Acetylcholine esterase mutant and application thereof

A technology of acetylcholinesterase and mutants, which is applied in the field of food testing and public health, and can solve the problems of complicated sample processing, inapplicability of rapid detection, and dependence on large-scale detection instruments.

Inactive Publication Date: 2013-12-04
上海福盈资产管理有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But at the same time, if the pesticide residues in the crops cannot be effectively removed, it will also have a negative impact on people's health. strict regulations
[0003] At present, the traditional detection methods of organophosphorus and carbamate pesticide residues are chromatographic methods and enzyme inhibition methods. Although the former has a low detection limit and high sensitivity, it relies on large-scale detection instruments, and the sample pretreatment is complicated. Suitable for on-site rapid detection

Method used

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  • Acetylcholine esterase mutant and application thereof
  • Acetylcholine esterase mutant and application thereof
  • Acetylcholine esterase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Embodiment 1, the construction of acetylcholinesterase mutant

[0112] The full length of the acetylcholinesterase (AChE) gene is 1950bp (SEQ ID NO: 1; GenBank accession number: NM_057605), which can encode 649 amino acids (SEQ ID NO: 2). The N-terminal of the protein sequence of the enzyme contains a signal peptide sequence, and its C The end contains its own GPI anchor sequence. The 408th Tyr of the wild-type protein shown in SEQ ID NO: 2 is the mutation site of the present invention.

[0113] In order to display and express on the surface of yeast cells, the inventors carried out a transformation of genetic engineering technology (see patent No. ZL 200810201894.5), and the signal peptide (1-38aa) at the N-terminus was replaced by the glucoamylase secretion signal peptide sequence , the C-terminal anchoring sequence (620-649aa) was replaced by the yeast's own α-lectin gene anchoring sequence, and a Flag epitope was added between the α-lectin gene anchoring sequence a...

Embodiment 2

[0122] Example 2, Induced expression of acetylcholinesterase and determination of enzyme activity

[0123] Transformed yeast cells (including yeast cells expressing mutant acetylcholinesterase and yeast cells expressing wild-type acetylcholinesterase) were screened for transformants on SC selection plates, and single clones were picked and cultured in 5ml SC medium at 30°C Overnight, the yeast cells were harvested by centrifugation, and the resulting yeast cells were induced to express protein, that is, the yeast cells cultured overnight were diluted to OD with SC medium with galactose as the carbon source 600 0.4 cell suspension, induced at 30°C for 4 hours, centrifuged at 10,000rpm at 4°C for 5min to harvest the cells.

[0124] Cells were resuspended in sodium phosphate buffer (pH 7.6) to make OD 600 After that, the substrate acetylcholine iodide (ATChI) and the chromogenic agent dithiodinitrobenzoic acid (DTNB) were sequentially added at a final concentration of 1 mM, and ...

Embodiment 3

[0128] Embodiment 3, the inhibition of organophosphorus and carbamate pesticides to acetylcholinesterase mutant

[0129] Yeast cells were induced to express mutant acetylcholinesterase or wild-type (non-mutated) acetylcholinesterase, collected by centrifugation, resuspended in sodium phosphate buffer (pH 7.6), added to a 96-well plate, and made OD 600 The value is 0.5, adding different final concentrations of organophosphorus and carbamate pesticides, and incubating at 37°C for 30 minutes to make the pesticides fully inhibit acetylcholine ester, and then adding the substrate ATChI and the display agent DTNB with a final concentration of 1mM, the total reaction system 200μL, react at 37°C for 15min, detect OD every 0.5min during the reaction 412 .

[0130] Taking the data without pesticide inhibition as a control, that is, the retained enzyme activity is 100%, the inhibition degree of 9 kinds of organophosphorus and carbamate pesticides to acetylcholinesterase can be from Fi...

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Abstract

The invention relates to an acetylcholine esterase mutant and application thereof and reveals an acetylcholine esterase mutant. The enzymatic activity of the acetylcholine esterase mutant is remarkably higher than the enzymatic activity of wild type under conditions of unchanged catalytic products. The acetylcholine esterase mutant is also displayed and expressed on surfaces of yeast cells, and used for detecting existence or not or existing amounts of organophosphorus or carbamates pesticides in samples to be detected. The sensitivity is raised greatly.

Description

technical field [0001] The invention belongs to the fields of food detection and public health; more specifically, the invention relates to a mutant of acetylcholinesterase and its application, which can be displayed on the surface of cells, so as to be used for the efficient and rapid removal of organophosphorus and carbamate pesticides detection. Background technique [0002] Organophosphorus and carbamate pesticides are commonly used in agricultural production at home and abroad. They have various types, wide insecticidal spectrum and low price. They are widely used in the production of agricultural crops such as grain and vegetables and cash crops such as tea. The mechanism by which these pesticides exert their insecticidal effect is that they reversibly inhibit the acetylcholinesterase of harmful insects and form neurotoxicity, thereby achieving the insecticidal effect. But at the same time, if the pesticide residues in the crops cannot be effectively removed, it will ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/81C12N1/19C12Q1/46C12Q1/02C12R1/84C12R1/865
Inventor 王慧尹珺李隽旸
Owner 上海福盈资产管理有限公司
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