CN102276718A - Process for extracting yolk immunoglobulin by water-caprylic acid two-step method - Google Patents
Process for extracting yolk immunoglobulin by water-caprylic acid two-step method Download PDFInfo
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- CN102276718A CN102276718A CN2011102104092A CN201110210409A CN102276718A CN 102276718 A CN102276718 A CN 102276718A CN 2011102104092 A CN2011102104092 A CN 2011102104092A CN 201110210409 A CN201110210409 A CN 201110210409A CN 102276718 A CN102276718 A CN 102276718A
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Abstract
The invention relates to a process for extracting yolk immunoglobulin by a water-caprylic acid two-step method, and belongs to the technical field of yolk immunoglobulin preparation in biotechnological pharmaceutics. The process comprises the following steps: step one, directly mixing water and a yolk stock solution, breaking an emulsification state by mechanical stirring and the effect of water to separate oil from an aqueous solution and form a precipitate; step two, mixing the aqueous solution extracted in step one with caprylic acid, allowing caprylic acid to react with other protein in the aqueous solution (wherein immunoglobulin does not react) to form a large-particle substance, separating from the immunoglobulin aqueous solution so as to reach the purpose of immunoglobulin extraction. The method has the advantages of low cost, high antibody recovery rate, simple operation, etc.
Description
One technical field
The present invention relates to water-sad two-step approach and extract immunoglobulin (Ig) technology, a kind of a kind of technology that is used for yolk extraction immunoglobulin (Ig) belongs to Yolk immunoglobulin preparing technical field in the biotechnological pharmaceutics.
Two background technologies
Immunoglobulin of Yolk G antibody is a kind of 7S immunoglobulin, and is slightly different with Mammals IgG.This protein molecular weight is about 180kDa, contains two subunits, and promptly the light chain of the heavy chain of 67～70kDa and 22～30kDa claims IgY(Yolk Immunoglobulin again), be present in the yolk of immunity back hen.
Experiment showed, be subjected to immunostimulation after, hen produces immune response, in the yolk ripening stage in uterine tube, IgY can optionally be transferred in the yolk in the blood, and is immunoglobulins unique in the yolk.As far back as the sixties, scientists just finds to exist in the Ovum Gallus domesticus Flavus IgY antibody, and its content is similar to chicken serum even higher.Yolk has many advantages as the source of specific antibody IgY.As: hen is easy to raise, and expense is not high; Collect egg instant, need not to twitch thing blood, not damaged meets modern protection of animal rule; It is little to produce the required antigen amount of effective immune response, and especially distance bird far away has stronger immunogenicity usually on the mammalian proteins confrontation phylogenetics of high conservative.
Because yolk is as many advantages of specific antibody IgY as previously mentioned, IgY is being subjected to using more and more widely aspect immunology detection.Aspect immunotherapy, acidproof, the heat-resisting and stable performance because of chicken antibody, but oral administration is used to prevent or treat young animal or human infectious intestinal disease, as baby's diseases prevention food etc.When particularly the use of microbiotic or other medicines had problems, IgY became first-selection.
Yolk is made up of lipid and other component of about 48.7% water, 16.6% protein and 32.6%.Owing to be difficult to IgY antibody is separated from abundant yolk lipid, make egg be restricted as the research of antibody sources.Up to the beginning of the eighties, external scientific research personnel has set up degreasing method in succession, as polyoxyethylene glycol (PEG) extraction method and the modification method (1980 and 1985) thereof of Polson, Jenseniusde T 500 extraction method (1981), and chloroform method and water dilution method etc.Domestic starting late, method also mainly are to improve on the above method basis.Though above method can obtain certain IgY rate of recovery, there is certain defective in using.At first, a large amount of toxic reagents of many needs in these methods, removing these reagent after the degreasing not so increases cost; Secondly these methods need be used instruments such as super-magnum centrifuge, and this will limit the suitability for industrialized production of IgY; And these method efficient and purity are not high, have influenced the quality of IgY.
In recent years, China's researcher has also carried out a lot of researchs to the extraction of IgY.As a kind of middle weighting degreasing method that adopts of novel method (application number 95110458.6) of extracting antibody from yolk of the patent of invention before the bavin man of Shandong Agricultural University.Phenol degreasing method is disclosed among the preparation technology of the patent of invention refined vitelline antibody of the normal Wei Shan of Shandong Agricultural University in 1998 (the ZL patent No. 9811025.8).The patent of invention vitelline polietilenglicol primary precipitation degreasing technology (application number 200810014515.1) that also has what unit dragon of academy of agricultural sciences, Shandong.More than these methods some shortcomings are all arranged, the reagent that has is poisonous, be difficult to remove in IgY liquid; The IgY rate of recovery that has is low, and purity is not high, influences the functional quality of antibody.
Three summary of the invention
Technical problem
The purpose of this invention is to provide a kind of preparation method who from yolk, extracts IgY.When guaranteeing the IgY antibody titer, adopt that dosage is few, the degreasing reagent of safety, low toxicity, convenient in the operating process, need not use expensive instrument, reduce production costs, and IgY purity is higher.
Technical scheme
In order to achieve the above object, the used technical scheme of the present invention is: water and yolk stoste are directly mixed, pass through mechanical stirring, break the emulsified state of yolk, make fat particles and protein separation in the yolk,, make the yolk grease mutual aggegation take place and natural sedimentation then by leaving standstill, the supernatant liquor that obtains mixes with sad, foreign protein reaction in sad and the aqueous solution forms irreversible precipitation, only the IgY antibody-like not with reaction, stay in the aqueous solution, thereby reach the purpose of extracting IgY.
Concrete grammar of the present invention is as follows:
Water-sad two-step approach method is separated the technology of Yolk immunoglobulin, it is characterized in that, the first step, pure water and yolk stoste are directly mixed fully stirring, leave standstill then, the mutual aggegation of yolk grease and natural sedimentation, the supernatant liquor of post precipitation is the yolk water soluble ingredient, i.e. the mixture of Yolk immunoglobulin IgY and other soluble proteins; In second step, the yolk water soluble ingredient that the first step is obtained mixes with sad or sad solution, leaves standstill after stirring, sad and albumen generation irreversible reaction, formation precipitates, IgY not with sad reaction, its aqueous solution is the aqueous solution of IgY.
In the described the first step, in 2 ℃ ~ 30 ℃ environment, abandon egg white and get yolk, add pure water dilution volume n doubly, wherein 0＜n≤10 stir, staticly settle, post precipitation is got supernatant liquor, and supernatant liquor is the yolk water soluble ingredient, i.e. the mixture of Yolk immunoglobulin IgY and other soluble proteins;
In described second step, add sad stirring in the yolk water soluble ingredient that obtains after the first step is handled, sad whole content volume is than being m, 0＜m≤3% wherein, standing and reacting, precipitation and upper strata floating matter are foreign protein, middle water is the IgY aqueous solution of acquisition.Can be dissolved in earlier in the pure water sad earlier, sad solution content volume is than being x, and 10%＜x＜100% wherein is again with sad solution and yolk water soluble ingredient thorough mixing.
Beneficial effect
Advantage of the present invention and prior art following advantage relatively arranged:
1, low toxicity: reagent of the present invention mainly is sad, and sad system dye, medicine, spices, softening agent, the lubricant etc. of can be used for are also as the raw material of sanitas, sterilant.Low toxicity stimulates less to body, compare with the formaldehyde of routine, phenol, chloroform etc., and is safer.
2, sterilization: sad effect with sterilization and anticorrosion, IgY was subjected to the pollution of bacterium during minimizing was produced, and prolonged the shelf time.
3, operation is simple and easy: this method does not need to use any instrument, and all natural sedimentations of yolk grease, foreign protein in the reaction process are operated the easiest.
4, organic efficiency height, active good: the water that the present invention uses and sadly can not exert an influence to IgY, most IgY obtain reclaiming.
5, with low cost, suitable scale operation: only relate to pure water and sad on a small quantity among the present invention, cost is all lower, and the equipment that needs is fairly simple, is fit to scale operation.
6, obtain IgY purity and reach more than 90%, sad content is lower than 0.1%, and antibody titer loses less than 15%.
Embodiment:
(1) with egg that laying hen produced as raw material, after the check sterilization, take craft or mechanical system to separate egg white and yolk.In 2 ℃ ~ 30 ℃ environment, yolk is directly mixed yolk with pure water: pure water is 1:3 ~ 1:8.Staticly settle, the bottom of post precipitation is the yolk grease, and supernatant liquor is the yolk water soluble component, i.e. the mixed solution of IgY and other foreign protein.
(2) isolating supernatant liquor adds 10% sad solution, and sad final concentration (v/v) is respectively 1.0%, 1.5%, 2%, 2.5%, 3%, fully stirs, and staticly settles, and aqueous portion is the IgY solution of extraction.
Dilute degreasing based on the first step water among the present invention, the rate of recovery height that this method advantage is IgY, easy to operate, with low cost; But non-comparatively speaking IgY material is many, cause irritated easily and stress, influence the result of use of IgY as the Antybody therapy agent.Sad system dye, medicine, spices, softening agent, the lubricants etc. of being mainly used in are also as the raw material of sanitas, sterilant.Its low toxicity is slightly soluble in water, forms precipitation with albumen test; Its anticorrosion, germicidal action can reduce the pollution of solution bacterium, and just little residual influences minimum to animal in the aqueous solution.
The ratio of water determines the efficient of preliminary degreasing among the present invention, and the quality of degreasing effect is embodied directly in color, volume and tire (the agar diffusion test result who tires with the gosling plague yolk antibody is an example) of supernatant liquor after the degreasing.See Table 1 in the water dilution method degreasing test, when yolk concentration was 1:3 and 1:4, layering did not take place in yolk liquid; Under 1: 5 the concentration, yolk liquid has begun to occur water fat separation phenomenon, but the more color of foreign protein content is yellow opaque in the supernatant liquor, and supernatant liquor and sedimentary fine jade expand the AGP that tires and be 1:8; Be that water fat separation phenomenon is fairly obvious under 1: 6 ~ 1: 8 the concentration when yolk, along with dilution increase, color is approaching more transparent, wherein when 1:6 and 1:7, supernatant liquor and sedimentary fine jade expand the AGP that tires and are all 1:8, when yolk concentration was 1:8, supernatant liquor and sedimentary fine jade expanded the AGP that tires and are all 1:4.In the water dilution method supernatant and precipitation fine jade expand tire identical.
The effect of table 1 water dilution method degreasing
| ? | Stoste | 1:3 | 1:4 | 1:5 | 1:6 | 1:7 | 1:8 |
| Layering | — | Not stratified | Not stratified | Layering | Layering | Layering | Layering |
| Supernatant fluid color | — | — | — | Yellow | Faint yellow | Faint yellow | Faint yellow |
| Supernatant liquor/precipitation | — | — | — | 5/1 | 6/1 | 7/1 | 8/1 |
| AGP（log2） | 1:64 | — | — | 1:8 | 1:8 | 1:8 | 1:4 |
| Precipitate A GP | — | — | —— | 1:8 | 1:8 | 1:8 | 1:4 |
Annotate: this result is the result after static 24 hours.
Sad consumption directly determines the content of foreign protein in the IgY solution among the present invention, removes the quality of foreign protein effect, is mainly reflected in the clarity of the aqueous solution and tires.See Table 2 in sad degreasing method, after the water dilution method degreasing, yolk concentration is that the supernatant liquor adding of 1:5 is sad, and during final concentration 3%, yolk liquid is divided into 3 layers, and with aseptic straw siphon middle layer transparent liquid, it is 1:8 that the mensuration fine jade expands the AGP that tires, and it is 0 that precipitation is tired; Yolk concentration is that the supernatant liquor adding of 1:6 and 1:7 is sad, and final concentration is 2% o'clock, the layering of yolk liquid, and it is 1:8 that middle layer yolk liquid fine jade expands chicken, precipitate A GP is 0; Yolk concentration is that the supernatant liquor adding of 1:8 is sad, and final concentration is 1.5% o'clock, the layering of yolk liquid, and it is 1:4 that middle layer yolk liquid fine jade expands chicken, precipitate A GP is 0.There is not yolk antibody in the precipitation.
The sad degreasing method of table 2 effect
| ? | Stoste | 1:5 | 1:6 | 1:7 | 1:8 |
| 1.5% | — | Not stratified | Layering/white | Layering/white | Layering/transparent |
| 2% | — | Not stratified | Layering/transparent | Layering/transparent | Layering/transparent |
| 2.5% | — | Layering/white | Layering/transparent | Layering/transparent | Layering/transparent |
| 3% | — | Layering/transparent | Layering/transparent | Layering/transparent | Layering/transparent |
| AGP（log2） | 1:64 | 1:8 | 1:8 | 1:8 | 1:4 |
| Precipitate A GP | — | 0 | 0 | 0 | 0 |
Annotate: this result is the result after static 24 hours.
Embodiment
Wipe away dirt and the ight soil of washing eggshell surface off with clear water, and rinse, put into 0.5% bromogeramine solution again and soak 20min, take out then and dry with tap water.With 75% alcohol swab wiping eggshell, eggshell is opened in aseptic technique, with egg white and yolk separately, yolk is put in the sterilization beaker, fully stirs to beat then and spares, make homogenate after packing standby, be yolk stoste.
Get yolk stoste 1L and pure water 6L, ratio between two is 1: 6, after directly mixing, stir 20 minutes abundant mixings, left standstill 8 hours under 2 ~ 30 ℃ of environment or spend the night, post precipitation is divided into 2 layers, the bottom is the yolk grease, and supernatant liquor is the yolk water soluble ingredient, collects supernatant liquor, it is sad slowly to add 140ml, and making sad final concentration is 2%, and the limit edged shakes up, after stirring, left standstill 24 hours, precipitation is divided into 3 layers, separate middle level liquid with the aseptic straw siphon, be IgY solution.Parallel test 3 times, the IgY solution degreasing rate of acquisition reaches more than 95%, and sad content is lower than 0.1%, and antibody titer loses less than 15%.
Claims (4)
1. water-sad two-step approach method is separated the technology of Yolk immunoglobulin, it is characterized in that, the first step, pure water and yolk stoste are directly mixed fully stirring, leave standstill then, the mutual aggegation of yolk grease and natural sedimentation, the supernatant liquor of post precipitation is the yolk water soluble ingredient, i.e. the mixture of Yolk immunoglobulin IgY and other soluble proteins; In second step, the yolk water soluble ingredient that the first step is obtained mixes with sad or sad solution, leaves standstill after stirring, sad and albumen generation irreversible reaction, formation precipitates, IgY not with sad reaction, its aqueous solution is the aqueous solution of IgY.
2. technology according to claim 1, it is characterized in that, in the described the first step, in 2 ℃ ~ 30 ℃ environment, get yolk, add n times of volume of pure water dilution, wherein 0＜n≤10 stir, staticly settle, post precipitation is got supernatant liquor, and supernatant liquor is the yolk water soluble ingredient, i.e. the mixture of Yolk immunoglobulin IgY and other soluble proteins.
3. technology according to claim 1 and 2, it is characterized in that, in described second step, add sad stirring in the yolk water soluble ingredient that after the first step is handled, obtains, sad whole content volume is than being m, 0＜m≤3% wherein, standing and reacting, precipitation and upper strata floating matter are foreign protein, and middle water is the IgY aqueous solution of acquisition.
4. according to the technology described in the claim 3, be dissolved in earlier in the pure water sad, sad solution deep volume ratio is x, and 10%＜x＜100% wherein is again with sad solution and yolk water soluble ingredient thorough mixing.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103102410A (en) * | 2013-01-23 | 2013-05-15 | 孙亮 | Method for separating anti-rotavirus immunoglobulin from chicken egg |
| CN103172732A (en) * | 2011-12-20 | 2013-06-26 | 普莱柯生物工程股份有限公司 | Anti-gosling plague egg yolk antibody and preparation method thereof |
| CN106866817A (en) * | 2017-03-01 | 2017-06-20 | 广州格雷特生物科技有限公司 | A kind of process for extracting duck tembusu virus disease Yolk antibody |
| CN106963956A (en) * | 2017-03-01 | 2017-07-21 | 广州格雷特生物科技有限公司 | The inactivation technology of duck tembusu virus viruses in yolk antibody |
| CN107383188A (en) * | 2017-08-07 | 2017-11-24 | 美国安泰健康投资管理公司 | A kind of yolk specific IgY and its preparation method and application |
| CN114644709A (en) * | 2022-03-02 | 2022-06-21 | 广东海大畜牧兽医研究院有限公司 | Method for mass production of yolk antibody capable of reducing residues of caprylic acid egg dregs |
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| CN1438244A (en) * | 2002-12-21 | 2003-08-27 | 郭占军 | Self-body immunity-disease related egg-yolk atibody preparation method and use thereof |
| CN1752105A (en) * | 2005-10-20 | 2006-03-29 | 上海交通大学 | Biomimetic affinity purification method of vitellus immune globulin |
| CN101845095A (en) * | 2010-05-10 | 2010-09-29 | 洛阳普莱柯生物工程有限公司 | Method for preparing double yolk antibody of porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1438244A (en) * | 2002-12-21 | 2003-08-27 | 郭占军 | Self-body immunity-disease related egg-yolk atibody preparation method and use thereof |
| CN1752105A (en) * | 2005-10-20 | 2006-03-29 | 上海交通大学 | Biomimetic affinity purification method of vitellus immune globulin |
| CN101845095A (en) * | 2010-05-10 | 2010-09-29 | 洛阳普莱柯生物工程有限公司 | Method for preparing double yolk antibody of porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103172732A (en) * | 2011-12-20 | 2013-06-26 | 普莱柯生物工程股份有限公司 | Anti-gosling plague egg yolk antibody and preparation method thereof |
| CN103172732B (en) * | 2011-12-20 | 2015-06-10 | 普莱柯生物工程股份有限公司 | Anti-gosling plague egg yolk antibody and preparation method thereof |
| CN103102410A (en) * | 2013-01-23 | 2013-05-15 | 孙亮 | Method for separating anti-rotavirus immunoglobulin from chicken egg |
| CN103102410B (en) * | 2013-01-23 | 2014-11-05 | 孙亮 | Method for separating anti-rotavirus immunoglobulin from chicken egg |
| CN106866817A (en) * | 2017-03-01 | 2017-06-20 | 广州格雷特生物科技有限公司 | A kind of process for extracting duck tembusu virus disease Yolk antibody |
| CN106963956A (en) * | 2017-03-01 | 2017-07-21 | 广州格雷特生物科技有限公司 | The inactivation technology of duck tembusu virus viruses in yolk antibody |
| CN107383188A (en) * | 2017-08-07 | 2017-11-24 | 美国安泰健康投资管理公司 | A kind of yolk specific IgY and its preparation method and application |
| CN114644709A (en) * | 2022-03-02 | 2022-06-21 | 广东海大畜牧兽医研究院有限公司 | Method for mass production of yolk antibody capable of reducing residues of caprylic acid egg dregs |
| CN114644709B (en) * | 2022-03-02 | 2024-05-07 | 广东海大畜牧兽医研究院有限公司 | Method for mass production of egg yolk antibody capable of reducing Xin Suandan slag residues |
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