Myclobutanil hapten MY-6, artificial antigen, nano antibody as well as preparation method and application of myclobutanil hapten MY-6 and artificial antigen

Abstract

The invention discloses a myclobutanil hapten MY-6, an artificial antigen, a nano antibody as well as a preparation method and application of the myclobutanil hapten MY-6 and the artificial antigen. The overlapping degree of the hapten MY-6 and the skeleton structure of myclobutanil to be detected is high, so that the immunogenicity of the hapten MY-6 is effectively improved; the hapten MY-6 is further utilized to prepare a nano antibody Nb MY-21 capable of specifically recognizing myclobutanil, and the antibody also has excellent organic solvent tolerance and acid-base resistance. According to the present invention, the ELISA detection method is established based on the nanometer antibody, the myclobutanil detection range of the method is 0.63-10.77 ng / mL, the half inhibitory concentration (IC50) is 2.61 ng / mL, the minimum detection limit is 0.34 ng / mL, and the detection sensitivity is high; therefore, the nano antibody Nb MY-21 can be well applied to actual sample detection of myclobutanil residues, and also has extremely high application value in preparation of myclobutanil detection products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular relates to a myclobutanil hapten MY-6, artificial antigen, nanobody and preparation method and application thereof. BACKGROUND

[0002] Myclobutanil is a triazole fungicide that can effectively prevent and control powdery mildew, rust, black disease, gray spot disease, brown spot disease and the like in fruits and vegetables, and has the characteristics of high efficiency, strong systemicity and broad spectrum. Myclobutanil has high persistence in the environment, can be enriched through the food chain, has toxic effects on non-target organisms (such as bees, aquatic organisms, etc.), and can interfere with the endocrine system of mammals, posing a potential threat to human health. Therefore, it is particularly important to develop a high-sensitivity myclobutanil rapid screening and detection method.

[0003] Currently, the research on detection technology for myclobutanil residues in food mainly includes chromatography, chromatography-mass spectrometry and spectrophotometry, etc. Although these instrumental analysis methods are standardized, highly sensitive and accurate, they are time-consuming, expensive and not suitable for rapid screening. In recent years, ELISA based on antigen-antibody reaction is an important supplement to instrumental analysis methods, which has the characteristics of high sensitivity, simple pretreatment and is more suitable for rapid detection on site, and can realize rapid screening of large quantities of samples in actual detection.

[0004] High-quality antibodies play a key role in the sensitivity and stability of ELISA detection methods. Currently, immunological analysis detection mainly relies on monoclonal antibodies as recognition elements. Patent CN117105875A discloses a myclobutanil hapten, which introduces an active group at the meta position of the chlorine atom on the benzene ring of myclobutanil. Compared with existing haptens, the hapten provided in the present application completely simulates the structure of myclobutanil, enhances the immunogenicity of the antigen, and after immunizing mice with the hapten, the produced antibody titer is 5.0×10 4 , and the half-inhibitory concentration (IC 50 ) is about 50 ppb; patent CN117466829A discloses a myclobutanil hapten, which replaces the chlorine atom on the benzene ring of myclobutanil. After immunizing mice with the hapten, the produced antibody IC 50The value is 254.44 ng / mL; it is also pointed out in the article "Preparation of monoclonal antibody of myclobutanil and establishment of colloidal gold detection method" that the half inhibition concentration of the colloidal gold test strip developed based on the monoclonal antibody prepared in the article is 2.10 μg / L, the linear range is 0.62-7.25 μg / L, and the detection limit is 50 μg / kg. Since the nature of the antibody is protein, the activity of the antibody will affect the accuracy and sensitivity of the detection result. In actual application, the biological recognition material is easy to be inactivated due to the storage and transportation conditions, and the sample pretreatment stage often needs to be treated with an organic solvent, and the residue of the organic solvent will greatly reduce the detection effect, and the organic tolerance and acid-base tolerance of the monoclonal antibody are poor. More importantly, the genetic stability of the monoclonal cell in the subculture process cannot be determined, and the separation of the monoclonal antibody from the animal increases the preparation cost. The camelid nanobody (Nb) prepared by genetic engineering has lower preparation cost and outstanding environmental adaptability than the traditional antibody, and gradually becomes a new force in the diagnostic reagent. At present, the development and application of the nanobody for specifically recognizing myclobutanil are less, and therefore, it has good application value to develop a method for rapidly detecting myclobutanil with good stability, high accuracy and strong sensitivity based on the nanobody. SUMMARY

[0005] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art, and provides a myclobutanil hapten MY-6.

[0006] The second object of the present application is to provide a preparation method of the above-mentioned myclobutanil hapten MY-6.

[0007] The third object of the present application is to provide a myclobutanil artificial antigen.

[0008] The fourth object of the present application is to provide a nanobody Nb MY-21 for specifically recognizing myclobutanil.

[0009] The fifth object of the present application is to provide a gene for encoding the above-mentioned nanobody Nb MY-21.

[0010] The sixth object of the present application is to provide a recombinant vector and a recombinant cell.

[0011] The seventh object of the present application is to provide an application of the above-mentioned nanobody Nb MY-21, the above-mentioned gene, the above-mentioned recombinant vector or the above-mentioned recombinant cell in detecting myclobutanil for non-disease diagnosis purpose or in preparing a product for detecting myclobutanil.

[0012] The eighth object of the present application is to provide a kit for detecting myclobutanil.

[0013] The ninth object of the present application is to provide an immunoassay method for detecting myclobutanil for non-disease diagnosis purpose.

[0014] The above object of the present application is achieved by the following technical solutions. The present application provides a myclobutanil hapten MY-6, whose structural formula is shown as formula (I): Formula (I).

[0015] The present application obtains the hapten MY-6 by reacting myclobutanil and 6-bromohexanoic acid, which has a high overlap degree with the skeleton structure of the analyte myclobutanil, effectively improving the immunogenicity of the hapten MY-6; further, the artificial antigen obtained by coupling the hapten MY-6 with keyhole limpet hemocyanin is used as an immunogen to immunize a llama, and a nanobody Nb MY-21 capable of specifically recognizing myclobutanil is screened; the amino acid sequence of the nanobody is shown as SEQ ID No. 1.

[0016] Therefore, the present application provides a preparation method of the above-mentioned myclobutanil hapten MY-6, which is obtained by reacting myclobutanil and 6-bromohexanoic acid.

[0017] Further, the preparation method of the myclobutanil hapten MY-6 is to dissolve myclobutanil in N,N-dimethylformamide, mix and react with 6-bromohexanoic acid, and purify the reactants by silica gel column chromatography to obtain a yellow solid, i.e. the myclobutanil hapten MY-6.

[0018] Further, the molar ratio of the myclobutanil and 6-bromohexanoic acid is 1:1.8-2.2.

[0019] Preferably, the molar ratio of the myclobutanil and 6-bromohexanoic acid is 1:2.

[0020] The present application provides the application of the myclobutanil hapten in the preparation of a myclobutanil artificial antigen or antibody.

[0021] The present application provides a myclobutanil artificial antigen, which is prepared by coupling the above-mentioned myclobutanil hapten MY-6 with a carrier protein; its structural formula is shown as formula (II): Formula (II).

[0022] Further, the hapten MY-6 is coupled with the carrier protein to prepare the artificial antigen by using the active ester method.

[0023] Further, the carrier protein is ovalbumin (OVA) or keyhole limpet hemocyanin (KLH).

[0024] Specifically, the preparation method is as follows: first, the myclobutanil hapten MY-6 is dissolved in DMF, EDC and NHS are added and stirred to activate the reaction, and the mixture is recorded as A liquid; second, the carrier protein is dissolved in phosphate buffer, and the mixture is recorded as B liquid; third, A and B liquids are mixed and stirred to react, and the mixture is dialyzed at 4 DEG C for 3 days to obtain the artificial antigen MY-6-KLH and MY-6-OVA.

[0025] The application provides application of the myclobutanil artificial antigen in preparation of an anti-myclobutanil antibody.

[0026] Further, the anti-myclobutanil antibody can be a monoclonal antibody, a polyclonal antibody or a nanobody.

[0027] The application provides a nanobody Nb MY-21 capable of specifically recognizing myclobutanil, which is prepared from the above-mentioned hapten MY-6 or the above-mentioned artificial antigen; the amino acid sequence of VHH of the nanobody Nb MY-21 is shown in SEQ ID No. 1.

[0028] Further, the preparation method of the nanobody Nb MY-21 is as follows: the artificial antigen is mixed and emulsified with an equal amount of Freund's adjuvant, a llama is immunized, and nanobodies capable of specifically binding to myclobutanil are screened from a camel-derived immunized nanobody library, and the nanobodies are prepared in a large amount by means of genetic engineering recombination expression.

[0029] Further, the nanobody Nb MY-21 comprises four framework regions FR1, FR2, FR3 and FR4 and three complementarity determining regions CDR1, CDR2 and CDR3, and 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.

[0030] The application provides a gene encoding the nanobody Nb MY-21, and the nucleotide sequence of VHH of the gene is shown in SEQ ID No. 9.

[0031] The application provides a recombinant vector containing the above-mentioned gene.

[0032] The present application also provides a recombinant cell containing the recombinant vector or the gene or expressing the nanobody Nb MY-21. Since the amino acid sequence of the nanobody Nb MY-21 and the gene sequence encoding the nanobody have been given in the present application, the nanobody described in the present application can be obtained by known recombinant DNA technology based on the amino acid sequence and the gene sequence of the nanobody. Therefore, any recombinant vector or recombinant cell that can be used to prepare the nanobody described in the present application should also be within the scope of protection of the present application.

[0033] The present application establishes an ELISA detection method based on the nanobody Nb MY-21, which has a detection range of 0.63-10.77 ng / mL for carbendazim, a half-inhibitory concentration (IC 50 ) of 2.61 ng / mL, and a minimum detection limit (LOD) of 0.34 ng / mL, and has high detection sensitivity. In addition, the nanobody also has excellent resistance to organic solvents and pH, and its antigen binding activity is still maintained at more than 75% in a 30% methanol solution, about half in a 50% methanol solution, and almost 100% in an acid-base environment with a pH of 3.4-11.4, and has good stability. Therefore, the antibody is less affected by organic solvents during the pretreatment process of actual sample detection, and can also be used for detection in an acid-base environment, and has the advantages of simple operation, short time consumption, high sensitivity, and high accuracy.

[0034] Therefore, the present application also provides the application of the nanobody Nb MY-21, the gene, the recombinant vector or the recombinant cell described above in the detection of carbendazim for non-diagnostic purposes or in the preparation of products for detecting carbendazim.

[0035] The present application also provides a kit for detecting carbendazim, containing the nanobody Nb MY-21 described above.

[0036] The present application also provides a method for detecting carbendazim for non-diagnostic purposes, using a complete antigen obtained by coupling the carbendazim hapten of formula (I) described above with a carrier protein as a coating agent, and using the nanobody Nb MY-21 described above or the kit described above for detection. The method has the advantages of simple operation, short time consumption, high sensitivity of detection results, and high accuracy.

[0037] Further, the carrier protein is ovalbumin.

[0038] Compared with the prior art, the present application has the following beneficial effects: The application provides a myclobutanil hapten MY-6, artificial antigen, nanobody and a preparation method and application thereof. The hapten MY-6 has high overlap with the skeleton structure of the to-be-detected myclobutanil, and effectively improves the immunogenicity of the hapten MY-6; further, the artificial antigen obtained by coupling the hapten MY-6 with a carrier protein is used as an immunogen to immunize a llama, and a nanobody Nb MY-21 capable of specifically recognizing myclobutanil is screened; the amino acid sequence of the VHH of the nanobody is shown as SEQ ID No. 1, and the nanobody has excellent organic solvent tolerance and acid-base tolerance. An ELISA detection method is established based on the nanobody, the detection range of the method for myclobutanil is 0.63-10.77 ng / mL, the half-inhibitory concentration (IC 50 ) is 2.61 ng / mL, the lowest detection limit (LOD) is 0.34 ng / mL, and the detection sensitivity is high; in the pretreatment process of actual sample detection, the nanobody is less affected by organic solvents, and can also be applied to the detection requirements in acid-base environments. Therefore, the nanobody Nb MY-21 can be well applied to the actual sample detection of myclobutanil residues, and has the advantages of simple operation, short time consumption, good stability of detection results, high sensitivity, high accuracy, and high application value in the detection of myclobutanil or the preparation of myclobutanil detection products. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 It is a mass spectrum identification diagram of the myclobutanil hapten MY-6.

[0040] Figure 2 It is an ultraviolet scanning diagram of the carrier protein (KLH, OVA), MY-6 and artificial antigens MY-6-KLH and MY-6-OVA.

[0041] Figure 3 It is an amino acid sequence and domain division schematic diagram of the specific nanobody Nb MY-21.

[0042] Figure 4 It is an indirect competitive ELISA standard curve diagram established based on the specific nanobody Nb MY-21.

[0043] Figure 5 It is an activity curve diagram of the nanobody Nb MY-21 when methanol / PBS with different proportions is used as a diluent.

[0044] Figure 6 It is an activity curve diagram of the nanobody Nb MY-21 when different pH values are used as a diluent. DETAILED DESCRIPTION

[0045] 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.

[0046] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0047] Example 1: Preparation of cyproconazole hapten MY-6 1. Experimental Methods 4 mmol of 6-bromohexanoic acid was added dropwise to a solution containing 2 mmol of cyproconazole in N,N-dimethylformamide, and the mixture was refluxed at 95 °C for 12 h. After the reaction was completed, the mixture was extracted with ethyl acetate and saturated NaCl to obtain a yellow viscous liquid. The filtrate was purified by vacuum distillation to remove the solvent, and the residue was purified by silica gel column chromatography to obtain a yellow solid, which was the cyproconazole hapten MY-6.

[0048] 2. Experimental Results The structural formula of the cyazofamid hapten MY-6 is shown in formula (I): Formula (I).

[0049] ESI-MS results are as follows Figure 1 As shown, 401.9 is the negative ion molecular peak of the hapten MY-6, and its relative molecular mass was calculated to be 402.9, which is consistent with the actual relative molecular mass, indicating that the cyproconazole hapten MY-6 was successfully prepared.

[0050] Example 2 Preparation of cyazofamid artificial antigen 1. Experimental Methods The cyproconazole hapten MY-6 prepared in Example 1 was prepared by coupling ovalbumin (OVA) and keyhole hemocyanin (KLH) with a carrier protein using the active ester method. The preparation method is as follows: 7.2 mg of myclobutanil hapten MY-6 was dissolved in DMF, 5.1 mg of EDC and 3.1 mg of NHS were added and stirred for 12 h to activate the reaction, and the mixture was recorded as A liquid. 10 mg of OVA was dissolved in phosphate buffer to form B liquid. A and B liquids were mixed and stirred for 12 h. After the reaction, PBS dialysis was performed at 4°C for 3 days. After dialysis, the artificial antigen MY-6-OVA was obtained. Similarly, 3.2 mg of myclobutanil hapten MY-6 was dissolved in DMF, 2.3 mg of EDC and 1.4 mg of NHS were added and stirred for 12 h to activate the reaction, and the mixture was recorded as A liquid. 10 mg of KLH was dissolved in phosphate buffer to form B liquid. A and B liquids were mixed and stirred for 12 h. After the reaction, PBS dialysis was performed at 4°C for 3 days. After dialysis, the artificial antigen MY-6-KLH was obtained. The structural formula of MY-6-OVA and MY-6-KLH is shown in formula (II): Formula (II).

[0051] In the formula, the carrier protein is ovalbumin (OVA) and keyhole limpet hemocyanin (KLH).

[0052] 2. Experimental results The carrier protein (KLH, OVA), MY-6, and artificial antigens MY-6-KLH and MY-6-OVA were subjected to ultraviolet scanning identification (190-450 nm), and the results are shown in Figure 2 The characteristic absorption peak of the prepared artificial antigen has a significant blue shift compared with the carrier protein (KLH, OVA) and MY-6, and the artificial antigen has the characteristic absorption peak of the hapten MY-6 and the carrier protein (KLH, OVA), indicating that the artificial antigen is successfully prepared.

[0053] Example 3. Construction of llama immune antibody library 1. Llama immunization scheme Healthy llamas were used for animal immunization, and MY-6-LF was used as an immunogen. The subcutaneous injection was performed on the back of the neck of the llama, and the immunization dose was 1.0 mg of immunogen each time. The first immunization was performed using 0.5 mL of complete Freund's adjuvant mixed with the immunogen for emulsification, and the subsequent booster immunization was performed using 0.5 mL of incomplete Freund's adjuvant mixed with the antigen for emulsification. Then, the booster immunization was performed every 2 weeks, and a total of 3 booster immunizations were performed.

[0054] Before immunization, 10 mL of blood was taken to separate the serum as a negative control. Starting from the second immunization, 10 mL of blood was taken after each immunization to detect the serum titer and competitive reaction. After the third and fourth immunizations, 50-100 mL of peripheral blood was collected to construct the nanobody library.

[0055] 2. Isolation of llama lymphocytes The llama peripheral blood needs to be quickly separated for lymphocytes, and the specific operation method is as follows: the llama peripheral blood is mixed and diluted with sterile normal saline in an RNase-free 50 mL centrifuge tube at 2:1. The diluted peripheral blood is centrifuged with a commercial lymphocyte separation medium, 15 mL of lymphocyte separation medium is added to a sterile 50 mL centrifuge tube, and 15 mL of diluted blood is slowly added along the wall of the test tube with a sterile Pasteur dropper. 800g centrifugation for 25min. Take the lymphocyte layer to a new 50mL centrifuge tube, dilute 2 times with normal saline, centrifuge at 1500g for 10min at 4℃, discard the supernatant. Blow the lymphocytes with 5mL of normal saline, centrifuge at 1500g for 10min again, discard the supernatant, and wash the lymphocytes thoroughly. Add lysis solution (TRNsol) to each portion of lymphocytes, 1mL per portion, and store in a 2mL centrifuge tube at -80℃ for standby.

[0056] 3. Extraction of total RNA The total RNA is extracted from the lymphocytes stored in the TRNsol lysis solution described above using a commercial RNA extraction kit, and the operation is performed according to the instructions.

[0057] After extracting the total RNA, a small amount of sample is taken for nucleic acid electrophoresis and determination of RNA concentration in a nanodrop. The ideal RNA sample should be complete and not degraded, with clear 28S and 18S bands visible on the nucleic acid electrophoresis gel, and no genomic DNA contamination, with the ultraviolet absorption value ratio (A 260 / 280 ) at 260nm and 280nm should be around 2.0. If genomic DNA contamination occurs, the genomic DNA should be removed with DNase before reverse transcription, and the removal of genomic DNA and the degradation of RNA during the process should be verified again by electrophoresis; if the RNA has been degraded, the RNA needs to be extracted again. The RNA should be reverse transcribed into cDNA as soon as possible or stored temporarily at -80℃.

[0058] 4. Synthesis of cDNA Using RNA as a template, the synthesis of cDNA first strand is carried out according to the instructions of Takara's first strand reverse transcription kit. The specific method is as follows: A. According to the first step reaction system of cDNA synthesis shown in Table 1, the reagents are mixed in a nuclease-free centrifuge tube, and operated in an ice bath; Table 1 First step reaction system of cDNA synthesis

[0059] B. Incubate the above reaction system at 65℃ for 5min, and cool on ice for 2min; C. The second step reaction system for cDNA synthesis shown in Table 2 was added to the system after step A reaction; Table 2 The second step reaction system for cDNA synthesis

[0060] D. Incubate at 42°C for 60 min and at 70°C for 5 min. The reverse transcription product cDNA was stored at -80°C.

[0061] 5. Amplification of the target gene of nanobody The target gene was amplified by two-step method using nested PCR.

[0062] The first round of PCR used cDNA as template and primer Q1 / Q2 for the first round of PCR reaction. The nucleotide sequence of primer Q1 / Q2 is shown in Table 5. The reaction system and reaction condition of the first round of PCR are shown in Table 3.

[0063] Table 3 The first step reaction system for nested PCR

[0064] The second round of PCR used the product of the first round of PCR reaction recovered by the kit as template after appropriate dilution, and primer Q3 / Q4 or Q3 / Q5 combination for the second round of PCR reaction. The nucleotide sequence of primer is shown in Table 5. The reaction system and reaction condition of the second round of PCR are shown in Table 4.

[0065] Table 4 The second step reaction system for nested PCR

[0066] Table 5 Primer and its nucleotide sequence used for amplification of the target gene of nanobody VHH

[0067] 6. Construction of gene library (1) Enzymatic digestion of VHH target gene and vector The VHH target gene and pComb3xss vector were subjected to enzymatic digestion using Sfil. The enzymatic digestion condition was 50°C for 16 h.

[0068] The 3500 bp band of the pComb3xss vector digestion product was recovered by agarose gel, and the VHH gene digestion product was directly cleaned and recovered by DNA recovery kit.

[0069] (2) Ligation of the digestion products The vector pComb3xss and VHH fragment were mixed (molar ratio 1:3), and after 16°C reaction for 16 h, they were cleaned and recovered by DNA recovery kit.

[0070] (3) Electroporation Take 5 μL ligation product into 50 μL electroporation competent E. coli TG1, mix gently and transfer to 0.2 cm electroporation cuvette for electroporation (voltage 1.8 kv), immediately after electroporation, add 800 μL and 150 μL preheated to 37°C SOC medium to the electroporation cuvette in two times, collect to a sterile centrifuge tube, cultivate at 37°C, 250 rpm for 1 h to recover the cells.

[0071] Take 50 μL recovered bacterial solution to gradient dilution, take 100 μL of each concentration gradient dilution bacterial solution to coat on LB-Amp culture dish with diameter of 90 mm as counting plate, cultivate at 37°C overnight. Coat the remaining undiluted recovered bacterial solution to LB-Amp culture dish with diameter of 120 mm, 1 mL bacterial solution coats 2-3 culture dishes as amplification plate, cultivate at 37°C overnight.

[0072] Count the number of bacteria on the counting plate, calculate the total number of bacteria in the recovered bacterial solution, carry out multiple electroporation to accumulate the total number of transformed colonies to 10 7 cfu or above, this number is the library capacity of nanobody gene library.

[0073] Scrape the transgenic E. coli colonies in the amplification plate with cell scraper, centrifuge to collect the bacterial bodies, discard the supernatant, add LB-Amp (0.5 mL per electroporation) to resuspend, mix uniformly, add sterile glycerol (v / v) with final concentration of 25%, take 50 μL bacterial solution to gradient dilution to determine the cell number, the remaining bacterial solution is aliquoted and stored at -80°C. This is the carbendazim nanobody gene library.

[0074] 7. Phage rescue According to the above determination of the number of transgenic E. coli cells, inoculate 10 times the library capacity of cells in 150 mL LB-Amp, control OD 600 <0.2, cultivate at 37°C, 250 rpm to logarithmic phase (OD 600 about 0.4-0.6); add 1 mL helper phage M 12 cfu / mL or above. 13 K 07, 37℃ static 30min, 250rpm culture 1h, add Kana (Kanamycin, working concentration 50μg / mL) 37℃, 250rpm culture overnight. Transfer the bacteria to centrifuge bottle, 12000rpm 4℃ 15min centrifugal, take supernatant, add 1 / 4 volume of PEG / NaCl, ice bath 2.5h or more. 12000rpm 4℃ 15min centrifugal, discard supernatant, the precipitate is resuspended with 750μL TBS, transfer to 1.5mL centrifuge tube, 4000rpm 25℃ 5min centrifugal, through 0.22μm polyether sulfone filter membrane. Take 10μL phage to determine the titer, the rest is mixed evenly after adding the final concentration of 50% sterile glycerol (v / v), -80℃ preservation, it is the carbendazim nanobody phage library, which can be directly used for affinity panning.

[0075] Example 4 Affinity panning of nanobody and its identification 1. Affinity panning of nanobody (1) Antigen and carrier protein immobilization Affinity panning uses strong adsorption enzyme-coated plates. Each round of panning is 1 column, and a total of 4 rounds of panning is 4 columns. AB wells are coated with 1 mg / mL of coated carrier protein OVA, and CDEF wells are coated with 10 μg / mL of detection antigen MY-6-OVA using CB coating solution, 100 μL per well, 37°C static overnight. In addition, because the llama may be immune to a variety of carrier proteins, three immune carrier proteins ConA (Concanavalin A), LF (Lactoferrin), and KLH (Keyhole limpet hemocyanin) were mixed and diluted to a final concentration of 2 mg / mL, and coated with 1 column of immunogen carrier protein alone. The next day, after washing the plate twice with PBST (0.01M PBS, 0.05% Tween-20), 120 μL of 1% fish gelatin solution was added to each well and incubated at 37°C for 3h. Pour out the liquid in the wells and pat dry on absorbent paper, 37°C dry for 1h, 4°C storage for later use.

[0076] (2) Positive phage screening Add the phage library in Example 1 to 2 immunogen carrier proteins, 150 μL per well, and incubate at 37°C for 1 h (only the first round needs this step, and the second, third, and fourth rounds start directly from the AB well). Transfer the free phage to the coated original carrier protein AB well, 150 μL per well, and incubate at 37°C for 1 h. Transfer the free phage to 3 micro-wells with immobilized antigen (MY-6-OVA), 100 μL per well, and incubate at 37°C for 1 h. Discard the free phage in the wells, wash the micro-wells with PBST (0.01M PBS, 0.05% Tween-20 (v / v)) for 10 times, and then wash with PBS for 5 times. Add 100 μL of 10 mg / mL trypsin-TBS solution to elute at 37°C for 30 min. Collect the phage, take 10 μL of the eluted phage to determine the titer, and use the rest to infect 5 mL of TG1 strain that is grown to the logarithmic phase. E. coli The next day, precipitate the amplified phage with PEG / NaCl and determine the titer of the phage.

[0077] In the second, third, and fourth rounds of the panning process, the MY-6-OVA coating concentration is reduced to 1000 ng / mL, 500 ng / mL, and 100 ng / mL, respectively. After incubation with the phage at 37°C for 1 h, wash with PBST (0.01M PBS, 0.05% Tween-20 (v / v)) and PBS, and then elute the phage by adding a certain concentration of drug, incubating at 37°C for 1 h, and then sucking out the liquid in the wells. Repeat the screening scheme in step (2). The drug elution concentration is 1000 ng / mL, 500 ng / mL, and 100 ng / mL, respectively. The above conditions can be adjusted according to the actual immunization situation. If the serum titer is low, the MY-6-OVA concentration for detection during panning can be appropriately increased; if the inhibition rate is low, the drug concentration for the competition reaction can be appropriately increased.

[0078] 2. Identification of positive clones An indirect enzyme-linked immunosorbent assay is used to identify positive phage clones. The specific method is as follows: (1) Antigen immobilization The detection antigen MY-6-OVA is diluted with coating solution to 1 μg / mL, 100 μL per well, and incubated at 37°C overnight. The next day, wash the plate twice with PBST (0.01M PBS, 0.05% Tween-20), add 120 μL of 2% skimmed milk powder solution to each well, and incubate at 37°C for 3 h. Discard the liquid in the wells and pat dry on absorbent paper, dry at 37°C for 1 h, and store at 4°C for later use.

[0079] (2) Small-scale expression of nanobodies 96 single colonies were randomly picked from each plate of the output titer determination of the 3rd and 4th round of panning, inoculated into 96-deep well plates with 0.5 mL LB-Amp in each well, and a TG1 monoclonal was inoculated as a negative control, incubated at 37°C, 180 rpm overnight, as a bacterial liquid "mother plate".

[0080] 10 μL bacterial liquid was taken from each well of the mother plate and inoculated into another 96-deep well plate with 1 mL LB-Amp in each well, the well number was corresponding to the mother plate, incubated at 37°C, 180 rpm for 4 h, and IPTG (1:1000 ratio, v / v) was added to each well, incubated at 37°C, 180 rpm overnight.

[0081] (3) Identification of positive clones by enzyme-linked immunoassay The supernatant was centrifuged at 4000 rpm on the third day, and 50 μL of the supernatant was added to the already coated enzyme-labeled plate, incubated at 37°C for 40 min, washed five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), patted dry the liquid in the well, added 100 μL of HRP-labeled anti-VHH secondary antibody diluted 1:5000, incubated at 37°C for 40 min, washed five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), patted dry the liquid in the well, added 100 μL of TMB substrate solution, developed color at 37°C for 10 min in the dark; added 50 μL of stop solution (10% H2SO4, v / v) to stop the reaction; measured the absorbance value at 450 nm with an enzyme-labeled instrument. Selected OD 450 Phage clones more than 3 times the negative were positive clones.

[0082] Selected OD 450 Phage clones more than 3 times the negative were positive clones for identification of positive nanobodies. Added titer group: 50 μL of supernatant of positive clones identified by indirect ELISA and 50 μL of PBS; inhibition group: 50 μL of supernatant of positive clones identified by indirect ELISA and 50 μL of carbendazim standard (concentration of 1 μg / mL), incubated at 37°C for 40 min, washed five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), patted dry the liquid in the well, added 100 μL of HRP-labeled anti-VHH secondary antibody diluted 1:5000, incubated at 37°C for 40 min, washed five times with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), patted dry the liquid in the well, added 100 μL of TMB substrate solution, developed color at 37°C for 10 min in the dark; added 50 μL of stop solution (10% H2SO4, v / v) to stop the reaction; measured the absorbance value at 450 nm with an enzyme-labeled instrument.

[0083] The OD value in the plate 1 is greater than 3 times the negative control hole, and the clone has obvious inhibition, and the corresponding hole number is recorded as Nb MY-21. The corresponding hole in the mother plate is transferred to a sterile centrifuge tube, and glycerol is added for storage.

[0084] That is, a nanobody capable of specifically recognizing carbendazim is obtained, which is named Nb MY-21.

[0085] Example 5 Sequencing of specific nanobody Nb MY-21 coding gene and determination of its amino acid sequence I. Experimental method The strain of the specific nanobody Nb MY-21 obtained by indirect competitive ELISA identification is sent to a sequencing company for sequencing to obtain the nucleotide sequence of the specific nanobody Nb MY-21; according to the DNA sequencing result and the codon table, the amino acid sequence of the specific nanobody Nb MY-21 is obtained.

[0086] II. Experimental results The amino acid sequence of the VHH of the specific nanobody Nb MY-21 is shown as SEQ ID No. 1: QVQLVESGGGLVQAGGSLRLTCAASRMVFSLNAAGWYRQPPGKERDLVASLSTSGASNYADSIEGRFSISWDPAKNTVSLQMNNLKPEATAVYYCSVGRGSARNYNPVHWGQGTQVTVSSEPKTPKPQD.

[0087] The amino acid sequence and domain division schematic diagram of the specific nanobody Nb MY-21 are shown as Figure 3 It can be seen that the specific nanobody Nb MY-21 includes four framework regions (Framework region, FR) and three complementarity-determining regions (Complementarity-determining region, CDR).

[0088] The framework regions (FR1-FR4) are as follows: SEQ ID No. 2: QVQLVESGGGLVQAGGSLRLTCAAS, SEQ ID No. 3: AGWYRQPPGKERDLVAS, SEQ ID No. 4: NYADSIEGRFSISWDPAKNTVSLQMNNLKPEATAVYYC, SEQ ID No. 5: WGQGTQVTVSSEPKTPKPQD. Complementarity determining regions (CDR1-CDR3) are as follows, respectively: SEQ ID No. 6: RMVFSLNA, SEQ ID No. 7: LSTSGAS, SEQ ID No. 8: SVGRGSARNYNPVH.

[0089] The nucleotide sequence of the VHH of the specific nanobody Nb MY-21 is shown as SEQ ID No. 9: CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGGCGGGGGGGTCTCTGAGACTCACCTGTGCAGCCTCAAGAATGGTCTTCAGTCTCAACGCCGCGGGCTGGTACCGCCAGCCTCCAGGGAAGGAGCGCGACTTGGTCGCAAGTCTGAGTACTAGTGGTGCTTCAAACTATGCAGACTCCATAGAGGGCCGATTCTCCATCTCGTGGGACCCCGCCAAGAACACAGTGTCCCTGCAAATGAACAATCTGAAACCTGAGGCCACGGCCGTCTACTATTGTAGTGTGGGTCGCGGCAGTGCCAGAAATTACAACCCCGTACACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAGAC Example 6 Mass production of specific nanobody Nb MY-21 The specific nanobody Nb MY-21 is prepared in the form of protein expression, and the specific method is as follows: The sequencing plasmid containing pComb3x-VHH is transformed into BL21 (DE3) by chemical transformation. E. coli A single colony is inoculated into 10 mL LB (Amp) medium, and cultured at 37°C, 250 rpm overnight. The overnight culture is inoculated into 750 mL LB (Amp) medium at a ratio of 1:100, and cultured at 37°C, 250 rpm until the OD 600 is about 0.4-0.6, IPTG (1:1000 ratio, v / v) is added, and the culture is incubated at 37°C, 250 rpm overnight. The next day, centrifugation is performed at 4°C, 12000 rpm for 5 min, and the bacterial pellet is collected. The osmotic pressure freeze-thaw method with sucrose is used, and centrifugation is performed at 12000 rpm for 10 min. The supernatant is collected to obtain the periplasmic cavity soluble protein.

[0090] Ni 2+ affinity chromatography column adsorption capacity difference, the separation and purification of IMZ nanobodies, the specific operation steps are as follows: the periplasmic cavity protein is filtered through 0.22 μm filter membrane, 1 mL Ni-NTA resin filling is added and pre-mixed overnight; the next day, it is transferred to a gravity column; 0.01 mol / L PBS is used to wash and balance the gravity column, the above mixed solution is added, the loading is repeated twice, PBS is added to balance and wash the gravity column, then 50 mmol / L imidazole-PBS is added for washing impurities, finally 200 mmol / L imidazole-PBS is used for elution of the target protein, and the eluate is collected as the myclobutanil nanobody Nb MY-21. The collected eluate is transferred to a 3 kDa dialysis bag and dialyzed in PBS at 4℃ for 3 days, a total of 6 times. After dialysis, it is stored at -20℃ for standby.

[0091] Example 7 Analysis of detection performance of specific nanobody Nb MY-21 I. Experimental method 1. Coating and blocking MY-6-OVA coating stock is diluted to 1 μg / mL with coating solution and coated at 37℃ overnight. The next day, after washing twice with PBST (0.01M PBS, 0.05% Tween-20 (v / v)), 1% fish gelatin solution is added at 120 μL per well, 37℃ blocking for 3h, 37℃ drying for 1h, and sealed in a bag at 4℃ for standby.

[0092] 2. Establishment of standard curve MY-6-OVA coating stock is diluted to 1 μg / mL with coating solution and coated at 37℃ overnight. The next day, after washing twice with PBST (0.01M PBS, 0.05% Tween-20 (v / v)), 120 μL / well of 2% skim milk powder solution is added, 37℃ blocking for 3h, discarding the blocking solution, 37℃ drying for 1h. 50 μL of nanobody and a series of different concentrations of 50 μL of myclobutanil standard are added to each well, 37℃ incubation for 40min, washing five times with PBST, patting the liquid in the well, adding 100 μL of 1:5000 diluted HRP-labeled anti-VHH secondary antibody, 37℃ incubation for 30min, washing five times with PBST, patting the liquid in the well, adding 100 μL of TMB substrate solution, 37℃ color development for 10min in the dark; adding 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; reading the absorbance value at 450nm with a microplate reader. The concentration of myclobutanil standard is taken as the abscissa, and B / B0 (OD of the well with myclobutanil / OD of the well without myclobutanil) is taken as the ordinate to establish an indirect competition standard curve. 450 450 2. Establishment of standard curve ​

[0093] II. Experimental results The standard curve graph of the indirect competitive ELISA established based on the specific nanobody Nb MY-21 is shown in Figure 4 It can be seen that the standard curve is S-shaped and has good linear correlation. The detection range of the method for myclobutanil is 0.63-10.77 ng / mL, the half-inhibitory concentration (IC 50 ) is 2.61 ng / mL, the minimum detection limit (LOD) is 0.34 ng / mL, and the detection sensitivity is high.

[0094] Example 8 Analysis of organic tolerance of nanobody Nb MY-21 I. Experimental method Different concentrations (10%, 20%, 30%, 40%, 50%) of methanol were used as diluents to dilute the nanobody Nb MY-21 to the same working concentration to determine the binding capacity of the antibody to the antigen. The binding capacity of the antibody diluted without organic solvent diluent to the antigen 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 as follows: Add 50 μL of the diluted nanobody Nb MY-21 and 50 μL of PBS to the coated enzyme-labeled plate, incubate at 37°C for 40 min, wash five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), tap dry the liquid in the well, add 100 μL of 1:5000 diluted HRP-labeled anti-VHH secondary antibody, incubate at 37°C for 30 min, wash five times with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), tap dry the liquid in the well, add 100 μL of TMB substrate solution, develop color at 37°C for 10 min in the dark; add 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; and read the absorbance value at 450 nm with an enzyme-labeled instrument.

[0095] II. Experimental results The activity curve of the nanobody Nb MY-21 when different proportions of methanol were used as diluents is shown in Figure 5 It can be seen that the antigen binding activity of the nanobody Nb MY-21 still maintains more than 75% in methanol solution below 30%, and still maintains about half in 50% methanol. Therefore, the nanobody Nb MY-21 has excellent organic solvent tolerance, and is less affected by organic solvents in the pretreatment process of actual sample detection, and has high detection accuracy.

[0096] Example 9 Analysis of acid-base tolerance of nanobody Nb MY-21 I. Experimental method Different pH PBS buffer solutions (pH 1.4, 2.4, 3.4, 4.4, 5.4, 6.4, 7.4, 8.4, 9.4, 10.4, 11.4, 12.4) were configured respectively, and the Nbs were diluted to the working concentration with the diluents, the biological activity of the antibody in the system was determined respectively, and the antigen-antibody binding capacity in the PBS buffer solution with pH 7.4 was taken as 100%, and the acid-base tolerance of the nanobody was evaluated. The specific method is as follows: 50 μL of diluted nanobody Nb MY-21 and 50 μL of PBS were added to the packaged enzyme-labeled plate, incubated at 37°C for 40 min, washed with PBST (0.01M PBS, 0.06% Tween-20 (v / v)) for five times, patted dry the liquid in the hole, added 100 μL of 1:5000 diluted HRP labeled anti-VHH secondary antibody, incubated at 37°C for 30 min, washed with PBST (0.01M PBS, 0.06% Tween-20 (v / v)) for five times, patted dry the liquid in the hole, added 100 μL of TMB substrate solution, 37°C color development for 10 min; added 50 μL of stop solution (10% H2SO4, v / v) to terminate the reaction; the absorbance value at 450 nm was read by the enzyme-labeled instrument.

[0097] II. Experimental results In this experiment, the stability of the nanobody Nb MY-21 of myclobutanil in the range of pH=1.4~12.4 was investigated, and the biological activity of the nanobody was determined by ELISA. Figure 6 It can be seen that the activity of the nanobody decreases to 30% in the over-acid (pH=1.4) and over-alkali environment (pH=12.4). But in the suitable acid-base environment (pH=3.4~11.4), the binding activity is almost maintained at 100%, which shows that the acid-base tolerance of Nb MY-21 is very high, and it can be applied to the detection requirements in the acid-base environment.

[0098] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and all are included in the protection scope of the present application.

Claims

1. A cyazofamid hapten MY-6, characterized in that, Its structural formula is shown in equation (I): Formula (I).

2. The method for preparing the cyazofamid hapten MY-6 according to claim 1, characterized in that, The product is obtained by reacting cyproconazole with 6-bromohexanoic acid.

3. A cyazofamid artificial antigen, characterized in that, It is prepared by coupling the cyproconazole hapten MY-6 of claim 1 with a carrier protein; its structural formula is shown in formula (II): Formula (II).

4. A nanobody Nb MY-21 that specifically recognizes cyazofamid, characterized in that, It is prepared from the hapten MY-6 of claim 1 or the artificial antigen of claim 3; the amino acid sequence of VHH of the nanobody Nb MY-21 is shown in SEQ ID No.

1.

5. The gene encoding the nanobody NbMY-21 of claim 4, characterized in that, The nucleotide sequence of the VHH of the gene is shown in SEQ ID No.

9.

6. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 5.

7. A recombinant cell, characterized in that, The recombinant cells contain the recombinant vector of claim 6 or the gene of claim 5 or express the nanobody NbMY-21 of claim 4.

8. The use of the nanobody NbMY-21 of claim 4, the gene of claim 5, the recombinant vector of claim 6, or the recombinant cell of claim 7 in the detection of cyazofamid for non-diagnostic purposes or in the preparation of products for the detection of cyazofamid.

9. A kit for detecting cyazofamid, characterized in that, It contains the nanobody NbMY-21 as described in claim 4.

10. A method for detecting cyazofamid for non-diagnostic purposes, characterized in that, Using the complete antigen obtained by conjugating the hapten of cyazofamid as described in claim 1 with the carrier protein as the coating antigen, detection was performed using the nanobody Nb MY-21 as described in claim 4 or the kit as described in claim 9.

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