A nanoantibody Nb Y-2-2-4 that specifically recognizes imazalil and its application

Abstract

The present invention discloses a nano antibody Nb Y-2-2-4 that specifically recognizes imazalil and its application. The amino acid sequence of the nano antibody is shown in SEQ ID No. 1. The nano antibody has excellent organic solvent tolerance and acid-base tolerance. During the pre-treatment process of actual sample detection, it is less affected by organic solvents and can also be applied to detection requirements in acid-base environments. The ELISA detection method established based on the nano antibody has a detection range of 3.70 to 20.65 ng / mL for imazalil, and a half inhibitory concentration (IC 50 ) is 8.74 ng / mL, the minimum detection limit is 1.85 ng / mL, and the detection sensitivity is high. When applied to the actual sample detection of imazalil residues, the operation is simple, the time consumed is short, the sensitivity is strong, and the accuracy is high. The nanoantibody has extremely high application value in detecting imazalil or preparing imazalil detection products.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a nano antibody Nb Y-2-2-4 that specifically recognizes imazalil and applications thereof. Background Art

[0002] Imazalil (IMZ), also known as imazalil, belongs to the imidazole class of fungicides and is a broad-spectrum, systemic fungicide. It inhibits the synthesis of lanosterol-14α-demethylase (CYP51) in fungi, inhibiting the demethylation reaction in ergosterol biosynthesis and resulting in the accumulation of 14α-demethylol. This inhibits the synthesis of ergosterol in cell membranes, altering cell membrane permeability and disrupting cell wall synthesis, leading to fungal death and achieving a bacteriostatic effect, thus protecting crops. IMZ is effective against many fungal diseases that attack fruits, vegetables, and grains. It is particularly effective against post-harvest rot in fruits such as citrus and bananas when sprayed or treated, making it a useful tool for controlling fungal damage to fruits and vegetables. For example, it has excellent control effects against green and blue mold infections in citrus, achieving antibacterial and fresh-keeping properties, leading to its widespread use. However, improper and misuse of IMZ is a common occurrence, posing potential risks to human health and the environment, including cytotoxicity, reproductive toxicity, DNA damage, and neurotoxicity.

[0003] Due to its wide application and high detection rate, IMZ is prone to the risk of exceeding the standard. Establishing a rapid, sensitive and efficient detection method to monitor pesticide residues in agricultural products such as fruits and vegetables is a key tool to ensure compliance with regulatory requirements, good agricultural practices and trade requirements stipulated by importing countries.

[0004] Currently, there are two methods for detecting IMZ residues in food: instrumental analysis and immunoassays. Instrumental analysis, as the mainstream method for food safety, is the standard method for determining IMZ residues in food and determining food safety. It offers high sensitivity, accuracy, and repeatability. However, it is complex to operate, requires tedious pre-treatment, and requires highly skilled personnel. Furthermore, the instrumentation is expensive and the cost is high. These objective factors preclude rapid, real-time, on-site testing. Immunoassays, based on the specific recognition of antigens and antibodies, enable qualitative and quantitative detection of target substances. They offer advantages such as high selectivity and sensitivity, simple operation, and low cost. In recent years, they have been increasingly used in market monitoring and on-site surveillance, complementing instrumental analysis methods to maintain food safety.

[0005] In immunoassay methods, the stability of antibodies plays a crucial role. Antibodies are the core of immunoassay methods. Currently, most immunoassay tests rely mainly on monoclonal antibodies as recognition elements. Since antibodies are essentially proteins, their activity will affect the accuracy and sensitivity of the test results. In practical applications, biorecognition materials are easily inactivated by storage and transportation conditions, and organic solvents are often required for sample pretreatment. The residual organic solvents will greatly reduce the detection effect, and monoclonal antibodies have poor organic tolerance and acid-base tolerance. More importantly, the genetic stability of monoclonal cells during the passage process cannot be determined, and the isolation of monoclonal antibodies from animals increases the preparation cost.

[0006] Nanobodies are a new type of genetically engineered antibody derived from camelids. They are small antigen-binding fragments with a crystal diameter of only 2.5 nm, a length of 4 nm, and a molecular weight of approximately 15 kDa. They are called VHH or nanobodies. Compared to traditional antibodies, nanobodies lack the Fc region, thus avoiding false-positive results caused by binding to non-target substances. Furthermore, they can be produced on a large scale through prokaryotic expression, have a high resistance to denaturants, and can be structurally modified through genetic engineering. These properties make nanobodies potentially valuable in immunoassays and better meet the market demand for rapid testing. Enzyme-linked immunosorbent assays (ELISAs), as rapid testing techniques, often require direct extraction with organic solvents as pretreatment. Nanobodies' superior tolerance to organic solvents makes them promising for rapid testing. Currently, there is no development and application of nanobodies that specifically recognize imazalil. Therefore, the development of a stable, accurate, and sensitive rapid detection method for imazalil based on nanobodies has great application value. Summary of the Invention

[0007] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a nanobody Nb Y-2-2-4 that specifically recognizes imazalil.

[0008] A second object of the present invention is to provide a gene encoding the Nanobody Nb Y-2-2-4.

[0009] The third object of the present invention is to provide a recombinant vector.

[0010] The fourth object of the present invention is to provide a recombinant cell.

[0011] A fifth object of the present invention is to provide the use of the nanobody Nb Y-2-2-4, the gene, the recombinant vector or the recombinant cell in detecting imazalil.

[0012] The sixth object of the present invention is to provide the use of the nanobody Nb Y-2-2-4, the gene, the recombinant vector or the recombinant cell in the preparation of a product for detecting imazalil.

[0013] The seventh object of the present invention is to provide a detection kit for imazalil.

[0014] The eighth object of the present invention is to provide an immunoassay method for detecting imazalil.

[0015] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0016] The present invention first provides a nanobody Nb Y-2-2-4 that specifically recognizes imazalil. The amino acid sequence of the nanobody NbY-2-2-4 is shown in SEQ ID No. 1.

[0017] The present invention prepares an artificial antigen to immunize alpacas to obtain a nanoantibody gene library. Phage display technology is then used to screen the alpaca immune antibody gene library through biopanning to obtain a nanoantibody Nb Y-2-2-4 that specifically recognizes imazalil. The preparation method of the nanoantibody Nb Y-2-2-4 is simple and has universal applicability. It can be used to screen and prepare nanoantibodies against other small molecules, and has high application value.

[0018] Furthermore, the Nanobody Nb Y-2-2-4 includes four framework regions FR1, FR2, FR3, FR4 and three complementary determining regions CDR1, CDR2, and CDR3, and the order of the four framework regions and the three complementary determining regions is FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; wherein the amino acid sequence of the FR1 is shown in SEQ ID No.2, the amino acid sequence of the FR2 is shown in SEQ ID No.3, the amino acid sequence of the FR3 is shown in SEQ ID No.4, the amino acid sequence of the FR4 is shown in SEQ ID No.5, the amino acid sequence of the CDR1 is shown in SEQ ID No.6, the amino acid sequence of the CDR2 is shown in SEQ ID No.7, and the amino acid sequence of the CDR3 is shown in SEQ ID No.8.

[0019] The present invention provides a gene encoding the nanobody Nb Y-2-2-4, characterized in that the nucleotide sequence of the gene is shown in SEQ ID No.9.

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

[0021] The present invention also provides a recombinant cell containing the recombinant vector. Since the present invention has provided the amino acid sequence of Nanobody Nb Y-2-2-4 and the gene sequence encoding the Nanobody, those skilled in the art can obtain the Nanobody described in this application by known recombinant DNA technology on this basis. Therefore, any recombinant vector or recombinant cell that can be used to prepare the Nanobody described in the present invention should also be within the scope of protection of the present invention.

[0022] Furthermore, the present invention conducted a detection performance and tolerance analysis on the nano antibody Nb Y-2-2-4. The results showed that the ELISA detection method based on the nano antibody was established, and the detection range of this method for imazalil was 3.70-20.65 ng / mL, and the half inhibitory concentration (IC 50 ) is 8.74ng / mL, and the minimum detection limit (LOD) is 1.85ng / mL, with high detection sensitivity. The antigen binding activity of the nanobody NbY-2-2-4 still maintains more than 80% activity in methanol, ethanol and acetonitrile solutions below 20%, and still maintains half of its activity in 40% methanol and ethanol, with excellent organic solvent tolerance; Nb Y-2-2-4 has better binding activity in a suitable acidic environment (pH = 2.4 ~ 6.4), and can even be increased to 150%. It still has high activity in alkaline conditions, and even at pH = 12.4, the activity remains at around 60%, with excellent acid-base tolerance. The above results show that the nanobody has high detection sensitivity and excellent organic tolerance and acid-base tolerance. During the pretreatment process of actual sample detection, it is less affected by organic solvents and can also be applied to detection needs in acid-base environments.

[0023] Therefore, the present invention also provides the use of the nanobody Nb Y-2-2-4, the gene, the recombinant vector or the recombinant cell in detecting imazalil or preparing a product for detecting imazalil.

[0024] The present invention provides a kit for detecting imazalil, wherein the kit contains the nano antibody Nb Y-2-2-4.

[0025] The invention provides an immunoassay method for detecting imazalil, which comprises using a complete antigen obtained by coupling an imazalil hapten with a carrier protein as a coating source, and using the nano antibody Nb Y-2-2-4 as a detection antibody for detection.

[0026] Furthermore, the structural formula of the imazalil hapten is shown in formula (I):

[0027]

[0028] Furthermore, the complete antigen is represented by formula (II):

[0029]

[0030] Furthermore, the carrier protein (Pro) is ovalbumin (OVA).

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a nano antibody Nb Y-2-2-4 that specifically recognizes imazalil, and its amino acid sequence is shown in SEQ ID No. 1. Based on the nano antibody, an ELISA detection method is established, which has a detection range of 3.70 to 20.65 ng / mL for imazalil, and a half inhibitory concentration (IC 50 ) is 8.74 ng / mL, the minimum detection limit (LOD) is 1.85 ng / mL, and the detection sensitivity is high. The nano antibody has excellent organic solvent tolerance and acid-base tolerance. It is less affected by organic solvents during the pretreatment process of actual sample detection and can also be applied to detection needs under acid-base environments. The present invention also provides an immunoassay method for detecting imazalil, which is simple to operate, short in time, and has strong sensitivity and high accuracy in the detection results. The preparation method of the nano antibody Nb Y-2-2-4 has universal applicability; therefore, the nano antibody Nb Y-2-2-4 can be well applied to the actual sample detection of imazalil residues, and has extremely high application value in detecting imazalil or preparing imazalil detection products. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the amino acid sequence and domain division of the specific nanoantibody Nb Y-2-2-4.

[0034] Figure 2 This is a standard curve of the indirect competitive ELISA established based on the specific nanobody Nb Y-2-2-4.

[0035] Figure 3 The activity curve of nanobody Nb Y-2-2-4 when different proportions of methanol, ethanol, and acetonitrile / PBS are used as diluents.

[0036] Figure 4 The activity curve of nanobody Nb Y-2-2-4 when different pH values ​​are used as diluents. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples 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 the art.

[0038] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0039] Example 1 Construction of alpaca immune antibody library

[0040] 1. Preparation of complete antigens IMZ-A-LF and IMZ-A-OVA

[0041] The structural formula of imazalil hapten is shown in formula (I):

[0042]

[0043] The imazalil hapten of the structural formula shown in formula (I) was coupled to lactoferrin (LF) and ovalbumin (OVA) by the active ester method to prepare the complete antigens IMZ-A-LF and IMZ-A-OVA. The complete antigen structure is shown in formula (II):

[0044]

[0045] Where Pro is lactoferrin (LF) or ovalbumin (OVA).

[0046] 2. Alpaca immunization program

[0047] Healthy alpacas were immunized with IMZ-A-LF as the immunizing antigen, administered subcutaneously in the neck. Each immunization dose was 0.5 mg. The first immunization was emulsified with 0.5 mL of complete Freund's adjuvant and the immunizing antigen. Subsequent booster immunizations were emulsified with 0.5 mL of incomplete Freund's adjuvant and the antigen. Thereafter, booster immunizations were administered every two weeks for a total of three booster immunizations.

[0048] Before immunization, 10 mL of blood was collected to separate serum as a negative control. Starting from the second immunization, 10 mL of blood was collected one week after immunization for serum titer and competition reaction detection. After the third and fourth immunizations, 50-100 mL of peripheral blood was collected for the construction of the nanobody library.

[0049] 3. Isolation of alpaca lymphocytes

[0050] Lymphocyte isolation should be performed as soon as possible after collecting alpaca peripheral blood. The specific procedure is as follows: dilute the alpaca peripheral blood with sterile saline in a 50mL centrifuge tube at a ratio of 2:1. Centrifuge the diluted peripheral blood with commercial lymphocyte separation buffer. Add 15mL of lymphocyte separation buffer to a sterile 50mL centrifuge tube and slowly add 15mL of diluted blood along the side of the tube using a sterile Pasteur pipette. Centrifuge at 800g for 25 minutes. Transfer the lymphocyte layer to a new 50mL centrifuge tube, dilute it 2-fold with saline, and centrifuge at 1500g for 10 minutes at 4°C. Discard the supernatant. Disperse the lymphocytes with 5mL of saline, centrifuge again at 1500g for 10 minutes, and discard the supernatant to thoroughly wash the lymphocytes. Add lysis buffer (TRNsol) to each lymphocyte aliquot, and aliquot 1mL into 2mL centrifuge tubes. Store at -80°C until needed.

[0051] 4. Total RNA Extraction

[0052] Total RNA was extracted from the lymphocytes stored in the TRNsol lysis solution using a commercial RNA extraction kit according to the instructions.

[0053] After total RNA extraction, a small sample was taken for nucleic acid electrophoresis and the RNA concentration was determined in a nanodrop spectrophotometer. The ideal RNA sample should be intact and undegraded, with clear 28S and 18S bands visible on the nucleic acid electrophoresis gel and no genomic DNA contamination. The ratio of the UV absorbance at 260 nm to 280 nm (A 260 / 280 ) should be around 2.0. If genomic DNA contamination is present, the genomic DNA should be removed with DNase before reverse transcription. Electrophoresis should be repeated to verify that the genomic DNA has been removed and that the RNA has not been degraded. If the RNA has been degraded, re-extraction is necessary. RNA should be reverse-transcribed into cDNA as quickly as possible or stored temporarily at -80°C.

[0054] 5. cDNA Synthesis

[0055] Using RNA as a template, synthesize the first strand of cDNA according to the instructions of Takara's first strand reverse transcription kit. The specific method is as follows:

[0056] A. 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;

[0057] Table 1. First step reaction system of cDNA synthesis

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

[0059] B. Incubate the above reaction system at 65°C for 5 min and cool on ice for 2 min.

[0060] C. According to the second step reaction system of cDNA synthesis shown in Table 2, add reagents to the system after the reaction in step A;

[0061] Table 2 The second step reaction system of cDNA synthesis

[0062] System after step A reaction 12 μL 5×Reaction Buffer 4μL RiboLock RNase Inhibitor(20U / μL) 1 μL 10mM dNTP Mix 2μL RevertAid M-MiLVRT (200 U / μL) 1 μL Total 20 μL

[0063] D. Incubate at 42°C for 60 minutes and then at 70°C for 5 minutes. Store the reverse transcribed cDNA at -80°C.

[0064] 6. Amplification of Nanobody Target Gene

[0065] Nested PCR was used to amplify the target gene in two steps.

[0066] The first round of PCR used cDNA as a template and primers Q1 / Q2 for the first round of PCR reaction. The nucleotide sequences of primers Q1 / Q2 are shown in Table 5 , and the reaction system and reaction system of the first round of PCR are shown in Table 3 .

[0067] Table 3 Nested PCR first step reaction system

[0068]

[0069] Second-round PCR: The first-round PCR reaction product was recovered using a kit and appropriately diluted as a template for the second-round PCR reaction. The second-round PCR reaction was performed using the primer combination Q3 / Q4 or Q3 / Q5. The nucleotide sequences of primers Q3 / Q4 are shown in Table 5. The reaction system and reaction conditions for the second-round PCR are shown in Table 4.

[0070] Table 4 Nested PCR second step reaction system

[0071]

[0072] Table 5 Primers and nucleotide sequences used for amplification of the target gene of Nanobody VHH

[0073]

[0074]

[0075] 7. Gene library construction

[0076] (1) Enzyme digestion of VHH target gene and vector

[0077] The VHH target gene and the pComb3xss vector were digested with SfiI enzyme at 50°C for 16 hours.

[0078] The pComb3xss vector digestion product was recovered by agarose gel as a band with a molecular weight of 3500 bp; the VHH gene digestion product was directly cleaned and recovered using a DNA recovery kit.

[0079] (2) Ligation of enzyme digestion products

[0080] The vector pComb3xss and the VHH fragment were mixed evenly (molar ratio 1:3), reacted at 16°C for 16 h, and then cleaned and recovered using a DNA recovery kit.

[0081] (3) Electric shock conversion

[0082] Take 5 μL of ligation product and add it to 50 μL of electroporation competent E. coli TG1. After gently mixing, transfer it to a 0.2 cm electroporation cup for electroporation (voltage is 1.8 kV). Immediately after electroporation, add 800 μL and 150 μL of SOC medium preheated to 37°C to the electroporation cup twice, collect the cells into a sterile centrifuge tube, and culture at 37°C, 250 rpm for 1 hour to recover the cells.

[0083] Prepare a serial dilution series with 50 μL of the recovered bacterial solution. Spread 100 μL of each serial dilution onto a 90 mm diameter LB-Amp dish to serve as a counting plate and incubate at 37°C overnight. Spread the remaining undiluted recovered bacterial solution onto a 120 mm diameter LB-Amp dish, spreading 2-3 dishes per 1 mL of solution as amplification plates. Incubate the culture plates at 37°C overnight.

[0084] Count the number of colonies on the culture dish, calculate the total number of bacteria in the resuscitated bacterial solution, and perform multiple electric shock transformations until the total number of transformed colonies reaches 10 7 This number is the library capacity of the nanobody gene library.

[0085] Scrape the transgenic E. coli colonies from the amplification plate with a cell scraper, collect the cells by centrifugation, discard the supernatant, and resuspend in LB-Amp (0.5 ml per tube of electroporation). Mix thoroughly, then add sterile glycerol to a final concentration of 25% (v / v). Take 50 μL of the bacterial solution for serial dilution to determine the cell count, and aliquot the remaining solution and freeze at -80°C. This is the imazalil nanobody gene library.

[0086] 8. Phage rescue

[0087] According to the above transgenic E. coli cell number determination results, 10 times more cells than the reservoir capacity were inoculated into 150 mL LB-Amp and the OD was controlled. 600<0.2, cultured at 37℃ and 250rpm until the logarithmic phase (OD 600 About 0.4~0.6); add 1mL titer of 10 12 Helper phage M above cfu / mL 13 K 07 , after standing at 37°C for 30 minutes, culture at 250rpm for 1 hour, add Kana (kanamycin, working concentration is 50μg / ml) and culture overnight at 37°C and 250rpm. Transfer the bacterial solution to a centrifuge bottle, centrifuge at 12000rpm at 4°C for 15 minutes, take the supernatant, add 1 / 4 volume of PEG / NaCl, and bathe on ice for more than 2.5 hours. Centrifuge at 12000rpm at 4°C for 15 minutes, discard the supernatant, resuspend the precipitate with 750μL TBS, transfer to a 1.5mL centrifuge tube, centrifuge at 4000rpm at 25°C for 5 minutes, and filter through a 0.22μm polyethersulfone membrane. Take 10μL of phage for titer determination, mix the rest evenly, add sterile glycerol (v / v) at a final concentration of 50%, and store at -80°C. This is the imazalil nanoantibody phage library, which can be directly used for affinity panning.

[0088] Example 2 Affinity panning and identification of nanobodies

[0089] 1. Experimental Methods

[0090] 1. Affinity panning of nanobodies

[0091] (1) Immobilization of antigens and carrier proteins

[0092] Affinity panning uses strong adsorption ELISA plates with strong adsorption capacity. One column of the plate is coated per round of panning, for a total of four rounds of coating. Wells AB are coated with the original carrier protein OVA diluted to 1 mg / mL, and wells CDEF are coated with the detection antigen IMZ-A-OVA diluted to 10 μg / mL using CB coating solution. 100 μL of this solution is added to the microwells of the strong adsorption plate, and the plate is incubated at 37°C overnight. Additionally, because alpacas may have been immunized with multiple carrier proteins, three immune carrier proteins—ConA (concanavalin), LF (lactoferrin), and KLH (keyhole limpet hemocyanin)—are mixed and diluted to a final concentration of 2 mg / mL. This is then coated on a separate column of wells containing the immunogen carrier protein. The next day, the plate is washed twice with PBST (0.01M PBS, 0.05% Tween-20), and 120 μL of a 1% fish gelatin solution is added to each well and incubated at 37°C for 3 hours. Pour out the liquid in the well and pat dry on absorbent paper, dry at 37℃ for 1h, and store at 4℃ for later use.

[0093] (2) Positive phage screening

[0094] Add the phage library from Example 1 to two wells coated with the immunogen carrier protein, adding 150 μL to each well and incubating at 37°C with shaking for 1 hour (this step is only required for the first round; rounds 2, 3, and 4 should begin directly in wells AB). Transfer free phage to wells coated with the original carrier protein AB, adding 150 μL to each well and incubating at 37°C with shaking for 1 hour. Transfer free phage to three wells containing immobilized antigen (IMZ-A-OVA), adding 100 μL to each well and incubating at 37°C with shaking for 1 hour. Discard free phage from the wells, wash the wells 10 times with PBST (0.01 M PBS, 0.05% Tween-20 (v / v)), and then wash the wells five times with PBS. Add 100 μL of 10 mg / mL trypsin-TBS solution and elute at 37°C for 30 minutes. Phage were collected and titered using 10 μL of eluted phage. The remainder was used to infect 5 mL of E. coli TG1 grown to logarithmic phase for amplification. The next day, the amplified phage were precipitated with PEG / NaCl and titered.

[0095] During the second, third, and fourth rounds of panning, the coating concentration of IMZ-A-OVA was reduced to 1000 ng / mL, 500 ng / mL, and 100 ng / mL, respectively. Phages were added and incubated at 37°C for 1 hour. After washing with PBST (0.01 MPBS, 0.05% Tween-20 (v / v)) and PBS, drug competitive elution was adopted, that is, a certain concentration of drug was added, incubated at 37°C for 1 hour, and the liquid in the well was aspirated to obtain the eluted phage. The screening scheme of step (2) was repeated. The drug elution concentrations were 1000 ng / mL, 500 ng / mL, and 100 ng / mL, respectively. The above conditions can be adjusted according to the actual immune situation. If the serum titer is low, the concentration of the detection antigen IMZ-A-OVA during panning can be appropriately increased; if the inhibition rate is low, the drug concentration of the competitive reaction needs to be appropriately increased.

[0096] 2. Identification of positive clones

[0097] Indirect enzyme-linked immunosorbent assay (ELISA) was used to identify positive phage clones. The specific method is as follows:

[0098] (1) Antigen immobilization

[0099] The detection antigen IMZ-A-OVA was diluted to 1 μg / mL with coating buffer, and 100 μL was added to each well. The plate was incubated at 37°C overnight. The next day, the plate was washed twice with PBST (0.01M PBS, 0.05% Tween-20). 120 μL of 2% skim milk solution was added to each well and incubated at 37°C for 3 hours. The liquid in the wells was poured out and patted dry on absorbent paper. The plate was then dried at 37°C for 1 hour and stored at 4°C until ready for use.

[0100] (2) Small-scale expression of nanoantibodies

[0101] In the third and fourth rounds of panning, 96 single colonies were randomly selected from the plate and inoculated into a 96-well deep-well plate containing 0.5 mL of LB-Amp per well. A single TG1 clone was also inoculated as a negative control. The plates were cultured overnight at 37°C and 180 rpm to serve as the bacterial solution "mother plate."

[0102] Take out 10 μL of bacterial solution from each well of the mother plate and inoculate it into another 96-well deep-well plate with 1 mL of LB-Amp in each well. The inoculated well numbers should correspond to those of the mother plate. Culture at 37°C, 180 rpm for 4 h. Add IPTG (1:1000 ratio, v / v) to each well and culture at 37°C, 180 rpm overnight.

[0103] (3) Enzyme-linked immunosorbent assay (ELISA) to identify positive clones

[0104] On the third day, centrifuge at 4000rpm to obtain the supernatant. Add 50 μL of supernatant to the coated ELISA plate, incubate at 37°C for 40 minutes, wash five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the well, add 100 μL of 1:5000 diluted HRP-labeled anti-VHH secondary antibody, incubate at 37°C for 40 minutes, wash five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the well, add 100 μL of TMB substrate solution, develop color at 37°C in the dark for 10 minutes; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; measure the absorbance at 450 nm with an enzyme-labeled instrument. Select OD 450 Phage clones with a count three times greater than that of negative clones were considered positive clones.

[0105] Select OD 450 Phage clones with a count greater than 3 times that of negative clones were positive clones for identification of positive nanoantibodies. Add titer group: 50 μL of supernatant identified as positive clones by indirect ELISA and 50 μL of PBS; inhibition group: 50 μL of supernatant identified as positive clones by indirect ELISA and 50 μL of imazalil standard (concentration of 1 μg / mL), incubate at 37°C for 40 min, wash five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), pat dry the liquid in the well, add 100 μL of 1:5000 diluted HRP-labeled anti-VHH secondary antibody, incubate at 37°C for 40 min, wash with PBST (0.01M The wells were washed five times with PBS, 0.06% Tween-20 (v / v), the liquid in the wells was patted dry, 100 μL of TMB substrate solution was added, and the cells were allowed to develop at 37°C in the dark for 10 min; 50 μL of stop solution (10% H2SO4, v / v) was added to terminate the reaction; and the absorbance at 450 nm was measured using a microplate reader.

[0106] Select the clone in plate 1 whose OD value is 3 times greater than that of the negative control well and has obvious inhibition, record the number of its corresponding well as Nb Y-2-2-4, and transfer the bacterial solution in the corresponding well of the mother plate to a sterile centrifuge tube, add glycerol and freeze for later use.

[0107] Example 3 Sequencing of the gene encoding the specific nanobody Nb Y-2-2-4 and determination of its amino acid sequence

[0108] 1. Experimental methods

[0109] The strain of specific nanoantibody Nb Y-2-2-4 obtained by indirect competitive ELISA identification was sent to a sequencing company for sequencing to obtain the nucleotide sequence of the specific nanoantibody Nb Y-2-2-4; based on the DNA sequencing results and the codon table, the amino acid sequence of the specific nanoantibody Nb Y-2-2-4 was obtained.

[0110] 2. Experimental results

[0111] The amino acid sequence of the VHH of the specific Nanobody Nb Y-2-2-4 is shown in SEQ ID No. 1:

[0112] QLQLVESGGGLVQAGGSLRLSCAASGRANILHTGAWFRQAPGEDREFVA AIRAIGHGTNYADSVKGRFIISGDNAKNTIYLEMNSLKPQDTAVYYCAASRVG ATTAVAEYEYWGQGTQVTVSSEPKTPKPQD.

[0113] The amino acid sequence and domain division diagram of the specific nanobody Nb Y-2-2-4 are shown in the figure. Figure 1As shown, it can be seen that the specific nanoantibody Nb Y-2-2-4 includes 4 framework regions (Framework region, FR) and 3 complementarity-determining regions (Complementarity-determining region, CDR); the framework regions (FR1~FR4) are respectively as shown in SEQ ID No.2: QLQLVESGGGLVQAGGSLRLSCAAS, SEQ ID No.3: GAWFRQAPGEDREFVAA, SEQ ID No.4: NYADSVKGRFIISGDNAKNTIYLEMNSLKPQDTAVYYC, SEQ ID No.5: WGQGTQVTVSSEPKTPKPQD; the complementarity determining regions (CDR1~CDR3) are respectively as shown in SEQ ID No.6: GRANILHT, SEQ ID No.7: IRAIGHGT, SEQ ID No.8: AASRVGATTAVAEYEY.

[0114] The nucleotide sequence of the VHH of the specific nanobody Nb Y-2-2-4 is shown in SEQ ID No.9:

[0115] CAGTTTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCGGGTCGCGCCAACATACTGCATACCGGCGCCTGGTTTCGCCAGGCTCCAGGGGAGGACCGTGAGTTTGTAGCAGCGATTAGGGCGATTGGTCATGGGACAAACTATGCAGACTCCGTGAAGGGT CGATTCATCATCTCCGGAGACAACGCCAAGAACACAATTTATCTTGAAATGAACAGCCTGAAACCTCAGGACACGGCCGTGTATTACTGTGCAGCATCTAGGGTGGGCGCTACTACGGCCGTGGCGGAGTATGAGTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAGAC.

[0116] Example 4 Large-scale preparation of specific nanobody Nb Y-2-2-4

[0117] The specific nanobody Nb Y-2-2-4 was prepared in the form of protein expression. The specific method is as follows:

[0118] The sequencing plasmid containing pComb3x-VHH was chemically transformed into E. coli BL21(DE3). A single colony was picked from the transformation plate and inoculated into 10 mL of LB(Amp) medium. The culture was inoculated into 750 mL of LB(Amp) medium at a ratio of 1:100 and incubated at 37°C, 250 rpm until the OD reached 0. 600 When the concentration was about 0.4-0.6, IPTG (1:1000 ratio, v / v) was added and cultured overnight at 37℃ and 250rpm. The next day, the cells were centrifuged at 12000rpm for 5min at 4℃ to collect the bacterial precipitate. The supernatant was collected to obtain the periplasmic soluble protein by freeze-thawing method with sucrose osmotic pressure and centrifuged at 12000rpm for 10min. The histidine tag carried by IMZ nanobody was used to bind to the miscellaneous protein. 2+ The IMZ nanobody was isolated and purified by affinity chromatography using the following steps: After filtering the periplasmic protein through a 0.22 μm filter, 1 mL of Ni-NTA resin was added and premixed overnight. The next day, the mixture was transferred to a gravity column. The column was washed and equilibrated with 0.01 mol / L PBS, and the mixture was added. The sample was loaded twice, and the column was then equilibrated and washed with PBS. The column was then washed with 50 mmol / L imidazole-PBS. Finally, the target protein was eluted with 200 mmol / L imidazole-PBS. The eluate was collected, which was the imazalil nanobody Nb Y-2-2-4. The collected eluate was transferred to a 3 kDa dialysis bag and dialyzed against PBS at 4°C for three days, a total of six times. After dialysis, the eluate was stored at -20°C until use.

[0119] Example 5 Detection performance analysis of specific nanoantibody Nb Y-2-2-4

[0120] 1. Coating and sealing

[0121] Dilute IMZ-A-OVA coating agent to 1 μg / mL with coating solution and coat overnight at 37°C. The next day, wash twice with PBST (0.01 M PBS, 0.05% Tween-20 (v / v)). Add 120 μL of 1% fish gelatin solution to each well, block at 37°C for 3 hours, dry at 37°C for 1 hour, and store in sealed bags at 4°C until use.

[0122] 2. Establishment of standard curve

[0123] (1) Experimental methods

[0124] Dilute IMZ-A-OVA coating agent to 1 μg / mL in coating solution and coat overnight at 37°C. The next day, wash twice with PBST (0.01 M PBS, 0.05% Tween-20 (v / v)). Add 120 μL / well of 2% skim milk solution and block at 37°C for 3 h. Discard the blocking solution and dry at 37°C for 1 h. Add 50 μL of nanoantibodies and a series of 50 μL of imazalil standards at different concentrations to each well, incubate at 37°C for 40 minutes, wash five times with PBST, pat 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 minutes, wash five times with PBST, pat dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop at 37°C in the dark for 10 minutes; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; read the absorbance at 450 nm with a microplate reader. The concentration of imazalil standard is plotted against the horizontal axis, B / B0 (OD value of the wells with imazalil added) 450 / OD of the well without imazalil 450 ) as the vertical axis, and establish an indirect competition standard curve.

[0125] (2) Experimental results

[0126] The standard curve of indirect competitive ELISA established based on the specific nanoantibody Nb Y-2-2-4 is shown in the figure below. Figure 2 As shown in the figure, it can be seen that the standard curve is S-shaped and has a good linear correlation. The detection range of this method for imazalil is 3.70-20.65 ng / mL, and the half inhibitory concentration (IC 50 ) was 8.74 ng / mL, and the limit of detection (LOD) was 1.85 ng / mL, indicating high detection sensitivity.

[0127] Example 6 Organic Tolerance Analysis of Nanobody Nb Y-2-2-4

[0128] (1) Experimental methods

[0129] Using different concentrations (10%, 20%, 30%, 40%, 50%) of ethanol / acetonitrile as diluent, the nanobody Nb Y-2-2-4 was diluted to the same working concentration to determine the binding ability of the antibody to the antigen. The antibody binding ability to the antigen without dilution with organic solvent diluent was taken as 100%, and the tolerance of the nanobody to different organic solvents and different concentrations of the same organic solvent was evaluated. The specific method is:

[0130] 50 μL of diluted nanoantibody Nb Y-2-2-4 and 50 μL of PBS were added to the coated ELISA plate, incubated at 37°C for 40 min, washed five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), patted the liquid in the wells dry, added 100 μL of 1:5000 diluted HRP-labeled anti-VHH secondary antibody, incubated at 37°C for 30 min, washed five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), patted the liquid in the wells dry, added 100 μL of TMB substrate solution, and developed at 37°C in the dark for 10 min; added 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; and read the absorbance value at 450 nm using a microplate reader.

[0131] (2) Experimental results

[0132] The activity curve of nanobody Nb Y-2-2-4 when different proportions of ethanol and acetonitrile were used as diluents is shown in the figure below. Figure 3 As shown, the antigen-binding activity of the nanobody Nb Y-2-2-4 remains above 80% in methanol, ethanol, and acetonitrile solutions with concentrations below 20%, and remains half as active in methanol and ethanol with concentrations below 40%. Therefore, the nanobody Nb Y-2-2-4 exhibits excellent tolerance to organic solvents. During pretreatment of actual sample testing, it is less affected by organic solvents, resulting in highly accurate test results.

[0133] Example 7 Acid-base tolerance analysis of nanobody Nb Y-2-2-4

[0134] (1) Experimental methods

[0135] PBS buffer solutions of different pH values ​​(pH 1.4, 2.4, 3.4, 4.4, 5.4, 6.4, 7.4, 8.4, 9.4, 10.4, 11.4, and 12.4) were prepared and used as diluents to dilute the Nbs to a working concentration. The biological activity of the antibodies in the system was measured, and the antigen-antibody binding capacity in a PBS buffer solution with a pH of 7.4 was taken as 100% to evaluate the acid-base tolerance of the nanobody. The specific method is as follows:

[0136] 50 μL of diluted nanoantibody Nb Y-2-2-4 and 50 μL of PBS were added to the coated ELISA plate, incubated at 37°C for 40 min, washed five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), patted the liquid in the wells dry, added 100 μL of 1:5000 diluted HRP-labeled anti-VHH secondary antibody, incubated at 37°C for 30 min, washed five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), patted the liquid in the wells dry, added 100 μL of TMB substrate solution, and developed at 37°C in the dark for 10 min; added 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; and read the absorbance value at 450 nm using a microplate reader.

[0137] (2) Experimental results

[0138] This study investigated the stability of the imazalil nanobody Nb Y-2-2-4 in the pH range of 1.4 to 12.4. Figure 4 The nanobody is inactivated in an overly acidic environment (pH = 1.4), but its binding activity is even better in a moderately acidic environment (pH = 2.4-6.4), increasing to 150%. Nb Y-2-2-4 maintains high activity in alkaline conditions, even maintaining around 60% activity at pH = 12.4. This demonstrates its high acidity and alkalinity tolerance, making it suitable for testing in both acidic and alkaline environments.

[0139] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A nanobody Nb Y-2-2-4 that specifically recognizes imazalil, characterized in that: The amino acid sequence of the nanobody Nb Y-2-2-4 is shown in SEQ ID No.

1.

2. The gene encoding the Nanobody Nb Y-2-2-4 according to 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 according to claim 2.

4. A recombinant cell, characterized in that The recombinant cell contains the recombinant vector according to claim 3.

5. Use of the nanobody Nb Y-2-2-4 according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3 or the recombinant cell according to claim 4 in detecting imazalil for non-diagnostic purposes.

6. Use of the nanobody Nb Y-2-2-4 according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3 or the recombinant cell according to claim 4 in the preparation of a product for detecting imazalil.

7. A kit for detecting imazalil, characterized in that: Contains the nanobody Nb Y-2-2-4 according to claim 1.

8. An immunoassay method for detecting imazalil for non-diagnostic purposes, characterized in that: The complete antigen obtained by coupling the imazalil hapten with the carrier protein is used as the coating source, and the nanobody Nb Y-2-2-4 according to claim 1 is used as the detection antibody for detection; the structural formula of the imazalil hapten is shown in formula (I): Formula (I).

9. The immunoassay method according to claim 8, characterized in that: The carrier protein is ovalbumin.

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