A nano-antibody of patulin and its application

Abstract

The present invention discloses a nano-antibody for patulin and its application. The present invention uses an artificial patulin antigen (a conjugate of patulin and a carrier protein BSA) as a coating source and utilizes a natural nano-antibody library to screen the patulin nano-antibody. This method eliminates the need to immunize camelids with the artificial patulin antigen, thereby avoiding the toxic effects of patulin on animals. The patulin nano-antibody is prepared and a patulin immunoassay method is established using the patulin nano-antibody. The detection limit for patulin is 0.12 μg / mL, the half-inhibitory concentration is 0.41 μg / mL, and the linear range is 0.17-0.97 μg / mL. The method is simple, rapid, highly specific, and highly sensitive, and can be used for the rapid detection of patulin in food.

Description

Technical Field

[0001] The present invention relates to the field of immunoassay technology, and more specifically to a nanometer antibody of patulin and its application. Background Art

[0002] Patulin (PAT), also known as patulin, is a polyacetyllactone mycotoxin produced primarily by fungi such as Penicillium, Aspergillus, and Byssochlamys. Food safety issues caused by mycotoxin contamination are becoming a global concern. Patulin has teratogenic, carcinogenic, mutagenic, immunocompromising, and neurotoxic properties. It has been detected in a variety of fruits and their products, vegetables, and grains, with contamination being particularly severe in fruits and their products.

[0003] Commonly used PAT methods domestically and internationally include thin-layer chromatography, high-performance liquid chromatography, and liquid chromatography-mass spectrometry. While instrumental methods offer advantages such as high accuracy and low detection limits, they suffer from complex pretreatment processes, long detection times, and high costs, making them difficult to meet the requirements of rapid testing.

[0004] Antibody-based immunoassays enable rapid detection of mycotoxins, offering advantages such as simplicity, high specificity, high sensitivity, and high throughput. However, because haptens are directly designed using PAT, the instability of PAT has resulted in poor results when using the resulting half-antibodies to prepare PAT antibodies. Polyclonal antibodies have been prepared using patulin hemiglutaric acid derivatives, but the antibody specificity and titer are low, limiting the application of immunoassays. Another derivatization strategy involves derivatizing patulin, resulting in polyclonal antibodies with some specificity, but is still limited in immunoassays by large variability between polyclonal batches. Patulin has problems such as small molecular weight, poor stability, and high toxicity; conventional antibodies have low specificity, thermal stability, and organic solvent tolerance; and the resulting half-antibodies have been ineffective in preparing PAT genetically engineered antibodies. Currently, there is no research on the preparation of nanoantibodies specifically for patulin analysis, so it is highly desirable to provide a patulin nanoantibody, its preparation method, and its application. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a nanobody of patulin and its application.

[0006] The first object of the present invention is to provide a nanobody against patulin.

[0007] The second object of the present invention is to provide a gene encoding a nanobody against patulin.

[0008] The third object of the present invention is to provide a recombinant vector.

[0009] The fourth object of the present invention is to provide a recombinant cell.

[0010] A fifth object of the present invention is to provide the use of any one or more of the nanobodies, the encoding genes, the recombinant vectors, and / or the recombinant cells in the preparation of an immunological detection kit for patulin.

[0011] A sixth object of the present invention is to provide a method for detecting patulin for non-diagnostic purposes.

[0012] The seventh object of the present invention is to provide an immunological detection kit for patulin.

[0013] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0014] The present invention claims protection for a nanobody against patulin, which comprises a framework region FR and a complementary determining region CDR, wherein the complementary determining region CDR is: CDR1 as shown in the amino acid sequence of SEQ ID NO: 6, CDR2 as shown in the amino acid sequence of SEQ ID NO: 7 and CDR3 as shown in the amino acid sequence of SEQ ID NO: 8.

[0015] Preferably, the framework region FR comprises: FR1 having an amino acid sequence as shown in SEQ ID NO: 2, FR2 having an amino acid sequence as shown in SEQ ID NO: 3, FR3 having an amino acid sequence as shown in SEQ ID NO: 4, and FR4 having an amino acid sequence as shown in SEQ ID NO: 5.

[0016] More preferably, the amino acid sequence of the Nanobody is as shown in SEQ ID NO: 1.

[0017] The present invention also claims the following:

[0018] A gene encoding a nanobody against patulin, wherein the gene encodes any nanobody as described.

[0019] A recombinant vector is connected to the encoding gene.

[0020] A recombinant cell, comprising the recombinant vector or capable of expressing the nanobody.

[0021] The use of any one or more of the nanobodies, the encoding genes, the recombinant vectors, and / or the recombinant cells in the preparation of an immunological detection kit for patulin also falls within the scope of protection of the present invention.

[0022] Also claimed is a method for detecting patulin for non-diagnostic purposes, using any of the Nanobodies described.

[0023] An immunological detection kit for patulin contains the nanobody.

[0024] Preferably, the method further comprises a solid phase carrier coated with a detection antigen, wherein the detection antigen is a complete patulin antigen obtained by coupling patulin with a carrier protein.

[0025] More preferably, the carrier protein includes but is not limited to BSA or OVA.

[0026] Specifically, the preparation method of the complete patulin antigen obtained by coupling patulin with a carrier protein is as follows: patulin, N,N'-carbonyldiimidazole and 4-dimethylaminopyridine are fully reacted in N,N-dimethylformamide, solid-liquid separation is performed, and the supernatant is obtained as liquid A; a PBS buffer solution of the carrier protein is obtained as liquid B; liquid A is gradually dripped into liquid B, fully reacted, solid-liquid separation is performed, and the supernatant is obtained overnight, and purification is performed to obtain the antigen.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention uses an artificial patulin antigen (a conjugate of patulin and a carrier protein BSA) as a coating source and utilizes a natural nanoantibody library to screen patulin nanoantibodies. This eliminates the need to immunize camelids with the artificial patulin antigen, thereby avoiding the toxic effects of patulin on animals. A patulin nanoantibody is prepared and a patulin immunoassay is established using the patulin nanoantibody. The detection limit for patulin is 0.12 μg / mL, the half-inhibitory concentration is 0.41 μg / mL, and the linear range is 0.17-0.97 μg / mL. The method is simple, rapid, highly specific, and highly sensitive, and can be used for the rapid detection of patulin in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Protein electrophoresis of patulin artificial antigen.

[0030] Figure 2 Monoclonal phage ELISA analysis for the screening of patulin nanobodies.

[0031] Figure 3 Protein electrophoresis of patulin nanobody.

[0032] Figure 4This is the standard curve of the patulin ELISA analysis method. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0034] Example 1 Preparation and identification of patulin artificial antigen

[0035] 1. Experimental Methods

[0036] 1. Synthesis of patulin artificial antigen

[0037] Dissolve 4.0 mg of patulin (PAT), 52.6 mg of N,N'-carbonyldiimidazole, and 39.6 mg of 4-dimethylaminopyridine in 600 μL of N,N-dimethylformamide and stir overnight. After centrifugation, the supernatant is Solution A. Dissolve 14 mg of bovine serum albumin (BSA) or egg albumin (OVA) in 6 mL of PBS buffer (Solution B). Add Solution A dropwise to Solution B and allow to react at 4°C for 12 hours. After centrifugation, remove the supernatant and dialyze against PBS buffer at 4°C. The resulting artificial antigen (PAT-BSA or PAT-OVA) is aliquoted into centrifuge tubes at a concentration of 1 mg / mL and stored frozen at -20°C until use.

[0038] 2. Protein gel electrophoresis

[0039] Prepare a 10% polyacrylamide gel, mix the sample to be tested with loading buffer, and heat denature in a boiling water bath for 10 minutes. Slowly add 6 μL of protein molecular weight marker and 10 μL of sample to the gel loading wells. Set the electrophoresis conditions to 35 mA for 30 minutes. After the electrophoresis is complete, carefully remove the gel, rinse twice with deionized water, add an appropriate amount of Coomassie Brilliant Blue stain, and continue staining on a shaker for 2 hours. Change the destaining buffer several times until clear protein bands are observed.

[0040] 2. Experimental Results

[0041] Figure 1This is a protein electrophoresis diagram of the patulin artificial antigen, showing the carrier proteins BSA and OVA, and the artificial antigens PAT-BSA and PAT-OVA, respectively, conjugated to patulin. The results show that BSA has a molecular weight of approximately 66 kDa, and OVA has a molecular weight of approximately 45 kDa. Both PAT-BSA and PAT-OVA display protein bands that are larger than those of the carrier proteins (BSA or OVA). This indicates that a conjugation reaction occurred between PAT and the carrier proteins (BSA or OVA), confirming the successful preparation of the artificial antigens PAT-BSA and PAT-OVA.

[0042] Example 2 Construction of a natural library of nanobodies

[0043] 1. Experimental Methods

[0044] 1. Isolation of Camelid Lymphocytes and Extraction of Total RNA

[0045] Collect 100 mL of fresh camelid blood using a sodium heparin anticoagulant tube. Slowly add 20 mL of blood diluted fivefold with normal saline to the top layer of 20 mL of human peripheral blood lymphocyte separation medium. Centrifuge at 1500 rpm for 20 minutes at room temperature. Remove the middle lymphocyte layer, mix thoroughly with 30 mL of normal saline, and centrifuge at 4000 rpm for 20 minutes. Collect the lymphocyte pellet, resuspend it in 1 mL of TRIZOL solution, and add 0.2 mL of chloroform. Total RNA is then extracted using an RNA extraction kit.

[0046] 2. Reverse transcription of camelid lymphocyte total RNA into cDNA

[0047] Total RNA was reverse transcribed into cDNA using a commercial reverse transcription kit. The following reaction mixture was prepared in a PCR tube: 6 μL of template RNA (2 μg), 1 μL of Oligo dT Primer (50 μM), 1 μL of dNTPs (50 μM), and 2 μL of RNase-free deionized water. The mixture was incubated at 65°C for 5 minutes and then rapidly cooled on ice for 5 minutes. The following reaction mixture was then added to the above mixture: 4 μL of 5× PrimerScript II buffer, 0.5 μL of RNase Inhibitor (40 U / μL), 1 μL of PrimerScript II RTase (200 U / μL), and 4.5 μL of RNase-free deionized water. The mixture was gently mixed and reverse transcribed at 42°C for 1 hour in a PCR instrument. The enzyme was inactivated at 95°C for 5 minutes and then rapidly cooled on ice for 5 minutes to obtain reverse-transcribed cDNA.

[0048] 3. Two-step PCR amplification of nanobody genes

[0049] The nanobody gene was amplified using cDNA as a template using a two-step PCR method. The first pair of primers CALL001 and CALL002 was used for the first PCR step, and the second pair of primers Fr1-sfiI and Fr4-sfiI was used for the second PCR step.

[0050] CALL001:GTCCTGGCTGCTCTTCTACAAGG,

[0051] CALL002:GGTACGTGCTGTTGAACTGTTCC;

[0052] Fr1-SfiI:ACTGGCCGGCCTGGCCTGAGGAGACGGTGACCWGGGTC,

[0053] Fr4-SfiI:ACTGGCCCAGGCGGCCGAGGTGCAGCTGSWGSAKTCKG;

[0054] The first step PCR reaction system: 25 μL of PCR premix, 2 μL of cDNA, 1.5 μL of primer CALL001 (10 μM), 1.5 μL of primer CALL002 (10 μM) and 20 μL of RNase-free deionized water. The PCR amplification program was pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles, and final extension at 72°C for 10 min. The first step PCR product was obtained using an agarose gel DNA recovery kit.

[0055] The second step PCR reaction system: 25 μL of PCR premix, 2 μL of the first step PCR product, 1.5 μL of primer Fr1-SfiI (10 μM), 1.5 μL of primer Fr4-SfiI (10 μM) and 20 μL of RNase-free deionized water. The PCR amplification program was pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles, and final extension at 72°C for 10 min. The nanoantibody gene fragment was obtained using an agarose gel DNA recovery kit.

[0056] 4. Construction of a two-step nanobody library

[0057] A two-step nanobody library was constructed using nanobody gene fragments amplified by two-step PCR. The double enzyme digestion system included 5 μL of vector pComb3XSS or 1.0 μg of nanobody gene fragments, 2 μL of SfiI endonuclease, 5 μL of 10× CutSmartBuffer, and 38 μL of RNase-free deionized water. The double enzyme digestion conditions were 50°C for 16 h.

[0058] A DNA recovery kit was used to obtain the digested pComb3XSS vector and the nanobody gene fragment. The digested pComb3XSS vector and nanobody gene fragment were ligated using T4 DNA ligase. The enzyme ligation system consisted of 3 μL of digested pComb3XSS (100 ng), 1 μL of digested nanobody gene fragment (30 ng), 1 μL of T4 DNA ligase, 2 μL of 10× Buffer, and 13 μL of RNase-free deionized water. The ligation conditions were 16°C for 16 h, resulting in the ligated product, pComb3XSS-VHH. The ligated product, pComb3XSS-VHH, was cleaned and recovered using a universal DNA purification and recovery kit.

[0059] Using E. coli ER2738 as the competent cell for electroporation, add 2 μL of the enzyme-linked product, pComb3XSS-VHH, to 100 μL of freshly thawed competent cells. The cells were quickly transferred to a 0.2 cm cuvette pre-chilled on ice and quickly placed in an electroporation tank for electroporation. After 2 minutes, 1 mL of SOC medium was added, and all electroporation products were transferred to a sterile 50 mL centrifuge tube. The tubes were shaken at 37°C and 250 rpm for 1 hour to recover. The number of single colonies on the plates at different dilutions was measured to calculate the library capacity of the nanobody library. The recovered bacterial suspension was evenly spread across multiple LB plates containing ampicillin resistance. All cells on the plates that had been incubated overnight were collected and resuspended in 10 mL of LB liquid medium. Glycerol was added, and the tubes were aliquoted and stored at -80°C to obtain the library.

[0060] 5. Calculation of the library capacity of the nanobody phage library

[0061] 1 mL of the bacterial library was inoculated into 200 mL of LB liquid medium and incubated at 37°C with shaking at 250 rpm for 2-3 hours. 1 mL of helper phage M13KO7 was added, and the culture was allowed to stand at 37°C for 30 minutes. Then, the culture was shaken at 37°C with shaking at 250 rpm for 2 hours. Kanamycin (final concentration 100 μg / mL) was added and the culture was shaken overnight. The overnight culture was centrifuged at 8000 rpm for 20 minutes, and the supernatant was collected. One-fifth volume of sterile polyethylene glycol / sodium chloride solution (mass ratio 100:73.1) was added and mixed thoroughly. The culture was allowed to stand on ice for 4 hours, and then centrifuged at 8000 rpm for 20 minutes at 4°C. The supernatant was discarded, and the culture was resuspended in 1 mL of TBS, filtered through a 0.22 μm microporous filter, and stored at -80°C. The number of single colonies on the plates at different dilutions was measured to calculate the library capacity of the nanobody phage library.

[0062] 2. Experimental Results

[0063] The nanobody gene fragment and pComb3xss were digested with SfiI and ligated with T4 DNA ligase, and then electroporated into E. coli ER2738 competent cells to obtain a library capacity of 3.60×10 8 cfu / mL of the nanobody natural library. Single colonies were randomly picked for colony PCR and sequencing. Gene sequencing revealed a 95% insertion rate of the nanobody gene fragment in the recombinant plasmid. Gene sequencing also confirmed a 100% diversity in the nanobody natural library, meeting the requirements for patulin nanobody biopanning.

[0064] Example 3 Bioscreening of Patulin Nanobodies

[0065] 1. Experimental Methods

[0066] Based on phage display technology, six rounds of screening were performed using the antigen solid phase coating method.

[0067] 1. Packing

[0068] The coating antigen (PAT-BSA or PAT-OVA) prepared in Example 1 at a certain concentration, 1 mg / mL of carrier protein BSA, and 1 mg / mL of carrier protein OVA were added to the microwells of the ELISA plate at 100 μL / well and coated overnight at 37°C. The cells were washed twice with PBST solution, patted dry, and then 120 μL / well of blocking solution (3% skim milk powder by mass volume fraction) was added and incubated in a 37°C water bath for 3 hours. The liquid in the microwells was discarded, patted dry, and then dried in a 37°C oven for 30 minutes and placed in a 4°C refrigerator for later use. The concentrations of the coating antigen (PAT-BSA or PAT-OVA) in rounds 1 to 6 were 15, 10, 5, 2.5, 0.5, and 0.1 μg / mL, respectively. The coating antigen PAT-BSA was used in the 1st, 3rd, and 5th rounds of screening, and the coating antigen PAT-OVA was used in the 2nd, 4th, and 6th rounds of screening.

[0069] 2. The first round of screening

[0070] Add 100 μL / well of the nanobody phage library solution to the BSA-coated microwells and incubate at 37°C with shaking for 1 hour. After the reaction is complete, transfer the unbound solution to the OVA-coated microwells and incubate at 37°C with shaking for 1 hour. Then, transfer the unbound solution to the microwells coated with the coating antigen PAT-BSA prepared in Example 1 and incubate at 37°C with shaking for 1 hour. After the reaction is complete, discard the unreacted supernatant and wash the microwells containing the coating antigen five times with sterile PBST and then 15 times with sterile PBS. Pat dry. Add 100 μL / well of 0.2 mol / L glycine-hydrochloric acid (Gly-HCl, pH 2.2) solution to the microwells and incubate at 37°C with shaking for 10 minutes. Neutralize with 50 μL / well of 1 mol / L Tris-HCl (pH 8.2) solution to obtain the eluted product from the first round of panning. The eluted product was serially diluted at 100-, 1000-, and 10,000-fold concentrations. The number of single colonies on the plates at different dilutions was measured to calculate the titer of the eluted product. Titer (pfu / mL) = number of colonies × dilution factor × 100.

[0071] 250 μL of the eluted product was inoculated into 100 mL of logarithmic-phase E. coli TG1 culture to obtain the phage solution for the second round of panning and determine its titer. The specific infection procedure was as follows:

[0072] Inoculate 250 μL of the eluted product into 100 mL of LB liquid medium and incubate at 37°C with shaking at 250 rpm for 2–3 hours. Add 1 mL of helper phage M13KO7, incubate at 37°C for 30 minutes, then incubate at 37°C with shaking at 250 rpm for 2 hours. Add kanamycin (final concentration 100 μg / mL) and continue shaking overnight. Centrifuge the overnight culture at 8000 rpm for 20 minutes, collect the supernatant, add 1 / 5 volume of sterile polyethylene glycol / sodium chloride solution (mass ratio 100:73.1), mix thoroughly, incubate on ice for 4 hours, and centrifuge at 8000 rpm for 20 minutes at 4°C. Discard the supernatant, resuspend in 1 mL of TBS, filter through a 0.22 μm microporous filter, and store at −80°C.

[0073] 3. Second to sixth rounds of screening

[0074] Following the above method, a gradient drug competitive elution method was used to screen specific phages in rounds 2 to 6 (the concentrations of the coating antigen PAT-BSA in rounds 2 to 6 were 10, 5, 2.5, 0.5, and 0.1 μg / mL, respectively). Single colonies from the elution product titer plate were picked and transferred to a deep-well plate and incubated overnight at 37°C on a shaker. 10 μL of the overnight culture was transferred to the deep-well plate and incubated at 37°C for 3 hours. 1 μL of IPTG solution was added and the culture was continued at 37°C overnight.

[0075] The supernatant was used as the expression supernatant for ic-ELISA analysis of the coating original binding ability and patulin specificity of the nanoantibody in the deep-well plate.

[0076] The specific method of ic-ELISA is as follows:

[0077] The coated original PAT-BSA was diluted to 1 μg / mL, added to the microwells of the ELISA plate (100 μL / well), and incubated in a 37°C water bath overnight; after washing twice, 120 μL / well of blocking solution was added, and the plate was incubated at 37°C for 3 h; the liquid in the wells was discarded, and the plate was patted dry and then dried in a 37°C oven for 30 min; the expression supernatant (50 μL / well) and PBS dilution (50 μL / well) were added to the microwells as titer wells, and the expression supernatant (50 μL / well) and 1 μg / mL test substance standard solution (50 μL / well) were added to the other microwells as inhibition wells, and the plate was incubated in a 37°C water bath for 40 min; the liquid in the wells was discarded, washed 5 times, and patted dry. After drying, add 100 μL / well of anti-His tag mouse monoclonal antibody dilution (5000-fold diluted with PBST) and incubate in a 37°C waterbath for 40 minutes. Discard the liquid in the wells, wash five times, pat dry, add 100 μL / well of goat anti-mouse-HRP antibody solution (5000-fold diluted with PBST), and incubate in a 37°C waterbath for 40 minutes. Discard the liquid in the wells, wash five times, pat dry, add 100 μL / well of TMB two-component colorimetric solution, incubate in a 37°C waterbath for 10 minutes, add 50 μL / well of stop solution, and measure the absorbance at 450 nm to analyze the nanoantibodies' coating binding ability and patulin specificity in the deep-well plate. The bacterial cultures of positive clones with antigen binding ability and patulin specificity were sent for sequencing analysis.

[0078] Positive clones were characterized by an absorbance value greater than 0.5 (coating agent binding capacity) in the titer well and an inhibition rate (%) greater than 30% (patulin specificity). Inhibition rate (%) = (absorbance value of the titer well - absorbance value of the inhibition well) / absorbance value of the titer well × 100.

[0079] 2. Experimental Results

[0080] like Figure 2 As shown, ELISA analysis of monoclonal phage from the patulin nanobody screening was performed. Using PAT-BSA as the coating agent, four clones were obtained after six rounds of solid-phase coating and panning. E9, F4, H11, and H12 all showed binding activity to the coating agent.

[0081] Furthermore, the supernatants of the deep-well plate mid-term expression corresponding to the above four clones were analyzed using ic-ELISA. The results are as follows: Figure 2As shown, the results showed that only Nanobody E9 had antigen binding activity and patulin specificity, with an inhibition rate of approximately 53.5% at 1 μg / mL patulin. Sequencing showed that the amino acid sequence of Nanobody E9 is shown in SEQ ID NO.1:

[0082] QLQLVESGGGLVQTGGSLRLSCAASARSARSFNSYTVGWFRQTPGKEREFVA

[0083] AISWSGGSTFYADSVKGRFTISREKSKNSVYLQMNSLKPEDTAVYTCKALPRG YRGQGTQVTVSS;

[0084] Among them, the amino acid sequence of FR1 of Nanobody E9 is:

[0085] QLQLVESGGGLVQTGGSLRLSCAAS(SED ID NO.2);

[0086] The amino acid sequence of FR2 of Nanobody E9 is:

[0087] VGWFRQTPGKEREFVAA(SED ID NO.3);

[0088] The amino acid sequence of FR3 of Nanobody E9 is:

[0089] FYADSVKGRFTISREKSKNSVYLQMNSLKPEDTAVYTC(SED ID NO.4);

[0090] The amino acid sequence of FR4 of Nanobody E9 is:

[0091] RGQGTQVTVSS (SED ID NO.5);

[0092] The amino acid sequence of CDR1 of Nanobody E9 is: ARSARSFNSYT (SED ID NO. 6);

[0093] The amino acid sequence of CDR2 of Nanobody E9 is: ISWSGGST (SED ID NO. 7);

[0094] The amino acid sequence of CDR3 of Nanobody E9 is: KALPRGY (SED ID NO. 8).

[0095] Example 4 Preparation of Nanobody E9

[0096] 1. Experimental Methods

[0097] The plasmid of the positive clone corresponding to the correctly sequenced nanobody E9 was transformed into E. coli BL21 (DE3) competent cells, and the plate was inverted and cultured at 37°C for 12 hours and shaken overnight;

[0098] Inoculate 7.5 mL of overnight cultured bacteria into 750 mL of LB liquid medium (final concentration of ampicillin solution is 100 μg / mL), and culture at 37°C with shaking at 250 rpm for 2 to 3 hours. 600 When the pH value is 0.6-0.8, add IPTG to a final concentration of 1 mmol / L and continue shaking culture at 37°C overnight;

[0099] After overnight culture, centrifuge at 8000 rpm for 20 min, collect the cells, resuspend them in 5 mL of TES solution, and freeze at -80°C for 6 h;

[0100] Thaw on ice, add 15 mL of 5-fold diluted TES solution, mix at 4°C for 4 h, and centrifuge at 12000 rpm for 30 min to obtain the periplasmic secretion expression solution containing soluble nanobody E9.

[0101] Add 1 mL of Ni-NTA filler to the periplasmic secretion expression solution, mix at 4°C for 12 h, then add to the gravity purification column, rinse with 20 mL of PBS, and then elute with a gradient concentration of imidazole solution (PBS solution containing 10, 20, 50, 100, 200 mM imidazole), and identify the nanoantibody using SDS-PAGE.

[0102] The eluate containing nanobody E9 was collected, dialyzed with PBS solution at 4°C for 3 days, its concentration was determined, and the eluate was aliquoted and stored at -20°C for later use.

[0103] 2. Experimental Results

[0104] like Figure 3 As shown, the purified nanobody E9 can be correctly expressed in Escherichia coli, and the protein molecular weight is about 17kDa, which is consistent with its theoretical amino acid molecular weight.

[0105] Example 5 Establishment of a Patulin ELISA Analysis Method

[0106] 1. Experimental Methods

[0107] S1. Coat the microwells of an ELISA plate with 5 μg / mL patulin-containing artificial antigen PAT-BSA prepared in Example 1. After overnight coating at 37°C, add 120 μL of a 5% (w / v) skim milk solution to each well and block at 37°C for 3 hours. After drying the wells, dry them at 37°C for 1 hour before use.

[0108] S2. Add 50 μL of a series of concentrations (6.25, 3.13, 0.78, 0.39, 0.2, 0.1, and 0 μg / mL) of patulin standard solution or the test sample solution into the microwells of the ELISA plate. Add 50 μL of a certain dilution of patulin nanobody (amino acid sequence shown in SEQ ID NO. 1) into the microwells, incubate at 37°C for a period of time, pour out the liquid in the wells, wash with washing solution five times, and pat dry on absorbent paper.

[0109] S3. Add 100 μL of 5000-fold diluted HRP-labeled goat anti-camel secondary antibody solution to the microwells, incubate at 37°C for a period of time, wash five times with washing buffer, and pat dry;

[0110] S4. Add 100 μL of substrate solution to the microwells, shake and mix, incubate at 37°C for a period of time, and measure the absorbance of each well at a wavelength of 450 nm. Use the absorbance value of each standard concentration well as the vertical axis and the log10 value of the drug standard solution concentration as the horizontal axis to draw a standard curve graph, and perform curve fitting y=(AD) / [1+(x / C)B]+D. Among them, A and D represent the absorbance values ​​of the minimum and maximum concentrations of the patulin standard, respectively, and C is the midpoint concentration; when the standard concentration is equal to C, the absorbance value is (A+D) / 2, which is at the inflection point of the curve, and the half-maximal inhibitory concentration is IC 50 , B represents the steepness of the curve, called the slope factor; IC10 is the detection limit, IC 20 ~IC 80 For the detection range.

[0111] S5. Based on the absorbance value of the sample to be tested, the content of patulin in the sample to be tested can be calculated.

[0112] 2. Experimental Results

[0113] The results are as follows Figure 4 As shown in the figure, the detection limit of the established ELISA analysis method for patulin is 0.12μg / mL, the half-inhibitory concentration is 0.41μg / mL, the linear range is 0.17-0.97μg / mL, and the inhibition rate and the logarithm of patulin show a significant S-shaped curve relationship, with a correlation coefficient R 2 The method can directly detect the content of patulin in food and has the advantages of high sensitivity and high specificity.

[0114] Example 6 A patulin ELISA detection kit

[0115] 1. Composition

[0116] The artificial patulin antigen PAT-BSA prepared in Example 1, the patulin nanobody with the amino acid sequence shown in SEQ ID NO.1, 5% skim milk powder (mass to volume ratio), patulin standard and washing solution.

[0117] 2. Usage

[0118] As in Example 5.

[0119] Example 7 Evaluation of the specificity of patulin nanobody by ELISA analysis

[0120] 1. Experimental Methods

[0121] According to the method of Example 5, other analogs (ochratoxin OTA, vomitoxin, aflatoxin B1) were used instead of patulin to establish the ELISA standard curve. The cross-reactivity rate (CR) was used to evaluate the specificity of the method. CR (%) = IC 50 (Patulin) / IC 50 (analog)×100.

[0122] 2. Experimental Results

[0123] Table 1 Specificity evaluation of ELISA analysis method

[0124]

[0125] The results are shown in Table 1. The results show that the patulin nanobody (amino acid sequence shown in SEQ ID NO. 1) has good detection specificity, and the cross-talk rate to patulin analogs is <1%.

Claims

1. A nanobody to patulin, characterized in that The nanobody comprises a framework region FR and a complementary determining region CDR, wherein the complementary determining region CDR is: CDR1 with an amino acid sequence as shown in SEQ ID NO: 6, CDR2 with an amino acid sequence as shown in SEQ ID NO: 7 and CDR3 with an amino acid sequence as shown in SEQ ID NO:

8.

2. The Nanobody according to claim 1, characterized in that The framework region FR includes: FR1 with an amino acid sequence as shown in SEQ ID NO: 2, FR2 with an amino acid sequence as shown in SEQ ID NO: 3, FR3 with an amino acid sequence as shown in SEQ ID NO: 4, and FR4 with an amino acid sequence as shown in SEQ ID NO:

5.

3. The Nanobody according to claim 1 or 2, characterized in that The amino acid sequence of the nanobody is shown in SEQ ID NO:

1.

4. A gene encoding a nanobody against patulin, characterized in that: The encoding gene encodes the Nanobody according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that The recombinant vector is connected to the coding gene according to claim 4.

6. A recombinant cell, characterized in that The recombinant cell contains the recombinant vector according to claim 5, or is capable of expressing the Nanobody according to any one of claims 1 to 3.

7. Use of one or more of the Nanobody according to any one of claims 1 to 3, the encoding gene according to claim 4, the recombinant vector according to claim 5, and / or the recombinant cell according to claim 6 in the preparation of an immunological detection kit for patulin.

8. A method for detecting patulin for non-diagnostic purposes, characterized in that: Utilize the Nanobody of any one of claims 1 to 3.

9. An immunological detection kit for patulin, characterized in that: Containing the Nanobody according to claim 1 or 2.

10. The immunological detection kit according to claim 9, characterized in that The invention also contains a solid phase carrier coated with a detection antigen, wherein the detection antigen is a complete patulin antigen obtained by coupling patulin with a carrier protein.

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