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Method for extracting rabbit liver metallothionein

A technology of metallothionein and extraction method, applied in metallothionein, specific peptides and other directions, can solve the problem of the use cost of toxic solvent chloroform, etc., and achieve the effects of high solubility, simple operation and simplified production process

Inactive Publication Date: 2011-09-07
SHAANXI INST OF ZOOLOGY NORTHWEST INSTOF ENDANGERED ZOOLOGICAL SPECIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for extracting rabbit liver metallothionein, to overcome the problems of the large amount of ethanol that exists in the prior art, the use of toxic solvent chloroform and the high cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] (1) Induction:

[0023] Take a rabbit and take subcutaneous injection of zinc sulfate aqueous solution in the abdomen every other day. The number of injections is 7 times, respectively 10mg / kg, 20mg / kg, 50mg / kg, 100mg / kg, 150mg / kg, 200mg / kg, 250mg / kg 24 hours after the seventh administration, the rabbit was slaughtered, and the liver was taken and stored in the refrigerator for use;

[0024] (2) Crushing of samples:

[0025] Fresh rabbit liver, weighed after cleaning the blood with normal saline, added 2.5 times the amount of 0.02M Tris-HCl pre-cooled in the refrigerator, pH8.6 buffer, and ground for 6 minutes;

[0026] (3) Heat treatment:

[0027] Put the grinding solution in a water bath preheated to 83°C, quickly heat it to 80°C, keep it for 5 minutes, then quickly cool it to 0~4°C in an ice-water bath, let it stand for 30 minutes, and then refrigerate and centrifuge. Centrifugation conditions: temperature 3°C, speed 10000rpm, 20min;

[0028] (4) acid to adjust t...

Embodiment 2

[0033] (1) Induction:

[0034] Take a rabbit and take subcutaneous injection of zinc sulfate aqueous solution in the abdomen every other day. The number of injections is 7 times, respectively 10mg / kg, 20mg / kg, 50mg / kg, 100mg / kg, 150mg / kg, 200mg / kg, 250mg / kg 24 hours after the seventh administration, the rabbit was slaughtered, and the liver was taken and stored in the refrigerator for use;

[0035] (2) Crushing of samples:

[0036] Fresh rabbit liver, weighed after cleaning the blood stains with normal saline, added 3 times the amount of 0.02M Tris-HCl, pH8.6 buffer solution pre-cooled in the refrigerator, and ground for 4 minutes;

[0037] (3) Heat treatment:

[0038] Put the grinding solution in a water bath preheated to 80°C, heat it quickly to 78°C, keep it for 8 minutes, then quickly cool it to 0~4°C in an ice-water bath, let it stand for 30 minutes, refrigerate and centrifuge, and centrifuge conditions: temperature 3 ℃, speed 10000rpm, 20min;

[0039] (4) acid to adj...

Embodiment 3

[0044] (1) Induction: Same as Example 1;

[0045] (2) Crushing of samples:

[0046] Fresh rabbits were cleaned of blood stains with normal saline, blotted dry with filter paper, accurately weighed 100g of rabbit liver, added 4 times the amount of 0.02M Tris-HCl pre-cooled in the refrigerator, 300ml of pH8.6 buffer, and thoroughly ground;

[0047] (3) Heat treatment:

[0048] Place the grinding solution in a water bath preheated to 83°C, heat it quickly to 80°C, keep it for 5 minutes, then quickly cool it in an ice-water bath to 0-4°C, let it stand for 20 minutes, and then refrigerate and centrifuge, the centrifugation conditions are the same as above;

[0049] (4) acid to adjust the pH for precipitation:

[0050] For the supernatant after centrifugation, adjust the pH value with dilute hydrochloric acid to control the pH value at 4.75, keep it in an ice-water bath for 60 minutes, refrigerate and centrifuge, and adjust the pH value to 7.0;

[0051] (5) Ultrafiltration, dialy...

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PUM

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Abstract

The invention relates to a method for extracting rabbit liver metallothionein. The traditional extracting process has high cost and pollutes the environment because a great deal of ethanol and chloroform are used, and the chloroform is harm to organs, such as liver, kidney and the like of people. The technical scheme adopted by the invention sequentially comprises the following steps of: (1) induction: injecting zinc sulfate into an adult rabbit for 7 times, wherein the dosage is in a sequential progressive increase form; (2) crushing of raw materials; (3) heat treatment: rapidly heating grinding liquid to 78-80 DEG C and maintaining the temperature for 5-10 minutes; then placing the grinding liquid into an ice water bath for rapidly cooling to 0-4 DEG C, freezing and centrifugally separating; (4) pH acid adjustment and impurity protein sedimentation: adjusting a pH value of supernatant fluid so that the pH is between 4 and 5.5 and the temperature is between 0 DEG C and 4 DEG C, maintaining the temperature for 30 minutes or so, and after freezing and centrifugally separating, adjusting the pH between 6.5 and 7.1; and (5) ultrafiltering and concentrating by a dialysis bag. The invention has the advantages of simple extraction process and low cost and can especially reduce the use of toxic solvents.

Description

technical field [0001] The invention relates to the technical field of metallothionein, in particular to a method for extracting rabbit liver metallothionein. Background technique [0002] Metallothionein (MT for short) is a class of low-molecular-weight proteins rich in cysteine ​​and metal ions. Studies and experiments have shown that MT has the functions of detoxifying heavy metals (cadmium, mercury and aluminum), anti-radiation (including ultraviolet rays), eliminating free radicals, and repairing damaged tissues in the human body. Its ability to scavenge hydroxyl free radicals (·OH) It is more than 10,000 times higher than SOD, and its ability to scavenge oxygen free radicals (O) is about 25 times that of glutathione (CSH), especially it can provide organisms with a variety of trace elements (such as Zn, Cu, Co, etc.) , Participate in the storage, transportation and metabolism of certain trace metal elements (such as Zn, Cu, Co, etc.) in the body. Therefore, it is wid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/825C07K1/36C07K1/34C07K1/30
Inventor 齐凡党芯叶蒋志武权清转
Owner SHAANXI INST OF ZOOLOGY NORTHWEST INSTOF ENDANGERED ZOOLOGICAL SPECIES
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