Recombinat acetylcholinesterase and its prepn process and usage in detecting presticide residue

A technology for the detection of acetylcholinesterase and pesticide residues, which is applied in the field of recombinant acetylcholinesterase and its preparation and use in the detection of pesticide residues, and can solve the problems affecting the repeatability, purity and stability of test results, etc.

Inactive Publication Date: 2007-01-03
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purity and stability of this type of enzyme products are not satisfactory, which affects the repeatability of the test results
At present, the application of recombinant acetylcholinesterase in the field of rapid detection of pesticide residues in my country has not been reported

Method used

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  • Recombinat acetylcholinesterase and its prepn process and usage in detecting presticide residue
  • Recombinat acetylcholinesterase and its prepn process and usage in detecting presticide residue
  • Recombinat acetylcholinesterase and its prepn process and usage in detecting presticide residue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1 - The activity of yeast-expressed recombinant AChE protein was inhibited by trace amounts of dichlorvos

[0098] 1. Solutions and reagents

[0099] (1) BMGY solution

[0100] Dissolve 10g of yeast extract and 20g of peptone in 700mL of water, and sterilize by autoclaving (20min); after the solution temperature drops to room temperature, add 100mL of potassium phosphate buffer (1M, pH 6.0), 100mL of 10×YNB, 2mL of 500×B, and 100mL, respectively. 10×GY; the shelf life of the solution at 4°C is two months.

[0101] (2) BMMY solution

[0102] The 10×GY in the BMGY solution was replaced with 10×M, and the rest of the components remained unchanged. The shelf life of the solution at 4°C is two months.

[0103] (3) Dichlorvos solution

[0104] 0.11 g of dichlorvos (MW=220.98) crystals were weighed and dissolved in 5 mL of acetone. At this time, the concentration of dichlorvos was 0.1M. Dilute the solution in steps of 10 to 10 -5 , 10 -6 , 10 -7 M.

[0105] (...

Embodiment 2

[0117] Example 2 - The activity of yeast-expressed recombinant AChE protein was inhibited by trace amounts of chlorpyrifos

[0118] 1. Solution

[0119] (1) Chlorpyrifos solution

[0120] 0.175 g of chlorpyrifos (MW=350.6) crystals were weighed, dissolved in 5 mL of acetone, and the concentration of chlorpyrifos was 0.1M at this time. Dilute the solution in steps of 10 to 10 -5 , 10 -6 , 10 -7 M.

[0121] Remaining solutions and reagent formulations are the same as those in Example 1.

[0122] 2. Operation steps

[0123] The operation is basically the same as in Example 1. Dissolve chlorpyrifos crystals in acetone to a concentration of 10 -5 , 10 -6 , 10 -7 M's solution. 3 μl and 50 μl of the crude enzyme solution were respectively mixed and incubated (25° C., 15 min). At the same time, a control group was set up, which was composed of 3 μl of acetone mixed with 50 μl of crude enzyme solution. Add the mixture into the well of the microtiter plate containing 250μl El...

Embodiment 3

[0126] Embodiment 3---recombinant AChE protein detects carbowei in spinach

[0127] 1. Solution

[0128] (1) gram Budweiser solution

[0129] Dilute the 100 mg / L standard solution of carbofuran with acetone to 0.5, 0.25 and 0.05 mg / L.

[0130] Remaining solutions and reagent formulations are the same as those in Example 1.

[0131] 2. Operation steps

[0132] Cut 4g spinach into about 1cm with scissors 2 As for the small pieces in the beaker, add 4 mL of a certain concentration of carbocarb solution. After the solvent in the solution—acetone is completely volatilized, add 20 mL of 0.25 mM sodium phosphate solution (pH 8.0), ultrasonically shake (10 min), and take 4 mL of the sample. The extract was centrifuged in a 5 mL centrifuge tube (room temperature, 3000 rpm, 3 min), and 2.4 mL of the supernatant was collected. Add 40 μl of enzyme solution (about 30 U) to 2.4 mL of supernatant and place it in a water bath at 25°C for 15 min, transfer the mixture to a cuvette, add 100...

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PUM

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Abstract

The present invention relates to pesticide technology, and is recombinant acetylcholinesterase and its preparation process and usage in detecting pesticide residue. Recombinant acetylcholinesterase may be dissolved in buffering sodium phosphate solution and after being maintained at 4 deg.c for 22 days, it has 75 % of its initial enzyme activity maintained. It has inhibiting constant on DDVP, omethoate, paraoxon, methyl paraoxon, aldicarb, carbaryl, carbofuran and aphox of 4.4E6, 2.2E4, 1.1E6, 4.2E5, 4.8E4, 5.9E5, 5.1E6 and 2.3E5 separately. It is prepared through selecting seed, shaking cultivation, centrifuging, filtering, depositing, leaching, stirring, dialyzing and freeze drying, and is maintained at -20 deg.c. The detection process includes preparing reagent, treating sample, detecting sample, blank testing, testing sample, and final calculating. The recombinant acetylcholinesterase has obvious sensitivity on organophosphorus pesticide and carbamate chemicals and may be used in detecting pesticide residue.

Description

technical field [0001] The invention relates to a recombinant enzyme in the technical field of pesticides, and a preparation method and detection method thereof, in particular to a recombinant acetylcholinesterase, a preparation method thereof, and a detection method for pesticide residues. Background technique [0002] my country is a major country in agricultural production and use in the world. At present, there are more than 2,500 designated pesticide production enterprises in my country, and more than 600 varieties of pesticides have been registered and can be produced. The production capacity of pesticides in my country is about 760,000 tons, and the total output is about 480,000 tons. The annual production of pesticides is second only to the United States. At the same time, my country is the country with the largest amount of pesticide application in the world, with an average annual application amount of more than 1.3 million tons, and the amount per unit area is twi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/09C12N1/19C12Q1/46
Inventor 张大兵武爱波许诵辞陈浩德
Owner SHANGHAI JIAO TONG UNIV
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