CN114621297B - Magnetic photoaffinity labeling probe molecule and preparation method thereof, preparation method of natural dihydrofolate synthase and competitive enzyme-linked detection method - Google Patents
Magnetic photoaffinity labeling probe molecule and preparation method thereof, preparation method of natural dihydrofolate synthase and competitive enzyme-linked detection method Download PDFInfo
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- 108030007223 Dihydrofolate synthases Proteins 0.000 title claims abstract description 51
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- 238000000034 method Methods 0.000 claims abstract description 33
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Abstract
The invention discloses a magnetic photoaffinity labeling probe molecule and a preparation method thereof, a preparation method of natural dihydrofolate synthase and a competitive enzyme-linked detection method. The magnetic photoaffinity labeling probe molecule is synthesized by a chemical method from 4-nitrobenzoyl chloride, absolute ethyl alcohol, 2 pieces of water of tin dichloride, methylene dichloride, triethylamine, glacial acetic acid, chromium trioxide, concentrated sulfuric acid, N-dimethylformamide, N-butylamine, isobutyl chloroformate and Fe 3O4@SiO2-NH2 magnetic particles. The synthesized magnetic photoaffinity labeling probe molecule has strong affinity, high selectivity and labeling function with a natural dihydrofolate synthase receptor, and can conveniently and rapidly capture the natural dihydrofolate synthase in escherichia coli; the obtained natural dihydrofolate synthase can identify all sulfanilamide medicines, can be used for multi-residue detection of all sulfanilamide medicines in animal meat tissues, and has the advantages of high accuracy, high detection efficiency and short detection time.
Description
Technical Field
The invention belongs to the field of food safety, and relates to a magnetic photoaffinity labeling probe molecule capable of capturing natural dihydrofolate synthase in bacteria, a preparation method thereof, a method for preparing natural dihydrofolate synthase in escherichia coli by using the probe molecule, and a direct competitive enzyme-linked detection method for detecting sulfonamides by using natural dihydrofolate synthase in escherichia coli prepared by using the probe molecule.
Background
The sulfonamides are artificially synthesized antibacterial agents with a p-aminobenzenesulfonamide structure, have the advantages of wide antibacterial spectrum, simple production, low price and the like, and are widely applied to the prevention and treatment of animal diseases and the promotion of growth. However, food safety problems caused by residues of sulfonamide drugs in animal foods are increasingly prominent, and the health of consumers is directly compromised. The maximum residue limit of sulfonamide in animal foods is clearly defined in China, the United states and the European Union. Therefore, the detection of the residue of the sulfonamides in animal foods is of great significance for guaranteeing the safety of foods and maintaining the health of human bodies.
At present, the main methods for detecting the residue of the sulfonamides in animal foods include an instrument detection method and an immunodetection method. The instrument analysis method is complex in operation, complicated in sample pretreatment process, long in time consumption, high in cost and unsuitable for popularization, and needs professional technicians to detect large batches of products. The core reagent of the immunodetection method is an antibody, so far, various antibodies aiming at sulfonamides are prepared and researched at home and abroad, including monoclonal antibodies, polyclonal antibodies, recombinant antibodies, nucleic acid aptamers, molecularly imprinted polymers and the like, but the results cannot meet the requirement of multi-residue detection.
The antibacterial mechanism of the sulfonamides is to combine with dihydrofolate synthase in bacteria to block the synthesis path of folic acid, thereby inhibiting the growth and reproduction of bacteria. The dihydrofolate synthase is therefore the receptor for sulfonamides. A specific receptor can be combined with all ligands in theory, so that the dihydrofolate synthase has broad-spectrum recognition on the sulfonamides, and the detection method based on the dihydrofolate synthase can realize multi-residue detection of the sulfonamides. In 2013 and 2019, liang et al expressed in vitro the dihydrofolate synthases from Streptococcus pneumoniae and Escherichia coli, respectively, and established a sulfanilamide multi-residue detection method based on the dihydrofolate synthases, so that 29 sulfanilamide drugs can be detected. However, liang et al are dihydrofolate synthases expressed in vitro by gene recombination techniques, which are different from natural dihydrofolate synthases in bacteria to some extent, and have high cost in the preparation process and high professional requirements for operators, and the obtained dihydrofolate synthases have a number of uncertain factors such as temperature and pH, which can cause the reduction of protein activity.
In recent years, activity-based protein profiling technology (Activity-Based Protein Profiling, ABPP) is attracting attention in the fields of receptor preparation and molecular mechanism research, and ABPP can directly obtain natural receptor proteins from organisms, so that the directionality of research is greatly improved, the error rate is reduced, and the high-affinity specific receptor is facilitated to be obtained. However, ABPP probes have the disadvantage that the forces between the active groups and the receptor are mainly ionic bonds, van der Waals forces, hydrogen bonds, etc., and the stability is poor, which may lead to loss in the isolation and purification of the target receptor. Thus, the photoaffinity labeling technique (Photoaffinity Labeling, PAL) was introduced into the ABPP technique to form a novel, more robust PAL-ABPP probe, however, the conventional PAL-ABPP probe has some disadvantages, such as the inability to reuse the probe, cumbersome and costly receptor preparation process, low purity of the prepared receptor, etc. The magnetic PAL-ABPP probe is used for preparing the receptor, the operation is simple, the time consumption is short, the cost is low, the purity of the obtained receptor is high, and the probe can be reused. Up to now, no patent and non-patent documents report that PAL-ABPP probe or magnetic PAL-ABPP probe is used for researching the sulfonamide drug receptor dihydrofolate synthetase at home and abroad, and no known practical application technology exists.
Disclosure of Invention
The first object of the invention is to provide a magnetic photoaffinity labeling probe molecule, which has strong affinity, high selectivity and labeling function with natural dihydrofolate synthase receptor, can be repeatedly used, and has the advantages of simple operation, short time, low cost and high purity of the obtained receptor.
In order to achieve the above purpose, the present invention adopts the following technical scheme: a magnetic photoaffinity labeling probe molecule has a molecular structural formula shown in formula 1:
Formula 1.
The second object of the present invention is to provide a preparation method of the magnetic photoaffinity labeling probe molecule, which has the advantages of simple operation, short time, low cost and high efficiency.
The preparation method of the magnetic photoaffinity labeling probe molecule comprises the following steps of, calculated according to the weight ratio,
(A) Dissolving 5-10 parts of 4-nitrobenzoyl chloride, namely a compound 1, in 10mL of toluene, stirring for 24-72 hours at room temperature under the protection of nitrogen, adding 10mL of deionized water, separating an organic phase, sequentially cleaning with 10mL of saturated NaHCO 3 and 10mL of saturated NaCl, drying 10 g of anhydrous MgSO 4, filtering, evaporating the solvent to obtain a compound 2, and carrying out a synthesis process of the following equation 2;
Equation 2;
(b) Dissolving 1-3 parts of compound 2 in 10mL of absolute ethyl alcohol, adding 5-10 parts of tin dichloride SnCl 2·2H2 O with 2 water, and stirring for 2-12 hours at 50 ℃ under the protection of nitrogen; vacuum drying and then adding 10mL of deionized water; the mixture was extracted with 20mL of dichloromethane, the organic phase was washed with 20mL of saturated NaCl, dried over 10 g of anhydrous MgSO 4, filtered, and the filtrate was dried in vacuo to give compound 3, which was synthesized as shown in equation 3 below;
equation 3;
(c) 1-5 parts of compound 3 is dissolved in 10mL of dichloromethane, 1-5 parts of compound 1 and 1-5mL of triethylamine are added, and the mixture is stirred for 2-12 hours at room temperature under the protection of nitrogen; the precipitate obtained by filtration after stirring is washed with 10mL of dichloromethane for several times and dried in vacuum to obtain a compound 4, and the synthesis process is shown in the following equation 4;
Equation 4;
(d) Taking 1-5 parts of compound 4, adding 30-50 mL parts of glacial acetic acid, 1-5 parts of chromium trioxide and 5-10 mL parts of concentrated sulfuric acid, and stirring at room temperature for 12 hours; pouring the reaction solution into 30-50 mL ice water, and extracting with 30-50 mL ethyl acetate; the obtained ethyl acetate solution is washed by 30-50 mL saturated NaCl, 10 g of anhydrous MgSO 4 is dried, filtered and dried in vacuum to obtain a compound 5, and the synthesis process is as shown in the following equation 5;
equation 5;
(e) Dissolving 1-5 parts of compound 5 in 10mL of absolute ethyl alcohol, adding 1-5 parts of SnCl 2·2H2 O, and stirring at 50 ℃ for 2-12 hours under the protection of nitrogen; after the mixture is dried in vacuum, 30-50mL of deionized water is added, 30-50mL of dichloromethane is used for extraction, the obtained dichloromethane solution is washed by 30-50mL of saturated NaCl, 10g of anhydrous MgSO 4 is dried, filtration and vacuum drying are carried out, and the compound 6 is obtained, wherein the synthesis process is as shown in the following equation 6;
equation 6;
Characterization data ：（IR: Fe-O 580 cm-1, Si-O-Si 1095 cm-1, Si-O-Si 794 cm-1, Si-O-Si 985 cm-1, Si-O 460 cm-1, H-N-H1650 and for compound 6 3415 cm-1,C=C 1420,C-H 3150 cm-1,C=O 1700cm-1）;1H NMR （DMSO-d6, 600 MHz） δ: 10.095 (s, 1H, COOH), 7.961-7.947 (d, J=8.4 Hz, 2H, ArH), 7.761-7.747 (d, J=8.4 Hz, 4H, ArH), 7.734-7.720 (d, J=8.4 Hz, 2H, ArH), 7.644-7.631 (d, J=7.8 Hz, 2H, ArH), 7.375-7.362 (d, J=7.8 Hz, 2H, ArH), 6.627-6.613 (d, J=8.4 Hz, 2H, ArH); 13C NMR (DMSO-d6, 150 MHz) δ: 194.2, 165.5, 152.4, 143.9, 142.4, 134.9, 132.3, 131.2, 130.9, 130.7, 129.5, 129.2, 129.0, 128.9, 128.6, 120.5, 119.0, 112.6, 112.5, 112.4. HRMS (ESI) m/z: [M-H]+ calcd for C21H15N2O4+: 359.1032; Found: 359.1038）;
(F) 1-5 parts of compound 6 is taken and dissolved in 10mL of N, N-dimethylformamide, 1-5 parts of N-butylamine and 1-5 parts of isobutyl chloroformate are added for reaction for 1-6 hours at 4 ℃; taking 100 mg Fe 3O4@SiO2-NH2 magnetic particles, suspending the particles in 10mL of N, N-dimethylformamide by ultrasonic, dropwise adding the reaction solution, and stirring for 12 hours at the temperature of 4 ℃; the obtained precipitate is washed by N, N-dimethylformamide, washed by water and dried in vacuum to obtain the magnetic photoaffinity labeling probe, and the synthesis process is shown in the following equation 7;
equation 7.
The third object of the present invention is to provide a method for preparing natural dihydrofolate synthase in E.coli using the above-mentioned magnetic photoaffinity labeling probe molecule; the preparation method has the advantages of simple operation, short time, low cost and high purity of the obtained natural dihydrofolate synthetase receptor.
A method for preparing natural dihydrofolate synthase in escherichia coli by using the magnetic photoaffinity labeling probe molecule comprises the following steps of, by weight,
(A) Inoculating a standard strain of escherichia coli ATCC25922 into an LB liquid culture medium, culturing for 24 hours at the temperature of 37 ℃ and the rotation speed of 220rmp to obtain a bacterial liquid, centrifuging the bacterial liquid at the temperature of 4 ℃, and collecting thalli;
(b) Re-suspending the thalli by using a phosphoric acid buffer solution, then carrying out ultrasonic crushing on the re-suspended thalli by using an ice bath, centrifuging at the temperature of 4 ℃ after ultrasonic treatment, collecting a supernatant I, and adding Soluble Bingding Buffer with an equal volume to obtain a supernatant II for later use;
(c) Adding 100mg of the magnetic photoaffinity labeling probe molecules into the supernatant II, fully stirring, incubating for 1-6 hours at 37 ℃, enriching magnetic substances at the bottom of a bottle by using a magnet, and pouring out the supernatant III;
(d) Washing the magnetic substance at the bottom of the bottle with water, eluting with 10mL of 20% methanol, and collecting the eluent, wherein the eluent is natural dihydrofolate synthase in Escherichia coli.
Further preferably, the magnetic substance remaining after the eluent is removed in the step (d), namely, the magnetic photoaffinity labeling probe molecule, is washed sequentially with 10mL of ethanol and 10mL of deionized water, and is stored at room temperature after vacuum drying for reuse. Can be repeatedly used for 7 times, and further reduces the preparation cost.
The fourth object of the present invention is to provide a direct competitive enzyme-linked assay for detecting sulfonamides using the above-described natural dihydrofolate synthase in E.coli, comprising the steps of, by weight,
(A) Dissolving the dihydrofolate synthase in a sodium carbonate buffer solution with the concentration of 0.1mol/L, pH value of 9.5, then adding the solution into each small hole of a 96-hole polystyrene titration plate, and standing at the temperature of 4 ℃ for 12 hours;
(b) Pouring out the solution in each small hole of the polystyrene titration plate, adding 1% of fetal bovine serum into each small hole of the polystyrene titration plate, and standing at 37 ℃ for 30 minutes;
(c) Mixing 50 mu L of sulfanilamide drug standard solution with concentration of 0, 0.1, 0.2, 0.5, 1,2, 5, 10, 20, 50 and 100ng/mL with 50 mu L of horseradish peroxidase-labeled sulfadiazine respectively, adding the mixture into each small hole of the treated polystyrene titration plate, competitively combining with the coated dihydrofolate synthase, and standing at 37 ℃ for 30 minutes;
(d) Washing the small holes of the polystyrene titration plate by using phosphate buffer solution with the concentration of 0.1 mol/L, pH and the value of 7.0-7.6, and removing the sulfonamide which is not adsorbed and the sulfadiazine marked by horseradish peroxidase;
(e) Adding a tetramethyl benzidine solution into the cleaned polystyrene titration plate small holes, developing for 15 minutes, and then adding a sulfuric acid solution of 2 mol/L; then placing the polystyrene titration plate in an enzyme-linked immunosorbent assay (ELISA) detector, and reading the optical density value of each small hole;
(f) Drawing a standard curve by taking the concentration of the sulfonamides as an abscissa and the optical density value obtained by each concentration as an ordinate; in this way, a standard curve was measured for each sulfa drug; obtaining half inhibition amount IC 50 of one sulfadrug according to the measured standard curve of the one sulfadrug, and calculating the cross reaction rate of the dihydrofolate synthase on the one sulfadrug, wherein the cross reaction rate (%) = (half inhibition amount of sulfadiazine/half inhibition amount of one sulfadrug) multiplied by 100%; the cross-reaction rate of each sulfa drug is greater than 10%, indicating that the dihydrofolate synthase can detect the presence of various sulfa drugs;
(g) Adding 5g of homogenate of animal meat sample into 10 mL g of 7.0-7.6 phosphate buffer solution with concentration of 0.1 mol/L, pH and value, oscillating for 5min, centrifuging for 5min, taking 50 mu L of supernatant, adding into the small holes of the polystyrene titration plate treated by the steps (a) and (b), and competitively combining with the coated dihydrofolate synthase; obtaining the optical density value of the small hole through the treatment of the steps (d) and (e); and comparing the optical density value with a standard curve of sulfadiazine to obtain the residual concentration of the sulfadiazine possibly existing in the animal meat sample.
The detection method uses natural dihydrofolate synthase in the escherichia coli as a core reagent, horseradish peroxidase as a catalytic reagent and tetramethyl benzidine as a chromogenic reagent, and detects the concentration of the object to be detected according to the principle that the chromogenic intensity is inversely proportional to the concentration of the object to be detected. The detection method can detect the residual concentration of the sulfa drug in pork, chicken, fish, beef and other animals using the sulfa drug, has high detection accuracy, can establish a standard curve for all sulfa drugs used at present, and can detect all sulfa drugs remained in animal meat.
The beneficial effects of the invention are as follows: a magnetic photoaffinity labeling probe molecule with strong affinity, high selectivity and labeling function with a natural dihydrofolate synthase receptor is synthesized by a chemical method, and according to the principle of receptor-ligand specific binding, the probe molecule can not only be specifically bound with the dihydrofolate synthase, but also can be used for conveniently and quickly obtaining the natural dihydrofolate synthase from bacteria by utilizing magnetism of the probe molecule. The dihydrofolate synthase prepared by the method retains natural activity, can identify all sulfonamide drugs, can detect all sulfonamide drugs remained in animal meat, has high detection accuracy, improves detection efficiency, shortens detection time, and reduces detection cost. Therefore, the method can be used for developing detection products aiming at the sulfanilamide drugs in animal foods, lays a foundation for realizing multi-residue rapid detection of the sulfanilamide drugs, and has important significance for guaranteeing the safety of animal foods and maintaining the physical health of consumers. Meanwhile, the method also provides data support for synthesizing magnetic photoaffinity labeling probes of other small molecular substances (human drugs, pesticides and veterinary drugs) and preparing corresponding receptors.
FIG. 1 is an electrophoretogram of a natural dihydrofolate synthase. Lanes 1 and 3 of the electrophoresis pattern of the whole mycoprotein solution of the control; lane 2 is an electrophoresis diagram of the mycoprotein liquid remaining after the dihydrofolate synthase in the whole mycoprotein liquid of lanes 1 and 3 is captured by using the magnetic photoaffinity labeling probe molecule of the present invention; lane 4 is an electrophoretogram of native dihydrofolate synthase captured from the whole mycoprotein solution of lanes 1 and 3; the magnetic photoaffinity labeling probe has strong affinity and high selectivity with a natural dihydrofolate synthase receptor, the dihydrofolate synthase in the whole bacterial protein liquid of lanes 1 and 3 is completely captured, and the purity of the captured natural dihydrofolate synthase is high.
Drawings
FIG. 1 is an electrophoretogram of a natural dihydrofolate synthase.
Detailed Description
The invention is further described below:
the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biological materials, unless otherwise specified, are available from commercial sources.
Example 1, a magnetic photoaffinity labeling probe molecule, the molecular structural formula of which is shown in formula 1:
Formula 1.
The preparation method of the magnetic photoaffinity labeling probe molecule comprises the following steps of, by weight,
(A) 5, 6, 8 or 10 parts of 4-nitrobenzoyl chloride, namely compound 1, are dissolved in 10mL of toluene, stirred for 24, 40, 60 or 72 hours at room temperature under the protection of nitrogen, 10mL of deionized water is added, an organic phase is separated, the mixture is sequentially washed with 10mL of saturated NaHCO 3 and 10mL of saturated NaCl, 10 g of anhydrous MgSO 4 is dried and then filtered, and the solvent is evaporated to obtain compound 2, and the synthesis process is as shown in the following equation 2;
Equation 2.
(B) 1, 2, 2.5 or 3 parts of compound 2 are dissolved in 10mL of absolute ethanol, 5, 7, 9 or 10 parts of 2 water of tin dichloride SnCl 2·2H2 O are added, and stirring is carried out for 2, 6, 9 or 12 hours at 50 ℃ under the protection of nitrogen. After drying in vacuo 10mL deionized water was added. The mixture was extracted with 20mL of dichloromethane, the organic phase was washed with 20mL of saturated NaCl, dried over 10 g of anhydrous MgSO 4, filtered, and the filtrate was dried in vacuo to give compound 3, which was synthesized as shown in equation 3 below;
Equation 3.
(C) 1, 3, 4 or 5 parts of compound 3 are dissolved in 10mL of dichloromethane, 1, 3, 4 or 5 parts of compound 1 and 1, 2, 3 or 5mL of triethylamine are added, and the mixture is stirred for 2, 6, 9 or 12 hours at room temperature under the protection of nitrogen; the precipitate obtained by filtration after stirring is washed with 10mL of dichloromethane for several times and dried in vacuum to obtain a compound 4, and the synthesis process is shown in the following equation 4;
Equation 4;
(d) 1, 3, 4 or 5 parts of compound 4 are taken, 30, 38, 45 or 50mL parts of glacial acetic acid, 1,2, 4 or 5 parts of chromium trioxide, 5, 6, 8 or 10mL of concentrated sulfuric acid are added, and the mixture is stirred at room temperature for 12 hours. The reaction solution was poured into 30, 38, 45 or 50mL of ice water and extracted with 30, 38, 45 or 50mL of ethyl acetate. The ethyl acetate solution obtained was washed with 30, 38, 45 or 50mL of saturated NaCl, dried over 10 g of anhydrous MgSO 4, filtered and dried in vacuo to give compound 5, which was synthesized as shown in the following equation 5;
Equation 5.
(E) 1, 3, 4 or 5 parts of compound 5 are dissolved in 10mL of absolute ethanol, 1, 2, 4 or 5 parts of SnCl 2·2H2 O are added, and stirring is carried out for 2,6, 9 or 12 hours at 50 ℃ under the protection of nitrogen. After the mixture is dried in vacuum, 30, 38, 45 or 50mL deionized water is added, 30, 38, 45 or 50mL dichloromethane is used for extraction, the obtained dichloromethane solution is washed by 30, 38, 45 or 50mL saturated NaCl, 10g anhydrous MgSO 4 is dried, filtered and dried in vacuum to obtain a compound 6, and the synthesis process is as shown in the following equation 6;
Equation 6.
Characterization data ：（IR: Fe-O 580 cm-1, Si-O-Si 1095 cm-1, Si-O-Si 794 cm-1, Si-O-Si 985 cm-1, Si-O 460 cm-1, H-N-H1650 and for compound 6 3415 cm-1,C=C 1420,C-H 3150 cm-1,C=O 1700cm-1）;1H NMR （DMSO-d6, 600 MHz） δ: 10.095 (s, 1H, COOH), 7.961-7.947 (d, J=8.4 Hz, 2H, ArH), 7.761-7.747 (d, J=8.4 Hz, 4H, ArH), 7.734-7.720 (d, J=8.4 Hz, 2H, ArH), 7.644-7.631 (d, J=7.8 Hz, 2H, ArH), 7.375-7.362 (d, J=7.8 Hz, 2H, ArH), 6.627-6.613 (d, J=8.4 Hz, 2H, ArH); 13C NMR (DMSO-d6, 150 MHz) δ: 194.2, 165.5, 152.4, 143.9, 142.4, 134.9, 132.3, 131.2, 130.9, 130.7, 129.5, 129.2, 129.0, 128.9, 128.6, 120.5, 119.0, 112.6, 112.5, 112.4. HRMS (ESI) m/z: [M-H]+ calcd for C21H15N2O4+: 359.1032; Found: 359.1038）.
(F) 1,3, 4 or 5 parts of compound 6 is taken and dissolved in 10mL of N, N-dimethylformamide, 1,3, 4 or 5 parts of N-butylamine and 1,3, 4 or 5 parts of isobutyl chloroformate are added for reaction at 4 ℃ for 1,3, 4 or 6 hours. Taking 100 mg Fe 3O4@SiO2-NH2 magnetic particles, suspending the particles in 10mL of N, N-dimethylformamide by ultrasonic, dropwise adding the reaction solution, and stirring for 12 hours at the temperature of 4 ℃; the obtained precipitate is washed by N, N-dimethylformamide, washed by water and dried in vacuum to obtain the magnetic photoaffinity labeling probe, and the synthesis process is shown in the following equation 7;
equation 7.
Example 2A method for preparing a natural dihydrofolate synthase in E.coli using the magnetic photoaffinity labeling probe molecule described in example 1, comprising the steps of, in weight ratios,
(A) Inoculating a standard strain of escherichia coli ATCC25922 into an LB liquid culture medium, culturing for 24 hours at the temperature of 37 ℃ and the rotation speed of 220rmp to obtain a bacterial liquid, centrifuging the bacterial liquid at the temperature of 4 ℃, and collecting bacterial bodies.
(B) And (3) re-suspending the thalli by using a phosphoric acid buffer solution, then carrying out ultrasonic crushing on the re-suspended thalli by using an ice bath, centrifuging at the temperature of 4 ℃ after ultrasonic treatment, collecting supernatant I, and adding Soluble Bingding Buffer with an equal volume to obtain supernatant II for standby.
(C) Adding 100mg of the magnetic photoaffinity labeling probe molecules into the supernatant II, fully stirring, incubating for any time between 1 and 6 hours at the temperature of 37 ℃, enriching magnetic substances at the bottom of a bottle by using a magnet, and pouring out the supernatant III;
(d) Washing the magnetic substance at the bottom of the bottle with water, eluting with 10mL of 20% methanol, and collecting the eluent, wherein the eluent is natural dihydrofolate synthase in Escherichia coli. The remaining magnetic substance, namely the magnetic photoaffinity labeling probe molecules, are washed sequentially with 10mL of ethanol and 10mL of deionized water, dried in vacuum and stored at room temperature for repeated use.
Example 3, this example is a direct competitive enzyme-linked assay for the detection of sulfonamides using the above-described natural dihydrofolate synthase of E.coli, comprising the steps of, by weight,
(A) The dihydrofolate synthase is dissolved in sodium carbonate buffer with a concentration of 0.1mol/L, pH value of 9.5 and then added to each well of a 96-well polystyrene titer plate and left at a temperature of 4℃for 12 hours.
(B) After pouring out the solution in each well of the polystyrene titer plate, 1% fetal bovine serum was added to each well of the polystyrene titer plate and left to stand at 37 ℃ for 30 minutes.
(C) 50. Mu.L of 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50 and 100ng/mL of sulfanilamide drug standard solution were mixed with 50. Mu.L of horseradish peroxidase-labeled sulfadiazine, respectively, and added to each well of the treated polystyrene titer plate, competitively bound to the coated dihydrofolate synthase, and left at 37℃for 30 minutes.
(D) Washing the wells of the treated polystyrene titer plate with phosphate buffer with a concentration of 0.1 mol/L, pH and a value of 7.0, 7.2, 7.4 or 7.6 to remove non-adsorbed sulfonamide and horseradish peroxidase labeled sulfadiazine.
(E) Adding a tetramethyl benzidine solution into the cleaned polystyrene titration plate small holes, developing for 15 minutes, and then adding a sulfuric acid solution of 2 mol/L; then the polystyrene titration plate is placed in an enzyme-linked immunosorbent assay instrument, and the optical density value of each small hole is read.
(F) Drawing a standard curve by taking the concentration of the sulfonamides as an abscissa and the optical density value obtained by each concentration as an ordinate; in this way, a standard curve was measured for each sulfa drug; obtaining half inhibition amount IC 50 of one sulfadrug according to the measured standard curve of the one sulfadrug, and calculating the cross reaction rate of the dihydrofolate synthase on the one sulfadrug, wherein the cross reaction rate (%) = (half inhibition amount of sulfadiazine/half inhibition amount of one sulfadrug) multiplied by 100%; the cross-reactivity of each sulfa drug was greater than 10%, indicating that the dihydrofolate synthase was able to detect the presence of various sulfa drugs.
(G) Adding 5 grams of homogenate of animal meat sample to 10 mL concentrations of 7.0, 7.2, 7.4 or 7.6 phosphate buffer with 0.1 mol/L, pH value, shaking for 5 minutes, centrifuging for 5 minutes, taking 50 μl of supernatant into wells of polystyrene titer plate treated in said steps (a) and (b), competitively binding to coated dihydrofolate synthase; and (3) obtaining the optical density value of the small hole through the treatment of the steps (d) and (e). And comparing the optical density value with a standard curve of sulfadiazine to obtain the residual concentration of the sulfadiazine possibly existing in the animal meat sample.
There are, of course, many other embodiments of the invention that will be apparent to those skilled in the art from consideration of this disclosure without departing from the spirit and scope of the invention, and that modifications can be made thereto without departing from the scope of the invention as defined in the appended claims.
Claims (4)
1. A magnetic photoaffinity labeling probe molecule is characterized in that the molecular structural formula is shown in formula 1:
Formula 1.
2. A method for preparing a magnetic photoaffinity labeling probe molecule according to claim 1, comprising the steps of, in weight ratio,
(A) Dissolving 5-10 parts of 4-nitrobenzoyl chloride, namely a compound 1, in 10mL of toluene, stirring for 24-72 hours at room temperature under the protection of nitrogen, adding 10mL of deionized water, separating an organic phase, sequentially cleaning with 10mL of saturated NaHCO 3 and 10mL of saturated NaCl, drying 10 g of anhydrous MgSO 4, filtering, evaporating the solvent to obtain a compound 2, and carrying out a synthesis process of the following equation 2;
Equation 2;
(b) Dissolving 1-3 parts of compound 2 in 10mL of absolute ethyl alcohol, adding 5-10 parts of tin dichloride SnCl 2·2H2 O with 2 water, and stirring for 2-12 hours at 50 ℃ under the protection of nitrogen; vacuum drying and then adding 10mL of deionized water; the mixture was extracted with 20mL of dichloromethane, the organic phase was washed with 20mL of saturated NaCl, dried over 10 g of anhydrous MgSO 4, filtered, and the filtrate was dried in vacuo to give compound 3, which was synthesized as shown in equation 3 below;
equation 3;
(c) 1-5 parts of compound 3 is dissolved in 10mL of dichloromethane, 1-5 parts of compound 1 and 1-5mL of triethylamine are added, and the mixture is stirred for 2-12 hours at room temperature under the protection of nitrogen; the precipitate obtained by filtration after stirring is washed with 10mL of dichloromethane for several times and dried in vacuum to obtain a compound 4, and the synthesis process is shown in the following equation 4;
Equation 4;
(d) Taking 1-5 parts of compound 4, adding 30-50 mL parts of glacial acetic acid, 1-5 parts of chromium trioxide and 5-10 mL parts of concentrated sulfuric acid, and stirring at room temperature for 12 hours; pouring the reaction solution into 30-50 mL ice water, and extracting with 30-50 mL ethyl acetate; the obtained ethyl acetate solution is washed by 30-50 mL saturated NaCl, 10 g of anhydrous MgSO 4 is dried, filtered and dried in vacuum to obtain a compound 5, and the synthesis process is as shown in the following equation 5;
equation 5;
(e) Dissolving 1-5 parts of compound 5 in 10mL of absolute ethyl alcohol, adding 1-5 parts of SnCl 2·2H2 O, and stirring at 50 ℃ for 2-12 hours under the protection of nitrogen; after the mixture is dried in vacuum, 30-50mL of deionized water is added, 30-50mL of dichloromethane is used for extraction, the obtained dichloromethane solution is washed by 30-50mL of saturated NaCl, 10g of anhydrous MgSO 4 is dried, filtration and vacuum drying are carried out, and the compound 6 is obtained, wherein the synthesis process is as shown in the following equation 6;
equation 6;
(f) 1-5 parts of compound 6 is taken and dissolved in 10mL of N, N-dimethylformamide, 1-5 parts of N-butylamine and 1-5 parts of isobutyl chloroformate are added for reaction for 1-6 hours at 4 ℃; taking 100 mg Fe 3O4@SiO2-NH2 magnetic particles, suspending the particles in 10mL of N, N-dimethylformamide by ultrasonic, dropwise adding the reaction solution, and stirring for 12 hours at the temperature of 4 ℃; the obtained precipitate is washed by N, N-dimethylformamide, washed by water and dried in vacuum to obtain the magnetic photoaffinity labeling probe, and the synthesis process is shown in the following equation 7;
equation 7.
3. A method for preparing natural dihydrofolate synthase in Escherichia coli by using the magnetic photoaffinity labeling probe molecule as described in claim 1, comprising the steps of, in weight ratio,
(A) Inoculating a standard strain of escherichia coli ATCC25922 into an LB liquid culture medium, culturing for 24 hours at the temperature of 37 ℃ and the rotation speed of 220rmp to obtain a bacterial liquid, centrifuging the bacterial liquid at the temperature of 4 ℃, and collecting thalli;
(b) Re-suspending the thalli by using a phosphoric acid buffer solution, then carrying out ultrasonic crushing on the re-suspended thalli by using an ice bath, centrifuging at the temperature of 4 ℃ after ultrasonic treatment, collecting a supernatant I, and adding Soluble Bingding Buffer with an equal volume to obtain a supernatant II for later use;
(c) Adding 100mg of the magnetic photoaffinity labeling probe molecules into the supernatant II, fully stirring, incubating for 1-6 hours at 37 ℃, enriching magnetic substances at the bottom of a bottle by using a magnet, and pouring out the supernatant III;
(d) Washing the magnetic substance at the bottom of the bottle with water, eluting with 10mL of 20% methanol, and collecting the eluent, wherein the eluent is natural dihydrofolate synthase in Escherichia coli.
4. The method for preparing natural dihydrofolate synthase according to claim 3, wherein said magnetic photoaffinity labeling probe molecules remaining after removing the eluent in said step (d) are sequentially washed with 10mL of ethanol, 10mL of deionized water, and vacuum-dried and stored at room temperature.
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