CN102210863A - Method for preparing yolk immune globulin nanometer liposome for preventing and controlling infectious bursal disease - Google Patents
Method for preparing yolk immune globulin nanometer liposome for preventing and controlling infectious bursal disease Download PDFInfo
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- CN102210863A CN102210863A CN201110095161XA CN201110095161A CN102210863A CN 102210863 A CN102210863 A CN 102210863A CN 201110095161X A CN201110095161X A CN 201110095161XA CN 201110095161 A CN201110095161 A CN 201110095161A CN 102210863 A CN102210863 A CN 102210863A
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Abstract
The invention relates to a method for preparing a yolk immune globulin nanometer liposome for preventing and controlling infectious bursal disease, and belongs to the technical field of medicine preparations of animals. The method for preparing the yolk immune globulin nanometer liposome for preventing and controlling chicken infectious bursa of Fabricius comprises the following steps of: 1, preparing a yolk antibody, namely performing vaccine inoculation on chickens by a multi-time and alternate immune method, and when the agar gel precipitate test (AGPT) detection valence of the yolk antibody is over 1:64, collecting yolk; 2, separating immune globulin, namely diluting yolk suspension by using acetic acid-sodium acetate buffer solution, stirring, standing, centrifuging and reserving supernate; and 3, preparing a liposome, namely preparing a liposome membrane by a membrane dispersion method, adding the yolk immune globulin, rotating and stirring to form a yolk immune globulin liposome capsule, and reforming the formed liposome capsule by using a homogenizer to obtain the nanometer liposome. In the method, the yolk immune globulin is enveloped, and the method has the advantages of simple process, economy, practicability, stable economic performance, good treatment effect and the like.
Description
Technical field
The invention belongs to the oral products technical field, particularly relate to a kind of method for making of preventing and treating infectious bursal disease Yolk immunoglobulin nanometer liposome.
Background technology
At present, (Infectious bursal disease IBD) is the viral infectious of the maincenter of the exempting from organ fabricius bursa of a kind of destruction chicken that is caused by double-stranded rna virus to infectious bursal disease.This disease increases owing to chicken mortality rate, mortality on the one hand, influences the chicken body weight gains, causes direct economic loss; Because IBDV brings out immunosuppressant, make immunne response ability drop or the non-responsiveness of chicken group on the other hand to multiple vaccine (as: chicken Newcastle disease live vaccine, infectious bronchitis of chicken live vaccine, chicken Marek's disease live-vaccine etc.).Simultaneously because human body immune function descends, and sick chicken increases the pathogen susceptibility, the concurrent or multiple other diseases of secondary easily is as the coliform disease etc.
The measure that this disease is taked is the immunity inoculation of carrying out vaccine, but immune effect is unsatisfactory, the phenomenon of immuning failure often occurs.In case the chicken group of immuning failure occurs, then mainly rely on the method for passive immunity, to chicken group injecting immune globulin.Present employed immunoglobulin mainly is a Yolk immunoglobulin.This immunoglobulin is very fast in vivo destroyed, and the half-life is short, and action time is short.
Summary of the invention
The present invention provides the method for making of a kind of control infectious bursal disease Yolk immunoglobulin (IgY) nanometer liposome for solving the technical problem that exists in the known technology.
The purpose of this invention is to provide a kind of liposome pharmaceutical technology that utilizes, Yolk immunoglobulin is sealed, thereby raising curative effect, it is simple to have technology, economical and practical, the preparation stable performance, therapeutic effect is good, the method for making of the control infectious bursal disease Yolk immunoglobulin nanometer liposome of characteristics such as applied range.
Liposome is made of oleophylic and hydrophilic parents' molecule, and oil loving end section is positioned at the centre of film in molecule, and the film forming surfaces externally and internally of hydrophilic head end part shape, medicine is contained within the bilayer; Cyst wall is made up of bimolecular film.Thereby liposome is a kind of vesicle with similar biofilm structure, can adsorb with cell, the exchange of fusion, fat directly brings the medicine of sealing into cell, makes medicine have cell targeted and improves therapeutic index or improve bioavailability.The destruction that can avoid various enzyme by the liposome drug coated; the time that prolong drug stops in blood circulation; thereby raising bioavailability of medicament; so medicine is had protective effect; make medicine avoid the destruction of biological enzyme and the interference of various factors, help medicine transportation in vivo.
The present invention utilizes liposome pharmacy new technique, Yolk immunoglobulin is sealed, thereby reached the purpose that improves curative effect.
The technical scheme that the present invention takes for the technical problem that exists in the solution known technology is:
The method for making of control infectious bursal disease Yolk immunoglobulin nanometer liposome, it is characterized in that: the preparation of yellow immunoglobulin nanometer liposome comprises following technical process,
1) preparation of yolk antibody: adopt repeatedly, alternative immunization method, chicken is carried out vaccination; Tire when 1:64 is above when chicken yolk antibody AGPT detects, collect yolk;
2) separating immune globulin: with acetic acid-sodium acetate buffer, the yolk suspension is diluted, stir, leave standstill, centrifugal, keep supernatant;
3) preparation liposome: adopt film dispersion method, make liposome membrane; Add Yolk immunoglobulin through rotation, stirring, form Yolk immunoglobulin liposome utricule; With refiner the liposome that forms is reformed, obtained nano level liposome.
The method for making that the present invention prevents and treats infectious bursal disease Yolk immunoglobulin nanometer liposome can also adopt following technical scheme:
The method for making of described control infectious bursal disease Yolk immunoglobulin nanometer liposome, be characterized in: during separating immune globulin, with acetic acid-sodium acetate buffer of pH 4.0-6.0, the yolk suspension is carried out 6-12 doubly dilute, the supernatant that obtains carries out concentration.
The method for making of described control infectious bursal disease Yolk immunoglobulin nanometer liposome is characterized in: supernatant adopts ultrafiltration to carry out concentration.
The method for making of described control infectious bursal disease Yolk immunoglobulin nanometer liposome is characterized in: during separating immune globulin, the condition of leaving standstill is 2-6 ℃, more than 12 hours; Centrifugal condition is 800-2000 * g, 10-30 min.
The method for making of described control infectious bursal disease Yolk immunoglobulin nanometer liposome is characterized in: vaccine is attenuated vaccine, inactivated vaccine and oil emulsion vaccine; Fundamental immunity is selected attenuated vaccine and inactivated vaccine for use; Booster immunization is selected inactivated vaccine and oil emulsion vaccine for use.
The method for making of described control infectious bursal disease Yolk immunoglobulin nanometer liposome, be characterized in: when making liposome membrane, the ratio of lecithin and cholesterol is 6-8:1, vitamin E is a stabilizing agent, consumption 3-7 ‰, procedure of processing is mixing, organic solvent dissolution, rotary evaporation, decompression, film forming.
The method for making of described control infectious bursal disease Yolk immunoglobulin nanometer liposome, be characterized in: during the preparation liposome, add Yolk immunoglobulin and be rotated in the rotary evaporation bottle of liposome membrane, time 10-35min forms Yolk immunoglobulin liposome utricule.
The method for making of described control infectious bursal disease Yolk immunoglobulin nanometer liposome is characterized in: preparation is during liposome, with refiner the liposome that forms is reformed comprise cutting, reformation, parcel obtains nano level liposome again; Refiner speed is 8000-20000rpm, and the time is 5-20min.
Advantage and good effect that the present invention has are:
The method for making of control infectious bursal disease Yolk immunoglobulin nanometer liposome is owing to adopted brand-new technology scheme of the present invention, compared with prior art, the method for making of control infectious bursal disease Yolk immunoglobulin nanometer liposome is utilized the liposome pharmaceutical technology, and Yolk immunoglobulin is sealed, thereby reach the purpose that improves curative effect, it is simple to have technology, economical and practical, and properties of product are stable, therapeutic effect is good, advantages such as applied range.
Description of drawings
Fig. 1 is a process flow diagram of the present invention.
The specific embodiment
For further understanding technology contents of the present invention, characteristics and effect, exemplify following examples now, and conjunction with figs. is described in detail as follows:
Embodiment 1
Consult accompanying drawing 1.
The present invention prevents and treats the preparation method of infectious bursal disease Yolk immunoglobulin nanometer liposome, comprises following processing step:
1, the preparation of yolk antibody: adopt repeatedly, alternative immunization protocol, experimental chicken is carried out vaccination.Tire more than 1:64 when experimental chicken yolk antibody AGPT detects, collect yolk.
2, separating immune globulin: with acetic acid-sodium acetate buffer of pH 5.0, the yolk suspension is carried out 9 times of dilutions, stir, leave standstill, centrifugal, keep supernatant.Supernatant is carried out concentration.
3, preparation liposome: adopt film dispersion method, make liposome membrane; Add Yolk immunoglobulin through rotation, stirring, form Yolk immunoglobulin liposome utricule.Reuse high-speed homogenization machine is reformed to the liposome that has formed, and obtains nano level liposome.
The concrete technological process of present embodiment comprises:
1, the preparation of yolk antibody:
Adopt repeatedly, alternative immunization protocol, experimental chicken is carried out vaccination.Used vaccine is attenuated vaccine, inactivated vaccine and oil emulsion vaccine.Fundamental immunity is selected attenuated vaccine and inactivated vaccine for use; Booster immunization is selected inactivated vaccine and oil emulsion vaccine for use; Being that the maintainability immunity keep high-caliber immunizing potency to carry out adopts being used alternatingly of above-mentioned 3 kinds of vaccines, is as according to determining according to the difference of antibody horizontal to the use of different vaccines.
Tire more than 1:64 when experimental chicken yolk antibody AGPT detects, can collect egg, it is carried out soaking disinfection, aseptic separation yolk and Ovum Gallus domesticus album with yolk homogenate and add an amount of antiseptic, are preserved standby.
2, separating immune globulin
Get above-mentioned yolk suspension, use 0.05 mol/L, acetic acid-sodium acetate buffer of pH 5.0 carries out 9 times of dilutions with the yolk suspension, after fully stirring, leaves standstill more than 12 hours under 4 ℃ of conditions.Centrifugal 20 min of 1 000 * g discard precipitation, keep supernatant.Adopt ultrafiltration that it is carried out concentration supernatant, the volume after the ultrafiltration is identical with former yolk suspension.
3, preparation liposome
Liposome preparation adopts film dispersion method, and the ratio of lecithin and cholesterol is about 7:1, is stabilizing agent with the vitamin E, and consumption is about 5 ‰.Tentatively make liposome membrane through steps such as mixing, organic solvent dissolution, rotary evaporation, decompression, film forming.
The Yolk immunoglobulin that above-mentioned separation is obtained joins in the rotary evaporation bottle of the liposome membrane of tentatively making and is rotated, liposome membrane is fully contacted and with its parcel to Yolk immunoglobulin, about about 20 minutes of rotational time, this moment, liquid presented uniform milky, began to take shape Yolk immunoglobulin liposome utricule.
In the high-speed homogenization machine that the Yolk immunoglobulin liposome utricule that begins to take shape is shifted, with the high-speed homogenization machine liposome that has formed is carried out cutting, reformation, process such as parcel again, to obtain nano level liposome.Homogenate speed is 10000rpm, and the time is about 10 minutes.
The control infectious bursal disease Yolk immunoglobulin nanometer liposome of present embodiment preparation has described advantage and good effect.
Claims (8)
1. prevent and treat the method for making of infectious bursal disease Yolk immunoglobulin nanometer liposome, it is characterized in that: the preparation of yellow immunoglobulin nanometer liposome comprises following technical process,
1) preparation of yolk antibody: adopt repeatedly, alternative immunization method, chicken is carried out vaccination; Tire when 1:64 is above when chicken yolk antibody AGPT detects, collect yolk;
2) separating immune globulin: with acetic acid-sodium acetate buffer, the yolk suspension is diluted, stir, leave standstill, centrifugal, keep supernatant;
3) preparation liposome: adopt film dispersion method, make liposome membrane; Add Yolk immunoglobulin through rotation, stirring, form Yolk immunoglobulin liposome utricule; With refiner the liposome that forms is reformed, obtained nano level liposome.
2. according to the method for making of the described control infectious bursal disease of claim 1 Yolk immunoglobulin nanometer liposome, it is characterized in that: during separating immune globulin, acetic acid-sodium acetate buffer with pH 4.0-6.0, the yolk suspension is carried out 6-12 doubly dilute, the supernatant that obtains carries out concentration.
3. according to the method for making of the described control infectious bursal disease of claim 2 Yolk immunoglobulin nanometer liposome, it is characterized in that: supernatant adopts ultrafiltration to carry out concentration.
4. according to the method for making of the described control infectious bursal disease of claim 1 Yolk immunoglobulin nanometer liposome, it is characterized in that: during separating immune globulin, the condition of leaving standstill is 2-6 ℃, more than 12 hours; Centrifugal condition is 2000 * g, 10-30 min.
5. according to the method for making of the described control infectious bursal disease of claim 1 Yolk immunoglobulin nanometer liposome, it is characterized in that: vaccine is attenuated vaccine, inactivated vaccine and oil emulsion vaccine; Fundamental immunity is selected attenuated vaccine and inactivated vaccine for use; Booster immunization is selected inactivated vaccine and oil emulsion vaccine for use.
6. according to the method for making of the described control infectious bursal disease of claim 1 Yolk immunoglobulin nanometer liposome, it is characterized in that: when making liposome membrane, the ratio of lecithin and cholesterol is 6-8:1, vitamin E is a stabilizing agent, consumption 3-7 ‰, procedure of processing is mixing, organic solvent dissolution, rotary evaporation, decompression, film forming.
7. according to the method for making of the described control infectious bursal disease of claim 1 Yolk immunoglobulin nanometer liposome, it is characterized in that: during the preparation liposome, adding Yolk immunoglobulin is rotated in the rotary evaporation bottle of liposome membrane, time 10-35min forms Yolk immunoglobulin liposome utricule.
8. according to the method for making of the described control infectious bursal disease of claim 1 Yolk immunoglobulin nanometer liposome, it is characterized in that: preparation is during liposome, with refiner the liposome that forms is reformed comprise cutting, reformation, parcel obtains nano level liposome again; Refiner speed is 8000-20000rpm, and the time is 5-20min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102716476A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Cell inactivated vaccine, egg yolk antibody injection, and preparation method of cell inactivated vaccine |
| CN111760022A (en) * | 2020-08-07 | 2020-10-13 | 山东泰邦生物制品有限公司 | Human immunoglobulin liposome pharmaceutical composition and preparation method thereof |
| CN112274638A (en) * | 2019-12-11 | 2021-01-29 | 安徽医科大学附属口腔医院 | Specific anti-porphyromonas gingivalis egg yolk antibody liposome solution and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101690715A (en) * | 2009-10-29 | 2010-04-07 | 东北农业大学 | Method for preparing immunoglobulin G liposome by using milk fat globule membrane phospholipids |
| CN101704889A (en) * | 2009-11-13 | 2010-05-12 | 烟台绿叶动物保健品有限公司 | Method for preparing chicken bursal antibody |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101690715A (en) * | 2009-10-29 | 2010-04-07 | 东北农业大学 | Method for preparing immunoglobulin G liposome by using milk fat globule membrane phospholipids |
| CN101704889A (en) * | 2009-11-13 | 2010-05-12 | 烟台绿叶动物保健品有限公司 | Method for preparing chicken bursal antibody |
Non-Patent Citations (1)
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| 张文生等: "3种卵黄抗体分离提取方法对其生物活性影响的比较", 《中国生化药物杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102716476A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Cell inactivated vaccine, egg yolk antibody injection, and preparation method of cell inactivated vaccine |
| CN102716476B (en) * | 2012-05-31 | 2015-04-22 | 郑州后羿制药有限公司 | Cell inactivated vaccine, egg yolk antibody injection, and preparation method of cell inactivated vaccine |
| CN112274638A (en) * | 2019-12-11 | 2021-01-29 | 安徽医科大学附属口腔医院 | Specific anti-porphyromonas gingivalis egg yolk antibody liposome solution and preparation method thereof |
| CN111760022A (en) * | 2020-08-07 | 2020-10-13 | 山东泰邦生物制品有限公司 | Human immunoglobulin liposome pharmaceutical composition and preparation method thereof |
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Application publication date: 20111012 |