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Method for extracting myoglobin in pig hearts and human total antioxidant status kit containing myoglobin

A myoglobin and kit technology, applied in the direction of biological testing, preparation of test samples, material inspection products, etc., can solve problems such as inability to meet the mass production requirements of kits, low yield of recombinant myoglobin, and difficulty in obtaining raw materials , to achieve good economic benefits, simple extraction process, and short processing time

Inactive Publication Date: 2017-02-15
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The prosthetic group of myoglobin is heme, which is composed of four pyrrole subunits to form a ring, and the center of the ring is a ferrous ion. The particularity and complexity of the structure of the substance make the recombinant myoglobin obtained through genetic engineering expression Red protein has problems such as low yield and poor stability, which limit its application in TAS kits
At present, it has been reported in the literature that two column chromatography methods can be used to extract myoglobin from animal tissues such as pig heart muscle and sardine muscle, but the method is cumbersome and the extraction efficiency is not high; The myoglobin extracted from myocardium has good stability, but the disadvantage is that the raw materials are difficult to obtain and the cost is high, which cannot meet the mass production requirements of the kit

Method used

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  • Method for extracting myoglobin in pig hearts and human total antioxidant status kit containing myoglobin
  • Method for extracting myoglobin in pig hearts and human total antioxidant status kit containing myoglobin
  • Method for extracting myoglobin in pig hearts and human total antioxidant status kit containing myoglobin

Examples

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example 1

[0044] Example 1: Extraction of myoglobin from pig heart

[0045] 1) After removing the fat and connective tissue of 500g fresh pig heart, add 1000ml 50mM phosphate buffer pH8.0, homogenize quickly with a tissue homogenizer for 5min, centrifuge at 12000rpm for 40min in a high-speed refrigerated centrifuge, remove the precipitate, and obtain the crude extract ;

[0046] 2) Add ammonium sulfate solids accounting for 40% of the mass of the crude liquid to the crude extract, stir gently, and wait for complete dissolution. After precipitation at room temperature for 1 hour, centrifuge with a high-speed refrigerated centrifuge at 12,000 rpm for 40 minutes, discard the precipitate, and keep the supernatant;

[0047] 3) Continue to add solid ammonium sulfate to the supernatant to saturation, stir slightly, wait for complete dissolution, and after precipitation at room temperature for 2 hours, centrifuge with a high-speed refrigerated centrifuge at 12,000 rpm for 40 minutes, discard th...

example 2

[0051] Example 2: Extraction of myoglobin from pig heart

[0052] 1) After removing the fat and connective tissue of 1000g fresh pig heart, add 2000ml 120mM Tris buffer pH8.8, homogenize quickly with a tissue homogenizer for 10min, centrifuge at 12000rpm for 40min in a high-speed refrigerated centrifuge, remove the precipitate, and obtain a crude extract ;

[0053] 2) Add 50% solid ammonium sulfate to the crude extract, stir slightly, wait for complete dissolution, settle at room temperature for 1 hour, centrifuge at 12,000 rpm for 40 minutes in a high-speed refrigerated centrifuge, and keep the supernatant;

[0054] 3) Add ammonium sulfate solid to the supernatant to a saturated state, stir slightly, and wait for complete dissolution. After precipitation at room temperature for 2 hours, centrifuge with a high-speed refrigerated centrifuge at 12,000 rpm for 40 minutes, discard the supernatant, and obtain the precipitate;

[0055] 4) The precipitate was redissolved in 35% ammo...

example 3

[0058] Example 3: Extraction of myoglobin from pig heart

[0059] 1) After removing the fat and connective tissue of 800g fresh pig heart, add 1400ml, 250mM boric acid buffer pH7.2, homogenize rapidly with a tissue homogenizer for 25min, centrifuge at 8000rpm in a high-speed refrigerated centrifuge for 40min, remove the precipitate, and obtain crude extract;

[0060] 2) Add 60% solid ammonium sulfate to the crude extract, stir gently, settle at room temperature for 1 hour, centrifuge at 8000 rpm for 40 minutes in a high-speed refrigerated centrifuge, and keep the supernatant;

[0061] 3) Add ammonium sulfate solid to the supernatant to a saturated state, precipitate at room temperature for 2 hours, centrifuge at 7500 rpm for 50 minutes in a high-speed refrigerated centrifuge, discard the supernatant, and obtain the precipitate;

[0062] 4) The precipitate was redissolved with 40% saturated ammonium sulfate buffer solution to 1400ml, filtered through a 0.8μm filter membrane an...

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Abstract

The invention provides a method for extracting myoglobin in pig hearts and a human total antioxidant status kit containing the myoglobin. The method comprises the following steps: a buffer is added into fresh pig hearts, homogenate and centrifugation are carried out, ammonium sulfate is added into a crude extract, stirring, precipitation, and centrifugation are carried out, and a supernatant is reserved; ammonium sulfate is added into the supernatant till a saturation state, precipitation is carried out for more than 2 hours, centrifugation is carried out, and the supernatant is removed; an ammonium sulfate containing buffer is used for redissolving the precipitate to an original volume, and filtering is carried out for waiting sampling; a hydrophobic filling material is filled into a column for sampling, balance is carried out till proteins do not flow out, the ammonium sulfate containing buffer is used for washing out, and target protein is collected according to an eluting peak. The method has high output and low extraction cost, obtained myoglobin has good stability and can be applied to a TAS kit, in order to detect concentration of total antioxidant substances in serum, urine and other components in body; at the same time the raw materials have abundant sources and are easy to obtain, and batch production requirements of the kit are satisfied.

Description

technical field [0001] The present invention relates to the fields of biochemistry and detection reagents, and specifically relates to a two-step method of ammonium sulfate precipitation and hydrophobic chromatography to obtain high-purity myoglobin from fresh pig hearts, which is made into dry powder by spray drying technology, and It is applied to the preparation of a human total antioxidant status (TAS) kit and a technology for detecting the human total antioxidant status; specifically, it is a method for extracting myoglobin from pig hearts and a human total antioxidant status kit containing the same. Background technique [0002] Reactive oxygen species (Reactive oxygen species, ROS) mainly include hydroxyl radicals, superoxide radicals and hydrogen peroxide. Active oxygen will be produced during the normal physiological metabolism of the body, and some environmental factors such as ultraviolet radiation, smoking, pollution, etc. will also induce the production of activ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N33/50G01N27/447
CPCG01N1/28G01N27/447G01N33/5005
Inventor 邹炳德邹继华张永章玉胜贾江花张帅
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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