CN109735505A - The amplification and application of one plant of canine parvovirus poison strain and its gene order - Google Patents
The amplification and application of one plant of canine parvovirus poison strain and its gene order Download PDFInfo
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Abstract
The present invention relates to virus technology fields, particularly relate to the amplification and application of one plant of canine parvovirus poison strain and its gene order.The deposit number of the strain is CCTCC NO:V201873, and depositary institution is China typical culture collection center, the overall length 5052bp of canine parvovirus poison strain gene order.The canine parvovirus poison strain is 1:256, the TCID of canine parvovirus poison strain to the agglutination titer that 1% swine erythrocyte generates50It is 10‑4.85/0.1mL.The amplification of the canine parvovirus poison strain gene order, the amplification of the canine parvovirus poison strain gene order, which is segmented, to be carried out, including the amplification of Y type hairpin structure, the amplification of U-shaped hairpin structure and coding region sequence amplification.This research provides gross data for researchs such as prevalence, the genetic evolutions of Henan CPV, while laying the foundation for the research of the infection mechanism of CPV.
Description
Technical field
The present invention relates to virus technology field, particularly relate to one plant of canine parvovirus poison strain and its gene order amplification and
Using.
Background technique
Canine parvovirus disease (Canine parvovirus) is that one kind caused by canine parvovirus (CPV) is acute, high contact
Sexually transmitted disease, using hemorrhagic enteritis and myocarditis as principal pathogenetic situation, and with hyperemesis, hematochezia, loss of appetite, essence
The mind clinical symptoms such as depressed.The disease separated in state foreign aggression enteritis dog for the first time in 1978, and then worldwide gradually
Report, the country formally report the prevalence of the disease in nineteen eighty-three Xu Hankun etc. for the first time.It is sick with canine distemper, canine coronavirus at home
And referred to as the canine farming and pet industry in China are arrived in the big infectious disease of canine three, serious harm, and cause huge economic damage every year
It loses.
CPV is no cyst membrane, single-stranded DNA viruses.Viral genome is by 5323 base compositions, containing there are two main openings
Property reading frame (ORF), is separately encoded two non-structural protein NS 1s and NS2 and three Structural protein VP1, VP2 and VP3.Viral clothing
Shell is made of 60 structural proteins, and wherein VP2 is most important structural proteins in viral capsid, accounts for the 90% of viral capsid, there are about
53 ~ 54.VP2 gene N-terminal and albumen corner regions LOOP1 and LOOP3 there are important epitope, therefore VP2
Amino acid substitution occurs for sequence, may cause the change of virus antigenicity.There are two hairpin structure (3 ' Y at its genome both ends
Type and 5 ' U-shaped) it plays an important role in virus replication, and the two hairpin structures also counteract viral genome
Complete sequence sequencing and analysis.
The prevention of CPV at present is mainly based on immunity inoculation, even if carrying out effective vaccine inoculation, the disease is still alive
It is widely current within the scope of boundary.From the virus in 1978 by since separating for the first time, virus has successively carried out multiple genetic mutation, generates
3 kinds of major antigens anomaly CPV-2a, CPV-2b and CPV-2c, wherein anomaly CPV-2a and CPV-2b is mainly in some Asias
Continent country is popular, and anomaly CPV-2c is mainly distributed and Europe and Americas.Research in recent years finds new anomaly New
CPV-2a, New CPV-2b and New CPV-2c start to occur on Asia, America and Europe and other places.The study found that VP2 base
The directional drift of cause causes the generation of various anomalies, and Met has occurred compared to CPV-2 in the VP2 albumen of CPV-2a87Leu、
Ile101Thr、Ala300Gly、Asp305Tyr and Val555Ile amino acid mutation;And CPV-2b is compared to its VP2 egg of CPV-2a
Much whiter Asn426Asp amino acid mutation, but there is no Val occurs555The amino acid mutation of Ile;CPV-2c is compared to CPV-2b
Its VP2 albumen is occurring outside the identical amino acid mutation of CPV-2b, also in Asp426Glu and Ser297Ala is mutated, these
Mutation change so as to cause some biological properties, such as cat transferrins bind receptor ability, antigen reactivity with
And cat is pathogenic.By finding compared with CPV-2a, CPV-2b, the VP2 for the quasi- New CPV-2a/2b that newly makes a variation is in aa297
Ser has occurred297The mutation of Ala, the amino acid mutation in the site can be used as the mark for judging New CPV-2a/2b.In addition, certain
Phe also has occurred in a little New CPV-2a and New CPV-2b267Tyr、Tyr324Ile and Thr440Ala mutation.Though CPV is as DNA
Virus, but its reproduction process still depends on host cell, and since it is as single stranded DNA, it may be in reproduction process
Middle duplicating efficiency and error correcting capability be not high, causes the viral genome base mutation rate to be higher than general dna virus, with certain RNA
It is viral suitable, it is 7.1 × 10-3~0.7×10-3.The feature causes virus to exempt from the adaptability of environment and host and escape host
The raising of epidemic disease responsibility greatly improves the efficiency of evolution of CPV and the transmission capacity in host animal.
There are the end hairpin structures that end palindromic sequence is formed for the end of CPV full-length genome 5 ' and 3 ' ends, in answering for virus
The important function such as starting and packaging signal are played during system.So far, because of the clone of both ends hairpin structure and sequencing
Difficulty causes to only exist several plants of complete CPV whole genome sequences in genome database, and it is complete that this seriously inhibits CPV
The research work of genome and infection mechanism.
This research is successfully separated out strain CPV from through the positive sick dog excrement of CPV colloid gold test paper identification, by the disease
Genome sequencing, physicochemical property and the hemagglutination-inhibition test etc. of poison are accredited as CPV, and are named as CPV-HN1.To dog base
Because of a group sequencing analysis, to understand the fashion trend of current Henan area, reference and foundation are provided for the treatment and prevention of the disease.
Summary of the invention
The present invention proposes the amplification and application of one plant of canine parvovirus poison strain and its gene order, and it is tiny to solve animal dog
The problem that Virus Sequencing is difficult, prevention is difficult.
The technical scheme of the present invention is realized as follows:
One plant of canine parvovirus poison strain(Canine Parvovirus, CPV), the deposit number of the strain is CCTCC NO:
V201873, preservation date: on December 18th, 2018, depositary institution are China typical culture collection center, preservation address: military
The Wuchang Han Shi Luo Jia Shan, the overall length 5052bp of canine parvovirus poison strain gene order is as shown in SEQ ID NO:1.
The canine parvovirus poison strain is 1:256 to the agglutination titer that 1% swine erythrocyte generates, canine parvovirus poison strain
TCID50It is 10-4.85/0.1mL。
The amplification of the canine parvovirus poison strain gene order, the amplification point of the canine parvovirus poison strain gene order
Duan Jinhang, including the amplification of Y type hairpin structure, the amplification of U-shaped hairpin structure and coding region sequence amplification.
The primer of Y type hairpin structure amplification is Y1 primer pair and Y2 primer pair；The primer of U-shaped hairpin structure amplification draws for U1
Object to and U2 primer pair；The primer of coding region sequence amplification is A1-5 primer pair.
The Y1 primer pair upstream primer is TTAGAACCAACTGACCAAGTTCACG, downstream primer is
CGTAGGCAGCGCGCGC；Y2 primer pair upstream primer is GCTGCGCGCGCTGCCTAC, downstream primer is
TACCGAACAAAGAGTCACCAACCA。
The U1 primer pair upstream primer is CAATAACTGTAAGAAATAGAAGAAC, downstream primer is
CATAGCGGTCTGGTTGATTAAGCA；U2 primer pair upstream primer is CTATGCGGTCTGGTTGATTAAGCG, downstream primer
For AAGTATCAATCTGTCTTTAAGGGGGG.
The upstream primer and downstream primer of the A1-A5 primer pair see the table below:
For the canine parvovirus poison strain as the application for preparing vaccine, the vaccine is subunit vaccine or inactivation epidemic disease
Seedling.
The subunit vaccine, which refers to, expresses it with antigenic structure by template of canine parvovirus poison strain CPV-HN1
Albumen is mixed with subunit vaccine with nanometer silica adjuvants.
The inactivated vaccine refers to that carrying out inactivation to canine parvovirus poison strain CPV-HN1 becomes no poison strain, and receives
Rice silica adjuvants is mixed with inactivated vaccine.
The beneficial effects of the present invention are: by its bioinformatic analysis, it is found that CPV-HN1 is one plant of completely new dog
Parvovirus strain, and direction is provided for the popular variation situation of current virus；And pass through the test such as infection cell, it was demonstrated that
Its growth rate is fast, virus titer is high, is the selection of good vaccine strain, has wide range of applications, and can not only establish and attack whereby
Malicious model is used for development test, and novel canine parvovirus vaccine can also be prepared on production development, opens for current vaccine
Good material foundation and selection space are provided.
The present invention is successfully separated out one plant of CPV strain from illness dog excrement, clones and the hair fastener knot of end two is sequenced out
Structure, successful stitch go out complete CPV whole genome sequence.By PCR to separation strains identification, blood clotting and hemagglutination-inhibition test,
Virus purification test and viral physicochemical property test prove that the separation strains are CPV, are named as CPV/HN1 plants.To the separation strains
Genome cloning and sequencing show that the separation strains are CPV-2a hypotype.CPV/HN1 strain is successfully separated, and is Henan CPV's
The researchs such as prevalence, genetic evolution provide gross data, while laying the foundation for the research of the infection mechanism of CPV.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 is identification 1% gel electrophoresis figure of primer PCR product, M:2000 DNA Mark；1,2:ns1, vp2 expand purpose
Segment；N: negative control.
Fig. 2 is variation of the CPV/HN-1 separation strains on F81 cell, A:F81 cytopathy B: normal cell controls.
Fig. 3 is 1% gel electrophoresis figure of code area PCR product.
Fig. 4 is U-shaped 1% gel electrophoresis figure of hairpin structure PCR product of Y type；M:2000 DNA Mark, 1,2,3,4:Y1, Y2,
U1, U2 expand target fragment, N: negative control.
Fig. 5 is CPV-HN1 and domestic 14 plants of separation strains nucleotide homologies are analyzed.
Fig. 6 is CPV-HN1 and domestic 14 plants of separation strains phylogenetic analysis.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described, it is clear that institute
The embodiment of description is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention,
Those of ordinary skill in the art's every other embodiment obtained under that premise of not paying creative labor, belongs to this hair
The range of bright protection.
Materials and methods
1.1 cells, reagent
Fetal calf serum is purchased from Hyclone company；DMEM high sugar cell culture fluid is purchased from Hyclone company；Proteinase K is purchased from
Promega company；Agarose is purchased from Promega company；DNA Marker DL 2000, pMT-18T carrier are raw purchased from Dalian treasured
Object company；DNA gel QIAquick Gel Extraction Kit and DH5 α competent cell are purchased from Beijing CoWin Bioscience Co., Ltd.；It goes
Plasmid extraction kit is purchased from OMEGA；DNA extraction kit is purchased from Shanghai Sheng Gong bioengineering Co., Ltd；This reality of F81 cell
Test room preservation.Other conventional reagents are that analysis is pure.
Pathological material of disease is collected
Pathological material of disease collects the sick dog excrement positive through the identification of CPV colloid gold test paper from Zhengzhou City's pets hospital.It is thin in F81
It is separated on born of the same parents.
Night soil-treatment
By the excrement of sick dog with PBS according to the dilution proportion of 1:10, then plus equivalent chloroform mix, be added it is dual anti-, 4 DEG C overnight.
8000r/min is centrifuged 10 minutes, takes supernatant, 0.22 μm of filter filtration sterilization, -80 DEG C save backup.
Sample DNA extracts
The sample handled well takes a part according to raw work DNA extraction kit operating instruction, extracts the full DNA of sample, and -40 DEG C
It saves backup.
Identification
The CPV-LZ2 (number of logging in: jq268284) logged according to GenBank, for the region virus N S1, VP2, with Primer
5.0 software designs two are to detection primer NS1, VP2(table 1).Primer is synthesized by the prosperous Biotechnology Co., Ltd of Wuhan AudioCodes.
Using the PCR amplification system of 50 μ L: 25 μ L of Taq Mix, each 2 μ L of upstream and downstream primer, 4 μ L of template, finally with going out
Bacterium distilled water is added to 50 μ L.Reaction condition are as follows: 94 DEG C of 5min；94℃ 30s；60℃ 30s；72 DEG C of 40s, 35 circulations；
72 DEG C of 10min, 4 DEG C of preservations.8 μ L of PCR product electroresis appraisal in 1% Ago-Gel is taken, and uses DNA gel QIAquick Gel Extraction Kit
Recovery purifying.Product after purification is connected on Pmd18-T carrier, is converted to DH-5 α competent cell, coated plate is in Amp+ agar
On plate, 37 DEG C of incubators are stayed overnight.It selects single bacterium colony and expands culture, the positive bacterium solution that bacterium solution PCR is accredited as is sent to Wuhan Austria
The prosperous Biotechnology Co., Ltd's sequencing of section.
Table 1.CPV identifies primer
1.6 isolated viral
10 times of the viral sample handled well dilutions synchronize and are inoculated in F81 cell, the cell negative control for not connecing poison are additionally provided with, in 37
DEG C cell incubator culture.Daily observation cytopathy simultaneously records, and when CPE reaches 80%, collects virus, and 5 generation of blind passage, disease
- 70 DEG C of venom preservations.
Test
It can be aggregated this characteristic of swine erythrocyte according to CPV, prepare 1% swine erythrocyte suspension, took for 5 generations viral, tested by HA-HI
Operating method identification virus it is coagulation.
50
Took for 5 generations viral, according to TCID50Operating method, with the TCID of Reed-Muench method measurement virus50。
Physicochemical property test
Virus, which is handled, with 20% ether carries out fatsolvent sensitization test[10]；Acid resisting test is carried out with the acid processing virus of PH=3；
56 DEG C of heating 30min carry out heat-resistance test to virus.Negative control group is all set up in every group of test simultaneously, and measurement is negative respectively
The TCID of control group and test group50。
Genome sequencing
The CPV-LZ2 (number of logging in: jq268284) logged according to GenBank draws with 5.0 software design of Primer, 9 pairs of sequencings
Object (table 2).Wherein Y1, Y2 are expanded for Y type hairpin structure (sequence is as shown in SEQ ID NO:2), and U1, U2 are used for U-shaped hair fastener
Structure (sequence is as shown in SEQ ID NO:3) amplification, A1-5 is expanded for coding region sequence, so primer is prosperous by Wuhan AudioCodes
Biotechnology Co., Ltd's synthesis.
Coding domain segment amplification uses the PCR amplification system of 50 μ L: 25 μ L of Taq Mix, each 2 μ L of upstream and downstream primer, mould
4 μ L of plate, is finally added to 50 μ L with sterilizing distilled water.Reaction condition are as follows: 94 DEG C of 5min；94℃ 30s；60℃ 30s；72℃
40s, 35 circulations；72 DEG C of 10min, 4 DEG C of preservations.8 μ L of PCR product electroresis appraisal in 1% Ago-Gel is taken, and uses DNA
Gel reclaims kit recovery purifying.Both ends hairpin structure fragment amplification reaction condition are as follows: 25 μ L reaction systems, template DNA2
μ L, 12 μ L, 5xGC Buffer of μ L, dNTP of upstream primer 5 μ L, 98 DEG C of 5min are immediately placed in 5min on ice, are added
PrimeSTAR HS DNA Polymerase 0.5 μ L, 72 DEG C of 3 min;1 μ L of downstream primer, supplement sterilizing distilled water is added
To 25 μ L.Amplification system 98 DEG C of 45s, 98 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 35 circulations, 72 DEG C of 10min, 4 DEG C
It saves.Take 8 μ L of PCR product electroresis appraisal in 1% Ago-Gel, and with DNA gel QIAquick Gel Extraction Kit recovery purifying.
Product after purification is connected on pMD18-T carrier, conversion to DH-5 α competent cell, and coated plate is flat in Amp+ agar
On plate, 37 DEG C of incubators are stayed overnight.It selects single bacterium colony and expands culture, bacterium solution PCR is accredited as positive bacterium solution and is sent to Wuhan AudioCodes
Prosperous Biotechnology Co., Ltd's sequencing.
Table 2.CPV full-length genome primer
1.11 complete genome sequencing
Amplified fragments are subjected to sequence assembly with DNAStar software, the whole genome sequence that has spliced (sequence such as SEQ ID NO:
Shown in 1) with log in 14 plants on GenBank domestic Reference Strains (table 3), carry out nucleotide homology ratio with MegAlign software
To analysis.
3 Reference strains information of table
1.12 prepared by subunit vaccine
1.12.1 the building of prokaryotic expression carrier
Using RT-PCR technology, canine parvovirus structural proteins vp2 gene is expanded and cloned, connects prokaryotic expression carrier pET-
28a is transferred in Escherichia coli and is expressed, and then carries out analysis of protein purifying.
The preparation of canine parvovirus nanometer silica adjuvants subunit vaccine
Proportional arrangement of the albumen after purification with 70nm nanometers of silica adjuvants according to 1:2 is taken, the final concentration of 1mg/mL of nano-silicon fills
Concussion is divided to mix, 4 DEG C of standings complete the preparation of canine parvovirus nano-silicon adjuvant inactivated vaccine.
Inactivated vaccine preparation
1.13.1 viral purification
Purified virus uses differential centrifugation, and the virus liquid 8000rpm after proliferation is centrifuged 30min, takes supernatant 30000rpm
Ultracentrifugation 3h abandons supernatant, and appropriate PBS is resuspended precipitating, is repeated twice, and appropriate PBS is resuspended precipitating and is placed in 8000rpm centrifugation
30min takes supernatant, harvests virus liquid after purification, and -20 DEG C of packing save.
The inactivation of virus
It takes 5mL virus liquid that 10uL formalin is added, mixes well and be put into 37 DEG C of insulating boxs and inactivated, every 2h rocks mixing.
Different time points (12h, 16h, 20h, for 24 hours, 36h, 48h) collect virus, be added 0.2% sodium pyrosulfite as terminator into
Row terminates inactivation.And virus is collected to different time points and carries out inactivating efficacy monitoring, determine inactivation time.
The preparation of canine parvovirus nano-silicon adjuvant inactivated vaccine
With 70nm nanometers of silica adjuvants according to the proportional arrangement of 1:2, the final concentration of 1mg/mL of nano-silicon fills the virus liquid inactivated
Concussion is divided to mix, 4 DEG C of standings complete the preparation of canine parvovirus nano-silicon adjuvant inactivated vaccine.
As a result
2.1 PCR qualification results
Pcr amplified fragment is detected through 1% agarose gel electrophoresis, and ns1 and vp2 shows the nov nucleic acid of 324bp and 388bp respectively
Swimming band, be consistent (Fig. 1) with desired value size, and sequencing result is successfully compared with Reference Strains.
Virus purification culture
After 10 times of viral sample handled well dilutions, synchronize and be inoculated in F81 cell, blind passage to 2 generation 48h, falling off occurs in cell,
The obvious cytopathies such as seine, aggregation, deformation continue blind passage to the 5th generation, generation typical case CPE (Fig. 2A), do not connect the blank of poison
Cell controls group is in shuttle-type, and well-grown, form is normal, and cell is completely embedded, and does not occur any cytopathy (Fig. 2 B).Harvest
Per generation virus, -80 DEG C of preservations are named as CPV-HN-1.
Hemagglutination test result
The 5th generation of CPV/HN-1 separation strains virus can generate agglutination to 1% swine erythrocyte, and agglutination titer is 1:256.
Viral TCID50Measurement result
The TCID of CPV-HN-1 separation strain virus is calculated according to Reed-Muench method50It is 10-4.85/0.1mL。
Physicochemical property test result
Through ether, acid (PH=3.0), heat treatment after, the TCID50 of separation strains has no significant change, processing group with it is corresponding not
Handle control group TCID50Without significant difference, illustrate that the separation strains all have stronger resistance to acid, heat and organic solvent, this
It is consistent with document report.
The preparation of nano silica
The nano silica of particle diameter 70nm examines biological Co., Ltd purchased from Nanjing east.
Separation strains genome cloning result and sequence analysis
CPV-HN-1 separation strains code area PCR amplification result such as Fig. 3, Y type, U-shaped hairpin structure PCR amplification result such as Fig. 4.Splicing
Obtaining whole genome sequence overall length is 5052bp, is successfully compared with template sequence, wherein U-shaped hairpin structure overall length 188bp, Y type
Hairpin structure overall length 120bp, ORF1 overall length 2007bp encodes non-structural protein NS 1, ORF2 overall length 2257bp coding structure albumen
VP1,VP2.By with 14 plants of the country CPV nucleotide sequence comparison that is logged on GenBank, nucleotide homology 96.4%~
Between 99.7%, wherein with Guangzhou separation strains CPV-SC02/JX660690.1/2012 homology minimum 96.4%, with Hebei point
It is up to 99.7% from strain CPV-HB3/KR002794.1/2013 homology, such as Fig. 5.In addition, the same He Beiji of CPV/HN1 separation strains
The northern areas separation strains such as woods homology with higher, thus speculate the isolated strain by the genetic evolution of northern area strain and
Come.
CPV full-length genome phylogenetic analysis is as shown in fig. 6, CPV/HN1 separation strains and Hebei strain CPV-HB3/
KR002794.1/2013, Jilin strain CPV-JL4/KR002797.1/2016, Lanzhou strain CPV-LZ1/JQ268283.1/
2017 in same branch and belong to CPV-2a hypotype, have closer affiliation, with the separation strains relationship such as domestic Shanghai
Relationship is farther out.In general, the separation strains are closer with the northern areas affiliation such as Hebei, with country southern area separation strains parent
Edge stands off, thus it is speculated that from the separation strains are evolved by northern area separation strains.
To sum up as a result, illustrating that CPV-HN1 separation strains may be due to HB-bp Strain CPV-HB3/KR002794.1/2013's
Genetic evolution.
Conclusion
There are the end hairpin structures that end palindromic sequence is formed for the end of CPV full-length genome 5 ' and 3 ' ends, in the copied of virus
The important function such as starting and packaging signal are played in journey.So far, because of the clone of both ends hairpin structure and the difficulty of sequencing
Property, cause to only exist several plants of complete CPV whole genome sequences in genome database, this seriously inhibits CPV full genomes
The research work of group and infection mechanism.In conjunction with document report, finally clones and the hairpin structure of end two, successful stitch is sequenced out
Complete CPV whole genome sequence out.
The present invention is successfully separated out one plant of CPV strain from illness dog excrement, passes through the PCR identification to the separation strains, blood
It is solidifying to prove that the separation strains are CPV with hemagglutination-inhibition test, virus purification test and viral physicochemical property test, it is named as CPV/
HN1 plants.Genome cloning and sequencing to the separation strains, show the separation strains be CPV-2a hypotype, by with 14 plants of country
CPV prevalence strain whole genome sequence nucleotide homology analysis, show homology between 96.4%~99.9%, wherein with
The HB-bp Strain CPV-HB3/KR002794.1/2013, HB-bp Strain homology is up to 99.7%.CPV/HN1 strain at
Function separation provides gross data for researchs such as prevalence, the genetic evolutions of Henan CPV, while establishing for the research of the infection mechanism of CPV
Basis.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
<110>Agricultural University Of He'nan
The amplification and application of<120>one plants of canine parvovirus poison strains and its gene order
<160> 3
<210> 1
<211> 5052
<212> dna
<213>canine parvovirus(canine parvovirus, cpv)
<220>
<221> gene
<222> (1)...(5052)
<400> 1
ttagaaccaa ctgaccaagt tcacgtacgt atgacgtgat gacgcgcgct gcgcgcgctg 60
cctacggcag tcacacgtca tacgtacgct ccttggtcag ttggttctaa agaatgatag 120
gcggtttgtg tgtttaaact tgggcgggaa aaggtggcgg gctaattgtg ggcgtggtta 180
aaggtataaa agacaaacca tagaccgtta ctgacattcg cttcttgtct ttgacagagt 240
gaacctctct tactttaact aaccatgtct ggcaaccagt atactgagga agttatggag 300
ggagtaaatt ggttaaagaa acatgcagaa aatgaagcat tttcgtttgt ttttaaatgt 360
gacaacgtcc aactaaatgg aaaggatgtt cgctggaata actataccaa accaattcaa 420
aatgaagagc taacatcttt aattagagga gcacaaacag caatggatca aaccgaagaa 480
gaagaaatgg actgggaatc ggaagttgat agtctcgcca aaaagcaagt acaaactttt 540
gatgcattaa ttaaaaaatg tctttttgaa gtctttgttt ctaaaaatat agaaccaaat 600
gaatgcgttt ggtttattca acatgaatgg ggaaaagatc aaggctggca ttgtcatgtt 660
ttacttcata gtaagaactt acaacaagca actggtaaat ggctacgcag acaaatgaat 720
atgtattgga gtagatggtt ggtgactctt tgttcggtaa acttaacacc aactgaaaag 780
attaagctca gagaaattgc agaagatagt gaatgggtga ctatattaac atacagacat 840
aagcaaacaa aaaaagacta tgttaaaatg gttcattttg gaaatatgat agcatattac 900
tttttaacaa agaaaaaaat tgtccacatg acaaaagaaa gtggctattt tttaagtact 960
gattctggtt ggaaatttaa ctttatgaag tatcaagaca gacaaattgt cagcacactt 1020
tacactgaac aaatgaaacc agaaaccgtt gaaaccacag tgacgacagc acaggaaaca 1080
aagcgcggga gaattcaaac taaaaaggaa gtgtcaatca aatgtacttt gcgggacttg 1140
gttagtaaaa gagtaacatc acctgaagac tggatgatgt tacaaccaga tagttatatt 1200
gaaatgatgg cacaaccagg aggtgaaaat cttttaaaaa atacacttga aatttgtact 1260
ttgactttag caagaacaaa aacagcattt gaattaatac ttgaaaaagc agataatact 1320
aaactaacta actttgatct tgcaaattct agaacatgtc aaatttttag aatgcacgga 1380
tggaattgga ttaaagtttg tcacgctata gcatgtgttt taaatagaca aggtggtaaa 1440
agaaatacag ttctttttca tggaccagca agtacaggaa aatctatcat tgctcaagcc 1500
atagcacaag ctgtgggtaa tgttggttgt tataatgcag caaatgtaaa ttttccattt 1560
aatgactgta ccaataaaaa tttaatttgg attgaagaag ctggtaactt tggtcaacaa 1620
gttaatcaat ttaaagcaat ttgttctgga caaacaatta gaattgatca aaaaggtaaa 1680
ggaagtaagc aaattgaacc aactccagta attatgacaa ctaatgaaaa tataacaatt 1740
gtgagaattg gatgtgaaga aagacctgaa catacacaac caataagaga cagaatgttg 1800
aacatcaagt tagtatgtaa gcttccagga gactttggtt tggttgataa agaagaatgg 1860
cctttaatat gtgcatggtt agttaaacat ggttatgaat caaccatggc taactataca 1920
catcattggg gaaaagtacc agaatgggat gaaaactggg cggagcctaa aatacaaaaa 1980
ggtataaatt caccaggttg caaagactta gagacgcaag cggcaagcaa tcctcagagt 2040
caagaccaag ttctaactcc tctgactccg gacgtagtgg accttgcact ggaaccgtgg 2100
agtactccag atacgcctat tgcagaaact gcaaagcaac aatcaaacca acttggcgtt 2160
actcacaaag acgtgcaagc gagtccaacg tggtccgaaa tagaggcaga cctgagagcc 2220
atctttactt ctgaacaatt ggaagaagat tttcgagacg acttggatta aggtacgatg 2280
gcacctccgg caaagagagc caggagaggt aagggtgtgt tagtaaggtg gggggagggg 2340
aaagatttga taacttaact aagtatgtgt tttttttata ggacttgtgc ctccaggtta 2400
taaatatctt gggcctggga acagtcttga ccaaggagaa ccaactaacc cttctgacgc 2460
cgctgcaaaa gaacacgacg aagcttacgc tgcttatctt cgctctggta aaaacccata 2520
cttatatttc tcgccagcag atcaacgctt tatagatcaa actaaggacg ctaaagattg 2580
gggggggaaa ataggacatt atttttttag agctaaaaag gcaattgctc cagtattaac 2640
tgatacacca gatcatccat caacatcaag accaacaaaa ccaactaaaa gaagtaaacc 2700
accacctcat attttcatca atcttgcaaa aaaaaaaaaa gccggtgcag gacaagtaaa 2760
aagagacaat cttgcaccaa tgagtgatgg agcagttcaa ccagacggtg gtcagcctgc 2820
tgtcagaaat gaaagagcta caggatctgg gaacgggtct ggaggcgggg gtggtggtgg 2880
ttctgggggt gtggggattt ctacgggtac tttcaataat cagacggaat ttaaattttt 2940
ggaaaacgga tgggtggaaa tcacagcaaa ctcaagcaga cttgtacatt taaatatgcc 3000
agaaagtgaa aattatagaa gagtggttgt aaataatttg gataaaactg cagttaacgg 3060
aaacatggct ttagatgata cccatgcaca aattgtaaca ccttggtcat tggttgatgc 3120
aaatgcttgg ggagtttggt ttaatccagg agattggcaa ctaattgtta atactatgag 3180
tgagttgcat ttagttagtt ttgaacaaga aatttttaat gttgttttaa agactgtttc 3240
agaatctgct actcagccac caactaaagt ttataataat gatttaactg catcattgat 3300
ggttgcatta gatagtaata atactatgcc atttactcca gcagctatga gatctgagac 3360
attgggtttt tatccatgga aaccaaccat accaactcca tggagatatt attttcaatg 3420
ggatagaaca ttaataccat ctcatactgg aactagtggc acaccaacaa atatatacca 3480
tggtacagat ccagatgatg ttcaatttta cactattgaa aattctgtgc cagtacactt 3540
actaagaaca ggtgatgaat ttgctacagg aacattttat tttgattgta aaccatgtag 3600
actaacacac acatggcaaa caaatagagc attgggctta ccaccatttc taaattcttt 3660
gcctcaagct gaaggaggta ctaactttgg ttatatagga gttcaacaag ataaaagacg 3720
tggtgtaact caaatgggaa atacaaacat tattactgaa gctactatta tgagaccagc 3780
tgaggttggt tatagtgcac catattattc ttttgaggcg tctacacaag ggccatttaa 3840
aacacctatt gcagcaggac gggggggagc gcaaacagat gaaaatcaag cagcagatgg 3900
tgatccaaga tatgcatttg gtagacaaca tggtcaaaaa actaccacaa caggagaaac 3960
acctgagaga tttacatata tagcacatca agatacagga agatatccag aaggagattg 4020
gattcaaaat attaacttta accttcctgt aacaaatgat aatgtattgc taccaacaga 4080
tccaattgga ggtaaagcag gaattaacta tactaatata tttaatactt atggtccttt 4140
aactgcatta aataatgtac caccagttta tccaaatggt caaatttggg ataaagaatt 4200
tgatactgac ttaaaaccaa gacttcatgt aaatgcacca tttgtttgtc aaaataattg 4260
tcctggtcaa ttatttgtaa aagttgcgcc taatttaaca aatgaatatg atcctgatgc 4320
atctgctaat atgtcaagaa ttgtaactta ctcagatttt tggtggaaag gtaaattagt 4380
atttaaagct aaactaagag cctctcatac ttggaatcca attcaacaaa tgagtattaa 4440
tgtagataac caatttaact atgtaccaag taatattgga ggtatgaaaa ttgtatatga 4500
aaaatctcaa ctagcaccta gaaaattata ctaacatact tactatgttt ttatgtttat 4560
tacatattat tttaagatta attaaattac agcatagaaa tattgtactt gtatttgata 4620
taggatttag aaggtttgtt atatggtata caataactgt aagaaataga agaacattta 4680
gatcatagtt agtagtttgt tttataaaat gtattgtaaa ccattaatgt atgttgttat 4740
ggtgtgggtg gttggttggt ttgcccttag aatatgttaa ggaccaaaaa aatcaataaa 4800
agacatttaa aactaaatgg cctcgtatac tgtctataag gtgaactaac cttaccatta 4860
gtatcaatct gtctttaagg gggggtgggt gggagatgca caatatcagt agactgactg 4920
gcctggttgg ttgcgcttaa tcaaccagac cgctatgcgg tctggttgat taagcggagc 4980
aaccaaccag gccagtcagt ctactgatat tgtgcatctc ccacccaccc ccccttaaag 5040
acagattgat actt 5052
<210> 2
<211> 120
<212> DNA
<213>canine parvovirus(canine parvovirus, cpv)
<220>
<221> misc_structure
<222> (1)...(120)
<400> 2
ttagaaccaa ctgaccaagt tcacgtacgt atgacgtgat gacgcgcgct gcgcgcgctg 60
cctacggcag tcacacgtca tacgtacgct ccttggtcag ttggttctaa agaatgatag 120
<210> 3
<211> 188
<212> DNA
<213>canine parvovirus(canine parvovirus, cpv)
<220>
<221> misc_structure
<222> (1)...(188)
<400> 3
caatctgtct ttaagggggg gtgggtggga gatgcacaat atcagtagac tgactggcct 60
ggttggttgc gcttaatcaa ccagaccgct atgcggtctg gttgattaag cggagcaacc 120
aaccaggcca gtcagtctac tgatattgtg catctcccac ccaccccccc ttaaagacag 180
attgatac 188
Claims (10)
1. one plant of canine parvovirus poison strain CPV-HN1, it is characterised in that: the deposit number of the canine parvovirus poison strain is CCTCC
NO:V201873, depositary institution are China typical culture collection center, the overall length of canine parvovirus poison strain gene order
5052bp。
2. canine parvovirus poison strain according to claim 1, it is characterised in that: the canine parvovirus poison strain is red to 1% pig
The agglutination titer that cell generates is 1:256, the TCID of canine parvovirus poison strain50It is 10-4.85/0.1mL。
3. the amplification of canine parvovirus poison strain gene order of any of claims 1 or 2, it is characterised in that: the canine parvovirus
The amplification of poison strain gene order, which is segmented, to be carried out, including the amplification of Y type hairpin structure, the amplification of U-shaped hairpin structure and coding region sequence
Amplification.
4. the amplification of canine parvovirus poison strain gene order according to claim 3, it is characterised in that: Y type hairpin structure
The primer of amplification is Y1 primer pair and Y2 primer pair；The primer of U-shaped hairpin structure amplification is U1 primer pair and U2 primer pair；Coding
The primer of region sequence amplification is A1-5 primer pair.
5. canine parvovirus poison strain according to claim 4, it is characterised in that: the Y1 primer pair upstream primer is
TTAGAACCAACTGACCAAGTTCACG, downstream primer CGTAGGCAGCGCGCGC；Y2 primer pair upstream primer is
GCTGCGCGCGCTGCCTAC, downstream primer TACCGAACAAAGAGTCACCAACCA.
6. canine parvovirus poison strain according to claim 4, it is characterised in that: the U1 primer pair upstream primer is
CAATAACTGTAAGAAATAGAAGAAC, downstream primer CATAGCGGTCTGGTTGATTAAGCA；U2 primer pair upstream primer
For CTATGCGGTCTGGTTGATTAAGCG, downstream primer AAGTATCAATCTGTCTTTAAGGGGGG.
7. canine parvovirus poison strain according to claim 4, which is characterized in that the upstream primer of the A1-A5 primer pair
It see the table below with downstream primer:
。
8. canine parvovirus poison strain of any of claims 1 or 2 is as the application for preparing vaccine, it is characterised in that: the vaccine
For subunit vaccine or inactivated vaccine.
9. canine parvovirus poison strain according to claim 8 is as the application for preparing vaccine, it is characterised in that: described sub- single
Position vaccine refers to its canine parvovirus poison strain CPV-HN1 with antigenic structural protein expression.
10. canine parvovirus poison strain according to claim 8 is as the application for preparing vaccine, it is characterised in that: described to go out
Live vaccine refers to the canine parvovirus poison strain CPV-HN1 of its active constituent inactivation.
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