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Method for separating immune globulin IgY(delta Fc) from goose blood

An immunoglobulin and goose blood technology is applied in the field of separation and purification of biologically active substances, and achieves the effects of high separation efficiency, simplified separation steps and large processing capacity

Inactive Publication Date: 2010-12-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there is no literature report on the separation and purification of high-purity IgY(ΔFc) from waterfowl blood such as goose blood

Method used

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  • Method for separating immune globulin IgY(delta Fc) from goose blood

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Experimental program
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Effect test

Embodiment 1

[0022] Take fresh goose blood, add 0.1M sodium citrate solution according to the ratio of 9:1 (volume ratio) for anticoagulation, centrifuge at 2500rpm for 20 minutes in a centrifuge, remove red blood cells, and let stand at 4°C for 5 hours to remove the upper layer of fat. , and then added 300 mg of silica per milliliter of plasma, stirred at room temperature for 30 minutes, and filtered to obtain defatted plasma. Take 5ml of defatted plasma, add 5ml of 60mM acetic acid-sodium acetate buffer (pH4) and mix well to obtain 10ml of diluted plasma, add 600μl of caprylic acid, stir well, let stand at 4°C for 2 hours, and when the impurities are completely precipitated, centrifuge at 8000rpm for 30 minutes to obtain 9.5 ml of supernatant. The supernatant was filtered with a 0.45 μm filter, adjusted to pH 7.0, and 1 ml was taken as the injection sample. The column (inner diameter 1 cm) was filled with 5 ml of MEP Hypercel mixed mode medium, the equilibration buffer was 20 mM disodiu...

Embodiment 2

[0024] Take fresh goose blood, add 0.1M sodium citrate solution for anticoagulation according to the ratio of 9:1 (volume ratio), centrifuge at 3000rpm for 15 minutes in a centrifuge, remove red blood cells, and let stand at 8°C for 12 hours to remove the upper layer of fat. , and then added 1000 mg of silica per milliliter of plasma, stirred at room temperature for 60 minutes, and filtered to obtain defatted plasma. Take 5 ml of defatted plasma, add 15 ml of 60 mM acetic acid-sodium acetate buffer (pH 4.5) and mix well to obtain 20 ml of diluted plasma, add 600 μl of caprylic acid, stir well, stand at 4°C for 4 hours, and wait for the precipitation of impurity proteins to complete. Centrifuge at 10,000 rpm for 20 minutes to obtain 19.6 ml of supernatant. The supernatant was filtered with a 0.45 μm filter, adjusted to pH 5.0, and 2 ml was taken as the injection sample. The column (inner diameter 1cm) was filled with 5ml of MEP Hypercel mixed mode medium, the equilibration buf...

Embodiment 3

[0026]Take fresh goose blood, add 0.1M sodium citrate solution in a ratio of 9:1 (volume ratio) for anticoagulation, centrifuge at 3000rpm for 10 minutes in a centrifuge, remove red blood cells, and let stand at 4°C for 8 hours to remove the upper layer of fat. , and then added 300 mg of silica per milliliter of plasma, stirred at room temperature for 60 minutes, and filtered to obtain defatted plasma. Take 5ml of defatted plasma, add 20ml of 60mM acetic acid-sodium acetate buffer (pH 5.0) and mix well to obtain 25ml of diluted plasma, add 2500μl of caprylic acid, stir evenly, stand at 8°C for 4 hours, and when the impurity protein is completely precipitated, run at 10000rpm Centrifuge for 20 minutes to obtain 23.8 ml of supernatant. The supernatant was filtered with a 0.45 μm filter, adjusted to pH 6.0, and 1 ml was taken as the injection sample. The column (inner diameter 1cm) was filled with 5ml of MEP Hypercel mixed mode medium, the equilibration buffer was 20mM disodium ...

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Abstract

The invention discloses a method for separating immune globulin IgY(delta Fc) from goose blood. The method comprises the following steps: 1) degreased plasm preparation, wherein fresh blood is selected to be centrifuged to remove erythrocyte, and then is degreased to obtain the degreased plasm; 2) octanoic acid precipitate, wherein octanoic acid is added to the plasm to reach a certain concentration, hybrid protein is precipitated and removed, and is centrifugally separated to obtain supernate; 3) column chromatography, wherein the supernate is separated by a chromatographic column which is filled with hybrid mode adsorbent to collect elution peak; and 4) desalination and drying, wherein the collected fluid is desalinated, refrigerated and dried to obtain the immune globulin IgY(delta Fc) with purity of over 95 percent. The method is characterized in that a new separation process is designed, the immune globulin IgY (delta Fc) can be separated from the goose blood; and the key of the method is that the immune globulin IgY (detal Fc) can be directly extracted from the supernate which is precipitated from the octanoic acid. The method has the advantages of simple operation steps and high separation and purification factors, and can be popularized and applied to treatment of blood of other waterfowls.

Description

technical field [0001] The present invention relates to a method for separating and purifying biologically active substances, in particular to a method for separating immunoglobulin IgY (ΔFc) from goose blood. Background technique [0002] my country is the main country for raising waterfowl such as ducks and geese, with an annual total of 4.3 billion birds, accounting for more than 75% of the world's total. Waterfowl blood is the leftovers of food processing. Some of the processed blood clots are eaten, and most of them are directly discharged into nature as waste, which not only wastes precious biological resources, but also causes certain pollution to the environment. Animal blood is an important source of protein and minerals. At present, pig blood, bovine blood and other animal blood resources have been partially used in my country to prepare blood meal, extract protein and other biologically active components, while waterfowl blood has not been fully utilized. Goose b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/06C07K1/36C07K1/30C07K1/22
Inventor 林东强童红飞姚善泾
Owner ZHEJIANG UNIV
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