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Method for protein affinity purification for IgY

A protein and affinity technology, applied in the field of biomedicine, can solve the problems of complicated steps, low recovery rate of IgY antibody, long time consumption, etc., and achieve the effect of simple process

Inactive Publication Date: 2015-07-08
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional methods used for IgY purification include ammonium sulfate precipitation, polyethylene glycol precipitation, water dilution, ultrafiltration, gel filtration, thiophilic gel chromatography, and ion exchange chromatography. These methods have complicated steps , takes a long time
In egg yolk, abundant lipoproteins are usually removed by chloroform denaturation and centrifugation with very low recovery of IgY antibodies

Method used

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Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0042] A method for protein affinity purification of IgY, comprising the following steps:

[0043] (1), gene cloning of protein M;

[0044] (2), construct expression vector, carry out soluble expression to protein M by expression vector, purify soluble protein M;

[0045] (3) Coupling the purified soluble protein M in step (2) with NHS-activated Sepharose 4FF according to a mass ratio of 1:1 to produce an affinity purification medium;

[0046] (4), the primary product of the IgY antibody is affinity purified through a protein M agarose gel column to obtain high-purity IgY, and the primary product of the IgY antibody is a mixture of egg yolk liquid and distilled water of equal quality;

[0047] (5) The IgY antibody solution eluted after purification was dialyzed overnight at 4° C. in PBS solution, and the pH of the PBS solution was 7.0;

[0048] (6) Collecting the liquid in the dialysis bag is the high-purity IgY antibody.

[0049] In this example, the template for gene clon...

specific Embodiment 2

[0071] A method for protein affinity purification of IgY, comprising the following steps:

[0072] (1), gene cloning of protein M;

[0073] (2), construct expression vector, carry out soluble expression to protein M by expression vector, purify soluble protein M;

[0074] (3) Coupling the purified soluble protein M in step (2) with NHS-activated Sepharose 4FF at a mass ratio of 1:3 to produce an affinity purification medium;

[0075] (4), the primary product of the IgY antibody is affinity purified through a protein M agarose gel column to obtain high-purity IgY, and the primary product of the IgY antibody is a mixture of egg yolk liquid and distilled water of equal quality;

[0076] (5) The IgY antibody solution eluted after purification was dialyzed overnight at 4° C. in PBS solution, and the pH of the PBS solution was 7.4;

[0077] (6) Collecting the liquid in the dialysis bag is the high-purity IgY antibody.

[0078] In this example, the template for gene cloning of pro...

specific Embodiment 3

[0100] A method for protein affinity purification of IgY, comprising the following steps:

[0101] (1), gene cloning of protein M;

[0102] (2), construct expression vector, carry out soluble expression to protein M by expression vector, purify soluble protein M;

[0103] (3) Coupling the purified soluble protein M in step (2) with NHS-activated Sepharose 4FF at a mass ratio of 1:2 to produce an affinity purification medium;

[0104] (4), the primary product of the IgY antibody is affinity purified through a protein M agarose gel column to obtain high-purity IgY, and the primary product of the IgY antibody is a mixture of egg yolk liquid and distilled water of equal quality;

[0105] (5) The IgY antibody solution eluted after purification was dialyzed overnight at 4° C. in PBS solution, and the pH of the PBS solution was 7.2;

[0106] (6) Collecting the liquid in the dialysis bag is the high-purity IgY antibody.

[0107] In this example, the template for gene cloning of pro...

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Abstract

The invention specifically relates to a method for protein affinity purification for IgY. The method comprises the following steps: 1. carrying out gene cloning for protein M; 2. constructing an expression vector, carrying out soluble expression on the protein M through the expression vector, and purifying the soluble protein M; 3. coupling the purified soluble protein M with NHS-activated agarose gel 4FF to produce an affinity purification medium; 4. carrying out affinity purification on an IgY antibody primary product through a protein M agarose gel column to obtain high-purity IgY, wherein the IgY antibody primary product is the mixture of equal-mass yolk solution and distilled water; 5. dialyzing the IgY antibody solution obtained by eluting after purification in PBS solution overnight at 4 DEG C, wherein the pH value of the PBS solution is 7.0-7.4; 6. collecting a liquid, that is, a high-purity IgY antibody in a dialysis bag. The method disclosed by the invention has the following beneficial effects: 1. the working procedures are simple; 2. the IgY purification efficiency is up to 95%; 3. the method is more environment-friendly, green and healthy.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for protein affinity purification of IgY. Background technique [0002] Egg yolk antibody (egg yolk antibody, IgY) is a specific antigen obtained by immunizing poultry (chicken) with specific antigens, thereby producing IgY in the body, passing from the mother to the egg yolk through the blood, enriching in the egg yolk, and then separating the egg yolk. Sexual polyclonal antibodies. Compared with obtaining antibodies from mammalian serum, it avoids routine animal blood collection and reduces the pain of animals. At the same time, antibodies are obtained through egg yolk, with large yield and simple preparation. 50-100mg of antibodies can be purified from each egg, and the hen It is easy to raise, low in cost, convenient for large-scale production of specific antibodies, and has obvious economic advantages. [0003] Similar to mammalian immunoglobulin G (immunogl...

Claims

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Application Information

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IPC IPC(8): C07K16/02C07K1/22
Inventor 张小莺江雪梅
Owner NORTHWEST A & F UNIV
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