Mutant enzyme for detecting organophosphorus pesticides and preparation method thereof

An organophosphorus pesticide and mutant enzyme technology, applied in the biological field, can solve the problems of false positive interference, inability to distinguish the type and content of organophosphorus, etc., and achieve the effect of small false positive interference, good mutation effect and good identification effect.

Inactive Publication Date: 2019-10-18
THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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Problems solved by technology

[0004] The purpose of the present invention is to provide a mutant enzyme for detecting organophosphorus pesticides and its preparation method and application, aiming to solve the problem of the inability to distinguish the type and content of organophosphorus when using acetylcholinesterase to detect organophosphorus pesticides, and there are false positives interference problem

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  • Mutant enzyme for detecting organophosphorus pesticides and preparation method thereof
  • Mutant enzyme for detecting organophosphorus pesticides and preparation method thereof
  • Mutant enzyme for detecting organophosphorus pesticides and preparation method thereof

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preparation example Construction

[0023] Another aspect of the present invention provides a kind of preparation method that detects the mutant enzyme of organophosphorus pesticide, comprises the following steps:

[0024] S01. Provide forward and reverse primers containing the planned site-directed mutagenesis sequence, use the recombinant plasmid as a template, use PCR site-directed mutagenesis technology for mutation, use DpnI to eliminate the template double-stranded and heterozygous template, and collect the mutated single-stranded circular DNA. The planned site-directed mutation sequence is human butyrylcholinesterase in which the A at position 328 is mutated to W.

[0025] In the above step S01, forward and reverse primers containing a planned site-directed mutation sequence are provided, wherein the planned site-directed mutation sequence is human butyrylcholinesterase in which A at position 328 is mutated to W. Add Pfu polymerase for PCR amplification. Preferably, the PCR amplification system for mutat...

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Abstract

The invention provides a mutant enzyme for detecting organophosphorus pesticides, and the mutant enzyme is a human butyryl cholinesterase mutant A328W. The mutant enzyme for detecting the organophosphorus pesticides provided by the invention has good identification effect on various organophosphorus pesticides and high kinetic constant, and can realize identification of multiple kinds of organophosphorus pesticides and detection of the content thereof at the same time. In addition, other organophosphorus compounds other than the organophosphorus pesticides have less inhibition on the human butyryl cholinesterase mutant A328W, the interference of false positives is small, and the detection accuracy is high.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a mutant enzyme for detecting organophosphorus pesticides and a preparation method thereof. Background technique [0002] Organophosphorus pesticides are widely used because of their high efficiency, variety, and wide application. The use of organophosphorus pesticides has played a huge role in crop protection in agricultural production, but at the same time, because organophosphorus is not easy to oxidize, decompose, and is difficult to be degraded by microorganisms, it can stay in the environment for a long time. Therefore, the large-scale use of organophosphorus pesticides , causing environmental pollution. In addition, some organophosphorus species are highly toxic, and are highly toxic to non-target organisms. The residues of organophosphorus pesticides in food and the environment not only threaten human health, but also cause harm to beneficial organisms in the ecosystem and h...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/85G01N33/573
CPCC12N9/18C12N15/85C12Y301/01008G01N33/573G01N2496/45
Inventor 潘雪华
Owner THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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