CN105504050A - Preparation method of hen egg-yolk antibodies resisting Angrara viruses - Google Patents
Preparation method of hen egg-yolk antibodies resisting Angrara viruses Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds
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Abstract
The invention relates to a preparation method of hen egg-yolk antibodies resisting Angrara viruses. The preparation method includes the steps of preparing Angrara immunizing antigens, immunizing healthy laying hens by the Angrara immunizing antigens to obtain qualified eggs, diluting egg yolk by an acetic acid-sodium acetate buffer solution, taking liquid supernatant, adding saturated ammonium sulfate to centrifuge, precipitate and filter, adding sterilized saline water for ultrafiltration, adding beta-cyclodextrin in a mass-to-volume ratio of 1:130-180, sodium glutamate in a mass-to-volume ratio of 1:150-230, skim milk powder in a mass-to-volume ratio of 1:5-20 and soluble starch in a mass-to-volume ratio of 1:10-30 with mixing uniformly and drying to obtain a dry egg-yolk antibody powder preparation. The preparation method has the advantages that filtering time is short and protein is less prone to denaturing and loss; the egg-yolk antibodies are remarkably effective in treatment of the Angrara disease, take action quickly and are good in use effect and low in sick hen death rate.
Description
Technical field
The invention belongs to biological technical field, particularly relate to a kind of preparation method of anti-Ankara chicken yolk antibody.
Background technology
Hydropericardium-hepatitis syndrome (Hydropericardiumhepatitissyndrome, HHS) is a kind of novel poultry disease caused by serum 4 type aviadenovirus (Fowladenovirusserotybe4, FAV4).Classical symptom is 3-5 week broiler chicken sudden death in age, and is attended by hydropericardium and hepatitis, therefore gains the name.Again because its Late Cambrian is in the place of Pakistan Karachi near Ankara, therefore have another name called Ankara disease (Angraradisease).
Ankara disease mechanically and pollutent horizontal transmission and contact between broiler chicken group propagate.Because Ankara disease does not occur special clinical symptom poultry, before symptom occurs, just make clinical diagnosis is difficult.This disease is at first in Pakistani 3-5 broiler chicken group discovery in age in week, and chicken group has more than 50% mortality ratio, and some chicken house mortality ratio are even up to 80%.So high mortality ratio provisions fowl industry brings great loss.
Because Ankara is sick temporarily without effective treatment plan, antiviral effect is undesirable.Can mortality ratio be reduced with oneself Formalin inactivation seedling prepared by the liver homogenate infected clinically and be employed in prevention.Vaccine prepared by the cell culture being used in chicken embryo liver cell growth virus also can produce protection.But control effect is still to be seen.
Yolk antibody (IgY), refers to the antibody for specific antigen extracted from immune eggs.Chicken IgY is functionally equivalent to Mammals IgG, but structurally different, because it has good stability, can acid and alkali-resistance, heat-resisting, proteolysisresistant, and even its enzymatic fragment also has partial antibody activity; And the antibody concentration energy long term maintenance in yolk is equivalent to the level even exceeding Serum antibody concentrations, from yolk, extract antibody without the need to blood sampling and damage, can greatly reduce stress simultaneously.Therefore, specific yolk antibody possesses easy to operate, that output is high feature.But existing extractive technique is mainly carried out dilution with acidified water and is stirred, and lipid and foreign protein deposition efficiency are not high, cause extraction time long, antibody activity decline.
Summary of the invention
The object of the invention is the preparation method providing a kind of efficient anti-Ankara chicken yolk antibody.
The object of the present invention is achieved like this: a kind of preparation method of anti-Ankara chicken yolk antibody, is characterized in that: its step is as follows:
The preparation of step 1), Ankara immunizing antigen: the pathological material of disease tissue being detected as Ankara positive is added sterilizing PBS liquid containing penicillin 1000U/mL and Streptomycin sulphate 1000U/mL by 1:5 (w/V); Being organized in stamp mill after the above-mentioned PBS of adding liquid smashed to pieces, in-20 DEG C of environment, multigelation 3 times, filters with aseptic hospital gauze; In the filtrate of gained, add the formaldehyde of cumulative volume 0.5% amount, be placed in 37 DEG C of thermostatic equipment deactivation 24h, with the bacterial filter filtration sterilization of 0.22 μm, Ankara immunizing antigen can be obtained;
Step 2), the immunity of laying hen: choose healthy laying hen, use when 140 age in days Ankara immunizing antigen to carry out first immunisation to healthy laying hen, after first immunisation, carry out booster immunization respectively after 14 days and 28 days; Carried out antibody test every 7 days after booster immunization first, collect and detect qualified egg;
The preparation of step 3), yolk antibody crude extract: a), by after egg yolk qualified for detection separation, according to the ratio of 1:8 add pH be 5.0 Acetic acid-sodium acetate damping fluid dilute, egg yolk liquid pH value after dilution is adjusted to 5.0, stirs 30min and make it form uniform egg yolk liquid; Under 4 DEG C of environment, leave standstill 4-6 hour, get supernatant liquor for subsequent use; B), under 20 DEG C of water bath condition, saturated ammonium sulphate solution is slowly added in supernatant liquor, limit edged stirs and makes the concentration of ammonium sulfate reach 40%, 1 hour is left standstill in 20 DEG C of water-baths, then under 4 DEG C of environment with the rotating speed of 3500r/min, to the centrifugal 15min of the supernatant liquor after ammonium sulfate be added, collecting precipitation; C), with the isopyknic sterile saline of initial yolk the precipitation obtained is dissolved, with the membrane filtration of 0.45 μm; D), by the filtrate obtained in c) add physiological saline, use the filter membrane that interception is 30kd to carry out ultrafiltration, detect and stop without after ammonium sulfate, gained solution is yolk antibody crude extract;
The drying of step 4), yolk antibody crude extract: after the detection of yolk antibody crude extract is qualified, mix after adding the Zulkovsky starch of beta-cyclodextrin that mass volume ratio is 1:130 ~ 180, the Sodium Glutamate of 1:150 ~ 230, the skim-milk of 1:5 ~ 20,1:10 ~ 30 respectively, spraying dry is carried out with the condition of 190 DEG C of inlet temperature, 70 DEG C of air outlet temperatures, final obtained yolk antibody dry powder formulations in spray-dryer.
The rotating speed of described stamp mill is 10000rpm, and the time of smashing to pieces is 2min.
Described yolk antibody crude extract detect qualified after, mix after adding the Zulkovsky starch of beta-cyclodextrin that mass volume ratio is 1:150, the Sodium Glutamate of 1:200, the skim-milk of 1:10,1:20 respectively.
The present invention extracts the sick chicken tissues in Ankara and makes homogenate, carries out sterilizing, as immunizing antigen after constant temperature deactivation with penicillin and Streptomycin sulphate; With this antigen reinforced immunological bird inlay, obtain high-immunity egg; It is effective that Acetic acid-sodium acetate damping fluid has grease removal, time is short, reduce protein-denatured advantage, therefore Acetic acid-sodium acetate damping fluid is utilized to extract anti-Ankara chicken yolk antibody crude product from high-immunity egg Huang, finally carry out uf processing, compare in dialysis, the time that ultrafiltration uses is shorter, yolk antibody through 30kd filter membrane, can not can not lose albumen.With beta-cyclodextrin, Sodium Glutamate, skim-milk, Zulkovsky starch, drying is carried out to yolk antibody crude extract, the activity of yolk antibody crude extract can be ensured preferably, reduce protein-denatured possibility.The yolk antibody of gained is remarkable to the result for the treatment of of Ankara disease, and responding time is fast, and result of use is good, and sick chicken death rate is low.
Embodiment
The preparation method of anti-Ankara chicken yolk antibody, its step is as follows:
The preparation of step 1), Ankara immunizing antigen: the pathological material of disease tissue being detected as Ankara positive is added in the sterilizing PBS liquid containing penicillin 1000U/mL and Streptomycin sulphate 1000U/mL by 1:5 (w/V); Being organized in stamp mill after the above-mentioned PBS of adding liquid is smashed to pieces, under the rotating speed of 10000rpm, smashs 2min to pieces; In-20 DEG C of environment, multigelation 3 times, filters with aseptic hospital gauze; In the filtrate of gained, add the formaldehyde of cumulative volume 0.5% amount, be placed in 37 DEG C of thermostatic equipment deactivation 24h, with the bacterial filter filtration sterilization of 0.22 μm, Ankara immunizing antigen can be obtained;
Step 2), the immunity of laying hen: choose healthy laying hen, use when 140 age in days Ankara immunizing antigen to carry out first immunisation to healthy laying hen, carry out booster immunization in first immunisation respectively after 14 days and 28 days; Carried out antibody test every 7 days after booster immunization first, collect and detect qualified egg;
The preparation of step 3), yolk antibody crude extract: a), by after egg yolk qualified for detection separation, according to the ratio of 1:8 add pH be 5.0 Acetic acid-sodium acetate damping fluid dilute, egg yolk liquid pH value after dilution is adjusted to 5.0, stirs 30min and make it form uniform egg yolk liquid; Under 4 DEG C of environment, leave standstill 4-6 hour, get supernatant liquor for subsequent use; B), under 20 DEG C of water bath condition, saturated ammonium sulphate solution is slowly added in supernatant liquor, limit edged stirs and makes the concentration of ammonium sulfate reach 40%, 1 hour is left standstill in 20 DEG C of water-baths, then under 4 DEG C of environment with the rotating speed of 3500r/min, to the centrifugal 15min of the supernatant liquor after ammonium sulfate be added, collecting precipitation; C), with the isopyknic sterile saline of initial yolk the precipitation obtained is dissolved, with the membrane filtration of 0.45 μm; D), by the filtrate obtained in c) add physiological saline, use the filter membrane that interception is 30kd to carry out ultrafiltration, detect and stop without after ammonium sulfate, gained solution is yolk antibody crude extract;
The drying of step 4), yolk antibody crude extract: after the detection of yolk antibody crude extract is qualified, mix after adding the Zulkovsky starch of beta-cyclodextrin that mass volume ratio is 1:150, the Sodium Glutamate of 1:200, the skim-milk of 1:10,1:20 respectively, spraying dry is carried out with the condition of 190 DEG C of inlet temperature, 70 DEG C of air outlet temperatures, final obtained yolk antibody dry powder formulations in spray-dryer.
Example one: the activity contrast of anti-Ankara chicken yolk antibody crude extract
Acidifying water law: the purified water of Ankara high-immunity yolk in 1:9 ratio pH5.0 diluted, after stirring, is 5.0-5.2 by HCl adjust ph, 4 DEG C of hold over night; With the centrifugal 25min of the rotating speed of 3500r/min under 4 DEG C of environment, discard precipitation, collect supernatant; Slowly add saturated ammonium sulphate solution in supernatant liquor, limit edged stirs, and makes the concentration of ammonium sulfate reach 40%; 1 hour is left standstill, the centrifugal 15min of 3500r/min at 4 DEG C, collecting precipitation in 20 DEG C of water-baths; The precipitation obtained is dissolved, with the membrane filtration of 0.45 μm with the isopyknic sterile saline of initial yolk; Use the filter membrane that interception is 30kd to carry out ultrafiltration, detect and stop without after ammonium sulfate, gained solution is yolk antibody crude extract.
The present invention: by Ankara high-immunity yolk according to the ratio of 1:8 add pH be 5.0 Acetic acid-sodium acetate damping fluid dilute, by dilution after egg yolk liquid pH value be adjusted to 5.0, stir 30min make it form uniform egg yolk liquid; Under 4 DEG C of environment, leave standstill 4-6 hour, get supernatant liquor for subsequent use; Under 20 DEG C of water bath condition, saturated ammonium sulphate solution is slowly added in supernatant liquor, limit edged stirs and makes the concentration of ammonium sulfate reach 40%, 1 hour is left standstill in 20 DEG C of water-baths, then under 4 DEG C of environment with the rotating speed of 3500r/min, to the centrifugal 15min of the supernatant liquor after ammonium sulfate be added, collecting precipitation; The precipitation obtained is dissolved, with the membrane filtration of 0.45 μm with the isopyknic sterile saline of initial yolk; Use the filter membrane that interception is 30kd to carry out ultrafiltration, detect and stop without after ammonium sulfate, gained solution is yolk antibody crude extract.
Utilize ultraviolet spectrophotometer to detect protein content in two kinds of crude extracts respectively, conventional ELISA method detects antibody titer respectively.Result is as follows:
| Acidifying water law | The present invention | |
| Protein content (mg/mL) | 4.32 | 6.78 |
| Antibody titer | 1：256 | 1：512 |
From experiment, the yolk antibody crude extract obtained with the method after the present invention improves, protein content and antibody titer have obvious raising.
Experimental example: the result for the treatment of of anti-Ankara chicken yolk antibody injection liquid:
The present invention being obtained yolk antibody dry powder formulations stroke-physiological saline solution, to be diluted to protein content be that 2.5mg/mL obtains anti-Ankara chicken yolk antibody injection liquid.
Linzhou City's chicken house, occurs that chicken morbidity is anxious this in December, 2014, and dead fast, diagnose through laboratory PCR, be diagnosed as hydropericardium-hepatitis syndrome, namely Ankara is sick.
This sick chicken about 7100, random choose 100 is only as blank, and all the other are equally divided into experimental group and control group two groups.Wherein experimental group injects the anti-Ankara chicken yolk antibody injection liquid prepared by the inventive method, every 0.5ml; Control group injection compound Radix Astragali polysaccharide injection, every 0.5ml; Blank group is left intact.Other are consistent in feeding and management.Observe and make a record.Test-results is as follows:
Table 1: dead quantity (only)
Table 2: average daily food consumption (g/ only)
As can be seen from Table 1 and Table 2, use the sick chicken of egg yolk antibody injection of the present invention, namely death toll obviously declines after injection first day, food consumption was recovered steadily from second day, substantially recovered normally, compared with control group to the 4th day chicken group, instant effect, effective, mortality ratio is low.Illustrate that this kind of egg yolk antibody injection has good therapeutic action for Ankara disease.
Claims (3)
1. a preparation method for anti-Ankara chicken yolk antibody, is characterized in that: its step is as follows:
The preparation of step 1), Ankara immunizing antigen: the pathological material of disease tissue being detected as Ankara positive is added sterilizing PBS liquid containing penicillin 1000U/mL and Streptomycin sulphate 1000U/mL by 1:5 (w/V); Being organized in stamp mill after the above-mentioned PBS of adding liquid smashed to pieces, in-20 DEG C of environment, multigelation 3 times, filters with aseptic hospital gauze; In the filtrate of gained, add the formaldehyde of cumulative volume 0.5% amount, be placed in 37 DEG C of thermostatic equipment deactivation 24h, with the bacterial filter filtration sterilization of 0.22 μm, Ankara immunizing antigen can be obtained;
Step 2), the immunity of laying hen: choose healthy laying hen, use when 140 age in days Ankara immunizing antigen to carry out first immunisation to healthy laying hen, after first immunisation, carry out booster immunization respectively after 14 days and 28 days; Carried out antibody test every 7 days after booster immunization first, collect and detect qualified egg;
The preparation of step 3), yolk antibody crude extract: a), by after egg yolk qualified for detection separation, according to the ratio of 1:8 add pH be 5.0 Acetic acid-sodium acetate damping fluid dilute, egg yolk liquid pH value after dilution is adjusted to 5.0, stirs 30min and make it form uniform egg yolk liquid; Under 4 DEG C of environment, leave standstill 4-6 hour, get supernatant liquor for subsequent use; B), under 20 DEG C of water bath condition, saturated ammonium sulphate solution is slowly added in supernatant liquor, limit edged stirs and makes the concentration of ammonium sulfate reach 40%, 1 hour is left standstill in 20 DEG C of water-baths, then under 4 DEG C of environment with the rotating speed of 3500r/min, to the centrifugal 15min of the supernatant liquor after ammonium sulfate be added, collecting precipitation; C), with the isopyknic sterile saline of initial yolk the precipitation obtained is dissolved, with the membrane filtration of 0.45 μm; D), by the filtrate obtained in c) add physiological saline, use the filter membrane that interception is 30kd to carry out ultrafiltration, detect and stop without after ammonium sulfate, gained solution is yolk antibody crude extract;
The drying of step 4), yolk antibody crude extract: after the detection of yolk antibody crude extract is qualified, mix after adding the Zulkovsky starch of beta-cyclodextrin that mass volume ratio is 1:130 ~ 180, the Sodium Glutamate of 1:150 ~ 230, the skim-milk of 1:5 ~ 20,1:10 ~ 30 respectively, spraying dry is carried out with the condition of 190 DEG C of inlet temperature, 70 DEG C of air outlet temperatures, final obtained yolk antibody dry powder formulations in spray-dryer.
2. the preparation method of anti-Ankara chicken yolk antibody according to claim 1, is characterized in that: the rotating speed of described stamp mill is 10000rpm, and the time of smashing to pieces is 2min.
3. the preparation method of anti-Ankara chicken yolk antibody according to claim 1, it is characterized in that: described yolk antibody crude extract detect qualified after, mix after adding the Zulkovsky starch of beta-cyclodextrin that mass volume ratio is 1:150, the Sodium Glutamate of 1:200, the skim-milk of 1:10,1:20 respectively.
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Cited By (6)
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| CN106065030A (en) * | 2016-08-02 | 2016-11-02 | 重庆三杰众鑫生物工程有限公司 | Yolk antibody of chicken inclusion body hepatitis and preparation method thereof |
| CN106954735A (en) * | 2017-03-10 | 2017-07-18 | 大连理工大学 | Special yolk solution additive by carrier of peanut shell powder and preparation method thereof |
| CN107279574A (en) * | 2017-07-25 | 2017-10-24 | 安徽华米生物科技有限公司 | It is a kind of to be used to prevent feed of duck Ankara disease and preparation method thereof |
| CN107383189A (en) * | 2017-08-11 | 2017-11-24 | 山东阿斯可来生物技术有限公司 | Method prepared by Yolk antibody xeraphium |
| CN110240648A (en) * | 2019-06-17 | 2019-09-17 | 长春西诺生物科技有限公司 | Cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy freeze-dried yolk antibody and preparation and application |
| CN111333721A (en) * | 2020-03-19 | 2020-06-26 | 河北科星药业有限公司 | Preparation method of yolk antibody for resisting hydropericardium syndrome and application of corresponding antibody |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106065030A (en) * | 2016-08-02 | 2016-11-02 | 重庆三杰众鑫生物工程有限公司 | Yolk antibody of chicken inclusion body hepatitis and preparation method thereof |
| CN106954735A (en) * | 2017-03-10 | 2017-07-18 | 大连理工大学 | Special yolk solution additive by carrier of peanut shell powder and preparation method thereof |
| CN107279574A (en) * | 2017-07-25 | 2017-10-24 | 安徽华米生物科技有限公司 | It is a kind of to be used to prevent feed of duck Ankara disease and preparation method thereof |
| CN107383189A (en) * | 2017-08-11 | 2017-11-24 | 山东阿斯可来生物技术有限公司 | Method prepared by Yolk antibody xeraphium |
| CN110240648A (en) * | 2019-06-17 | 2019-09-17 | 长春西诺生物科技有限公司 | Cat infectiousness nose conjunctivitis and feline panleukopenia bigeminy freeze-dried yolk antibody and preparation and application |
| CN111333721A (en) * | 2020-03-19 | 2020-06-26 | 河北科星药业有限公司 | Preparation method of yolk antibody for resisting hydropericardium syndrome and application of corresponding antibody |
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