CN101948536B - Porcine transmissible gastroenteritis virus (PTGEV) and porcine epidemic diarrhea virus (PEDV) dual yolk antibody and preparation method thereof - Google Patents
Porcine transmissible gastroenteritis virus (PTGEV) and porcine epidemic diarrhea virus (PEDV) dual yolk antibody and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a porcine transmissible gastroenteritis virus (PTGEV) and porcine epidemic diarrhea virus (PEDV) dual yolk antibody and a preparation method thereof. The preparation method comprises the following steps of: 1) preparing vaccines for immunization; 2) performing immunization; and 3) extracting the porcine PTGEV and PEDV dual yolk antibody, and purifying and freeze-drying the antibody. By adopting a special immunization program, the porcine PTGEV and PEDV dual yolk antibody has high potency. The porcine PTGEV and PEDV dual yolk antibody prepared by the method has the advantages of obvious effect, high specificity, quick treatment effect, accurate curative effect, low production cost and the like, and has good application prospect.
Description
Technical field
The invention belongs to the veterinary biologics technical field, be specifically related to a kind of transmissible gastro-enteritis virus, Porcine epidemic diarrhea virus bigeminy yolk antibody and preparation method thereof.
Background technology
Transmissible gastroenteritis of swine, porcine epizootic diarrhea are that two important viral spreading venereal diseasess of diarrhoea take place the pig that causes that confirms at present.Mainly the influence to piglet is bigger for these two kinds of diseases, is the main diseases diarrhea virus that causes piglet death.These two kinds of usually polyinfections of disease; all closely similar in epidemiology, clinical symptom, pathological anatomy variation; very difficult difference is any virus infection on earth; and morbidity rapidly; nearly several hrs just can involve full group after morbidity; mainly be the large quantities of death that cause nursery-age pig, cause tremendous loss for the production of raising pigs.
Transmissible gastroenteritis of swine (Porcine Transmissible Gastroenteritis, TGE) be transmissible gastro-enteritis virus (Porcine Transmissible Gastroenteritis virus by coronaviridae (Coronaviridae), coronavirus genus (Coronavirus), what TGEV) cause is the transmissible disease of feature with pig vomiting, diarrhoea, dehydration, newborn piglet is had the height lethality rate, be generally 100%.Though the pig of different ages is to the equal susceptible of this virus, after 5 ages in week, above pig infected, mortality ratio was very low.Doyle in 1945 in U.S.'s reported first after this disease, many countries have all reported transmissible gastroenteritis of swine in succession in the world.China from 1956 after this disease takes place in Guangdong Province first, the generation that also there is transmissible gastroenteritis of swine in national most of provinces has brought serious harm to pig industry.
Porcine epizootic diarrhea (Porcine Epidemic Diarrhae, PED) be Porcine epidemic diarrhea virus (the Porcine Epidemic Diarrhae virus of coronaviridae (Coronaviridae), coronavirus genus (Coronavirus), PEDV) a kind of chitling road transmission disease that causes is feature with watery diarrhea, vomiting and dehydration.The equal susceptible of the pig at various ages, the sickness rate of sucking piglets, feeder pig or growing and fattening pigs can reach 100%, and especially sucking piglets is injured the most serious.Porcine epizootic diarrhea mainly occurs in winter and rasputitsa comparatively cold season, but also can take place in summer.Expect the generation that multinational family has reported porcine epizootic diarrhea in succession the beginning of the seventies in last century, China begins that the report that porcine epizootic diarrhea takes place is arranged successively the eighties in last century.
At present mainly prevent the generation of these two kinds of diseases with the vaccine immunity sow.Because also not at the specific medicament of virus disease, therefore on clinical treatment, mainly take symptomatic treatment and with the generation of antibiotics complication prevention, its result for the treatment of is undesirable.In addition, because these two kinds of disease incidences rapidly, after occurring suffering from diarrhoea, piglet dewaters owing to diarrhoea mostly, will soon die of exhaustion and die.Therefore, be badly in need of aborning a kind of at transmissible gastroenteritis of swine, epidemic diarrhea virus disease all have specificity, result for the treatment of is good and curative effect is treated preparation rapidly.
Summary of the invention
In view of this, main purpose of the present invention is to provide a kind of transmissible gastro-enteritis virus, Porcine epidemic diarrhea virus bigeminy yolk antibody and preparation method thereof.
Technical scheme
Among the present invention, described " PK15 " is porcine kidney cell, and " Vero " is African green monkey kidney cell.
Described " saturated formalin " is commercially available 40% formalin.
One aspect of the present invention provides the preparation method of a kind of transmissible gastroenteritis of swine, epidemic diarrhea virus bigeminy yolk antibody.
For realizing the object of the invention, the technical solution of the present invention step is as follows:
1) the immunity preparation of vaccine:
Cultivate transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus with pig kidney passage cell, African green monkey kidney passage cell respectively, concentrate the back respectively as antigen, two kinds of antigens are fully mixed the back deactivation, add immunostimulant and white-oil adjuvant, preparation water-in-oil-type transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus bivalent inactivated vaccine are as the immunity vaccine;
2) immunity:
Immunity with the step 1) preparation is carried out repeatedly immunity with vaccine to non-immunization laying hen, immune time 〉=2, preferably, and immune time of the present invention 〉=4, immune time is 4 times in the embodiment of the invention.
3) from step 2) collect egg yolk liquid the immunization laying hen output egg, extract transmissible gastroenteritis of swine and epidemic diarrhea virus bigeminy yolk antibody, behind the purifying, freeze-drying.
Preferably, the preparation of Transmissible gastroenteritis virus antigen of the present invention may further comprise the steps: Transmissible gastroenteritis virus liquid is seeded on the PK15 monolayer cell, sets up simultaneously not meet poison cell contrast, CO under 37 ℃ of temperature
2Cultivate in the incubator, receive poison when cytopathy occurring to 75% cell, neutralization test is measured malicious valency, concentrates the back as antigen.
Preferably, the preparation of epidemic diarrhea virus antigen of the present invention may further comprise the steps: epidemic diarrhea virus liquid is inoculated on the Vero monolayer cell, sets up simultaneously not meet poison cell contrast, CO under 37 ℃ of temperature
2Cultivate in the incubator, receive poison when cytopathy occurring to 75% cell, neutralization test is measured malicious valency, concentrates the back as antigen.
Preferably, transmissible gastro-enteritis virus and epidemic diarrhea virus mixed in 1: 0.8 by volume～1: 1.2 in the step 1) of the present invention, mixed the malicious valency of back two kinds of viruses all 〉=1.0 * 10
6.0TCID
50/ ml.
Preferably, in the step 1) of the present invention with after transmissible gastro-enteritis virus and the deactivation of epidemic diarrhea virus antigen, add yeast glucan that lipopolysaccharides that final concentration is volume ratio 0.01%～0.05% and final concentration be volume ratio 0.2%～0.8% respectively as immunostimulant, with the white-oil adjuvant emulsification of 2 times of amounts of the cumulative volume of described viral liquid, preparation water-in-oil-type transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus bivalent inactivated vaccine are as the immunity vaccine.
Preferably, step 2 of the present invention) immune programme for children is in: exempted from preceding 10 days at head, drink for chicken with the astragalus polysaccharides of volume ratio 1%～5% every day, promote chicken body overall immunity, head exempts to inject mycobacterium tuberculosis var bovis polyoses nucleic acid 0.1～0.5ml, Freund's complete adjuvant 100～300 μ g under every plumage cock skin in preceding 7 days, carries out immunity and irritates.
Chest muscle multi-point injection immunity vaccine 1～2ml when head exempts from; Carry out two after 2 weeks and exempt from, injecting immune vaccine 1～2ml and mycobacterium tuberculosis var bovis polyoses nucleic acid 0.1～0.5ml; After 2 weeks, carry out three again and exempt from, injecting immune vaccine 2～4ml and mycobacterium tuberculosis var bovis polyoses nucleic acid 0.2～0.8ml; After 4～8 weeks, carry out four again and exempt from, injecting immune vaccine 2～4ml.
Preferably, two exempt from back 1 week beginning and measure height every sampling in 7 days and exempt from the egg yolk anti-swine infectious enterogastritis virus and Porcine epidemic diarrhea virus specific antibody and tire in the step 3) of the present invention; Antibody titer all reaches 1: the 128 above yolk of collecting, for the preparation of transmissible gastro-enteritis virus and epidemic diarrhea virus bigeminy yolk antibody.
Preferably, the abundant stirring makes yolk be even paste in the step 3) of the present invention, adds equal-volume through the distilled water of sterilization and cooling, stirring and evenly mixing, deactivation; Add the pH value be equivalent to 6 times of volumes of former yolk and be 4.2 sterile purified water in the interlayer retort, and be cooled to 1～4 ℃, the egg yolk liquid with deactivation adds then, the stirring of limit edged, and 4～8 ℃ leave standstill 4h; The centrifugation supernatant liquor also changes in another retort, is that 0.2% adding of total amount is sad by volume, stirs, and room temperature is placed 2～4h; After the filter cloth filtration, be filtered to clarification with K type multilayer sheet frame again, be that 0.1% of total amount adds saturated formalin in above-mentioned filtrate by volume, abundant stirring and evenly mixing, room temperature is placed 24h, during jolting for several times, filtration sterilization, with molecular weight cut-off be 1000KDa ultrafiltration membrance filter except virus, the transmissible gastro-enteritis virus that can obtain purifying and epidemic diarrhea virus bigeminy yolk antibody.
Preferably, antibody titer detection step is as follows in the step 3) of the present invention:
Be to add agar gel behind 7.2 the phosphate buffered saline buffer doubling dilution on every side in the hole for 0.01mol/L pH with the egg yolk liquid concentration of collecting, add in the step 1 through centrifugal concentrated and purified transmissible gastro-enteritis virus (or epidemic diarrhea virus) antigen in the interstitial hole, putting 37 ℃ of incubators reaction 24h in the wet box, is the antibody titer of the transmissible gastro-enteritis virus (or epidemic diarrhea virus) of this egg yolk liquid with the egg yolk liquid of high dilution that the precipitation band occurs.
Preferably, transmissible gastro-enteritis virus of the present invention and epidemic diarrhea virus bigeminy yolk antibody are freeze-drying bigeminy yolk antibody.
Preferably, transmissible gastro-enteritis virus and epidemic diarrhea virus bigeminy yolk antibody freeze-drying program are in the step 3) of the present invention: will purify and the yolk antibody liquid through being up to the standards is sub-packed in cillin bottle in a small amount, freeze-drying is spent the night on Freeze Drying Equipment, temperature is minimum in the freeze-drying process is down to-38 ℃, keep 3～4h to freeze-drying fully, return to room temperature then gradually, take out and be stored in 4 ℃.
The present invention has provided a kind of transmissible gastro-enteritis virus, epidemic diarrhea virus bigeminy yolk antibody on the other hand.
Another aspect of the present invention is transmissible gastroenteritis of swine and the application of epidemic diarrhea virus bigeminy yolk antibody in transmissible gastro-enteritis virus and epidemic diarrhea disease clinical treatment and prevention.
Technique effect
Compared with prior art, transmissible gastro-enteritis virus of the present invention and epidemic diarrhea virus bigeminy yolk antibody and preparation method thereof have following beneficial effect:
The immune programme for children that the present invention adopts, guaranteed that exempting from back 12～16 all transmissible gastro-enteritis viruss and epidemic diarrhea virus bigeminy yolk antibody at head all can reach 1: 512, two kinds antibody titer obtains higher level simultaneously, thereby realizes two kinds of diseases---and transmissible gastroenteritis of swine and epidemic diarrhea play treatment and preventive effect simultaneously.Secondly, owing to obtain yolk two len antibodies, compare with the yolk antibody of independent virus, it is simple also to have a production sequence, the advantage that production cost reduces.
Transmissible gastro-enteritis virus of the present invention and epidemic diarrhea virus bigeminy yolk antibody carry out animal experiment, laboratory animal is the piglet that 1 age in days is not eaten colostrum, test group animal transmissible gastro-enteritis virus and epidemic diarrhea virus oral challenge, oral transmissible gastro-enteritis virus of the present invention and the epidemic diarrhea virus bigeminy yolk antibody of feeding then.Other establish attack poison not with the antibody group, do not attack poison not with the antibody group, do not attack the poison test in contrast for 3 groups with the antibody group; the result shows: transmissible gastro-enteritis virus of the present invention and epidemic diarrhea virus bigeminy yolk antibody have the obvious treatment effect to the grice diarrhoea that transmissible gastro-enteritis virus and epidemic diarrhea virus cause; being diluted to feeds after 1: 32 attacks 1 malicious age in days piggy, to piggy protection ratio 100%.
Porcine epidemic diarrhea virus and transmissible gastro-enteritis virus belong to coronaviridae together, and the two clinical symptom of causing a disease is extremely similar again, and generally is polyinfection, the pig at each age all can infect, obvious characteristics is exactly watery diarrhea, and sickness rate is higher, and the piglet mortality ratio is higher.Its mechanism of action generally all is to be adsorbed on the mucous membrane of small intestine, and infects epithelial and make little intestinal absorption and metabolism disorder causes the generation of diarrhoea.Therefore, improve chitterlings local immunity ability, suppressing cause of disease is the key for the treatment of and infection prevention gastro-enteritis and epidemic diarrhea in absorption and the propagation of mucous membrane of small intestine.Transmissible gastro-enteritis virus of the present invention and epidemic diarrhea virus bigeminy yolk antibody directly by little intestinal absorption, have improved the local immunity power of small intestine by oral, have suppressed absorption and the propagation of cause of disease to mucous membrane of small intestine.
As seen from the above, compared with the prior art, provide the preparation method of a kind of transmissible gastro-enteritis virus and epidemic diarrhea virus bigeminy yolk antibody with the inventive method.Transmissible gastro-enteritis virus and the epidemic diarrhea virus bigeminy yolk antibody of the inventive method preparation, be used for prevention and treatment transmissible gastroenteritis of swine, these two kinds of diseases of porcine epizootic diarrhea, attacking malicious contrast and experiment shows: yolk antibody treatment group grice diarrhoea symptom then obviously alleviates, recover gradually about one week, death condition do not occur.Transmissible gastro-enteritis virus of the present invention and epidemic diarrhea virus bigeminy yolk antibody have result for the treatment of advantage rapid, evident in efficacy.
Embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention that NM concrete experimental technique in the following example carries out according to the normal experiment method usually.
The preparation of embodiment 1 transmissible gastro-enteritis virus and epidemic diarrhea virus bigeminy yolk antibody
1. the immunity preparation of vaccine:
1.1 the preparation of Transmissible gastroenteritis virus propagation and antigen
With 3 bottles of PK15 cells of Transmissible gastroenteritis virus liquid inoculation, every bottle of cell inoculation 1ml virus liquid, bottle floorage 25cm
2Build bottle cap, accumbency bottle pipe rotates gently, viral liquid is fully contacted with whole cell monolayer, under 37 ℃ of temperature, adsorb 40min, rock 1 time every 10min therebetween, need not to inhale and abandon viral liquid, directly add DMEM (Dulbecco ' s Modified Eagle Medium) the cell maintenance culture solution 5ml that contains 2% foetal calf serum, set up the cell contrast simultaneously, CO under 37 ℃ of temperature
2Cultivate in the incubator.Outwell behind the 24h and keep liquid and change liquid.Day by day observation of cell pathology, if 72h postoperative infection cytopathy is not obvious, then behind harvested cell suspension-20～20 ℃ multigelation 3 times under 4 ℃ of conditions the centrifugal 30min of 3000rpm get supernatant liquid and continue blind passage on cell, cytopathy appears in blind passage to the s-generation, receives poison when cytopathy occurring to 75% cell,-20～20 ℃ of multigelations 3 times, under 4 ℃ of conditions, the centrifugal 30min of 3000rpm removes cell debris then, collect supernatant liquid, concentrate the back as antigen.
1.2 the preparation of epidemic diarrhea virus propagation and antigen
Epidemic diarrhea virus liquid is inoculated on the Vero cell monolayer, sets up the cell contrast simultaneously, 37 ℃ of CO
2Cultivate in the incubator.Day by day observation of cell pathology, if 72h postoperative infection cytopathy is not obvious, then behind harvested cell suspension-20～20 ℃ multigelation 3 times under 4 ℃ of conditions the centrifugal 30min of 3000rpm, get supernatant liquid and continue blind passage on cell, cytopathy appears in blind passage to the third generation, when cytopathy occurring to 75% cell, shows as cytogamy and circle contracting, plaqueization appears in the part cell detachment.This moment the harvested cell suspension, under 4 ℃ of conditions, the centrifugal 30min of 3000rpm removes cell debris, collects supernatant liquor, concentrates the back as antigen behind-20～20 ℃ of multigelations 3 times.
1.3 the preparation of vaccine:
Transmissible gastro-enteritis virus antigen and epidemic diarrhea virus antigen mixed in 1: 1.03 by volume, and the malicious valency that mixes back two kinds of virus antigens is 1.0 * 10
6.3TCID
50/ ml.Add yeast glucan that lipopolysaccharides that final concentration is volume ratio 0.03% and final concentration be volume ratio 0.5% respectively as immunostimulant, with the white-oil adjuvant emulsification of 2 times of amounts, preparation water-in-oil-type transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus bivalent inactivated vaccine are as the immunity vaccine.
2. immune non-immunization laying hen
Immune programme for children is: exempted from preceding 10 days at head, drink for chicken with the astragalus polysaccharides of volume ratio 3% every day, promote chicken body overall immunity, head exempts to inject " mycobacterium tuberculosis var bovis polyoses nucleic acid " 0.2ml, Freund's complete adjuvant 200 μ g under every plumage cock skin in preceding 7 days, carries out immunity and irritates.
Chest muscle multi-point injection immunity vaccine 1ml when head exempts from; Carry out two after 2 weeks and exempt from, injecting immune reaches " mycobacterium tuberculosis var bovis polyoses nucleic acid " 0.2ml with vaccine 1ml; Carry out three again and exempt from after 2 weeks, injecting immune reaches " mycobacterium tuberculosis var bovis polyoses nucleic acid " 0.4ml with vaccine 2ml; After 6 weeks, carry out four again and exempt from injecting immune vaccine 2ml.
3. bigeminy yolk antibody preparation
3.1 the results of yolk and deactivation:
Two exempt from then to begin in 1 week to collect through inspecting qualified height that immune chicken produces by random samples to exempt from egg, with the eggshell sterilization, take craft or machinery to beat eggs.Fully remove egg white (in vain), blastodisc and frenulum, collect yolk.Fully stirring makes yolk be even paste, adds equal-volume through the distilled water of 121 ℃ of 30min sterilizations and cooling, stirring and evenly mixing, 62.5 ℃ of heat inactivation 30min.
3.2 the acidifying of antibody distillation water law is purified:
In the interlayer retort, add earlier the pH value that is equivalent to 6 times of volumes of former yolk and be 4.2 sterile purified water, and be cooled to 4 ℃.Then, with the egg yolk liquid adding of deactivation, the limit edged stirs, and 4 ℃ leave standstill 4h.Tubular type low temperature continuous centrifuge 14000rpm centrifugation supernatant liquor also changes in another retort.
3.3 the sad method of antibody is purified:
0.2% adding that is total amount by volume is sad, stirs, and room temperature is placed 4h.After filtering with filter cloth, being filtered to clarification with K type multilayer sheet frame again, be that 0.1% of total amount adds saturated formaldehyde solution in above-mentioned filtrate more by volume, abundant stirring and evenly mixing, room temperature placement 24h, during the jolting several.
3.4 filtration sterilization:
With 0.22 μ m micropore filter element filtration sterilization.With molecular weight cut-off be 1000KDa ultrafiltration membrance filter except virus.
4. the yolk antibody freeze-drying of Ti Chuning
Purification and the yolk antibody liquid through being up to the standards are sub-packed in cillin bottle in a small amount, and freeze-drying is spent the night on Freeze Drying Equipment, and temperature is minimum in the freeze-drying process is down to-38 ℃, keeps 4h to freeze-drying fully, returns to room temperature then gradually, takes out and is stored in 4 ℃.
The detection of tiring of embodiment 2 transmissible gastro-enteritis viruss and epidemic diarrhea virus bigeminy yolk antibody
1. transmissible gastro-enteritis virus and epidemic diarrhea virus standard positive serum are prepared in experiment, are provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture; Antigen is by embodiment 1 preparation; Agarose, punch tool, 16# syringe needle, scissors, tweezers, spirit lamp, marking pen, clean plate, water-bath.
2. the preparation of transmissible gastro-enteritis virus (or epidemic diarrhea virus) agp antigen: carry out antigen with polyoxyethylene glycol (PEG-20000) dry powder embedding dialysis tubing and concentrate.
The treatment process of dialysis tubing: 0.5mol/L EDTA-2Na 1ml adds the 10g sodium bicarbonate, mends distilled water to 500ml, and the pH value transfers to 8.0; Dialysis tubing is put to state and is boiled 10min in the liquid; Take out dialysis tubing distilled water and clean up, put again and boil 10min in the distilled water; 40ml virus liquid is poured in the dialysis tubing after the processing, tightened sack, be placed in the clean aluminium box, with polyoxyethylene glycol (PEG-20000) dry powder embedding dialysis tubing, put 4 ℃ of dialysis.Observe when liquid remains 3～4ml in the dialysis tubing and take out, the antigen that sucking-off concentrates is the agp antigen of preparation.
The preparation of 3.1% agarose plate
Getting concentration is 0.01mol/L, and pH is 7.2 phosphate buffer 1 00ml, adds the 1g agarose, and the NaCl of 8.0g boils and melts back elimination precipitation, and it is anticorrosion to add 0.01% Thiomersalate, is cooled to 50 ℃ of left and right sides glue agar plates processed.In the plate of diameter 90mm, annotate the agarose 16ml that adds thawing.Agarose thickness is no less than 2.8mm, punches after 4 ℃ of coolings.
4. operation steps
Punch with sexangle seven apertures in the human head mould 4.1 get agarose plate, aperture 5mm, pitch-row is 3mm.Agar is crossed at the spirit lamp flame envelope after punching with punch tool, the bottom in hole is sealed, in case the egg yolk liquid seepage.
4.2 antigen is demarcated
Interstitial hole adds transmissible gastro-enteritis virus (or epidemic diarrhea virus) standard positive serum, and the hole begins to add successively the antigen of the antigen of the antigen of the antigen of antigen stock, 2 times of dilutions, 4 times of dilutions, 8 times of dilutions, 16 times of dilutions, the antigen of 32 times of dilutions from the hole, the top on every side.Liquid feeding 40 μ l in every hole after application of sample finishes, are placed on the agarose plate in the lidded container that is covered with wet gauze, and with upholder agarose plate are put surely, put the wet box internal reaction 24～72h of 37 ℃ of incubators, produce until precipitation line.The most tangible dilution antigen of precipitation line to occur as standard antigen.
4.3 the mensuration of antibody titer
Be that 7.2 phosphate buffered saline buffer is made doubling dilution with the egg yolk liquid of the antibody titer to be determined of results with 0.01mol/L pH, extent of dilution is respectively 1 times, 4 times, 8 times, 16 times, 32 times, 64 times and reaches higher extension rate.Add transmissible gastro-enteritis virus (or epidemic diarrhea virus) antigen of demarcating in the interstitial hole, add different dilution antibody on every side in the hole, wherein 1 hole adds standard positive serum, every hole liquid feeding 40 μ l, after application of sample finishes, the agarose plate is placed in the lidded container that is covered with wet gauze, and with upholder agarose plate is put surely, put the wet box internal reaction 24～72h of 37 ℃ of incubators, produce until precipitation line.Be the antibody titer of test sample with the antibody maximum dilution multiple of observing precipitation line.
5. the result judges
During clear, the fine and close precipitation line of of positive serum and corresponding antigen hole intermediate formation, just can judge.
Positive: tested egg yolk liquid and antigen hole intermediate formation precipitation line, and with the crooked ring of positive serum precipitation line company, be judged to the positive (+).
Suspicious: the unintelligible or positive control precipitation line of precipitation line is little when curved to tested serum hole, is judged to suspicious (±).Suspicious specimen should heavily be examined, and heavily inspection should be judged to the positive (+) when coming to the same thing.
Negative: no precipitation line appears as feminine gender.When precipitation line intersects or do not link to each other with the positive control precipitation line, all belong to nonspecific reaction.Be judged to feminine gender (-).
6. detected result:
Two exempt from 1 week of back to gather egg yolk liquid detects, and detects once every 7 days later on, until head exempts from 24 weeks of back.The result shows, adopt immune programme for children of the present invention, 12～16 all transmissible gastro-enteritis viruss and epidemic diarrhea virus bigeminy yolk antibody are tired and all can be reached 1: 512 after head exempts from, head exempts from 3～24 weeks of back, and transmissible gastro-enteritis virus and epidemic diarrhea virus bigeminy yolk antibody tire all 〉=and 1: 256.
Embodiment 3 transmissible gastro-enteritis viruss and the therapeutic test of epidemic diarrhea virus bigeminy yolk antibody
Get 1 age in days and do not eat 16 of first suckling piglet, the temperature control of experimental animal room is at 25～30 ℃, adopt the artificial suckling method, every the 2h milk of feeding 1 time, feeding to observe after 1 day is divided into 4 groups at random, is respectively: attack malicious treatment group (4), attack poison and do not treat group (4), treatment and do not attack poison group (4) and do not treat and do not attack malicious organize (4).Transmissible gastro-enteritis virus and epidemic diarrhea virus that to attack malicious used virus be ultrafiltration, be directly oral after 0 times of dilution of phosphate buffer 1 of 7.2 through concentration for 0.01mol/L pH, attack the toxic agent amount and be minimum morbidity amount 5 times, it is identical that every piglet is attacked the toxic agent amount, observes the diarrhoea situation after piglet is attacked poison.Feed transmissible gastro-enteritis virus and epidemic diarrhea virus bigeminy yolk antibody for the treatment of group treated used yolk antibody and is the level sample of purifying when treating that piglet has just begun symptom of diarrhea to occur, and it is 1: 32 that agar diffusion test measures that yolk antibody tires.Do not treat and organize the equivalent physiological saline of feeding, observe every grice diarrhoea symptom and transmissible gastro-enteritis virus and epidemic diarrhea virus bigeminy yolk antibody result for the treatment of day by day, judge result for the treatment of with 2 indexs of ight soil exponential sum mortality ratio.The ight soil index is according to (Sherman D M such as Sherman, Acres S D, Sadowski P L, et al.Protection of calves against fatal enteric colibacillosis by orally administered Escherichia coli K99-specipic monoclonal antibody[J] .Infect Immun, 1974,10:770-782.) standard determination formulated, namely 0 be divided into normal, the ight soil solid molding; 1 is divided into laxativeness, the soft shaping of loose and watery stool; 2 are divided into moderate diarrhoea, and ight soil is yellow water sample; 3 are divided into severe diarrhoea, and ight soil is water sample and sprays.
Attack poison back all piglets of 12～24h and in various degree yellow watery diarrhea all occurs, attack malicious treatment group at oral transmissible gastro-enteritis virus and the epidemic diarrhea virus bigeminy yolk antibody sx of suffering from diarrhoea after 1 day, become yellow loose stool after 2 days, ight soil becomes sticky thickly after 3 days, becomes the soft excrement of shaping after 4～5 days; Not treat the group piglet mental status abnormal with not attacking poison not attack malicious treatment group, no symptom of diarrhea; Attack poison and do not treat the group grice diarrhoea and be water sample and spray, after 2～3 days because extremely dehydration is dead.The results are shown in Table 1.
Table 1 piglet is attacked poison treatment back different number of days death condition and ight soil index
Annotate: denominator is survival piglet number, and molecule is diarrhoea piglet number; Be average ight soil index in the bracket
Test-results shows: symptom of diarrhea does not appear in two groups of piglets of not attacking poison; Attack poison and do not treat the group piglet and serious symptom of diarrhea just occurred at first day, ight soil is watery injection, and piglet becomes thin rapidly, and piglet 3 days is all dead; Attack malicious treatment group piglet and symptom of diarrhea occurring, with after the yolk antibody treatment, this group grice diarrhoea symptom then obviously alleviates, and ight soil becomes yellow loose stool by yellow watery diarrhea, after shaping gradually, get well about a week, death condition does not appear.
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. the preparation method of a transmissible gastro-enteritis virus and epidemic diarrhea virus bigeminy yolk antibody is characterized in that, may further comprise the steps:
1) the immunity preparation of vaccine:
Cultivate transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus with pig kidney passage cell, African green monkey kidney passage cell respectively, concentrate the back respectively as antigen, two kinds of antigens are fully mixed the back deactivation, add immunostimulant and white-oil adjuvant, preparation water-in-oil-type transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus bivalent inactivated vaccine are as the immunity vaccine;
2) immunity:
Immunity with the step 1) preparation is carried out immunity with vaccine to non-immunization laying hen, immune time 〉=2;
3) from step 2) collect egg yolk liquid the immunization laying hen output egg, extract transmissible gastroenteritis of swine and epidemic diarrhea virus bigeminy yolk antibody, behind the purifying, freeze-drying, wherein, two exempt from back 1 week beginning and measure height every sampling in 7 days and exempt from the egg yolk anti-swine infectious enterogastritis virus and Porcine epidemic diarrhea virus specific antibody and tire; Antibody titer all reaches 1: the 128 above yolk of collecting, for the preparation of transmissible gastro-enteritis virus and epidemic diarrhea virus bigeminy yolk antibody;
Wherein, transmissible gastro-enteritis virus antigen and epidemic diarrhea virus antigen mixed in 1: 0.8 by volume～1: 1.2 in the described step 1), mixed the malicious valency of back two kinds of virus antigens all 〉=1.0 * 10
6.0TCID
50/ ml.
2. method according to claim 1, it is characterized in that, the preparation of transmissible gastro-enteritis virus antigen may further comprise the steps in the described step 1): Transmissible gastroenteritis virus liquid is seeded on the PK15 monolayer cell, sets up simultaneously not meet poison cell contrast, CO under 37 ℃ of temperature
2Cultivate in the incubator, receive poison when cytopathy occurring to 75% cell, neutralization test is measured malicious valency, concentrates the back as antigen.
3. according to the described method of claim 1, it is characterized in that, the preparation of epidemic diarrhea virus antigen may further comprise the steps in the described step 1): epidemic diarrhea virus liquid is inoculated on the Vero monolayer cell, sets up simultaneously not meet poison cell contrast, CO under 37 ℃ of temperature
2Cultivate in the incubator, receive poison when cytopathy occurring to 75% cell, neutralization test is measured malicious valency, concentrates the back as antigen.
4. method according to claim 1, it is characterized in that, in the described step 1) with after transmissible gastro-enteritis virus and the deactivation of epidemic diarrhea virus hybrid antigen, add the yeast glucan of the lipopolysaccharides of volume ratio 0.01%～0.05% and volume ratio 0.2%～0.8% respectively as immunostimulant, with the white-oil adjuvant emulsification of 2 times of amounts of cumulative volume of described viral liquid, preparation water-in-oil-type transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus bivalent inactivated vaccine are as the immunity vaccine.
5. method according to claim 1, it is characterized in that, described step 2) immune programme for children is in: exempted from preceding 10 days at head, drink for chicken with the astragalus polysaccharides of volume ratio 1%～5% every day, promote chicken body overall immunity, head exempts to inject mycobacterium tuberculosis var bovis polyoses nucleic acid 0.1～0.5ml, Freund's complete adjuvant 100～300 μ g under every plumage cock skin in preceding 7 days, carries out immunostimulation;
Chest muscle multi-point injection immunity vaccine 1～2ml when head exempts from; Carry out two after 2 weeks and exempt from, injecting immune vaccine 1～2ml and mycobacterium tuberculosis var bovis polyoses nucleic acid 0.1～0.5ml; After 2 weeks, carry out three again and exempt from, injecting immune vaccine 2～4ml and mycobacterium tuberculosis var bovis polyoses nucleic acid 0.2～0.8ml; After 4～8 weeks, carry out four again and exempt from, injecting immune vaccine 2～4ml.
6. method according to claim 1 is characterized in that, fully stirs in the described step 3) to make yolk be even paste, adds equal-volume through the distilled water of sterilization and cooling, stirring and evenly mixing, deactivation; Add the pH value be equivalent to 6 times of volumes of former yolk and be 4.2 sterile purified water in the interlayer retort, and be cooled to 1～4 ℃, the egg yolk liquid with deactivation adds then, the stirring of limit edged, and 4～8 ℃ leave standstill 4h; The centrifugation supernatant liquor also changes in another retort, is that 0.2% adding of total amount is sad by volume, stirs, and room temperature is placed 2～4h; After the filter cloth filtration, be filtered to clarification with K type multilayer sheet frame again, be that 0.1% of total amount adds saturated formalin in above-mentioned filtrate by volume, abundant stirring and evenly mixing, room temperature is placed 24h, during jolting for several times, filtration sterilization, with molecular weight cut-off be 1000KDa ultrafiltration membrance filter except virus, the transmissible gastro-enteritis virus that can obtain purifying and epidemic diarrhea virus bigeminy yolk antibody.
7. transmissible gastro-enteritis virus and epidemic diarrhea virus bigeminy yolk antibody by any method preparation of claim 1～6.
8. transmissible gastro-enteritis virus according to claim 7 and epidemic diarrhea virus bigeminy yolk antibody is characterized in that, described bigeminy yolk antibody is freeze-drying bigeminy yolk antibody.
9. transmissible gastro-enteritis virus as claimed in claim 7 and the epidemic diarrhea virus bigeminy yolk antibody application in the medicine of preparation treatment and prevention transmissible gastroenteritis of swine and epidemic diarrhea disease.
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| CN 201010273191 CN101948536B (en) | 2010-05-10 | 2010-09-02 | Porcine transmissible gastroenteritis virus (PTGEV) and porcine epidemic diarrhea virus (PEDV) dual yolk antibody and preparation method thereof |
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| CN102276718A (en) * | 2011-07-26 | 2011-12-14 | 广州格雷特生物科技有限公司 | Process for extracting yolk immunoglobulin by water-caprylic acid two-step method |
| CN103127497B (en) * | 2011-11-25 | 2015-12-09 | 普莱柯生物工程股份有限公司 | Porcine circovirus 2 type, mycoplasma pneumoniae bivalent inactivated vaccine and preparation method thereof |
| CN103865884A (en) * | 2012-12-11 | 2014-06-18 | 普莱柯生物工程股份有限公司 | Duck viral hepatitis bivalent yolk antibody, preparation method and application thereof |
| CN103041385B (en) * | 2013-01-29 | 2014-02-19 | 齐鲁动物保健品有限公司 | Porcine transmissible gastroenteritis and epidemic diarrhea combined live vaccine and preparation method thereof |
| CN103263666B (en) * | 2013-05-24 | 2019-07-16 | 北京大北农科技集团股份有限公司动物医学研究中心 | Porcine circovirus 2 type, porcine mycoplasmal pneumonia bivalent inactivated vaccine and preparation method thereof |
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