CN107815440A - Separation, the preparation and application of inactivated vaccine of pig Delta coronavirus strain - Google Patents
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Abstract
The invention belongs to animal virology and epizootiology technical field.The preparation and application of separation, inactivated vaccine more particularly to pig Delta coronavirus strain.Disclose the preparation and its application of the isolated culture method, inactivated vaccine of pig Delta coronavirus strain.2014 plants of the pig Delta coronavirus CHN HN that the present invention separates are deposited in China typical culture collection center, and deposit number is：CCTCC NO:V201650.The inactivated vaccine prepared using the pig Delta coronavirus of preservation of the present invention has good immunogenicity, and piglet can be induced to produce the neutralizing antibody of higher level, and can be effectively protected the attack of immune piglet resistance pig Delta coronavirus.
Description
Technical field
The invention belongs to animal virology and epizootiology technical field.More particularly to pig Delta coronavirus strain
Separation, the preparation and application of inactivated vaccine.
Background technology
Pig Delta coronavirus (porcine deltacoronavirus, PDCoV) is a kind of newfound chitling road
Coronavirus, it can cause piglet that diarrhea disease occurs.2011, Hong Kong scholar was carrying out molecular flow to birds and mammal
Row disease detects PDCoV in swine excrement first when learning investigation, but to its it is pathogenic do not furtherd investigate (Woo,
P.C.,et al.,Discovery of seven novel Mammalian and avian coronaviruses in the
genus deltacoronavirus supports bat coronaviruses as the gene source of
alphacoronavirus and betacoronavirus and avian coronaviruses as the gene
source of gammacoronavirus and deltacoronavirus.J Virol,2012,86(7):3995-
4008).At 2 months 2014, large-scale diarrhea disease occurred for Ohio, USA selected swine farms, and testing result shows PDCoV
For the positive, and subsequent animal returns experiment confirms that PDCoV can cause severe diarrhea (Wang, L., et in piglet really
al,Detection and genetic characterization of deltacoronavirus in pigs,Ohio,
USA,2014.Energ Infect Dis,2014,20(7):1227-1230；Jung et al.,Pathogenicity of
2porcine deltacoronavirus strains in gnotobiotic pigs.Energ Infect Dis,2015,
21(4):650-654).In view of influence of the diarrhea disease to U.S.'s pig aquaculture is serious, and on June 5th, 2014, United States Department of Agriculture's hair
The government decree of cloth federation, the cause diarrhoea enteric coronavirus virus case report mechanism including PEDV, PDCoV is set up, and appropriated funds
26200000 dollars for coherent detection preventing and controlling (www.aasv.org).The U.S., Canada, South Korea, Mexico, Thailand at present
The country such as state, Laos reports PDCoV appearance (Lee, J.H., et al., Detection and Phylogenetic successively
Analysis of Porcine Deltacoronavirus in Korean Swine Farms,2015.Transbound
Emerg Dis,2016,63(3):248-252；Wang,L.,et al,Porcine coronavirus HKU15detected
in 9US states,2014.Energ Infect Dis,2014,20(9):1594-1595；Homwong,N.,et al.,
Characterization and evolution of porcine deltacoronavirus in the United
States.Prev Vet Med,2016,123:168-74；Janetanakit,T.,et al.,Porcine
Deltacoronavirus,Thailand,2015.Energ Infect Dis,2016,22(4):757-759；
Lorsirigool,A.,.et al,The first detection and full-length genome sequence of
porcine deltacoronavirus isolated in Lao PDR Arch Virol,2016,161:2909).Nearest state
Interior different research unit also reports prevalences of the PDCoV in the different provinces of China in succession, and it is left that total positives rate can reach 30%
The right side, and PDCoV and Porcine epidemic diarrhea virus (PEDV), transmissible gastro-enteritis virus (TGEV) coinfection situation compared with
Generally (Chen, F., et al., Full-Length Genome Characterization of Chinese Porcine
Deltacoronavirus Strain CH/SXD1/2015.Genome Announcements,2015,3(5)；Song,D.,
et al.,Newly Emerged Porcine Deltacoronavirus Associated With Diarrhoea in
Swine in China:Identification,Prevalence and Full-Length Genome Sequence
Analysis.Transbound Emerg Dis,2015,62(6):575-580；Dong,N.,et al.,Porcine
Deltacoronavirus in Mainland China.Energ Infect Dis,2015,21(12):2254-2255.)。
Typical intestines problem can be caused after PDCoV infection piglets, cardinal symptom is vomiting, diarrhoea, and with food
It is intended to decline and (Ma, Y., et al., Origin, evolution, and virulence of porcine such as tired mind
deltacoronaviruses in the United States.MBio,2015,6(2):e00064；Hu,H.,et al.,
Isolation and characterization of porcine deltacoronavirus from pigs with
diarrhea in the United States.J Clin Microbiol,2015,53(5):1537-1548).Current people
It is pathogenic that work animalbioassay confirms that this virus is respectively provided with to the piglet of the age in days of 5 age in days -21, and with the increase of piglet age in days
Symptom gradually mitigates caused by virus, and the clinical report of nearest Thailand shows that this virus equally can be in sow and growing and fattening pigs in addition
In cause serious diarrhoea, and cause it is dead (Janetanakit, T., et al., Porcine Deltacoronavirus,
Thailand,2015.Energ Infect Dis,2016,22(4):757-759).The metainfective chitling roads of PDCoV can produce
The raw typical cytopathic based on enterocyte enlargement and vacuolation, and lesion is mainly in jejunum and ileum, artificial challenge's
Piglet can be more than 2 weeks by excrement toxin expelling, have stronger infectiousness.
Currently for pig enteric coronavirus virus such as PEDV, TGEV preventing and treating mainly by inactivated vaccine and causes weak live vaccine
Immunoprotection.And there is no particularly effective prevention and controls also currently for the pig Delta coronavirus of kainogenesis, therefore
The development of PDCoV vaccines is extremely urgent.
The content of the invention
First purpose of the present invention is for problem described in existing background technology, there is provided one plant of immunogenicity is stronger
Pig Delta coronavirus (porcine deltacoronavirus, abbreviation PDCoV) separation strains.
Second purpose of the invention is that providing pig Delta coronavirus prepared by a pig Delta coronavirus goes out
Live vaccine.
Third object of the present invention is the application for providing pig Delta coronavirus inactivated vaccine.
Realize that technical scheme is as described below：
Isolated one plant of viral candidates strain during the present invention falls ill pig farm pathological material of disease from Henan Province, is named as pig moral after identification
You are tower coronavirus (porcine deltacoroanvirus) abbreviation PDCoV, and the strain is named as pig Delta by applicant
Coronavirus CHN-HN-2014 strains, porcine deltacoroanvirus CHN-HN-2014 strains, in September in 2016 13 days
The China typical culture collection center preservation of censorship China Wuhan Wuhan Universitys, deposit number are：CCTCC NO:V201650.
The strain whole genome sequence has been committed to GenBank databases, and GenBank searching numbers are KT336560.1.
Biological function verification experiment shows that PDCoV CHN-HN-2014 strains of the invention are inoculated with energy after LLC-PK1 cells
Typical cells lesion is enough produced, and breeds on LLC-PK1 cells stable, virus titer is up to 108.0TCID50/ more than ml.
PDCoV CHN-HN-2014 strains are enlarged propagation on LLC-PK1 cells, collect virus liquid, are added dense eventually
Spend and inactivated for 0.1% formaldehyde, by the antigen inactivated and Montanide ISA 201 (SEPPIC) adjuvant according to quality
Than for 1：1 is mixed and is emulsified pig Delta coronavirus (PDCoV) inactivated vaccine is prepared.
The positive effect of the present invention is：
(1) inactivated vaccine prepared by pig Delta coronavirus CHN-HN-2014 strains of the invention has good safety
Performance, the adverse reaction of immune piglet will not be caused.
(2) inactivated vaccine prepared by pig Delta coronavirus CHN-HN-2014 strains of the present invention has preferable immunogene
Property, piglet immunological the present invention vaccine after can 21 days produce higher level serum neutralizing antibody.
(3) inactivated vaccine prepared by pig Delta coronavirus CHN-HN-2014 strains of the invention can after piglet is immunized
Protect piglet from the attack of pig Delta coronavirus, protective rate is up to more than 80%.
(4) pig Delta coronavirus CHN-HN-2014 strain applications of the invention are wider, can both be made single seedling or
Connection seedling can be prepared into other antigen combinations.
Embodiment
Further the present invention is explained with reference to specific embodiment, so that those skilled in the art can be more preferably geographical
The solution present invention can be simultaneously practiced, but embodiment is not as a limitation of the invention.
Embodiment 1：The acquisition of pig Delta coronavirus PDCoV CHN-HN-2014 strains
1. the collection and processing of pathological material of disease：
From the piglet of the appearance severe diarrhea disease of Henan, China pig farm censorship, small intestine contents is gathered, with 10 times
Volume DMEM culture mediums dilute content, and fully homogenate concussion, 4 DEG C of 4200 × g are centrifuged 10 minutes, take supernatant micro- with 0.45
Frozen after rice filtering with microporous membrane is degerming as inoculation liquid standby in -70 DEG C.
2. pathological material of disease RT-PCR is detected：
200 microlitres of the inoculation liquid of gained in step 1 is taken, (kit is limited purchased from precious bioengineering Dalian with Trizol methods
Company, operated according to kit specification) the total serum IgE reverse transcription of extraction is cDNA using RNA PCR Kit by extraction total serum IgE
Laggard performing PCR amplification, PCR upstream and downstream primers are respectively F：TTTCAGGTGCTCAAAGCTCA R：
GCGAAAAGCATTTCCTGAAC, PCR system are 10 × Buffer5 μ l, μ l of dNTPs (10mM) 1, upstream, each 1 μ of anti-sense primer
L, the μ l of Taq archaeal dna polymerases 0.5, cDNA 1 μ l, ddH2O 40.5μl.PCR reaction conditions are：Enter after 95 DEG C of pre-degeneration 5min
Circulation：95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 45s, 72 DEG C of extension 5min after 35 circulations.PCR is set up when expanding
ddH2O is as negative control.After PCR reactions terminate, 10 μ l PCR primers are taken to carry out 0.8% agarose gel electrophoresis detection, hair
Existing positive has a specific band at 694bp.The positive samples of PDCoV will be detected as and carry out other common diarrhoeas disease
(such as PRV (PRV), porcine circovirus 2 type (PCV2), Porcine epidemic diarrhea virus (PEDV), pig pass for former detection
Metachromia marcy agent (TGEV) etc.), testing result PDCoV is the positive, and the negative sample of other cause of diseases carries out follow-up virus
Separation.
3. virus purification and passage：
LLC-PK1 cells are inoculated with 6 porocyte culture plates, is inhaled after cell length to individual layer and abandons cell growth medium, addition contains 10 μ
DMEM maintaining liquids (source company) 2ml of g/ml pancreatin, add in step 2 and be detected as the positive μ l of inoculum 20 of PDCoV, sense
The new DMEM maintaining liquids containing 10 μ g/ml pancreatin are changed to after making 1 hour, 37 DEG C is put and contains 5%CO2Incubator culture.Treat cell
(about 24-36 hours) collects virus liquid after there is notable lesion, the isolated coronal disease of pig Delta from the virus liquid collected
Malicious PDCoV CHN-HN-2014 strains.
4.PDCoV CHN-HN-2014 strain sequencings
(1) prepared by the extraction of PDCoV viral RNAs and cDNA
The μ l of PDCoV CHN-HN-2014 venom 200 of freeze thawing are taken to 1.5ml centrifuge tubes, often pipe adds 1ml
Reagent RNA Isolation Solvent, overturn and mix, be stored at room temperature 5~10min.Often pipe adds 200 μ l chloroforms, whirlpool
15sec, strict ice bath 10min are revolved, 4 DEG C of 12,000r/min centrifuge 10min.80% supernatant is taken to go to new 1.5ml centrifugations
Pipe, 500 μ l isopropanols are added, mixed, be stored at room temperature 10min, room temperature 12,000r/min centrifugations 10min.Supernatant is abandoned, is added
1.2ml absolute ethyl alcohols and 300 μ l DEPC water, it is slight to be vortexed, 7500r/min centrifugation 5min, supernatant is abandoned, residual is blotted with pipette tips
Moisture content, 5~10min is air-dried, be eventually adding the dissolving of 20 μ l DEPC water.Using the RNA PCR of precious bioengineering Dalian Co., Ltd
Kit (AMV) kit carries out RT-PCR amplifications (the specification operation provided by the kit).The total serum IgE of extraction is with following anti-
It is cDNA to answer system and reaction condition reverse transcription：Under specific reaction system：1.0μl 10×RT Buffer、2.0μl MgCl2、
1.0μl dNTPs(10mM)、0.25μl RNase Inhibitor、0.5μl ReverTra Ace、1.0μl Oligo(dT)
20th, 1.0 μ l RNA and 3.75 μ l RNase-Free H2O, mix in rearmounted PCR instrument, reaction is carried out as follows：30℃
10min、42℃30min、95℃5min。
(2) PDCoV CHN-HN-2014 strains genome sequencing
Using the cDNA prepared in step (1) as masterplate, with following 16 pairs of primers amplicon virus whole genome sequence
(3) genetic fragment pcr amplification reaction condition：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 40s, 50 DEG C of 40s that anneal, 72
DEG C extension 2.5min program, 34 circulation；72 DEG C of extension 10min.The PCR primer of each fragment is coagulated with 0.8% agarose
Gel electrophoresis reclaim, and are sequenced after being cloned according to conventional TA, PDCoV CHN-HN-2014 strain full genomes are obtained after each fragment assembly
Group sequence, the genome sequence have been committed to GenBank databases, and GenBank searching numbers are KT336560.1.
Embodiment 2：The coronavirus CHN-HN-2014 strains of pig Delta are in pig Delta coronavirus inactivated vaccine is prepared
Application：
1. take PDCoV CHN-HN-2014 strain (deposit numbers：CCTCC NO:V201650 LLC-PK1 cells) are inoculated in
(being purchased from ATCC cell banks, the U.S.), by 2 collection virus liquids of cell freeze thawing after cytopathy, according to conventional TCID50Assay method
Virus titer is determined, and viral level is adjusted to 108.0TCID50/ ml, after final concentration of 0.1% formalin-inactivated with phase
Montanide ISA 201 (being purchased from SEPPIC companies, France) adjuvant of homogenous quantities is mixed, is thoroughly mixed under aseptic condition
And emulsify, emulsifying temperature is 31 DEG C, and emulsification times are 15 minutes, do aseptic subpackaged after emulsification completely, are sealed standby.
(1) pig Delta coronavirus inactivated vaccine safety verification
Test method：Choose 6 3-5 age in days sodium selenites, pig Delta prepared by every piglet intramuscular injection present invention
Coronavirus inactivated vaccine 2ml, breeding observing 14 days after injection, observation result show, be immunized afterwards the piglet state of mind, body temperature,
Appetite, defecation etc. are normal, and vaccine injection site is without adverse reactions such as swelling necrosis.
(2) pig Delta coronavirus inactivated vaccine immuning effect test：
Test method：PDCoV serum neutralizing antibody is not higher than 1:4 sows produce 3~5 age in days sodium selenite 10 and are divided into
2 groups, every group 5, one of which is immunized through intramuscular injection 1ml vaccines, and another group is used as negative control, be immunized after 7 days, 14
My god, take a blood sample respectively within 21 days, separate serum, determine PDCoV neutralizing antibodies.Every Piglet by Oral 10ml PDCoV after the completion of blood sampling
CHN-HN-2014 strain virus liquid (108.0TCID50/ ml), attack Continuous Observation 7 days after poison.
Neutralizing antibody testing result：The inactivated vaccine prepared by PDCoV CHN-HN-2014 strains 21 days can after piglet is immunized
To produce the PDCoV neutralizing antibodies (table 1) of higher level, it was demonstrated that pig Delta coronavirus inactivated vaccine tool prepared by the present invention
There is preferable immunogenicity.
Neutralizing antibody testing result after the age in days piglet immunological of table 1 3~5
Attack malicious result：Inactivated vaccine is immunized piglet and mild diarrhea occurs for only one after PDCoV strong virus attacks, and very
Fast to recover, remaining 4 pig does not occur any adverse reaction within the whole observation period, illustrates prepared by PDCoV CHN-HN-2014 strains
Inactivated vaccine can effectively protect the attack of the strong poison of immune swine resistance PDCoV, protective rate reaches 80%, and control group is all fallen ill
(being shown in Table 2).
Challenge test result after the pig Delta coronavirus inactivated vaccine of table 2 is immune
As known to the result of Tables 1 and 2, pig Delta coronavirus prepared by PDCoV CHN-HN-2014 strains inactivates epidemic disease
Seedling has preferable immunogenicity, and immune swine can produce the serum neutralizing antibody of higher level after 21 days, is carried for immune swinery
For preferably protection.
Claims (3)
1. the pig Delta coronavirus CHN-HN-2014 strains of one plant of separation screening, are deposited in China typical culture collection
The heart, deposit number are CCTCC NO:V201650.
2. the pig Delta coronavirus inactivation comprising the pig Delta coronavirus CHN-HN-2014 strains described in claim 1
Vaccine.
3. the pig Delta coronavirus CHN-HN-2014 strains described in claim 1 are preparing the inactivation of pig Delta coronavirus
Application in vaccine.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110144332A (en) * | 2019-05-13 | 2019-08-20 | 中国农业科学院兰州兽医研究所 | One plant of pig Delta Coronavirus Strain and its application |
| CN110747175A (en) * | 2019-12-09 | 2020-02-04 | 中牧实业股份有限公司 | Pig delta coronavirus and application thereof |
| CN111073863A (en) * | 2019-12-25 | 2020-04-28 | 武汉科前生物股份有限公司 | Porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine and preparation method thereof |
| CN113493775A (en) * | 2021-06-21 | 2021-10-12 | 江西正邦科技股份有限公司 | Porcine delta coronavirus strain and application thereof |
| CN114790448A (en) * | 2022-03-21 | 2022-07-26 | 华中农业大学 | Porcine delta coronavirus virulent strain and application thereof in vaccine preparation |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110144332A (en) * | 2019-05-13 | 2019-08-20 | 中国农业科学院兰州兽医研究所 | One plant of pig Delta Coronavirus Strain and its application |
| CN110747175A (en) * | 2019-12-09 | 2020-02-04 | 中牧实业股份有限公司 | Pig delta coronavirus and application thereof |
| CN110747175B (en) * | 2019-12-09 | 2021-07-06 | 中牧实业股份有限公司 | Pig delta coronavirus and application thereof |
| CN111073863A (en) * | 2019-12-25 | 2020-04-28 | 武汉科前生物股份有限公司 | Porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine and preparation method thereof |
| CN113493775A (en) * | 2021-06-21 | 2021-10-12 | 江西正邦科技股份有限公司 | Porcine delta coronavirus strain and application thereof |
| CN113493775B (en) * | 2021-06-21 | 2022-12-06 | 江西正邦科技股份有限公司 | Porcine delta coronavirus strain and application thereof |
| CN114790448A (en) * | 2022-03-21 | 2022-07-26 | 华中农业大学 | Porcine delta coronavirus virulent strain and application thereof in vaccine preparation |
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