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Method for extracting gamma-livetin (IgY) from yolk

A technology for yolk globulin and self-yolk, which is applied in the field of extracting γ-vitelloglobulin from egg yolk, can solve problems such as time-consuming, difficult purification of IgY, difference in amino acid sequence, etc., and achieves simple steps, low reagent cost, and recovery. High efficiency and purity

Inactive Publication Date: 2014-02-12
王玉麒
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Since the antibodies induced and expressed in animals are mostly distributed in the blood, medium and large-sized rabbits and sheep are usually used for the production of polyclonal antibodies. The accompanying disadvantages are: space requirements and feeding costs for raising animals Higher, and mammals are closer to each other, not suitable for making antibodies to mammalian conserved proteins
[0005] However, due to the rich lipid (up to 34%) and other proteins in the yolk, the purification of IgY is not easy, and the application of IgY is still not common enough.
Furthermore, due to the difference in the amino acid sequence of IgY and IgG at the Fc site, the A / G protein column, which is widely used in the purification of IgG, cannot be used to adsorb and separate IgY.
The existing extraction methods of egg yolk IgY all start with the degreasing step, including water dilution method, PEG precipitation method, chloroform precipitation method, glue precipitation method, etc.; followed by salting out, or filtration, or ion exchange column, or IgY is further purified by specific adsorption columns, or different combinations of the above methods; the process steps are not only cumbersome and time-consuming

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  • Method for extracting gamma-livetin (IgY) from yolk
  • Method for extracting gamma-livetin (IgY) from yolk
  • Method for extracting gamma-livetin (IgY) from yolk

Examples

Experimental program
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Effect test

Embodiment 1

[0042] [Example 1-The stirring time test of crudely extracted egg yolk IgY]

[0043] [crude extraction of egg yolk IgY]

[0044] Take a quantitative volume of egg yolk liquid, add 9 times the volume of 0.1M, pH5.0 sodium acetate solution, mix well, place it at 4°C for four different stirring times (0.5h, 1h, 3h, 6h), Then centrifuge at 12,000×g at 4°C for 30 minutes, and the collected supernatant is the crude extract of egg yolk. After measuring the volume, add NaN 3 to a final concentration of 0.02% (w / v), and stored at 4°C for subsequent analysis.

[0045] [Quantitative analysis of IgY]

[0046] Enzyme-linked immuno-sorbent assay (ELISA) was used to measure the IgY content in the analyzed samples. First, serially dilute the IgY standard (Jackson, 003-000-003) and the sample to be tested with 0.2M borate, and then dispense 50 μL each into the sample well of the ELISA plate (Costar EW-01959-20) After 6h (room temperature) or overnight (4°C) adsorption, wash with wash buffe...

Embodiment 2

[0055] [Extraction effect of embodiment 2-pH value on egg yolk IgY]

[0056] Four 0.1M sodium acetate solutions with pH values ​​of 4.2, 4.6, 5.0, and 5.4 were prepared, and then used to carry out the same process as [crude extraction of egg yolk IgY] in Example 1, wherein the stirring time was 30 minutes. After centrifugation, the figure 2 As shown, there are obvious differences in the appearance of the crude egg yolk extracts of the four different pH conditions: the crude extract of pH 4.2 is the most yellow and turbid, followed by the crude extract of pH 5.4, and the crude extract of pH 5.0 is relatively the clearest . Since the turbid yellow color of the egg yolk extract is mainly affected by the content of water-insoluble lipids, it can be deduced that the fat-removing effect of the sodium acetate solution is pH dependent.

[0057] In addition, the above-mentioned crude egg yolk extracts under four different pH conditions were subjected to the same analysis as [quantit...

Embodiment 3

[0061] [pH value change of embodiment 3-egg yolk crude extract]

[0062] In order to explore why the use of sodium acetate solution can have a higher extraction efficiency of egg yolk IgY than commonly used acid water, this experiment design is divided into 3 groups: the first group takes quantitative egg yolk and adds 9 times the volume of 0.1M sodium acetate solution (pH 5.0), the initial pH of the diluted sample is 5.1 (sodium acetate dilution group); the second group gets the same amount of egg yolk as the first group, and adds 9 times the volume of secondary water, the initial pH of the diluted sample is 6.2, Then immediately add a small amount of 0.1N HCl (<2% total volume) to adjust its pH value to 5.0 (acid water dilution group); the third group is simple secondary water, and add a small amount of 0.1N HCl to adjust its pH value to 5.0 (acid water control group). After the preparation of the three groups of samples was completed, they were transferred to 4°C for stirr...

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Abstract

The invention discloses a method for extracting gamma-livetin (IgY) from yolk. The method comprises the following steps: (A) providing a buffer salt solution, a yolk sample and an inorganic salt; (B) diluting the yolk sample by using the buffer salt solution, stirring for a preset time, and centrifuging to obtain a supernatant; and (C) performing salting-out precipitation on the supernatant by using the inorganic salt solution, so that a high-purity gamma-livetin (IgY) is obtained, wherein a pH value of the buffer salt solution is in a range from 4.6 to 5.4, the concentration of the buffer salt solution is in a range from 0.05 to 0.15M, and the saturation level of the inorganic salt is 30 to 60 percent.

Description

technical field [0001] The invention relates to a method for extracting gamma-yolk globulin (IgY) from egg yolk, in particular to a method for simply and quickly extracting high-purity gamma-yolk globulin. Background technique [0002] When the immune system of a vertebrate encounters a specific foreign antigen, it often induces the production of a variety of antibody molecules that recognize different epitopes (epitopes) of the antigen. The collection of these antibody molecules is collectively called polyclonal antibodies; Antigen epitopes have strong binding force and high specificity, so they are often used in the identification, analysis and purification of specific antigen molecules. At present, polyclonal antibodies have become one of the indispensable tools in basic research, general testing, and medical treatment. [0003] Since the antibodies induced and expressed in animals are mostly distributed in the blood, medium and large-sized rabbits and sheep are usually ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/02C07K1/30
Inventor 王玉麒汪宗明
Owner 王玉麒
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