Formica sinensis AChE gene, formica sinensis AChE and preparation method thereof

A technology of acetylation and genes of red forest ants, applied in the field of genetic engineering, can solve the problems of unreasonable and unseen pesticide sensitivity, achieve high activity and sensitivity, and good application prospects

Active Publication Date: 2015-05-06
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, although there are a large number of studies and reports on the eukaryotic expression system prepared by using the insect acetylcholinesterase gene, the sensitivity to pesticides is still n...

Method used

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  • Formica sinensis AChE gene, formica sinensis AChE and preparation method thereof
  • Formica sinensis AChE gene, formica sinensis AChE and preparation method thereof
  • Formica sinensis AChE gene, formica sinensis AChE and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0069] Red forest ant acetylcholinesterase (AChE) gene, the base sequence is shown in SEQ ID NO.1 in the sequence table.

[0070] The amino acid sequence of the acetylcholinesterase (AChE) encoded by the red forest ant acetylcholinesterase (AChE) gene is shown in SEQ ID NO.2.

[0071] The technical principle of preparing the acetylcholinesterase of the red forest ant is: use recombinant DNA technology to clone the acetylcholinesterase gene of the red forest ant into a eukaryotic expression vector and express it in a large amount in Pichia pastoris. Further purification can obtain its expression product Acetylcholinesterase.

[0072] The preparation method of red forest ant acetylcholinesterase specifically includes the following steps:

[0073] 1. Construction and PCR amplification of the complete sequence of the acetylcholinesterase (AChE) gene of the red forest ant

[0074] (1) Extract total RNA, reverse transcription to obtain cDNA

[0075] A. Extract total RNA, Using the capture...

Embodiment 2

[0203] For the acetylcholinesterase (AChE) of the red wood ant prepared in Example 1, the inventors further carried out activity detection and inhibition rate detection, which are briefly introduced as follows.

Embodiment 1

[0204] Example 1 Detection of the activity of the acetylcholinesterase (AChE) of the red forest ants after purification

[0205] Prepare test reagents according to GB / T 5009.199-2003:

[0206] PH=8.0 buffer solution: Take 11.9 g of anhydrous dipotassium hydrogen phosphate and 3.2 g of potassium dihydrogen phosphate, respectively, and dissolve in 1000 mL of distilled water;

[0207] Chromogenic reagent: Take 160 mg of dithiodinitrobenzoic acid (DTNB) and 15.6 mg of sodium bicarbonate respectively, dissolve them with 20 mL of the pH=8.0 buffer prepared above, and store at 4°C.

[0208] Substrate: Take 25 mg of S-acetylthioacetylcholine, dissolve it in 3.0 mL of distilled water, and store at 4°C.

[0209] According to Ellman's method, take 100 μL of the red wood ant acetylcholinesterase (AChE) enzyme solution purified in Example 1, add 2.5 mL of potassium phosphate buffer (1M, PH=8.0), and bathe in water at 37°C for 15 min, then add 100 μL After mixing the color reagent quickly, add 100 ...

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Abstract

The invention belongs to the technical field of gene engineering, and in particular relates to a formica sinensis acetylcholin esterase (AChE) gene, encoded formica sinensis acetylcholin esterase (AChE) and a method for preparing the enzyme. The base sequence and the amino acid sequence of the formica sinensis AChE gene and the encoded formica sinensis AChE are as shown in a sequence list. The preparation method of the enzyme comprises the following steps: (1) constructing a formica sinensis AChE gene complete sequence and carrying out polymerase chain reaction (PCR) amplification; (2) constructing an eukaryotic expression recombinant vector of the formica sinensis AChE gene complete sequence; (3) converting a pichia pastoris host cell, cultivating and inducing expression; and (4) purifying. Experiments prove that the prepared formica sinensis AChE, compared with the existing drosophila melanogaster AChE, is higher in activity and sensitivity; therefore, the formica sinensis AChE has a relatively good application prospect in the technical field of future pesticide detection reagent preparation.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a red forest ants acetylcholinesterase (AChE) gene, the coded red forest ants acetylcholinesterase (AChE), and a method for preparing the enzyme. Background technique [0002] Now that global environmental pollution, especially pesticide pollution, is becoming more and more serious, it is particularly urgent to quickly and easily detect pesticide residues. Most pesticides are organophosphorus and carbamates, which have been widely used in the production of various crops. However, such pesticides have caused serious pollution to the environment and poisoned humans and animals. Therefore, it is of great practical significance to develop sensitive and rapid monitoring of pesticide residues. [0003] At present, the widely used method for detecting pesticide residues is to use the inhibitory reaction between acetylcholinesterase (AChE) and organophosphorus and...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/18C12N15/81
Inventor 翁海波张佳丽李倩张欢安秀丽
Owner ZHENGZHOU UNIV
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