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Encoded gene sequence of nucleotide of acetylcholine esterase in fruit fly

A technology of acetylcholinesterase and nucleotide genes, applied in genetic engineering, plant gene improvement, hydrolytic enzymes, etc., can solve undiscovered problems

Inactive Publication Date: 2006-02-01
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Regarding the recombinant expression of the Drosophila AChE gene and its application in the detection of pesticide residues, a large amount of research work has been carried out abroad since the 1990s, but no reports of documents and patents closely related to the subject of the present invention have been found so far

Method used

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  • Encoded gene sequence of nucleotide of acetylcholine esterase in fruit fly
  • Encoded gene sequence of nucleotide of acetylcholine esterase in fruit fly
  • Encoded gene sequence of nucleotide of acetylcholine esterase in fruit fly

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1——The activity of the recombinant AChE protein expressed by yeast is inhibited by a small amount of dichlorvos

[0090] 1. Solutions and reagents

[0091] (1) BMGY solution

[0092] Dissolve 10g of yeast extract and 20g of peptone in 700ml of water, and autoclave (20min); after the temperature of the solution drops to room temperature, add 100ml of potassium phosphate buffer (1M, pH6.0), 100ml of 10×YNB, 2ml of 500×B, 100ml 10×GY; the shelf life of the solution at 4°C is two months.

[0093] (2) BMMY solution

[0094] Change the 10×GY in the BMGY solution to 10×M, and keep the other components unchanged. The solution has a shelf life of two months at 4°C.

[0095] (3) Dichlorvos solution

[0096] 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 stepwise by a factor of 10 to 10 -5 、10 -6 、10 -7 M.

[0097] (4) Ellman's reagent

[0098] W...

Embodiment 2

[0107] Example 2——The activity of recombinant AChE protein expressed by yeast is inhibited by trace amounts of chlorpyrifos

[0108] 1. Solution

[0109] (1) Chlorpyrifos solution

[0110] Weigh 0.175g of chlorpyrifos (MW=350.6) crystals and dissolve in 5ml of acetone. At this time, the concentration of chlorpyrifos is 0.1M. Dilute the solution stepwise by a factor of 10 to 10 -5 、10 -6 、10 -7 M.

[0111] All the other solutions and reagent formulations are the same as in Example 1.

[0112] 2. Operation steps

[0113]Operation is basically the same as in Example 1. Dissolve chlorpyrifos crystals with acetone to prepare a concentration of 10 -5 , 10 -6 , 10 -7 M's solution. Take 3 μl and 50 μl of crude enzyme solution, mix them, and incubate (25°C, 15min). At the same time, a control group was set up, which was formed by mixing 3 μl of acetone and 50 μl of crude enzyme solution. Add the mixed solution into the microplate well containing 250 μl Ellman reagent, shak...

Embodiment 3

[0116] Example 3——The activity of the recombinant AChE protein expressed by yeast is inhibited by a small amount of pirimicarb

[0117] 1. Solution

[0118] (1) Anti-pirimicarb solution

[0119] Weigh 0.119g of pirimicarb (MW=238.33) crystals and dissolve it in 5ml of acetone. At this time, the concentration of pirimicarb is 0.1M. Dilute the solution stepwise by a factor of 10 to 10 -5 、10 -6 、10 -7 M.

[0120] All the other solutions and reagent formulations are the same as in Example 1.

[0121] 2. Operation steps

[0122] Operation is basically the same as in Example 1. Dissolve anti-pirimicarb crystals with acetone, and prepare a concentration of 10 -5 , 10 -6 , 10 -7 M's solution. Take 3 μl and 50 μl of crude enzyme solution, mix them, and incubate (25°C, 15min). At the same time, a control group was set up, which was formed by mixing 3 μl of acetone and 50 μl of crude enzyme solution. Add the mixed solution into the microwell containing 250 μl Ellman reagent...

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PUM

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Abstract

A nucleotide sequence for coding the acetylcholinesterase of fruit fly is same as the sequence shown by SEQ ID No.5. Said coding is to cut off the signal peptide of acetylcholinesterase and the nucleotide of glucophosphatide anchoring signal. The residual nucleotide sequence is same as that shown by SEQ ID No.6. Its product has high sensitivity to low-concentration organophosphorus and aminoformate, so it can be used to detect the residual agricultural chemicals in agricultural products.

Description

technical field [0001] The present invention relates to a gene sequence in the field of molecular biological gene technology, in particular to a nucleotide gene sequence encoding Drosophila acetylcholinesterase. Background technique [0002] In multicellular animals, acetylcholinesterase (AChE for short) is located on the chemical synapses between neurons and between neurons and muscle cells. It is the main signal transmission molecule and performs the function of hydrolyzing choline ester. Studies have shown that a variety of compounds can inhibit AChE activity, resulting in excessive choline-like metabolic pathways in organisms, choline esters cannot be effectively hydrolyzed, and accumulate in large amounts in nerve endings. The organisms show symptoms such as muscle twitching and central nervous system dysfunction. even death. Among many inhibitors, organophosphorus and carbamates are considered to be specific inhibitors of AChE, which can phosphorylate or carbamoylate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/16
Inventor 张大兵许诵辞武爱波梁婉琪潘爱虎
Owner SHANGHAI JIAO TONG UNIV
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