Method for preparing recombinant bombyx mori acetylcholinesterase for detecting pesticide residue

A technology for acetylcholinesterase and pesticide residues, which is applied in the direction of material analysis by observing the impact on chemical indicators, and analysis by making materials undergo chemical reactions, can solve the problem of low activity of recombinant housefly acetylcholinesterase, acetylcholinesterase There are no problems such as activity and large sensitivity differences between species, and achieve the effect of low price, easy storage and short time consumption

Active Publication Date: 2015-08-26
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, most of the commercially available pesticide residue rapid detection kits or test strips, whose core material cholinesterase is directly extracted from housefly, horse serum, fish tissue, pig liver, sheep red blood cells, plant tissue, etc., these The cholinesterase directly isolated from organisms has disadvantages such as low extraction amount, difficult purification, large sensitivity difference among species, poor stability, etc. Therefore, false positives are very easy to occur in actual detection, and there are many shortcomings in application.
Wang Dong and others constructed the housefly acetylcholinesterase gene into prokaryotic and eukaryotic expression systems respectively, and carried out prokaryotic and eukaryotic expression respectively. However, it was found that the activity of the recombinant housefly acetylcholinesterase expressed in prokaryotic was very low, while that of eukaryotic The expressed recombinant housefly acetylcholinesterase has no activity

Method used

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  • Method for preparing recombinant bombyx mori acetylcholinesterase for detecting pesticide residue
  • Method for preparing recombinant bombyx mori acetylcholinesterase for detecting pesticide residue
  • Method for preparing recombinant bombyx mori acetylcholinesterase for detecting pesticide residue

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Experimental program
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Effect test

Embodiment 1

[0036] ankyrin gene AGα1 Insert it into the yeast expression vector pPIC9K to construct a surface display vector. The whole vector construction process is as follows: figure 1 shown. The constructed surface display vector was transformed into Pichia pastoris GS115, and positive recombinants were screened.

[0037] Specific steps are as follows:

[0038] 1. Use Trizol reagent to extract the total RNA of the silkworm head; use the RT-PCR kit to use the total RNA of the silkworm head as a template, Oligo(dT) 18 As a primer, the first strand of cDNA was synthesized by reverse transcription under the action of M-MuLV reverse transcriptase; the silkworm acetylcholinesterase gene was amplified by PCR using the first strand cDNA as a template bmace (The full-length sequence of the gene is shown as SEQ ID NO.1), and the bmace The gene was connected into the PMD-18-T vector, and the connection product was transformed into Escherichia coli DH5α; the recombinant plasmid PMD-18-T- b...

Embodiment 2

[0045] Preparation of recombinant silkworm acetylcholinesterase: yeast expression engineering strain GS115 / pPIC9K -bmace-AGα1 The expression was induced for 4 days, the supernatant was removed by centrifugation, and the recombinant silkworm acetylcholinesterase was obtained after the bacteria were freeze-dried. The recombinant silkworm acetylcholinesterase was subpackaged in plastic centrifuge tubes and stored under low temperature and light-proof conditions. When in use, take 0.1 g of freeze-dried solid enzyme preparation, add 50 mL of auxiliary reagent C, shake and mix evenly to make a liquid enzyme preparation.

[0046] Activity determination of recombinant silkworm acetylcholinesterase:

[0047] Take 800 μL of recombinant silkworm acetylcholinesterase, add 100 μL of substrate and 100 μL of chromogen to react for 5 min, and replace the surface-displayed recombinant silkworm acetylcholinesterase suspension with empty bacteria GS115 / pPIC9K suspension as the For the blank co...

Embodiment 3

[0053] Application of the enzyme preparation described in Example 2 in detecting pesticide residues in each sample.

[0054] Fruit: Chop a little fruit, take about 2 g of fruit pieces and put them in a 50 mL Erlenmeyer flask, add 10 mL of auxiliary reagent C, shake for 1 minute, and stand still. Take an empty centrifuge tube and add 100 μL of recombinant enzyme preparation and 2.5 mL of fruit extract, mix well and incubate for 2 minutes, then add 100 μL of auxiliary reagent A and auxiliary reagent B, and develop color for 2 to 3 minutes, observe the liquid in the centrifuge tube s color. If it appears purple, it means that the pesticide residues in the fruit have not exceeded the standard, and if it appears light red or yellow, it means that the pesticide residues in the fruit exceed the standard. Compared with the standard color chart, you can roughly know the amount of pesticide residues.

[0055] Vegetables: Rinse off the dirt on the surface of the vegetables, cut t...

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Abstract

The invention discloses a method for preparing recombinant bombyx mori acetylcholinesterase for detecting pesticide residues. The method comprises the following steps: connecting a bombyx mori acetylcholinesterase gene with an anchoring protein gene Agalpha1, inserting into a yeast expression vector pPIC9K to establish a surface display vector and convert pichia pastoris GS115, screening positive recon, performing inductive expression on positive recon by using methanol, centrifuging to remove the supernate after the expression is ended, and performing freeze-drying on the bacterium, thereby obtaining recombinant bombyx mori acetylcholinesterase. The prepared recombinase is good in stability, simple to operate, low in detection limit, high in sensitivity, low in cost, capable of performing in-situ detection in families, markets, vegetable production bases and the like, very applicable to screening of a great amount of samples, and has significant practical application and popularization meanings.

Description

technical field [0001] The invention relates to the technical field of rapid detection of pesticide residues, in particular to a method for preparing recombinant silkworm acetylcholinesterase for detecting pesticide residues, and more specifically to a method for detecting organic phosphorus and carbamate pesticide residues The preparation method of the recombinant silkworm acetylcholinesterase. Background technique [0002] While the use of pesticides brings huge economic benefits to agricultural production, it also causes serious pollution to the environment and products, and has a great impact on people's physical and mental health. At present, the detection methods of pesticide residues mainly include instrumental analysis, immunoassay and enzyme inhibition. The instrumental analysis method has high detection sensitivity, good selectivity, qualitative and quantitative results can be carried out at the same time, and the results are accurate and reliable. It is currently...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78
Inventor 王弘杨金易崔孟姣谢曦何永盛卢临萍孙远明徐振林
Owner SOUTH CHINA AGRI UNIV
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