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

A technology of immunoglobulin and goose blood, which is applied in the field of separation and purification of biologically active substances, to achieve the effect of large processing capacity, improved separation efficiency and high purity

Inactive Publication Date: 2012-08-08
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 for anticoagulation according to the ratio of 9:1 (volume ratio), centrifuge with a centrifuge at 2500rpm for 20 minutes, remove red blood cells, and let stand at 4°C for 5 hours to remove the fat in the upper layer , and then add 300 mg of silicon dioxide per milliliter of plasma, stir at room temperature for 30 minutes, and filter to obtain delipidated plasma. Take 5ml of defatted plasma, add 5ml of 60mM acetic acid-sodium acetate buffer (pH4) and mix evenly to obtain 10ml of diluted plasma, add 600μl octanoic acid, stir evenly, let stand at 4°C for 2 hours, and centrifuge at 8000rpm for 30 Minutes, 9.5ml supernatant was obtained. The supernatant was filtered with a 0.45 μm filter membrane, the pH value was adjusted to 7.0, and 1 ml was taken as an injection sample. The chromatographic column (1cm inner diameter) is filled with 5ml MEP Hypercel mixed mode medium, the equilibration buffer is 20mM disodium hydrog...

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 with a centrifuge at 3000rpm for 15 minutes to remove red blood cells, and let stand at 8°C for 12 hours to remove the fat in the upper layer , and then add 1000 mg of silicon dioxide per milliliter of plasma, stir at room temperature for 60 minutes, and filter to obtain delipidated plasma. Take 5ml of defatted plasma, add 15ml of 60mM acetic acid-sodium acetate buffer solution (pH4.5) and mix evenly to obtain 20ml of diluted plasma, add 600μl octanoic acid, stir evenly, let stand at 4°C for 4 hours, and wait for the precipitation of miscellaneous 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 membrane, the pH value was adjusted to 5.0, and 2 ml was taken as an injection sample. The chromatographic column (1cm inner diameter) is filled wit...

Embodiment 3

[0026]Take fresh goose blood, add 0.1M sodium citrate solution for anticoagulation according to the ratio of 9:1 (volume ratio), centrifuge with a centrifuge at 3000rpm for 10 minutes, remove red blood cells, let stand at 4°C for 8 hours, and remove the fat in the upper layer , and then add 300 mg of silicon dioxide per milliliter of plasma, stir at room temperature for 60 minutes, and filter to obtain delipidated plasma. Take 5ml of defatted plasma, add 20ml of 60mM acetic acid-sodium acetate buffer solution (pH 5.0) and mix evenly to obtain 25ml of diluted plasma, add 2500μl octanoic acid, stir well, let stand at 8°C for 4 hours, wait for the precipitation of miscellaneous proteins to complete, and then use 10000rpm Centrifuge for 20 minutes to obtain 23.8 ml of supernatant. The supernatant was filtered with a 0.45 μm filter membrane, the pH value was adjusted to 6.0, and 1 ml was taken as an injection sample. The chromatographic column (1cm inner diameter) is filled with 5...

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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 andhigh separation and purification factors, and can be popularized and applied to treatment of blood of other waterfowls.

Description

technical field [0001] The invention relates to a method for separating and purifying biologically active substances, in particular to a method for isolating immunoglobulin IgY (ΔFc) from goose blood. Background technique [0002] my country is the main country that raises waterfowl such as ducks and geese, and the annual breeding total reaches 4.3 billion, accounting for more than 75% of the world's total. Waterfowl blood is the leftovers of the food processing process, some of which are processed into mature blood clots and 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, cow blood and other animal blood resources have been partially utilized in my country to prepare blood meal, extract protein and other bioactive components, while waterfowl blood has not been full...

Claims

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Application Information

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Patent Type & Authority Patents(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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