CN101845095A - Method for preparing double yolk antibody of porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus - Google Patents
Method for preparing double yolk antibody of porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus Download PDFInfo
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Abstract
The invention discloses a method for preparing a double yolk antibody of a porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus. The method comprises the following steps of: performing porcine transmissible gastroenteritis virus multiplication on porcine kidney cells (PK15); performing porcine epidemic diarrhea virus multiplication on African green monkey kidney cells (Vero); emulsifying the two cell cultures used as antigen with an oil emulsion adjuvant to prepare immunogen, namely, mixing the two kinds of viruses in a ratio of (1-3):(1-3) to prepare the immunogen; immunizing non-immunologic laying hens; and obtaining the double yolk antibody which can prevent and treat porcine transmissible gastroenteritis and porcine epidemic diarrhea based on the collection and purification of the yolk. When the double yolk antibody is used for curing experimental pigs, the clinical symptoms in the experiment are obviously reduced compared with a control group, and the death rate of the experimental group is obviously lower than that of the control group. The double yolk antibody has obvious preventing and treating functions when applied in a pig farm with high incidence rate of the porcine transmissible gastroenteritis and the porcine epidemic diarrhea.
Description
Technical field
The invention belongs to the veterinary biologics technical field, be specifically related to transmissible gastro-enteritis virus and 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 in the world countries have all reported transmissible gastroenteritis of swine in succession.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 a 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 of treatment 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 a kind of specificity that both had aborning, curative effect is certain again, the treatment preparation that result of treatment manifests rapidly.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of transmissible gastroenteritis of swine, epidemic diarrhea virus bigeminy yolk antibody.
Immunogen of the present invention adopts cell culture oiling adjuvant emulsion to be prepared from, and wherein transmissible gastro-enteritis virus is cultivated with porcine kidney cell (PK15) passage cell; Porcine epidemic diarrhea virus is cultivated with African green monkey kidney cell (Vero) passage cell; The viral level of these two kinds of viruses all 〉=10
6.0TCID
50/ ml, with these two kinds of viruses with volume ratio (1～3): after the mixed of (1～3) with oily adjuvant with volume ratio emulsification in 1: 2 after as immunogen, the non-immunization laying hen of immunity is collected yolk and is carried out yolk antibody detections of tiring, the yolk purification yolk antibody of selecting height to tire.Directly act on intestine of young pigs after this antibody oral administration, with treatment and infection prevention marcy agent (TGEV) and epidemic diarrhea virus (PEDV) infection.
For achieving the above object, the present invention adopts following technological step:
One, the antigenic preparation of TGEV
Transmissible gastroenteritis virus liquid is seeded on the PK15 monolayer cell, sets up the cell contrast simultaneously, 37 ℃ of CO
2Cultivate in the incubator, outwell behind the 24h and keep liquid and change liquid, day by day the observation of cell pathology, if 72h postoperative infection cytopathy is not obvious, then centrifugation after the freeze thawing of harvested cell suspension is got supernatant liquid and is continued blind passage on cell, and cytopathy appears in blind passage to the s-generation, when appearring in 75% cell, cytopathy receives poison, freeze thawing, cell debris is removed in centrifugation, collect supernatant liquid, concentrate the back as antigen.
Two, the antigenic preparation of PEDV
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 after the freeze thawing of harvested cell suspension in centrifugation, get supernatant liquid and continue blind passage on cell, cytopathy appears in blind passage to the third generation, when cytopathy appears in 75% cell, the harvested cell suspension, after the freeze thawing in centrifugation, collect supernatant liquor, concentrate the back as antigen.
Three, the preparation of yolk antibody
1. animal immune
(1) immunity is prepared: with TGEV and the PEDV antigen mixed with 1: 1, behind 37 ℃ of deactivation 24h, exempt from use immunogen as head by the 0.1% saturated formalin of adding (being commercially available 40% formalin) of total amount; Get an amount of TGEV and PEDV antigen with 1: 1 mixed, add formaldehyde solution (40% aqueous solution) behind 37 ℃ of deactivation 24h, behind the oily adjuvant emulsion of 2 times of amounts, as two exempting from, three exempting from and four exempt to use immunogen by 0.1% of total amount.
(2) immunity: the immunogen with above-mentioned preparation is carried out immunity to non-immunization laying hen, and other establishes not immune blank group.
Immune programme for children is: to the thoracic muscle multi-point injection, collunarium, eye droppings were total to 1ml simultaneously when head exempted from; Carry out two after 2 weeks and exempt from, immunizing dose is identical with approach when just exempting from; After 2 weeks, carry out three again and exempt from (the immunity amount is 2ml, and approach is identical), after 4～8 weeks, carry out the 4th immunity (the immunity amount is 2ml, and approach is identical) again.
2. egg yolk liquid preparation
Two exempt from back 1 week beginning every sampling in 3 days with agar diffusion method mensuration height exempt from anti-TGEV in the egg yolk, the PEDV specific antibody is tired.If antibody titer reaches and gets final product egg collection more than 1: 8, egg yolk liquid is used to prepare TGEV, PEDV bigeminy yolk antibody.
3. antibody titer detects
The TGEV that obtains, PEDV bigeminy yolk antibody prove by the antigen antibody reaction in the agar diffusion test.Antigen in the agar diffusion test is spissated virus, because the immunogen of laying hen immunity usefulness is from the cell infection thing, wherein impure composition may be cell debris, and there is not cellular constituent in the detection antigen, if this detection antigen and yolk antibody react, containing in the provable yolk antibody at antigen is the antibody of TGEV and PEDV.
The preparation agar gel: the egg yolk liquid that step 2 is collected is with adding behind PBS (pH7.4) doubling dilution of 0.01mol/L around the agar gel in the hole, add in the interstitial hole in the step 1, two through centrifugal concentrated and purified TGEV, PEDV antigen, putting 37 ℃ of incubators reaction 24h in the wet box, is the antibody titer of this egg yolk liquid with the high dilution egg yolk liquid that the precipitation band occurs.
Detected result: two exempt to gather in 1 week of back that egg yolk liquid detects TGEV and the PEDV antibody titer can both reach 1: 8, regather yolk and detect antibody titer in one month and all peaked 1: 64.
4. the purification of yolk antibody
The method that acidifying distillation water law is purified and sad method is purified and combined is adopted in the purification of yolk antibody.Collection is exempted from egg through the qualified height of sampling Detection, with the eggshell sterilization, adopts 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 121 ℃ of 30min sterilizations and refrigerative distilled water, stirring and evenly mixing, 62.5 ℃ of heat inactivation 30min.In the interlayer retort, add earlier the pH value that is equivalent to 6 times of volumes of former yolk then and be 4.2 sterile purified water, and be cooled to 1～4 ℃.Then, with the egg yolk liquid adding of deactivation, the limit edged stirs, and 4～8 ℃ leave standstill 4h.Tubular type low temperature continuous centrifuge 14000rpm centrifugation supernatant liquor also changes in another retort.It is sad to add by 0.2% of total amount, stirs, and room temperature is placed 2～4h.After filtering with filter cloth, be filtered to clarification with K type multilayer sheet frame again, add formaldehyde solution (40% aqueous solution) in above-mentioned filtrate by 0.1% of total amount, abundant stirring and evenly mixing, room temperature is placed 24h, during the jolting several.Use 0.22 μ m micropore filter element filtration sterilization at last, the ultrafiltration membrance filter that with molecular weight cut-off is 1000KDa is except that virus.The yolk antibody that obtains purifying, i.e. TGEV, PEDV bigeminy yolk antibody.
5. with the yolk antibody liquid freeze-drying of purifying, be stored in 4 ℃.
Four, TGEV, the therapeutic test of PEDV bigeminy yolk antibody
Laboratory animal is the piglet that 1 age in days is not eaten colostrum, and the test group animal is used TGEV and PEDV virus oral challenge, the oral then yolk antibody of the present invention of feeding.Other establish attack poison not with the antibody group, do not attack poison not with the antibody group, do not attack poison in contrast with 3 groups of antibody groups.Judge the result of treatment of TGEV and PEDV bigeminy yolk antibody according to each group clinical symptom and mortality ratio.
Test-results shows: TGEV provided by the invention and PEDV bigeminy yolk antibody have the obvious treatment effect to the grice diarrhoea that TGEV and PEDV cause, any toxic side effect do not occur.
Porcine epidemic diarrhea virus and transmissible gastro-enteritis virus belong to coronaviridae together, and the two morbific clinical symptom is extremely similar again, and generally are 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, the inhibition cause of disease is the key of treatment and infection prevention gastro-enteritis and epidemic diarrhea to the absorption and the increment of mucous membrane of small intestine.This TGEV-PEDV bigeminy yolk antibody directly by little intestinal absorption, has improved the local immunity power of small intestine by oral, has suppressed absorption and the propagation of cause of disease to mucous membrane of small intestine.
Transmissible gastroenteritis of swine, the epidemic diarrhea virus bigeminy yolk antibody of the inventive method preparation, adopt two kinds of immunogen preparing preventions and treatment transmissible gastroenteritis of swine, these two kinds of diseases of porcine epizootic diarrhea, has the specificity height, characteristics with strong points, significant to treatment and prevention transmissible gastroenteritis of swine, epidemic diarrhea.
Embodiment
Explanation provides the following examples in nonrestrictive mode for example.
Embodiment 1
Transmissible gastro-enteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV) propagation and antigen prepd
1.TGEV virus multiplication and antigenic preparation
With 3 bottles of PK15 cells of Transmissible gastroenteritis virus liquid inoculation (bottle floorage 25cm
2), every bottle of cell inoculation 1ml virus liquid, build bottle cap, accumbency bottle pipe rotates gently, and viral liquid is fully contacted with whole cell monolayer, 37 ℃ of absorption 40min, rock 1 time every 10min therebetween, need not to inhale and abandon viral liquid, directly add the DMEM cell maintenance culture solution 5ml that contains 2% foetal calf serum, set up the cell contrast simultaneously, 37 ℃ of CO
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 (CPE) is not obvious, then behind the 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 appears in 75% cell,-20 ℃～20 ℃ multigelations 3 times, under 4 ℃ of conditions, the centrifugal 30min of 3000rpm removes cell debris then, collect supernatant liquid, concentrate the back as antigen.
2.PEDV virus multiplication and antigenic preparation
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 the 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 appears in 75% cell, shows as cytogamy and circle contracts, 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 ℃ multigelations 3 times.
The preparation of embodiment 2 TGEV-PEDV bigeminy yolk antibodies
1. immunogen preparing
Get the TGEV antigen that obtains and PEDV antigen with 1: 1 mixed after, add formaldehyde solution (40% aqueous solution) by 0.1% of total amount, 37 ℃ of deactivation 24h exempt to use immunogen as head.The antigen of above-mentioned deactivation is added 4 parts of tween-80s mixing backs as water (now joining with preceding) for 96 parts; The oil phase preparation: 94 parts of white oil+6 part Arlacel-80s, the amount of pressing 0.015g/ml again adds aluminum stearate, after heating in water bath mixes, 121 ℃ of autoclaving 30min, standby; Emulsification: add earlier oil phase, 3000rpm stirs 5min in advance, slowly adds water then, and the limit edged rocks, stir 30s after, rotating speed is transferred to 12000rpm, stir 5min and get final product, put 4 ℃ standby.Get deactivation vaccine 10ml after the emulsification in the 15ml centrifuge tube, the centrifugal 15min of 3000rpm, lower floor's stratified liquid amount promptly shows the emulsification success less than 0.5ml.Immunogen after the emulsification success is as two exempting from, three exempting from and four exempt to use immunogen.
2. immunity
To thoracic muscle, leg flesh multi-point injection, collunarium, eye droppings were total to 1ml simultaneously when head exempted from; Carry out two after 2 weeks and exempt from, with the oil seepage after the emulsification, immunizing dose is identical with approach when just exempting from; Exempt from (the immunity amount is 2ml, and approach is identical) every carrying out 32 weeks again, after 6 weeks, carry out the 4th immunity (the immunity amount is 2ml, and approach is identical) again.
3. the preparation of egg yolk liquid
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 121 ℃ of 30min sterilizations and refrigerative distilled water, 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 1～4 ℃.Then, with the egg yolk liquid adding of deactivation, the limit edged stirs, and 4～8 ℃ 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:
It is sad to add by 0.2% of total amount, stirs, and room temperature is placed 4h.After filtering with filter cloth, be filtered to clarification with K type multilayer sheet frame again, add formaldehyde solution (40% aqueous solution) in above-mentioned filtrate by 0.1% of total amount again, abundant stirring and evenly mixing, room temperature is placed 24h, during the jolting several.
3.4. filtration sterilization:
With 0.22 μ m micropore filter element filtration sterilization.With molecular weight cut-off is that the ultrafiltration membrance filter of 1000KDa removes virus.
4. antibody titer detects (agar diffusion method)
4.1. experiment is prepared: TGEV and PEDV standard positive serum 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.
4.2.TGEV the preparation of agp antigen: carry out antigen with polyoxyethylene glycol (PEG-20000) dry powder embedding dialysis tubing and concentrate.The treatment process of dialysis tubing:
4.2.1.0.5mol/LEDTA-2Na1ml add the 10g sodium bicarbonate, mend distilled water to 500ml, transfer pH to 8.0.
4.2.2. putting to state, dialysis tubing boils 10min in the liquid.
Clean up 4.2.3. take out dialysis tubing distilled water, put again and boil 10min in the distilled water.
4.2.3. the cooling back is available.(if temporarily need not, can put in sodium benzoate or the sodiumazide and preserve)
4.2.4. the exhausted dialysis tubing is handled with method, puts in 50% aqueous glycerin solution or 50% aqueous ethanolic solution to preserve, clean with preceding distilled water and get final product next time.
4.2.5. notice that getting dialysis tubing must wear gloves.
40ml virus liquid is poured in the dialysis tubing after the processing, tightened sack, be placed in the clean aluminium box,, put 4 ℃ of dialysis with polyoxyethylene glycol (PEG-20000) dry powder embedding dialysis tubing.Observe when liquid remains 3-4ml in the dialysis tubing and take out, the spissated antigen of sucking-off is the agp antigen of preparation.Preparation PEDV agp antigen uses the same method.
4.3.1% the preparation of agarose plate
Get PBS (pH7.0) the liquid 100ml of 0.01mol/L, add agarose 1g, Nacl 8.0g boils and melts back elimination precipitation, and it is anticorrosion to add 0.01% Thiomersalate, is cooled to 50 ℃ of side-inverted agar plates.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.
4.4. operation steps
Punch with sexangle seven apertures in the human head mould 4.4.1. get agarose plate, aperture 5mm, pitch-row is 3mm.Agar is crossed on the spirit lamp flame envelope after punching with punch tool, the bottom in hole is sealed, in case the egg yolk liquid seepage.
4.4.2. antigen is demarcated
Interstitial hole adds TGEV (or PEDV) 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.
4.4.3. the mensuration of antibody titer
The yolk of the antibody titer to be determined of results is made doubling dilution with the PBS (pH7.0) of 0.01mol/L, extent of dilution is respectively 1 *, 4 *, 8 *, 16 *, 32 *, 64 * reach higher extension rate.Add TGEV (or PEDV) 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 agarose plate is put surely with upholder, put the wet box internal reaction 24～72h of 37 ℃ of incubators, produce until precipitation line.With the antibody maximum dilution multiple of observing precipitation line is the antibody titer of test sample.
4.5. 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 (-).
5. 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 reduces to-38 ℃, keeps 3～4h to freeze-drying fully, returns to room temperature then gradually, takes out and is stored in 4 ℃.
Embodiment 3 TGEV-PEDV bigeminy yolk antibody therapeutic tests
Get 1 age in days and do not eat 16 of the piglets that colostrum does not have TGEV and PEDV morbidity history, experimental animal room temperature is controlled 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).TGEV and PEDV that to attack malicious used virus be ultrafiltration are directly oral after 10 times of dilutions of the PBS of 0.01mol/L (pH7.4), and 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.The treatment group TGEV-PEDV bigeminy yolk antibody of feeding is 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: 64 that agar diffusion test measures that yolk antibody tires.Do not lift the treatment group equivalent physiological saline of feeding, observe every grice diarrhoea symptom and TGEV-PEDV bigeminy yolk antibody result of treatment day by day, judge the result of treatment of TGEV-PEDV bigeminy yolk antibody 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 calvesagainst fatal enteric colibacillosis by orally administered Escherichiacoli K99-specipic monoclonal antibody[J] .InfectImmun, 1974,10:770-782.) standard determination formulated, promptly 0 be divided into normal; 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 the oral TGEV-PEDV 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 of 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; In the bracket is average ight soil index
Test-results shows: it is normal neither to attack poison group piglet growth, symptom of diarrhea do not occur, and whether the TGEV-PEDV bigeminy of feeding yolk antibody is to not influence of its growth; And two attack poison group piglet and serious symptom of diarrhea all occurs, show as yellow watery diarrhea, piglet becomes thin rapidly, without all death in 3 days of yolk antibody treatment group piglet, yolk antibody treatment group grice diarrhoea symptom then obviously alleviates, and ight soil becomes soft excrement by watery diarrhea, and is shaped gradually, recover gradually about one week, death condition do not occur.
Conclusion
The TGEV-PEDV bigeminy yolk antibody made by the present invention carries out therapeutic test; the result shows reduction piglet mortality ratio and the symptom of diarrhea that energy is effective, and progressively takes a turn for the better, until rehabilitation; have provide protection completely, have a significant effect for the control diarrhea of pigs.
Above-listed detailed description system specifies at one of the present invention possible embodiments, and only this embodiment is not the claim in order to restriction the present invention, all do not break away from skill spirit of the present invention institute for it equivalence implement or change, all should be contained in the claim of this case.
Claims (10)
1. the preparation method of a transmissible gastroenteritis of swine, epidemic diarrhea virus bigeminy yolk antibody, transmissible gastro-enteritis virus is cultivated with the porcine kidney cell passage cell, Porcine epidemic diarrhea virus is cultivated with the African green monkey kidney cell passage cell, with these two kinds of viruses with volume ratio (1～3): after the mixed of (1～3) with oily adjuvant with volume ratio emulsification in 1: 2 after as immunogen, the non-immunization laying hen of immunity extracts transmissible gastroenteritis of swine, epidemic diarrhea virus yolk antibody from its yolk.
2. the preparation method of transmissible gastroenteritis of swine according to claim 1, Porcine epidemic diarrhea virus bigeminy yolk antibody is characterized in that may further comprise the steps:
(1) the antigenic preparation of TGEV
Transmissible gastroenteritis virus liquid is seeded on the PK15 monolayer cell, sets up the cell contrast simultaneously, 37 ℃ of CO
2Cultivate in the incubator, outwell behind the 24h and keep liquid and change liquid, day by day the observation of cell pathology, if 72h postoperative infection cytopathy is not obvious, then centrifugation after the freeze thawing of harvested cell suspension is got supernatant liquid and is continued blind passage on cell, and cytopathy appears in blind passage to the s-generation, when appearring in 75% cell, cytopathy receives poison, freeze thawing, cell debris is removed in centrifugation, collect supernatant liquid, concentrate the back as antigen;
(2) the antigenic preparation of PEDV
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 centrifugation after the freeze thawing of harvested cell suspension is got supernatant liquid and is continued blind passage on cell, cytopathy appears in blind passage to the third generation, when cytopathy appears in 75% cell, harvested cell suspension, centrifugation after the freeze thawing, collect supernatant liquor, concentrate the back as antigen;
(3) preparation of yolk antibody
With TGEV and PEDV antigen mixed, behind the deactivation 24h, exempt to use immunogen as head with 1: 1; Get an amount of TGEV and PEDV antigen with 1: 1 mixed, behind the deactivation 24h, with the oily adjuvant emulsion of 2 times of amounts, as two exempting from, three exempting from and four exempt to use immunogen; Immunogen with above-mentioned preparation is carried out immunity to non-immunization laying hen, and other establishes not immune blank group; Two exempt from back 1 week beginning measures every sampling in 3 days that height is exempted from anti-TGEV in the egg yolk, the PEDV specific antibody is tired; If antibody titer reaches and gets final product egg collection more than 1: 8, collect yolk; Fully stirring makes yolk be even paste, adds equal-volume through sterilization and refrigerative distilled water, stirring and evenly mixing, deactivation; Add earlier the pH value that is equivalent to 6 times of volumes of former yolk then 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, and it is sad to add by 0.2% of total amount, stirs, and room temperature is placed 2～4h; After the filter cloth filtration, be filtered to clarification with K type multilayer sheet frame again, add saturated formalin in above-mentioned filtrate by 0.1% of total amount, abundant stirring and evenly mixing, room temperature is placed 24h, during jolting for several times, filtration sterilization, with molecular weight cut-off is that the ultrafiltration membrance filter of 1000KDa removes virus, the yolk antibody that obtains purifying, i.e. TGEV, PEDV bigeminy yolk antibody;
(4) the yolk antibody liquid freeze-drying of purifying is stored
3. the preparation method of transmissible gastroenteritis of swine according to claim 2, Porcine epidemic diarrhea virus bigeminy yolk antibody, the viral level that it is characterized in that used transmissible gastro-enteritis virus and Porcine epidemic diarrhea virus all 〉=10
6.0TCID
50/ ml.
4. the preparation method of transmissible gastroenteritis of swine according to claim 2, Porcine epidemic diarrhea virus bigeminy yolk antibody is characterized in that the antigenic preparation procedure of TGEV is: with 3 bottles of PK15 cells of Transmissible gastroenteritis virus liquid inoculation, and bottle floorage 25cm
2Every bottle of cell inoculation 1ml virus liquid, build bottle cap, accumbency bottle pipe rotates gently, and viral liquid is fully contacted with whole cell monolayer, 37 ℃ of absorption 40min, rock 1 time every 10min therebetween, need not to inhale and abandon viral liquid, directly add the DMEM cell maintenance culture solution 5ml that contains 2% foetal calf serum, set up the cell contrast simultaneously, 37 ℃ of CO
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 (CPE) is not obvious, then behind the 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, when appearring in 75% cell, cytopathy receives poison,-20 ℃～20 ℃ multigelations 3 times, then under 4 ℃ of conditions, the centrifugal 30min of 3000rpm, remove cell debris, collect supernatant liquid, concentrate the back as antigen.
5. the preparation method of transmissible gastroenteritis of swine according to claim 2, Porcine epidemic diarrhea virus bigeminy yolk antibody, it is characterized in that the antigenic preparation procedure of PEDV is: epidemic diarrhea virus liquid is inoculated on the Vero cell monolayer, set 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 the 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 appears in 75% cell, shows as cytogamy and circle contracts, 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 ℃ multigelations 3 times.
6. the preparation method of transmissible gastroenteritis of swine according to claim 2, Porcine epidemic diarrhea virus bigeminy yolk antibody, it is characterized in that immunogenic preparation procedure is: get the TGEV antigen that obtains and PEDV antigen with 1: 1 mixed after, add saturated formalin by 0.1% of total amount, 37 ℃ of deactivation 24h exempt to use immunogen as head; The antigen of above-mentioned deactivation is added 4 parts of tween-80s mixing backs as water for 96 parts, now with the current; The oil phase preparation: 94 parts of white oil+6 part Arlacel-80s, the amount of pressing 0.015g/ml again adds aluminum stearate, after heating in water bath mixes, 121 ℃ of autoclaving 30min, standby; Emulsification: add earlier oil phase, 3000rpm stirs 5min in advance, slowly adds water then, and the limit edged rocks, stir 30s after, rotating speed is transferred to 12000rpm, stir 5min and get final product, put 4 ℃ standby; Get deactivation vaccine 10ml after the emulsification in the 15ml centrifuge tube, the centrifugal 15min of 3000rpm, lower floor's stratified liquid amount promptly shows the emulsification success less than 0.5ml; Immunogen after the emulsification success is as two exempting from, three exempting from and four exempt to use immunogen.
7. the preparation method of transmissible gastroenteritis of swine according to claim 2, Porcine epidemic diarrhea virus bigeminy yolk antibody, used immune programme for children is when it is characterized in that the non-immune chicken of immunity: chest muscle multi-point injection immunogen when head exempts from, collunarium, eye droppings 1ml altogether simultaneously; Carry out two after 2 weeks and exempt from, the former 1ml of injecting immune; After 2 weeks, carry out three again and exempt from immunogen 2ml; After 4～8 weeks, carry out four again and exempt from immunogen 2ml.
8. the preparation method of transmissible gastroenteritis of swine according to claim 2, Porcine epidemic diarrhea virus bigeminy yolk antibody, the preparation that it is characterized in that egg yolk liquid is two to exempt from the back and began in 1 week to collect through inspecting qualified height that immune chicken produces by random samples and exempt from egg, eggshell is sterilized, 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, add equal-volume through 121 ℃ of 30min sterilizations and refrigerative distilled water, stirring and evenly mixing, 62.5 ℃ of heat inactivation 30min.
9. the preparation method of transmissible gastroenteritis of swine according to claim 2, Porcine epidemic diarrhea virus bigeminy yolk antibody, it is characterized in that two len antibody methods of purification are: 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 1～4 ℃; Then, with the egg yolk liquid adding of deactivation, the limit edged stirs, and 4～8 ℃ leave standstill 4h; Tubular type low temperature continuous centrifuge 14000rpm centrifugation supernatant liquor also changes in another retort; It is sad to add by 0.2% of total amount, stirs, and room temperature is placed 4h.After filtering with filter cloth, be filtered to clarification with K type multilayer sheet frame again, add saturated formalin in above-mentioned filtrate by 0.1% of total amount again, abundant stirring and evenly mixing, room temperature is placed 24h, during the jolting several; With 0.22 μ m micropore filter element filtration sterilization; With molecular weight cut-off is that the ultrafiltration membrance filter of 1000KDa removes virus.
10. the preparation method of transmissible gastroenteritis of swine according to claim 2, Porcine epidemic diarrhea virus bigeminy yolk antibody, it is characterized in that the bigeminy yolk antibody freeze-drying stored program of purifying is: 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 reduces to-38 ℃, keep 3～4h to freeze-drying fully, return to room temperature then gradually, take out and be stored in 4 ℃.
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