a schematic diagram comparing the amino acid sequences of wild-type Nb3 and generated Nb3-P-3 nanobodies.

[0032] Figure 2Standard curves for indirect competitive ELISA based on wild-type Nb3 and generative Nb3-P-3.

[0033] Figure 3 Activity curves of nanobody Nb3-P-3 when methanol, acetonitrile, and acetone / PBS were used as diluents in different proportions. 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: AI-driven affinity maturation of Nb3 nanobodies 1. Structural modeling and identification of key residues of the initial nanobody Nb3 The three-dimensional structure of the initial nanobody Nb3 (amino acid sequence: EVQLEDSGGGSVQAGGSLRLSCTGSGSTSPFNCVAWFRQAPGLEREGVAAISTASGSTYYADSVKGRFAISRDYAKRTVYLQMNNLKPDDIAMYYCAAIVGRECLGSWPQAARYNYWGQGTLVTVSSGQAG) (SEQ ID No. 12) was predicted using AlphaFold3, obtaining a high-confidence model. Fluocinolone molecules were docked to the Nb3 model via molecular docking, and combined with alanine scanning mutations, six residues contributing to binding were confirmed: Thr28, Pro30, Phe31, Arg72, Tyr74, and Arg77. Among them, Arg72 was the most critical (the affinity decreased by 5.38-fold after mutation). In subsequent affinity maturation designs, Arg72 was retained, and five sites—Thr28, Pro30, Phe31, Tyr74, and Arg77—were synergistically optimized. IC50 was then measured using icELISA. 50 The results are shown in Table 1.

[0037] Table 1. Results of icELISA identification of alanine mutants

[0038] 2. Construction of Virtual Mutant Library and Selection of Optimal Mutants Using the known PocketGen protein pocket design model based on graph neural networks, the design scope was strictly limited to the five key sites mentioned above by modifying its generation logic, and a library containing 200 virtual mutants was constructed. Based on the predicted binding affinity scores, 20 candidate sequences were initially screened. Using drug design principles, the interaction between the mutants and fluopyram was simulated through molecular docking. The optimal mutant sequence Nb3-P-3 was selected as the mutant with the most significant affinity enhancement. Its mutation site combination is: Thr28 retention, Pro30→Thr, Phe31→Ser, Tyr74 retention, and Arg77 retention. Nb3-P-3 will be subsequently expressed and identified.

[0039] Example 2: Gene recombination expression and preliminary identification of mutant nanobodies 1. Construction of homologous recombination expression vectors Using the plasmid of the initial nanobody Nb3 as a template, the Nb3-P-3 mutation site obtained in Example 1 was introduced into the expression vector using homologous recombination technology. The specific steps are as follows: (1) Plasmid extraction: E. coli TG1 strain containing wild-type Nb3 plasmid was inoculated into LB-Amp liquid medium and cultured overnight at 37°C and 250 rpm. The bacterial cells were collected the next day, and plasmids were extracted using a plasmid extraction kit. After the concentration was determined, the plasmids were stored at -20°C for later use.

[0040] (2) Primer design: Based on the mutation site of Nb3-P-3, homologous recombination primers were designed, and the primer sequences are shown in Table 2. The primers contain sequences (15-20 bp) homologous to the vector at both ends, and the mutation site is in the middle.

[0041] Table 2. Primer sequences for homologous recombination

[0042] (3) PCR amplification of the linearized vector fragment: Using the wild-type Nb3 plasmid as a template, PCR amplification was performed using the primers described above. Reaction system (50 μL): template plasmid (50 ng), 1 μL each of forward and reverse primers (10 μmol / L), 25 μL of 2×TransStartFastPfu Fly Reaction Mix, 1 μL of TransStartFastPfu Fly DNA Polymerase, and ddH2O added to 50 μL. Reaction conditions: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 10 s, 55℃ annealing for 5 s, 72℃ extension for 40 s, 30 cycles; 72℃ final extension for 1 min. After the reaction, 5 μL of the product was taken for agarose gel electrophoresis verification. After a specific band of about 4000 bp appeared, the target product was excised and the concentration was determined.

[0043] (4) DpnⅠ digestion: To remove the methylated template plasmid, add 1 μL of DpnⅠ enzyme to the recovered product and incubate at 37°C for 1 h. The digestion product does not require purification and can be used directly for homologous recombination.

[0044] (5) Homologous recombination reaction: The recombination reaction was performed using the Mut Express II Fast Mutagenesis Kit V2. The reaction system (20 μL) consisted of: linearized vector fragment (50 ng), 5×CE Buffer 4 μL, Exnase II 2 μL, and ddH2O added to a final volume of 20 μL. After gentle mixing, the mixture was incubated at 37°C for 30 min and immediately cooled on ice.

[0045] (6) Transformation and culture: 10 μL of recombinant product was transformed into E.coli BL21(DE3) competent cells, incubated on ice for 25 min, heat-shocked at 42℃ for 45 s, incubated on ice for 2 min, added 500 μL of SOC medium, and recovered at 37℃ and 250 rpm for 1 h. The cells were then spread on LB-Amp plates and incubated upside down at 37℃ overnight.

[0046] (7) Sequencing verification: Three single colonies were randomly selected from the plate and inoculated into LB-Amp liquid medium. The culture was carried out overnight at 37°C and 250 rpm. The plasmid was extracted and sent to a sequencing company for sequencing. The sequencing results were consistent with the target sequence, indicating that the expression plasmid of Nb3-P-3 was successfully constructed.

[0047] 2. Induction and Preliminary Activity Identification of Nanobodies (1) Transformation of expression host: The Nb3-P-3 expression plasmid with correct sequencing was transformed into E.coli BL21(DE3) competent cells, plated on LB-Amp plates, and incubated overnight at 37°C with the plates inverted.

[0048] (2) Small-scale induction expression: Inoculated into 10 mL LB-Amp liquid medium, cultured at 37℃ and 250 rpm for 12 h on a shaker, collected cells by centrifugation at 4000 rpm for 10 min, added 200 μL TES, vortexed thoroughly, centrifuged at 12000 rpm for 3 min, collected the supernatant and transferred it to a 1.5 mL centrifuge tube to obtain the periplasmic cavity protein supernatant, added 400 μL deionized water, and the activity of the nanobody was identified by icELISA.

[0049] (3) Periplasmic cavity protein extraction: Collect bacterial cells and extract soluble proteins from the periplasmic cavity using the sucrose osmotic pressure freeze-thaw method. Specific operation: Centrifuge the bacterial solution at 4℃ and 4000 rpm for 10 min, discard the supernatant, resuspend the precipitate in 1 mL TES buffer, vortex thoroughly, incubate on ice for 30 min, centrifuge at 4℃ and 12000 rpm for 10 min, and the supernatant is the periplasmic cavity protein. Dilute with an equal volume of ddH2O for later use.

[0050] (4) Preliminary identification by icELISA: The activity of nanobodies in the periplasmic cavity supernatant was detected by indirect competitive ELISA. Fluocinolone artificial antigen (FD-H1-OVA) was used as the coating agent (1 μg / mL, 100 μL / well, overnight coating at 37℃). After blocking, 50 μL of periplasmic cavity supernatant and 50 μL of PBS (titer well) or 50 μL of 100 ng / mL fluocinolone standard (inhibition well) were added to each well, and incubated at 37℃ for 40 min. After washing, HRP-labeled anti-VHH secondary antibody (1:5000 dilution) was added, and incubated at 37℃ for 30 min. After color development for 10 min, OD was measured. 450 Select the valence well OD 450 Clones that are more than 3 times larger than the negative control and have an inhibition rate of more than 90% in the inhibition well are considered positive.

[0051] Preliminary identification showed that the obtained Nb3-P-3 pericyte supernatant significantly inhibited 100 ng / mL fludioxonil (inhibition rate: 90%), indicating that the recombinant nanobody has specific binding activity.

[0052] Example 3: Sequencing and determination of the amino acid sequence of the encoding gene for the specific nanobody Nb3-P-3. 1. Experimental Methods The strain of Nb3-P-3, a specific nanobody obtained through indirect competitive ELISA, was sent to a sequencing company for sequencing to obtain the nucleotide sequence of the specific nanobody Nb3-P-3; based on the DNA sequencing results and codon table, the amino acid sequence of the specific nanobody Nb3-P-3 was obtained.

[0053] 2. Experimental Results The amino acid sequence of VHH of the specific nanobody Nb3-P-3 is shown in SEQ ID No. 1: EVQLEDSGGGSVQAGGSLRLSCTGSGSTSTSNCVAWFRQAPGLEREGVAAISTASGSTYYADSVKGRFAISRDYAKRTVYLQMNNLKPDDIAMYYCAAIVGRECLGSWPQAARYNYWGQGTLVTVSSGQAG.

[0054] like Figure 1 As shown, the amino acid sequence of the generated specific nanobody Nb3-P-3 differs from that of the wild-type nanobody Nb-3 only at amino acid positions 30 and 31. This specific nanobody Nb3-P-3 includes four framework regions (FRs) and three complementarity-determining regions (CDRs).

[0055] The frame regions (FR1-FR4) are as follows: SEQ ID No. 2: EVQLEDSGGGSVQAGGSLRLSCTGS (amino acids 1-25); SEQ ID No. 3: VAWFRQAPGLEREGVAA (amino acids 34-50); SEQ ID No. 4: YYADSVKGRFAISRDYAKRTVYLQMNNLKPDDIAMYYC (amino acids 59-96); SEQ ID No. 5: WGQGTLVTVSSGQAG (amino acids 117-131) is shown.

[0056] The complementarity determinants (CDR1-CDR3) are as follows: SEQ ID No. 6: GSTSTSNC (amino acids 26-33); SEQ ID No. 7: ISTASGST (amino acids 51-58); SEQ ID No. 8: AAIVGRECLGSWPQAARYNY (amino acids 97-116) is shown.

[0057] The nucleotide sequence of the gene encoding Nanobody Nb3-P-3 is as SEQ ID Shown in No.9: GAGGTGCAGCTGGAGGATTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTCAGACTCTCCTGTACAGGCTCTGGATCGACCAGCACCTCTAACTGCGTGGCCTGGTTCCGCCAGGCTCCAGGCCTAGAGCGCGAGGGGGTCGCAGCTATTAGTACTGCTAGTGGTAGCACATACTATGCCGACTCCGTGAA GGGCCGATTCGCCATCTCCCGAGACTACGCCAAGAGAACGGTGTATCTGCAAATGAACAACCTGAAACCTGACGACATTGCCATGTACTACTGTGCGGCAATAGTGGGGCGGAATGTCTTGGTTCATGGCCCCAAGCGGCTCGGTATAACTACTGGGGCCAGGGGGACCCTGGTCACCGTCTCCTCAGGCCAGGCCGGCC.

[0058] Example 4: Large-scale preparation of specific nanobody Nb3-P-3 The specific nanobody Nb3-P-3 was prepared by protein expression, and the specific method is as follows: 750 mL of bacterial cells were expressed in large quantities, and soluble nanobodies in the periplasmic cavity were extracted using the sucrose osmotic pressure method. Single colonies cultured overnight in Example 2 were inoculated into 10 mL of LB-Amp liquid medium and cultured at 37°C and 250 rpm for 12 h. The bacterial culture was then transferred to 750 mL of LB-Amp liquid medium and cultured at 37°C and 250 rpm for 4 h. IPTG was added to a final concentration of 1 mmol / L and cultured overnight. The bacterial culture was centrifuged at 12000 rpm for 3 min, the supernatant was discarded, 10 mL of TES was added, and the mixture was vortexed thoroughly. The mixture was then frozen at -80°C for at least 3 h. After thawing at room temperature, 20 mL of ddH2O was added, and the mixture was stirred at 4°C for 1 h. The mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected to obtain the periplasmic cavity soluble protein. The nanobodies Nb3-P-3 were purified by nickel column chromatography.

[0059] Example 5: Application of the specific nanobody Nb3-P-3 I. Experimental Methods 1. Wrapping and sealing Dilute the coating antigen FD-H1-OVA to the optimal working concentration using coating buffer, add 100 μL to each well of the ELISA plate, and incubate overnight at 37°C. Wash the overnight coated ELISA plate twice with PBST and blot dry. Add 120 μL of blocking buffer to each well, incubate at 37°C for 3 h, then dry in a 37°C oven for 1 h. After drying, seal and store at 4°C for later use.

[0060] 2. Establishment of the standard curve A standard curve was established using an indirect competitive enzyme-linked immunosorbent assay (icELISA). The nanobody Nb3-P-3 was diluted to its optimal working concentration (initial concentration 1.30 mg / mL, dilution factor 6K). Fluocinolone standards were serially diluted (0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 ng / mL). 50 μL of nanobody and 50 μL of different concentrations of fluocinolone standards were added to each well, and the mixture was incubated at 37°C for 40 min. After washing, HRP-labeled anti-VHH secondary antibody (1:5000 dilution) was added, and the mixture was incubated at 37°C for 30 min. After 10 min of color development, the A450 was measured. The logarithm of the fluocinolone standard concentration was plotted on the x-axis, and B / B0 (OD450 of the well containing fluocinolone) was plotted on the y-axis. 450 OD of pores without fluopyram 450 Using y as the ordinate, the data is fitted using the four-parameter fitting function of Origin software to establish an indirect competitive standard curve.

[0061] II. Experimental Results The indirect competitive ELISA standard curve based on the specific nanobody Nb3-P-3 is shown in the figure. Figure 2 As shown, the standard curve exhibits an S-shape, indicating good linear correlation. The half-maximal inhibitory concentration (IC50) of the nanobody Nb3-P-3 against fludioxonil was determined. 50 The limit of detection (LOD) was 1.95 ng / mL, the limit of detection (LOD) was 0.09 ng / mL, and the linear range (IC50) was [not specified]. 20 -IC 80 The concentration range is 0.29-19.30 ng / mL, with high detection sensitivity compared to wild-type nanobody Nb3 (IC50). 50 (19.61 ng / mL), with a 10-fold increase in sensitivity, making it the best-performing nanobody among the currently published immunoassay methods for fludioxonil.

[0062] Example 6 Specificity analysis of nanobody Nb3-P-3 I. Experimental Methods Prepare standard solutions of drugs with similar structures and functions to fludioxonil, including seed dressing fludioxonil, prothioconazole, imazalil, carbendazim, fenpyraclostrobin, dimethomorph, azoxystrobin, pyrimethanil, chlorothalonil, and iprodione. Determine the IC50 of each drug using the icELISA method. 50 The cross-reactivity ratio (CR) is calculated using the IC value. The formula for calculating the cross-reactivity ratio is: CR(%) = ICi 50 (Cyclopyralid) / IC 50 (Similar substances) × 100%.

[0063] II. Experimental Results The specificity assay results are shown in Table 3. The cross-reactivity rate of the nanobody Nb3-P-3 with the 10 structural and functional analogs tested was less than 0.1%, indicating that the nanobody Nb3-P-3 can specifically recognize fludioxonil.

[0064] Table 3. Cross-reactivity of icELISA (n=3)

[0065] Example 7: Analysis of the organic tolerance of the nanobody Nb3-P-3 I. Experimental Methods The nanobody Nb3-P-3 was diluted to its optimal working concentration using methanol, acetonitrile, and acetone at different concentrations (10%, 20%, 30%, 40%, and 50%) to determine its binding ability to the antigen. PBS dilution without organic solvents was used as a control, with its binding ability to the antigen considered 100%. 50 μL of the diluted Nb3-P-3 nanobody and 50 μL of PBS were added to a pre-coated ELISA plate and incubated at 37°C for 40 min. After washing, HRP-labeled anti-VHH secondary antibody (1:5000 dilution) was added, and the plate was incubated at 37°C for 30 min. After 10 min of color development, the A450 was measured, and the relative activity of the antibody at different organic solvent concentrations was calculated.

[0066] II. Experimental Results The activity curves of the nanobody Nb3-P-3 with different proportions of methanol, acetonitrile, and acetone as diluents are shown in the figure. Figure 3 As shown in the figure. Experimental results indicate that the Nb3-P-3 nanobody retains over 75% activity in 10% methanol, acetonitrile, and acetone solutions, and maintains approximately 50% bioactivity in 50% methanol, 50% acetonitrile, and 40% acetone solutions. Therefore, the Nb3-P-3 nanobody exhibits excellent resistance to organic solvents (methanol, acetonitrile, acetone), and is not affected by organic solvents during the pretreatment process for actual sample detection, resulting in high accuracy of the detection results.

[0067] 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 Nb3-P-3 that specifically recognizes fludioxonil, characterized in that, The amino acid sequence of the nanobody Nb3-P-3 is shown in SEQ ID No.

1.

2. The gene encoding the nanobody Nb3-P-3 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 cell contains the gene of claim 2 or the recombinant vector of claim 3.

5. The nanobody Nb3-P-3 according to claim 1, characterized in that, The screening method for the nanobody Nb3-P-3 includes the following steps: S1. The three-dimensional structure of fludioxonil nanobody was predicted to obtain a high-confidence model; S2. The fluopyram molecules are docked to the high-confidence model of step S1, and amino acid scanning mutations are used to identify the amino acid residues that contribute to the binding. S3. Perform synergistic optimization on the amino acid residues obtained in step S2 to obtain the final product.

6. The application of the nanobody Nb3-P-3 of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the recombinant cell of claim 4 in the detection of fludioxonil.

7. The use of the nanobody Nb3-P-3 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 fludioxonil detection reagent / kit.

8. A kit for detecting fludioxonil, characterized in that, The kit includes the nanobody Nb3-P-3 as described in claim 1.

9. A method for detecting fludioxonil, characterized in that, The method involves using a complete antigen obtained by conjugating fluocinolone hapten with a carrier protein as a coating antigen, and using the nanobody Nb3-P-3 described in claim 1 as a detection antibody for detection.

10. The method according to claim 9, characterized in that, The structural formula of the fludioxonil hapten is shown in formula (Ⅰ): 。

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