CN106636011A - Porcine Epidemic Diarrhea Virus and application thereof - Google Patents
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Abstract
Disclosed is a Porcine Epidemic Diarrhea Virus strain PEDV-KB2013-4. The microbial preservation number is CGMCC No.12663. The classification name is Porcine Epidemic Diarrhea Virus, PEDV. The preservation time is August 23, 2016. The preservation organization is China General Microbiological Culture Collection Center which is located in Institute of Microbiology, Chinese Academy of Sciences, No.3, yard 1, Beichen West Road, Chaoyang District, Beijing, China.
Description
Technical field
The invention belongs to biological technical field, particularly belongs to field of biological pharmacy for animals, it is related to a kind of porcine epizootic diarrhea
Virus and its application.
Background technology
Porcine epizootic diarrhea（Porcine Epidemic Diarrhea, PED）It is by Porcine epidemic diarrhea virus
（Porcine Epidemic Diarrhea Virus, PEDV）Cause a kind of viral infectious intestinal disease of acute, contact, its
Disease is to vomit, be common Intestinum Sus domestica road transmission disease based on watery diarrhea, loss of appetite and dehydration.Porcine epizootic diarrhea
Virus（PEDV）It is coronaviridae coronavirus genuses member, Porcine epidemic diarrhea virus（PEDV）Directly enter after oral nose infection
Enter small intestinal, in Proliferation of Rat Intestinal Crypt Cells, cause intestinal villi absorptive epithelium cytopathy, damage, coming off withered with intestinal villi
Contracting, causes enteral enzymatic activity to reduce, malabsorption, and osmotic pressure increases in intestinal, causes osmotic diarrhea, ultimately causes
Fat, reduction feedstuff absorption rate.PEDV has had a strong impact on the pig industry of China, and its infectious strong, hazardness is big, is to affect complete
One of most important virus of ball pig industry, up to 100%, sow does not often show disease to mortality rate with band poison after suckling pig infection
Shape form occurs.Save outburst PED more China in recent years, to pig industry serious economic loss is caused.
The specific medicament for the treatment of porcine epizootic diarrhea, conventional therapy effect on driving birds is not good, therefore still with vaccine prevention are there is no at present
Based on, and the success or not of Strain and its culture is the key factor of vaccine development.At present, the vaccine that foreign countries use is mainly
Korea's low virulent strain, the country is mainly the porcine epizootic diarrhea of Harbin veterinary institute development, transmissible gastroenteritis of swine bigeminy and goes out
Seedling living etc., the strain used in this vaccine is Porcine epidemic diarrhea virus PEDV CV777 strains, and the isolation of strains age is more early, is led to
Cross the gene analysiss to existing epidemic isolates, it is found that epidemic isolates are sent out with the CV777 virulence genes that twentieth century is separated to the seventies
Larger change is given birth to, so this may is that one of the reason for being failed with the vaccine immunity containing original strain.Therefore screen new
Strain, preparing the popular diarrhoea vaccine of row pig is used to prevent porcine epizootic diarrhea to be very necessary.
The content of the invention
To solve the above problems, the present invention is intended to provide a kind of Porcine epidemic diarrhea virus and its application, the strain heredity
Stable, immunogenicity can be good, the vaccine prepared with it Vaccine effectiveness good for porcine epizootic diarrhea has.
To solve above technical problem, the purpose of the present invention is reached, the present invention is adopted the following technical scheme that：
The invention provides a kind of Porcine epidemic diarrhea virus strain (PEDV-KB2013-4 strains), its microbial preservation numbering is
CGMCC No.12663；Classification And Nomenclature：Porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus,
PEDV)；The preservation time is on 08 23rd, 2016；Depositary institution：The common micro- life of China Committee for Culture Collection of Microorganisms
Thing center；Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
The present invention is also claimed Porcine epidemic diarrhea virus strain PEDV-KB2013-4 strains and is preparing treatment pig popularity
Application in diarrhea virus infection medicine.
Specifically, the present invention is claimed Porcine epidemic diarrhea virus strain PEDV-KB2013-4 strains and is preparing prevention pig
Application in the vaccine of epidemic diarrhea virus infection.Preferably, the vaccine is inactivated vaccine.
Compared with existing commercialized vaccine strain, the Porcine epidemic diarrhea virus PEDV-KB2013- that the present invention is screened
4 plants have immunogenicity good, and antibody produces fast, and the antibody titer of generation is high and length of holding time, and immunizing dose is little, antenatal 42
The inoculation of inactivated vaccine is carried out before it can make the farrowing sow pig that farrows obtain preferable passive immunity, can be effective against
The attack of virulent strain, improves the survival rate of piglet.
Description of the drawings：
Fig. 1：PEDV-KB2013-4 strains are inoculated with CPE picture of the Vero cells in different times, wherein Fig. 1-A, Fig. 1-C, Fig. 1-F
Cell image after Vero cell culture 24h, 36h, 48h of respectively non-virus inoculation；Fig. 1-B, Fig. 1-D, Fig. 1-F are respectively
CPE images after inoculation PEDV-KB2013-4 strain 24h, 36h, 48h.
Fig. 2：PEDV RT-PCR testing results, passage 1：Takara DNA Maker；Passage 2：Positive control；Passage 3：
9th generation virus liquid；Passage 4：Negative control.
Fig. 3：Antibody horizontal testing result after sow immunity difference vaccine.
Specific embodiment：
Embodiment 1：The isolation identification of Porcine epidemic diarrhea virus PEDV-KB2013-4 strains
（1）The separation of Porcine epidemic diarrhea virus PEDV-KB2013-4 strains
In Epidemiological study, censorship diarrhoea Small Intestine of Piglets in Shaanxi pig farm scrapes intestinal mucosa and content, according to 1:5
Ratio（Weight：Volume）Add PBS, multigelation 3 times, centrifuging and taking supernatant, 0.22 μm of membrane filtration that final concentration is added in filtrate
For the pancreatin of 20 μ g/ml, 37 DEG C are processed 1.5 hours.
The Vero cells of monolayer are covered with inoculation according to a conventional method（Three times are washed before inoculation with the PBS of pH7.4）, according to 10%
Ratio virus inoculation, 37 DEG C adsorb 1 hour, supply cell maintenance medium（Pancreatin containing 10 g/ml）, 37 DEG C of incubator cultures.So
Operation blind passage sees whether to produce CPE to the 10th generation, while arranging blanc cell as control.
There are slight CPE changes in cell when blind passage reached for 5 generation, then occurs obvious, stable CPE when reaching for 9 generation
Change, cell circle contracting, granule increases, and poly- heap is in grape cluster sample, and damaging and coming off occur in cell, i.e., isolated PEDV-
KB2013-4 strains.
Figure of description Fig. 1 illustrates CPE picture of the PEDV-KB2013-4 strains inoculation Vero cells in different times, its
Middle Fig. 1-A, Fig. 1-C, Fig. 1-F are matched group, and its image is respectively after Vero cell culture 24h, 36h, 48h of non-virus inoculation
Cell image, as seen from the figure, until Vero cell culture to 48h, cell still keeps fine and close monolayer, only a small amount of cell take off
Fall；Fig. 1-B, Fig. 1-D, Fig. 1-F are respectively inoculated with the CPE images after PEDV-KB2013-4 strain 24h, 36h, 48h, and Fig. 1-B are to connect
The pathological changes that PEDV-KB2013-4 strains 24h is produced are planted, wherein there is a small amount of cell to start circle contracting, come off, Fig. 1-D are inoculation PEDV-
The pathological changes produced after KB2013-4 strain 36h, have more cell to start shedding off, and intercellular space becomes big；Fig. 1-F are inoculations
The pathological changes produced after PEDV-KB2013-4 strain 48h, cell starts to come off in a large number, reaches more than 75%, and the poly- heap of iuntercellular is in Portugal
Grape string sample, shows as typical netted CPE symptoms.
（2）Viral PT-PCR detections
Explanation according to OMEGA test kits carries out the extraction of RNA, product carry out reverse transcription immediately or be stored in -80 DEG C it is standby.
And synthesize detection primer PEDVF：5’-aacggttctattcccgttgatg-3’；PEDVR：5’-
taaatgaagcactttctcactatc-3’。
Take the L of total serum IgE 5.25, the L of 5 × Buffer 2, dNTP (10 mM each) 0.5 L, downstream primer（PEDV-R）1
The L of L, AMV reverse transcription 0.5, the L of RNase inhibitor 0.25, the L of cumulative volume 10.Reaction condition：37 DEG C of 10min, 42 DEG C
Reverse transcription 1h, ice bath 2min.
Take the L of RT products 1,2 × Promega Mix 12.5ul, each 1ul of primer PEDV-F, PEDV- R, autoclaving water
9.5ul, cumulative volume 25ul.Reaction condition：94 DEG C of 3min, 94 DEG C of 30s, 60 DEG C of 45s, 72 DEG C of 45s, 35 circulations, 72 DEG C
7min。
Product after amplification, enters row agarose gel electrophoresis, amplifies purpose band 645bp, referring to Figure of description figure
2, Jing sequencing identifications, the virus purification strain is PEDV.
（3）Virus drop poison is determined
The 10th generation virus is taken, 10 times is carried out with cell maintenance medium and is serially diluted, take 10-4、10-5、10-6、10-7Four dilution factors,
Each dilution factor is inoculated with respectively the hole of 96 porocyte plates 6 for covering with Vero cell monolayers, 100 μ l/ holes, while setting negative control cell
6 holes, 37 DEG C of 5% CO2Incubator culture 72～120 hours, observation of cell pathological changes, cell granulationses increase, circle contracting, cell damage,
Come off and be judged to infection.Simultaneously negative control group cell hole should occur without cytopathy.Using Reed-Muench methods, calculate
TCID50.Jing is determined, and viral level is 5 × 105TCID50/ml。
（4）Animal Orthogonal Rotational Regressive Tests
Take PEDV neutralizing antibodies and be respectively less than 1：3～5 age in days piglet 10 that 8 sow is produced, per the oral generations of 2ml the 10th disease
Poison, observes 7 days, counts the incidence of Pigs Inoculated, and cut open inspection is carried out to test pig, observes pathological change.Scraping morbidity Intestinum Sus domestica glues
Film and content, extract RNA, by the detection primer that the step of embodiment 1 the 2nd is mentioned, carry out RT-PCR detections, and by PCR products
Carry out sequencing.As a result piglet 8/10 falls ill, and morbidity pig shows as diarrhoea, and vomiting occur in indivedual pigs, and cut open inspection observation finds stomach
There is typical pathological change with small intestinal.From morbidity pig intestinal mucosa and content in it is amplifiable go out 645bp fragment, Jing sequences
Row analysis is PEDV S genes.
Jing laboratory isolation identification, is successfully separated to pig popular from the diarrhoea pig small intestine content of Shaanxi pig farm
Diarrhea viruses, are named as PEDV-KB2013-4 strains, and its microbial preservation numbering is CGMCC No.12663；Classification And Nomenclature：Pig is flowed
Row diarrhea viruses (Porcine Epidemic Diarrhea Virus, PEDV)；The preservation time is on 08 23rd, 2016；
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center；Preservation address：The Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica.
Embodiment 2：Vaccine is prepared with PEDV-KB2013-4 strains
（1）The preparation of PEDV vaccines
A. Vero cells are conventionally prepared（Vero cells are purchased from U.S. ATCC）, growth-promoting media is containing 10% new-born calf serum
DMEM, cultivate to cell grow up to monolayer when inoculation PEDV-KB2013-4 it is viral；
B. PEDV-KB2013-4 strains are taken（5×105TCID50/ml）By the Vero cell monolayers that 1% inoculation is grown fine, 37 are put
DEG C absorption 1 hour, discard adsorption liquid, add containing the tryptic DMEM of 10 μ g/mL as maintaining liquid continue cultivate；
C. connect after poison, daily observation 2 times, record cytopathy situation, cytopathy will be harvested up to virus is harvested when more than 80%
Virus at -80 DEG C, multigelation three times, 6000 rpm are centrifuged 20 min, collect supernatant, as virus liquid；
D. virus liquid adds 0.2% formaldehyde, 37 DEG C of inactivations to obtain PEDV-KB2013-4 vaccinogens in 24 hours Jing after 20 times of concentrations
Liquid；
E. adjuvant is prepared, the formula of the adjuvant contains 5% squalane, 1% Oleic acid, 1% polysorbate60,93% 0.005M sodium citrates,
Its compound method is：By the Tween 80 of recipe quantity and sodium citrate buffer solution stirring and evenly mixing, Oleic acid, the spiny dogfish of recipe quantity are added
Alkane, high pressure homogenizer 1200bar carries out 5 circulations, then collects feed liquid, 0.22 μm of PTFE film filtration sterilization.
F. PEDV-KB2013-4 strain vaccines stock solution and adjuvant are pressed into 1.5:1 volume ratio, in gnotobasiss, stirring mixing
Uniformly, that is, porcine epizootic diarrhea inactivated vaccine is obtained.
（2）Vaccine test method and result
3 batches of vaccines are prepared according to the method described above, and lot number is respectively 20131201,20131202,20131203.
2.1 character check 3 batches of inactivated vaccine outward appearance pinkiness emulsion states.
2.2 inactivated vaccines of steriling test 3 batches are according to existing《Republic of China Veterinary Pharmacopoeia》The 3rd annex of version in 2010
Test, T.G, G.P pipe and G.A slant mediums do not observe bacterium colony.
2.3 mycoplasmas check 3 batches of inactivated vaccines according to《Republic of China Veterinary Pharmacopoeia》The 3rd annex of version in 2010
Test, do not it is found that significant change occur in bottle and tubule culture color, the liquid culture of transplanting is in solid medium
Upper nothing " fried egg " shape mycoplasma bacterium colony.
2.4 exogenous viruses check 3 batches of inactivated vaccines according to《Republic of China Veterinary Pharmacopoeia》Version the 3rd is attached within 2010
Record is tested, without swine fever virus, bovine viral diarrhea virus, pig parvoviral, PRV (Pseudorabies virus), rotavirus, pig
Transmissible gastroenteritis virus etc. pollute.Prove that seed culture of viruses is pure.
2.5 safety verifications take the 3 age in days pig 24 that porcine epizootic diarrhea neutralizing antibody, antigen are feminine gender, are randomly divided into
4 groups, 6 per group, 10 part vaccines of intramuscular injection, clinical observation 14 days, equal 100% strong work has no that untoward reaction occurs.
Table 1：3 batches of porcine epizootic diarrhea inactivated vaccine assays
| Inspection project | 20131201 | 20131202 | 20131203 |
| Character is checked | Pink emulsion | Pink emulsion | Pink emulsion |
| Steriling test | Asepsis growth | Asepsis growth | Asepsis growth |
| Mycoplasma is checked | Without mycoplasma growth | Without mycoplasma growth | Without mycoplasma growth |
| Exogenous viruses are checked | Without exogenous viruses pollution | Without exogenous viruses pollution | Without exogenous viruses pollution |
| Safety detection | 100% strong work, has no untoward reaction | 100% strong work, has no untoward reaction | 100% strong work, has no untoward reaction |
The safety test of the Porcine epidemic diarrhea virus PEDV-KB2013-4 strain inactivated vaccines of embodiment 3
1. materials and methods
The single dose experiment of 1.1 pairs of in-pigs
The in-pig 10 that antenatal 5-6 weeks porcine epizootic diarrhea neutralizing antibody, antigen are feminine gender is taken, 2 groups is randomly divided into, often
Group 5, first group of intramuscular injection, 20131201 batches of Porcine epidemic diarrhea virus PEDV-KB2013-4 strains 1 part/heads of inactivated vaccine,
Second group is not injected as a control group, is observed to sows farrowing.
The single dose of 1.2 pairs of in-pigs repeats to test
The in-pig 10 that antenatal 5-6 weeks porcine epizootic diarrhea neutralizing antibody, antigen are feminine gender is taken, 2 groups are randomly divided into,
5 per group, first group of intramuscular injection, 20131201 batches of Porcine epidemic diarrhea virus PEDV-KB2013-4 strains 1 parts of inactivated vaccine/
Head, second group does not carry out injecting immune.1st group of intramuscular injection in 2 weeks, 20131201 crowdes of Porcine epidemic diarrhea virus PEDV- after immunity
1 part/head of KB2013-4 strains inactivated vaccine, observes to sows farrowing.
The overdose test of 1.3 pairs of in-pigs
The in-pig 16 that antenatal 5-6 week porcine epizootic diarrhea neutralizing antibody, antigen are feminine gender is taken, 4 groups are randomly divided into, the
1st, 2,3 groups of difference intramuscular injection, 20131201,20131202,20131203 crowdes of Porcine epidemic diarrhea virus PEDV-KB2013-4
The each 2 part/heads of strain inactivated vaccine, the 4th group does not carry out injection and compares group, observes to sows farrowing.
2. result
2.1 single dose experiment results
Immune group compares by 5 in-pigs with matched group, search for food, drink water, gestation farrowing be showed no exception, injection site is showed no
Untoward reaction.
2.2 single doses repeat result of the test
Immune group compares by 5 in-pigs with matched group, search for food, drink water, gestation farrowing be showed no exception, injection site is showed no
Untoward reaction.
2.3 overdose result of the tests
3 batches of vaccine immunity groups compare by 12 in-pigs with matched group, search for food, drink water, gestation farrowing be showed no exception, injection part
Position is showed no untoward reaction.
Antibody horizontal detection test after the sow of embodiment 4 injection porcine epizootic diarrhea inactivated vaccine
1 materials and methods
The in-pig 20 that antenatal 42 days porcine epizootic diarrhea neutralizing antibodies, antigen are feminine gender is taken, 4 groups is randomly divided into, often
Group 5,20131201 batches of Porcine epidemic diarrhea virus PEDV-KB2013-4 strains 1 part/heads of inactivated vaccine of the 1st group of intramuscular injection, the
2 groups, the 3rd group are injected respectively existing commercialization PEDV-TGEV bigeminy vaccines on market（It is respectively labeled as vaccine A, vaccine B）,
4th group is not injected as a control group.Each group is the 1st week after immunity, adopt within the 2nd week, the 3rd week, the 4th week, the 5th week, the 6th week
Blood, separating serum carries out virus neutralization tests.
2 results
Test result indicate that, the vaccine prepared by the present invention produces antibody soon, and the antibody titer for producing is high, length of holding time,
Referring to Figure of description 3.
The potency test of the Porcine epidemic diarrhea virus PEDV-KB2013-4 strain inactivated vaccines of embodiment 5
1 materials and methods
The in-pig 30 that antenatal 5-6 weeks porcine epizootic diarrhea neutralizing antibody, antigen are feminine gender is taken, 3 groups is randomly divided into, often
Group 10,20131201 batches of Porcine epidemic diarrhea virus PEDV-KB2013-4 strains 1 part/heads of inactivated vaccine of the 1st group of intramuscular injection,
1 part/head of attenuated vaccine prepared by the 2nd group of injection PEDV attenuated vaccine strains CV777（It is commercial, 1ml/ head parts, viral level
105TCID50/ml）, the 3rd group of same amount of normal saline of injection as a control group, after sows farrowing, 3 ages in days, 7 ages in days, 14 days
Age piglet respectively takes 24, is randomly divided into 3 groups, and 8 per group, the strong poison 5ml of oral porcine epizootic diarrhea observes piglet after counteracting toxic substances
Clinical manifestation.
2 results
The piglet that 1st group of PEDV-KB2013-4 strains inactivated vaccine immunity sow is produced, after 3 age in days counteracting toxic substances, 8 immune piglet sucklings,
Spirit, feces no abnormality seen, are good for and live；The piglet that 2nd group of PEDV attenuated vaccine strain CV777 attenuated vaccine immunity sow is produced, 3
After age in days counteracting toxic substances, 3 show typical porcine epizootic diarrhea symptom, and then dead, 5 piglet sucklings, spirit, feces have no
It is abnormal, be good for and live, i.e., 5/8 shows PED symptoms；After 3rd group of 8 control piglet counteracting toxic substances, the typical pig of 8/8 performance is popular
Symptom of diarrhea, 8/8 is dead.
After 7 age in days counteracting toxic substances, the 1st group of 8 immune piglet sucklings, spirit, feces no abnormality seens are good for and are lived；The 8 of 2nd group
Head piglet, after 8 matched group piglet counteracting toxic substances, 3 show PED symptoms and die of illness to die, i.e., mortality rate is 3/8；3rd group of piglet is complete
Portion shows typical porcine epizootic diarrhea symptom, and 8/8 is dead.
After 14 age in days counteracting toxic substances, the 1st group of 8 immune piglet sucklings, spirit, feces no abnormality seens are good for and are lived；The 8 of 2nd group
Head piglet, after 8 matched group piglet counteracting toxic substances, 2 show PED symptoms and die of illness to die, i.e., mortality rate is 2/8；3rd group of 8 controls
After group piglet counteracting toxic substances, the typical porcine epizootic diarrhea symptom of 8/8 performance, 5/8 is dead.
The piglet counteracting toxic substances protective rate that the immune sow of table 2 is produced
As shown in Table 2, the PEDV-KB2013-4 strains that the present invention is obtained have good immunogenicity, and it is to animal
Protective rate close 100%, higher than commercially available attenuated vaccine.
Vaccine immunogenicity prepared by the Porcine epidemic diarrhea virus PEDV-KB2013-4 strains that the present invention is screened is good, resists
Body produces fast, and the antibody titer of generation is high and length of holding time, and immunizing dose is little, and carrying out inoculation before antenatal 42 days can make
The farrowing sow pig that farrows obtains preferable passive immunity, can be effective against the attack of virulent strain, improves the survival rate of piglet.
Claims (7)
1. a kind of Porcine epidemic diarrhea virus strain PEDV-KB2013-4 strains, its microbial preservation numbering is CGMCC
No.12663；Classification And Nomenclature：Porcine epidemic diarrhea virus Porcine Epidemic Diarrhea Virus, PEDV；Preservation
Time is on 08 23rd, 2016；Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center；Preservation
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
2. the Porcine epidemic diarrhea virus strain PEDV-KB2013-4 strains described in claim 1 are preparing prevention or are treating pig stream
Application in row diarrhea virus infection medicine.
3. the Porcine epidemic diarrhea virus strain PEDV-KB2013-4 strains described in claim 1 are preparing prevention pig epidemic
Application in the vaccine for rushing down.
4. application according to claim 3, it is characterised in that the vaccine is inactivated vaccine, it is by adjuvant and inactivation
PEDV-KB2013-4 strain vaccine stock solutions are prepared from.
5. application according to claim 3, it is characterised in that described adjuvant is by 5% spiny dogfish in terms of weight/mass percentage composition
Alkane, 1% Oleic acid, 1% polysorbate60,93% 0.005M sodium citrates composition.
6. application according to claim 3, it is characterised in that the vaccine is prepared via following methods：
A. Vero cells are conventionally prepared, growth-promoting media is the DMEM containing 10% new-born calf serum, cultivates to cell and grows up to
PEDV-KB2013-4 is inoculated with during monolayer viral；
B. content is taken for 5 × 105TCID50The Vero monolayers that the PEDV-KB2013-4 strains of/ml grow fine by 1% inoculation
Cell, puts 37 DEG C and adsorbs 1 hour, discards adsorption liquid, adds and continues as maintaining liquid containing the tryptic DMEM of 10 μ g/mL
Culture；
C. connect after poison, daily observation 2 times, record cytopathy situation, cytopathy will be received up to virus is harvested when more than 80%
At -80 DEG C, multigelation three times, 6000 rpm are centrifuged 20 min to the virus for obtaining, and collect supernatant, as virus liquid；
D. virus liquid adds 0.2% formaldehyde, 37 DEG C of inactivations to obtain PEDV-KB2013-4 vaccinogens in 24 hours Jing after 20 times of concentrations
Liquid；
E. adjuvant is prepared, the formula of the adjuvant contains 5% squalane, 1% Oleic acid, 1% polysorbate60,93% 0.005M sodium citrates,
Its compound method is：By the Tween 80 of recipe quantity and sodium citrate buffer solution stirring and evenly mixing, Oleic acid, the spiny dogfish of recipe quantity are added
Alkane, high pressure homogenizer 1200bar carries out 5 circulations, then collects feed liquid, 0.22 μm of PTFE film filtration sterilization；
F. PEDV-KB2013-4 strain vaccines stock solution and adjuvant are pressed into 1.5:1 volume ratio, in gnotobasiss, stirring mixing is equal
It is even, that is, the vaccine is obtained.
7. the preparation-obtained vaccine of claim 6 is used to prevent Porcine epidemic diarrhea virus to infect, it is characterised in that by flesh
The mode of meat injection is used.
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