CN110144332A - One plant of pig Delta Coronavirus Strain and its application - Google Patents
One plant of pig Delta Coronavirus Strain and its application Download PDFInfo
- Publication number
- CN110144332A CN110144332A CN201910394213.XA CN201910394213A CN110144332A CN 110144332 A CN110144332 A CN 110144332A CN 201910394213 A CN201910394213 A CN 201910394213A CN 110144332 A CN110144332 A CN 110144332A
- Authority
- CN
- China
- Prior art keywords
- pig
- strain
- delta coronavirus
- vaccine
- coronavirus strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention provides a boar Delta Coronavirus Strain and its applications, belong to the immunoprophylaxis technical field of pig, and the pig Delta Coronavirus Strain is preserved in China General Microbiological culture presevation administrative center, and deposit number is CGMCC No.17383.The strong toxicity of the pig Delta Coronavirus Strain；Porcine epidemic diarrhea virus, good immune effect will be prepared after pig Delta Coronavirus Strain inactivation.
Description
Technical field
The invention belongs to the immunoprophylaxis technical field of pig more particularly to a boar Delta Coronavirus Strain and its answer
With.
Background technique
Pig Delta coronavirus (Porcine Deltacoronavirus, PDCoV) is coronaviridae δ coronavirus
The newcomer belonged to, can cause diarrhea, the vomiting, dehydration of sow and piglet, seriously can be lethal, make to pig farm and aquaculture
At serious economic loss.2012, which was found for the first time in Hong-Kong.At the beginning of 2014, Russia, the U.S.
Sow and piglet diarrhoea have been broken out in the pig farm in last of the twelve Earthly Branches Russia state, are detected as the PDCoV positive, and at least 18 states in the U.S.
Also the Novel pig coronavirus is detected in succession.Then, in the fecal specimens of South Korea and Canadian diarrhea piglet
Detect PDCo V.In China, 2013~2014, to the pig raising from provinces and cities such as Jiangsu, Guangdong, Anhui, Hubei, Henan
The 258 parts of fecal samples acquired in find that PDCoV positive rate reaches 14.3%, confirms in China for the first time after being detected
There are PDCoV on ground.2015 so far, and in other provinces and cities, China, such as Shaanxi, Gansu, Zhejiang, Hebei, Tianjin area are also successively
Detect PDCoV.Currently, the porcine diarrhea disease caused by PDCoV is in world pop and the trend gradually spread.With pig popularity
Diarrhea virus (PEDV) and transmissible gastro-enteritis virus (TGEV) are identical, and PDCoV is also the common disease for causing porcine diarrhea disease
One of original.The virus can cause a disease to the pig of different days, have to piglet it is stronger pathogenic, show as persistent diarrhea, vomiting,
Can be lethal because being dehydrated when serious, case fatality rate is generally 30%~40%.But PDCoV is a kind of novel disease just found in recent years
Original worldwide not yet develops effective vaccine or drug for the disease at present.
Summary of the invention
In view of this, it is an object of the invention to a boar Delta Coronavirus Strain and its applications；The pig Dare
Tower Coronavirus Strain immune protective effect is good, can be used in preparing vaccine.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
The present invention provides a boar Delta Coronavirus Strain, during the pig Delta Coronavirus Strain is preserved in
State's General Microbiological Culture preservation administrative center, deposit number are CGMCC No.17383.
The present invention provides the recombinant vectors including the pig Delta Coronavirus Strain S genome.
The present invention provides the pig Delta Coronavirus Strains to prepare the application in pig epidemic diarrhea vaccine.
Preferably, the pig epidemic diarrhea vaccine includes inactivated vaccine and attenuated vaccine.
Preferably, the inactivated vaccine includes the pig Delta coronavirus and the adjuvant of inactivation.
The present invention provides the recombinant vectors to prepare the application in pig epidemic diarrhea vaccine.
Preferably, the pig epidemic diarrhea vaccine includes subunit vaccine.
Beneficial effects of the present invention: pig Delta Coronavirus Strain provided by the invention, the infected cell first generation just
There is typical induced cytopathic effect CPE, strong toxicity；Preparation pig after pig Delta Coronavirus Strain inactivation is popular
Property diarrhea virus, good immune effect.It records according to embodiments of the present invention, with the pig Delta Coronavirus Strain inactivated vaccine
Immune piglet can cause the significant raising of IgG antibody for 7 days after being immunized, and the lasting raising in 14,21 days after immune, and anti-
Body level is significantly higher than negative control group (PBS)；Further protest test is the results show that the coronal disease of pig Delta
Poison strain inactivated vaccine can protect 4/5 piglet to resist 100 TCID50The attack of homologous strain, and negative control group is then whole
Morbidity (0/5 protection), it is seen that the pig Delta Coronavirus Strain inactivated vaccine good immune effect.
Detailed description of the invention
Fig. 1 is the microscope photograph of the pig Delta Coronavirus Strain 70 generation CPE；
Fig. 2 is the systematic evolution tree of the pig Delta Coronavirus Strain；
Fig. 3 is the growth curve of the pig Delta Coronavirus Strain；
Fig. 4 is the IFA qualification figure of the pig Delta Coronavirus Strain；
Fig. 5 is the viral particle morphology figure of the pig Delta Coronavirus Strain；
Fig. 6 is the pathogenic analysis of the pig Delta Coronavirus Strain, and wherein A is Clinical symptoms figure, and B is dissect disease
Become figure；
Fig. 7 is the Histopathological Studies of the intestinal lesion of piglet after infecting the pig Delta Coronavirus Strain
Figure；
Fig. 8 is that the IgG antibody after piglet is immunized with the pig Delta Coronavirus Strain inactivated vaccine is horizontal.
Biological deposits explanation
Pig Delta coronavirus CH/XJYN03/2016 provided by the invention was preserved in China on February 28th, 2019
General Microbiological Culture preservation administrative center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number:
CGMCC No.17383。
Specific embodiment
The present invention provides a boar Delta Coronavirus Strain, during the pig Delta Coronavirus Strain is preserved in
State's General Microbiological Culture preservation administrative center, deposit number are CGMCC No.17383.In the present invention, the pig Delta hat
Shape virus stain is isolated from Xinjiang, China, the pig Delta Coronavirus Strain and other PDCoV Reference strains in same
One branch, and affiliation is close, belongs to δ coronavirus genus；And with TGEV the and PEDV otherness of α coronavirus genus compared with
Greatly, not in same branch.In the present invention, the pig Delta coronavirus particles shape is substantially oblong-shaped, diameter about 100nm
Left and right, surrounding have the apparent fibre like crown-shaped prominent, are the characteristic feature of coronavirus.
Pig Delta Coronavirus Strain positive sample inoculation Vero cell passage training is carried out passage training by the present invention
It supports, there have been typical CPE in the first generation for the pig Delta Coronavirus Strain；In the present invention, the pig Delta is coronal
Virus stain P0 generation and P20 generation malicious valence are respectively 104.65TCID50/ mL and 105.57TCID50/mL。
The present invention provides the recombinant vectors including the pig Delta Coronavirus Strain S genome.In the present invention
In, the pig Delta Coronavirus Strain S genome is prepared preferably through following methods: A) extracting includes the pig
The total serum IgE of the sample of Delta Coronavirus Strain；B S genome specificity primer) is utilized, is carried out by template of the total serum IgE
RT-PCR reaction obtains the pig Delta Coronavirus Strain S genome.In the present invention, including the pig Delta is coronal
The sample of virus stain is preferably field fecal specimens or cell culture and virus liquid；In the present invention, the side of the extraction of the total serum IgE
Method uses the virus total RNA extracting method of this field routine, without other particular/special requirements.In the present invention, the S genome specificity
Primer include A fragment primer to, B fragment primer to and C fragment primer pair；The A fragment primer to, B fragment primer to and C piece
The nucleotide sequence of section primer pair is as shown in NO:1~6 SEQ ID.In the present invention, the RT-PCR reaction is preferred to be usedGXL DNA Polymerase kit carries out；The amplification system of the RT-PCR reaction is excellent in terms of 50 μ L
Choosing includes following component:
The amplification program of the RT-PCR reaction is preferably as follows: 98 DEG C of 1min；95 DEG C of 30s, 60 DEG C of 40s, 68 DEG C of 1min,
34 circulations；68℃1min；4 DEG C of preservations.
In the present invention, the product of the RT-PCR amplified reaction is the pig Delta Coronavirus Strain S genome.
In the present invention, it is preferred to the RT-PCR amplified production and carrier linked obtain recombinant vector.In the present invention, the load
Body and connection method use this field routine carrier and connection method, without other particular/special requirements.Of the invention specific real
During applying, the carrier is preferably blunt end cloning carrier pCR-XL-2TOPO carrier.Heretofore described recombinant vector energy
It is enough in clone and the identification of the pig Delta S gene in coronavirus group；The recombinant vector can also be used to subunit's epidemic disease
The preparation of seedling.
The present invention also provides the pig Delta Coronavirus Strains to prepare the application in pig epidemic diarrhea vaccine.
In the present invention, the pig epidemic diarrhea vaccine preferably includes inactivated vaccine and attenuated vaccine；The inactivated vaccine is preferred
The pig Delta coronavirus and adjuvant including inactivation；The present invention is not special to the type and dosage of the adjuvant
It limits, using the vaccine adjuvant of this field routine.In the present invention, the attenuated vaccine is preferably through to the pig moral
You carry out attenuation acquisition by tower coronavirus poison；The method of the attenuation uses the virus attenuation method of this field routine.
The present invention also provides the recombinant vectors to prepare the application in pig epidemic diarrhea vaccine.In the present invention,
The pig epidemic diarrhea vaccine preferably includes subunit vaccine, more preferably genetic engineering subunit vaccine.The present invention couple
The preparation method of the vaccine does not have particular/special requirement, using the vaccine preparation method of this field routine.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
1 pathological material of disease and cell
Pathological material of disease acquisition is identified from Xinjiang, China, separation of 12 parts of samples containing PDCoV positive strain for PDCoV；
LLC-PK-1 cell is by this laboratory freezen protective.The cell is passed on by the dual anti-MEM culture medium containing 10%FBS and 1%
Culture.
2 PDCoV CH/XJYN03/2016 strain S genome sequence determinations
Conservative region is chosen according to the PDCoV sequence alignment result announced, corresponding amplimer is designed and carries out PDCoV's
The amplification of S genome sequence, primer situation are as shown in table 1.CH/XJYN03/2016 strain field fecal specimens and cell are trained
After the venom RNA that recuperates is extracted, RT-PCR reaction is carried out using S genome specificity primer.Select blunt end cloning carrier pCR-
XL-2TOPO carrier carries out the subclone of PCR fragment, and digestion is identified that correct plasmid is sent to company and is sequenced.
The S genome amplification primer of 1 PEDV of table
RNA extracts (Qiagen RNeasy Mini Kit)
1) the 350 μ L of virus liquid after taking 3 multigelations is slowly mixed after 350 μ L RLT lysates are added, is stood on ice
5min；
2) 700 μ L dehydrated alcohols are added, stand 5min on ice after slowly mixing；
3) mixed liquor of 700 μ L is added in centrifugal column, 12000rpm is centrifuged 30s, discards liquid in collecting pipe simultaneously
Centrifugal column is put back in collecting pipe；
4) the RW1 buffer of 700 μ L is added into centrifugal column, stands 1min on ice, 12000rpm will centrifugation after being centrifuged 30s
Column is placed into new collecting pipe；
5) 500 μ L RPE buffers are drawn, discard liquid in collecting pipe after 12000rpm centrifugation 15；
6) liquid 12000rpm sky is discarded from 1min after 500 μ L, 12000rpm centrifugation 2min of RPE buffer is added again；
7) empty that pillar is put into sterilized 1.5mL eppendorf pipe from after, 30 water of the μ L without RNA enzyme are added, it is quiet
Viral RNA is collected by centrifugation after setting 1min, -20 DEG C store for future use.
RT-PCR reaction
The cDNA obtained using reverse transcription is utilized as templateGXL DNA Polymerase kit into
Row pcr amplification reaction.
2 RT-PCR reaction system of table
PCR response procedures: 98 DEG C of 1min；95 DEG C of 30s, 60 DEG C of 40s, 68 DEG C of 1min, 34 circulations；68℃1min；4 DEG C of guarantors
It deposits.
3 PDCoV CH/XJYN03/2016 strain phylogenetic analysis
More plants of domestic and international representative PDCoV strain whole genome sequences are obtained from Genebank, with this experiment CH/
The whole genome sequence of XJYN03/2016 strain carries out sequence alignment and adjacent method (Bootstrap using MEGA6.0 software
Replication:1000 systematic evolution tree) is constructed.
Using the S genome sequence of the primer amplified PDCoV CH/XJYN03/2016 of design, sequence will be measured
It is spliced into complete whole genome sequence, is compared using MEGA6.0 software and known PEDV strain whole genome sequence and structure
Build systematic evolution tree.As a result such as Fig. 2 is shown, the CH/XJYN03/2016 strain is with other PDCoV Reference strains in same
A branch, and affiliation is close, belongs to δ coronavirus genus；And with TGEV the and PEDV otherness of α coronavirus genus compared with
Greatly, not in same branch.
The separation of 4.PDCoV CH/XJYN03/2016 and secondary culture
1, the virus liquid for taking 1.5mL to freeze, with 0.22um filter filtration sterilization.The virus liquid for drawing the above-mentioned filtering of 1mL, adds
Enter 2mLMMT (Trypsin final concentration is then 20ug/mL), it is spare that concussion is placed at room temperature for 10min after mixing.
2, growth conditions are taken out well and are almost paved with the single layer LLC-PK-1 cell of cell bottle, with the DPBS close to room temperature
After washing 3 times, the venom prepared in step 2 is added, 37 DEG C of incubation 1h containing 5%CO2 are subsequently placed in, every 20min
It shakes up primary.
3, the fresh MMT of 5mL (Trypsin concentration is 20ug/mL) is added after being incubated for is placed on incubation culture.
4, whether observation cell there is CPE at regular intervals, and receipts poison is subsequent to resume generation.
As a result the sample inoculation Vero cell secondary culture by 12 parts containing PDCoV positive strain as shown in Figure 1:, wherein
PDCoV CH/XJYN03/2016 strain, there have been typical CPE, selects the strain to continue secondary culture, mesh in the first generation
Preceding to reach for 45 generations, the cause for being committed to strain is weak.There is CPE in cell since HPI12 in the strain, cell shrinkage and
It is rounded, assembles agglomerating, formation plasomidum；As infection time increases, cytopathy area increases, and HPI48 cell is complete
Lesion, and there is obscission, form cell vacuole.
5.PDCoV CH/XJYN03/2016 strain growth curve
By the 60dish of confluent monolayers cell clear 3 times with PBS, it is added 500 μ L virus liquids (20 μ g/mL of pancreas enzyme concentration),
After 37 DEG C of incubation 1h, the MM of 1.5mL is added, is placed in and is cultivated containing 5%CO237 DEG C.Different time sections collect venom after infection,
It is placed in -80 DEG C of refrigerators and saves backup.Viral level is measured with QPCR, draws different generation PDCoV CH/XJYN03/2016
The growth curve of strain, it is primary every the measurement of 10 generations.
The MM culture medium isMaintenancemedium (MM), preparation method: in 500mLDMEM culture solution
1wt%Penicillin/Streptomycin (Invitrogen/GIBCO is added in (Invitrogen Cat#12430054)
Cat#15140122), 0.3wt%tryptose phosphate broth (TPB, Sigma Cat#T8159-100mL) and end
After concentration is the pancreatin (Invitrogen cat#15090-046) of 10 μ g/mL, it is uniformly mixed.
As a result from the figure 3, it may be seen that in 0~48h, gradually increase as infection time increases viral level, after connecing poison
PEDV viral level highest in 48h culture medium, corresponding normal cell almost 90% lesion；After peak value, with the time
Extend cell to start shedding off to which malicious particle content is begun to decline.
6 PDCoV CH/XJYN03/2016 strain TCID50Measurement
1, the virus liquid room temperature collected through freeze thawing is melted, 3500rpm is centrifuged 2min, takes supernatant
2, virus is made continuous 10 times with DMEM culture medium (2020 μ g/mL of pancreas enzyme concentration) to dilute, i.e., 10-1,10-2... ....Diluted multiple is determined according to the rough titre of virus.
3, cell culture fluid is discarded, PBS is cleaned 2 times；Again with maintaining culture medium (MM) to wash once, discards and (guarantee nothing in hole
Liquid residual).
4, each dilution takes 100 μ L that 96 orifice plates are added, and each dilution makees 8 repetitions.Adding 100 μ L MM, whole body
Product is 200 μ L.And blank control is set, i.e. 200 μ L MM.
5, it is placed in containing 5%CO237 DEG C of cultures.Day by day cytopathy is observed, and records cytopathy hole count.Generally need to see
It examines 5~7 days.As a result calculating Reed-Muench Liang Shi method.
PDCoV CH/XJYN03/2016 strain P0 generation and P20 generation malicious valence are respectively 10 as the result is shown4.65TCID50/ mL and
105.57TCID50/mL。
7 PDCoV CH/XJYN03/2016 immunofluorescents (IFA)
1, it is inoculated with venom in 6 orifice plates, after CPE occurs in cell, discards maintenance culture medium；
2,1ml fixative i.e. 4% paraformaldehyde is added in every hole, is placed in 4 DEG C of refrigerators, fixed 30min；
3, fixer is discarded, every hole is added penetrating dose of 1ml i.e. 0.25%Tripon-100, room temperature penetrating 10min, and PBS is washed
Plate 3 times, each 5min；
4,5%BSA closes 60min or 10%BSA and closes 30min, every hole 1ml, after closing, PBS board-washing 3 times, every time
5min；
5,1ml is added with the diluted anti-PDCoV N protein monoclonal antibody of 1:2000 or 1:5000,37 DEG C of incubations in every hole
60min (or 4 DEG C overnight).PBS board-washing 3 times, each 5min；
6, under the conditions of being protected from light, 1ml is added with the anti-mouse secondary antibody of diluted 488 label of 1:2000,37 DEG C of incubations in every hole
60min, PBS board-washing 3 times, each 5min；
7, every hole is added two drop DAPI (with a little PBS dilution), and room temperature 5min, PBS board-washing 3 times, each 5min, rear fluorescence
Micro- sem observation.
As a result as shown in figure 4, detecting specific green fluorescence after PDCoV infection cell.Meanwhile when with infection
Between increase, visual field Green fluorescence is more, shows that PDCoV content is higher.
8 PDCoV CH/XJYN03/2016 Electronic Speculum detect virion
1,400mL virus liquid is collected, three times, 10000rpm collects cell supernatant in 4 DEG C of centrifugation 1h to multigelation.It receives
The cell supernatant of collection crosses 0.22 μM of filter membrane.Will 50%PEG-8000 be added vial supernatant in, make its final concentration of 10%, mistake
Night precipitate virus, 4 DEG C of gentle agitations are stayed overnight.
2, above-mentioned virus liquid-PEG8000 mixed liquor is put into 250mL centrifuge tube, 12000rpm is in 4 DEG C of centrifugation 2h precipitating diseases
Poison.
3, TBS suspension viral pellet prepares virus-TBS mixed liquor, 4 DEG C of stirring 30min.
4,0.5M sucrose solution first time Sucrose density centrifugation: is added to the bottom of vial supernatant, ultracentrifugation
30000rpm 3h, 4 DEG C.Supernatant is discarded, precipitating is sufficiently resuspended with pH 7.5TBS, then 4 DEG C of 3000rpm are centrifuged 10min.
5, second of Sucrose density centrifugation: above-mentioned TBS re-suspension liquid is added in centrifuge tube and (40% and 50% is added in advance
Sucrose solution), 30000rpm, 4 DEG C, be centrifuged 3h.Viral band is drawn after centrifugation.
6, it takes copper mesh to be placed on sealed membrane (copper mesh faces up) with fine-pointed forceps sub-folder, draws about 10 μ L venom drop in copper mesh
On, it adsorbs about 15min (operating process wants the moment to pay attention to that front and back sides cannot be obscured).
7, adsorption time blots venom above copper mesh with filter paper after, then air-dries about 10min；
8, the water-soluble drop of 10 μ L, 2% sodium phosphotungstate is drawn in the about 20min of negative staining on copper mesh after air-drying；
9, the negative staining time blots the solution on copper mesh to filter paper, air-dries about 10min, and clamping copper mesh is placed in inside copper mesh box
Send to Electronic Speculum.
As a result such as Fig. 5 shows that PDCoV CH/XJYN03/2016 virion shape is substantially oblong-shaped, diameter about 100nm
Left and right, surrounding have the apparent fibre like crown-shaped prominent, are the characteristic feature of coronavirus.
The purifying of 9 PDCoV CH/XJYN03/2016 strains
LLC-PK1 cell in good condition to be grown is cleaned 3 times, every Kong Yi when 6 orifice plates single layer is paved with 80% with DPBS
The secondary virus liquid (pancreas enzyme concentration: 20 μ g/mL) that 500 μ L10 times gradient dilutions are added, is placed in containing 5%CO237 DEG C of incubators
It is incubated for 1h.Culture solution is discarded after incubation, DPBS is cleaned 1 time, and every hole is added 2mL and contains MEM's (pancreas enzyme concentration: 20 μ g/mL)
1.5% eutectic dispensing.15min is placed at room temperature, and incubator is inverted into after being gelled admittedly and continues culture observation.To six holes
When occurring CPE in plate, with the minimum dilution plaque of sterile pipette tips picking in 1.5mLEP pipe.
10 PDCoV CH/XJYN03/2016 strains are to the pathogenic analysis of piglet
Serum and fecal specimens are acquired in large-scale pig farm, whether there is using ELISA kit detection piglet serum
PDCoV antibody, and using QPCR detection piglet with the presence or absence of the infection of PDCoV.7 qualified age in days piglets of detection are chosen to exist in advance
Animal house is raised 3 days, it is ensured that experimental animal adapts to surrounding enviroment, reduces stress reaction.7 age in days piglets of selection are randomly divided into
2 groups, i.e. G1, G2.Every group of 4 piglets, every group of piglet are numbered to distinguish.G1 is experimental group, and G2 is control group.It will
PDCoV CH/XJYN03/2016 strain P10 is diluted for venom with PBS10 times, every part 2mL venom, oral challenge.Control group
Oral PBS.From self tapping poison day, the observation piglet state of mind, whether diet is found changes, if symptom of diarrhea occurs.Every natural gift
Three period (9:00,15:00 and 21:00) cotton stick swabs acquire fecal specimens, and the fecal specimens of acquisition are with 2mL containing dual anti-
MEM processing, 3500rpm is centrifuged 30min, and Aspirate supernatant is placed in -80 DEG C and saves backup.Extract RNA, real time fluorescent quantitative
PCR detects piglet and infects with the presence or absence of PEDV.When piglet frequency is died on one's deathbed, heart is put to death, at once dissect, and observation enteron aisle macroscopic view becomes
Change.Enteron aisle is taken into 2~3cm by duodenum, jejunum, ileum, caecum and colon respectively, is placed in the fixed guarantor of 10% formalin
It deposits, carries out Histopathological Studies.
It is normal and active that piglet attacks hair color before poison, feeding, weight.24~48h piglet starts to fall ill after attacking poison, excrement
Sample is thinning, and individual piglets are apathetic, appetite stimulator, rubescent at anus.48h after poison is attacked, all piglets of experimental group fall ill,
There is serious yellow watery diarrhea, is in spurting, and breath malodor；It is rubescent that swelling occurs in anus area, hind leg fecal pollution
Seriously；There is different degrees of vomiting phenomenon in part piglet appetite abolish after feed；There is serious syntexis, quilt in all piglets
Hair is dirty and messy random, and happiness is sleeping, hind limb weakness, astasia, and serious person's hind leg paralysis is poured on ground, can only slide by forelimb (see figure
6A).The control group experiment piglet state of mind is good, and diet is normal.Frequently the piglet heart died on one's deathbed is put to death, dissect is carried out.Such as
Shown in Fig. 6 B, intestinal mucosa hyperemia swelling, intestinal wall is thinning to become bright, includes a large amount of yellow water sample contents；Mesenteric lymph is born
Now different degrees of enlargement.Histopathological Studies result is as shown in Figure 7.
11, PDCoV CH/XJYN03/2016 strain immune protective effect is evaluated
1) prepared by PDCoV CH/XJYN03/2016 inactivated vaccine:
(1) liquid configures
0.2N sodium hydroxide: it is sterile to weigh sodium hydroxide 0.8g, add aqua sterilisa to 100mL, sufficiently shakes up.Or press above-mentioned amount
After preparing, high pressure sterilization.0.2M BEI configuration: it is sterile to weigh BEA4.1g/0.41g, it is molten that sterilized 0.2N sodium hydroxide is added
Liquid 100mL/10mL sufficiently dissolves, makes BEA concentration 0.2M, set in 37 DEG C of water-baths, when BEA solution temperature equilibrates to 37 DEG C,
Every 15min shake is primary, amounts to 1h, and BEA cyclisation at this time is 0.2M BEI (ready-to-use).
(2) it inactivates
By P4,30, P70 for cell toxicant, cell fragment is removed through 3500rmp centrifugation 10min, it is molten with 7.5% sodium bicarbonate
Liquid tune pH value is 7.6~7.8 (150 μ L are added in test strips 7.5,15mL cell toxicant), and 1%Penicillin/ is added
Streptomycin(GIBCO Cat#15140122).The formalin of final concentration 0.05% is added, i.e., adds in 100mL virus liquid
The formalin (1mL:1.35 μ L) for entering 0.135mL37~40%, sufficiently shakes up.Then it is molten that the 0.2M BEI prepared is added
Liquid makes the final concentration of 0.002M of BEI, i.e., 0.2M BEI solution 1mL (1mL:10 μ L) is added in 100mL virus liquid, sufficiently shakes up.
30 DEG C of calculating inactivation times are warming up to, temperature is maintained at 30 ± 1 DEG C, 12h is inactivated, therebetween every 2h shake 1 time.
(3) it emulsifies
Cell toxicant inactivation antigen 15mL+ Freund's complete adjuvant 15mL (1:1 mixing), inhaled repeatedly with 10mL syringe blow until
Mix (at least 10~15 times) completely.
2) design of protest test
PDCoV negative antibody piglet 10 is chosen, two groups, i.e. immune group (n=5) and negative control group (PBS, n=are divided into
5).The inactivated vaccine prepared in immune group musculi colli injection 2mL above-mentioned steps, control group musculi colli inject 2mLPBS.?
0,7,14,21 day serum of being taken a blood sample and separated by vena cava anterior respectively after immune passes through the immune rear blood of indirect ELISA method detection
Clear IgG antibody is horizontal.Further, the 21st day after head exempts from, with 100 TCID50Homologous strain to immune group and control group into
Row protest test, and then the immune protective effect of the strain is evaluated.
3) indirect ELISA method
The PDCoVN proteantigen of eukaryotic expression coating buffer (0.1M carbonate buffer solution, pH 9.0) is diluted to
Final concentration of 0.1 μ g/mL is coated in the 96 hole polystyrene titer plates in 50 holes μ L/, and is incubated overnight at 4 DEG C.With
PBS-T wash 5 times after, with containing 2% bovine serum albumin(BSA) 100 holes μ L/ in PBS-T 37 DEG C of closing 2h, then washed with PBS-T
It washs 5 times, immune piglet blood serum sample (1:100 dilution), 37 DEG C of incubation 60min then is added with 50 holes μ L/, while establishing sun
Property, feminine gender and blank control.After incubation, washed 5 times with PBS-T.Then the diluted HRP label in 50 20000 times of the holes μ L/ is added
The anti-pig IgG monoclonal antibody of goat.Then it is incubated for 30min at 37 DEG C, is then washed 5 times with PBS-T, 50 μ L/ are added later
The TMB one pack system substrate solution in hole, is incubated for 15min at 37 DEG C.The OD value in each hole is measured at 450nm using ELISA Plate.
In immune rear IgG antibody testing result as shown in figure 8, CH/XJYN03/2016 strain inactivated vaccine is 7 days after immune
Can cause the significant raising of IgG antibody, and after immune 14,21 days it is lasting increase, and antibody level be significantly higher than it is negative right
According to group (PBS group).Further protest test is the results show that CH/XJYN03/2016 strain inactivated vaccine can protect 4/5
Piglet resist 100 TCID50The attack of homologous strain, and negative control group then all morbidity (0/5 protections).Therefore, it is immunized
Protection the result shows that, CH/XJYN03/2016 strain after being prepared into inactivated vaccine to piglet have good immunoprotection effect
Fruit is one plant of good, effective candidate vaccine strain.
It can be seen that the pig Delta Coronavirus Strain provided by the invention, virulence is strong, good immune effect, can
It is used to prepare pig epidemic diarrhea vaccine, prevents and treats pig epidemic diarrhea.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>one plants of pig Delta Coronavirus Strains and its applications
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
attatctatg gtgatgattc ca 22
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
acaattcagc atgagtggtt 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
caccagaatg cagagagctc 20
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ccaaatacca tagctccagt aa 22
<210> 5
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ggtggcagat ctgctattga a 21
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gaccgctctt gtagatcatt aat 23
Claims (7)
1. a boar Delta Coronavirus Strain, which is characterized in that the pig Delta Coronavirus Strain is preserved in China
General Microbiological Culture preservation administrative center, deposit number are CGMCC No.17383.
2. including the recombinant vector of pig Delta Coronavirus Strain S genome described in claim 1.
3. pig Delta Coronavirus Strain described in claim 1 is preparing the application in pig epidemic diarrhea vaccine.
4. applying according to claim 3, which is characterized in that the pig epidemic diarrhea vaccine includes inactivated vaccine and attenuation
Vaccine.
5. applying according to claim 4, which is characterized in that the inactivated vaccine includes that the pig Delta of inactivation is coronal
Virus and adjuvant.
6. recombinant vector as claimed in claim 2 is preparing the application in pig epidemic diarrhea vaccine.
7. application according to claim 6, which is characterized in that the pig epidemic diarrhea vaccine includes subunit vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910394213.XA CN110144332B (en) | 2019-05-13 | 2019-05-13 | Porcine delta coronavirus strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910394213.XA CN110144332B (en) | 2019-05-13 | 2019-05-13 | Porcine delta coronavirus strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110144332A true CN110144332A (en) | 2019-08-20 |
| CN110144332B CN110144332B (en) | 2020-12-08 |
Family
ID=67595349
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910394213.XA Active CN110144332B (en) | 2019-05-13 | 2019-05-13 | Porcine delta coronavirus strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110144332B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114107223A (en) * | 2021-11-25 | 2022-03-01 | 湖北省疾病预防控制中心（湖北省预防医学科学院） | Method for improving culture titer of SARS-CoV-2 virus cells by TPCK pancreatin |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104513827A (en) * | 2013-09-30 | 2015-04-15 | 普莱柯生物工程股份有限公司 | Porcine epizootic diarrhea virus strain, attenuated vaccine strain thereof and application thereof |
| US20170202951A1 (en) * | 2014-07-11 | 2017-07-20 | Zoetis Services Llc | Novel Vaccine Compositions for Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus |
| CN107815440A (en) * | 2016-09-14 | 2018-03-20 | 华中农业大学 | Separation, the preparation and application of inactivated vaccine of pig Delta coronavirus strain |
| WO2018160977A1 (en) * | 2017-03-03 | 2018-09-07 | Loyola University Chicago | Coronaviruses, vaccines comprising the same, and methods for preventing disease |
| CN109022371A (en) * | 2018-09-14 | 2018-12-18 | 中国农业科学院兰州兽医研究所 | One plant of Porcine epidemic diarrhea virus strain and its preparing the application in pig epidemic diarrhea vaccine |
-
2019
- 2019-05-13 CN CN201910394213.XA patent/CN110144332B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104513827A (en) * | 2013-09-30 | 2015-04-15 | 普莱柯生物工程股份有限公司 | Porcine epizootic diarrhea virus strain, attenuated vaccine strain thereof and application thereof |
| US20170202951A1 (en) * | 2014-07-11 | 2017-07-20 | Zoetis Services Llc | Novel Vaccine Compositions for Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus |
| CN107815440A (en) * | 2016-09-14 | 2018-03-20 | 华中农业大学 | Separation, the preparation and application of inactivated vaccine of pig Delta coronavirus strain |
| WO2018160977A1 (en) * | 2017-03-03 | 2018-09-07 | Loyola University Chicago | Coronaviruses, vaccines comprising the same, and methods for preventing disease |
| CN109022371A (en) * | 2018-09-14 | 2018-12-18 | 中国农业科学院兰州兽医研究所 | One plant of Porcine epidemic diarrhea virus strain and its preparing the application in pig epidemic diarrhea vaccine |
Non-Patent Citations (1)
| Title |
|---|
| ZHANG Q等: "Detection and phylogenetic analyses of spike genes in porcine epidemic diarrhea virus strains circulating in China in 2016-2017", 《VIROL J.》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114107223A (en) * | 2021-11-25 | 2022-03-01 | 湖北省疾病预防控制中心（湖北省预防医学科学院） | Method for improving culture titer of SARS-CoV-2 virus cells by TPCK pancreatin |
| CN114107223B (en) * | 2021-11-25 | 2024-03-26 | 湖北省疾病预防控制中心（湖北省预防医学科学院） | Method for improving SARS-CoV-2 virus cell culture titer by using TPCK pancreatin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110144332B (en) | 2020-12-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110144332A (en) | One plant of pig Delta Coronavirus Strain and its application | |
| CN109182278A (en) | Seneca Valley virus strain and its application | |
| CN106591242B (en) | One plant of canine parvovirus poison strain CPV-YH and its application | |
| CN101935637B (en) | Recombinant low-virulent vaccine strain of chicken infectious bursal disease viruses (IBDV) and application thereof | |
| CN107513524A (en) | One plant of pig Sai Neijia paddy virus stain and its application | |
| CN106636012B (en) | One boar lid his virus stain, vaccine combination and its preparation method and application | |
| CN107619822B (en) | Porcine epidemic diarrhea virus low virulent strain, and culture method and application thereof | |
| CN104826103B (en) | A kind of porcine pseudorabies virus vaccine | |
| CN108486067A (en) | The inactivated vaccine and application of Porcine epidemic diarrhea virus variant and its preparation | |
| CN107384874A (en) | Pseudorabies virus epidemic strain gI/gE gene deletion mutants and structure and application | |
| CN107988170A (en) | The inactivated vaccine and application of porcine rotavirus strain and its preparation | |
| CN103509761B (en) | Recombinant porcine pseudorabies virus strain used for expression of porcine circovirus type II (PCV2) ORF2 gene, and preparation method thereof | |
| CN104152416A (en) | Pseudorabies virus gene-deleted attenuated strain as well as preparation method and application thereof | |
| CN109517806A (en) | A kind of Tilapia mossambica lake Virus strain and its RPA detection primer and method | |
| CN107815440A (en) | Separation, the preparation and application of inactivated vaccine of pig Delta coronavirus strain | |
| CN106282132A (en) | The low virulent strain of Pseudorabies virus variant and application thereof | |
| CN102732486B (en) | Porcine circovirus type2 strain and application thereof | |
| CN104928260A (en) | Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof | |
| CN111073862B (en) | Bovine viral diarrhea type2 attenuated strain and application thereof | |
| CN101914502B (en) | I-type canine adenovirus attenuated vaccine strain and application thereof | |
| CN109022371A (en) | One plant of Porcine epidemic diarrhea virus strain and its preparing the application in pig epidemic diarrhea vaccine | |
| CN104419686B (en) | recombinant PRRS virus HV-nsp9 and application thereof | |
| CN107653230A (en) | A kind of II type pseudoabies poison strain and its application | |
| CN107304416A (en) | One plant of Porcine epidemic diarrhea virus and its cultural method and application | |
| CN105200015B (en) | A kind of porcine pseudorabies strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |