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Application of ascorbic acid peroxidase 1 in catalysis of luminol chemiluminescence reaction

A peroxidase, ascorbic acid technology, applied in the direction of analysis, chemiluminescence/bioluminescence, etc. by chemical reaction of materials, which can solve problems such as limitations

Pending Publication Date: 2022-02-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since horseradish peroxidase cannot enter the cell, the luminol chemiluminescence method can only be used to measure the accumulation of intercellular active oxygen, or the active oxygen released from the intracellular to the extracellular, so that it can be used in the measurement of living Application of intracellular ROS content is limited

Method used

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  • Application of ascorbic acid peroxidase 1 in catalysis of luminol chemiluminescence reaction
  • Application of ascorbic acid peroxidase 1 in catalysis of luminol chemiluminescence reaction
  • Application of ascorbic acid peroxidase 1 in catalysis of luminol chemiluminescence reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Obtaining cAPX1 protein using prokaryotic expression system

[0044] 1. Reagent materials and equipment

[0045] Vector: pMAL-c2x-GW (gateway) - used for expression of MBP tagged protein;

[0046] Escherichia coli: BL21(DE3);

[0047] Column material: Amylose Resin, NEB, Cat # E8021L, for purification of MBP-tagged proteins;

[0048] IPTG: Solarbio, Cat # 18070, configured as a 240mg / mL (1M) aqueous solution, filtered and sterilized before storage, the use concentration is generally 0.2-0.5mM;

[0049] PBS buffer: store the stock solution at 20×, pH 7.2-7.6, Sangon Biotech, dilute to 1× with water when used;

[0050] Protease inhibitors: Merck, Cat # 4693116001, now add protein extract;

[0051] Empty chromatography column: 20mL, Sangon Biotech;

[0052] Protein electrophoresis apparatus, protein tank transfer apparatus, high pressure crusher, 4°C centrifuge, horizontal shaker (Shanghai Zhichu Instrument Co., Ltd.).

[0053] 2. Experimental steps

[...

Embodiment 2c

[0072] Example 2 cAPX1 protein catalyzes luminol and H in vitro 2 o 2 Reaction

[0073] The cAPX1 protein purified in Example 1 was used to detect the luminescence intensity after catalyzing luminol.

[0074] 1. Reagent materials and equipment

[0075] 96-well plate (white) (Wohong Bio, Cat # WHB-96);

[0076] Purification of maltose binding protein MBP from Escherichia coli;

[0077] Purify MBP-tagged MBP-cAPX1 recombinant protein from Escherichia coli;

[0078] 10mg / mL HRP (Peroxidase from horseradish, Sigma-Aldrich): Weigh 20mg HRP powder and dissolve in 2mL ddH 2 O, then aliquoted into 1.5mL light-proof centrifuge tubes (50μL per tube) and stored at -20°C as a storage solution;

[0079] 30%H 2 o 2 (Shanghai test, 30% equivalent to 10M after conversion): Dilute it to a final concentration of 100mM when used;

[0080] 100mM luminol (Luminol, Sigma-Aldrich, S-A4685-25G): Weigh 177mg of luminol powder (MW=177.16) and dissolve it in 10mL KOH, then dispense it into 1.5...

Embodiment 3c

[0093] Example 3 cAPX1 is used for luminol to monitor the dynamics of active oxygen generation in plant cells

[0094] In order to test whether cAPX1 can catalyze the interaction of luminol and H 2 o 2 response, we applied lipopolysaccharide LPS to induce plant intracellular H 2 o 2 accumulation.

[0095] Lipopolysaccharide is the main component of the outer membrane of Gram-negative bacteria. After treating the plant leaves, it was observed under a microscope by fluorescent staining that lipopolysaccharide induced the accumulation of intracellular reactive oxygen species, which were mainly located at the periphery of the chloroplast in the cytoplasm.

[0096] Monitoring by luminol chemiluminescence method found that lipopolysaccharide can cause biphasic ROS burst, the first ROS burst rapidly increased within 30min and lasted for a short time; the second ROS burst (later ROS burst) at It started after 3 hours of lipopolysaccharide treatment, reached the maximum value at ab...

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Abstract

The invention discloses application of ascorbic acid peroxidase 1 in catalysis of luminol chemiluminescence reaction, and belongs to the technical field of plant immunity. The invention discloses the activity of ascorbic acid peroxidase 1 for catalyzing oxidation of luminol for the first time, and the ascorbic acid peroxidase 1 can be developed into a luminol-H2O2 chemiluminescence reaction catalyst. Based on the characteristic of stable expression of ascorbic acid peroxidase 1 in plant cells, the dynamic state and content of intracellular active oxygen can be monitored in real time by using a luminol chemiluminescence method, and the model is used for screening induction factors capable of causing burst-out of the intracellular active oxygen.

Description

technical field [0001] The invention relates to the technical field of plant immunity, in particular to a method for monitoring active oxygen in plants based on ascorbic acid peroxidase 1 catalyzing luminol chemiluminescent reaction. Background technique [0002] ROS (Reactive oxygen species) is a type of oxygen-containing group produced during the evolution of aerobic organisms and has high biological activity. Active oxygen mainly includes singlet oxygen ( 1 o 2 ), superoxide anion (O 2 -· ), hydrogen peroxide (H 2 o 2 ) and hydroxyl radicals (OH·) etc. In plant cells, the main sites that produce reactive oxygen species are the plasma membrane, chloroplast, mitochondria, and peroxisomes. [0003] Reactive oxygen species play a dual role in the life activities of plants: on the one hand, low-concentration reactive oxygen species play a signal regulation role in physiological processes such as cell division, programmed death, organ development, and biotic and abiotic ...

Claims

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Application Information

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IPC IPC(8): G01N21/76
CPCG01N21/76
Inventor 洪秀芳贾志怡梁岩
Owner ZHEJIANG UNIV
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