CN110468155A - It is a kind of for saving the system, method and application of chitling road A type coronavirus - Google Patents
It is a kind of for saving the system, method and application of chitling road A type coronavirus Download PDFInfo
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Abstract
The invention discloses a kind of for saving the system, method and application of chitling road A type coronavirus.The system comprises: the recombination transcription vector comprising chitling road A type coronavirus complete genome DNA；Helper plasmid comprising SeACoV-N gene.The present invention successfully constructs SADS-CoV/SeACoV reverse genetic operating system, the virus of generation can continuous passage, Revive virus and parental virus have consistent biological characteristics.Reverse genetics system is established by the unstability of virus genome sequence and the obstruction of toxicity, the rescue method has potential using value in the research of pig coronavirus reverse genetics and vaccine initiative, has wide practical use in terms of known viral gene function.
Description
Technical field
The present invention relates to field of biotechnology, are for save chitling road A type coronavirus more particularly to a kind of
System, method and application.
Background technique
China is the first in the world pig raising big country, and large-scale cultivation is quickly grown.But with the fast development of large scale of pig farm,
Coronavirus infection is increasing to the harm of pig breeding industry.Therefore, reinforce the infection of pig coronavirus and the understanding of pathogenesis,
New theory and technical support will be provided for these control and prevention of disease.
Chitling road A type coronavirus (the swine enteric found for the first time in south China in 2017
Alphacoronavirus, SeACoV) it is also known as pig acute diarrhea syndrome virus (Swine acute Diarrhoea
Syndrome Coronavirus, SADS-CoV) cause piglet severe diarrhea, be evolutionary source be bat HKU2-CoV it is novel
Coronavirus.Compared with HKU2, there is height and make a variation in the S protein of SADS-CoV/SeACoV, and be concentrated mainly on N-terminal region
The 1-238 amino acids of (NTD, domain 0): there is the insertion of 75 amino acid point mutants and 2 amino acid in the region.We
Speculating the height variation on NTD to have may cause from bat coronavirus HKU2 variation is pig SeACoV, thus across kind from bat
Bat travels to pig.But at present to the research of the virus almost blank out.
Diarrhea of pigs correlated virus generates huge threat to global pig breeding industry, it is therefore desirable to gain more insight into this kind of disease
Poison.The main problem faced at present is to lack the genetic tool for being suitable for studying pig coronavirus SADS-CoV/SeACoV.
The virus of coronaviridae is single strand plus RNA virus, and there is genome the ORF of overlapping to encode replicase
(ORF1a, 1b), non-structural protein and ORF3, spike protein (S), envelope protein (E), memebrane protein (M) and nucleocapsid protein
(N).The pathogenesis of research coronavirus is largely segregated culture at present and shortage can operational virus genetic tool
Limitation.Compared to other virus, coronavirus have length about 27-30kb genome, be it is maximum in RNA virus,
There are significant challenge in the engineered vector for generating infection clones, natural difference between amplification related mutation and
Sequencing mistake may be extremely difficult.In addition, establishing the trial of reverse genetics system by the unstable of virus genome sequence
The obstruction of property and toxicity.
Reverse Genetics are a kind of useful platforms for carrying out virus research, are illustrating viral pathogenesis mechanism and vaccine development
In can play a significant role.Equally, pig coronavirus SADS-CoV/SeACoV infective molecule cloning is constructed, as base
What plinth can study the more efficiently research of the specific gene of SADS-CoV/SeACoV expansion and SADS-CoV/SeACoV
One useful platform.
Currently, there is no the report of the reverse genetics system of SADS-CoV/SeACoV successfully constructed both at home and abroad.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, and providing one kind, can successfully to save out chitling road A type coronal
The virus rescue system of virus.
A kind of system for saving chitling road A type coronavirus, comprising:
Recombination transcription vector comprising chitling road A type coronavirus complete genome DNA；
Helper plasmid comprising SeACoV-N gene,
Wherein, the chitling road A type coronavirus is single strand plus RNA virus, and the chitling road A type coronavirus is complete
Genomic DNA refers to the corresponding DNA sequence dna of chitling road A type coronavirus positive chain RNA sequence, the sequence of the SeACoV-N gene
It is classified as the corresponding DNA sequence dna of RNA sequence that chitling road A type coronavirus encodes the gene.
Since chitling road A type coronavirus is single strand plus RNA virus, so using when rescue is viral gene
The corresponding DNA sequence dna of group RNA, i.e., only become T for the base U in RNA sequence.
The sequence size of SeACoV-N gene is 1125bp (as shown in SEQ ID NO.1), expresses SeACoV-N albumen, warp
Research find can by way of inhibiting interferon expression helper virus rescue.
The recombination transcription vector is pSB using plasmid, and the plasmid that the helper plasmid uses is pRK5.The two plasmids
It is the commercially available plasmid of commercialization.
Revive virus and wild virus, the rescue chitling road A type coronavirus full-length genome are distinguished for the ease of detection
The 24222-24224 bit base of cDNA sports TCT by AGT, the two codons correspond to encoding serine (Ser), institute
It is remained unchanged with amino acid Ser.
The invention also discloses a kind of methods for saving chitling road A type coronavirus, comprising the following steps:
(1) by the system recombination transcription vector and helper plasmid cotransfection enter in host cell；
(2) chitling road A type coronavirus obtained by the rescue in cell and supernatant is collected after cultivating.
The mass ratio that recombination transcription vector and helper plasmid are added in step (1) is 2: 1.The host cell is BHK-21
Cell.
The present invention also provides the chitling road A type coronavirus for using the method rescue to obtain.
The present invention also provides the chitling road A type coronavirus of the rescue acquisition as the coronal disease of chitling road A type
Application in malicious research model.
The present invention also provides the chitling road A type coronavirus that the rescue obtains to prevent in diarrhea of pigs vaccine in preparation
Application.
The present invention successfully constructs SADS-CoV/SeACoV reverse genetic operating system, the virus of generation can continuous passage,
Revive virus and parental virus have consistent biological characteristics.Reverse genetics system is established by virus genome sequence not
The obstruction of stability and toxicity, the rescue method have potential answer in the research of pig coronavirus reverse genetics and vaccine initiative
With value, have wide practical use in terms of known viral gene function.
Detailed description of the invention
Fig. 1 is 16 sections of genetic fragment structural schematic diagrams of chitling road A type coronavirus SADS-CoV/SeACoV.
Fig. 2 is 16 sections of gene fragment amplification result detection figures of chitling road A type coronavirus SADS-CoV/SeACoV.
Fig. 3 is that chitling road A type coronavirus SADS-CoV/SeACoV infective cloned plasmids pSB-SeACoV building is shown
It is intended to.
Fig. 4 is the 23 sections of sequencing fragment signals of chitling road A type coronavirus SADS-CoV/SeACoV infective cloned plasmids
Figure.
Fig. 5 is the 23 sections of fragment amplification identifications of chitling road A type coronavirus SADS-CoV/SeACoV infective cloned plasmids
Result figure.
Fig. 6 is that chitling road A type coronavirus SADS-CoV/SeACoV infection clones Revive virus utilizes SeACoV-N
Protein antibodies Immunofluorescence test result figure, figure A are individually to transfect infective cloned plasmids pSB-SeACoV Revive virus group, figure
B is infective cloned plasmids pSB-SeACoV and pRK5-SeACoV-N plasmid co-transfection Revive virus group.
Fig. 7 is that molecular labeling is located at TF21 segment PCR testing result figure in the ORF3 gene of Revive virus rSeACoV,
Middle swimming lane 1 is control, and supernatant when swimming lane 2 is virus rescue, swimming lane 3 is intracellular when being virus rescue, and swimming lane M is standard molecular weight
Marker。
Fig. 8 is that Revive virus rSeACoV is located at Ser molecular labeling sequencing result figure on ORF3.
Fig. 9 is detection Revive virus and parental virus growth curve comparison result figure.
Figure 10 is that the virion of Revive virus observes result figure under an electron microscope.
Specific embodiment
Embodiment 1
The amplification of the full-length infectious clone's target fragment of 1.SADS-CoV/SeACoV
According to the chitling road A type coronavirus SeACoV CH/GD-01/2017/P2 pnca gene sequence announced on Genbank
It arranges (GenBank accession no.MF370205, Fig. 1), full length viral genome 27155nt, with 10 years preceding areals
It was found that homology of the bat enteron aisle A type coronavirus HKU2 in full-length genome nucleic acid level of infection Rhinolophus sinicus be
94.9%.Cytotostatic passes on the cell infection Supernatant samples of strain SADS-CoV/SeACoV-P10, and Trizol method extracts RNA
Reverse transcription obtains cDNA afterwards, and using 16 pairs of primers (primer sequence is shown in Table 1) provided by the invention, amplification obtains corresponding 16 bases
Because of a group full length fragment, by pCR-Blunt carrier cloning, 3 monoclonal colonies of picking are extracted plasmid order-checking splicing, are determined
The full-length genome sequence of SADS-CoV/SeACoV-P10.Using corresponding 16 pairs of primers, having with preservation respectively
The pCR-Blunt plasmid of 16 segments of SADS-CoV/SeACoV is template, expands point truncation of corresponding full-length genome overall length
Segment (Fig. 2).
Table 1
For that can connect each segment of SADS-CoV/SeACoV overall length on carrier, that is, the 5 ' of SeACoV-F1 segment is required to hold
With carrier segments 3 ' end, SeACoV-F15 segment 3 ' end with carrier segments 5 ' end ' at least homologous region of 20bp, in order to homologous
Recombination.Design primer adds 5 ' and 3 ' homologous sequences to SeACoV-F1 and two segment of SeACoV-F15 respectively.Primer sequence is such as
Under:
SeACoV-F1 upstream primer SeACoV-F1-F:
5'-ATAAGCAGAGCTCGTTTAGTGAACCGTGACTTAAAGATATAA-3'；
SeACoV-F1 downstream primer SeACoV-F1-R:
5'-GTCATCACAGAGGGCAGTAAAGC-3'；
SeACoV-F15 upstream primer SeACoV-F15-F:
5'-ATGGCATCAGAATTGCTACTGGTGT-3'；
SeACoV-F15 downstream primer SeACoV-F15-R:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTGTGTATCACTGTCAA-3’。
The same sense mutation for introducing an amino acid in ORF3 gene simultaneously is marked as the detection molecules of Revive virus, i.e.,
24222-24224 bit base sports TCT by AGT, and amino acid Ser is remained unchanged (Fig. 3), expands chitling road A type coronavirus
Genetic fragment between SeACoV-F14 23403-24239 bit base and 24207-25298 bit base passes through over-lap PCR
Expanded 2 genetic fragments are merged using overlapping PCR method, fusion segment SeACoV-F14-Marker is obtained, is being located at
Molecular labeling is introduced in the ORF3 gene of SeACoV-F14 segment, primer sequence is as follows:
SeACoV-F14-Marker-a sections of upstream primer SeACoV-F14-F:
5'-ATTTGCTAATGTCATTGCCGTTTCC-3'；
SeACoV-F14-Marker-a sections of downstream primer SeACoV-Marker-a-R:
5'-GAGAACAAAAGCAAAAGACCTG-3'；
SeACoV-F14-Marker-b sections of upstream primer SeACoV-Marker-b-F:
5'-GCTTTTTGTTACAGGTCTTTTGC-3'；
SeACoV-F14-Marker-b sections of downstream primer SeACoV-F14-R:
5’-GGCGACAGTCACAAATTGCGGTA-3’。
The splicing of the full-length infectious clone of 2.SADS-CoV/SeACoV
It is assembled by Gibson assembly method, with the seamless Cloning Kit of GBclonart by SADS-CoV/SeACoV
Each DNA fragmentation and the pSB carrier of linearisation (insertion point is in CMV promoter downstream) are connected by way of external homologous recombination
It is connected into full-length infectious cloning recombinant plasmids.It is screened by conversion connection product to DH10B and expands positive recombinant plasmid clone.
By the SeACoV-F1~F15 expanded, centrifuge tube is added in 16 segments and linearized vector segment equal proportion in total
In, operation is required the step of Cloning Kit seamless according to GBclonart, reaction system is as follows:
After mixing simultaneously 45 DEG C of incubation 2h, it is transferred on ice.DH10B competent cell is converted immediately.
The verifying of the full-length infectious clone of 3.SADS-CoV/SeACoV
Random picking is individually cloned, and is inoculated into the LB liquid medium that 5mL contains chloramphenicol (30mg/mL) resistance respectively
In, 37 DEG C of shaking table cultures extract the SADS-CoV/ for cloning and obtaining according to AxyPrep Plasmid DNA small volume of reagent box specification
The full-length infectious cloned plasmids of SeACoV.To carry out PCR amplification verifying and subsequent sequence verification to full-length infectious clone, originally grind
Study carefully and full-length genome sequence is divided into 23 sections, every section of 1400bp or so, upstream and downstream two reactions are only needed when in order to be sequenced
Survey logical (Fig. 4).
The full-length infectious cloned plasmids that 23 sections of PCR sequencing primers are shown in Table 2 pairs of extractions carry out PCR verifying.5 μ L PCR are taken to produce
Object is detected in 1% agarose gel electrophoresis, chooses positive colony, and 1~23 each section of PCR product of the clone is sent to still sub- biological skill
The sequencing of art Co., Ltd saves and correctly full-length infectious clone's glycerol stock (Fig. 5) is sequenced.By the recombination BAC of acquisition, it is named as
PSB-SeACoV obtains the full-length infectious cloned plasmids of chitling road A type coronavirus SADS-CoV/SeACoV of molecular labeling.
Table 2
4. virus rescue
4.1SADS-CoV/SeACoV infective cloned plasmids mention greatly
It is equipped with 2 × YT of 1L fluid nutrient medium, glycerol stock is connected to containing chlorine according to the ratio of bacterium solution and culture medium 1: 100
2 × YT fluid nutrient medium of mycin (30mg/mL) resistance, 37 DEG C of 200rpm shaking table culture 12h.According to BAC/PAC DNA
Isolation Maxi Kit specification extracts SeACoV infective cloned plasmids, and -20 DEG C save backup.
4.2pRK5-SeACoV-N plasmid construction
According to upstream primer SeACoV-N-F:
5'-ACCTCGGTTCTATCGATTGGCCACCATGGCCACTGTTAATTGG-3'；
Downstream primer SeACoV-N-myc-R:
5'-CAGATCCTCTTCAGAGATGAGTTTCTGCTCATTAATAATCTCATC-3'；
Using pSB-SeACoV plasmid as template, amplified fragments SeACoV-N uses the two digestion positions EcoR I and Xba I
Point linearizes pRK5 plasmid by double enzyme digestion reaction.Using seamless Cloning Kit, connection linearisation pRK5 plasmid and
Connection product conversion is entered Top10 competence by segment SeACoV-N, and picking monoclonal colonies extract plasmid order-checking verifying, at
Function constructs pRK5-SeACoV-N plasmid.
4.3SADS-CoV/SeACoV transfection rescue
BHK-21 cell is spread in 12 orifice plates before transfection, is placed in 5% CO2, cultivate in 37 DEG C of incubators, reached to cell density
It is transfected to 70% or so.Cell culture fluid is discarded when transfection, is washed twice with Opti-MEM, transfection reagent is made negative right
According to, infective cloned plasmids and expression SADS-CoV/SeACoV-N albumen plasmid according to3000 transfection examinations
Agent box specification carries out cotransfection, and wherein infective cloned plasmids pSB-SeACoV is added according to 2 holes μ g/, pRK5-SeACoV-N
Plasmid is added according to 1 hole μ g/, is changed liquid after transfecting 7h, is observed day by day, and until there is cytopathy, multigelation collection is saved
Vial supernatant is rescued, rSeACoV is named as.Another transfection experiment group individually adds the infective cloned plasmids in 2 holes μ g/ simultaneously
PSB-SeACoV does not add pRK5-SeACoV-N plasmid as control.
Vero cell is spread in 12 orifice plates, is placed in 5%CO2, cultivate in 37 DEG C of incubators, reach 70% or so to cell density
It is infected, uses SADS-CoV/SeACoV-N albumen rabbit polyclonal antibody after infection 3 days, pass through indirect immunofluorescene assay
(IFA) virus rescue situation (Fig. 6) is detected.The individually rescue disease of transfection infective cloned plasmids pSB-SeACoV Revive virus group
Malicious supernatant vero cells infection, and the expression of SADS-CoV/SeACoV-N albumen is not detected, and infective cloned plasmids
The Revive virus supernatant of pSB-SeACoV and pRK5-SeACoV-N plasmid co-transfection group is used for vero cells infection, exempts from indirectly
Epidemic disease fluorescence detection can significantly observe the green florescent signal of SADS-CoV/SeACoV-N protein expression, it was demonstrated that rSeACoV
The successful rescue of virus.
The RT-PCR qualification result and sequence verification of 5.rSeACoV
Supernatant and cell sample to rSeACoV vero cells infection extract total serum IgE, and reverse transcription is at cDNA.According to drawing
The same sense mutation for entering an amino acid is located at full-length genome sequence as the ORF3 gene of the detection molecules label of Revive virus
It is divided into 23 sections of TF21 segment, using the primer amplification segment of TF21 segment,
Wherein, upstream primer SeACoV-TF21:5 '-TACTGGATGTTGTGGCATGT-3 '；
Downstream primer SeACoV-TR21:5 '-TTCCACTTAAAATCGTCAGA-3 ',
PCR product is sent to Shang Ya Bioisystech Co., Ltd sequencing (Fig. 7, Fig. 8).According to sequencing result, successfully rescue is obtained
Obtain the chitling road A type coronavirus SADS-CoV/SeACoV with molecular labeling that can stablize passage.
The growth curve of protovirus SADS-CoV/SeACoV and Revive virus rSeACoV, the two are detected by TCID50
Growth kinetics characteristic having the same, guarantee the virus generated can continuous passage, Revive virus and parental virus have consistent
Biological characteristics (Fig. 9).After expanding cultivation separation and purification to Revive virus, virion is observed under an electron microscope, it can be with
Observe typical complete coronavirus particles (Figure 10).
Sequence table
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<120>a kind of for saving the system, method and application of chitling road A type coronavirus
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atgcctagaa atggagttcc gacaggtaaa ggcactccag atcaacagat tggttattgg 180
gttgaacaaa aacgctggcg aatgcaaaaa ggccaacgta aagatcagcc ttctaactgg 240
cacttttatt accttggtac tggtcctcac gcagatgctc ctttcaggaa acggattcag 300
ggtgtgcatt gggtcgctgt tgacggtgct aaaactagcc ccacaggtct tggtgttcgc 360
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gttgctgcag ttaaacaagc tttggcagac ttgggcatag cttctagcca gtccaggcct 600
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aaaccggctc gtaaacagat ggacaaacct gaatggaagc gtgttcctaa ttctggggag 720
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gctgcactag cctttggtag tgaaatcaca accaaagagt ctggtgaatt tgtagaagtc 900
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caagtctcgg cttactctaa acccagtcaa attaggagat ctcaatctca acaagaccta 1020
aatgctgatg ccccagtgtt cactccggca cctccagcta ctccagtttc ccaaaatcct 1080
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<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
cgtatgttag gtttgcagac 20
<210> 23
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
accattaacg ccttctagtg 20
<210> 24
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
gtgcctattt tggaactgta atg 23
<210> 25
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
cataatagtg gaattgcgcc 20
<210> 26
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
cgctatggct gttaagatta ccg 23
<210> 27
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
caatggcatt tctgtgtacc tctc 24
<210> 28
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
gctagttacg cacctaatga cacc 24
<210> 29
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
cattagggtc aagtttagca gctc 24
<210> 30
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
atttgctaat gtcattgccg tttcc 25
<210> 31
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
ggcgacagtc acaaattgcg gta 23
<210> 32
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
atggcatcag aattgctact ggtgt 25
<210> 33
<211> 42
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 33
tttttttttt tttttttttt tttttttgtg tatcactgtc aa 42
<210> 34
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
gagaacaaaa gcaaaagacc tg 22
<210> 35
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
gctttttgtt acaggtcttt tgc 23
<210> 36
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
tccaaaatgt cgtaacaact 20
<210> 37
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
tccaagctca taacatgatt 20
<210> 38
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
tccactggta aagttacgac 20
<210> 39
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
agtaccctta agctcaccaa 20
<210> 40
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
tggttcagac tgttgccaat 20
<210> 41
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
taacaagatt atgtgtgcca 20
<210> 42
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
tcattgaggt aaacaaggct 20
<210> 43
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
acatgtcaga taacaagcca 20
<210> 44
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
ttgaaacacc tgtggttgaa 20
<210> 45
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
accattacgg ataacaactg 20
<210> 46
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
cagcggtagc ttattaaacg 20
<210> 47
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
agataaacac acgcattctg 20
<210> 48
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
agggttgagt ttagtgatgg 20
<210> 49
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
tataccatcc acctgtctgc 20
<210> 50
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
ggtgctatga cttatggtga 20
<210> 51
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
tgaccattca taacaaaacc 20
<210> 52
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
ctgtgtcaca ggctaatgtt 20
<210> 53
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
gagtcaaaag gacctcttgg 20
<210> 54
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
tggacttatc tcctttgttg 20
<210> 55
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
catgctagct cagtctgttt 20
<210> 56
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
ttaaaggttg tcaagtggga 20
<210> 57
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
ccgtaaaatc atattcaagc 20
<210> 58
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
gcgtcttaac attggacaac 20
<210> 59
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
ccgttagaat gcacaacctc 20
<210> 60
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
tgaatgatgt cgataatggt 20
<210> 61
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
gacttaacac tctcctccct 20
<210> 62
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
ccacccatag gtttatcttg 20
<210> 63
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
gctccattta gggttctttg 20
<210> 64
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
taggacctga tgtgtttttg 20
<210> 65
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
tgagactgaa ctattcgtcc 20
<210> 66
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
gcgactcatg cgtactattg 20
<210> 67
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
agcattacga ccctgtgagt 20
<210> 68
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
gaggacgtgt gtacttgctt 20
<210> 69
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
agcactagaa aagtcaccga 20
<210> 70
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
ttatacgtga aaaacttgcg 20
<210> 71
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
atcaacaatt ctgttcaacc 20
<210> 72
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
gtggttctaa ttcgtgccct 20
<210> 73
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
ccatagcaac agaccatgtg 20
<210> 74
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
ggcttttacg gtgactatta 20
<210> 75
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
ttcgtcatta gggtcaagtt 20
<210> 76
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 76
tactggatgt tgtggcatgt 20
<210> 77
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 77
ttccacttaa aatcgtcaga 20
<210> 78
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 78
gttcttctcc ttcagcattc 20
<210> 79
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 79
ttatcagtct tggggacctt 20
<210> 80
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 80
tggtcctcgc tcagtttcta 20
<210> 81
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 81
tagaaggcac agtcgagtcc 20
<210> 82
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 82
acctcggttc tatcgattgg ccaccatggc cactgttaat tgg 43
<210> 83
<211> 45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 83
cagatcctct tcagagatga gtttctgctc attaataatc tcatc 45
Claims (9)
1. a kind of system for saving chitling road A type coronavirus characterized by comprising
Recombination transcription vector comprising chitling road A type coronavirus complete genome DNA；
Helper plasmid comprising SeACoV-N gene,
Wherein, the chitling road A type coronavirus is single strand plus RNA virus, the chitling road A type coronavirus full genome
Group DNA refers to the corresponding DNA sequence dna of chitling road A type coronavirus positive chain RNA sequence, and the sequence of the SeACoV-N gene is
Chitling road A type coronavirus encodes the corresponding DNA sequence dna of RNA sequence of the gene.
2. the system as claimed in claim 1, which is characterized in that the recombination transcription vector is pSB, the auxiliary using plasmid
The plasmid that plasmid uses is pRK5.
3. the system as claimed in claim 1, which is characterized in that the rescue chitling road A type coronavirus full-length genome cDNA
24222-24224 bit base TCT is sported by AGT.
4. a method of rescue chitling road A type coronavirus, which comprises the following steps:
(1) by any system of claims 1 to 3 recombination transcription vector and helper plasmid cotransfection enter host cell
In；
(2) chitling road A type coronavirus obtained by the rescue in cell and supernatant is collected after cultivating.
5. method as claimed in claim 4, which is characterized in that recombination transcription vector and helper plasmid are added in step (1)
Mass ratio is 2: 1.
6. method as claimed in claim 4, which is characterized in that the host cell is BHK-21 cell.
7. the chitling road A type coronavirus obtained using method as claimed in claim 4 rescue.
8. the chitling road A type coronavirus that rescue obtains as claimed in claim 7 is studied as chitling road A type coronavirus
Application in model.
9. the chitling road A type coronavirus that rescue obtains as claimed in claim 7 answering in preparation prevention diarrhea of pigs vaccine
With.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114657213A (en) * | 2022-05-23 | 2022-06-24 | 华南农业大学 | Porcine acute diarrhea syndrome coronavirus artificial chromosome recombinant vector and construction method and application thereof |
| CN114752710A (en) * | 2022-05-26 | 2022-07-15 | 中国农业科学院都市农业研究所 | CrRNA, kit and method for rapid visual detection of SADS-CoV |
| CN115287268A (en) * | 2022-05-19 | 2022-11-04 | 浙江大学 | Method for proliferating porcine acute diarrhea syndrome coronavirus on porcine intestinal organoid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107630109A (en) * | 2017-10-27 | 2018-01-26 | 华南农业大学 | A kind of fluorescence quantification PCR primer and kit for detecting Novel pig acute diarrhea syndrome coronavirus |
| CN108384762A (en) * | 2018-01-24 | 2018-08-10 | 广东温氏食品集团股份有限公司 | Pig α enteric coronavirus virus and its cultural method and application |
| CN108411040A (en) * | 2018-05-21 | 2018-08-17 | 浙江大学 | Pig acute diarrhea syndrome coronavirus Primer composition and its kit and method |
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2019
- 2019-07-17 CN CN201910645552.0A patent/CN110468155B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107630109A (en) * | 2017-10-27 | 2018-01-26 | 华南农业大学 | A kind of fluorescence quantification PCR primer and kit for detecting Novel pig acute diarrhea syndrome coronavirus |
| CN108384762A (en) * | 2018-01-24 | 2018-08-10 | 广东温氏食品集团股份有限公司 | Pig α enteric coronavirus virus and its cultural method and application |
| CN108411040A (en) * | 2018-05-21 | 2018-08-17 | 浙江大学 | Pig acute diarrhea syndrome coronavirus Primer composition and its kit and method |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115287268A (en) * | 2022-05-19 | 2022-11-04 | 浙江大学 | Method for proliferating porcine acute diarrhea syndrome coronavirus on porcine intestinal organoid |
| CN115287268B (en) * | 2022-05-19 | 2023-08-25 | 浙江大学 | Method for proliferation of porcine acute diarrhea syndrome coronavirus on porcine intestinal organoids |
| CN114657213A (en) * | 2022-05-23 | 2022-06-24 | 华南农业大学 | Porcine acute diarrhea syndrome coronavirus artificial chromosome recombinant vector and construction method and application thereof |
| CN114657213B (en) * | 2022-05-23 | 2022-08-02 | 华南农业大学 | Porcine acute diarrhea syndrome coronavirus artificial chromosome recombinant vector and construction method and application thereof |
| CN114752710A (en) * | 2022-05-26 | 2022-07-15 | 中国农业科学院都市农业研究所 | CrRNA, kit and method for rapid visual detection of SADS-CoV |
| CN114752710B (en) * | 2022-05-26 | 2022-12-06 | 中国农业科学院都市农业研究所 | CrRNA, kit and method for rapid visual detection of SADS-CoV |
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| CN110468155B (en) | 2021-05-14 |
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