A bongkrekic acid hapten, its preparation method and application

Abstract

The present invention relates to the technical field of haptens, and particularly to a bongkrekic acid hapten, a preparation method thereof and an application. On the basis of the structure of bongkrekic acid, the present invention selects two intermediates with partial structures similar to the two ends thereof respectively for modification and coupling, adds an alanine coupling arm at a specific site, and prepares a bongkrekic acid hapten with an active amino group. After biological coupling with a carrier protein, the corresponding artificial complete antigen of bongkrekic acid can be obtained; by using the artificial complete antigen of bongkrekic acid, an antibody against bongkrekic acid can be prepared, which can be used to quickly and accurately detect bongkrekic acid in food.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and particularly relates to a bongkrekic acid hapten, a preparation method thereof and an application thereof. Background Art

[0002] Bongkrekic Acid (BA, C 28 H 38 O 7 ) is a toxin that can cause food poisoning and is produced by Burkholderia gladioli (formerly named "Pseudomonas cocovenenans", "Pseudomonas cocovenenans subsp. farinofermentans", "Burkholderia cocovenenans", etc.). Bongkrekic Acid is highly toxic and can cause acute poisoning in humans and various animals, resulting in damage to the nervous system, digestive system and urinary system. It is the main cause of food poisoning caused by fermented corn flour products, spoiled fresh tremella and other spoiled starch products. Since bongkrekic acid is stable and heat-resistant, its toxicity cannot be destroyed by general cooking methods, and there is currently no effective rapid detection reagent for bongkrekic acid.

[0003] Currently, the detection methods for bongkrekic acid are mainly instrumental methods. These methods are accurate and reliable and are often used as confirmation methods. However, due to reasons such as expensive equipment, high operation requirements, and long detection time, it is difficult to achieve rapid detection of a large number of samples in actual applications. Immunological analysis techniques based on the specific binding of antigens and antibodies can meet the needs of on-site rapid screening. Therefore, the research on the preparation of monoclonal antibodies against bongkrekic acid has very important economic and social significance for the rapid detection of bongkrekic acid. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a bongkrekic acid hapten, a preparation method thereof and an application thereof. By bioconjugating the hapten with a carrier protein, an artificial complete antigen can be obtained, and then an antibody against bongkrekic acid can be prepared, which can be used for rapid detection of bongkrekic acid in food.

[0005] In the first aspect, the present invention provides a bongkrekic acid hapten, and the chemical formula of the bongkrekic acid hapten is shown in Formula I:

[0006]

[0007] In the second aspect, the present invention provides an artificial antigen of bongkrekic acid, which is prepared from the hapten.

[0008] Furthermore, it is obtained by conjugating the bongkrekic acid hapten with a carrier protein, and the carrier protein preferably includes one or more of bovine serum albumin, keyhole limpet hemocyanin, bovine thyroglobulin or ovalbumin.

[0009] The structure of the artificial antigen of bongkrekic acid is as follows, where Carrier protein is the carrier protein.

[0010]

[0011] The present invention further provides a kit comprising the hapten of bongkrekic acid or the artificial antigen of bongkrekic acid.

[0012] In a third aspect, the present invention provides a hybridoma cell line, which is prepared by using the artificial antigen of bongkrekic acid as an immunogen.

[0013] As a preferred specific embodiment, the present invention provides a method for preparing a hybridoma cell line, comprising:

[0014] Using the artificial antigen of bongkrekic acid as an immunogen to immunize Balb / c mice. After analysis and determination, B cells are collected from the mice with better immune effects for cell fusion to obtain hybridoma cells, and after multiple rounds of screening and cloning, a hybridoma cell line with better effects is obtained.

[0015] Further, the carrier protein used in the artificial antigen of bongkrekic acid is bovine thyroglobulin.

[0016] Further, the artificial antigen of bongkrekic acid is obtained by coupling the hapten and the carrier protein by the active ester method.

[0017] Further, the number of Balb / c mice is 8.

[0018] Further, the method for immunizing Balb / c mice is as follows: The immunogen is emulsified with an equal volume of Freund's complete adjuvant and used to immunize Balb / c mice for the first time. Then, the dose of the immunogen used in the first immunization is halved, and it is emulsified with an equal volume of Freund's incomplete adjuvant and used to boost the immunization of the Balb / c mice immunized for the first time.

[0019] Further, the immunization method is multi-point immunization on the back of the neck, with 4 - 8 immunization points.

[0020] Further, the immunogen is diluted with PBS solution;

[0021] The concentration of the PBS solution is specifically 0.01 mol / L, and the pH is 7.4.

[0022] Further, the dose of the immunogen for the first immunization is 200 μg / animal, and the boost immunization dose is 100 μg / animal;

[0023] The number of boost immunizations is 2 - 3 times, preferably 3 times;

[0024] The booster immunization is preferably carried out once on the 21st day after the primary immunization.

[0025] Furthermore, the obtained hybridoma cell line of the present invention is submitted for biological preservation, and the specific information is as follows:

[0026] Preservation number: CGMCC No.21905, preservation date: March 5, 2021, classification and naming: mouse hybridoma cell line, preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms, preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101.

[0027] The present invention further provides a monoclonal antibody secreted by the said hybridoma cell line.

[0028] Furthermore, the heavy chain subtype of the monoclonal antibody is IgG1 type, and the light chain subtype is λ type.

[0029] Fourthly, the present invention provides a preparation method of the said bongkrekic acid hapten, including:

[0030] (1) Using compound 1 as the starting product, compound 3 is obtained through saponification reaction;

[0031] (2) Compound 3 and 3-amino-1-propanol are subjected to esterification reaction to obtain compound 4;

[0032] (3) Compound 2 is obtained through methylation reaction and saponification reaction to obtain compound 5;

[0033] (4) Compound 4 and compound 5 react to obtain the said bongkrekic acid hapten;

[0034] Among them, the chemical formula of compound 1 is shown in formula II:

[0035]

[0036] The chemical formula of compound 2 is shown in formula III:

[0037]

[0038] The chemical formula of compound 3 is shown in formula IV:

[0039]

[0040] The chemical formula of compound 4 is shown in formula V:

[0041]

[0042]

[0043] The chemical formula of Compound 5 is shown in Formula VI:

[0044]

[0045] Furthermore, the methylation reaction in step (3) is carried out at 55 - 65 °C under the action of methyl trifluoromethanesulfonate and 2,6 - di - tert - butylpyridine.

[0046] As a preferred specific embodiment, the present invention provides a method for preparing the bongkrekic acid hapten, including:

[0047] (1) Starting from Compound 1 dissolved in an organic solvent, adding KOH and then carrying out a thermal reflux reaction for 2 - 3 hours, followed by salting - out and drying;

[0048] (2) Mixing the product of step (1) with 3 - amino - 1 - propanol, adding concentrated sulfuric acid and then carrying out a thermal reflux for 1 - 2 hours, followed by salting - out, washing, centrifugation and drying to obtain Compound 4;

[0049] (3) Starting from Compound 2 dissolved in an organic solvent, carrying out a methylation reaction at 55 - 65 °C under the action of methyl trifluoromethanesulfonate and 2,6 - di - tert - butylpyridine, and then carrying out a saponification reaction to obtain Compound 5;

[0050] (4) Carrying out a Sonogashira coupling reaction on Compound 4 and Compound 5 in a basic solvent, and obtaining the bongkrekic acid hapten through filtration, concentration and purification.

[0051] The present invention further provides the application of the bongkrekic acid hapten, the bongkrekic acid artificial antigen, the kit or the monoclonal antibody in detecting bongkrekic acid or its derivatives.

[0052] Furthermore, the detection is an enzyme - linked immunosorbent assay, preferably detecting the titer of the bongkrekic acid antiserum by the indirect ELISA method; or, determining the sensitivity of the antibody by the indirect competitive ELISA method.

[0053] The present invention has the following beneficial effects:

[0054] Based on the structure of bongkrekic acid, the present invention obtains a bongkrekic acid hapten. After coupling it with a carrier protein, a bongkrekic acid artificial antigen can be obtained, and an effective monoclonal antibody against bongkrekic acid is prepared using it as an immunogen.

[0055] The bongkrekic acid antibody prepared by the method provided by the present invention is simple and easy to operate, and the antibody titer screened is 2.5×10 -6 , and the sensitivity (IC 50 ) of the ELISA method established with this antibody is 1.7 ng / mL, and the linear range is 0.1 - 26.5 ng / mL.

[0056] Preparing the monoclonal antibody of bongkrekic acid by using the method provided by the present invention has unique advantages such as simple preparation process, economy, high sensitivity, high practical value, etc., and has good application prospects in food safety detection. Description of the Drawings

[0057] Figure 1 It is a schematic structural diagram of bongkrekic acid provided in Example 1 of the present invention.

[0058] Figure 2 It is a schematic structural diagram of the hapten of bongkrekic acid provided in Example 1 of the present invention.

[0059] Figure 3 It is a schematic structural diagram of the artificial antigen of bongkrekic acid provided in Example 1 of the present invention.

[0060] Figure 4 It is the MALDI-TOF identification result of the artificial antigen of bongkrekic acid provided in Example 1 of the present invention.

[0061] Figure 5 It is the ELISA standard curve of the monoclonal antibody of bongkrekic acid provided in Example 2 of the present invention. Detailed Embodiments

[0062] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0063] Example 1. Preparation of the hapten, antigen and antibody of bongkrekic acid and their preparation

[0064] I. Synthesis of the hapten and artificial antigen of bongkrekic acid and their preparation

[0065] 1. Synthesis of the hapten of bongkrekic acid

[0066] To avoid the mutual interference of the same carboxyl groups at both ends of the bongkrekic acid structure (the structure is as Figure 1 shown), the present invention uses two compounds (Compound 1 and Compound 2) similar to the structures at both ends of bongkrekic acid as raw materials, respectively performs structural modification and then couples them to obtain the target hapten.

[0067] The structural formula of the Compound 1 is:

[0068]

[0069] The structural formula of the Compound 2 is:

[0070]

[0071] The specific process is as follows:

[0072] 1.1 Modification of Compound 1

[0073] Compound 1 first removes the methyl ester protecting group through saponification reaction to obtain a carboxyl product, and the obtained compound 3 then undergoes an esterification reaction with 3-amino-1-propanol to introduce an amino spacer arm to obtain compound 4.

[0074] Specifically: Dissolve compound 1 with an aqueous solution of ethylene glycol dimethyl ether (DME:H 2 O 4:1), add KOH (2eq) for reaction, heat under reflux for 2 hours, pour the solution into saturated brine after the reaction, stir for salting out, filter and dry with anhydrous calcium chloride;

[0075] Take 10 mg of the above product in a round-bottom flask, add 10 mL of 3-amino-1-propanol solution, then slowly add 0.5 mL of concentrated sulfuric acid, add zeolite, install a condenser, heat under reflux for 1-2 hours, pour the obtained solution into 200 mL of warm saturated brine to precipitate for 20 min, discard the salt layer, and then wash with 100 mL of 0.5% Na 2 CO 3 solution, centrifuge, add anhydrous sodium sulfate for drying to obtain compound 4.

[0076] The structural formula of compound 3 in the above process is:

[0077]

[0078] The structural formula of compound 4 is:

[0079]

[0080] 1.2 Modification of compound 2

[0081] Dissolve compound 2 with dichloromethane, react at 60 °C under the action of methyl trifluoromethanesulfonate and 2,6-di-tert-butylpyridine to achieve methylation. Then dissolve the methyl ether product with DME / H 2 O (4:1) and carry out saponification reaction to obtain compound 5.

[0082] The structural formula of compound 5 is:

[0083]

[0084] Finally, carry out a Sonogashira coupling reaction on the two modified products, compound 4 and compound 5, in the basic solvent triethylamine.

[0085] Specifically include: 36.3 mg of compound 4, Pd(PPh 3 ) 2 C1 2(10 mol%) and CuI (2 mol%) were added to a 25 mL flask. After evacuation, nitrogen was filled, and this was repeated three times. Then, 21.9 mg of Compound 5 and 4 mL of triethylamine were injected. The mixture was stirred at room temperature for 3 h, and TLC was used for detection until the reaction was complete. Finally, the insoluble substances were removed by filtration, and the filtrate was concentrated. Using petroleum ether:dichloromethane = 5:1 as the developing solvent, column chromatography was performed for separation, and finally, the bongkrekic acid hapten was obtained. The structure of the hapten is (as Figure 2 shown):

[0086]

[0087] 2. Synthesis of bongkrekic acid immunogen

[0088] The bongkrekic acid hapten is a small molecule and lacks immunogenicity. To prepare antibodies against bongkrekic acid, it must be conjugated to a protein carrier with a relatively large molecular weight (such as BSA, KLH, etc., the structure is as Figure 3 shown), to stimulate the immune system to produce a response. In the present invention, after activating the carboxyl group of the carrier protein with a carbodiimide reagent, it was connected to the amino group of the bongkrekic acid hapten to prepare an artificial antigen. The specific process is as follows:

[0089] 5.36 mg of bovine thyroglobulin was weighed and dissolved in 5 mL of 0.1 M MES (pH 6.0, containing 0.5 M NaCl). 1.16 mg of EDC and 0.7 mg of NHS were added, and the reaction was carried out at room temperature for 3 h. 3.5 μL of 2-mercaptoethanol (final concentration 20 mM) was added to quench EDC to obtain the activation solution.

[0090] 1.8 mg of the hapten raw material was weighed and dissolved in 0.5 mL of DMF, and then added to the above activation solution. The mixture was stirred at room temperature overnight. After the reaction was completed, it was taken out after dialysis in 0.1 moI / L phosphate buffer (PBS) at pH 7.2 for 3 d at 4 °C, and the bongkrekic acid immunogen (BA-BTG) was obtained.

[0091] 3. Synthesis of bongkrekic acid coating antigen

[0092] The method is the same as the above method for preparing the immunogen, except that the carrier protein used is bovine serum albumin. The obtained bongkrekic acid coating antigen (BA-BSA) was identified by MALDI-TOF, and the coupling ratio of the hapten to the carrier protein was 6.46:1. The mass spectrometry results are as Figure 4 shown.

[0093] II. Preparation of bongkrekic acid monoclonal antibody

[0094] 1. Preparation of monoclonal antibody

[0095] (1) Animal immunization

[0096] Dilute the above immunogen BA-BTG with PBS solution, emulsify it with an equal volume of Freund's complete adjuvant and immunize Balb / c mice for the first time. The dose of immunogen used for the first immunization is 200 μg / mouse. Strengthen the immunization every 21 days by emulsifying the immunogen with Freund's incomplete adjuvant, and the dose is 100 μg / mouse. A total of 3 booster immunizations are carried out.

[0097] (2) Cell fusion and cloning

[0098] Take the spleen cells of immunized Balb / c mice, fuse them with sp2 / 0 myeloma cells at a ratio of 5:1 (quantity ratio), and use indirect competitive ELISA to measure the cell supernatant to screen positive wells. Clone the positive wells by the limiting dilution method until a hybridoma cell line that stably secretes the monoclonal antibody against bongkrekic acid is obtained. The name of this cell line is BA-BTG-4G1, and the present invention has carried out biological preservation on it. The preservation information is as follows:

[0099] Preservation number: CGMCC No.21905, preservation date: March 05, 2021, classification name: mouse hybridoma cell line, preservation unit: General Microbiology Center of China Committee for Culture Collection of Microorganisms, preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing 100101.

[0100] (3) Cell cryopreservation and resuscitation

[0101] Prepare the monoclonal antibody hybridoma cell line BA-BTG-4G1 of bongkrekic acid into a cell suspension of 1×10 6 cell / mL with cryopreservation solution and store it in liquid nitrogen for a long time. When resuscitating, take out the cryopreservation tube, immediately put it into a 37°C water bath for rapid thawing, remove the cryopreservation solution by centrifugation, and transfer it into a culture flask for culture.

[0102] (4) Preparation and purification of monoclonal antibody

[0103] Incremental culture method: Place the monoclonal antibody hybridoma cell line of bongkrekic acid in a cell culture medium and culture it at 37°C. Purify the obtained culture solution by the caprylic acid-ammonium sulfate saturation method to obtain the monoclonal antibody and store it at -20°C.

[0104] The cell culture medium is prepared by adding calf serum and sodium bicarbonate to RPM1-1640 medium, so that the final concentration of calf serum in the cell culture medium is 20% (mass percentage), and the final concentration of sodium bicarbonate in the cell culture medium is 0.2% (mass percentage); the pH of the cell culture medium is 7.4.

[0105] The above monoclonal antibody can also be prepared by the following method: Inject 0.4 mL of sterilized paraffin oil into the abdominal cavity of each Balb / c mouse. After 7 days, inject 5×10 5 cells of cell line BA-BTG-4G1 into the abdominal cavity of each mouse. After 7 days, collect the ascites. Purify it by the caprylic acid-ammonium sulfate saturation method, and store the purified ascites in an environment of -20°C.

[0106] Example 2. Detection of bongkrekic acid antibody

[0107] I. Determination of antibody titer

[0108] The titer of the monoclonal antibody was determined by indirect ELISA and checkerboard method (the standard curve is as Figure 5 shown). The results showed that the titer of the bongkrekic acid monoclonal antibody was 2.5×10 -6 , and the half inhibitory concentration (IC 50 ) was 1.7 ng / mL.

[0109] The steps of the indirect ELISA method are as follows:

[0110] (1) Coating: Dilute the coating antigen BA-BSA with 0.05 mol / L carbonate buffer (pH 9.6) to 0.2 μg / mL, add 100 μL to each well in a 96-well transparent ELISA plate, incubate in a 37°C incubator for 2 h, and wash the plate 3 times with PBST buffer (pH 7.2).

[0111] (2) Blocking: Add 150 μL / well of blocking solution (5% skim milk powder), incubate in a 37°C incubator for 1 h, discard the blocking solution, wash 3 times with PBST buffer (pH 7.2), and pat dry.

[0112] (3) Adding the antibody to be tested: Add 50 μL of 0.01 mol / L PBS (pH 7.4) to each well in each column, and then add 50 μL of the bongkrekic acid antibody obtained in Example 1 after dilution. The antibody is diluted with 0.01 mol / L PBS starting from 1:2500 with a gradient of 2, and a total of 8 gradients are diluted. The sample addition volume is 50 μL per well, react in a 37°C incubator for 30 min, wash 3 times with PBST buffer (pH 7.2), and pat dry.

[0113] At the same time, set the antiserum of non-immunized mice as a negative control.

[0114] (4) Adding the enzyme-labeled secondary antibody: Add the HRP-labeled goat anti-mouse IgG antibody diluted 1:5000 by volume with the enzyme-labeled secondary antibody dilution solution, 100 μL per well, react in a 37°C incubator for 30 min, wash 3 times with PBST buffer (pH 7.2), and pat dry.

[0115] (5) Color development: horseradish peroxidase substrate 3,3',5,5'-tetramethylbenzidine solution and 30% hydrogen peroxide were mixed in a volume ratio of 1:1, 100 μL was added to each well, and color was developed in an incubator at 37°C for 15 min.

[0116] (6) Termination: Add 50 μL of 2 mol / L concentrated sulfuric acid to each well.

[0117] (7) Reading: Measure the OD of each well 450 The antibody titer was determined by taking the negative OD value no greater than 0.15 and the antibody concentration corresponding to the maximum OD value between 1.0 and 1.8.

[0118] 2. Determination of Antibody Sensitivity

[0119] The antibody titer was tested by indirect competitive ELISA. The steps of indirect competitive ELISA are as follows:

[0120] (1) Coating: Dilute the coating original BA-BSA to 0.2 μg / mL with 0.05 mol / L carbonate buffer (pH 9.6), add 100 μL to each well of a 96-well transparent ELISA plate, incubate at 37°C for 2 h, and wash the plate three times with PBST buffer (pH 7.2).

[0121] (2) Blocking: Add 150 μL / well of blocking solution (5% skim milk powder), incubate at 37°C for 1 h, discard the blocking solution, wash three times with PBST buffer (pH 7.2), and pat dry.

[0122] (3) Adding standards and antibodies: 50 μL of fumonisin standards with concentrations of 0, 0.8, 2.5, 7.4, 22.2, 66.7, 200 and 400 ng / mL were added to each column of wells in sequence, with three replicates for each concentration; then 50 μL of the antibody working solution with an OD value between 1.5 and 1.8 in Example 2 was added to each well, reacted in an incubator at 37°C for 30 min, washed three times with PBST buffer (pH 7.2), and patted dry.

[0123] Among them, the fumonisin standard and the coating original BA-BSA competitively bind to the fumonisin antibody.

[0124] (4) Adding enzyme-labeled secondary antibody: Add HRP-labeled goat anti-rabbit IgG antibody diluted with enzyme-labeled secondary antibody diluent at a volume ratio of 1:5000, 100 μL per well, react in a 37°C incubator for 30 min, wash three times with PBST buffer (pH 7.2), and pat dry.

[0125] (5) Color development: horseradish peroxidase substrate 3,3',5,5'-tetramethylbenzidine solution and 30% hydrogen peroxide were mixed in a volume ratio of 1:1, 100 μL / well was added, and color development was carried out in a 37°C incubator for 15 min.

[0126] (6) Termination: Add 50 μL of 2 mol / L concentrated sulfuric acid to each well.

[0127] (7) Reading: Measure the OD value of each well at 450 nm.

[0128] The -log (concentration of fumonisin) is the horizontal axis, and the OD value (OD 450 ) as the ordinate, the four-parameter equation of Origin 8.0 was used for fitting, and the standard curve was established to obtain the half inhibition concentration (IC 50 After screening, an antibody with good sensitivity was finally obtained. The half inhibition concentration (IC 50 ) is 1.7ng / mL.

[0129] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A bongkrekic acid hapten, characterized in that, the chemical formula of the bongkrekic acid hapten is shown as Formula I: 。 2. A bongkrekic acid artificial antigen, characterized in that, it is prepared from the bongkrekic acid hapten described in Claim 1.

3. The bongkrekic acid artificial antigen according to Claim 2, characterized in that, it is obtained by conjugating the bongkrekic acid hapten described in Claim 1 with a carrier protein.

4. The bongkrekic acid artificial antigen according to Claim 3, characterized in that, the carrier protein includes one or more of bovine serum albumin, keyhole limpet hemocyanin, bovine thyroglobulin or ovalbumin.

5. A kit, characterized in that, the kit includes the bongkrekic acid hapten described in Claim 1 or the bongkrekic acid artificial antigen described in any one of Claims 2-4.

6. A hybridoma cell line, characterized in that, the hybridoma cell line is prepared by using the bongkrekic acid artificial antigen described in Claim 2 or 3 as an immunogen; the preservation number of the hybridoma cell line is CGMCC 21905.

7. A monoclonal antibody, characterized in that, the monoclonal antibody is secreted by the hybridoma cell line described in Claim 6.

8. The preparation method of the bongkrekic acid hapten described in Claim 1, characterized in that, it includes: (1) Using compound 1 as a starting product, obtaining compound 3 through a saponification reaction; (2) Performing an esterification reaction between compound 3 and 3-amino-1-propanol to obtain compound 4; (3) Obtaining compound 5 from compound 2 through a methylation reaction and a saponification reaction; (4) Reacting compound 4 and compound 5 to obtain the bongkrekic acid hapten; wherein, the chemical formula of compound 1 is shown as Formula II: ， the chemical formula of compound 2 is shown as Formula III: ， the chemical formula of compound 3 is shown as Formula IV: ， the chemical formula of compound 4 is shown as Formula V: ， the chemical formula of compound 5 is shown as Formula VI: 。 9. The preparation method according to Claim 8, characterized in that, in step (3), the methylation reaction is carried out at 55-65 °C under the action of methyl trifluoromethanesulfonate and 2,6-di-tert-butylpyridine.

10. The application of the bongkrekic acid hapten described in Claim 1, or the bongkrekic acid artificial antigen described in any one of Claims 2-4, or the kit described in Claim 5, or the monoclonal antibody described in Claim 7 in the preparation of a drug for detecting bongkrekic acid or its derivatives.

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