CN1634989A - Bacillus coli resisting chicken yolk antibody, its preparation and use - Google Patents
Bacillus coli resisting chicken yolk antibody, its preparation and use Download PDFInfo
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- CN1634989A CN1634989A CNA2003101129806A CN200310112980A CN1634989A CN 1634989 A CN1634989 A CN 1634989A CN A2003101129806 A CNA2003101129806 A CN A2003101129806A CN 200310112980 A CN200310112980 A CN 200310112980A CN 1634989 A CN1634989 A CN 1634989A
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Abstract
This invention discloses an anti-colibacillus egg yolk antibody and its process method and application, wherein the antibody is processed by the following steps: a, collecting thalline and extracting antigen for solving into PBS buffer liquid and adding assistant agent to get the raw antigen of colibacillus; b, to make hen immune by use of the antigen processed by step an and then collecting its eggs for extracting yolk antibody. The antibody in this invention molecular weight is about 180KD.
Description
Technical field
The present invention relates to a kind of immunoglobulin (Ig) and preparation method thereof and application in the biological field, particularly relate to a kind of Chinese People's Anti-Japanese Military and Political College enterobacteria chicken yolk antibody and preparation method thereof and application.
Background technology
Coliform diarrhea in piglets is a kind of common transmittable disease that is caused by the pig pathogenic colon bacillus, usually causes piglet death or grows slowly the development of serious harm pig industry.Since the e. coli serotype complexity, the immunodeficiency specific aim, so, mostly used the method for microbiotic and vaccine immunity sow to prevent and treat this disease in the past.But because of pathogenic colon bacillus is easy to generate resistance to microbiotic, the active immunity effect is also undesirable, and therefore preventing and treating colibacillosis of piglet just becomes one of outstanding problem in the Swine Production.
Chicken yolk antibody (IgY) is a kind of immunoglobulin (Ig) (Ig) of chicken, and its structure is similar to IgG, has the space conformation of typical immunoglobulin (Ig).Studies show that IgY has the stability of height, heat, acid, alkali environment are had good tolerance.Utilize chicken production specific immunity antibody, have advantages such as be produced on a large scale, cost is low, specific antibody content height, security are good, than under the laboratory condition by experiment animal prepare limited hyper-immune serum practicality more arranged.The present domestic yolk antibody of successfully developing and use avian viral transmissible diseases such as newcastle disease, infections bursal disease, duck viral hepatitis, but less to the yolk antibody research of bacillary livestock and poultry transmissible disease.
The innovation and creation content
The purpose of this invention is to provide a kind of anticolibacillary chicken yolk antibody and preparation method thereof.
The Chinese People's Anti-Japanese Military and Political College provided by the present invention enterobacteria chicken yolk antibody, available following method obtains:
(1) collects coli common pili antigen;
(2) with coli common pili antigen immune healthy hens, collect it then and lay eggs, from yolk, extract Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody.
The antigenic concrete steps of described collection coli common pili are: collect thalline, extract antigen, it is dissolved in PBS buffered soln, add adjuvant, obtain coli common pili antigen; Described adjuvant is preferably freund's adjuvant, and general head exempts to use Freund's complete adjuvant, and two exempt to use Freund's incomplete adjuvant; The method of extracting Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody in the step (2) from ovum yolk can be with sterile purified water or sterile saline, be preferably and use distilled water diluting yolk, transfer pH to 5.0-7.1, be preferably pH7.0,4 ℃ left standstill 2-6 hour, be preferably and leave standstill 2 hours, centrifugal 30 minutes of 6000 rev/mins of levels are removed a large amount of lipids, collect supernatant, by 0.45 μ m membrane filtration degerming, spraying drying or lyophilize obtain Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody powder.The molecular weight of described Chinese People's Anti-Japanese Military and Political College enterobacteria chicken yolk antibody is about 180KD.
Preparation method's scope of application of the Chinese People's Anti-Japanese Military and Political College of the present invention enterobacteria chicken yolk antibody is extensive, can be used for preparing various colibacillary antibody, resulting antibody can be used for preparing medicine, healthcare products, foodstuff additive and the fodder additives that prevents and/or treats the relative disease that coli-infection causes, also can be used for preparing the preparation that detects the relative disease that coli-infection causes.
The Chinese People's Anti-Japanese Military and Political College provided by the present invention enterobacteria chicken yolk antibody is laid eggs hen by the bacillus coli antigen immunity and is got, and has specificity, and the digestive tract diseases that control animal large intestine coli infections is caused has good result.Antibody of the present invention adopts hen lay eggs to be carrier, and safety non-toxic is easy to industrialization.
Embodiment
Embodiment 1, Chinese People's Anti-Japanese Military and Political College enterobacteria (Escherichia coli) K
88, K
99, 987P bacterial strain chicken yolk antibody preparation
Chinese People's Anti-Japanese Military and Political College enterobacteria (Escherichia coli) K
88, K
99, 987P bacterial strain chicken yolk antibody technical process be microbial culture → antigen extraction → immunization laying hen → collection egg → separation yolk antibody → lyophilize → raw material finished product, concrete steps are as follows:
1, the preparation of pili antigen
Intestinal bacteria (Escherichia cold) K
88, K
99The 987P bacterial strain, in the TSB liquid nutrient medium, be cultured to logarithmic phase, under 4 ℃ of conditions, centrifugal 15 minutes of 3000r/min level, precipitation is dissolved in the PBS buffered soln, under similarity condition centrifugal 15 minutes then, collect thalline, after its deactivation, stir 10min in ice bath high speed (8000r/min), again under 4 ℃ of conditions, centrifugal 15 minutes of 10000g level is collected the supernatant liquor that contains pili antigen, with 0.45 μ m filter filtering supernatant, filtrate is transferred to after pH is 4.7-4.9, the 10000g level is centrifugal 15 minutes under 4 ℃ of conditions, and collecting precipitation is dissolved in the PBS buffered soln, is pili antigen solution, concentration is about 2.25mg/ml, in-20 ℃ of freezing preservations.Incomplete antigen solution added sterilized complete Freund's adjuvant (head exempts to use) in 1: 1 by volume, and incomplete antigen solution added sterilized incomplete Freund's adjuvant (two exempt to use) in 1: 1 by volume, and emulsification gets O antigen.Above all operations all carries out under aseptic condition.
2, Chinese People's Anti-Japanese Military and Political College enterobacteria (Escherichia coli) K
88, K
99, 987P bacterial strain chicken yolk antibody preparation
(1) to the immunity of the hen that lays eggs
A. fundamental immunity: select the health non-immunization laying hen isolated rearing of laying eggs for use.The complete Freund's adjuvant O antigen 1ml/ that carries out under the cock skin preparation in the multi-point injection step 1 only.
B. reinforced immunological: fundamental immunity is carried out reinforced immunological after two weeks, and the incomplete Freund's adjuvant O antigen 1ml/ of injecting step 1 preparation only.
Begin to collect egg after two weeks, 4 ℃ store for future use.
(2) Chinese People's Anti-Japanese Military and Political College enterobacteria (Escherichia coli) K
88, K
99, 987P bacterial strain chicken yolk antibody extraction
From immune ovum gallinaceum, separate yolk, with 8 times of distilled water dilutings, transfer to pH to 5.0-7.1, be preferably pH7.0,4 ℃ left standstill 2-6 hour, be preferably and leave standstill 2 hours, centrifugal 30 minutes of 6000 rev/mins of levels are removed a large amount of lipids, collect supernatant, by 0.45 μ m membrane filtration degerming, spraying drying or lyophilize obtain Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody powder.
Embodiment 2, Chinese People's Anti-Japanese Military and Political College enterobacteria (Escherichia coli) K
88, K
99, 987P bacterial strain chicken yolk antibody detection
(1) mensuration of molecular weight and purity check
With the Chinese People's Anti-Japanese Military and Political College enterobacteria that obtains among the SDS-PAGE electrophoresis detection embodiment 1 (Escherichia coli) K
88, K
99, 987P bacterial strain chicken yolk antibody purity, detailed process is as follows: the SDS-PAGE electrophoresis, resolving gel concentration 8%, concentrate gum concentration 5%, after current stabilization electrophoresis 4 hours, Marker adopt low relative molecular mass standard protein electrophoresis to finish, taking off gel dyes with Coomassie brilliant blue, an electrophoresis band only appears in result's demonstration, and antibody purity height is described, its molecular weight is about 180KD.
(2) concentration determination
Measure Chinese People's Anti-Japanese Military and Political College enterobacteria (Escherichiacoli) K that obtains among the embodiment 1 with Bradford determination of protein concentration method
88, K
99, 987P bacterial strain chicken yolk antibody concentration.
(3) the active detection:
1. ELISA indirect test
Every hole adds the antigenic solution wrapper sheet of preparation among the 100 μ l embodiment 1, and 37 ℃ of temperature were bathed 2 hours; Outwell coating buffer, wash plate 5 times, pat dry with PBST solution; Every hole adds 100 μ l confining liquids (1% bovine serum albumin solution), and 37 ℃ left standstill 1 hour, washed plate 5 times with PBST solution, patted dry; Every hole adds the goat-anti chicken IgY (working dilution is 1: 5000) of 100 μ l horseradish peroxidase-labeled, and 37 ℃ left standstill 1 hour, washed plate 5 times with PBST solution, patted dry; Every hole adds the TMB that 100 μ l contain hydrogen peroxide, reacts after 15 minutes, and every hole adds the 2MH of 100 μ l
2SO
4Termination reaction, displaing yellow is surveyed light absorption value OD with DG3022 type enzyme-linked immunosorbent assay instrument
450nm
The result shows, recording with enzyme linked immunosorbent assay that Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody tires is 1: 14000-16000.
2. agar diffusion method
A. the preparation of agar plate: 100ml physiological saline adds 1.2 gram agar powders, and heating and melting is poured glass dish into, makes the thick agar plate of 2-3mm, and after room temperature was solidified, 4 ℃ of inversions were preserved stand-by.
B. the dilution of antibody: adopt doubling dilution to dilute yolk antibody with continuous normal saline, its extension rate is respectively 2 *, 4 *, 8 *, 16 *, 32 *, 64 * and highly diluted multiple more.
C. agar diffusion reaction: with punch tool agar plate is broken into seven apertures in the human head blossom type hole, spirit lamp heating back cover.During test sample, add the antigenic solution that obtains among the embodiment 1 at medium pore, periphery holes adds the antibody of different extension rates in certain sequence respectively, and every hole adds 20 μ l samples; Be inverted plate in 37 ℃ of airtight wet boxes 24 to 72 hours, produce, observe antibody titer, with maximum dilution multiple the tiring that precipitation line occurs for this yolk antibody until precipitation line.
The result shows that the Chinese People's Anti-Japanese Military and Political College of the present invention enterobacteria chicken yolk antibody records antibody titer more than 1:128 with agar diffusion method.
Embodiment 3, experimentation on animals
Piglet is attacked poison and therapeutic test: get 1 age in days and do not eat just 12 of suckling piglet, be divided into two groups at random, adopt the artificial suckling method milk of feeding one time every two hours, observe and attack poison after piglet conforms, attacking the toxic agent amount is 1.0 * 10
9The CFU/ head, when treating that piglet has just begun symptom of diarrhea to occur, the treatment group refined vitelline antibody solution 4ml that feeds, twice of every day; Control group is fed equivalent physiological saline, observes the yolk antibody result of treatment, is the index reflection treatment group and the control group significance of difference with ight soil exponential sum mortality ratio.
Test-results shows, attacks poison back all piglets of 12-24h and in various degree diarrhoea all occurs, and the treatment group sx of behind oral yolk antibody liquid 24h, suffering from diarrhoea, ight soil becomes thickness behind the 48h, recovers normal substantially behind the 72h; And control group diarrhoea is the water sample injection, and death mainly appears at 48-72h, and symptom is tending towards relaxing afterwards, and mortality ratio is 36%-65%.
Claims (10)
1, a kind of Chinese People's Anti-Japanese Military and Political College enterobacteria chicken yolk antibody, available following method obtains:
(1) collects coli common pili antigen;
(2) with coli common pili antigen immune healthy hens, collect it then and lay eggs, from yolk, extract Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody.
2, Chinese People's Anti-Japanese Military and Political College according to claim 1 enterobacteria chicken yolk antibody, it is characterized in that: the antigenic concrete steps of described collection coli common pili are: collect thalline, extract antigen, it is dissolved in PBS buffered soln, add adjuvant, obtain coli common pili antigen; Described adjuvant is a freund's adjuvant.
3, Chinese People's Anti-Japanese Military and Political College according to claim 1 and 2 enterobacteria chicken yolk antibody, it is characterized in that: the extracting method that extracts Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody described in the step (2) from ovum yolk is for separating yolk from immune ovum gallinaceum, with 8 times of distilled water dilutings, transfer to pH to 5.0-7.1, be preferably pH7.0,4 ℃ left standstill 2-6 hour, be preferably and leave standstill 2 hours, centrifugal 30 minutes of 6000 rev/mins of levels, remove a large amount of lipids, collect supernatant, by 0.45 μ m membrane filtration degerming, spraying drying or lyophilize obtain Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody powder.The molecular weight of described Chinese People's Anti-Japanese Military and Political College enterobacteria chicken yolk antibody is about 180KD.
4, a kind of method for preparing Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody may further comprise the steps:
(1) collects coli common pili antigen;
(2) with coli common pili antigen immune healthy hens, collect it then and lay eggs, from yolk, extract Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody.
5, method according to claim 4 is characterized in that: the antigenic concrete steps of described collection coli common pili are: collect thalline, extract antigen, it is dissolved in PBS buffered soln, add adjuvant, obtain coli common pili antigen; Described adjuvant is a freund's adjuvant.
6, according to claim 4 or 5 described methods, it is characterized in that: the extracting method that extracts Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody described in the step (2) from ovum yolk is for separating yolk from immune ovum gallinaceum, with 8 times of distilled water dilutings, transfer to pH to 5.0-7.1, be preferably pH7.0,4 ℃ left standstill 2-6 hour, be preferably and leave standstill 2 hours, centrifugal 30 minutes of 6000 rev/mins of levels, remove a large amount of lipids, collect supernatant, by 0.45 μ m membrane filtration degerming, spraying drying or lyophilize obtain Chinese People's Anti-Japanese Military and Political College's enterobacteria chicken yolk antibody powder.
7, method according to claim 6 is characterized in that: described thinner is a sterile purified water.
8, method according to claim 6 is characterized in that: described pH is 7.0; Described temperature is 4 ℃.
9, the application of the described Chinese People's Anti-Japanese Military and Political College of claim 1 enterobacteria chicken yolk antibody in the preparation of the relative disease that preparation detection coli-infection causes.
10, the described Chinese People's Anti-Japanese Military and Political College of claim 1 enterobacteria chicken yolk antibody prevents and/or treats application in medicine, healthcare products, foodstuff additive and the fodder additives of the relative disease that coli-infection causes in preparation.
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| CNB2003101129806A CN100412092C (en) | 2003-12-29 | 2003-12-29 | Bacillus coli resisting chicken yolk antibody, its preparation and use |
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|---|---|---|---|
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| CN1634989A true CN1634989A (en) | 2005-07-06 |
| CN100412092C CN100412092C (en) | 2008-08-20 |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948538A (en) * | 2010-10-19 | 2011-01-19 | 郑州后羿制药有限公司 | Method for refining chicken egg yolk antibodies against Escherichia coli |
| CN102188449A (en) * | 2011-04-15 | 2011-09-21 | 广西壮族自治区兽医研究所 | Preparation method of high-immune whole-egg powder for resisting piglet diarrhea |
| CN102205122A (en) * | 2011-05-05 | 2011-10-05 | 广西壮族自治区兽医研究所 | Method for preparing piglet diarrhea-resisting egg yolk antibody microcapsules by spray drying method |
| 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 |
| CN102914647A (en) * | 2012-05-29 | 2013-02-06 | 安徽农业大学 | Method for detecting duck flavivirus antibody by using agar diffusion method |
| CN104017060A (en) * | 2014-05-22 | 2014-09-03 | 深圳市海王英特龙生物技术股份有限公司 | Extraction method for escherichia coli pilus antigen used for preparing yolk antibody, and method for preparing yolk antibody |
| CN105175536A (en) * | 2015-09-30 | 2015-12-23 | 大连理工大学 | Egg yolk antibody extraction method suitable for large-scale industrial production |
| CN105713088A (en) * | 2016-04-01 | 2016-06-29 | 佛山科学技术学院 | ETEC (enterotoxigenic escherichla coli) yolk antibody powder and preparation method thereof |
| CN106035672A (en) * | 2016-05-27 | 2016-10-26 | 佛山科学技术学院 | Functional egg-milk powder for resisting piglet ETEC (Enterotoxigenic Escherichla coli) diarrhea and preparation method thereof |
| CN108030916A (en) * | 2015-07-03 | 2018-05-15 | 刘永庆 | The Th2 immune responses inhibitor of anti-curing oncoma and/or chronic tuberculosis disease and its application |
-
2003
- 2003-12-29 CN CNB2003101129806A patent/CN100412092C/en not_active Expired - Fee Related
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948538A (en) * | 2010-10-19 | 2011-01-19 | 郑州后羿制药有限公司 | Method for refining chicken egg yolk antibodies against Escherichia coli |
| CN102188449A (en) * | 2011-04-15 | 2011-09-21 | 广西壮族自治区兽医研究所 | Preparation method of high-immune whole-egg powder for resisting piglet diarrhea |
| CN102188449B (en) * | 2011-04-15 | 2012-12-19 | 广西壮族自治区兽医研究所 | Preparation method of high-immune whole-egg powder for resisting piglet diarrhea |
| CN102205122A (en) * | 2011-05-05 | 2011-10-05 | 广西壮族自治区兽医研究所 | Method for preparing piglet diarrhea-resisting egg yolk antibody microcapsules by spray drying method |
| 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 |
| CN102914647A (en) * | 2012-05-29 | 2013-02-06 | 安徽农业大学 | Method for detecting duck flavivirus antibody by using agar diffusion method |
| CN104017060A (en) * | 2014-05-22 | 2014-09-03 | 深圳市海王英特龙生物技术股份有限公司 | Extraction method for escherichia coli pilus antigen used for preparing yolk antibody, and method for preparing yolk antibody |
| CN104017060B (en) * | 2014-05-22 | 2017-01-11 | 深圳市海王英特龙生物技术股份有限公司 | Extraction method for escherichia coli pilus antigen used for preparing yolk antibody, and method for preparing yolk antibody |
| CN108030916A (en) * | 2015-07-03 | 2018-05-15 | 刘永庆 | The Th2 immune responses inhibitor of anti-curing oncoma and/or chronic tuberculosis disease and its application |
| CN105175536A (en) * | 2015-09-30 | 2015-12-23 | 大连理工大学 | Egg yolk antibody extraction method suitable for large-scale industrial production |
| CN105713088A (en) * | 2016-04-01 | 2016-06-29 | 佛山科学技术学院 | ETEC (enterotoxigenic escherichla coli) yolk antibody powder and preparation method thereof |
| CN106035672A (en) * | 2016-05-27 | 2016-10-26 | 佛山科学技术学院 | Functional egg-milk powder for resisting piglet ETEC (Enterotoxigenic Escherichla coli) diarrhea and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN100412092C (en) | 2008-08-20 |
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Effective date of registration: 20060127 Address after: No. 12 feed station, South Street, Haidian District, Beijing, Zhongguancun Applicant after: Institute of fodder, Beijing Academy of Agricultural Sciences Address before: Beijing City, Haidian District Zhongguancun South Street No. 12 feed Ruisi JIAYE Applicant before: Beijing Ruisijiaye Fodder Technology Research Center |
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Assignee: Telefield agro-industrial Ltd. Assignor: Feed Research Institute Chinese Academy of Agricultural Sciences Contract record no.: 2010990001007 Denomination of invention: Bacillus coli resisting chicken yolk antibody, its preparation and use Granted publication date: 20080820 License type: Exclusive License Open date: 20050706 Record date: 20101224 |
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