CN112649600A

CN112649600A - DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergist drugs

Info

Publication number: CN112649600A
Application number: CN202110039762.2A
Authority: CN
Inventors: 梁晓; 于万鹏
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2021-01-13
Filing date: 2021-01-13
Publication date: 2021-04-13

Abstract

The invention provides a DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergists, wherein DHFR is derived from sulfonamide synergist drug sensitive strains including staphylococcus aureus, streptococcus pneumoniae and mycobacterium tuberculosis. The invention establishes a sulfanilamide synergist drug multi-residue fluorescence polarization immunoassay method based on sulfanilamide synergist drug receptor protein dihydrofolate reductase for the first time, applies the established method to the sulfanilamide synergist drug multi-residue detection in milk, and provides a method technical support for the sulfanilamide synergist drug screening in food. The invention creatively adopts DHFR derived from sulfonamide synergist drug sensitive strains, and belongs to the initiative in the field. The method breaks through the limitation of the conventional method, expands the application range, and has the advantages of rapidness, simplicity, accuracy and the like.

Description

DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergist drugs

Technical Field

The invention belongs to the technical field of veterinary drug residue detection, and particularly relates to a DHFR-based multi-residue fluorescence polarization immunoassay method for sulfanilamide synergist drugs.

Background

Sulfonamide synergists (ASGs) are 2, 4-diaminopyrimidines and are Dihydrofolate Reductase (DHFR) inhibitors for blocking bacterial folate metabolism. The common structure of the sulfonamide potentiators is diaminopyrimidine, and the common drugs mainly include Trimethoprim (TMP), Diaveridine (DVD), Ormoprim (OMP), Bromopramine (BDP), Baquiloprim (Baquiloprim, BQP), and the like. The sulfanilamide synergist has various types of medicines and wide application range, and can be used together with other antibiotics to expand antibacterial spectrum. If the composition is used with sulfonamides, the sulfonamides can inhibit dihydrofolate synthetase, thus doubly blocking folic acid metabolism of bacteria, hindering biosynthesis of thallus nucleic acid and protein, enhancing antibacterial effect and even generating bactericidal effect. The sulfonamide synergist is used together with fluoroquinolone medicines, beta-lactam medicines or aminoglycoside medicines, and has an inhibiting effect on gram-positive bacteria and gram-negative bacteria. Because the sulfonamide synergist has low price, definite curative effect and wide antibacterial spectrum, the sulfonamide synergist is often combined with other antibacterial drugs to be applied to the livestock breeding industry.

Just because the sulfanilamide synergist is widely applied to livestock and poultry breeding, drug residues easily exist, food safety problems caused by the residues seriously harm human health and cause potential public health safety problems. Therefore, it is necessary to establish a safe, effective and rapid method for detecting the drug residue.

At present, the detection method of the sulfonamide synergist drug residue mainly comprises a microbiological method, an instrumental method, an immunoassay method and the like. The microbiological assay is based on the inhibition of the metabolism of microorganisms by antibiotics to determine the antibiotic residues in a sample. The microbiological method does not require highly specialized and expensive equipment, but it lacks rapidity and specificity and only measures the residual biological activity. The detection result of the instrumental method is accurate, but the method needs complicated instruments and professionals, and the related cost is high. At present, the immunoassay technology for sulfonamide potentiators is reported less, and mainly refers to an enzyme-linked immunosorbent assay (ELISA) detection method. The ELISA method is a solid-phase immunoassay method, and needs a plurality of separation and washing steps in the detection process, so that the time is long. The Fluorescence Polarization Immunoassay (FPIA) is a representative homogeneous Fluorescence Immunoassay, and has the characteristics of simplicity, reliability, rapidness and high efficiency. Compared with ELISA method, FPIA method does not need washing to separate bound and unbound antibody, thus greatly simplifying operation steps and shortening operation time, and antigen-antibody reaction in homogeneous system is fast, and detection time mainly depends on sample adding speed, therefore, only several minutes or even less are needed in the field generally, and there is no related report of using FPIA to detect sulfa synergist drug residue.

The FPIA method for establishing the sulfanilamide synergist drug residue in the animal food can greatly improve the detection efficiency, is particularly suitable for the rapid screening and analysis of a large number of samples, and has important significance for realizing the rapid screening and detection of the sulfanilamide synergist drug residue in the animal food and the standard use in the livestock and poultry production in China.

Disclosure of Invention

Aiming at the defects of complex operation, long time consumption and the like of the existing method for detecting the sulfanilamide synergist medicines, the invention establishes a fast, efficient and accurate FPIA method for detecting the sulfanilamide synergist medicine residue.

The method is a multi-residue Fluorescence Polarization Immunoassay (FPIA) method for sulfanilamide synergist drugs based on dihydrofolate reductase (DHFR). Because the sulfonamide synergist drugs are dihydrofolate reductase inhibitors, DHFR derived from the sulfonamide synergist drug sensitive strains has the potential ability of recognizing the drugs. Therefore, the invention prepares the broad-spectrum identification material receptor protein DHFR, incubates the sample or standard substance to be detected, the fluorescent marker and the DHFR solution for a short time according to the fluorescence polarization principle, detects the FP value, calculates the concentration of the substance to be detected according to the established standard curve, and finally establishes the detection method.

Based on the above, the invention provides a DHFR sulfonamide synergist-based multi-residue fluorescence polarization immunoassay method, which is established based on dihydrofolate reductase (DHFR); the dihydrofolate reductase (DHFR) is obtained by inducing a dihydrofolate reductase recombinant expression strain. The recombinant expression strain is derived from sulfonamide synergist drug sensitive strains, including staphylococcus aureus, streptococcus pneumoniae and mycobacterium tuberculosis.

Preferably, the dihydrofolate reductase (DHFR) is obtained by the following method: extracting genes of sulfanilamide synergist drug sensitive strains (such as staphylococcus aureus, streptococcus pneumoniae, mycobacterium tuberculosis and the like), and performing PCR amplification to obtain dihydrofolate reductase genes; constructing a recombinant clone strain, and further obtaining a recombinant expression strain for efficiently expressing dihydrofolate reductase; and finally inducing the recombinant expression strain to express to obtain the recombinant dihydrofolate reductase. The conditions for expressing the recombinant dihydrofolate reductase by the induced recombinant expression strain are that the temperature is 20 ℃ and the induction is carried out for 13-16 h by 0.5mM IPTG.

The invention discloses a DHFR-based sulfonamide synergist drug multi-residue fluorescence polarization immunoassay method, which comprises the following steps:

s1, mixing a series of sulfanilamide synergist drug standard solutions with known concentrations with a fluorescent marker solution and a dihydrofolate reductase solution respectively, incubating for a competitive reaction, and determining the fluorescence polarization value of the obtained system;

s2, drawing a standard curve by taking the measured fluorescence polarization value as a vertical coordinate and taking the logarithm value of the concentration of the series of sulfanilamide synergist drug standard products with known concentrations as a horizontal coordinate;

s3, replacing the sulfanilamide synergist drug standard in the step S1 with a sample to be detected, mixing the sample to be detected with the fluorescent marker solution and the dihydrofolate reductase solution in equal volume, incubating for a competitive reaction, and determining the fluorescence polarization value of the obtained system;

s4, calculating the concentration of the sulfanilamide synergist drug in the sample to be detected according to the standard curve in the step S2.

The fluorescent marker is an amide conjugate of sulfonamide synergist drug hapten and fluorescein isothiocyanate. Preferably, the sulfonamide potentiator hapten has the structural formula:

preferably, the fluorescein isothiocyanate is butanediamine fluorescein isothiocyanate (BDF).

The buffer solution used in the method is MTEN buffer solution; preferably, the buffer contains 25mM MES, 125mM Tris, 25mM ethanolamine, 100mM NaCl, 100. mu.M NADPH.

The concentration of the fluorescent marker solution is working concentration, and the working concentration of the fluorescent marker is concentration corresponding to 10 times of the total fluorescence intensity of the fluorescent marker to the fluorescence intensity of the reaction buffer solution; the concentration of the dihydrofolate reductase solution is working concentration, and the working concentration is DHFR dilution times when the dihydrofolate reductase solution is combined with the fluorescent marker by 50 percent and is taken as the working concentration.

The incubation competition reaction is carried out at the temperature of 20-25 ℃ for 2-20 min; the measurement conditions of the fluorescence polarization value are as follows: an optical filter with excitation wavelength of 485nm, emission wavelength of 530nm and emission wavelength of 515 nm.

Drawing the standard curve by using Origin 8.5 software, fitting the curve by using a four-parameter equation model, taking FP as a vertical coordinate and taking a logarithmic value of the concentration of the standard substance as a horizontal coordinate, and establishing the curve; the standard curve IC₅₀The value was 14.9. mu.g/L, the LOD value was 2.9. mu.g/L, IC₂₀～IC₈₀5.3 to 41.5 μ g/L.

The invention also provides application of the DHFR-based sulfanilamide synergist drug multi-residue fluorescence polarization immunoassay method in detection of the content of milk sulfanilamide synergist drugs. And when the sample to be detected is milk, carrying out sample pretreatment by using 55% saturated ammonium sulfate solution.

The invention also provides application of the DHFR-based sulfanilamide synergist drug multi-residue fluorescence polarization immunoassay method in preparation of a fluorescence polarization immunoassay kit for detecting sulfanilamide synergist drugs.

The invention also provides a fluorescence polarization immunoassay kit for detecting the sulfanilamide synergist drug, which comprises a fluorescence marker solution and DHFR derived from sulfanilamide synergist drug sensitive strains. Preferably, the fluorescent marker is a conjugate of TMP hapten and BDF; the DHFR is recombinant staphylococcus aureus DHFR. The kit also comprises a standard solution of sulfanilamide synergist drugs, an MTEN buffer solution, a 96-hole black fluorescent plate and the like.

Compared with the prior art, the invention has the following advantages:

1. the invention establishes a sulfanilamide synergist drug multi-residue fluorescence polarization immunoassay method based on sulfanilamide synergist drug receptor protein dihydrofolate reductase for the first time, applies the established method to the sulfanilamide synergist drug multi-residue detection in milk, and provides a method technical support for the sulfanilamide synergist drug screening in food. At present, no related report for establishing a fluorescence polarization immunoassay method based on a sulfonamide synergist drug receptor protein dihydrofolate reductase exists in the field, and the method is originated in the field.

2. The method creatively adopts DHFR from the sulfanilamide synergist drug sensitive strain, breaks through the limitation of the method and expands the application range. The dihydrofolate reductase catalyzes the reduction of 7, 8-Dihydrofolate (DHF) to 5, 6, 7, 8-Tetrahydrofolate (THF) using NADPH as a coenzyme. Since THF and its metabolites are precursors to purine and pyrimidine bases, the normal function of this enzyme is critical for cell proliferation. This makes DHFR an ideal target for antifolates. The sulfanilamide synergist drug is a dihydrofolate reductase inhibitor, plays a role by blocking folic acid metabolism in bacteria, and DHFR derived from sulfanilamide synergist drug sensitive strains has potential capability of recognizing the drugs. Therefore, the invention creatively adopts the receptor protein, theoretically can combine with all corresponding ligands, can simultaneously detect a plurality of sulfanilamide synergist drugs, has no cross reaction, and breaks through the limitation of the existing detection method.

3. The method of the invention also has the advantages of rapidness, simplicity, accuracy and the like. According to the detection method of the sulfonamide synergist medicaments established by the invention, the IC of 5 sulfonamide synergist medicaments is detected₅₀The values are respectively 14.9, 38.7, 18.6, 16.0 and 20.2 mu g/L, and the detection limit is 1.3-6.2 mu g/L.

Drawings

FIG. 1 shows the result of dhfr gene amplification in Staphylococcus aureus ATCC 29213 of example one.

FIG. 2 shows the results of PCR identification of pET-28a-S.A-dhfr as an expression vector in the example. Lane M: DNAmarker; lane 1-8: 8 clones were randomly selected.

FIG. 3 shows the results of double digestion and identification of the expression vector pET-28a-S.A-dhfr in the first example. Lane M: DNAmarker; lane 1: performing double enzyme digestion on Nhe I and Xho I; lane 2: a plasmid.

FIG. 4 shows the SDS-PAGE and Western-Blotting detection results of the fusion protein of the example. Lane M: marker; lane 1: crushing the precipitate; lane 2: adding IPTG pre-full mycoprotein; lane 3: IPTG induced whole mycoprotein; lane 4: purifying the protein; lane 5: washing buffer eluent;

FIG. 5 is an example one DHFR induction condition optimized SDS-PAGE analysis. Lane M: a protein Marker; (A) lane 1-8: the induction time is 3-10 h in sequence; (B) lane 1-8: the induction time is 3-10 h in sequence; (C) lane 1-9: the induction time is 8-16 h in sequence; (D) lane 1-9: the induction time is 8-16 h in sequence.

FIG. 6 is a standard curve of the multi-residue fluorescence polarization detection method based on Staphylococcus aureus-DHFR sulfonamide potentiators in accordance with the second embodiment of the present invention.

Fig. 7 is a cross-curve of five sulfanilamide synergists based on the staphylococcus aureus-DHFR sulfanilamide synergist drug multi-residue fluorescence polarization detection method of the second embodiment of the present invention.

FIG. 8 shows the third embodiment of the present invention, which uses the FPIA method for detecting five sulfanilamide synergists ASG based on DHFR sulfanilamide synergists_SCalibration curves in pure and skim milk. (A) Five ASG_SAdding a standard curve to the pure milk substrate; (B) five ASG_SThe skim milk base is plotted.

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the reagents, biological materials and the like used are commercially available unless otherwise specified.

EXAMPLE one preparation of dihydrofolate reductase DHFR derived from a drug sensitive strain of the sulfonamide potentiator class

The method is suitable for sulfonamide synergist drug sensitive strains including staphylococcus aureus, streptococcus pneumoniae, mycobacterium tuberculosis and the like. The process of the present invention is described in detail below, taking Staphylococcus aureus ATCC 29213 as an example. The strain is commercially available.

1. Primer design

According to the dhfr gene sequence of staphylococcus aureus ATCC 29213 in GeenBank in NCBI, primers are designed according to the principle of homologous arms, wherein an upstream primer is inserted into a NheI enzyme cutting site, and a downstream primer is inserted into a XhoI enzyme cutting site.

TABLE 1.1 Staphylococcus aureus ATCC 29213dhfr Gene amplification primer sequences

The primer used by the invention has high specificity, the selected enzyme cutting site is convenient for multiple enzyme cutting in the cloning and expression processes of the dihydrofolate reductase, the enzyme cutting efficiency is high, and the dihydrofolate reductase gene is not damaged.

2. Whole genome extraction and target gene amplification

Staphylococcus aureus ATCC 29213DNA was extracted using the Novokean Biotechnology Co., Ltd.

The dhfr gene is amplified by adopting a PCR technology, the reaction system is 20 mu L, and the system design is as follows:

TABLE 1.2 PCR reaction System

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 ℃ for 7 min. And recovering and sequencing the target gene after the amplification is finished.

The dhfr fragment was amplified based on the extracted staphylococcus aureus ATCC 29213 genome as a template. The result in FIG. 1 shows that there is a significant band around 500bp, which is identical with 480bp of the dhfr gene of Staphylococcus aureus.

3. Construction of recombinant expression vector pET-28a-S.A-dhfr

(1) Plasmid digestion

Plasmid vector pET-28a was double digested according to the system in Table 1.3, with Nhe I and Xho I sites. The temperature of the enzyme digestion reaction is 37 ℃, the water bath is carried out for 3h, and the total reaction volume is 120 mu L. And (4) carrying out 1% gel electrophoresis on the enzyme digestion product, and recovering the enzyme digestion product.

TABLE 1.3 plasmid cleavage System

(2) Recombination reactions

The recombinant reaction system was formulated on ice according to table 1.4. The reaction solution was mixed well, centrifuged at 5000rpm for 1min, and the reaction system was collected. Water bath at 50 deg.c for 5min, and cooling on ice immediately.

TABLE 1.4 recombination reaction systems

4. Identification of expression vector pET-28a-S.A-dhfr

And connecting the target fragment and an expression vector, transforming into DH5 alpha, and performing PCR and double enzyme digestion to identify the transformation result. FIG. 2 shows that, in addition to clone No. 5, there is a significant band around 500 bp. pET-28a is 5369bp in size, FIG. 3 shows that strips appear around 5000bp and 500bp respectively, and the sequencing result is consistent with the target gene sequence through comparison.

5. Expression and purification in Escherichia coli BL21(DE3)

The constructed recombinant expression vector pET-28a-S.A-dhfr is transformed into competent cell Escherichia coli BL21(DE3) for protein expression and purification.

(1) Expression of recombinant protein DHFR

BL21(DE3) single colonies were picked from LB agar plates and cultured overnight at 37 ℃ and 160rpm in 1mL LB broth containing 30. mu.g/mL kanamycin. The bacterial liquid was inoculated into 100mL LB broth containing 30. mu.g/mL kanamycin, and shaken at 250rpm at 37 ℃ for about 4 hours to an OD600 of about 0.6. 1mL of the bacterial solution was taken out and placed at 4 ℃ for non-induced control. IPTG was added to the remaining bacterial solution to a final concentration of 0.5 mM. The bacterial solution was induced at 30 ℃ and 160rpm for 3 h. After induction, the bacterial liquid is centrifuged at 10000rpm and 4 ℃ for 10min, supernatant is discarded, and bacterial liquid precipitate is collected and stored at-80 ℃.

(2) Preparation of protein samples

10mL of Columns binding buffer is used for resuspending the bacteria, and cells are crushed for 20min (ultrasonic 3s and stop for 2s) at a power of 240w until the bacteria liquid is clarified. Centrifuging at 10000rpm and 4 deg.C for 10min, and recovering supernatant to obtain DHFR extractive solution of Staphylococcus aureus.

(3) SDS-PAGE and Western-Blotting detection of DHFR

Taking the non-induced bacterial liquid, the induced bacterial liquid, crushing and precipitating, washing buffer eluent, adding 60 mu L of DHFR purified by a nickel ion affinity chromatography column, adding 5 multiplied by protein sample buffer solution, carrying out boiling water bath for 10min, centrifuging at 10000rpm for 2min, and collecting the supernatant for later use. SDS-PAGE and Western-Blotting assays were performed.

The DHFR of the staphylococcus aureus target protein is about 18 KD. The induction expression of BL21(DE3) containing expression vector pET-28a-S.A-dhfr resulted in more obvious target protein band after IPTG induction than 18KD band of whole bacterial protein without IPTG induction, as shown in FIG. 4.

Optimization of DHFR recombinant protein induced expression conditions

The recombinant expression vector pET-28a-S.A-dhfr is transformed into BL21(DE3), and IPTG 0.2mM and 0.5mM are added respectively for induction for 8-16 h when the induction temperature is 20 ℃. When the induction temperature is 30 ℃, IPTG (isopropyl thiogalactoside) 0.2mM and IPTG (isopropyl thiogalactoside) 0.5mM are respectively added for induction for 3-10 h, and the protein expression amount is observed. As shown in FIG. 5, the target protein expression was performed under various conditions, and the final induction was carried out at 20 ℃ for 13-16 hours with 0.5mM IPTG.

Example II establishment of Multi-residual fluorescence polarization detection method based on receptor DHFR sulfonamide potentiator

1. Primary reagent

Sulfonamide potentiators Trimethoprim (TMP), fluorescein isothiocyanate BDF, N-hydroxysuccinimide (NHS), ethyldimethylaminocarbodiimide hydrochloride (EDC. HCl), N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 2- (N-morpholine) ethanesulfonic acid MES, Tris, and the like are commercially available.

Dihydrofolate reductase DHFR was obtained by the method of example 1. The method is suitable for sulfonamide synergist drug sensitive strain recombinant dihydrofolate reductase including staphylococcus aureus, streptococcus pneumoniae, mycobacterium tuberculosis and the like, and the method is explained in detail in the second embodiment by taking staphylococcus aureus-DHFR obtained in the first embodiment as an example.

The TMP hapten (TMP-CS) is from Chinese agriculture university and is described in patent number ZL201810450544.6, namely sulfonamide synergist hapten and holoantigen, and the invention patent of preparation method and application thereof. The structural formula of the TMP hapten is shown as the following formula:

2. preparation, purification and identification of sulfanilamide synergist drug fluorescent marker

2.1 preparation of fluorescent marker TMP-CS-BDF

(1) Weighing 10mg TMP-CS in a 2mL centrifuge tube;

(2) 40mg EDC and 30mg NHS were dissolved in 500 μ LDMF;

(3) adding the solution into hapten, quickly shaking to dissolve, clarifying after dissolving, adding into rotor, stirring with magnetic stirrer, standing overnight, and keeping out of the sun;

(4) centrifuge at 5000rpm for 10 min. Centrifuging to remove the precipitate to obtain well-activated TMP-CS active ester, and keeping a small amount of the active ester as a control;

(5) weighing 10mg BDF, adding 500 mu LDMF to dissolve;

(6) dropwise adding the dissolved fluorescein into the activated hapten, placing the mixture into a magnetic stirrer for stirring, and keeping the mixture overnight in the dark;

(7) a small amount of reaction solution (about 50 μ L) was taken and separated using a thin layer chromatography plate, using EDF as a control, pre-run with chloroform, then chloroform: methanol: glacial acetic acid (6:1:0.01, v: v: v) was isolated as a developing agent;

(8) a yellow band different from BDF was scraped off, extracted with 200. mu.L of methanol, and the resulting yellow solution was stored at 4 ℃ in the dark.

The fluorescent marker TMP-CS-BDF has the following structure:

2.2 identification of the fluorescent marker TMP-CS-BDF

(1) Measuring the FP value of TMP-CS-BDF at the working concentration;

(2) mixing 70 mu LTMP-CS-BDF with 70 mu LDHFR with working concentration, and measuring FP value after 5min incubation;

(3) after specific binding to DHFR, FP values for free TMP-CS-BDF and bound TMP-CS-BDF were compared.

The concentration of the fluorescent marker corresponding to the fluorescence value of 10 times the reaction buffer was defined as the working concentration based on the fluorescence value of 50mM MES reaction buffer. Diluting the protein by 50 times, identifying the fluorescent marker, respectively measuring mP values of the free fluorescent marker and the fluorescent marker DHFR, and calculating a delta mP value. The mP value is obviously increased. The success of the tracer synthesis is shown. Illustrating the method of establishing the receptor DHFR with which the present invention can be used.

The fluorescent marker and the receptor protein are used for establishing a protein dilution curve, and the titer of the receptor protein is the dilution concentration of 50 percent of the protein in the curve when the fluorescent marker is combined with the protein. A high receptor titer indicates a higher affinity of the receptor for the marker.

TABLE 2.1 dilution Curve parameters based on Staphylococcus aureus-DHFR

3. FPIA research of sulfonamide potentiators based on DHFR

3.1 determination of working concentration of fluorescent marker

Working concentration of TMP-CS-BDF fluorescent marker, with reaction buffer as reference solution, total fluorescence intensity of fluorescent marker is about 10 times (about 200 Relative Fluorescence Units (RFU)) of the fluorescence intensity of reaction buffer.

3.2 determination of optimal DHFR dilution

Receptor DHFR was diluted at 1/12.5, 1/25, 1/50, 1/100, 1/200, 1/400, 1/800, 1/1600, and 1/3200 in 70 μ L volume. And respectively adding TMP-CS-BDF into the proteins with different dilution multiples, and reacting for 2min to determine the FP value. The DHFR dilution at 50% binding was taken as the working concentration (preferably 20. mu.g/ml).

3.3 procedure for competing for FPIA

To a 96-well black fluorescent plate, 70. mu.L of the standard was added, followed by 70. mu.L of the fluorescent marker at the working concentration, followed by mixing and adding the receptor DHFR solution at the working concentration. And replacing the standard substance with buffer solution in the control hole, shaking and mixing uniformly, reacting for 2min in a dark place, detecting the FP value on a computer, wherein the excitation wavelength is 485nm, the emission wavelength is 530nm, and a 515nm optical filter is selected.

3.4 creation of Standard Curve

And (3) utilizing Origin 8.5 software, fitting a curve by using a four-parameter equation model, taking FP as a vertical coordinate, and taking a logarithmic value of the concentration of the standard substance as a horizontal coordinate, and establishing the curve. The four parameter equation is:

Y＝(A–D)/[1+(x/C)B]+D

wherein Y is a fluorescence polarization value corresponding to different target (standard) concentrations, and A represents an FP value corresponding to the lowest drug concentration or zero concentration, i.e. the highest value of a standard curve; d represents the FP value corresponding to the highest drug concentration, i.e. the lowest point of the standard curve. C is an abscissa value corresponding to the FP value of (A + D)/2, i.e., 50% inhibitionDrug concentration (IC) of₅₀) And B represents the slope of the straight line part of the standard curve; with IC₁₀To the detection limit of the method, IC₂₀～IC₈₀Is the detection range of the method.

3.5FPIA kinetic Studies

Adding 70 μ L TMP standard substance with concentration of 0, 1.4, 4.1, 12.3, 37.0, 111.1, 333.3 and 1000 μ g/L into the micropore, adding 70 μ L fluorescent marker, mixing well, adding DHFR solution, measuring FP value at 0, 2, 5, 10, 15, 20, 30 and 60min, establishing standard curve, and calculating IC₅₀Value according to Δ mP and IC₅₀And (6) drawing a kinetic curve.

TABLE 2.2 Standard Curve parameters based on Staphylococcus aureus-DHFR

4. Optimization of reaction buffer

The fluorescence polarization method based on the receptor DHFR is established to detect the multi-residue of the sulfanilamide synergist drugs, and the DHFR needs to be combined with the drugs under the conditions of specific salt ion concentration, pH, NADPH and the like, which is different from the fluorescence polarization method based on the antibody. Therefore, the concentrations of NaCl and Tris in the reaction buffer, the amounts of pH and NADPH used, Δ mP and IC, were further investigated₅₀Optimizing reaction conditions, selecting IC₅₀The condition corresponding to the lowest Δ mP being greater than 80 is the optimum condition.

4.1 optimization of NaCl concentration

Fixing the concentration and pH value of Tris and NADPH in buffer solution, configuring the concentration of NaCl to be 0, 50, 100, 200, 400 and 800mM, establishing a standard curve by using TMP as a standard substance, and calculating IC₅₀And Δ mP.

The concentration of NaCl in the buffer was adjusted to 0, 50, 100, 200, 400 and 800mM, respectively, and the results showed that the sensitivity of the receptor DHFR was the highest and the IC was the highest when the concentration of NaCl was 100mM₅₀13.8 mug/L and Δ mP meets the requirement.

4.2 optimization of Tris concentration

Method for immobilizing buffers NaCl, NADPHConcentration and pH value, configuring the concentration of Tris to be 25, 50, 75, 100, 125, 150, 175 and 200mM, establishing a standard curve by taking TMP as a standard substance, and calculating IC₅₀And Δ mP. The results show that the IC of the receptor DHFR is at a Tris concentration of 125mM₅₀Minimum, 12.0 μ g/L,. DELTA.mP 96;

4.3 optimization of buffer pH

Fixing the concentrations of NaCl, Tris and NADPH in the buffer, adjusting the pH of the buffer to 6, 7, 7.5, 8, 8.5 and 9, establishing a calibration curve using TMP as a standard, and calculating IC₅₀And Δ mP. The results show that the sensitivity of the receptor DHFR is highest at pH 7, IC₅₀The concentration is 13.8 mu g/L, and the delta mP meets the screening requirement.

4.4 optimization of NADPH concentration

Fixing buffer NaCl, Tris and pH value, adjusting NADPH concentration to 0, 25, 50, 100, 200 and 400 μ M, establishing standard curve with TMP as standard, and calculating IC₅₀And Δ mP.

The amounts of NADPH used were adjusted to 0, 25, 50, 100, 200 and 400. mu.M, respectively, and the results showed that the sensitivity of the DHFR receptor protein was the highest and the IC was the highest when the amount of NADPH was 100. mu.M₅₀The concentration is 14.8 mu g/L, and the delta mP meets the screening requirement.

Finally, the optimized staphylococcus aureus-DHFR buffer solution is 25mM 2- (N-morpholine) ethanesulfonic acid MES, 125mM Tris, 25mM ethanolamine, 100mM NaCl and 100 mu M NADPH, and a standard curve and IC are established after optimization₅₀The value is reduced and the sensitivity is improved.

5. Organic solvent to detection sensitivity (IC)₅₀) And detecting the effect of the signal window (Δ mP)

Adding methanol and acetonitrile with final concentration of 0%, 2.5%, 5%, 10%, 20% and 40% respectively into buffer solution, diluting TMP standard substance, fluorescent marker and receptor protein, establishing standard curve, and studying organic solvent pair IC₅₀The effect of the value and Δ mP was used to examine the tolerance of the protein to organic solvents.

The methanol concentrations were adjusted to 0, 2.5, 5, 10, 20 and 40%, respectively, and the results showed that both the protein sensitivity and Δ mP decreased with increasing methanol concentration, and Δ mP decreased by half at 20% methanol concentration and almost no binding at 40%.

The acetonitrile concentrations were adjusted to 0, 2.5, 5, 10 and 20%, respectively, and the results showed that both protein sensitivity and Δ mP decreased with increasing acetonitrile concentration, with little binding at 20%.

6. Establishment of optimized standard curve

Using the optimized conditions described above (NaCl concentration 100mM, Tris concentration 125mM, buffer pH 7, NADPH concentration 100. mu.M), a TMP standard curve was constructed, as shown in FIG. 6, along which IC is the curve₅₀The value was 14.9. mu.g/L, the LOD value was 2.9. mu.g/L, IC₂₀～IC₈₀5.3 to 41.5 μ g/L.

7. Determination of the Cross-reactivity

After the FPIA method is optimized, the competitive inhibition curves of 5 sulfanilamide synergist medicines are detected under the same condition, and the corresponding IC of each medicine is calculated₅₀. IC corresponding to TMP₅₀The value was 100%, and the cross-reactivity (CR) was calculated by the following formula.

CR(％)＝(IC₅₀ ofTMP/IC₅₀ oftested compound)×100％

FIG. 7 is a cross-curve of five sulfanilamide synergists based on the staphylococcus aureus-DHFR sulfanilamide synergist drug multi-residue fluorescence polarization detection method of the present invention.

According to the established standard curve of the sulfanilamide synergist medicaments, the medicament IC is calculated₅₀Working concentration (IC)₂₀～IC₈₀) And cross-reactivity, as shown in the table below. IC of 5 sulfanilamide synergist medicines₅₀The values were all below 50. mu.g/L, with the sensitivity of DVD being slightly worse. IC of five medicines of TMP, DVD, OMP, BDP and BQP₅₀The values are respectively 14.9, 38.7, 18.6, 16.0 and 20.2 mu g/L, and the detection limit is 1.3-6.2 mu g/L.

The parameters of the standard curve of 5 sulfanilamide synergist drugs established based on the receptor DHFR of the invention are shown in the following table:

TABLE 2.3 Staphylococcus aureus-DHFR based drug cross-reactivity rates

Example III application of DHFR sulfonamide synergist-based medicine multi-residue fluorescence polarization detection method in milk

1. Milk sample pretreatment

The inventor finds that the matrix effect can be eliminated by extracting with 55% saturated ammonium sulfate and diluting by 5 times.

4mL of pure milk and skimmed milk and 4mL of 55% saturated ammonium sulfate solution are respectively put into a 10mL centrifuge tube, vortexed for 2min, ultrasonically treated for 5min, centrifuged at 4 ℃ and 8000rpm for 10min, supernate is extracted, 2mL of the supernate is mixed with 8mL of reaction buffer solution, and the FP value is detected.

2. FPIA (fluorescence immunoassay) method for detecting sulfonamide synergist based on DHFR (dihydrofolate reductase)

Five ASG_SThe calibration curves in pure and skim milk are shown in fig. 8, and the specific test results are shown in tables 3.1 and 3.2. Five ASG in pure milk_SThe detection limit of (2) is 10.1-40.7 mug/L, and the detection range is 22.7-832.6 mug/L. Five ASG in skimmed milk_SThe detection limit of (2) is 10.2-32.9 μ g/L, and the detection range is 23.4-949.5 μ g/L. Five ASG_SThe detection limit in both pure milk and skim milk was below the residual standard.

The parameters of the spiking curves of the five sulfonamide potentiators in pure and skim milk are shown in the table below.

TABLE 3.1 spiking Curve parameters of five sulfonamide boosters in pure milk samples

TABLE 3.2 spiking Curve parameters of five sulfonamide potentiators in skim milk samples

3. Addition recovery experiment

In pure milk and defatted cattle respectivelyTMP, DVD, OMP, BDP and BQP were added to the milk samples. According to the standard curve of the five medicines, the IC in pure milk and skim milk samples₂₀、IC₅₀、IC₈₀Additions, 5 replicates per sample, were made 3 times per addition. FP values were measured and recovery was calculated.

The recovery rate (measured concentration/added concentration) × 100%

The average addition recovery is shown in table 3.3. The adding recovery rate of the pure milk is 73.9-98.9%, the variation coefficient is less than 14.6%, the adding recovery rate of the skim milk is 75.2-106.3%, and the variation coefficient is less than 14.4%.

The addition recovery rate is the ratio of the detected amount to the added amount after a sample is treated by a certain method, and is one of the standards for judging whether the test method is accurate and reliable. The high recovery rate indicates that the reliability of the test method is high, and the recovery rate range of the general quantitative method is 70-120%. In the test, the sulfonamide synergist is added into a blank milk sample, the FPIA recovery rate is evaluated, and the result shows that the addition recovery rate and the variation coefficient both meet the requirement of rapid detection.

Table 3.3 percent recovery of the five sulfonamide potentiators in milk (n ═ 3)

Sequence listing

<110> Qingdao agricultural university

<120> DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergists

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<170> SIPOSequenceListing 1.0

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<213> Artificial sequence (Artificial sequence)

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<210> 2

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<212> DNA

<213> Artificial sequence (Artificial sequence)

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gtggtggtgg tggtgctcga gttagacttc ctttctcttg cggta 45

Claims

1. A multi-residue fluorescence polarization immunoassay method of sulfanilamide synergist drugs based on DHFR is characterized in that the method is established based on dihydrofolate reductase DHFR; the dihydrofolate reductase DHFR is obtained by inducing a dihydrofolate reductase recombinant expression strain.

2. The method according to claim 1, wherein the dihydrofolate reductase is obtained by inducing the dihydrofolate reductase recombinant expression strain as follows: extracting sulfanilamide synergist drug sensitive strain genes, and performing PCR amplification to obtain dihydrofolate reductase genes; constructing a recombinant clone strain, and further obtaining a recombinant expression strain for efficiently expressing dihydrofolate reductase; and finally inducing the recombinant expression strain to express to obtain the recombinant dihydrofolate reductase.

3. The method of claim 2, wherein the sulfonamide-potentiator drug-sensitive strains comprise staphylococcus aureus, streptococcus pneumoniae, mycobacterium tuberculosis; the conditions for expressing the recombinant dihydrofolate reductase by the induced recombinant expression strain are that the temperature is 20 ℃ and the induction is carried out for 13-16 h by 0.5mM IPTG.

4. The method of claim 1, comprising the steps of:

5. The method of claim 4, wherein the fluorescent marker is an amide conjugate of a sulfonamide potentiator hapten and fluorescein isothiocyanate.

6. The method according to claim 4, wherein the buffer used in the method is MTEN buffer containing 25mM MES, 125mM Tris, 25mM ethanolamine, 100mM NaCl and 100. mu.M NADPH.

7. The method of claim 4, wherein the standard curve is plotted by using Origin 8.5 software, fitting the curve with a four-parameter equation model, using FP as ordinate and logarithm of standard concentration as abscissa, and establishing the curve; the standard curve IC₅₀The value was 14.9. mu.g/L, the LOD value was 2.9. mu.g/L, IC₂₀～IC₈₀5.3 to 41.5 μ g/L.

8. The method as claimed in claim 4, wherein when the sample to be tested is milk, the sample is pretreated with 55% saturated ammonium sulfate solution.

9. A fluorescence polarization immunoassay kit for detecting sulfanilamide synergist drugs is characterized by comprising a fluorescent marker solution and DHFR derived from sulfanilamide synergist drug sensitive strains.

10. The kit of claim 9, wherein the fluorescent marker is a conjugate of TMP hapten and BDF; the DHFR is recombinant staphylococcus aureus DHFR.