CN112321724A - Rapid detection method for detecting glyphosate based on EPSP synthase and GFP fusion protein - Google Patents

Rapid detection method for detecting glyphosate based on EPSP synthase and GFP fusion protein Download PDF

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CN112321724A
CN112321724A CN202011410606.4A CN202011410606A CN112321724A CN 112321724 A CN112321724 A CN 112321724A CN 202011410606 A CN202011410606 A CN 202011410606A CN 112321724 A CN112321724 A CN 112321724A
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glyphosate
fusion protein
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epsp synthase
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CN112321724B (en
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王淼
王静
佘永新
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Institute of Agricultural Quality Standards and Testing Technology for Agro Products of CAAS
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    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1092—3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract

The invention discloses a rapid detection method for detecting glyphosate based on EPSP synthase and GFP fusion protein, which comprises that the cloned glyphosate main target EPSP synthase is used as the receptor protein of glyphosate; the receptor protein and the green fluorescent protein are fused to construct a new protein EPSPS-GFP which contains a glyphosate binding site and is also provided with a fluorescent label, and the new protein is subjected to prokaryotic expression and purification in escherichia coli and is used for detecting glyphosate in water. The invention adopts a fluorescence photometer to detect the change of the fluorescence response value by constructing the fusion protein, establishes a standard correction curve and realizes the qualitative and quantitative detection of the glyphosate herbicide.

Description

Rapid detection method for detecting glyphosate based on EPSP synthase and GFP fusion protein
Technical Field
The invention relates to the field of glyphosate detection and extraction, in particular to a rapid detection method for detecting glyphosate based on EPSP synthase and GFP fusion protein.
Background
Glyphosate, english name glyphosate, chemical name N-phosphomethylglycine, is an systemic conductive type broad-spectrum biocidal herbicide, and the action mechanism of the herbicide is mainly to inhibit enol pyruvyl shikimate phosphate synthase (EPSP synthase) in plants, so as to inhibit the conversion of shikimate to phenylalanine, tyrosine and tryptophan, so that the protein synthesis is interfered, and the plants die. The glyphosate is the pesticide variety with the widest application and the largest yield in the world, and the annual sale amount is higher than that of the pesticide. The Maximum Residual Limit (MRL) of the glyphosate in the drinking water is set by countries in the world, and the MRL of the glyphosate in the drinking water is set to be 700 mug/L by the American Environmental Protection Association (EPA); the maximum acceptable limit (MAC) of glyphosate in the Canada specified drinking water is 280 mug/L; the limit of the glyphosate in drinking water is regulated to 700 mug/L in the sanitary Standard for Drinking Water (GB5749-2006) of China.
Because the glyphosate has higher polarity, is difficult to dissolve in an organic solvent and lacks functional groups which can be used for detection, derivatization is needed for detection by adopting chromatographic methods such as liquid phase, gas phase and the like; the ion chromatography interference factors are more, and the influence on the detection result is serious; the immunoassay methods such as ELISA and colloidal gold detection are limited by antibodies, and the stability and sensitivity are not high. Therefore, it is important to develop a rapid glyphosate detection method with simple operation, low cost, high sensitivity and good specificity. Therefore, a rapid detection method for detecting glyphosate based on EPSP synthase and GFP fusion protein is needed to effectively solve the above problems.
Disclosure of Invention
The invention aims to provide a rapid detection method for detecting glyphosate based on EPSP synthase and GFP fusion protein.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention comprises the following steps:
the glyphosate main target EPSP synthase of A clone is used as the receptor protein of glyphosate;
b, fusing the receptor protein and the green fluorescent protein to obtain a fusion protein;
and C, carrying out prokaryotic expression and purification on the fusion protein in escherichia coli, and using the fusion protein for detecting the glyphosate in water.
Further, the fusion method of the receptor protein and the green fluorescent protein comprises the following steps:
cloning an enolpyruvylshikimate phosphate synthetase (EPSPS) gene from tobacco, synthesizing a cDNA sequence of the gene after sequencing, and connecting the cDNA sequence into a prokaryotic expression vector pET-28a-eGFP capable of expressing enhanced green fluorescent protein (eGFP) by adopting a homologous recombination method to enable the EPSPS and the eGFP to form fusion protein so as to obtain a new recombinant expression vector.
b, introducing the plasmid vector into an escherichia coli expression strain BL21 by adopting a heat shock transformation method, shaking the strain on a shaker at 37 ℃ and 180rpm for 2h, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, and continuing shaking the strain on the shaker for culturing for 16 h.
Centrifuging at C12000 r for 2min, discarding supernatant, collecting thallus, resuspending with PBS (50mM, pH7.5) buffer solution, ultrasonic breaking bacteria under working condition for 2s, intermittent for 8s, and total time of 30min, centrifuging to obtain supernatant, and collecting crude protein.
Further, the prokaryotic expression purification conditions are as follows:
and a, carrying out affinity chromatography by using a His label on the fusion protein and adopting a Ni column to obtain the purified fusion protein.
And b, centrifuging again for 30min under the condition of 12000r, and collecting thalli. The pellet was washed 3 times with washing solution (20mM Tris, 1mM EDTA, 2M urea, 1M NaCl, 1% Triton X-100, pH 8.0). Dissolving the precipitate with dissolving buffer solution (20mM Tris, 5mM DTT, 8M urea pH8.0) at a certain ratio, and standing at 4 deg.C overnight; centrifuge at 15000rpm for 15min at room temperature. Then, the solution was added dropwise to 20mM Tris-HCl, 100mM NaCl, pH8.0 buffer solution, gradually diluted in a stepwise gradient with slow stirring, and the protein solution was dialyzed overnight in 20mM Tris-HCl, 100mM NaCl, pH8.0 solution in a dialysis bag.
Further, the detection condition of glyphosate detection
Under the condition of room temperature (about 25 ℃), 10 mu L of 20 mu g/mL recombinant protein working solution is added into each hole of a 96-hole black enzyme label plate, then 200 mu L of glyphosate standard solution containing 0.2 mu g/mL-100 mu g/mL is respectively added, and detection is carried out under a fluorescence enzyme label instrument at the excitation wavelength of 480nm and the emission wavelength of 515 nm. The glyphosate concentration is used as the abscissa and the fluorescence response value is used as the ordinate, and a standard quantitative calibration curve is prepared and is shown in figure 2.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts a fluorescence photometer to detect the change of the fluorescence response value by constructing the fusion protein, establishes a standard correction curve and realizes the qualitative and quantitative detection of the glyphosate herbicide.
Drawings
FIG. 1 collection of electrophoresis identification chart of purified protein
FIG. 2 is a graph of a standard glyphosate detection calibration curve based on fluorescent recombinant protein according to the present invention;
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be in a manner including, but not limited to, the following examples.
As shown in fig. 1, the present invention comprises the steps of:
the glyphosate main target EPSP synthase of A clone is used as the receptor protein of glyphosate;
b, fusing the receptor protein and the green fluorescent protein to obtain a fusion protein;
construction and expression of the fusion protein:
cloning an enolpyruvylshikimate phosphate synthetase (EPSPS) gene from tobacco, synthesizing a cDNA sequence of the gene after sequencing, connecting the cDNA sequence into a prokaryotic expression vector pET-28a-eGFP capable of expressing enhanced green fluorescent protein (eGFP) by adopting a homologous recombination method, and forming a fusion protein by the EPSPS and the eGFP to obtain a new recombinant expression vector.
The plasmid vector is introduced into an escherichia coli expression strain BL21 by adopting a heat shock transformation method, the strain is shaken at 180rpm on a shaker at 37 ℃ for 2h, IPTG is added for induction expression, and the strain is continuously shaken on the shaker for culturing for 16 h.
Centrifuging at C12000 r for 2min, discarding supernatant, collecting thallus, resuspending with PBS (50mM, pH7.5) buffer solution, ultrasonic breaking bacteria under working condition for 2s, intermittent for 8s, and total time of 30min, centrifuging to obtain supernatant, and collecting crude protein.
The prokaryotic expression purification conditions are as follows:
and carrying out affinity chromatography by using a His label on the fusion protein and adopting a Ni column to obtain the purified fusion protein.
Or centrifuging again for 30min under 12000r, and collecting thallus. The pellet was washed 3 times with washing solution (20mM Tris, 1mM EDTA, 2M urea, 1M NaCl, 1% Triton X-100, pH 8.0). Dissolving the precipitate with dissolving buffer solution (20mM Tris, 5mM DTT, 8M urea pH8.0) at a certain ratio, and standing at 4 deg.C overnight; centrifuge at 15000rpm for 15min at room temperature. Then, the solution was added dropwise to 20mM Tris-HCl, 100mM NaCl, pH8.0 buffer solution, gradually diluted in a stepwise gradient with slow stirring, and the protein solution was dialyzed overnight in 20mM Tris-HCl, 100mM NaCl, pH8.0 solution in a dialysis bag.
Detection conditions for detecting glyphosate
Under the condition of room temperature (about 25 ℃), 10 mu L of 20 mu g/mL recombinant protein working solution is added into each hole of a 96-hole black enzyme label plate, then 200 mu L of glyphosate standard solution containing 0 mu g/mL-100 mu g/mL is respectively added, and detection is carried out under a fluorescence enzyme label instrument at the excitation wavelength of 480nm and the emission wavelength of 515 nm. The glyphosate concentration is used as the abscissa and the fluorescence response value is used as the ordinate, and a standard quantitative calibration curve is prepared and is shown in figure 2.
The fusion protein is used for detecting glyphosate: when the detection system does not contain glyphosate, the detection system is characterized by the fluorescent signal of eGFP, and the protein solution shows green fluorescence (Ex 480nm, Em515nm) under the excitation of blue light; when the detection system contains glyphosate, the detection system is characterized by glyphosate binding characteristics of EPSPS, and after the EPSPS is bound with the glyphosate, three-dimensional space conformation changes, so that eGFP conformation connected in series at the rear end is driven to change, and then fluorescence spectrum change of the fusion protein is initiated. As shown in figure 2, the qualitative and quantitative detection of the glyphosate herbicide is realized by adopting a fluorescence photometer to detect the change of the fluorescence response value and establishing a standard correction curve,
in another embodiment, distilled water with 0.7 μ g/mL is added as a simulated sample to be tested, the fluorescence response value is 3220, and the value y is substituted into the calibration curve equation of-30.808 x +3242.1, so that the detection concentration x is 0.714(μ g/mL), the recovery rate is 102%, and the requirement of detection accuracy is met.
The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or changes made within the spirit and scope of the main design of the present invention, which still solve the technical problems consistent with the present invention, should be included in the scope of the present invention.

Claims (4)

1. A rapid detection method for detecting glyphosate based on EPSP synthase and GFP fusion protein is characterized by comprising the following steps:
the glyphosate main target EPSP synthase of A clone is used as the receptor protein of glyphosate;
b, fusing the receptor protein and the green fluorescent protein to obtain a fusion protein;
and C, carrying out prokaryotic expression and purification on the fusion protein in escherichia coli, and using the fusion protein for detecting the glyphosate in water.
2. The method for rapidly detecting glyphosate based on the EPSP synthase and GFP fusion protein as claimed in claim 1, wherein the fusion method for detecting glyphosate based on the EPSP synthase and GFP fusion protein comprises a cloning of an enolpyruvylshikimate phosphate synthase (EPSPS) gene from tobacco, synthesis of a cDNA sequence of the gene after sequencing, and joining into a prokaryotic expression vector pET-28a-eGFP capable of expressing enhanced green fluorescent protein (eGFP) by adopting a homologous recombination method, so that the EPSPS and the eGFP form the fusion protein to obtain a new recombinant expression vector.
b, introducing the plasmid vector into an escherichia coli expression strain BL21 by adopting a heat shock transformation method, shaking the strain on a shaker at 37 ℃ and 180rpm for 2h, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, and continuing shaking the strain on the shaker for culturing for 16 h.
And C, centrifugally collecting the thalli, re-suspending the thalli by using PBS (50mM, pH7.5) buffer solution, then breaking the bacteria by adopting ultrasonic waves, centrifugally taking supernatant, and collecting crude protein.
And d, carrying out affinity chromatography by using a His label on the fusion protein and adopting a Ni column to obtain the purified fusion protein.
3. The rapid detection method for detecting glyphosate based on the fusion protein of EPSP synthase and GFP according to claim 1, characterized in that the prokaryotic expression is purified.
4. The rapid detection method for detecting glyphosate based on the fusion protein of EPSP synthase and GFP as claimed in claim 1, which is characterized in that the detection condition of glyphosate detection is described.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004331A2 (en) * 1999-07-09 2001-01-18 Calgene Llc Enhanced expression of proteins using gfp
KR20140094035A (en) * 2012-12-12 2014-07-30 고려대학교 산학협력단 DNA labeled Core/Shell nanoparticle having fluorescence, and method of detecting a bioactive material
CN107478828A (en) * 2017-09-25 2017-12-15 河南科技大学 The kit of quick detection glyphosate residual and detection method and the application of glyphosate residual
CN107748156A (en) * 2017-12-14 2018-03-02 重庆大学 A kind of detection method of glyphosate
CN111518113A (en) * 2020-05-11 2020-08-11 西北农林科技大学 Fluorescent probe for detecting glyphosate, detection test paper and preparation method thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004331A2 (en) * 1999-07-09 2001-01-18 Calgene Llc Enhanced expression of proteins using gfp
KR20140094035A (en) * 2012-12-12 2014-07-30 고려대학교 산학협력단 DNA labeled Core/Shell nanoparticle having fluorescence, and method of detecting a bioactive material
CN107478828A (en) * 2017-09-25 2017-12-15 河南科技大学 The kit of quick detection glyphosate residual and detection method and the application of glyphosate residual
CN107748156A (en) * 2017-12-14 2018-03-02 重庆大学 A kind of detection method of glyphosate
CN111518113A (en) * 2020-05-11 2020-08-11 西北农林科技大学 Fluorescent probe for detecting glyphosate, detection test paper and preparation method thereof

Non-Patent Citations (4)

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Title
JIANPING GUAN，等: "Employing a fluorescent and colorimetric picolyl-functionalized rhodamine for the detection of glyphosate pesticide", 《TALANTA》 *
PIERRE-EMMANUEL Y.N’GUETTA，等: "Engineering a fluorescence biosensor for the herbicide glyphosate", 《PROTEIN ENGINEERING, DESIGN AND SELECTION》 *
张超，等: "分子印迹电化学传感器的制备及其快速检测饮水中草甘膦残留的应用研究", 《分析测试学报》 *
李燕虹，等: "草甘膦农药残留检测方法研究进展", 《农产品加工》 *

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