CN101955529A - Method for extracting yolk immune globulin by one-way ultrafiltration - Google Patents
Method for extracting yolk immune globulin by one-way ultrafiltration Download PDFInfo
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- CN101955529A CN101955529A CN2010105044578A CN201010504457A CN101955529A CN 101955529 A CN101955529 A CN 101955529A CN 2010105044578 A CN2010105044578 A CN 2010105044578A CN 201010504457 A CN201010504457 A CN 201010504457A CN 101955529 A CN101955529 A CN 101955529A
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Abstract
The invention provides a method for simply and rapidly extracting yolk immune globulin, which belongs to the technical field of bioseparation engineering. The yolk immune globulin can be extracted from yolk mainly by applying an ultrafiltration separation technology through four steps of dilution, frozen thawing, centrifugation and one-way ultrafiltration; and the invention does not use an organic solvent in the extracting process, effectively avoids albuminous degeneration caused by the organic solvent and completely eradicates product potential safety hazards caused by solvent residual.
Description
Technical field
The invention belongs to bioseparation engineering and technical field, specifically is the method for using one way ultra-filtration technique rapid extraction Yolk immunoglobulin from Ovum Gallus domesticus Flavus, and product purity can reach 85%.
Background technology
Immunoglobulin (Ig) is that body is subjected to antigen (as pathogenic agent) to stimulate the back to produce, and it mainly acts on is to play immune response with antigen, generates antigen-antibody complex, thereby the blocking-up pathogenic agent makes pathogenic agent lose pathogenic effects to the harm of body.
Yolk immunoglobulin (IgY) is meant the main immunoglobulin that exists in the Ovum Gallus domesticus Flavus, is hen 7～10 days after accepting immunity, i.e. yolk ripening stage, optionally transferred in the yolk by the immunoglobulin IgG in the hen blood and form.So IgY is equivalent to mammiferous IgG.Simultaneously, compare with IgG, Yolk immunoglobulin (IgY) has plurality of advantages: (1) IgY does not combine with Rheumatoid factors, polyclonal in the serum, does not activate the Mammals complement system, also not with the Fc receptors bind, help improving specificity and susceptibility that immunodiagnosis detects; (2) stable in properties of IgY, the active maintenance well,, below 75oC, the IgY activity is unaffected substantially, and between pH3～12, activity remains unchanged; (3) need not blood sampling, meet the international animal conservation regulation; (4) cheap and easy to get, the amount of the IgY that contains in one piece of egg is equivalent to the amount extracted from 200mL blood; (5), can not intersect serological reaction between bird IgY and the mammalian immune sphaeroprotein owing to plant system and distance takes place differ greatly.Therefore, in recent years, many scholars and biological products company are all getting down to the separation of IgY, purifying research.
At present, the main method of separating IgY has from yolk: salting-out process, polyethylene glycol precipitation, chloroform extraction method, medium chain fatty acid method, surfactant method etc.Yet all there is certain limitation in these technology, and wherein, salting-out process and polyethylene glycol precipitation cause proteinic inactivation and sex change easily; The processing cycle of chloroform extraction method, medium chain fatty acid method, surfactant method is longer, has residue problem, is unfavorable for that IgY's is medicinal.
Membrane separation process is often referred to the film that utilizes natural or synthetic, is impellent with outside energy or chemical potential difference, to two components or multi-component mixture separate, the method for purification and enrichment.Membrane separation technique is a high efficient separation technology that grows up the 1950's, compares with traditional isolation technique, but has separation efficiency height, the low operate continuously of energy consumption, is easy to amplify, advantage such as pollution-free.
Ultra-filtration membrane is meant membrane pore size between 1～100nm, has the composite membrane of unsymmetrical structure.Because the size of its aperture size and biomacromolecule is comparatively approaching, so can be used for the isolation and purification of biomacromolecule such as protein etc.Ultra-filtration and separation is a molecular level, can hold back the macromole solute in the solution, and the small molecules solute is seen through; Sometimes also can make the protein molecule of pre-separation and film surface have electric charge of different nature, by the electrostatic interaction between the protein molecule, between protein and the film surface, reach isolating purpose again by microenvironment in the regulator solution.At present, in the biological processing field, ultra-filtration technique is usually used in protein purification and concentrates, in industrial existing more application, but because kinds of protein complexity in the natural system, and there is more isozyme, uses the direct examples of many successful that obtains highly purified functional protein of separating of ultrafiltration process few.
Summary of the invention
The method that the purpose of this invention is to provide a kind of rapid extraction Yolk immunoglobulin.
The technological line that the present invention takes is:
1. eggshell, egg white and the vitelline membrane of egg are removed, got egg yolk liquid;
2. use deionized water or ionic strength yolk liquid to be diluted to 6-15 doubly less than the buffered soln of 500mM, pH4.0-10.0, the best be 7-10 doubly, fully stir more than 20 minutes, in-20 ℃ or more freezing more than 8 hours under the low temperature, thaw, centrifugal, get supernatant liquor;
3. utilize molecular weight cut-off for the ultra-filtration membrane of 100-150kDa the supernatant liquor that step 2 obtains to be carried out ultra-filtration and separation, separation condition is: pH value of solution value 4-10, ionic strength 0-1M, trapped fluid are highly purified Yolk immunoglobulin solution;
4. with the freeze-drying of resulting Yolk immunoglobulin (IgY) solution, promptly obtain Yolk immunoglobulin (IgY) dry powder.
Embodiment
Further describe the present invention below in conjunction with embodiment.
Embodiment 1, extracts the method for Yolk immunoglobulin (IgY) from the Calusena lansium egg, step:
1. get four fresh Calusena lansium eggs, clean eggshell, dry with paper again, behind removal eggshell and the egg white, gently membrane of yolk is punctured, collect egg yolk liquid, about 50mL with syringe needle with tap water;
2. use deionized water that the 50mL yolk liquid that above-mentioned steps obtains is diluted to 350mL, fully stir 20 minutes after, place-20 ℃ freezing 8 hours, thaw, thawing solution under 4 ℃, centrifugal with 3000rpm, get supernatant liquor, about 300mL;
3. utilize molecular weight cut-off for the polysulfone membrane of 100kDa the supernatant liquor that step 2 obtains to be carried out ultra-filtration and separation, separation condition is: 4 ℃, pH6.0, trapped fluid are Yolk immunoglobulin (IgY) solution (about 160mL), purity 82.5%;
4. the IgY solution that obtains is placed-60 ℃ of cold-trap freeze-drying 8 hours, obtain the about 541mg of IgY dry powder.
Embodiment 2, extract the method for Yolk immunoglobulin (IgY) from white skin egg, step:
1. get three fresh white skin eggs, clean eggshell, dry with paper again, behind removal eggshell and the egg white, gently membrane of yolk is punctured, collect egg yolk liquid, about 38mL with syringe needle with tap water;
2. use the phosphate buffer soln of pH4.0,100mM that the yolk liquid that above-mentioned steps obtains is diluted to 380mL, fully stir 30 minutes after, place-20 ℃ freezing 12 hours, thaw, with thawing solution under 4 ℃, centrifugal with 6000rpm, get supernatant liquor, about 320mL;
3. utilize molecular weight cut-off for the regenerated cellulose film of 100kDa the supernatant liquor that step 2 obtains to be carried out ultra-filtration and separation, separation condition is 4 ℃, pH8.0, gets to hold back to be Yolk immunoglobulin (IgY) solution liquid (about 170mL), purity 84%;
4. the IgY solution that obtains is placed-60 ℃ of cold-trap freeze-drying 12 hours, obtain the about 415mg of IgY dry powder.
Embodiment 3, extract the method for Yolk immunoglobulin (IgY) from egg, step:
1. get five fresh eggs, clean eggshell, dry with paper again, behind removal eggshell and the egg white, gently membrane of yolk is punctured, collect egg yolk liquid, about 60mL with syringe needle with tap water;
2. use the phosphate buffer soln of pH10.0,400mM that the yolk liquid that above-mentioned steps obtains is diluted to 420mL, fully stir 25 minutes after, place-20 ℃ freezing 10 hours, thaw, with thawing solution 4 ℃ centrifugal with 5000rpm down, get supernatant liquor, about 360mL;
3. utilize molecular weight cut-off for the poly (ether sulfone) film of 150kDa the supernatant liquor that step 2 obtains to be carried out ultra-filtration and separation, separation condition is 4 ℃, pH7.0, gets trapped fluid and is Yolk immunoglobulin (IgY) solution liquid (about 200mL), purity 80%;
4. the IgY solution that obtains is placed-60 ℃ of cold-trap freeze-drying 11 hours, obtain the about 642mg of IgY dry powder.
Claims (3)
1. the method for a rapid extraction Yolk immunoglobulin is to be the crude enzyme liquid of the ultra-filtration membrane uf processing Yolk immunoglobulin of 100-150kDa with the molecular weight cut-off, finally obtains the yellow immunoglobulin (Ig) of higher degree; The crude enzyme liquid of described yellow immunoglobulin (Ig) is the supernatant liquor that egg yolk liquid is obtained through dilution, freeze thawing, after centrifugal.
2. the method for the yellow immunoglobulin (Ig) of extraction according to claim 1 is characterized in that: described when being the ultrafiltration membrane treatment of 100-150kDa with molecular weight cut-off, and pH value of solution value 4-10, ionic strength 0-1M.
3. the method for the yellow immunoglobulin (Ig) of extraction according to claim 1 and 2 is characterized in that: described is that the kit form of the ultra-filtration membrane of 100-150kDa is hollow cellulose film, flat sheet membrane or rolled film with molecular weight cut-off.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105044578A CN101955529A (en) | 2010-10-13 | 2010-10-13 | Method for extracting yolk immune globulin by one-way ultrafiltration |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105044578A CN101955529A (en) | 2010-10-13 | 2010-10-13 | Method for extracting yolk immune globulin by one-way ultrafiltration |
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| CN101955529A true CN101955529A (en) | 2011-01-26 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102584994A (en) * | 2012-03-26 | 2012-07-18 | 广东紫金正天药业有限公司 | Egg yolk antibody for curing colibacillosis and preparation method thereof |
| CN111718410A (en) * | 2020-06-05 | 2020-09-29 | 江南大学 | Method for preparing yolk immunoglobulin |
-
2010
- 2010-10-13 CN CN2010105044578A patent/CN101955529A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102584994A (en) * | 2012-03-26 | 2012-07-18 | 广东紫金正天药业有限公司 | Egg yolk antibody for curing colibacillosis and preparation method thereof |
| CN111718410A (en) * | 2020-06-05 | 2020-09-29 | 江南大学 | Method for preparing yolk immunoglobulin |
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Application publication date: 20110126 |