CN103724429A - Preparation method of anti-piglet diarrhea IgY (immunoglobulin of yolk) - Google Patents
Preparation method of anti-piglet diarrhea IgY (immunoglobulin of yolk) Download PDFInfo
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- CN103724429A CN103724429A CN201310718916.6A CN201310718916A CN103724429A CN 103724429 A CN103724429 A CN 103724429A CN 201310718916 A CN201310718916 A CN 201310718916A CN 103724429 A CN103724429 A CN 103724429A
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Abstract
The invention discloses a preparation method of anti-piglet diarrhea IgY (immunoglobulin of yolk). The preparation method comprises the following steps: separating and purifying a transmissible gastroenteritis virus (TGEV); preparing immunogen through the TGEV and a freund's adjuvant; immunizing a 8-week-old laying hen, injecting 1mL of the immunogen into chicken breast muscle at different points, immunizing for the second time after 10 days, and immunizing for the third time after 10 days; after immunizing for the third time, collecting eggs of the laying hen after 15 days, and separating the yolk; adding dextran sulfate into the yolk, fully stirring, and centrifuging; adding 30% cold ethanol into centrifugal supernatant, fully stirring, and centrifuging to obtain deposits, namely the anti-piglet diarrhea IgY. The anti-piglet diarrhea IgY can be used for effectively treating swine transmissible gastroenteritis, is quick-acting and has a good effect.
Description
Technical field
The invention belongs to medical preparing technical field, relate in particular to the preparation method of a kind of anti-grice diarrhoea IgY.
Background technology
Transmissible gastroenteritis of swine claims again the gastro-enteritis of young pig, is a kind of height contagiousness, propagates rapidly, can spread full group in a few days, vomitting, severe diarrhea, dehydration, causes the viral infectious that two weeks age, interior piglet high mortality was feature.
Just there is relevant report the Illinois of these disease 1933 Nian U.S., and nineteen forty-six determines that pathogenic agent is virus, and this has been carried out to relevant research.Subsequently, in Japan, Britain, Central and South America, Canada, Korea and Philippines, all in succession reported TGEV.Temperate zone to the north of north latitude 30 degree, to refrigerant latitudes, all has TGE to occur at present.Also there is the generation of this disease in the provinces and cities such as the Sichuan of China, Shaanxi, Hubei, Beijing, Jilin, Guangzhou, Taiwan.Piglet mortality ratio is very high in 2 week age, and age in days is less, and death is faster, and within 5 weeks, pig mortality ratio more than age is lower, and larger or Adult Pig is fallen ill and almost be there is no death, but these pigs often become cad pig, and the price of deed is low.TGEV usually mixes generation or secondary with other diseases, be one of major reason causing the grice diarrhoea death of large scale of pig farm field, to aquaculture, has brought huge financial loss.
TGEV is swallowed and is entered digestive tube while infecting, and hydrochloric acid in gastric juice and proteolytic ferment in opposing digestive tube enter small intestine, and small intestine is the main target organ of TGEV.TGEV infects after intestinal epithelial cells, destroys rapidly functional epithelial cell, hinders the decomposition of nutritive substance in small intestine.The nutritive substance that can not be decomposed, as a large amount of gatherings in enteric cavity such as lactose, causes osmotic pressure in enteric cavity to raise, thereby causes diarrhoea and dehydration.
Prior art fails to provide a kind of medicine of effective treatment transmissible gastroenteritis of swine.
Summary of the invention
The invention provides the preparation method of a kind of anti-grice diarrhoea IgY, be intended to solve the problem that prior art fails to provide a kind of medicine of effective treatment transmissible gastroenteritis of swine.
The object of the present invention is to provide the preparation method of a kind of anti-grice diarrhoea IgY, the preparation method of this anti-grice diarrhoea IgY comprises the following steps:
Step 1, separation and purification transmissible gastro-enteritis virus TGEV: jejunum and the mesenteric lymph nodes of getting morbidity piglet are placed in mortar, shred, and repeatedly grind, adding pH value is that 7.0 PBS makes the tissue homogenate of 1: 5 ,-70 ℃ of multigelations 3 times; 5000 revs/min centrifugal 20 minutes, get supernatant liquor through the filtering with microporous membrane of 0.22 μ m, then add dual anti-100IU/mL ,-70 ℃ of preservations, standby;
By processing pathological material of disease, be inoculated in the ST cell that grows up to individual layer, 3 bottles of each sample inoculations, 37 ℃ adsorb 1 hour; The venom of preventing or cure a disease that inclines, adds the DMEM containing 5% serum to continue to cultivate, and observes 4d, does not occur that pathology person continues 5 generations of blind passage; There is cytopathy person to continue to go down to posterity, until cytopathy is stable;
Appearance 80% cytopathy person is arised to-70 ℃ of multigelations 3 times together with substratum one; 5000 revs/min centrifugal 20 minutes, get supernatant liquor through the filtering with microporous membrane of 0.22 μ m, then add dual anti-100IU/mL, for making TGEV immunogen;
Step 2, TGEV and Fu Shi completely or Freund's incomplete adjuvant according to the ratio of 1: 1, vibrator fully shakes and mixes, and is immunogen;
Step 3, the laying hen in 8 weeks age of immunity, the former 1mL of chicken chest muscle branch injecting immune, the full adjuvant immunity that toos many or too much for use after 10 days is former carries out immunity for the second time, and the former teaching of full adjuvant immunity of toing many or too much for use after 10 days of immunity is for the second time immune for the third time;
Step 4, after immunity, collects egg that laying hen produces, separated yolk for 15 days for the third time;
Step 5, yolk adds T 500 concentration 3.5%, fully stir, 5000 revs/min centrifugal 30 minutes, get supernatant;
Step 6, supernatant thing adds 30% ethanol, fully stirs, and after centrifugal treating, gained precipitation is anti-grice diarrhoea IgY.
Technique effect:
The preparation method of anti-grice diarrhoea IgY provided by the invention, the preparation method of this anti-grice diarrhoea IgY comprises the following steps: separation and purification transmissible gastro-enteritis virus TGEV; TGEV+ freund's adjuvant is prepared immunogen; The laying hen in immune 8 week age, the former 1mL of chicken chest muscle branch injecting immune, immunity for the second time after 10 days, immunity is for the second time immunity for the third time after 10 days; After immunity, within 15 days, collect egg that laying hen produces, separated yolk for the third time; Yolk adds T 500, fully stirs centrifugal treating; Get centrifugal supernatant, add 30% ethanol, fully stir, after centrifugal treating, gained precipitation is anti-grice diarrhoea IgY; Anti-grice diarrhoea IgY can effectively treat transmissible gastroenteritis of swine, and instant effect is effective.
Accompanying drawing explanation
Fig. 1 is the preparation method's of the anti-grice diarrhoea IgY that provides of the embodiment of the present invention realization flow figure.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is described in further detail.Should be appreciated that specific embodiment described herein is only in order to explain the present invention, and be not used in restriction invention.
Fig. 1 shows the preparation method's of the anti-grice diarrhoea IgY that the embodiment of the present invention provides realization flow.
The preparation method of this anti-grice diarrhoea IgY comprises the following steps:
Step S101, separation and purification transmissible gastro-enteritis virus TGEV;
Step S102, TGEV+ freund's adjuvant is prepared immunogen;
Step S103, the laying hen in immune 8 week age, the former 1mL of chicken chest muscle branch injecting immune, immunity for the second time after 10 days, immunity is for the second time immunity for the third time after 10 days;
Step S104, after immunity, collects egg that laying hen produces, separated yolk for 15 days for the third time;
Step S105, yolk adds T 500, fully stirs centrifugal treating;
Step S106, gets centrifugal supernatant, adds 30% ethanol, fully stirs, and after centrifugal treating, gained precipitation is anti-grice diarrhoea IgY.
Concrete grammar of the present invention is:
Step 1, separation and purification transmissible gastro-enteritis virus TGEV: jejunum and the mesenteric lymph nodes of getting morbidity piglet are placed in mortar, shred, and repeatedly grind, adding pH value is that 7.0 PBS makes the tissue homogenate of 1: 5 ,-70 ℃ of multigelations 3 times; 5000 revs/min centrifugal 20 minutes, get supernatant liquor through the filtering with microporous membrane of 0.22 μ m, then add dual anti-100IU/mL ,-70 ℃ of preservations, standby;
By processing pathological material of disease, be inoculated in the ST cell that grows up to individual layer, 3 bottles of each sample inoculations, 37 ℃ adsorb 1 hour; The venom of preventing or cure a disease that inclines, adds the DMEM containing 5% serum to continue to cultivate, and observes 4d, does not occur that pathology person continues 5 generations of blind passage; There is cytopathy person to continue to go down to posterity, until cytopathy is stable;
Appearance 80% cytopathy person is arised to-70 ℃ of multigelations 3 times together with substratum one; 5000 revs/min centrifugal 20 minutes, get supernatant liquor through the filtering with microporous membrane of 0.22 μ m, then add dual anti-100IU/mL, for making TGEV immunogen;
Step 2, TGEV and Fu Shi completely or Freund's incomplete adjuvant according to the ratio of 1: 1, vibrator fully shakes and mixes, and is immunogen;
Step 3, the laying hen in 8 weeks age of immunity, the former 1mL of chicken chest muscle branch injecting immune, the full adjuvant immunity that toos many or too much for use after 10 days is former carries out immunity for the second time, and the former teaching of full adjuvant immunity of toing many or too much for use after 10 days of immunity is for the second time immune for the third time;
Step 4, after immunity, collects egg that laying hen produces, separated yolk for 15 days for the third time;
Step 5, yolk adds T 500 concentration 3.5%, fully stir, 5000 revs/min centrifugal 30 minutes, get supernatant;
Step 6, supernatant thing adds 30% ethanol, fully stirs, and after centrifugal treating, gained precipitation is anti-grice diarrhoea IgY.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
Example one: 5000 of Shangnan County Rui Sheng limited industrial company pig livestock on hands, 300 of suitable numerous sows, within 2012, appearance 7 ages in days are suffered from diarrhoea in a large number with interior piglet, sickness rate 53%, mortality ratio starts trial edition engineering test preparation in March, 37%, 2013, to June, sickness rate is down to 13%, and mortality ratio is down to 5.3%.
10000 of the numerous cultivation of starlight breeding pig company limited pig livestock on hands, 800 of suitable numerous sows, start trial edition test preparation in May, 2012, compare with early stage, newborn piglet diarrhea disease percentage reduces 26%, mortality ratio reduces by 17%, 7 age in days piglet survival ratio and reaches 94%, has significantly improved the economic benefit of company.
7000 of the recessed Ecodevelopment of Danfeng County corrie company limited pig livestock on hands, 500 of suitable numerous sows, in May, 2013 trial edition engineering test preparation, 7 ages in days reduce 30.6% with interior grice diarrhoea sickness rate, mortality ratio reduces 25.3%, this preparation can make newborn piglet obtain the strong immunizing power of transmissible gastro-enteritis virus, effectively reduces the mortality causing because of transmissible gastroenteritis of swine.
2000 of new origin animal husbandry company limited pig livestock on hands, 1000 of suitable numerous sows, in September, 2012 trial edition technology test preparation after, M & M significantly reduces, and than reducing by 36% and 30% early stage, has significantly prevented the generation of the diarrhoea of piglet respectively.
The preparation method of the anti-grice diarrhoea IgY that the embodiment of the present invention provides, the preparation method of this anti-grice diarrhoea IgY comprises the following steps: separation and purification transmissible gastro-enteritis virus TGEV; TGEV+ freund's adjuvant is prepared immunogen; The laying hen in immune 8 week age, the former 1mL of chicken chest muscle branch injecting immune, immunity for the second time after 10 days, immunity is for the second time immunity for the third time after 10 days; After immunity, within 15 days, collect egg that laying hen produces, separated yolk for the third time; Yolk adds T 500, fully stirs centrifugal treating; Get centrifugal supernatant, add 30% ethanol, fully stir, after centrifugal treating, gained precipitation is anti-grice diarrhoea IgY; Anti-grice diarrhoea IgY can effectively treat transmissible gastroenteritis of swine, and instant effect is effective.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (3)
1. an anti-grice diarrhoea IgY, is characterized in that, the formula of this anti-grice diarrhoea IgY forms: according to the ratio of 1: 5, by stroke-physiological saline solution, powdery yolk (purity > 95%) is diluted.
2. anti-grice diarrhoea IgY as claimed in claim 1, is characterized in that, the using method of this anti-grice diarrhoea IgY is: after piglet birth, oral 1.0mL, allows piglet to eat colostrum again after 3 hours immediately.
3. a preparation method of anti-grice diarrhoea IgY, is characterized in that, the preparation method of this anti-grice diarrhoea IgY comprises the following steps:
Step 1, separation and purification transmissible gastro-enteritis virus TGEV: jejunum and the mesenteric lymph nodes of getting morbidity piglet are placed in mortar, shred, and repeatedly grind, adding pH value is that 7.0 PBS makes the tissue homogenate of 1: 5 ,-70 ℃ of multigelations 3 times; 5000 revs/min centrifugal 20 minutes, get supernatant liquor through the filtering with microporous membrane of 0.22 μ m, then add dual anti-100IU/mL ,-70 ℃ of preservations, standby;
By processing pathological material of disease, be inoculated in the ST cell that grows up to individual layer, 3 bottles of each sample inoculations, 37 ℃ adsorb 1 hour; The venom of preventing or cure a disease that inclines, adds the DMEM containing 5% serum to continue to cultivate, and observes 4d, does not occur that pathology person continues 5 generations of blind passage; There is cytopathy person to continue to go down to posterity, until cytopathy is stable;
Appearance 80% cytopathy person is arised to-70 ℃ of multigelations 3 times together with substratum one; 5000 revs/min centrifugal 20 minutes, get supernatant liquor through the filtering with microporous membrane of 0.22 μ m, then add dual anti-100IU/mL, for making TGEV immunogen;
Step 2, TGEV and Fu Shi completely or Freund's incomplete adjuvant according to the ratio of 1: 1, vibrator fully shakes and mixes, and is immunogen;
Step 3, the laying hen in 8 weeks age of immunity, the former 1mL of chicken chest muscle branch injecting immune, the full adjuvant immunity that toos many or too much for use after 10 days is former carries out immunity for the second time, and the former teaching of full adjuvant immunity of toing many or too much for use after 10 days of immunity is for the second time immune for the third time;
Step 4, after immunity, collects egg that laying hen produces, separated yolk for 15 days for the third time;
Step 5, yolk adds T 500 concentration 3.5%, fully stir, 5000 revs/min centrifugal 30 minutes, get supernatant;
Step 6, supernatant thing adds 30% ethanol, fully stirs, and after centrifugal treating, gained precipitation is anti-grice diarrhoea IgY.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104001171A (en) * | 2014-05-26 | 2014-08-27 | 黄光东 | Preparation method of compound IgY for preventing various diarrheas of piglets |
Citations (4)
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| CN1509765A (en) * | 2002-12-20 | 2004-07-07 | 北京市农林科学院畜牧兽医研究所 | Freezed dry preparation of lecithal antibody and its producing method |
| CN1569895A (en) * | 2003-07-11 | 2005-01-26 | 华中农业大学 | Yelk antibody specific for preventing pigling diarrhea and its preparing method and application |
| CN101081866A (en) * | 2006-05-29 | 2007-12-05 | 雅臣药业集团（远东）有限公司 | Domestic animal and aquatic product causal agent resistant specific IgY or compound IgY and application thereof |
| KR20110000854A (en) * | 2009-06-29 | 2011-01-06 | (주)애드바이오텍 | Composition of food additives of piglets containing igy from egg yolk for preventing of porcine epidemic diarrhea or transmissible gastroenteritis |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1509765A (en) * | 2002-12-20 | 2004-07-07 | 北京市农林科学院畜牧兽医研究所 | Freezed dry preparation of lecithal antibody and its producing method |
| CN1569895A (en) * | 2003-07-11 | 2005-01-26 | 华中农业大学 | Yelk antibody specific for preventing pigling diarrhea and its preparing method and application |
| CN101081866A (en) * | 2006-05-29 | 2007-12-05 | 雅臣药业集团（远东）有限公司 | Domestic animal and aquatic product causal agent resistant specific IgY or compound IgY and application thereof |
| KR20110000854A (en) * | 2009-06-29 | 2011-01-06 | (주)애드바이오텍 | Composition of food additives of piglets containing igy from egg yolk for preventing of porcine epidemic diarrhea or transmissible gastroenteritis |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104001171A (en) * | 2014-05-26 | 2014-08-27 | 黄光东 | Preparation method of compound IgY for preventing various diarrheas of piglets |
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Application publication date: 20140416 |