CN110468155B - System, method and application for rescuing porcine intestinal tract type A coronavirus - Google Patents
System, method and application for rescuing porcine intestinal tract type A coronavirus Download PDFInfo
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Abstract
The invention discloses a system, a method and application for saving porcine intestinal tract type A coronavirus. The system comprises: a recombinant transcription vector comprising porcine entero-A coronavirus whole genome DNA; a helper plasmid comprising the SeACoV-N gene. The invention successfully constructs the SADS-CoV/SeACoV reverse genetic operating system, the generated virus can be continuously passaged, and the rescued virus and the parental virus have consistent biological characteristics. The establishment of a reverse genetics system is hindered by the instability and toxicity of a virus genome sequence, and the rescue method has potential application value in the research of the reverse genetics of the porcine coronavirus and the creation of a vaccine, and has wide application prospect in the aspects of the research of virus gene functions and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a system, a method and application for rescuing porcine intestinal tract type A coronavirus.
Background
China is the first pig-raising kingdom in the world, and large-scale cultivation and development are rapid. However, along with the rapid development of large-scale pig raising, the harm of coronavirus infection to the pig raising industry is larger and larger. Therefore, the understanding of infection and pathogenesis of the porcine coronavirus is strengthened, and a new theory and technical support is provided for the prevention and control of the epidemic diseases.
The porcine enteric Coronavirus (SeACoV) which is discovered for the first time in the south China in 2017 is also called porcine acute diarrhea Syndrome virus (SADS-CoV) which causes severe diarrhea of piglets and is a novel Coronavirus with the evolutionary origin of bat HKU 2-CoV. The S protein of SADS-CoV/SeACoV is highly variant compared to HKU2 and is mainly concentrated in amino acids 1-238 of the N-terminal region (NTD, domain 0): this region had 75 amino acid point mutations and 2 amino acid insertions. We speculate that a high degree of variation in NTD may result in a variation from bat coronavirus HKU2 to pig SeACoV, thereby spreading from bat to pig across species. However, the current study of this virus is almost blank.
Porcine diarrhea-associated viruses pose a significant threat to the global swine industry, and a greater understanding of such viruses is needed. The major problem faced at present is the lack of genetic tools suitable for studying porcine coronavirus SADS-CoV/SeACoV.
Viruses of the family coronaviridae are single-stranded positive-strand RNA viruses whose genomes have overlapping ORFs encoding replicase (ORF1a, 1b), nonstructural proteins and ORF3, spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The study of the pathogenesis of coronaviruses is currently largely limited by the isolation of cultures and the lack of tools for the inheritance of operable viruses. Compared to other viruses, coronaviruses have genomes of about 27-30kb in length, the largest of the RNA viruses, which present significant challenges in engineering vectors for generating infectious clones, and discrimination between natural and amplification-related mutations and sequencing errors can be extremely difficult. Furthermore, attempts to establish reverse genetics systems have been hampered by the instability and toxicity of the viral genomic sequences.
The reverse genetic technology is a useful platform for developing virus research and can play an important role in the elucidation of virus pathogenic mechanisms and vaccine development. Similarly, the construction of the infectious molecular clone of the porcine coronavirus SADS-CoV/SeACoV can be used for carrying out more effective research on the specific gene of the SADS-CoV/SeACoV and is a useful platform for the research of the SADS-CoV/SeACoV.
At present, no report that the reverse genetic system of SADS-CoV/SeACoV is successfully constructed exists at home and abroad.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a virus rescue system capable of successfully rescuing the porcine intestinal tract type A coronavirus.
A system for rescuing porcine enteric coronavirus a, comprising:
a recombinant transcription vector comprising porcine entero-A coronavirus whole genome DNA;
helper plasmid comprising the SeACoV-N gene,
the porcine entero-A coronavirus is a single-strand positive-strand RNA virus, the porcine entero-A coronavirus complete genome DNA is a DNA sequence corresponding to a porcine entero-A coronavirus positive-strand RNA sequence, and the SeACoV-N gene sequence is a DNA sequence corresponding to an RNA sequence of a porcine entero-A coronavirus coding gene.
Since porcine entero-A coronavirus is a single-stranded positive-strand RNA virus, the DNA sequence corresponding to the genomic RNA of the virus used for rescue, i.e., only the base U in the RNA sequence was changed to T.
The sequence size of the SeACoV-N gene is 1125bp (shown as SEQ ID NO. 1), the SeACoV-N protein is expressed, and researches show that the SeACoV-N protein can assist the rescue of the virus in a mode of inhibiting the expression of interferon.
The plasmid used for the recombinant transcription vector is pSB, and the plasmid used for the helper plasmid is pRK 5. Both plasmids are commercially available.
In order to facilitate the detection and the differentiation of the rescued virus and the wild virus, the base 24222 th-24224 th site of the cDNA of the rescued porcine intestinal tract A type coronavirus is mutated from AGT to TCT, and the two codons correspond to coding serine (Ser), so the amino acid Ser is kept unchanged.
The invention also discloses a method for rescuing the porcine intestinal tract type A coronavirus, which comprises the following steps:
(1) co-transfecting the recombinant transcription vector and helper plasmid in the system into a host cell;
(2) collecting the cells after culture and rescuing the obtained porcine intestinal tract type A coronavirus from the supernatant.
The mass ratio of the recombinant transcription vector and the helper plasmid added in the step (1) is 2: 1. The host cell is BHK-21 cell.
The invention also provides a porcine intestinal coronavirus rescued by the method.
The invention also provides application of the rescued and obtained porcine intestinal tract type A coronavirus in a research model of the porcine intestinal tract type A coronavirus.
The invention also provides application of the rescued and obtained porcine intestinal tract type A coronavirus in preparation of a vaccine for preventing porcine diarrhea.
The invention successfully constructs the SADS-CoV/SeACoV reverse genetic operating system, the generated virus can be continuously passaged, and the rescued virus and the parental virus have consistent biological characteristics. The establishment of a reverse genetics system is hindered by the instability and toxicity of a virus genome sequence, and the rescue method has potential application value in the research of the reverse genetics of the porcine coronavirus and the creation of a vaccine, and has wide application prospect in the aspects of the research of virus gene functions and the like.
Drawings
FIG. 1 is a schematic diagram of the structure of a 16-segment gene fragment of porcine enterovirus A-CoV/SeACoV.
FIG. 2 is a diagram showing the amplification result of the gene fragment of porcine enterovirus A SADS-CoV/SeACoV 16.
FIG. 3 is a schematic diagram of construction of porcine enterovirus A SADS-CoV/SeACoV infectious clone plasmid pSB-SeACoV.
FIG. 4 is a schematic diagram of 23 sequencing fragments of porcine enterovirus A-type coronavirus SADS-CoV/SeACoV infectious clone plasmid.
FIG. 5 is a diagram showing the result of amplification and identification of 23 segments of porcine enteric coronavirus SADS-CoV/SeACoV infectious clone plasmid.
FIG. 6 shows the results of immunofluorescence assay of porcine enterovirus A, SADS-CoV/SeACoV infectious clone rescue virus using SeACoV-N protein antibody, wherein A shows the group of viruses rescued by separately transfecting infectious clone plasmid pSB-SeACoV, and B shows the group of viruses rescued by co-transfecting infectious clone plasmids pSB-SeACoV and pRK 5-SeACoV-N.
FIG. 7 is a PCR detection result chart of the molecular Marker in ORF3 gene of rescued virus rSeACoV located in TF21 fragment, in which lane 1 is a control, lane 2 is the supernatant at the time of virus rescue, lane 3 is the intracellular at the time of virus rescue, and lane M is a standard molecular weight Marker.
FIG. 8 is a diagram showing the sequencing result of the Ser molecular marker of rescued virus rSeACoV located on ORF 3.
FIG. 9 is a graph showing the results of comparison of growth curves for detecting rescued viruses and parental viruses.
FIG. 10 is a graph showing the results of observation of virus-rescued virus particles under an electron microscope.
Detailed Description
Example 1
Amplification of fragments of interest of full-Length infectious clones of SADS-CoV/SeACoV
According to the gene sequence of the pig enterovirus A SeACoV CH/GD-01/2017/P2 strain published on Genbank (Genbank accession No. MF370205, figure 1), the full length of the viral genome is 27155nt, and the homology of HKU2 of the bat enterovirus A of the Chinese horsetail bats, which is found in the same region 10 years ago and infected with the bats, is 94.9% at the whole genome nucleic acid level. Cell infection supernatant samples of cell stable passage strains SADS-CoV/SeACoV-P10 are subjected to reverse transcription after RNA is extracted by a Trizol method to obtain cDNA, 16 pairs of primers (the primer sequences are shown in Table 1) provided by the invention are used for amplification to obtain corresponding 16 genome full-length fragments, cloning is carried out through a pCR-Blunt vector, 3 monoclonal colonies are picked, plasmid sequencing and splicing are extracted, and the genome full-length sequence of the SADS-CoV/SeACoV-P10 is determined. Corresponding 16 primer pairs were used to amplify the corresponding full-length fragmented segments of the whole genome using the preserved pCR-Blunt plasmid with 16 SADS-CoV/SeACoV fragments as template (FIG. 2).
TABLE 1
In order to ligate the full-length SADS-CoV/SeACoV fragments into the vector, it is required that the 5 'end of the SeACoV-F1 fragment and the 3' end of the vector fragment, and the 3 'end of the SeACoV-F15 fragment and the 5' end of the vector fragment have at least 20bp of homologous regions for homologous recombination. Primer pairs SeACoV-F1 and SeACoV-F15 were designed with 5 'and 3' homologous sequences added to the fragments, respectively. The primer sequences are as follows:
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’。
meanwhile, a synonymous mutation of an amino acid is introduced into the ORF3 gene as a detection molecular Marker for rescuing the virus, namely, the base 24222-24224 is mutated from AGT to TCT, the amino acid Ser is kept unchanged (figure 3), a gene segment between the base 23403-24239 and the base 24207-25298 of the porcine intestinal tract A coronavirus SeACoV-F14 is amplified, the amplified 2 gene segments are fused by an overlapping PCR method to obtain a fusion segment SeACoV-F14-Marker, and a molecular Marker is introduced into the ORF3 gene positioned in the SeACoV-F14 segment, wherein the primer sequence is as follows:
SeACoV-F14-Marker-a segment upstream primer SeACoV-F14-F:
5’-ATTTGCTAATGTCATTGCCGTTTCC-3’；
SeACoV-F14-Marker-a segment downstream primer SeACoV-Marker-a-R:
5’-GAGAACAAAAGCAAAAGACCTG-3’；
SeACoV-F14-Marker-b segment upstream primer SeACoV-Marker-b-F:
5’-GCTTTTTGTTACAGGTCTTTTGC-3’；
SeACoV-F14-Marker-b segment downstream primer SeACoV-F14-R:
5’-GGCGACAGTCACAAATTGCGGTA-3’。
splicing of full-Length infectious clones of SADS-CoV/SeACoV
The SADS-CoV/SeACoV DNA fragments and the linearized pSB vector (with the insertion site at the downstream of the CMV promoter) are connected into a full-length infectious clone recombinant plasmid by means of in vitro homologous recombination by using a GBclonart seamless cloning kit. Positive recombinant plasmid clones were screened and amplified by transformation of the ligation products into DH 10B.
Adding the total 16 amplified SeACoV-F1-F15 fragments and linearized vector fragments into a centrifuge tube in equal proportion, and operating according to the step requirements of the GBclonart seamless cloning kit, wherein the reaction system is as follows:
after mixing and incubation at 45 ℃ for 2h, transfer to ice. DH10B competent cells were transformed immediately.
Validation of full-Length infectious clones of SADS-CoV/SeACoV
Individual clones were randomly picked, inoculated into 5mL LB liquid medium containing chloramphenicol (30mg/mL) resistance, shake-cultured at 37 ℃ and extracted with SADS-CoV/SeACoV full-length infectious clone plasmid obtained by cloning according to AxyPrep plasmid DNA minikit instructions. In order to perform PCR amplification verification and subsequent sequencing verification on the full-length infectious clone, the full-length genome sequence is divided into 23 segments, each of which is about 1400bp, so that the sequencing can be performed only by two upstream and downstream reactions (FIG. 4).
The PCR verification of the extracted full-length infectious clone plasmid was performed with the 23 PCR sequencing primers shown in Table 2. Taking 5 mu L of PCR product to carry out electrophoresis detection on 1% agarose gel, selecting positive clone, sending each section of PCR product of the clone 1-23 to Hipposhu biotechnology limited company for sequencing, and storing the full-length infectious clone glycerobacteria with correct sequencing (figure 5). The obtained recombinant BAC is named pSB-SeACoV, and the porcine intestinal coronavirus SADS-CoV/SeACoV full-length infectious clone plasmid with the molecular marker is obtained.
TABLE 2
4. Virus rescue
4.1 Large extract of infectious clone plasmid of SADS-CoV/SeACoV
Preparing 1L of 2 XYT liquid medium, inoculating glycerol to 2 XYT liquid medium containing chloramphenicol (30mg/mL) resistance at a ratio of 1: 100, and shake-culturing at 37 deg.C and 200rpm for 12 h. SeACoV infectious clone plasmids were extracted according to the BAC/PAC DNA Isolation Maxi Kit instructions and stored at-20 ℃ until use.
4.2 construction of pRK5-SeACoV-N plasmid
According to the upstream primer SeACoV-N-F:
5’-ACCTCGGTTCTATCGATTGGCCACCATGGCCACTGTTAATTGG-3’；
the downstream primer SeACoV-N-myc-R:
5’-CAGATCCTCTTCAGAGATGAGTTTCTGCTCATTAATAATCTCATC-3’；
the plasmid pSB-SeACoV was used as a template to amplify a SeACoV-N fragment, and the plasmid pRK5 was linearized by a double digestion reaction using two digestion sites, EcoR I and Xba I. And (3) connecting the linearized pRK5 plasmid and the SeACoV-N fragment by using a seamless cloning kit, transforming the connection product into Top10 competence, selecting a single clone colony, extracting plasmid sequencing verification, and successfully constructing the pRK5-SeACoV-N plasmid.
4.3SADS-CoV/SeACoV transfection rescue
BHK-21 cells were plated in 12-well plates and placed in 5% CO prior to transfection2And culturing in an incubator at 37 ℃ until the cell density reaches about 70 percent, and performing transfection. Discarding the cell culture solution during transfection, washing twice with Opti-MEM, using transfection reagent as negative control, and mixing infectious clone plasmid with SADS-CoV/SeACoV-N protein expression plasmid
3000 transfection kit instructions for cotransfection, in which infectious clone plasmid pSB-SeACoV was added at 2. mu.g/well, plasmid pRK5-SeACoV-N was added at 1. mu.g/well, fluid was changed 7h after transfection, day by day observation until cytopathic effect appeared, repeated freeze-thawing to collect the rescued virus supernatant, named rSeACoV. Meanwhile, another transfection experiment group was supplemented with 2. mu.g/well of infectious clone plasmid pSB-SeACoV alone, and without pRK5-SeACoV-N plasmid as a control.
Vero cells were plated on 12-well plates and placed in 5% CO2Culturing at 37 deg.C in incubatorInfection was carried out until the cell density reached about 70%, and 3 days after infection, virus rescue was detected by indirect immunofluorescence assay (IFA) using a SADS-CoV/SeACoV-N protein rabbit polyclonal antibody (FIG. 6). The Vero cells are infected by the supernatant of the rescued viruses of the infectious clone plasmid pSB-SeACoV rescued virus group alone, the expression of the SADS-CoV/SeACoV-N protein is not detected, while the Vero cells are infected by the supernatant of the rescued viruses of the infectious clone plasmid pSB-SeACoV and pRK5-SeACoV-N plasmid cotransfection group, and the indirect immunofluorescence detection can obviously observe the green fluorescence signal of the expression of the SADS-CoV/SeACoV-N protein, thereby proving the successful rescue of the rSeACoV virus.
RT-PCR identification result and sequencing verification of rSeACoV
Total RNA was extracted from the supernatant and cell samples of rSeACoV-infected Vero cells and reverse-transcribed to cDNA. According to the ORF3 gene which is used as a detection molecular marker for rescuing viruses and introduces a synonymous mutation of one amino acid, the fragment is located in the TF21 fragment with the full-length genome sequence divided into 23 segments, the primer of the TF21 fragment is used for amplifying the fragment,
wherein, the upstream primer SeACoV-TF 21: 5'-TACTGGATGTTGTGGCATGT-3', respectively;
the downstream primer SeACoV-TR 21: 5'-TTCCACTTAAAATCGTCAGA-3' the flow of the air in the air conditioner,
the PCR products were sent to the Shanyabiotech Co., Ltd for sequencing (FIG. 7, FIG. 8). According to the sequencing result, the porcine intestinal coronavirus SADS-CoV/SeACoV with the molecular marker, which can be stably passaged, is successfully rescued and obtained.
The growth curves of the original virus SADS-CoV/SeACoV and the rescued virus rSeACoV were tested by TCID50, and both have the same growth kinetics, thus ensuring that the generated virus can be continuously passaged and the rescued virus and the parental virus have the same biological characteristics (FIG. 9). After separation and purification of the rescued virus by scale-up culture, typical whole coronavirus particles were observed by observing the virions under an electron microscope (FIG. 10).
Sequence listing
<110> Zhejiang university
<120> system, method and application for rescuing porcine intestinal tract type A coronavirus
<160> 83
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1125
<212> DNA
<213> porcine enteric coronavirus (swine enterovirus alphacoronavirus)
<400> 1
atggccactg ttaattgggg tgacgctgtt gaacaggcgg aatctcgtgg tcgtaaaaga 60
attccattgt cactctttgc gcctttgcgt gttatagatg gcaaaaactt ttggaatgtc 120
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
aatcgtaaca aagaacctgc tacacctcag tttgggtttc aattaccacc agacctgact 420
gttgttgagg ttacttctag aagtgcttca cgttcacagt ctcgttctcg caatcaaagt 480
caaagccgca gtggtgctca gacacctcgt gctcaacagc cgtcacagtc tgttgacatt 540
gttgctgcag ttaaacaagc tttggcagac ttgggcatag cttctagcca gtccaggcct 600
caaagtggta aaaatacacc caaaccaaga agcagagctg tctcacctgc acctgcccct 660
aaaccggctc gtaaacagat ggacaaacct gaatggaagc gtgttcctaa ttctggggag 720
gacgtgcgta aatgctttgg tcctcgctca gtttctagaa attttggtga cagtgacctc 780
gttcagcacg gtgttgaagc taagcacttt ccaacaattg ctgagttgct tccgacacaa 840
gctgcactag cctttggtag tgaaatcaca accaaagagt ctggtgaatt tgtagaagtc 900
acctatcact atgtaatgaa ggtccccaag actgataaaa atctacccag atttcttgag 960
caagtctcgg cttactctaa acccagtcaa attaggagat ctcaatctca acaagaccta 1020
aatgctgatg ccccagtgtt cactccggca cctccagcta ctccagtttc ccaaaatcct 1080
gcttttcttg aggaggaggt tgagatggtg gatgagatta ttaat 1125
<210> 2
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ataagcagag ctcgtttagt gaaccgtgac ttaaagatat aa 42
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gtcatcacag agggcagtaa agc 23
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gcattcagtg ttgttgacgg c 21
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gcaccgctaa gttcttcgaa g 21
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gatgttgcac attgtttaga ggta 24
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gtaacttatt ccacaaatcc tcc 23
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
caattgctgg gttaatgcga c 21
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aaatgcctta tgcaaagcac c 21
<210> 10
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gtttatctct cacaacttct gtgt 24
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
attgataaga cgctcataag aac 23
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gccatggtgg ttgcttacat 20
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gcacaacatt ggcacactta ag 22
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gtccttttga ctctgtatta cttag 25
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tttgttatac atggactgct cgt 23
<210> 16
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
caagcacgat gccttctttg ttatt 25
<210> 17
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tttgaaccga gaaccatagc agc 23
<210> 18
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tcctaaatgt gatagagcta tgcct 25
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aataatacgt gagcatctgt cta 23
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gtggcaaatc acattgtgtt 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gttcatgtca aaacggaagc 20
<210> 22
<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 system for rescuing porcine enteric coronavirus a, comprising:
a recombinant transcription vector comprising porcine entero-A coronavirus whole genome DNA;
helper plasmid comprising the SeACoV-N gene,
the porcine entero-A coronavirus is a single-strand positive-strand RNA virus, the porcine entero-A coronavirus complete genome DNA is a DNA sequence corresponding to a porcine entero-A coronavirus positive-strand RNA sequence, the SeACoV-N gene sequence is a DNA sequence corresponding to an RNA sequence of the porcine entero-A coronavirus coding gene, and the SeACoV-N gene sequence is shown as SEQ ID NO. 1.
2. The system of claim 1, wherein the recombinant transcription vector uses a plasmid pSB and the helper plasmid uses a plasmid pRK 5.
3. The system of claim 1, wherein the whole genomic DNA of porcine Enterovirus A has the mutation at position 24222-24224 from AGT to TCT.
4. A method for rescuing porcine enteric coronavirus a, comprising the steps of:
(1) co-transfecting the recombinant transcription vector and helper plasmid of the system of any one of claims 1 to 3 into a host cell;
(2) collecting the cells after culture and rescuing the obtained porcine intestinal tract type A coronavirus from the supernatant.
5. The method of claim 4, wherein the recombinant transcription vector and the helper plasmid are added in step (1) in a mass ratio of 2: 1.
6. The method of claim 4, wherein the host cell is a BHK-21 cell.
7. The method for rescuing the porcine enteric coronavirus A is used for rescuing the porcine enteric coronavirus A, wherein the method comprises the following steps:
(1) co-transfecting the recombinant transcription vector and helper plasmid of the system of claim 3 into a host cell;
(2) collecting the cells after culture and rescuing the obtained porcine intestinal tract type A coronavirus from the supernatant.
8. The application of the rescued porcine entero-type A coronavirus of claim 7 as a research model of the porcine entero-type A coronavirus.
9. The use of the rescued porcine entero-type A coronavirus of claim 7 in the preparation of a vaccine for the prevention of porcine diarrhea.
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