Nanometer antibody of patulin and application

Abstract

The invention discloses a patulin nano antibody and application thereof, patulin artificial antigen (conjugate of patulin and carrier protein BSA) is used as coating antigen, a nano antibody natural library is used for screening the patulin nano antibody, and the patulin artificial antigen does not need to be used for immunizing camelidae animals. The toxic effect of patulin on animals is avoided; according to the present invention, the patulin nano-antibody is prepared by using the patulin nano-antibody, the patulin immunodetection method is established by using the patulin nano-antibody, the patulin detection limit of the patulin nano-antibody is 0.12 [mu] g / mL, the half inhibition concentration is 0.41 [mu] g / mL, and the linear range is 0.17-0.97 [mu] g / mL, and the method has characteristics of simpleness, rapidness, strong specificity and high sensitivity, and can be used for 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 nano antibody of patulin and its application Background Art

[0002] Patulin (PAT), also known as patulin, is a type of polyacetyl lactone mycotoxin, mainly produced by fungi such as Penicillium, Aspergillus and Byssochlamys. Food safety issues caused by mycotoxin contamination are becoming the focus of global attention. Patulin has teratogenic, carcinogenic, mutagenic, immune-damaging and neuro-damaging toxicities. It has been detected in a variety of fruits and their products, vegetables, grains, etc., with the most serious contamination in fruits and their products.

[0003] The PAT detection methods commonly used at home and abroad mainly include thin layer chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry, etc. Although the instrument detection method has advantages such as high accuracy and low detection limit, it has problems such as complex pre-treatment process, long detection time and high cost, and it is difficult to meet the requirements of rapid detection.

[0004] The antibody-based immunoassay can achieve rapid detection of mycotoxins, and has the advantages of simple operation, high specificity, high sensitivity and high throughput. However, since the hapten is directly designed using PAT, due to the instability of PAT, the preparation of PAT antibodies using the designed half-antibodies has poor results. Among them, polyclonal antibodies were prepared using semi-glutaric acid patulin derivatives, but the antibody specificity and titer were not high, which limited the application of immunoassay methods; another derivatization strategy was used to derive patulin, and the prepared polyclonal antibodies had certain specificity, but were still limited by the large differences between different batches of polyclonal antibodies in immunoassay. Patulin has problems such as small molecular weight, poor stability, and high toxicity; conventional antibodies have low specificity, thermal stability, and organic solvent tolerance; the preparation of PAT genetic engineering antibodies using the designed half-antibodies has poor results. At present, there is no research on the preparation of nanoantibodies for patulin analysis, so it is very necessary to provide a patulin nanoantibody and its preparation method and application. Summary of the invention

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

[0006] The first object of the present invention is to provide a nanobody of 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 by the following technical solutions:

[0014] The present invention claims protection for a nanobody of patulin, the nanobody comprising a framework region FR and a complementary determining region CDR, the complementary determining region CDR comprising: 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.

[0015] Preferably, the framework region FR is: 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 according to any one of claims 1 to 3.

[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, it 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, and the supernatant is separated by solid-liquid separation to obtain liquid A; the PBS buffer solution of the carrier protein is liquid B; liquid A is gradually dripped into liquid B, fully reacted, the supernatant is separated by solid-liquid separation, and purified to obtain the antigen.

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

[0028] The invention uses artificial patulin antigen (a conjugate of patulin and carrier protein BSA) as a coating source, uses a natural library of nanoantibodies to screen patulin nanoantibodies, does not need to use artificial patulin antigen to immunize camelids, and avoids the toxic effect of patulin on animals; a patulin nanoantibody is prepared, and a patulin immunoassay method is established using the patulin nanoantibody, the detection limit of which for patulin is 0.12 μg / mL, the half inhibition 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 in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels 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] Take 4.0 mg of patulin (PAT), 52.6 mg of N, N'-carbonyldiimidazole, and 39.6 mg of 4-dimethylaminopyridine and dissolve them in 600 μL of N, N-dimethylformamide and stir to react overnight. After centrifugation, the supernatant is liquid A. Dissolve 14 mg of bovine serum albumin (BSA) or chicken egg protein (OVA) in 6 mL of PBS buffer (liquid B). Liquid A is gradually dripped into liquid B and reacted at 4°C for 12 hours. After centrifugation, take the supernatant overnight and dialyze it with PBS buffer at 4°C. The obtained artificial antigen (PAT-BSA or PAT-OVA) is divided into centrifuge tubes at a concentration of 1 mg / mL and frozen at -20°C for use.

[0038] 2. Protein gel electrophoresis

[0039] Prepare 10% polyacrylamide gel, mix the sample to be tested with the loading buffer, and heat denature in a boiling water bath for 10 minutes. Take 6μL of protein standard molecular weight marker and 10μL of sample and slowly add them to the protein gel loading well, and set the electrophoresis conditions to 35mA and 30min. After the electrophoresis is completed, carefully remove the protein gel, rinse it twice with deionized water, add an appropriate amount of Coomassie Brilliant Blue staining solution, place it on a shaker and continue staining for 2h, then change the destaining solution several times until clear protein bands can be observed.

[0040] 2. Experimental Results

[0041] Figure 1This is the protein electrophoresis diagram of the artificial patulin antigen, showing the carrier proteins of BSA and OVA and the artificial antigens PAT-BSA and PAT-OVA obtained by coupling with patulin, respectively, with different properties. The results show that the molecular weight of BSA is about 66kDa, the molecular weight of OVA is about 45kDa, and both PAT-BSA and PAT-OVA show protein bands larger than the carrier protein (BSA or OVA), which indicates that a coupling reaction occurs between PAT and the carrier protein (BSA or OVA), indicating that the artificial antigens PAT-BSA and PAT-OVA are successfully prepared.

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

[0043] 1. Experimental Methods

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

[0045] 100 mL of fresh camelid blood was collected using a sodium heparin anticoagulation tube, and 20 mL of blood diluted 5 times with normal saline was slowly added to the upper layer of 20 mL of human peripheral blood lymphocyte separation solution, and centrifuged at 1500 rpm for 20 min at room temperature. The middle lymphocyte layer was taken out and mixed evenly with 30 mL of normal saline, and centrifuged at 4000 rpm for 20 min. The lymphocyte precipitate was collected, resuspended with 1 mL of TRIZOL solution, and 0.2 mL of chloroform was added thereto, and total RNA was prepared using an RNA extraction kit.

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

[0047] The total RNA obtained 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 dNTP (50 μM), and 2 μL of RNase-free deionized water. The mixture was kept at 65°C for 5 minutes and then quickly cooled on ice for 5 minutes. The following reaction mixture was prepared in 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 slowly mixed and reverse transcribed at 42°C for 1 hour on a PCR instrument. After the enzyme was inactivated at 95°C for 5 minutes, the reverse transcribed cDNA was obtained by quickly cooling on ice for 5 minutes.

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

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

[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 nanoantibody library

[0057] A two-step nanoantibody library was constructed using nanoantibody gene fragments amplified by two-step PCR. The double enzyme digestion system included 5 μL of vector pComb3XSS or 1.0 μg of nanoantibody gene fragment, 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] The pComb3XSS vector and the nanobody gene fragment after restriction digestion were obtained using a DNA recovery kit. The pComb3XSS vector and the nanobody gene fragment after restriction digestion were connected using T4 DNA ligase. The enzyme connection system was: 3 μL of restriction digested pComb3XSS (100 ng), 1 μL of restriction 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 enzyme connection conditions were 16°C for 16 h, and the enzyme-linked product pComb3XSS-VHH was obtained. The enzyme-linked product pComb3XSS-VHH was cleaned and recovered using a universal DNA purification and recovery kit.

[0059] Using E.coli ER2738 as the electroporation competent cell, add 2 μL of the enzyme-linked product pComb3XSS-VHH to 100 μL of the freshly thawed competent cells and quickly transfer to a 0.2 cm electroporation cup precooled in ice, and quickly place the electroporation cup into the electroporation tank for electroporation. After 2 minutes, add 1 mL of SOC medium, and transfer all electroporation products to a 50 mL sterile centrifuge tube, place it at 37 ° C, 250 rpm shaker for 1 hour of recovery culture, and determine the number of single colonies on the plate at different dilutions to calculate the library capacity of the nanoantibody bacterial library. All the recovered bacterial liquids were evenly spread on multiple LB culture plates containing ampicillin resistance, all the bacteria on the plates cultured overnight were collected, resuspended in 10 mL of LB liquid culture medium, glycerol was added, and stored at -80 ° C after aliquoting to obtain a bacterial library.

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

[0061] 1mL of bacterial library was inoculated into 200mL of LB liquid culture medium and placed at 37℃, 250rpm shaking culture for 2-3h. 1mL of helper phage M13KO7 was added, and it was allowed to stand at 37℃ for 30min, then shaken and cultured at 37℃, 250rpm for 2h, and then kanamycin (final concentration of 100μg / mL) was added and continued to shake and culture overnight. The overnight cultured bacterial solution was centrifuged at 8000rpm for 20min, the supernatant was collected, 1 / 5 volume of sterile polyethylene glycol / sodium chloride (mass ratio of the two was 100:73.1) solution was added, mixed evenly, and allowed to stand in ice for 4h, centrifuged at 8000rpm for 20min at 4℃, the supernatant was discarded, and it was resuspended with 1mL of TBS solution, filtered through a 0.22μm microporous filter membrane, and stored at -80℃. The number of single colonies on the plate at different dilutions was determined to calculate the library capacity of the nanoantibody phage library.

[0062] 2. Experimental Results

[0063] The nanobody gene fragment and pComb3xss were double-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 nanoantibody natural library. Single colonies were randomly picked for colony PCR and sequencing. Gene sequencing revealed that the insertion rate of the nanoantibody gene fragment in the recombinant plasmid was 95%, and gene sequencing revealed that the diversity of the nanoantibody natural library was 100%, which could meet the requirements of patulin nanoantibody bioscreening.

[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. Coating

[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. Washed twice with PBST washing solution, patted dry, added 120 μL / well of blocking solution (mass volume fraction of 3% skim milk powder), and incubated in a 37°C water bath for 3h; poured out the liquid in the microwell, patted dry, dried in a 37°C oven for 30min, and placed in a 4°C refrigerator for standby use; the concentrations of the coating antigen (PAT-BSA or PAT-OVA) in the 1st to 6th rounds were 15, 10, 5, 2.5, 0.5, 0.1 μg / mL, respectively, and 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 nano antibody phage library solution to the microwells coated with BSA, and shake at 37°C for 1 hour. After the reaction is completed, transfer the unbound solution to the microwells coated with OVA, and shake at 37°C for 1 hour. Then, transfer the unbound solution to the microwells coated with the coating antigen PAT-BSA prepared in Example 1, and shake at 37°C for 1 hour. After the reaction is completed, discard the unreacted supernatant, wash the microwells containing the coating antigen 5 times with sterilized PBST solution, and then wash 15 times with sterilized PBS solution. After patting dry. Add 100 μL / well of 0.2 mol / L glycine-hydrochloric acid (Gly-HCl, pH 2.2) solution to the microwells, shake at 37°C for 10 minutes, and neutralize with 50 μL / well of 1 mol / L Tris-HCl (pH 8.2) solution to obtain the eluted product of the first round of panning. The eluted product was diluted in 100-, 1000-, and 10000-fold concentration series, and the number of single colonies on the plate 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 taken to infect 100 mL of E. coli TG1 bacterial solution in the logarithmic phase to obtain the phage solution for the second round of panning and determine its titer. The specific method of infection was as follows:

[0072] 250 μL of eluted product was inoculated into 100 mL of LB liquid medium and placed at 37°C, 250 rpm shaking culture 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, and then 37°C, 250 rpm shaking culture was carried out for 2 hours. Kanamycin (final concentration of 100 μg / mL) was added and continued to shake culture overnight. The overnight cultured bacterial solution was centrifuged at 8000 rpm for 20 minutes, the supernatant was collected, 1 / 5 volume of sterile polyethylene glycol / sodium chloride (mass ratio of the two was 100:73.1) solution was added, mixed evenly, placed in ice for 4 hours, centrifuged at 8000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the culture was resuspended with 1 mL of TBS solution, filtered through a 0.22 μm microporous filter membrane, and stored at -80°C.

[0073] 3. Second to sixth rounds of screening

[0074] According to the above method, the gradient drug competitive elution method was used to screen the specific phages in the second to sixth rounds (the concentrations of the coating antigen PAT-BSA in the second to sixth rounds were 10, 5, 2.5, 0.5, and 0.1 μg / mL, respectively). Pick the single colony on the plate for titer determination of each round of elution products into a deep-well plate and culture it in a shaking incubator at 37°C overnight. Pipette 10 μL of the overnight cultured bacterial solution into the deep-well plate, culture it in a shaking incubator at 37°C for 3 hours, add 1 μL of IPTG solution, and continue to culture it in a shaking incubator at 37°C overnight.

[0075] The supernatant was taken as the expression supernatant and used 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 incubated at 37°C for 3 hours; the liquid in the wells was discarded, and the wells were dried in a 37°C oven for 30 minutes after being patted dry; the expression supernatant (50 μL / well) and PBS diluent (50 μL / well) were added to the microwells as titer wells, and the expression supernatant (50 μL / well) and 1 μg / mL of the test substance standard solution (50 μL / well) were added to the other microwells as inhibition wells, and incubated in a 37°C water bath for 40 minutes; 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 diluent (5000 times diluted with PBST), incubate in a 37°C water bath for 40 minutes; discard the liquid in the well, wash 5 times, pat dry, add 100 μL / well of goat anti-mouse-HRP antibody solution (5000 times diluted with PBST), incubate in a 37°C water bath for 40 minutes; discard the liquid in the well, wash 5 times, pat dry, add 100 μL / well of TMB two-component colorimetric solution, incubate in a 37°C water bath for 10 minutes, add 50 μL / well of stop solution, measure the absorbance at a wavelength of 450nm, and analyze the coating original binding ability and patulin specificity of the nanoantibody in the deep well plate. The bacterial solution of the positive clone with antigen binding ability and patulin specificity was sent for sequencing analysis.

[0078] Positive clones are: the absorbance value of the titer well is greater than 0.5 (coating original binding ability), and the inhibition rate (%) is greater than 30% (patulin specificity), inhibition rate (%) = (titer well absorbance value - inhibition well absorbance value) / titer well absorbance value × 100.

[0079] 2. Experimental Results

[0080] like Figure 2 As shown, ELISA analysis was performed on monoclonal phages in the screening of patulin nanoantibodies. Using PAT-BSA as the coating source, four clones were obtained after six rounds of solid phase coating and panning, and E9, F4, H11, and H12 all had binding activity with the coating source.

[0081] Furthermore, the supernatants of the deep-well plate mid-term expression corresponding to the above four clones were analyzed by 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 about 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: ISWGGST (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 h, with shaking 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 incubate at 37°C with shaking at 250 rpm for 2-3 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 gradient concentrations of imidazole solution (PBS solution containing 10, 20, 50, 100, and 200 mM imidazole), and use SDS-PAGE to identify the nanoantibody.

[0102] The eluate containing nanobody E9 was collected, dialyzed with PBS solution at 4°C for 3 days, its concentration was determined, and it was stored at -20°C after being aliquoted 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. 5 μg / mL patulin artificial antigen PAT-BSA prepared in Example 1 was coated in the microwells of the ELISA plate. After overnight coating at 37°C, 120 μL of 5% (mass volume ratio) skimmed milk powder solution was added to each well and blocked at 37°C for 3 hours. After drying the liquid in the wells, the wells were dried at 37°C for 1 hour for use;

[0108] S2. Add 50 μL of a series of concentrations (6.25, 3.13, 0.78, 0.39, 0.2, 0.1, 0 μg / mL) of patulin standard solution or the sample solution to be tested into the microwells of the ELISA plate, add 50 μL of a certain dilution of patulin nanobody (amino acid sequence as 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 5 times, and pat dry on absorbent paper;

[0109] S3. Add 100 μL of 5000-fold diluted HRP-labeled goat anti-camel secondary antibody solution into the microwells, incubate at 37°C for a period of time, wash 5 times with washing solution, 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 coordinate and the log10 value of the drug standard solution concentration as the horizontal coordinate to draw a standard curve, 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 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. According to 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 inhibition concentration is 0.41μg / mL, the linear range is 0.17-0.97μg / mL, and the inhibition rate and the logarithmic value of patulin have a significant S-shaped curve relationship, and the correlation coefficient R 2 The value was 0.987. This 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-volume ratio), patulin standard, patulin standard and washing solution.

[0117] 2. Usage

[0118] As in Example 5.

[0119] Example 7 Evaluation of the Specificity of Patulin Nanobodies by ELISA Analysis

[0120] 1. Experimental Methods

[0121] According to the method of Example 5, other analogs (ochratoxin OTA, vomitoxin, aflatoxin B1) were used to replace patulin to establish the ELISA standard curve, and 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 Evaluation of specificity of ELISA analysis method

[0124]

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

Claims

1. A nanobody of 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 FRs are: 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 of 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 of claim 5, or is capable of expressing the Nanobody of 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.

Images