CN113957067B - TetR protein steric hindrance and gene shearing technology-based tetracycline drug detection method - Google Patents

TetR protein steric hindrance and gene shearing technology-based tetracycline drug detection method Download PDF

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CN113957067B
CN113957067B CN202111189870.4A CN202111189870A CN113957067B CN 113957067 B CN113957067 B CN 113957067B CN 202111189870 A CN202111189870 A CN 202111189870A CN 113957067 B CN113957067 B CN 113957067B
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CN113957067A (en
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王战辉
沈建忠
于雪芝
温凯
江海洋
余文博
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China Agricultural University
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Abstract

The application discloses a tetracycline drug detection method based on TetR protein steric hindrance and gene shearing technology, a double-stranded DNA molecule for detecting the tetracycline drug, a composition, a kit and application thereof. The double-stranded DNA molecule comprises a tetracycline manipulation gene tetO and a restriction endonuclease recognition site, wherein one end of the double-stranded DNA molecule is connected with a fluorescent group, and the other end of the double-stranded DNA molecule is connected with biotin. The double-stranded DNA molecule can realize the detection of tetracycline drugs.

Description

TetR protein steric hindrance and gene shearing technology-based tetracycline drug detection method
Technical Field
The application relates to the technical field of biology, in particular to a tetracycline drug detection method based on TetR protein steric hindrance and gene shearing technology.
Background
The tetracycline medicine is a broad-spectrum antibiotic, has wide application in livestock breeding industry, and can effectively improve the utilization rate of feed, promote growth and prevent and treat diseases. However, the medicine is used in a large amount, abused or even forbidden, which can cause the medicine to remain in animal-derived foods and environments and harm human health, and the efficient, rapid, sensitive and multi-target detection of the medicine is an important means for guaranteeing the food safety.
The currently reported methods for detecting tetracycline drug residues mainly comprise a microbiological method, a spectroscopic method, a chromatographic method, a liquid chromatography-mass spectrometry method, a capillary electrophoresis method, an immunological method and the like. The high performance liquid chromatography and the liquid chromatography have higher sensitivity and specificity, but have more complex operation, require large-scale precise instruments and trained operators, and have long detection time and high detection cost. The enzyme-linked immunosorbent assay (ELISA) combines the antigen-antibody binding reaction and the amplification effect of enzyme labeling, and has the characteristics of simple operation, high sensitivity, strong specificity and suitability for rapid screening of a large number of samples. However, because of the large structural difference of tetracyclines, it is difficult to prepare antibodies capable of recognizing various tetracyclines, and the broad-spectrum property or sensitivity is not required. Therefore, a novel biological identification material is necessary to be prepared, and all tetracycline drugs can be identified with high sensitivity.
The tetracycline repressor TetR can uniformly identify tetracycline drugs with high affinity, and is an ideal biological identification material. Analysis of the three-dimensional structure of TetR it was found that TetR homodimers consisted of two identical monomers, the N-terminus of each of which was divided into two DNA domains and a regulatory core structure, involved in dimerization and ligand binding. Thus, the tetracycline repressor TetR has DNA binding properties, and when magnesium complexed tetracycline binds to TetR, the spatial conformation of the tetracycline repressor TetR changes, disassociating from the DNA (tetO). ELISA methods based on TetR and tetO have been reported, but the traditional ELISA methods need multi-step incubation, and catalytic substrates need to generate detection signals, so that the operation is complex, and popularization and application are not facilitated, and therefore, development of a new detection technology is urgently needed.
Disclosure of Invention
The present application provides a double-stranded DNA molecule comprising a tetracycline manipulation gene tetO and a restriction endonuclease recognition site, said double-stranded DNA molecule having a fluorescent group attached at one end and Biotin (Biotin) attached at the other end.
The double stranded DNA molecule was designated tetO-X.
Alternatively, according to the double-stranded DNA molecule described above, one end of one strand of the double-stranded DNA molecule is linked to a fluorescent group, and one end of the other strand is linked to biotin. For example, one strand of the double-stranded DNA molecule is linked at its 3 'end to a fluorophore and the other strand is linked at its 3' end to biotin, the biotin and the fluorophore being located at both ends of the double-stranded DNA molecule.
The restriction endonuclease may be QuickCut TM All restriction enzymes such as EcoR I; the fluorescent group may be any fluorescent group such as TAMRA.
Alternatively, according to the double-stranded DNA molecule described above, the sequence of the tetracycline manipulation gene tetO is shown at positions 22-40 in SEQ ID No. 1.
Alternatively, according to the double-stranded DNA molecule described above, one strand sequence of the double-stranded DNA molecule is shown as SEQ ID No.1, and the other strand sequence of the double-stranded DNA molecule is shown as SEQ ID No. 2.
The double-stranded DNA molecule may be a double-stranded DNA molecule obtained by complementary hybridization of tetOF with TAMRA attached at the 3 'and biotin attached at the 3' when the single strand shown by SEQ ID No.1 is designated as tetOF and the single strand shown by SEQ ID No.2 is designated as tetOR.
The present application also provides a composition for detecting tetracycline and/or derivatives thereof, the composition comprising the double stranded DNA molecule described above and at least one of the following: tetracycline repressor (TetR), the restriction endonuclease and a streptavidin-coated solid support, the double stranded DNA molecule being immobilized on the streptavidin-coated solid support. The amino acid sequence of the tetracycline repressor protein may be as shown in SEQ ID No. 3.
A large number of materials are available as the solid support, such as polystyrene, cellulose, polyacrylamide, polyethylene, polypropylene, cross-linked dextran, glass, silicone rubber, agarose gel, and the like. The solid phase carrier may be in the form of a test tube, microplate well, bead, wafer, etc.
The principle of the composition for detecting the tetracycline and/or the derivatives thereof is as follows: the tetracycline repressor TetR specifically binds to the tetracycline operator tetO; restriction endonucleases can specifically cleave target gene sequences. tetO-X was immobilized to a solid support via the streptavidin-biotin system. When the TetR and restriction endonuclease are close in distance, only one protein binds to tetO-X due to steric hindrance, and thus the TetR and restriction endonuclease competitively bind to tetO-X. When the tetracycline drug exists in the sample, the drug is combined with the TetR to cause the conformational change of the protein, so that the TetR is separated from tetO-X, the steric hindrance is relieved, and at the moment, the restriction endonuclease can be combined with tetO-X to generate gene cleavage, so that the fluorescent signal at one end of tetO-X is reduced. The content of the tetracycline drugs and/or the tetracycline drugs in the sample can be characterized by utilizing the intensity of the fluorescence signal.
The application also provides a kit for detecting tetracycline and/or derivatives thereof, comprising the composition described above and tetracycline or derivatives thereof as a standard.
The use of the double-stranded DNA molecules described above or the compositions described above or the kits described above is also within the scope of the application. The application may specifically be an application in at least one of:
1) Use in the detection or co-detection of tetracycline or a derivative thereof;
2) Use in the manufacture of a product for detecting or aiding in the detection of tetracycline or a derivative thereof;
3) The application in detecting or assisting in detecting the content of tetracycline or derivatives thereof;
4) The application of the method in preparing products for detecting or assisting in detecting the content of the tetracycline or the derivatives thereof.
Optionally, according to the above application, said detecting or aiding in the detection of tetracycline or derivatives and/or amounts thereof comprises mixing the above-mentioned double stranded DNA molecule, the tetracycline repressor protein, the restriction endonuclease and the test sample, and performing a fluorescent detection after washing. For example, it may comprise diluting 10. Mu.M tetO-X with water at 1:2500, adding to streptavidin-coated ELISA plates, incubating for 1h, and washing with PBST 3 times. TetR and QuickCut diluted with 10Xbuffer were added simultaneously TM EcoR I, tetR dilution 1:2700, quickCu TM EcoR I was added in an amount of 0.8. Mu.L. And simultaneously adding the sample to be tested. After 1 hour of room temperature treatment, PBST was washed 3 times for fluorescence detection.
Alternatively, according to the above application, the product is a kit.
In the above, the derivative of tetracycline may be a tetracycline, for example, doxycycline, aureomycin, minocycline, demeclocycline, meclocycline, oxytetracycline, rolicycline.
Experiments prove that after the fluorescence value of tetO-X is stable and successfully fixed on a streptavidin-coated microplate, the fluorescence value when the restriction endonuclease acts on tetO-X alone and the fluorescence value when the restriction endonuclease and the TetR act on tetO-X together are detected, and the TetR prevents the restriction endonuclease from combining with tetO-X, so that the fluorescence value of tetO-X is kept stable.
The method detects the minimum concentration of the tetracycline drug for preventing the combination of the tetR and the tetO-X, specifically, the tetracycline standard substance is diluted by a multiple ratio, the intensity of a tetO-X fluorescence signal when the tetracycline drug with different concentrations exists is detected, the detected minimum concentration is obtained, and a standard curve is drawn to determine the range.
The double-stranded DNA molecule provided by the application can be used for detecting tetracycline drugs. The embodiment of the application establishes a rapid detection method of the tetracycline, the half inhibition concentration (IC 50) value of the method for the tetracycline is 0.09ng/mL, the linear range is 0.04-0.23ng/mL, the cross reaction rate for other tetracycline is 29-987%, and the multi-residue detection of the tetracycline can be realized.
According to the embodiment of the application, the tetracycline repressor TetR and tetracycline control gene tetO combination property, the restriction endonuclease specificity shear target sequence and the steric hindrance between proteins are utilized, the content of the tetracycline drugs is detected through the intensity of fluorescent signals, the operation steps and the reaction time of the conventional ELSIA are reduced, and the detection method with simple operation and high sensitivity is obtained.
Drawings
FIG. 1 is a tetracycline standard curve for example 2.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Data were processed using Origin 95 statistical software and experimental results were expressed as averages.
QuickCut used in the examples below TM EcoR I (cat. No. 1611), available from Takara doctor materials technology (Beijing) Inc. (Takara China), including 10Xbuffer.
TetR is obtained by artificial synthesis, and the amino acid sequence of the TetR is shown as SEQ ID No.3, and the concentration is 2.5mg/kg.
Example 1 competitive binding of tetR and restriction endonuclease to tetO-X
1. Preparation of double-stranded DNA molecule tetO-X for detection of tetracycline and its derivatives
A DNA fragment (tetO-X) containing the tetracycline operator tetO and restriction endonuclease recognition site was designed and synthesized, with biotin coupled to the 3 'end of one strand and fluorescein coupled to the 3' end of the other strand. The two strands are complementarily hybridized into a section of double-stranded DNA for standby in vitro.
the sequences of the two chains of tetO-X are as follows:
tetOF：5’-GTTATTTTACCACGGAATTCCTCCCTATCAGTGATAGAGAAA-3’-TAMRA(SEQ ID No.1)
tetOR：5’-TTTCTCTATCACTGATAGGGAGGAATTCCGTGGTAAAATAACTTTTTTTTTTTTTT-3’-biotin(SEQ ID No.2)。
wherein the sequence of the tetracycline manipulation gene tetO is shown as 22-40 bits in SEQ ID No.1, and the sequence of the recognition site of the restriction endonuclease EcoRI is 5' -)GAATTC-3’。
Mixing the two chains at a ratio of 1:1, heating at 95deg.C for 5min, and cooling to room temperature. Specific procedures are shown in Table 1, with single-stranded tetOF 22.6. Mu.l added water to 226. Mu.l designated tube 1, and single-stranded tetOR18.9. Mu.l added water to 189. Mu.l designated tube 2; taking 189 mu l of each of the tube 1 and tube 2 reagents, and uniformly mixing to obtain a double-strand pre-hybridization solution; heating the solution before double-strand hybridization at 95 ℃ for 5min in a constant-temperature metal bath to obtain a solution after double-strand hybridization; the solution after double-strand hybridization was gradually cooled to room temperature to obtain a double-strand DNA molecule tetO-X (concentration: 10. Mu.M) for detection of tetracycline and its derivatives.
2. Mu.l of the solution before double-strand hybridization and 10. Mu.l of water were added to each of the two-strand DNA molecule tetO-X, and the mixture was mixed uniformly to perform fluorescence detection. The blank is single-stranded tetOR.
TABLE 1 tetO-X double-strand hybridization method
The fluorescence detection results are shown in Table 2, wherein the fluorescence value before hybridization is the fluorescence detection result of the solution before double-strand hybridization, the fluorescence value after hybridization is the fluorescence detection result of the double-strand DNA molecule tetO-X, 1 and 2 respectively represent two parallel tests, A, B, C and D respectively represent the fluorescence values of the solution before double-strand hybridization or the tetO-X under different dilution factors. The detection result proves that the heating at 95 ℃ for 5min has no influence on the fluorescence value of the fluorescein connected with the DNA fragment.
TABLE 2 influence of 5min at 95℃on the tetO-X fluorescence values
tetR and restriction endonuclease competitively bind to tetO-X
In the absence of TetR, there is no steric hindrance between proteins
(1) The experiments were divided into A1-H18 groups, wherein A1 and B1 were diluted 20 times with water, C1 and D1 were diluted 100 times with water, E1 and F1 were diluted 500 times with water, respectively, and G1 and H1 were diluted 2500 times with water, respectively, and diluted tetO-X was added to the streptavidin-coated enzyme-labeled plate in an amount of 100. Mu.l/well. After incubation at room temperature for 1 hour, PBST was washed 3 times for fluorescence detection.
(2) Adding 1. Mu.l/well of restriction enzyme and 100. Mu.l/well of buffer solution to the experimental group treated in the step (1), wherein the restriction endonuclease is QuickCut TM EcoR I, buffer solution is QuickCut TM 10Xbuffer dilution of EcoR I was obtained. After 5min of treatment at 37℃PBST was washed 3 times for fluorescence detection.
The blank is single-stranded tetOR.
See table 3 for specific treatment methods and fluorescence detection results. Experiments prove that the restriction endonuclease can be combined with tetO-X, specifically recognizes the cleavage site, and then cuts the tetO-X, so that the fluorescence value is reduced.
TABLE 3 variation of fluorescence values in the absence of steric hindrance
When TetR is added first, tetR binds to a specific site on tetO-X first and forms a steric hindrance with the restriction endonuclease
(1) The experiments were divided into A1-H18 groups, wherein A1 and B1 were diluted 20 times with water, C1 and D1 were diluted 100 times with water, respectively, E1 and F1 were diluted 500 times with water, respectively, and G1 and H1 were diluted 2500 times with water, respectively, and diluted tetO-X was added to the streptavidin-coated ELISA plate in an amount of 100. Mu.l/well. After incubation at room temperature for 1 hour, PBST was washed 3 times for fluorescence detection.
(2) To each of the test groups A1, C1, E1 and G1 treated in step (1), tetR diluted 1000-fold with water was added, and to each of the test groups B1, D1, F1 and H1 treated in step (1), PBS (solvent) having a pH of 7.4 at 10mM was added. The amount of the reagent added was 100. Mu.l/well, and after 1 hour of room temperature treatment, PBST was washed 3 times for fluorescence detection.
(3) Respectively to the step (2)The treated experimental groups A1, C1, E1 and G1 were added with QuickCut TM EcoR I1. Mu.l/well and buffer 100. Mu.l/well were obtained by 10-fold dilution with 10Xbuffer, and 10mM PBS (solvent) at pH 7.4 was added to each of the experimental groups B1, D1, F1, H1 treated in step (2) at 100. Mu.l/well. After 5min of treatment at 37℃PBST was washed 3 times for fluorescence detection.
See table 4 for specific treatment methods and fluorescence detection results. Experiments have shown that when TetR is added first, it binds to a specific site on tetO-X first, forming a steric hindrance with the restriction endonuclease. When the restriction endonuclease is added, the restriction endonuclease is prevented from combining with tetO-X, a specific enzyme cutting site cannot be cut, and the fluorescence value is unchanged.
TABLE 4 variation of fluorescence values in the presence of steric hindrance
In the table, + is added, -is not added.
Example 2 detection of Tetracycline and derivatives thereof Using tetO-X
1. Determination of optimal working concentrations of TetR and EcoRI
The tetO-X prepared in example 1 was diluted 1:2500 in water, added to a streptavidin-coated black ELISA plate, incubated for 1h, and PBST washed 3 times. Simultaneously adding 100 μl/well of TetR solution diluted with 10xbuffer series and QuickCut respectively TM EcoRI solution, ELISA plate left to right, tetR dilutions were: 1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24300. ELISA plate from top to bottom, quickCut TM EcoR I was added in amounts of 0.2. Mu.L, 0.4. Mu.L, 0.6. Mu.L, 0.8. Mu.L, 1.0. Mu.L, 1.2. Mu.L, 1.4. Mu.L, and 1.6. Mu.L, respectively. While 10ng/mL tetracycline was added. After 1 hour of room temperature treatment, PBST was washed 3 times for fluorescence detection.
Chessboard method for determining TetR and QuickCut TM EcoR I was diluted at an optimal working concentration of TetR 1:2700 fold, quickCut TM EcoR I was added in an amount of 0.8. Mu.L/well.
2. Drawing a tetracycline standard curve
The tetO-X prepared in example 1 was diluted 1:2500 in water and incubated in a streptavidin-coated black ELISA plate for 1h and washed 3 times with PBST. 100 μl/well of TetR solution diluted with 10Xbuffer and QuickCut were added simultaneously TM EcoRI solution, tetR dilution 1:2700, ecoRI was added in an amount of 0.8. Mu.L. At the same time, add the concentration of 9.72ng/mL, 3.24ng/mL, 1.08ng/mL, 0.36ng/mL, 0.12ng/mL, 0.04ng/mL, 0.013ng/mL tetracycline 100 u l/hole. After 1 hour of room temperature treatment, PBST was washed 3 times for fluorescence detection.
The standard curve of tetracycline drawn according to the experimental results is shown in FIG. 1, IC 50 The value is 0.09ng/mL, and the linear range is 0.04-0.23ng/mL.
3. Detection of tetracyclines
The tetO-X prepared in example 1 was diluted 1:2500 in water and incubated in a streptavidin-coated black ELISA plate for 1h and washed 3 times with PBST. TetR and QuickCut diluted with 10Xbuffer were added simultaneously TM EcoR I, tetR dilution 1:2700, quickCut TM EcoR I was added in an amount of 0.8. Mu.L. Meanwhile, tetracyclines (tetracycline, doxycycline, aureomycin, minocycline, demeclocycline, meclocycline, oxytetracycline and rolicycline) are respectively added. After 1 hour of room temperature treatment, PBST was washed 3 times for fluorescence detection.
Tetracyclines IC obtained according to experimental results 50 Values and cross-reactivity are shown in table 5. The calculation formula of the cross-reaction rate is tetracycline cross-reaction rate=tetracycline IC 50 Tetracycline IC 50 ×100％。
TABLE 5 Cross-reactivity Table
Drug name IC 50 Value (ng/mL) Cross reaction Rate (%)
Doxycycline 0.01 987
Aureomycin 0.04 214
Minocycline 0.06 159
Tetracycline 0.09 100
Dimei cyclic extract 0.29 31
Mechlorethamine 0.11 83
Terramycin 0.20 45
Rollerocycline 0.31 29
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Sequence listing
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<120> TetR protein steric hindrance and gene shearing technology-based tetracycline drug detection method
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His Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu
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Gly Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu
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Thr Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu
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Glu Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly
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Cys Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu
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Thr Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu
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Leu Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu
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Leu Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser
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Claims (6)

1. A double-stranded DNA molecule characterized by: the double-stranded DNA molecule comprises a tetracycline manipulation gene tetO and a restriction endonuclease recognition site, wherein the 3 'end of one strand of the double-stranded DNA molecule is connected with a fluorescent group, the 3' end of the other strand of the double-stranded DNA molecule is connected with biotin, and the biotin and the fluorescent group are positioned at two ends of the double-stranded DNA molecule;
one strand sequence of the double-stranded DNA molecule is shown as SEQ ID No.1, and the other strand sequence of the double-stranded DNA molecule is shown as SEQ ID No. 2.
2. A composition for detecting tetracyclines, characterized in that: the composition comprises the double stranded DNA molecule of claim 1 and the restriction endonuclease, tetracycline repressor, and streptavidin-coated solid support, the double stranded DNA molecule being immobilized on the streptavidin-coated solid support.
3. A kit for detecting tetracycline, its characterized in that: the kit comprises the composition of claim 2 and a tetracycline as a standard.
4. The application is characterized in that: the use of the double stranded DNA molecule of claim 1 or the composition of claim 2 or the kit of claim 3 in at least one of:
1) The application in the detection or auxiliary detection of the non-disease diagnosis and treatment purpose in the tetracycline drugs;
2) The application in preparing products for detecting or assisting in detecting tetracycline drugs;
3) The application in the detection or auxiliary detection of the tetracycline drug content for the purpose of non-disease diagnosis and treatment;
4) The application of the method in preparing products for detecting or assisting in detecting the content of the tetracycline drugs.
5. The use according to claim 4, characterized in that: wherein in said 1) or 3), said use comprises mixing said double stranded DNA molecule of claim 1 with said restriction endonuclease, tetracycline repressor, and test sample, and washing followed by fluorescence detection.
6. The use according to claim 5, characterized in that: the product is a kit.
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CN101059518A (en) * 2007-05-15 2007-10-24 东南大学 DNA binding protein detection method based on moving endonuclease
CN102680711A (en) * 2012-05-25 2012-09-19 北京维德维康生物技术有限公司 Application of protein TetR combined with tetracycline in tetracycline antibiotic detection
CN102690338A (en) * 2012-05-25 2012-09-26 中国农业大学 Protein TetR combinable with tetracycline and coding genes and applications of protein TetR combinable with tetracycline

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