CN101698680A - PEG-gelchromatography combined purification method for egg yolk immunoglobulins (IgY) - Google Patents
PEG-gelchromatography combined purification method for egg yolk immunoglobulins (IgY) Download PDFInfo
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- CN101698680A CN101698680A CN200910041330A CN200910041330A CN101698680A CN 101698680 A CN101698680 A CN 101698680A CN 200910041330 A CN200910041330 A CN 200910041330A CN 200910041330 A CN200910041330 A CN 200910041330A CN 101698680 A CN101698680 A CN 101698680A
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Abstract
The invention relates to a purification method of a biological antiviral and antiseptic egg yolk immunoglobulin (IgY) antibody, which comprises the following steps: (1) coarsely extracting an IgY egg yolk antibody by using PEG ( polyethylene glycol) for settlement and using chloroform for extraction and removing hybrid proteins and fat from egg yolks; (2) subjecting the antibody settle sediment obtained after coarse extraction to gelchromatography purification; and (3), testing the purity and the sterilizing performance of the IgY egg yolk antibody which is purified, wherein the purity is 99 percent, the Escherichia coli inhibition rate is 95.8 percent and the Staphylococcus aureus inhibition rate is more than 99 percent.
Description
Technical field: what the present invention relates to is the purification process of the antiviral antibiotic IgY immunoglobulin (Ig) of a kind of biology, the coupling PEG precipitator method, chloroform extraction method and gel chromatography.
Background technology
In general, with the specific antigens immune hen and after immunne response, can from its egg yolk, constantly obtain specific immune globulin antibody (Immunoglobulin of egg yolk, IgY), this immune globulin antibody is similar to the unlimitedness of monoclonal antibody, again because of its antibody sources in same individuality, so the homogeneity of antibody also is similar to monoclonal antibody.In addition, the purity height of IgY antibody, high specificity can not resemble and can intersect sero-reaction or anaphylaxis the antibody that other Mammalss produce, thereby it is safest biotechnological formulation.
At present the purifying of IgY immunoglobulin (Ig) generally is to dilute by PEG precipitation, chloroform extracting, water earlier and method such as freeze thawing carries out passing through polishing purifications such as ultrafiltration, ion exchange chromatography, affinity chromatography again after antibody slightly carries.The bio-affinity purifying method of the IgY of the Li Rong of Shanghai Communications University show and Liu Haoran invention is to utilize the synthetic a kind of bionical affine parting material of basic chromatography media to be prepared into the separation and purification that affinity column carries out yolk antibody.And this patent is that coupling PEG precipitates, the chloroform extracting is carried out carrying out polishing purification by the gel chromatography chromatography again after antibody is slightly carried, and is with low cost, and the purifying time is short, can be fit to large-scale operation.
Summary of the invention
The present invention comes the antiviral antibiotic IgY immunoglobulin (Ig) of purifying biological after gel chromatographic columns after coupling PEG precipitation and chloroform extracting are slightly carried yolk antibody.This purification process cost is low, the cycle is short, effective, can be fit on a large scale, quick, easy separation and purification immunoglobulin (Ig) in enormous quantities.
This IgY immunoglobulin (Ig) is a kind of passive immunization technology of using antibody engineering, a kind of immunoglobulin (Ig) that extracts the yolk of extraordinary breeding chicken after immunity, have antibody titer height, specificity strong (only killing pathogenic bacterium), can not produce and intersect advantages such as serum or anaphylaxis, homogeneity are good, and the yolk antibody good stability, can not produce the disadvantage of using microbiotic, anti-inflammatory enzyme to occur.
The present invention is achieved by the following technical solutions, with traditional method yolk antibody is slightly proposed separation after, directly go up the gel chromatography column purification, have that pre-treatment is simple to operate, purifying fast, characteristics such as antibody purity and active height.
The present invention includes following steps:
(1) the IgY yolk antibody slightly carries
Contain 30% lipid in the yolk approximately, many forms with lipoprotein exist, so will select suitable method to remove foreign protein.Polyoxyethylene glycol (PEG) is the non-ionic type water-soluble polymers, and very strong wetting ability is arranged, and is fit to the separation and Extraction of IgY water-soluble protein, and sedimentation time is short, does not influence centrifugal treating, even and high density do not cause protein denaturation yet.Add the chloroform extracting, can remove lipid more completely, and supernatant liquor compares clear, antibody rate of recovery height.So coupling polyethylene glycol precipitation of the present invention and chloroform extracting are slightly carried the IgY yolk antibody.
(2) gel chromatography IgY yolk antibody
The antibody precipitation of slightly carrying directly goes up Sephadex G-75 pillar with an amount of pH 7.4PBS dissolving.
(3) mensuration of IgY yolk antibody purity
Purity by antibody after the SDS-PAGE gel electrophoresis purification Identification.
(4) IgY yolk antibody bactericidal property is measured
The intestinal bacteria of IgY yolk antibody behind the purifying and proper concn and streptococcus aureus are fallen dull and stereotyped the cultivation jointly, the bactericidal property of the size detection IgY antibody by inhibition zone.
Embodiment
Embodiment 1 slightly carries the IgY yolk antibody
Get yolk (removing egg white and membrane of yolk), add 4%PEG6000 while stirring, volume is 3 times of yolk volume, when also remaining 100ml PEG6000, adds the chloroform of 1/4 yolk volume, is continuing to add remaining PEG6000 under the slow speed agitation condition.With 6000rpm, 10 ℃ centrifugal 10 minutes, supernatant liquor with filtered through gauze after, dripping volume while stirring is the 40%PEG6000 of 0.3 times of supernatant liquor, and again with 6000rpm, 10 ℃ centrifugal 10 minutes, abandon supernatant, be all over the gained precipitation with distilled water flushing 4-5 and slightly carry antibody.
Embodiment 2 gel chromatography IgY yolk antibodies
The above-mentioned isolated antibody precipitation of slightly carrying with an amount of pH7.4PBS dissolving, is directly gone up Sephadex G-75 pillar and filters.With pH7.40.01mol/L PBS wash-out, flow velocity is 0.3ml/min, applied sample amount 3-5ml.
The mensuration of IgY yolk antibody purity behind embodiment 3 purifying
Gel chromatography is collected the pure product of IgY, carries out the SDS-PAGE gel electrophoresis and identifies that resolving gel concentration is 15%, and concentrated glue is 5%, and voltage is respectively 110V and 80V during electrophoresis.Applied sample amount is about 20 μ l.Coomassie brilliant blue staining 2h, the shaking table decolouring.
IgY yolk antibody bactericidal property test behind embodiment 4 purifying
Get in the suspension that two parts of above-mentioned IgY yolk antibodies of 5mlPBS dissolved join the intestinal bacteria of proper concn of 100 μ l and streptococcus aureus respectively, mix, add 40～50 ℃ of PDA liquid nutrient mediums of 15ml place the sterilization plate in 33～35 ℃ of incubators cultivate 24h.The bacterium colony result shows that the IgY yolk antibody all has the good restraining effect to intestinal bacteria and streptococcus aureus, and inhibiting rate is respectively more than 95.8% and 99%.
Claims (6)
1. the PEG of a Yolk immunoglobulin and gel chromatography coupling purification process is characterized in that through after PEG precipitation and the chloroform extracting, and the above-mentioned antibody precipitation of slightly carrying is directly gone up gel chromatographic columns after the PBS dissolving.IgY immunoglobulin (Ig) behind the purifying is carried out purity and bactericidal property mensuration.
2. the PEG of Yolk immunoglobulin according to claim 1 and gel chromatography coupling purification process is characterized in that, the first step is slightly put forward process and is: get yolk (removing egg white and membrane of yolk), add PEG6000 and stir, can remove the lipoprotein in the yolk.Use the chloroform extracting simultaneously, promptly add the chloroform of 1/4 yolk volume, after fully mixing, the centrifuging and taking supernatant.
3. the PEG of Yolk immunoglobulin according to claim 1 and gel chromatography coupling purification process, it is characterized in that, the second step gel chromatography process is: varying in size according to the volume of solute molecule in mobile phase solvent reaches isolating purpose, and we directly go up Sephadex G-75 pillar with above-mentioned isolating IgY antibody after the PBS dilution.By obtaining highly purified IgY antibody behind the gel chromatography chromatography.
4. the PEG of Yolk immunoglobulin according to claim 1 and gel chromatography coupling purification process is characterized in that, through antibody slightly carry with the gel chromatography chromatography after, the IgY antibody of purifying is carried out purity and bactericidal property is measured.
5. the PEG of Yolk immunoglobulin according to claim 4 and gel chromatography coupling purification process is characterized in that, by the purity of antibody after the SDS-PAGE gel electrophoresis purification Identification.
6. the PEG of Yolk immunoglobulin according to claim 4 and gel chromatography coupling purification process, it is characterized in that, get the intestinal bacteria of this an amount of IgY yolk antibody and proper concn and streptococcus aureus and fall dull and stereotyped the cultivation jointly, the fungicidal activity of the size detection IgY antibody by inhibition zone.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200910041330A CN101698680A (en) | 2009-07-22 | 2009-07-22 | PEG-gelchromatography combined purification method for egg yolk immunoglobulins (IgY) |
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| CN200910041330A CN101698680A (en) | 2009-07-22 | 2009-07-22 | PEG-gelchromatography combined purification method for egg yolk immunoglobulins (IgY) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304182A (en) * | 2011-08-24 | 2012-01-04 | 双胞胎饲料有限公司 | Preparation method and use of enterotoxigenic Escherichia coli egg yolk antibody powder |
| CN102850453A (en) * | 2012-09-03 | 2013-01-02 | 华中农业大学 | Method for extracting and separating immunoglobulin IgY from egg yolk |
| CN103554251A (en) * | 2013-11-12 | 2014-02-05 | 福州大北农生物技术有限公司 | Simple method for extracting egg yolk antibodies |
-
2009
- 2009-07-22 CN CN200910041330A patent/CN101698680A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304182A (en) * | 2011-08-24 | 2012-01-04 | 双胞胎饲料有限公司 | Preparation method and use of enterotoxigenic Escherichia coli egg yolk antibody powder |
| CN102850453A (en) * | 2012-09-03 | 2013-01-02 | 华中农业大学 | Method for extracting and separating immunoglobulin IgY from egg yolk |
| CN103554251A (en) * | 2013-11-12 | 2014-02-05 | 福州大北农生物技术有限公司 | Simple method for extracting egg yolk antibodies |
| CN103554251B (en) * | 2013-11-12 | 2016-01-20 | 福州大北农生物技术有限公司 | A kind of method of simple and easy extraction yolk antibody |
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Application publication date: 20100428 |