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A rapid detection method for detecting glyphosate based on the fusion protein of EPSP synthase and GFP

An EPSP synthase, fusion protein technology, applied in chemical instruments and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of ion chromatography with many interference factors, low stability and sensitivity, and insoluble in organic solvents.

Active Publication Date: 2021-08-10
INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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Problems solved by technology

[0003] Due to the high polarity of glyphosate, it is difficult to dissolve in organic solvents, and lacks functional groups that can be used for detection, derivatization is required for detection by liquid phase and gas phase chromatography; there are many interference factors in ion chromatography, which seriously affect the detection results; ELISA Immunoassay methods such as colloidal gold detection and colloidal gold detection are limited by antibodies, and the stability and sensitivity are not high

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  • A rapid detection method for detecting glyphosate based on the fusion protein of EPSP synthase and GFP
  • A rapid detection method for detecting glyphosate based on the fusion protein of EPSP synthase and GFP

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Embodiment Construction

[0023] The present invention will be further described according to the following examples, and the mode of the present invention includes but not limited to the following examples.

[0024] Such as figure 1 Shown, the present invention comprises the following steps:

[0025] The glyphosate main target EPSP synthase of clone A is used as the receptor protein of glyphosate;

[0026] B fuse the receptor protein with the green fluorescent protein to obtain the fusion protein;

[0027] Fusion protein construction expression:

[0028] The enolpyruvylshikimate phosphate synthase (EPSPS) gene was cloned from tobacco, and the cDNA sequence of the gene was synthesized after sequencing, and connected into the prokaryotic expression vector pET-28a capable of expressing enhanced green fluorescent protein (eGFP) by homologous recombination In -eGFP, EPSPS and eGFP are made to form a fusion protein to obtain a new recombinant expression vector.

[0029] The plasmid vector was introduce...

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Abstract

The invention discloses a rapid detection method for detecting glyphosate based on EPSP synthase and GFP fusion protein, including the cloned glyphosate main target EPSP synthase as the receptor protein of glyphosate; the receptor protein and the green Fluorescent protein was fused to construct EPSPS‑GFP, a new protein containing both glyphosate-binding sites and fluorescent labels. It was prokaryotically expressed and purified in Escherichia coli, and used for the detection of glyphosate in water. The invention realizes the qualitative and quantitative detection of the glyphosate herbicide by constructing the fusion protein and using a fluorescence photometer to detect the change of the fluorescence response value and establishing a standard calibration curve.

Description

technical field [0001] The invention relates to the field of glyphosate detection and extraction, in particular to a rapid detection method for glyphosate detection based on EPSP synthase and GFP fusion protein. Background technique [0002] Glyphosate, English name glyphosate, chemical name N-phosphomethylglycine, is a systemic conduction type broad-spectrum herbicide, its mechanism of action is mainly to inhibit enolpyruvylshikimate phosphate synthase in plants (EPSP synthase), thereby inhibiting the conversion of shikime to phenylalanine, tyrosine and tryptophan, interfering with protein synthesis and causing plant death. Glyphosate is the most widely used pesticide variety with the largest output in the world, and its annual sales volume ranks first among pesticides. All countries in the world have set limits on the maximum residue limit (MRL) of glyphosate in drinking water. The US Environmental Protection Association (EPA) has set the MRL for glyphosate in drinking wa...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/70G01N33/543
CPCC07K14/43595C12N9/1092C12N15/70C12Y205/01019G01N33/543G01N2430/20
Inventor 王淼王静佘永新
Owner INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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