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Method for separating and purifying scallop phenol oxidase

A scallop phenol oxidase, separation and purification technology, applied in the field of shellfish immunology, can solve problems such as no relevant reports, and achieve the effects of improving purification efficiency, simple equipment requirements, and short time consumption

Active Publication Date: 2010-12-01
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no relevant reports on the separation and purification of invertebrate PO by the combination of continuous gradient non-denaturing electrophoresis-specific substrate color development-electroelution and other technologies.

Method used

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  • Method for separating and purifying scallop phenol oxidase
  • Method for separating and purifying scallop phenol oxidase
  • Method for separating and purifying scallop phenol oxidase

Examples

Experimental program
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Effect test

example 1

[0024] Example 1: Separation and purification of bay scallop (Argopectenirradians) PO by continuous gradient non-denaturing electrophoresis-specific substrate color development-electroelution

[0025] (1) Prepare the supernatant of blood cell lysate

[0026] ①Use a sterilized syringe to collect blood from the adductor muscle of bay scallop, centrifuge (4°C, 3000rpm, 15min), collect the blood cell pellet, and use 1.5ml of 20mM Tris- Resuspend in HCl buffer (pH 7.0);

[0027] ② Disrupt the blood cell suspension with an ultrasonic disruptor at 4°C, centrifuge (4°C, 14,000 rpm, 30 min), and take the supernatant, which is the supernatant of the blood cell disruption solution.

[0028] (2) Continuous gradient non-denaturing electrophoresis using the supernatant of blood cell lysate as a sample

[0029] ① Prepare pH 8.8 Tris-Glycin non-denaturing electrophoresis buffer (6.06g Tris, 28.82g Glycin, 2L DW), and pre-cool to 4°C.

[0030] ② Use a constant flow pump to prepare a continu...

example 2

[0072] Example 2: Separation and purification of Chlamysfarreri PO by continuous gradient non-denaturing electrophoresis-specific substrate color development-electroelution

[0073] (1) Prepare the supernatant of blood cell lysate

[0074] ①Use a sterilized syringe to take blood from the adductor muscle of Chlamys farreri, centrifuge (4°C, 3000rpm, 15min) to collect the blood cell pellet, and use 1.5ml of 20mM Tris pre-cooled to 4°C for the blood cell pellet obtained by centrifuging about 100ml of hemolymph -HCl buffer (pH 7.0) resuspended;

[0075] ② Disrupt the blood cell suspension with an ultrasonic disruptor at 4°C, centrifuge (4°C, 14,000 rpm, 30 min), and take the supernatant, which is the supernatant of the blood cell disruption solution.

[0076] (2) Continuous gradient non-denaturing electrophoresis using the supernatant of blood cell lysate as a sample

[0077] Same as step (2) in Example 1, replace the sample with the supernatant of blood cell disruption liquid o...

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Abstract

The invention discloses a method for separating and purifying scallop phenol oxidase. The method is characterized by comprising the following steps of: (1) obtaining haemolymph from adductor muscles of scallops, centrifuging, suspending and crushing the haemolymph, then centrifuging the crushed haemolymph, and taking the supernate, namely obtaining blood cell crushed supernate; (2) performing continuous gradient non-denaturing electrophoresis on the blood cell crushed supernate serving as a sample; (3) positioning a phenol oxidase protein band of non-denaturing electrophoresis by using a specific substrate coloring method; and (4) performing glue cutting on a destination band, reclaiming the phenol oxidase protein band by an electro-elution method, and obtaining purified scallop phenol oxidase protein after dialysis and freeze-drying. The method has the advantages that: (1) the method has short purification time; (2) the method has simple and convenient operation, simple equipment requirement and low cost, and greatly reduces the treatment steps of the sample; and (3) the purified sample has high purity, only presents one band by non-denaturing and denaturing electrophoresis detection, keeps the bioactivity of enzyme well and improves the purifying efficiency.

Description

technical field [0001] The invention relates to the improvement of the separation and purification technology of phenol oxidase of bivalve mollusk, in particular to a separation and purification method of scallop phenol oxidase, which belongs to the technical field of shellfish immunology. Background technique [0002] Scallops are lower invertebrates that resist the invasion of foreign pathogens through non-specific immunity. Phenoloxidase (PO) is one of the important factors of non-specific immunity. It is activated under the induction of microbial polysaccharides, catalyzes the production of quinones from related substrates, and finally produces melanin. A series of products generated by the PO catalyzed reaction can limit and isolate foreign substances; inhibit the activity of pathogenic bacteria extracellular proteins and chitin; kill pathogens; heal wounds; play an opsonizing role; promote blood cell phagocytosis and encapsulation; mediate agglutination, Coagulation a...

Claims

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Application Information

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IPC IPC(8): C12N9/02
Inventor 邢婧蒋经伟战文斌绳秀珍
Owner OCEAN UNIV OF CHINA
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