CN106636011B - A kind of Porcine epidemic diarrhea virus and its application - Google Patents
A kind of Porcine epidemic diarrhea virus and its application Download PDFInfo
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Abstract
A kind of 4 plants of Porcine epidemic diarrhea virus strain PEDV KB2013, microbial preservation number 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：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.
Description
Technical field
The invention belongs to biotechnologies, particularly belong to field of biological pharmacy for animals, are related to a kind of pig epidemic diarrhea
Virus and its application.
Background technology
Pig epidemic diarrhea（Porcine Epidemic Diarrhea, PED）It is by Porcine epidemic diarrhea virus
（Porcine Epidemic Diarrhea Virus, PEDV）Cause a kind of acute, viral enteric infectious disease of contact,
Illness is to vomit, based on watery diarrhea, loss of appetite and dehydration, is common chitling road transmission disease.Pig epidemic diarrhea
Virus（PEDV）It is coronaviridae coronavirus genus member, Porcine epidemic diarrhea virus（PEDV）After mouth and nose infect directly into
Enter small intestine, in Proliferation of Rat Intestinal Crypt Cells, cause intestinal villi absorptive epithelium cytopathy, damage, come off and wither with intestinal villi
Contracting causes enteral enzymatic activity to reduce, disturbance of absorptive function, and osmotic pressure increases in enteron aisle, causes osmotic diarrhea, ultimately causes
Fat reduces feed absorption rate.PEDV has seriously affected the pig breeding industry in China, and infectivity is strong, harmfulness is big, is to influence entirely
One of most important virus of ball pig breeding industry, the death rate is up to 100% after suckling pig infection, and sow is often with band poison without showing disease
Shape form occurs.China saved outburst PED more in recent years, and serious economic loss is caused to pig breeding industry.
It there is no the specific medicament for the treatment of pig epidemic diarrhea at present, conventional therapy is ineffective, therefore still with vaccine prevention
Based on, and the success or not of Strain and its culture is the key factor of vaccine development.At present, the external vaccine that uses is mainly
South Korea's low virulent strain, the country mainly pig epidemic diarrhea of Harbin veterinary institute development, transmissible gastroenteritis of swine bigeminy are gone out
Live seedling etc., the strain used in this vaccine are CV777 plant of Porcine epidemic diarrhea virus PEDV, and the isolation of strains age is more early, logical
The genetic analysis to existing popular strain is crossed, it is found that popular strain is 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 of being failed with the vaccine immunity containing original strain.Therefore it screens new
Strain, it is very necessary to prepare row pig prevalence diarrhea vaccine for preventing pig epidemic diarrhea.
Invention content
To solve the above problems, the present invention is intended to provide a kind of Porcine epidemic diarrhea virus and its application, strain heredity
Stablize, immunogenicity can be good, has good Vaccine effectiveness for pig epidemic diarrhea with the vaccine of its preparation.
For solution more than technical problem, reach the purpose of the present invention, the present invention adopts the following technical scheme that：
The present invention provides a kind of Porcine epidemic diarrhea virus strain (PEDV-KB2013-4 plants), microbial preservation is compiled
Number be 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：China Committee for Culture Collection of Microorganisms's commonly micro- life
Object center；Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
The present invention is also claimed PEDV-KB2013-4 plants of Porcine epidemic diarrhea virus strain and is preparing treatment pig popularity
Application in diarrhea virus infection drug.
Specifically, the present invention is claimed PEDV-KB2013-4 plants of Porcine epidemic diarrhea virus strain 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- of the invention screened
4 plants good with immunogenicity, and antibody generates soon, and the antibody titer height of generation and length of holding time, immunizing dose is small, antenatal 42
Inoculation of inactivated vaccine is carried out before it can make farrowing sow farrow the preferable passive immunity of pig acquisition, can be effective against
The attack of velogen strain improves the survival rate of piglet.
Description of the drawings：
Fig. 1：PEDV-KB2013-4 plants of inoculation Vero cells are in the CPE pictures of different times, wherein Fig. 1-A, Fig. 1-C, figure
1-F is respectively cell image of the Vero cell culture of non-virus inoculation for 24 hours, after 36h, 48h；Fig. 1-B, Fig. 1-D ,-F points of Fig. 1
The PEDV-KB2013-4 plants of CPE images for 24 hours, after 36h, 48h Wei be inoculated with.
Fig. 2：PEDV RT-PCR testing results, channel 1：Takara DNA Maker；Channel 2：Positive control；Channel 3：
9th generation virus liquid；Channel 4：Negative control.
Fig. 3：The antibody level testing result after different vaccines is immunized in sow.
Specific embodiment：
Embodiment 1：The separation identification that PEDV-KB2013-4 plants of Porcine epidemic diarrhea virus
（1）The separation that PEDV-KB2013-4 plants of Porcine epidemic diarrhea virus
In epidemiological survey, inspection diarrhea Small Intestine of Piglets in Shaanxi pig farm scrapes intestinal mucosa and content, according to 1:
5 ratio（Weight：Volume）Add in PBS, multigelation 3 times, centrifuging and taking supernatant, 0.22 μm of membrane filtration added in eventually in filtrate
The pancreatin of a concentration of 20 μ g/ml, 37 DEG C are handled 1.5 hours.
The Vero cells of individual layer are covered in inoculation according to a conventional method（It is washed three times with the PBS of pH7.4 before inoculation）, according to 10%
Ratio virus inoculation, 37 DEG C adsorb 1 hour, supply cell maintenance medium（Pancreatin containing 10 μ g/ml）, 37 DEG C of incubator cultures.So
Blind passage is operated to the 10th generation, sees whether to generate CPE, while set blanc cell as control.
There are slight CPE variations in cell when blind passage reached for 5 generation, then occurs CPE that is apparent, stablizing when reaching for 9 generation
Variation, cell circle contracting, particle increase, and poly- heap is in grape cluster sample, and cell occurs damaging and come off, i.e., isolated PEDV-
KB2013-4 plants.
Figure of description Fig. 1 illustrates CPE picture of the PEDV-KB2013-4 plants of inoculation Vero cells in different times,
Middle Fig. 1-A, Fig. 1-C, Fig. 1-F be control group, image be respectively non-virus inoculation Vero cell culture for 24 hours, after 36h, 48h
Cell image, as seen from the figure, until Vero cell culture to 48h, cell still keeps fine and close individual layer, and only a small amount of cell takes off
It falls；Fig. 1-B, Fig. 1-D, Fig. 1-F are respectively to be inoculated with the PEDV-KB2013-4 plants of CPE images for 24 hours, after 36h, 48h, and Fig. 1-B are to connect
The PEDV-KB2013-4 plants of lesions generated for 24 hours of kind, wherein there is a small amount of cell to start circle contracting, come off, Fig. 1-D are inoculation PEDV-
The lesion generated after KB2013-4 plants of 36h, has more cell to start shedding off, and intercellular gap becomes larger；Fig. 1-F are inoculations
The lesion generated after PEDV-KB2013-4 plants of 48h, cell start largely to come off, and reach 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
According to OMEGA kits illustrate carry out RNA extraction, product carry out reverse transcription immediately or be stored in -80 DEG C it is standby
With.And synthesize detection primer PEDVF：5’-aacggttctattcccgttgatg-3’；PEDVR：5’-
taaatgaagcactttctcactatc-3’。
Take total serum IgE 5.25 μ L, 5 × Buffer 2 μ L, dNTP (10 mM each) 0.5 μ L, downstream primer（PEDV-R）1
μ L, AMV reverse transcriptase 0.5 μ L, 0.25 μ L of RNase inhibitor, 10 μ L of total volume.Reaction condition：37 DEG C of 10min, 42 DEG C
Reverse transcription 1h, ice bath 2min.
Take RT products 1 μ L, 2 × Promega Mix 12.5ul, primer PEDV-F, each 1ul of PEDV- R, high pressure sterilization water
9.5ul, total 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 cycles, 72 DEG C
7min。
Product after amplification into row agarose gel electrophoresis, amplifies purpose band 645bp, referring to Figure of description figure
2, it is identified through sequencing, the virus purification strain is PEDV.
（3）Virus drop poison measures
It took for the 10th generation viral, carries out 10 times with cell maintenance medium and be serially diluted, take 10-4、10-5、10-6、10-7Four dilutions
Degree, each dilution is inoculated with 96 porocyte plates, 6 hole for covering with Vero cell monolayers, 100 μ l/ holes respectively, while sets negative control
6 hole of cell, 37 DEG C of 5% CO2Cytopathy is observed in incubator culture 72~120 hours, and cell granulations increase, circle contracting, cell
Breakage comes off and is judged to infect.Cytopathy should not occur in negative control group cell hole simultaneously.Using Reed-Muench methods, calculate
TCID50.After measured, viral level is 5 × 105TCID50/ml。
（4）Animal Orthogonal Rotational Regressive Tests
PEDV neutralizing antibodies is taken to be respectively less than 1：8 produced 3~5 age in days piglet of sow 10, every oral the 10th generation of 2ml
Virus is observed 7 days, counts the incidence of Pigs Inoculated, is carried out dissect to test pig, is observed pathological change.Scraping morbidity chitling
Mucous membrane and content extract RNA, by the detection primer that the 2nd step of embodiment 1 is mentioned, carry out RT-PCR detections, and PCR is produced
Object carries out sequencing.As a result piglet 8/10 falls ill, and morbid pig shows as diarrhea, and indivedual pigs vomit, and dissect observation is found
There is typical pathological change in stomach and small intestine.From the intestinal mucosa and content of morbid pig it is amplifiable go out 645bp segment, warp
Sequence analysis is PEDV S genes.
It detaches and identifies through laboratory, be successfully separated to pig popularity from the diarrhea chitterlings content of Shaanxi pig farm
Diarrhea virus is named as PEDV-KB2013-4 plants, and microbial preservation number is CGMCC No.12663；Classification And Nomenclature：Pig is flowed
Row diarrhea virus (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 plants
（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 bovine
The DMEM of serum is cultivated and PEDV-KB2013-4 viruses is inoculated with when growing up to individual layer to cell；
B. PEDV-KB2013-4 strains are taken（5×105TCID50/ml）The Vero cell monolayers to grow fine are inoculated with by 1%,
It puts 37 DEG C to adsorb 1 hour, discards adsorption liquid, add the DMEM for containing 10 μ g/mL trypsase and continue to cultivate as maintaining liquid；
C. it is daily to observe 2 times after connecing poison, cytopathy situation is recorded, cytopathy, will up to virus is harvested when more than 80%
For the virus of harvest at -80 DEG C, three times, 6000 rpm centrifuge 20 min to multigelation, collect supernatant, as virus liquid；
D. virus liquid adds in 0.2% formaldehyde, 37 DEG C of inactivations and obtains PEDV-KB2013-4 vaccines in 24 hours after 20 times of concentrations
Stoste；
E. adjuvant is prepared, the formula of the adjuvant is containing 5% saualane, 1% oleic acid, 1% polysorbate60,93% 0.005M citric acids
Sodium, preparation method are：The Tween 80 of recipe quantity is stirred and evenly mixed with sodium citrate buffer solution, add recipe quantity oleic acid,
Saualane, high pressure homogenizer 1200bar carry out 5 cycles, then collect feed liquid, 0.22 μm of PTFE film filtration sterilization.
F. PEDV-KB2013-4 strain vaccines stoste and adjuvant are pressed 1.5:1 volume ratio in gnotobasis, is stirred
Uniformly, pig epidemic 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 characters examine 3 batches of inactivated vaccine appearance pinkiness emulsion states.
2.2 inactivated vaccines of steriling test 3 batches are according to existing《Republic of China Veterinary Pharmacopoeia》Version third portion annex in 2010
It tests, T.G, G.P pipe and G.A slant mediums do not observe bacterium colony.
2.3 mycoplasmas examine 3 batches of inactivated vaccines according to《Republic of China Veterinary Pharmacopoeia》Version third portion annex in 2010
It tests, does not find 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 virus examine 3 batches of inactivated vaccines according to《Republic of China Veterinary Pharmacopoeia》Version third portion is attached within 2010
Record is tested, without swine fever virus, bovine viral diarrhea virus, pig parvoviral, porcine pseudorabies virus, rotavirus, pig
The pollutions such as infectious gastroenteritis virus.Prove that seed culture of viruses is pure.
It is negative 3 age in days pig 24 that 2.5 safety verifications, which take pig epidemic diarrhea neutralizing antibody, antigen, is randomly divided into
4 groups, every group 6,10 part vaccines of intramuscular injection, clinical observation 14 days, equal 100% is good for work, has no adverse reaction.
Table 1：3 batches of pig epidemic diarrhea inactivated vaccine inspection results
| Inspection project | 20131201 | 20131202 | 20131203 |
| Character is examined | Pink emulsion | Pink emulsion | Pink emulsion |
| Steriling test | Asepsis growth | Asepsis growth | Asepsis growth |
| Mycoplasma is examined | No mycoplasma growth | No mycoplasma growth | No mycoplasma growth |
| Exogenous virus is examined | No exogenous virus pollution | No exogenous virus pollution | No exogenous virus pollution |
| Safety detection | 100% strong work, has no adverse reaction | 100% strong work, has no adverse reaction | 100% strong work, has no adverse reaction |
The safety test of 3 PEDV-KB2013-4 plants of inactivated vaccines of Porcine epidemic diarrhea virus of embodiment
1. materials and methods
The single dose experiment of 1.1 pairs of pregnant sows
It is negative pregnant sow 10 to take antenatal 5-6 weeks pig epidemic diarrhea neutralizing antibody, antigen, is randomly divided into 2
Group, every group 5, first group of PEDV-KB2013-4 plants of Porcine epidemic diarrhea virus inactivated vaccine 1 of intramuscular injection 20131201 batches
Part/head, second group, without injecting as a control group, is observed to sows farrowing.
The single dose of 1.2 pairs of pregnant sows repeats to test
It is negative pregnant sow 10 to take antenatal 5-6 weeks pig epidemic diarrhea neutralizing antibody, antigen, is randomly divided into 2
Group, every group 5, first group of PEDV-KB2013-4 plants of Porcine epidemic diarrhea virus inactivated vaccine 1 of intramuscular injection 20131201 batches
Part/head, second group without injecting immune.1st group of intramuscular injection in 2 weeks, 20131201 batches of Porcine epidemic diarrhea virus after immune
1 part/head of PEDV-KB2013-4 plants of inactivated vaccines, is observed to sows farrowing.
The overdose experiment of 1.3 pairs of pregnant sows
It is negative pregnant sow 16 to take antenatal 5-6 weeks pig epidemic diarrhea neutralizing antibody, antigen, is randomly divided into 4
Group, the 1st, 2,3 group of difference intramuscular injection 20131201,20131202,20131203 crowdes of Porcine epidemic diarrhea virus PEDV-
Each 2 part/heads of KB2013-4 plants of inactivated vaccines, the 4th group compares group without injection, observes to sows farrowing.
2. result
2.1 single dose experiment results
Compared with the control group, feeding, drinking-water, gestation farrowing are showed no exception to 5 pregnant sows of immune group, and injection site is equal
Have no adverse reaction.
2.2 single doses repeat result of the test
Compared with the control group, feeding, drinking-water, gestation farrowing are showed no exception to 5 pregnant sows of immune group, and injection site is equal
Have no adverse reaction.
2.3 overdose result of the tests
Compared with the control group, feeding, drinking-water, gestation farrowing are showed no exception to 3 batches of vaccine immunity groups, 12 pregnant sows, note
It penetrates position and is showed no adverse reaction.
Antibody level detection experiment after 4 sow of embodiment injection pig epidemic diarrhea inactivated vaccine
1 materials and methods
It is negative pregnant sow 20 to take antenatal 42 days pig epidemic diarrhea neutralizing antibodies, antigen, is randomly divided into 4
Group, every group 5,1 part of the 1st group of PEDV-KB2013-4 plants of the Porcine epidemic diarrhea virus inactivated vaccine of intramuscular injection 20131201 batches/
Head, the 2nd group, the 3rd group is injected existing commercialization PEDV-TGEV bigeminy vaccines in the market respectively（It is respectively labeled as vaccine A, epidemic disease
Seedling B）, the 4th group without injecting as a control group.Each group is 1st week after immune, the 2nd week, the 3rd week, the 4th week, the 5th week, the 6th
Week blood sampling, separation serum carry out virus neutralization tests.
2 results
The experimental results showed that the vaccine prepared by the present invention, generate that antibody is fast, and the antibody titer generated is high, during maintenance
Between it is long, referring to Figure of description 3.
The potency test of 5 PEDV-KB2013-4 plants of inactivated vaccines of Porcine epidemic diarrhea virus of embodiment
1 materials and methods
It is negative pregnant sow 30 to take antenatal 5-6 weeks pig epidemic diarrhea neutralizing antibody, antigen, is randomly divided into 3
Group, every group 10, the 1st group of PEDV-KB2013-4 plants of the Porcine epidemic diarrhea virus inactivated vaccine 1 of intramuscular injection 20131201 batches
Part/head, 1 part/head of attenuated vaccine prepared by the 2nd group of injection PEDV attenuated vaccine strains CV777（It is purchased in market, 1ml/ parts, virus
Content 105TCID50/ml）, the 3rd group of same amount of physiological saline of injection as a control group, after sows farrowing, 3 ages in days, 7 ages in days,
14 age in days piglets respectively take 24, are randomly divided into 3 groups, every group 8, son after poison is attacked in poison 5ml, observation to oral pig epidemic diarrhea by force
The clinical manifestation of pig.
2 results
The produced piglet of sow is immunized in 1st group of PEDV-KB2013-4 plants of inactivated vaccines, after 3 ages in days attack poison, 8 immune piglets
Lactation, spirit, excrement no abnormality seen, it is strong to live；2nd group of PEDV attenuated vaccine strain CV777 attenuated vaccine immunity sow is produced
Piglet, after 3 ages in days attack poison, 3 show typical pig epidemic diarrhea symptom, then dead, 5 piglet lactations, spirit, excrement
Just no abnormality seen, strong to live, i.e., 5/8 shows PED symptoms；After 3rd group of 8 control piglets attack poison, the 8/8 typical pig of performance
Epidemic diarrhea symptom, 8/8 is dead.
After 7 ages in days attack poison, the 1st group of 8 immune piglet lactations, spirit, excrement no abnormality seens are strong to live；The 8 of 2nd group
Head piglet, after 8 control group piglets attack poison, 3 show PED symptoms and die of illness to die, i.e., the death rate is 3/8；3rd group of piglet is complete
Portion shows typical pig epidemic diarrhea symptom, and 8/8 is dead.
After 14 ages in days attack poison, the 1st group of 8 immune piglet lactations, spirit, excrement no abnormality seens are strong to live；The 8 of 2nd group
Head piglet, after 8 control group piglets attack poison, 2 show PED symptoms and die of illness to die, i.e., the death rate is 2/8；3rd group of 8 controls
After group piglet attacks poison, the 8/8 typical pig epidemic diarrhea symptom of performance, 5/8 is dead.
Table 2 is immunized the produced piglet of sow and attacks malicious protective rate
As shown in Table 2, the PEDV-KB2013-4 strains that the present invention obtains have good immunogenicity, to dynamic
The protective rate of object is close to 100%, higher than commercially available attenuated vaccine.
The vaccine immunogenicity of PEDV-KB2013-4 plants of preparations of Porcine epidemic diarrhea virus that the present invention is screened is good, resists
Body generates fast, the antibody titer height of generation and length of holding time, and immunizing dose is small, and inoculation was carried out before antenatal 42 days to be made
The farrowing sow pig that farrows obtains preferable passive immunity, can be effective against the attack of velogen strain, improve the survival rate of piglet.
Claims (6)
1. a kind of PEDV-KB2013-4 plants of Porcine epidemic diarrhea virus strain, microbial preservation number 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. PEDV-KB2013-4 plants of Porcine epidemic diarrhea virus strain described in claim 1 is preparing prevention or treatment pig stream
Application in row diarrhea virus infection drug.
3. PEDV-KB2013-4 plants of Porcine epidemic diarrhea virus strain described in claim 1 is preparing prevention pig epidemic
Application in the vaccine rushed down.
4. application according to claim 3, which is characterized in that the vaccine is inactivated vaccine, by adjuvant and inactivation
PEDV-KB2013-4 strain vaccine stostes are prepared.
5. application according to claim 3, which is characterized in that the adjuvant is by 5% spiny dogfish in terms of mass percentage
Alkane, 1% oleic acid, 1% polysorbate60,93% 0.005M sodium citrates composition.
6. application according to claim 3, which is characterized in that the vaccine is prepared via following methods：
A. Vero cells are conventionally prepared, growth-promoting media is the DMEM containing 10% newborn bovine serum, cultivates to cell and grows up to
PEDV-KB2013-4 viruses are inoculated with during individual layer；
B. it is 5 × 10 to take content5TCID50The Vero individual layers that the PEDV-KB2013-4 strains of/ml grow fine by 1% inoculation
Cell is put 37 DEG C and is adsorbed 1 hour, discards adsorption liquid, adds the DMEM for containing 10 μ g/mL trypsase and continues as maintaining liquid
Culture；
C. it is daily to observe 2 times after connecing poison, cytopathy situation is recorded, cytopathy will be received up to virus is harvested when more than 80%
For the virus obtained at -80 DEG C, three times, 6000 rpm centrifuge 20 min to multigelation, collect supernatant, as virus liquid；
D. virus liquid adds in 0.2% formaldehyde, 37 DEG C of inactivations and obtains PEDV-KB2013-4 vaccinogens in 24 hours after 20 times of concentrations
Liquid；
E. adjuvant is prepared, the formula of the adjuvant contains 5% saualane, 1% oleic acid, 1% polysorbate60,93% 0.005M sodium citrates,
Its preparation method is：The Tween 80 of recipe quantity with sodium citrate buffer solution is stirred and evenly mixed, adds oleic acid, the spiny dogfish of recipe quantity
Alkane, high pressure homogenizer 1200bar carry out 5 cycles, then collect feed liquid, 0.22 μm of PTFE film filtration sterilization；
F. PEDV-KB2013-4 strain vaccines stoste and adjuvant are pressed 1.5:1 volume ratio in gnotobasis, is stirred
It is even, obtain the vaccine.
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| CN108588036A (en) * | 2018-04-23 | 2018-09-28 | 南京农业大学 | Porcine epidemic diarrhea virus and its application |
| CN109810950A (en) * | 2019-03-15 | 2019-05-28 | 扬州大学 | The pig epidemic diarrhea virus attenuated strain of one plant of G2b hypotype and its application |
| CN111308071B (en) * | 2020-03-13 | 2021-06-18 | 长春生物制品研究所有限责任公司 | Desorption agent for antigen in aluminium salt desorption type vaccine and antigen content detection method |
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