CN105175539A - Production method for refined yolk antibody for chicken infectious bursal disease - Google Patents
Production method for refined yolk antibody for chicken infectious bursal disease Download PDFInfo
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- CN105175539A CN105175539A CN201510617627.6A CN201510617627A CN105175539A CN 105175539 A CN105175539 A CN 105175539A CN 201510617627 A CN201510617627 A CN 201510617627A CN 105175539 A CN105175539 A CN 105175539A
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Abstract
The invention provides a production method for a refined yolk antibody for the chicken infectious bursal disease. The method includes the complete technology from early laying hen immunization to follow-up egg yolk antibody separation and purification, substances such as impurities and a heat source can be removed in the purification process, the risk of introducing other exogenous pathogenic microorganisms in the production process is reduced, and product quality safety is ensured. More importantly, accumulation of the antibody in yolk can be remarkably promoted by the immunization method, the combined purification technology is effective, and therefore the product titer is obviously improved compared with the prior art. The production method for the refined yolk antibody for the chicken infectious bursal disease is obtained by the inventor through groping by means of rigorous experiment measures, and the method is prominent in effect, low in cost and easy to realize and has good popularization prospects.
Description
Technical field
The present invention relates to veterinary biologics technical field, be specifically related to a kind of infectious bursal disease refined vitelline antibody production method.
Background technology
Yolk antibody refers to the antibody for specific antigen extracted from immune eggs, owing to only having IgG antibody-like in yolk, is therefore called Yolk immunoglobulin IgG (yolkantibody), referred to as IgY.
After body is subject to exotic antigen stimulation, B cell differentiation in the fabricius bursa becomes plasmocyte, secreting specificity antibody enters blood circulation, when blood flow is through ovary, specific antibody (mainly IgG) is accumulated gradually in ovum, forms yolk antibody, IgG divides a word with a hyphen at the end of a line and enters the result that ovum is receptor acting, thus IgG can accumulate in a large number in ovum, and concentration is higher than the IgG in blood, and in single yolk (about 15ml), yolk antibody content can reach about 200mg.From yolk, extract antibody in addition easy compared with extracting antibody from animal serum, therefore yolk antibody has obvious technical superiority.
In addition, yolk antibody has good stability in multiple environment.Lower than under 75 DEG C of conditions, yolk antibody has good thermostability, and after 90 DEG C of process 15min, most of yolk antibody loses binding activities, when PH<4, only has a small amount of yolk antibody to lose activity.In the scope of pH4 ~ 12, the activity of yolk antibody is influenced hardly, and when pH>12, yolk antibody loses binding activities rapidly.Experiment shows, yolk antibody has the characteristic of tolerance multigelation, even if through 5 freeze thawing, its antigen-binding activity is influenced hardly.At room temperature can preserve 6 months, 4 DEG C of preservations can reach more than 5 years, and activity only declines about 5%.
Owing to possessing above-mentioned superior biology, physicochemical property, therefore the preparation and application of yolk antibody receive increasing concern.But therefore have impact on product quality greatly due to the yolk antibody production technique existing defects of prior art; such as some rough antibody products not only antibody titer be difficult to ensure, and usually can pollute Avianreovirus, fowl cell leukemia virus, RE outgrowth factor, intestinal bacteria, Salmonellas, mycoplasma etc. exogenous cause a disease former.When injection of antibodies prevention or disease therapy, very likely cause the diffusion even outburst of disease of vertical transmission cause of disease or some other bacterium, virus, bring serious financial loss.In addition, producer due to these rough antibody often attempts the quality guaranteed period extending its product by adding heavy dose of measure such as formaldehyde, penicillin and streptomycin, increase so-called use " security ", but result cause often larger stress with serious drug residue.And containing the macromolecular substance such as a large amount of lipoprotein and Yelkin TTS in rough antibody, these materials are without any therapeutic action and volume is large, and viscosity is high, injections difficult and be difficult to absorb, to body after injection stress be large, easily causing anaphylaxis, is EP.Easily cause after injection that injection site local organization is hemorrhage, swelling, necrosis, downright bad tissue can cause organism fever, and heating can cause the disorder of the functions such as body neuroregulation, Humoral immunity, thus causes morbidity colony food consumption to reduce, resistibility declines, secondary other diseases.
Summary of the invention
The present invention is intended to the technological deficiency for prior art, provides a kind of infectious bursal disease refined vitelline antibody production method, to tire lower technical problem with the yolk antibody prepared by the yolk antibody production method solving prior art.
Another technical problem that the present invention solves is that the yolk antibody production method of prior art can exist impurities left.
The technical problem again that the present invention solves is that the yolk antibody production method of prior art may be mixed into exogenous pathogenic microorganism.
For realizing above technical purpose, the present invention by the following technical solutions:
A kind of infectious bursal disease refined vitelline antibody production method, comprises the following steps:
1) bursal disease vaccine immunity chicken is utilized;
2) get height and exempt from egg yolk, broken after removing vitelline membrane and frenulum;
3) with 1:(2 ~ 3) ratio of (w/w) is to step 2) add that temperature is 50 ~ 56 DEG C, pH is the acetate buffer of 4.8 ~ 5.2 in yolk after fragmentation, stir 25 ~ 35min, obtain mixed solution;
4) to step 3) add the n-caprylic acid of 3 ~ 5% (v/v) in described mixed solution, stir 30 ~ 180min;
5) filter removal solid substance, namely obtain yolk antibody.
Preferably, step 1) described in chicken be the hy-line brown hen of 120 ages in days.
Preferably, step 1) described in immunity be first utilize infectious bursal disease live-vaccine B87 strain immunity chicken 1 time, then recycle infectious bursal disease oil emulsion inactivated vaccine immunity 2 times.Can perform preferably following on this basis further: utilize the vaccine consumption of infectious bursal disease live-vaccine B87 strain immunity chicken 1 time to be 6 ~ 8 plumage parts/only; Utilize the vaccine consumption of infectious bursal disease oil emulsion inactivated vaccine immunity chicken to be 2 ~ 5 plumage parts/only at every turn; Often the timed interval of adjacent twice immunity is 14d.
Preferably, step 2) in vitelline membrane and frenulum utilize 60 order nylon mesh to filter to remove.
Preferably, step 2) described in broken utilize colloidal mill to realize.
Preferably, step 3) described acetate buffer prepared by following methods: dissolves 0.886g sodium acetate and 2.82ml glacial acetic acid in every 1000mL purified water, then utilizes the NaOH of 4% (w/w) concentration to regulate pH.
Preferably, step 5) described in filter utilize polypropylene fibre 750B filter cloth to realize.
In above technical scheme, the approach of described immunity is subcutaneous injection.Described plumage part refers to the specification sheets consumption of this vaccine product for immunization, and that is, 1 plumage part refers to the vaccination amount for immunization 1 chicken.Selected chicken should be the chicken be in a good state of health.Selected infectious bursal disease live-vaccine B87 strain belongs to mesogenic.
The inventive method contains the complete process from laying hen immunity in early stage to follow-up yolk antibody separation and purification, purge process can remove the material such as impurity, thermal source, reduce production process simultaneously and open the risk introducing other exogenous pathogenic microorganisms, ensure that Product quality and safety.What is more important, immunization method of the present invention can the significantly accumulation of enhancing antibody in yolk, then it is effective to add purifying process, and product comparatively prior art of tiring obviously is promoted.Contriver gropes to obtain infectious bursal disease refined vitelline antibody production method of the present invention with rigorous laboratory facilities, and the outstanding cost simultaneously of the method technique effect is lower, be easy to realize, and therefore has good promotion prospect.
Embodiment
Below will be described in detail the specific embodiment of the present invention.In order to avoid too much unnecessary details, in the examples below to belonging to known structure or function will not be described in detail.Apart from outside definition, technology used in following examples and scientific terminology have the identical meanings generally understood with those skilled in the art of the invention.
Embodiment 1
A kind of infectious bursal disease refined vitelline antibody production method, comprises the following steps:
1) bursal disease vaccine immunity chicken is utilized;
2) get height and exempt from egg yolk, broken after removing vitelline membrane and frenulum;
3) with the ratio of 1:2 (w/w) to step 2) add that temperature is 50 DEG C, pH is the acetate buffer of 4.8 in yolk after fragmentation, stir 25min, obtain mixed solution;
4) to step 3) add the n-caprylic acid of 3% (v/v) in described mixed solution, stir 30min;
5) filter removal solid substance, namely obtain yolk antibody.
On the basis of above technical scheme, meet the following conditions:
Step 1) described in chicken be the hy-line brown hen of 120 ages in days.
Step 1) described in immunity be first utilize infectious bursal disease live-vaccine B87 strain immunity chicken 1 time, then recycle infectious bursal disease oil emulsion inactivated vaccine immunity 2 times.The vaccine consumption of infectious bursal disease live-vaccine B87 strain immunity chicken 1 time is wherein utilized to be 6 plumage parts/only; Utilize the vaccine consumption of infectious bursal disease oil emulsion inactivated vaccine immunity chicken to be 2 plumage parts/only at every turn; Often the timed interval of adjacent twice immunity is 14d.
Step 2) in vitelline membrane and frenulum utilize 60 order nylon mesh to filter to remove.
Step 2) described in fragmentation be utilize colloidal mill grind realize crushing effect.
Step 3) described acetate buffer prepared by following methods: dissolves 0.886g sodium acetate and 2.82ml glacial acetic acid in every 1000mL purified water, then utilizes the NaOH of 4% (w/w) concentration to regulate pH.
Step 5) described in filter utilize polypropylene fibre 750B filter-cloth filtering.
Embodiment 2
A kind of infectious bursal disease refined vitelline antibody production method, comprises the following steps:
1) bursal disease vaccine immunity chicken is utilized;
2) get height and exempt from egg yolk, broken after removing vitelline membrane and frenulum;
3) with the ratio of 1:2 (w/w) to step 2) add that temperature is 56 DEG C, pH is the acetate buffer of 5.2 in yolk after fragmentation, stir 35min, obtain mixed solution;
4) to step 3) add the n-caprylic acid of 5% (v/v) in described mixed solution, stir 180min;
5) filter removal solid substance, namely obtain yolk antibody.
On the basis of above technical scheme, meet the following conditions:
Step 1) described in immunity be first utilize infectious bursal disease live-vaccine B87 strain immunity chicken 1 time, then recycle infectious bursal disease oil emulsion inactivated vaccine immunity 2 times.The vaccine consumption of infectious bursal disease live-vaccine B87 strain immunity chicken 1 time is wherein utilized to be 8 plumage parts/only; Utilize the vaccine consumption of infectious bursal disease oil emulsion inactivated vaccine immunity chicken to be 5 plumage parts/only at every turn; Often the timed interval of adjacent twice immunity is 14d.
Step 2) described in fragmentation be utilize colloidal mill grind realize crushing effect.
Step 5) described in filter utilize polypropylene fibre 750B filter-cloth filtering.
Embodiment 3
A kind of infectious bursal disease refined vitelline antibody production method, comprises the following steps:
1) bursal disease vaccine immunity chicken is utilized;
2) get height and exempt from egg yolk, broken after removing vitelline membrane and frenulum;
3) with the ratio of 1:2.5 (w/w) to step 2) add that temperature is 53 DEG C, pH is the acetate buffer of 5.0 in yolk after fragmentation, stir 30min, obtain mixed solution;
4) to step 3) add the n-caprylic acid of 4% (v/v) in described mixed solution, stir 105min;
5) filter removal solid substance, namely obtain yolk antibody.
Embodiment 4
A kind of infectious bursal disease refined vitelline antibody production method, comprises the following steps:
1) bursal disease vaccine immunity chicken 3 times are utilized;
2) get height and exempt from egg yolk, broken after removing vitelline membrane and frenulum;
3) with the ratio of 1:2 (w/w) to step 2) add that temperature is 50 DEG C, pH is the acetate buffer of 4.8 in yolk after fragmentation, stir 25min, obtain mixed solution;
4) to step 3) add the n-caprylic acid of 3% (v/v) in described mixed solution, stir 30min;
5) filter removal solid substance, namely obtain yolk antibody.
On the basis of above technical scheme, meet the following conditions:
Step 1) described in chicken be the hy-line brown hen of 120 ages in days.
Step 2) in vitelline membrane and frenulum utilize 60 order nylon mesh to filter to remove.
Step 2) described in fragmentation be utilize colloidal mill grind realize crushing effect.
Step 3) described acetate buffer prepared by following methods: dissolves 0.886g sodium acetate and 2.82ml glacial acetic acid in every 1000mL purified water, then utilizes the NaOH of 4% (w/w) concentration to regulate pH.
Step 5) described in filter utilize polypropylene fibre 750B filter-cloth filtering.
Arrange 3 kinds of immunization wayses, wherein the 1st group of experiment 1 is exempted from IBD living vaccine 2,3 and is exempted from IBD deactivation vaccine, and IBD living vaccine is exempted from the 2nd group of experiment 1,2,3, and IBD inactivated vaccine is exempted from the 3rd group of experiment 1,2,3, and prepared yolk antibody is tired as shown in table 1:
The impact that the different immunization ways of table 1 is tired on yolk antibody
| Grouping | Laying hen quantity | Immune programme for children | Antibody titer |
| Immune group (1) | 10 | 1 exempts from IBD living vaccine 2,3 exempts from IBD deactivation vaccine | 2 8～2 9 |
| Immune group (2) | 10 | 1,2,3 IBD living vaccine is exempted from | 2 4～2 5 |
| Immune group (3) | 10 | 1,2,3 IBD inactivated vaccine is exempted from | 2 5～2 6 |
As shown in table 1, first utilize the immunity of IBD living vaccine then to contribute to lifting antibody titer by the IBD inactivated vaccine immunity mode of 2 times again 1 time.
Above embodiments of the invention have been described in detail, but described content is only preferred embodiment of the present invention, not in order to limit the present invention.All make in application range of the present invention any amendment, equivalent to replace and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. an infectious bursal disease refined vitelline antibody production method, is characterized in that comprising the following steps:
1) bursal disease vaccine immunity chicken is utilized;
2) get height and exempt from egg yolk, broken after removing vitelline membrane and frenulum;
3) with 1:(2 ~ 3) ratio of (w/w) is to step 2) add that temperature is 50 ~ 56 DEG C, pH is the acetate buffer of 4.8 ~ 5.2 in yolk after fragmentation, stir 25 ~ 35min, obtain mixed solution;
4) to step 3) add the n-caprylic acid of 3 ~ 5% (v/v) in described mixed solution, stir 30 ~ 180min;
5) filter removal solid substance, namely obtain yolk antibody.
2. production method according to claim 1, is characterized in that step 1) described in chicken be the hy-line brown hen of 120 ages in days.
3. production method according to claim 1, is characterized in that step 1) described in immunity be first utilize infectious bursal disease live-vaccine B87 strain immunity chicken 1 time, then recycle infectious bursal disease oil emulsion inactivated vaccine immunity 2 times.
4. production method according to claim 3, is characterized in that utilizing the vaccine consumption of infectious bursal disease live-vaccine B87 strain immunity chicken 1 time to be 6 ~ 8 plumage parts/only.
5. production method according to claim 3, is characterized in that utilizing the vaccine consumption of infectious bursal disease oil emulsion inactivated vaccine immunity chicken to be 2 ~ 5 plumage parts/only at every turn.
6. production method according to claim 3, is characterized in that the timed interval of often adjacent twice immunity is 14d.
7. production method according to claim 1, is characterized in that step 2) in vitelline membrane and frenulum utilize 60 order nylon mesh to filter to remove.
8. production method according to claim 1, is characterized in that step 2) described in broken utilize colloidal mill to realize.
9. production method according to claim 1, it is characterized in that step 3) described acetate buffer prepared by following methods: dissolves 0.886g sodium acetate and 2.82ml glacial acetic acid in every 1000mL purified water, then utilizes the NaOH of 4% (w/w) concentration to regulate pH.
10. production method according to claim 1, is characterized in that step 5) described in filter utilize polypropylene fibre 750B filter cloth to realize.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955532A (en) * | 2010-10-21 | 2011-01-26 | 郑州后羿制药有限公司 | Method for extracting chicken egg yolk antibodies against infectious bursal disease |
| CN103341164A (en) * | 2013-07-19 | 2013-10-09 | 天津瑞普生物技术股份有限公司 | Infectious bursal disease antigen-antibody complex as well as preparation and preparation method thereof |
| CN104479012A (en) * | 2014-11-18 | 2015-04-01 | 中国农业科学院北京畜牧兽医研究所 | Preparation method of peste des petits ruminants virus yolk antibody |
| CN104530231A (en) * | 2014-12-22 | 2015-04-22 | 天津瑞普生物技术股份有限公司 | Method for extracting refined egg yolk antibody by utilizing chick infectious bursal disease virus |
| CN104548093A (en) * | 2014-12-22 | 2015-04-29 | 天津瑞普生物技术股份有限公司 | Composition for treating piglet diarrhea and preparation method of composition |
-
2015
- 2015-09-23 CN CN201510617627.6A patent/CN105175539A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955532A (en) * | 2010-10-21 | 2011-01-26 | 郑州后羿制药有限公司 | Method for extracting chicken egg yolk antibodies against infectious bursal disease |
| CN103341164A (en) * | 2013-07-19 | 2013-10-09 | 天津瑞普生物技术股份有限公司 | Infectious bursal disease antigen-antibody complex as well as preparation and preparation method thereof |
| CN104479012A (en) * | 2014-11-18 | 2015-04-01 | 中国农业科学院北京畜牧兽医研究所 | Preparation method of peste des petits ruminants virus yolk antibody |
| CN104530231A (en) * | 2014-12-22 | 2015-04-22 | 天津瑞普生物技术股份有限公司 | Method for extracting refined egg yolk antibody by utilizing chick infectious bursal disease virus |
| CN104548093A (en) * | 2014-12-22 | 2015-04-29 | 天津瑞普生物技术股份有限公司 | Composition for treating piglet diarrhea and preparation method of composition |
Non-Patent Citations (1)
| Title |
|---|
| 欧阳琴等: "鸡传染性法氏囊病卵黄抗体制备——免疫、效价检测和沉降分离", 《福州大学学报（自然科学版）》 * |
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