CN1268642C - Yelk antibody specific for preventing pigling diarrhea and its preparing method and application - Google Patents
Yelk antibody specific for preventing pigling diarrhea and its preparing method and application Download PDFInfo
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- CN1268642C CN1268642C CNB031283101A CN03128310A CN1268642C CN 1268642 C CN1268642 C CN 1268642C CN B031283101 A CNB031283101 A CN B031283101A CN 03128310 A CN03128310 A CN 03128310A CN 1268642 C CN1268642 C CN 1268642C
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Abstract
The present invention relates to a specificity yolk antibody for preventing piglet diarrhea, and a preparation method and the application thereof. In the method, TSB liquid culture media are used for culturing strains and purifying and obtaining specificity pilus adhesin proteins which are mixed with Freund's adjuvant agents for preparing immunogens; the immunogens are used for immunizing laying hens for obtaining the K88 ac specificity yolk antibody for preventing piglet diarrhea. The yolk antibody of the present invention can very remarkably inhibit the sticking effect of Escherichia coli K88 ac generating enterotoxin on enteric epithelium cells (p is smaller than 0.01); the addition of yolk antibody powder with the concentration of 1% can remarkably reduce the material meat ratio and the diarrhea rate (p is smaller than 0.05), and simultaneously, the addition of yolk antibody powder with the concentration of 1% has a certain promotion function to the growth of piglets.
Description
Technical field
The invention belongs to disease of domestic animals prevention and control techniques field, be specifically related to the application of genetic engineering technique, relate to enterotoxigenic intestinal bacteria K88ac pili antigen of preparation and special pili antigen yolk antibody, their preparation method and yolk antibody are used to prevent grice diarrhoea, and be relevant with the nutrition immune of piglet.
Technical background
Intestinal bacteria are the bacterium that normally lives away from home in the humans and animals enteron aisle, can cause disease again.In large scale of pig farm was produced, caused by intestinal bacteria was that the financial loss that gastrointestinal illness caused of cardinal symptom can not be ignored with the grice diarrhoea.It is reported, the M ﹠ M of the grice diarrhoea that intestinal bacteria cause accounts for the whole piglet of China respectively and rushes down 56.2% and 24.7% of dysentery M ﹠ M, bring tremendous loss (Shi Qishun to pig industry, the sick breeding for disease resistance research of chitling toxicity intestinal bacteria (ETEC). external livestock technology, 1999,26 (4): 51～54).And enteron aisle produces enterotoxigenic escherichia coli (Enterotoxigenic Escherichia coli, ETEC) be to cause that cub diarrhoea is the gastrointestinal illness The main pathogenic fungi of cardinal symptom (Hampson.D.L., Escherichia coil in domestic animals and humans.CAB.InternationalWallingford UK, pp:629～647,1994).The ratio that cub ETEC diarrhoea such as China pig, ox, sheep take place is respectively 35%, 26% and 17%, mortality ratio be 10%～30% (the poplar timing., zoonosis originality intestinal bacteria and colibacillosis.China animal doctor science and technology, 1987 (6): 25～29).Pig source ETEC colonizing factor (colonization factor) or adhesin (adhesion) mainly comprise K88 (F4), K99 (F5), 987P (F6) and F41, and wherein K88 is main virulence factor.K88 has three kinds of varient K88ab, K88ac, K88ad.From Wuhan and the diarrhoea piglet excrement sample gathered of surrounding area large-scale pig farm, identify that by molecular biology method intestinal bacteria K88 accounts for 9.3% of sum, and is the K88ac varient.
Yokoyama etc. carry out controlling colibacillosis of piglet with yolk antibody first, they make the cilia protein vaccine with ETEC host specificity bacterium K88, K99 and the 987P that purifies, chest muscle injecting immune bird inlay, antibody horizontal reaches height tire after, collect egg, separate and the spraying drying yolk antibody, carry out yolk antibody and treat infecting the newborn newborn piglet of not eating of different ETEC bacterial strains, the result shows that all piglets accept different strains and infect in back 12 hours and all suffer from diarrhoea; Be that 625 and 2500 Antybody therapy piglet all survives with tiring, and control group piglet mortality ratio surpasses 80% (Passive protective effect of chicken egg-yolk immunoglobulins against experimentalenterotoxigenic Escherichia coil infection in neonatal pigs.Infect Immun, 1992,60:998～1007).(Prophylactic effect of specific egg yolk antibodies in diarrhea of weaned piglets caused by Escherichia coil K88.J Vet Med A such as Erhard, 1996,43:217～223), (chicken egg yolk antibodies against F18ab fimbriae ofEscherichia coil inhibit shedding of F18 positive E.coli by experimentally infected pigs.Vet.Microbiol.1997 such as Imberechts, 54:329-341) and Zuniga etc. (Reduced intestinal colonization with F18-positive Escherichia coil in weaned pigsfed chicken egg antibody against the fimbriae.FEMS.Immunology and Medical Microbiology 1997.18:153-161) has carried out relevant research and has all obtained similar conclusion.(the developments of the anti-swine escherichia coli yolk antibody of chicken such as beard letter, China's livestock and poultry transmissible disease, 1994,2:26-27) manufactured experimently the four batches of anti-pig ETEC of chicken yolk antibodies with K88, K99,987P trivalent deactivation vaccine, in nearly 900 piglets in 9 pig farms, five counties, the suburbs, Beijing, prevent and treat test.For being diagnosed as the yellow scour of piglet that intestinal bacteria cause, the treatment of dysentery characterized by white mucous stool and prevent efficiently to be 100% (development of the anti-swine escherichia coli yolk antibody of chicken, Chinese livestock and poultry transmissible disease, 1994,2:26-27).
Summary of the invention
The objective of the invention is to develop a kind of special yolk antibody, it can prevent the grice diarrhoea that caused by enterotoxigenic escherichia coli K88ac.The preparation of this antibody has advantages such as output height, high specificity, simple to operate, environmentally safe, is a kind of microbiotic substitute of green.
The present invention is achieved through the following technical solutions:
A kind of special yolk antibody that prevents grice diarrhoea, be with enterotoxigenic escherichia coli (Enterotoxigenic Escherichiacoli, ETEC) K88ac pili adhesin albumen and freund's adjuvant are mixed and made into the specific immune source, open the product hen with this immunogenic immune health, and collect by the egg that chicken produced of immunity, contain the special yolk antibody that prevents grice diarrhoea in the described egg yolk.
The method of the special yolk antibody of preparation prevention grice diarrhoea, the method that wherein prepares the specific immune source adopts the TSB liquid nutrient medium, 37 ℃ of shaking culture enterotoxigenic escherichia coli K88ac bacterial strains, after 18 hours, extract and purifying pili adhesin albumen with 60 ℃ of heating-ice bath homogenate methods and iso-electric point continuous precipitation, mix with freund's adjuvant then.
Described from the egg that healthy bird inlay produced of specific immune source immunity, separate yolk, spraying drying is made powdery yolk, contains the described special yolk antibody that can prevent grice diarrhoea in this egg yolk.
The immune step of described immune bird inlay is: opening laying hen chest muscle both sides in health, to inject 500 μ l concentration respectively be that the specific immune of 400 μ g/ml is former, and it is that the specific immune of 600 μ g/ml is former that chest muscle both sides, 2 week back are injected 500 μ l concentration more respectively.
A kind of special yolk antibody that prevents grice diarrhoea, it is used as antibiotic substitute and is applied to feed, the grice diarrhoea that prevention is caused by enterotoxigenic escherichia coli K88ac.
Details of the present invention is by shown in the following step:
One, the preparation of K88ac pili antigen
1. cultivate the K88ac bacterial strain, measure the pili expression amount
Enterotoxigenic escherichia coli K88ac bacterial strain (C83715 (O
s: K
88ac) available from China Veterinary Drugs Supervisory Inst..After bacterial strain brings back to life, inoculum size with 1% is inoculated in respectively 300ml TSB substratum is housed (is trysinization soybean broth substratum, referring to: the Fang Hai chief editor, " colon bacillus ", the Hebei science and technology is published Du, 1997,) medium component is as follows: pancreas casein peptone 17g, soy peptone 3g, sodium-chlor 5g, dipotassium hydrogen phosphate 3.27g, glucose 2.5g, pH 7.4, and bi-distilled water is fixed molten to the triangular flask of 1000ml, cultivate in 37 ℃ of concussions.Took out the 3ml sample after 5 times of dilutions every 2 hours, in 420nm place colorimetric, the record light absorption value.Cultivate after 18 hours, the centrifugal bacterium liquid of collecting, the blood counting chamber microscopic count, adjusting bacterial concentration is 5,000,000,000 bacterium/milliliters.(Mannose-resistant hemagglutination MRHA) checks the adhesin expression to adopt seminose opposing hemagglutination.The MRHA method is pressed the Fang Hai chief editor, and " colon bacillus ", Hebei science tech publishing house, 1997,409～429 reported method are measured the pili expression amount, require every 1L bacterium liquid to extract more than the pure adhesin albumen 1.0mg.
2. the extraction of enterotoxigenic escherichia coli K88ac pili antigen and purifying
The positive K88ac bacterial strain of seminose opposing hemagglutination that select to bring back to life is equipped with by 0.5% inoculum size inoculation after 37 ℃ of concussions are cultivated 18 hours in the 1000ml triangular flask of 500ml TSB (trysinization soybean broth) substratum, by 1% inoculum size inoculation 37 ℃ of concussions is housed in the 5000ml triangular flask of 2500mlTSB substratum again and cultivates 18 hours.Collect bacterium liquid, 4 ℃ centrifugal, and (6,000 * g) 15 minutes, supernatant discarded was with an amount of PBS (phosphoric acid buffer) suspension lawn.Bacterial suspension is put in 60 ℃ of water-baths hatching 30 minutes, be interrupted and rock suspension: then bacterium liquid is placed ice bath low speed homogenate 5 minutes; Under 4 ℃, removed thalline in centrifugal 15 minutes, collect the supernatant liquor of no thalline with 14000 * g.
Supernatant liquor is through the membrane filtration of 0.4um, the citric acid of adding 2.5% is adjusted pH value to 4.0 in filtrate, placed 2 hours down or spend the night at 4 ℃, under 4 ℃, got precipitation in centrifugal 15 minutes then, be suspended in again in PBS1 (phosphoric acid buffer 1) damping fluid with 14000 * g; Above-mentioned steps repeats 2-3 time, at last throw out is dissolved among the 1ml 0.1M PBS2 (phosphoric acid buffer 2), with SDS-PAGE (sodium lauryl sulphate-polyacrylamide gel electrophoresis) method identification of protein purity.Measure test kit (available from glad biotechnology research institute of Shanghai section) with total protein and measure protein content.-20 ℃ of preservations behind the purifying.
Two, the preparation of yolk antibody
1.K88ac the extraction of pili antigen
Be inoculated in the 50ml triangular flask that the 10mlTSB substratum is housed with the positive bacterium colony of transfering loop picking K88ac MRHA, 37 ℃ of concussions were cultivated 18 hours; Getting the 1ml nutrient solution respectively is inoculated in 37 ℃ of concussions of 6 5000ml triangular flasks that the 2500mlTSB substratum is housed and cultivated 18 hours; Repeat batch cultivation, collect bacterium liquid, press (In vitro inhibition of adhesion of enterotoxigenic Escherichia coli K such as Jin
88To pigletintestinal mucus by egg-yolk antibodies.FEMS Immunology and Medical Microbiology, 1,998 21 (4): 313-321) the improved method of report is extracted and purifying K88 adhesin.Measure test kit (available from glad biotechnology research institute of Shanghai section) with total protein and measure protein content.
2. steriling test
Getting said extracted pili liquid 0.5ml is inoculated in the nutrient broth 37 ℃ of concussions and cultivates to observe in 18 hours and have or not bacterial growth.The K88ac pili suspension of no bacterial growth is used for the preparation of the newborn vaccine of following oil.
3. the preparation of oily newborn vaccine
K88ac pili suspension and isopyknic freund's adjuvant (Freund's complete adjuvant is available from Sigma company, and Freund's incomplete adjuvant is available from Beijing ancient cooking vessel state biotech development center) thorough mixing is emulsified into the newborn vaccine of water-in-oil-type oil that protein concn is 400 μ g/ml and 600 μ g/ml.
4. laboratory animal and immunity
To inject 500 μ l concentration respectively be the immunogen of 400 μ g/ml opening laying hen chest muscle both sides 20 weeks ages, and 22 ages in week, to inject 500 μ l concentration more respectively be the immunogen of 600 μ g/ml in the chest muscle both sides.Two exempt from the back began to collect egg in one week.
5. the yolk antibody mensuration of tiring
Adopt indirect ELISA method: 100 μ l K88ac adhesins (being diluted to 15 μ g/ml with coating buffer) wrap by 96 hole polyethylene boards, 4 ℃ spend the night (18h-24h), discard, with washings flushing 3 times, 37 ℃ of bags of diluent of using 150 μ l then are by 1h, discard, as preceding washing 3 times, every hole added 100 μ l yolk liquid (1: 10,1: 20,1: 40,1: 80,1: 160 ... diluting with PBSII) 37 ℃ of bags are by 1h, discard, as preceding washing 5 times, every hole added 100 μ l mouse-anti chicken IgG horseradish peroxidases (1: 15,000, the washings dilution) 25 ℃ of bags are discarded, as preceding washing 5 times by 30min, last every hole adds 100 μ l substrate solutions, and colour developing is back with 50 μ l stop buffer termination reactions.Be deeper than control group with color reaction and be judged to the positive, and be antibody titer with the high dilution that positive reaction occurs.
Three, the evaluation of yolk antibody effect
1. yolk antibody vitro inhibition ETEC (enterotoxigenic escherichia coli) K88ac sticks test
The preparation of jejunal epithelium cell: butcher and get about 50cm jejunum in back 30 minutes, remove the enteric cavity content for several times with 37 ℃ of normal saline flushings,, pour into sodium citrate buffer solution (the Nacl 96mM of pH7.4 then again with the normal saline flushing 2 times that contains the 1Mm dithiothreitol (DTT), Kcl 1.5mM, KH
2PO
48mM, Na
2HPO
45.6mM, Trisodium Citrate 27mM), 38 ℃ of following incubations 10 minutes, the EDTA solution (1.5mM EDTA+PBS liquid) of using pH7.4 again made cell precipitation 38 ℃ of following incubations 15 minutes with the cell centrifugation of collecting 5 minutes (900g).Use PBS damping fluid (Nacl 130mM, Kcl 8.1mM, the KH of pH7.4 again
2PO
41.5mM, Na
2HPO
48.1mM) flushing twice, at last with the cell suspension collected in PBS liquid (containing 0.2% glucose, 200IU/ml penicillin, 200IU/ml Streptomycin sulphate).The blood counting chamber counting, adjusting cell concn is 10
6Individual/ml.
The preparation of bacterial cultures: bacterial cultures is used the PBS washed twice through centrifugation, and throw out is suspended among the PBS, regulates cell concentration and is about 10
9Individual/ml.
sticks test: 1ml epithelial cell suspension+1ml somatic cells suspension, 37 ℃ act on 30 minutes, get a droplet and make a pressure sample sheet, observe in microscopically.Write down the total count that adsorbs on 20 epithelial cells, calculate the bacterial count of average each cell adhesion, above-mentioned test triplicate.
yolk antibody vitro inhibition is sticked test: 1ml bacterium liquid is mixed (tiring: 1: 1280) with the 1ml yolk antibody, 37oC effect 30 minutes adds 1ml epithelial cell suspension again, 37oC effect 30 minutes, and as preceding observation, record result, above-mentioned test triplicate.
2. prevention diarrhea of weaned piglets test
Select 120 of 30 ± 2 age in days weanling pigs, the principle consistent relatively by blood relationship, body weight, parity, age in days etc. is divided into 4 groups, 30 every group at random.Give 0%, 0.5%, 1%, 2% spraying drying powdery yolk in the daily ration respectively.Though after the off-test there being than remarkably influenced (p=0.059) to the growth in piglets performance yolk antibody powder of interpolation 1%, has significantly reduced feedstuff-meat ratio and diarrhea rate (p＜0.05), the late growing stage to piglet has certain promotion simultaneously.
Description of drawings
Fig. 1: be process flow sheet of the present invention.
Embodiment
Embodiment 1: preparation enterotoxigenic escherichia coli K88ac pili antigen
One, materials and methods
1.1. bacterial strain
The K88ac bacterial strain is available from veterinary drug supervision institute of the Chinese Academy of Agricultural Sciences
1.2. substratum and reagent
TSB substratum (pH7.4): pancreas casein peptone 17g, soy peptone 3g, sodium-chlor 5g, dipotassium hydrogen phosphate 3.27g, glucose 2.5g.Bi-distilled water is fixed molten to 1000ml;
Damping fluid PBS1 (pH7.2): NaCL 10g, KH
2PO
40.25g, Na
2HPO
412H
2O 3.58g, KCL 0.25g, bi-distilled water is fixed molten to 1000ml;
0.1M damping fluid PBS2 (pH7.2): NaH
2PO
42H
2O4.37g, Na
2HPO
412H
2O 25.81g, bi-distilled water is fixed molten to 1000ml;
1.3. the mensuration of enterotoxigenic escherichia coli K88ac bacterial strain pili adhesin expression effect
After bacterial strain brought back to life, the inoculum size with 1% was inoculated in the 1000ml triangular flask that 300ml TSB substratum is housed respectively, cultivated in 37 ℃ of concussions.Took out the 3ml sample after 5 times of dilutions every 2 hours, in 420nm place colorimetric, the record light absorption value.Cultivate after 18 hours, the centrifugal bacterium liquid of collecting, the blood counting chamber microscopic count, adjusting bacterial concentration is 5,000,000,000 bacterium/milliliters.(Mannose-resistanthemagglutination MRHA) checks the adhesin expression to adopt seminose opposing hemagglutination.The MRHA method is pressed sea, room (colon bacillus.Hebei science tech publishing house, 1997,409～429) report method mensuration pili expression amount, require every 1L bacterium liquid to extract more than the pure adhesin albumen 1.0mg.
2. the extraction of enterotoxigenic escherichia coli K88ac pili antigen and purifying
The positive K88ac bacterial strain of seminose opposing hemagglutination that select to bring back to life is equipped with by 0.5% inoculum size inoculation after 37 ℃ of concussions are cultivated 18 hours in the 1000ml triangular flask of 500mlTSB substratum, is inoculated in the 5000ml triangular flask that the 2500mlTSB substratum is housed 37 ℃ of concussions by 1% inoculum size again and cultivates 18 hours.Collect bacterium liquid, 4 ℃ centrifugal, and (6,000 * g) 15 minutes, supernatant discarded was with an amount of PBS suspension lawn.Bacterial suspension is put in 60 ℃ of water-baths hatching 30 minutes, be interrupted and rock suspension; Then bacterium liquid is placed ice bath low speed homogenate 5 minutes; Under 4 ℃, removed thalline in centrifugal 15 minutes, collect the supernatant liquor of no thalline with 14000 * g.
Supernatant liquor is through the membrane filtration of 0.4um, the citric acid of adding 2.5% is adjusted pH value to 4.0 in filtrate, placed 2 hours down or spend the night at 4 ℃, under 4 ℃, got precipitation in centrifugal 15 minutes then with 14000 * g, again be suspended in the PBS1 damping fluid: above-mentioned steps repeats 2-3 time, last throw out is dissolved among the 1ml 0.1M PBS2, with SDS-PAGE identification of protein purity.Biuret method is measured protein content ,-20 ℃ of preservations.
Two, implementation result
Under the shaking culture condition, K88ac entered logarithmic phase in 4-6 hour after inoculation, and 16-20 hour finishes; Cultivating its pili hemagglutinative titer of thalline of collecting after 18 hours is 2
6
Adopted TSB liquid nutrient medium shaking culture 18 hours, with 60 ℃ of heating and ice bath homogenate method and iso-electric point continuous precipitation extract, the purifying pilin, extract 3.75 milligrams of the pilins of purifying from 2.5 milliliters bacterium liquid, average one milliliter of bacterium liquid is gathered in the crops 1.5 milligrams of pure pilins.
Embodiment 2: prepare the special pili antigen yolk antibody of anti-enterotoxigenic escherichia coli K88ac
1.1 reagent
Freund's complete adjuvant is available from Sigma company
Freund's incomplete adjuvant is available from Beijing ancient cooking vessel state biotech development center
0.02M PBS (pH7.4); Nacl8g, KH
2PO
40.2g, Na
2HPO
412H
2O2.9g, Kcl0.2g, distilled water is fixed molten to 500ml;
0.01M PBS (pH7.4): Nacl 8g, KH
2PO
40.2g, Na
2HPO
412H
2O 2.9g, Kcl 0.2g, distilled water is fixed molten to 1000ml;
Coating buffer: 0.05M carbonate buffer solution (pH9.6, NaHCO
32.93g, Na
2CO
31.95g distilled water is fixed molten to 1000ml);
Washings (PBS-T): the 0.01M PBS that contains 0.05% tween 20;
Diluent (PBS-BSA: 0.01 MPBS that contains 3% bovine serum albumin;
Substrate solution: O-Phenylene Diamine 40mg is dissolved in phosphoric acid salt-citrate buffer solution (0.2M Na of pH5.0
2HPO
4(28.4g/l) 51.4ml, 0.1M citric acid (19.2g/l) 48.6ml adds bi-distilled water 100ml mixing) 100ml, adding 30%H
2O
20.15ml get final product, now with the current.
Stop buffer: 2M H
2SO
4Solution (mixing gets final product for vitriol oil 22.2ml, distilled water 177.8ml)
1.2K88ac the extraction of pili antigen
Be inoculated in the 50ml triangular flask that the 10mlTSB substratum is housed with the positive bacterium colony of transfering loop picking K88acMRHA, 37 ℃ of concussions were cultivated 18 hours; Getting the 1ml nutrient solution respectively is inoculated in 37 ℃ of concussions of 6 5000ml triangular flasks that the 2500mlTSB substratum is housed and cultivated 18 hours; Repeat batch cultivation, collect bacterium liquid, press (In vitro inhibition of adhesion of enterotoxigenic Escherichia coli K such as Jin
88To pigletintestinal mucus by egg-yolk antibodies.FEMS Immunology and Medical Microbiology, 199821 (4): 313-321) the improved method of report is extracted and purifying K88 adhesin.Measure test kit (available from glad biotechnology research institute of Shanghai section) with total protein and measure protein content.
1.3 steriling test
Getting said extracted pili liquid 0.5ml is inoculated in the nutrient broth 37 ℃ of concussions and cultivates to observe in 18 hours and have or not bacterial growth.The K88ac pili suspension of no bacterial growth carries out the preparation of oily newborn vaccine
1.4 the preparation of oily newborn vaccine
K88ac pili suspension and isopyknic freund's adjuvant (Freund's complete adjuvant is available from Sigma company, and Freund's incomplete adjuvant is available from Beijing ancient cooking vessel state biotech development center) thorough mixing is emulsified into the newborn vaccine of water-in-oil-type oil that protein concn is 400 μ g/ml and 600 μ g/ml.
1.5 laboratory animal and immunity
Opening laying hen chest muscle both sides 20 weeks ages, to inject 500 μ l concentration respectively be the immunogen of 400 μ g/ml, and 22 ages in week, to inject 500 μ l concentration more respectively be the immunogen of 600 μ g/ml in the chest muscle both sides.Two exempt from the back began to collect egg in one week.
1.6 the mensuration that yolk antibody is tired
Adopt indirect ELISA method: 100ulK88ac adhesin (being diluted to 15 μ g/ml with coating buffer) wraps by 96 hole polyethylene boards, 4 ℃ spend the night (18h-24h), discard, with washings flushing 3 times, 37 ℃ of bags of diluent of using 150 μ l then are by 1h, discard, as preceding washing 3 times, every hole adds 100 μ l yolk liquid, with PBSII dilution (extension rate 1: 10,1: 20,1: 40,1: 80,1: 160 ...), 37 ℃ of bags are by 1h, discard, as preceding washing 5 times, every hole added 100 μ l mouse-anti chicken IgG horseradish peroxidases (1: 15,000, the washings dilution) 25 ℃ of bags are discarded, as preceding washing 5 times by 30min, last every hole adds 100 μ l substrate solutions, and colour developing is back with 50 μ l stop buffer termination reactions.Be deeper than control group with color reaction and be judged to the positive, and be antibody titer with the high dilution that positive reaction occurs.
Embodiment 3: the evaluation of yolk antibody effect
One, materials and methods
1. yolk antibody vitro inhibition ETEC K88ac sticks test
1.1 the preparation of jejunal epithelium cell: butcher and get about 50cm jejunum in back 30 minutes, remove the enteric cavity content for several times with 37 ℃ of normal saline flushings, again with the normal saline flushing 2 times that contains 1Mm dithiothreitol (DTT) (DTT), pour into sodium citrate buffer solution (the Nacl 96mM of pH7.4 then, Kcl1.5mM, KH
2PO
48mM, Na
2HPO
45.6mM, Trisodium Citrate 27mM), 38 ℃ of following incubations 10 minutes, the EDTA solution (1.5mM EDTA+PBS liquid) of using pH7.4 again made cell precipitation 38 ℃ of following incubations 15 minutes with the cell centrifugation of collecting 5 minutes (900g).Use PBS damping fluid (Nacl 130mM, Kcl 8.1mM, the KH of pH7.4 again
2PO
41.5mM, Na
2HPO
48.1mM) flushing twice, at last with the cell suspension collected in PBS liquid (containing 0.2% glucose, 200IU/ml penicillin, 200IU/ml Streptomycin sulphate).The blood counting chamber counting, adjusting cell concn is 10
6Individual/ml
1.2 the preparation of bacterial cultures: bacterial cultures is used the PBS washed twice through centrifugation, and throw out is suspended among the PBS, regulates cell concentration and is about 10
9Individual/ml
1.3 stick test: 1ml epithelial cell suspension+1ml somatic cells suspension, 37 ℃ act on 30 minutes, get a droplet and make a pressure sample sheet, observe in microscopically.Write down the total count that adsorbs on 20 epithelial cells, calculate the bacterial count of average each cell adhesion, above-mentioned test triplicate.
1.4 the yolk antibody vitro inhibition is sticked test: 1ml bacterium liquid is mixed (tiring: 1: 1280) with the 1ml yolk antibody, 37 ℃ act on 30 minutes, add 1ml epithelial cell suspension again, 37 ℃ act on 30 minutes, as preceding observation, record result, above-mentioned test triplicate.
3. prevention diarrhea of weaned piglets test
4.2.1 selection and the grouping of examination pig
Select 120 of 30 ± 2 age in days weanling pigs, the principle consistent relatively by blood relationship, body weight, parity, age in days etc. is divided into 4 groups at random, and 30 every group, raise on every component three hurdles.
2.2 test design and test daily ration are formed
The single-factor design is adopted in test.Control group commodity daily ration adds the test daily ration that 0.5%, 1%, 2% spraying drying powdery yolk forms two groups of one group of tests, test and three groups respectively in basic components.
2.3 feeding and management
Water fountain is freely drunk water, the dry mash free choice feeding, and day feeds 4 times, claims the residue material morning next day, presses hurdle statistics material amount.Other management more solitos such as castrating, immunization inocultation are carried out.The course of disease surpasses 3 days, and the pig that degree seriously can not continue to participate in test in time withdraws from test, and deducts its feed consumption rate.
2.4 weigh
The weaned piglet recast weaned back 14 days and 21 days for going into test mass, weighed 1 time on an empty stomach by head, and nominal weighs 3 times.
2.5 diarrhoea situation
Write down piglet ight soil situation morning every day, be divided into 0,1,2,3 level Four records by dried, soft, rare, water sample.Do just reaching soft stool during statistics, rarely just be designated as diarrhoea with watery stool for normal.
2.6. data processing and analysis
Average daily gain, food consumption and feedstuff-meat ratio are carried out variance analysis, the diarrhea rate chi square test
Two, result and analysis
1 yolk antibody is to the restraining effect of the outer cell adhesion of bacterial body
Stick in the test at the K88ac cell in vitro as can be seen from Table 1, on average each epithelial cell sticks 16.4 K88ac; Suppress to stick in the test at yolk antibody, antibody titer is respectively 640 and 1280 yolk antibody and has all extremely significantly suppressed the stick effect of K88ac to intestinal epithelial cells than control group, average each epithelial cell only sticks 8.5 and 2.4 bacteriums (p＜0.01), also has utmost point significant difference (p＜0.01) between the two.Antibody titer is that 320 antibody group relative comparison group has the effect of certain inhibition bacterial adhesion, but difference not significantly (p=0.15).
Table 1 yolk antibody is to the restraining effect of the outer cell adhesion of bacterial body
| Anti-K88ac antibody titers | Stick bacterial count |
| 0 320 640 1280 | 16.4±9.6aA 13.7±7.4aAB 8.5±5.7bB 2.4±1.1C |
2 yolk antibodies are to the influence of diarrhea of weaned piglets situation
As can be seen from Table 2, test two groups and test three groups of diarrhoea number and a head time number average and be lower than one group of control group and test; One group of relative comparison group and test are tested two groups and three groups diarrhea rates of test and are extremely significantly reduced (p＜0.01), and one group of control group and test are tested two groups and test between three groups diarrhea rate difference not remarkable (p＞0.05).
Table 2 trial period, respectively organized the diarrhoea situation
| Group | The pig's head number | The diarrhoea head | Diarrhea rate |
| Control group 123 | 28 28 28 28 | 54(22 *) 48(21) 20(14) 26(14) | 13.8％A 12.2％A 5.1％B 6.7％B |
*: the piglet number of diarrhoea for taking place in bracket inner digital
3 yolk antibodies are to the influence of weanling pig growth performance
3.1. yolk antibody is to the influence of weanling pig average daily gain
As can be seen from Table 3, interim in whole test, each organizes average daily gain does not all have significant difference; But test test two groups of relative comparison groups, one group of test the later stage and test three groups of average daily gains all have some improvement (p=0.078, p=0.059, p=0.132).
Table 3 trial period, respectively organized average daily gain (g/d)
| Group | The pig's head number | In earlier stage | The wean latter stage later stage | The full phase |
| Contrast 123 | 28 28 28 28 | 306.2±62.4 290.8±50.6 295.1±73.9 291.6±51.4 | 469.2±108.4 466.4±92.2 522.1±80.4 476.9±105.6 | 385.4±56.5 378.6±49.1 409.5±61.2 384.3±67.3 |
3.2 yolk antibody is to the influence of the average food consumption of weanling pig
As can be seen from Table 4, no matter be test early stage, later stage or full phase, each organizes average food consumption does not all have significant difference, but along with the increase of yolk antibody addition, average food consumption has decline trend.The test later stage, test three groups of relative comparison groups and the test one group have obvious minimizing (p=0.071, p=0.195).
Table 4 trial period, respectively organized average food consumption (g/d)
| Group | The pig's head number | In earlier stage | The wean latter stage later stage | The full phase |
| Contrast 123 | 28 28 28 28 | 435.5±45.8 433.0±50.3 429.1±38.8 411.0±11.7 | 873.1±78.3 845.5±44.9 797.3±18.8 787.1±41.4 | 646.9±53.6 639.3±47.6 618.7±22.3 597.5±22.4 |
2.3. yolk antibody is to the influence of weanling pig feedstuff-meat ratio
As can be seen from Table 5, each group test early stage (wean two weeks of back) feedstuff-meat ratio of test does not have significant difference; But from test later stage and full phase of test, test two groups and three groups of relative other two groups of feedstuff-meat ratios of test all significantly reduction (wherein test two groups and reach utmost point conspicuous level (p＜0.01).
Table 5 trial period, respectively organized feedstuff-meat ratio
| Group | The pig's head number | In earlier stage | The wean latter stage later stage | The full phase |
| Contrast 123 | 28 28 28 28 | 1.40±0.07 1.48±0.06 1.40±0.15 1.47±0.17 | 1.82±0.04aA 1.80±0.05aAC 1.54±0.05bB 1.68±0.06bC | 1.70±0.02A 1.71±0.05A 1.51±0.03aB 1.60±0.05bB |
Claims (3)
1, a kind of special yolk antibody that prevents grice diarrhoea, it is characterized in that, it is former earlier enterotoxigenic escherichia coli K88ac pili adhesin albumen and freund's adjuvant to be mixed and made into specific immune, open with this immunogen immune health again and produce hen, collect the egg that is produced by immune chicken then, obtain containing the special yolk antibody that prevents grice diarrhoea.
2, the preparation method of the special yolk antibody of the described prevention grice diarrhoea of claim 1, its step comprises:
1) preparation immunogen: earlier enterotoxigenic escherichia coli K88ac bacterial strain is placed on the TSB liquid nutrient medium, in 37 ℃ of shaking culture 18 hours, extract and purifying pili adhesin albumen with 60 ℃ of heating-ice bath homogenate methods and iso-electric point continuous precipitation again, then this pili adhesin albumen is mixed with freund's adjuvant, it is former to obtain described specific immune;
2) immune health hen: opening laying hen chest muscle both sides in health, to inject 500 μ l concentration respectively be the former by the resulting specific immune of step 1) of 400 μ g/ml, it is that the prepared specific immune of 600 μ g/ml step 1) is former that 2 week backs are injected 500 μ l concentration respectively again in the chest muscle both sides that described health is opened laying hen, collects the egg by immune chicken produced;
3) obtain special yolk antibody: with step 2) in the resulting egg, separate yolk, spray-dried, obtain containing the powdery yolk of the special yolk antibody that prevents grice diarrhoea.
3, the application of the described yolk antibody of claim 1 in preparation prevention grice diarrhoea feed.
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| 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 |
| CN102908620A (en) * | 2011-08-04 | 2013-02-06 | 广州格拉姆生物科技有限公司 | Preparation method of egg yolk antibody injection for treating piglet diarrhea |
| CN103724429A (en) * | 2013-12-24 | 2014-04-16 | 黄光东 | Preparation method of anti-piglet diarrhea IgY (immunoglobulin of yolk) |
| 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 |
| 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 |
| CN107686518B (en) * | 2016-08-05 | 2019-12-06 | 中国农业大学 | Single-chain antibody of anti-Escherichia coli k88ac, and coding gene and application thereof |
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