CN112852873A - Construction method of porcine delta coronavirus infectious clone plasmid - Google Patents

Abstract

The invention discloses a construction method of porcine delta coronavirus infectious clone plasmid, belonging to the field of animal infectious disease control and biotechnology. The invention is based on a BAC system and a homologous recombination one-step cloning method to save a reverse genetic system of a PDCoV CHN-HN-2014 strain recombinant virus, 5 gene segments of a strain are cloned into an intermediate vector pBAC-M-PDCoV in one step to obtain a recombinant plasmid pBAC-CHN-HN-2014 containing the PDCoV CHN-HN-2014 strain full-length cDNA, and the recombinant virus rCHN-HN-2014 is saved after cells are transfected, thereby establishing a porcine delta coronavirus reverse genetic operation platform and laying a foundation for deeply researching the infection and pathogenic mechanism of the PDCoV. Meanwhile, aiming at the actual problem that no available PDCoV vaccine exists in China at present, the PDCoV infectious clone platform can be used for developing a safer and more efficient novel PDCoV genetic engineering vaccine, and can also be used as a live virus vector for developing a multi-connected vaccine, so that the PDCoV infectious clone platform has great value for the research related to the virus.

Description

Construction method of porcine delta coronavirus infectious clone plasmid

Technical Field

The invention belongs to the field of animal infectious disease control and biology. In particular to a construction method of porcine delta coronavirus infectious clone plasmid.

Background

Porcine delta coronavirus (PDCoV) belongs to the family of coronaviridae, belongs to the genus of delta coronavirus, has a cyst membrane, has single-stranded positive-strand RNA as a genome, and is the only delta coronavirus which is successfully separated at present. The porcine delta coronavirus was first discovered by Woo et al when samples collected from different animals in 2010-2011 in hong Kong area, China (Woo P.C.Y., Discovery of seven novel mammalia and alias coronaviruses in the gene deltacoronaviruses subports of genes as the gene source of alphacoronaviruses and aconaviruses and alias as the gene source of gamma-oronaviruses and deltacoronaviruses. J.Virol.2012,86: 95-4008). PDCoV infection first breaks out in the United states in early 2014 and quickly reaches a plurality of states in the United states, and then countries such as Korea, Canada, China, Thailand, Vietnam, Laos, Japan also successively report that PDCoV is detected (Wang L et al, Port coronavirus HKU15 detected in 9US states,2014, Emerg Infect Dis 2014,20(9): 1594-. Until now, a plurality of topic components are separated to PDCoV at home and abroad, animal experiments prove that the PDCoV has stronger pathogenicity to piglets, can cause the piglets to have typical intestinal disease symptoms such as vomit, diarrhea and the like, and the cesarean examination shows that the intestinal wall of the infected piglets becomes thin and transparent, and cecum and colon are dilated and accompanied by a large amount of yellow liquid; histopathological analysis found severe atrophy of the villi of the duodenum, jejunum, ileum (Ma Y M et al, Origin, evolution, and vision of soil deltacononavirus in the United states. MBIO 2015,6(2): e 00064; Dong et al, Isolation, genetic characterization, and pathology of a Chinese soil deltacononavirus strain CHN-HN-2014.Vet Microbiol.2016,196: 98-106). Although the mortality rate caused by the PDCoV infection is lower compared with the Porcine Epidemic Diarrhea Virus (PEDV) and the transmissible gastroenteritis virus (TGEV), the PDCoV infection and the PEDV and the TGEV have mixed infection phenomena, thereby causing more serious clinical symptoms and threatening the healthy development of the pig industry. As a newly discovered porcine enterocoronavirus in recent years, the number of PDCoV strains passaged in vitro is still small relative to PEDV, the mechanism of PDCoV infection and pathogenicity is not completely understood, and no available vaccine or specific antiviral drug exists, so that the method has important value in further increasing epidemiological investigation, separating clinical strains and researching pathogenic mechanisms.

The reverse genetic operation system of the virus can realize virus modification by modifying a target gene, including mutation, deletion or insertion of foreign genes and other operations, research the function of virus coding protein, determine virulence genes and provide a technical platform for research and development of novel vaccines and live virus vector vaccines. The current methods for constructing reverse genetic operation systems of positive-strand RNA viruses mainly comprise 4 methods: 1) constructing a reverse genetic operation system of the virus by a targeted RNA recombination technology; 2) using a Bacterial Artificial Chromosome (BAC) system; 3) constructing a reverse genetic operation system of the virus by using an in vitro cDNA connection method; 4) infectious clones of the virus were constructed using vaccinia virus as a vector (Almaz n F, Coronavir reverse genetic systems: infectious clones and replication. Virus Res.2014,189: 262-; masters PS, Coronavir reverse genetics by targeted RNA recombination. curr Top Microbiol Immunol.2005,287: 133-. However, the construction of PDCoV infectious clone mainly adopts an in vitro cDNA connection method, which connects a plurality of cDNA fragments of virus by using type II restriction enzyme, obtains full-length RNA by using T7 RNA polymerase in vitro transcription, and electrically transfers the full-length RNA to a sensitive cell line to save recombinant virus. The method for rescuing the virus generally needs to simultaneously electrically transfer the N gene transcript of the corresponding virus so as to improve the rescuing efficiency, and meanwhile, T7 RNA polymerase can generate mutation during working, which influences the rescuing efficiency of the method to a certain extent. The BAC system does not involve in vitro connection and transcription, the rescue efficiency of the recombinant virus is high, and the mature CRISPR/Cas9 technology is added, so that the recombinant plasmid can be efficiently reconstructed, and the system has stronger advantages. In addition, the existing reverse genetic operation system constructed based on the BAC system mostly adopts a multi-step homologous recombination method for construction strategies, and the period is relatively long. Based on the defects and shortcomings of the construction of the PDCoV reverse genetic operation system, a BAC system and a homologous recombination one-step cloning method are adopted to quickly construct PDCoV infectious clone plasmids, transfect susceptible cells to save recombinant viruses, and establish a good foundation for the subsequent research of PDCoV.

Disclosure of Invention

The invention mainly aims to provide a construction method of porcine delta coronavirus infectious clone plasmids. The invention aims at porcine delta coronavirus CHN-HN-2014 strain (GenBank accession number: KT336560) (Dong N, Fan L, Yang H, Liu H, Du T, Fan P, Wang D, Chen H, Xiao S.isolation, genomic characterization, and pathogenesis of a porcine gene of porcine gene transcriptional and transcriptional mutant strain CHN-HN-2014.Vet Microbiol.2016,196:98-106), and BAC system and homologous recombination one-step cloning method are adopted to obtain porcine delta coronavirus CHN-HN-2014 strain infectious clone plasmid pBAC-CHN-HN-PDC and recombinant virus rCHN-HN-2014, thereby providing a very valuable technical platform for the subsequent research of the oV.

The technical scheme of the invention is as follows:

a method for constructing infectious clone plasmids of porcine delta coronavirus comprises the following steps:

(1) extracting RNA of the porcine delta coronavirus and performing reverse transcription to obtain cDNA;

(2) using the cDNA as a template, designing primers to carry out PCR amplification respectively to obtain a 5 'end gene sequence, a 3' end gene sequence and 5 gene fragments of the porcine delta coronavirus, wherein the 5 gene fragments contain a homologous sequence of 20bp, and in addition, a 15121 site C silencing mutation is changed into A, so that a ClaI endonuclease sequence is eliminated and is used as a genetic marker;

(3) taking pBAC-AJ1102 plasmid as a template, designing primers, and respectively carrying out PCR amplification to obtain a CMV promoter and an HDV-BGH gene sequence;

(4) cloning a CMV promoter, a 5 'end gene sequence, a 3' end gene sequence, a poly (A) structure of 27 adenine deoxynucleotides (A), a hepatitis C virus (HDV) ribozyme self-cleavage site and a bovine growth hormone termination sequence (BGH) into a low-copy vector pBeloBAC11 by using an ApaLI and HindIII restriction endonuclease and a multi-step fusion PCR method by using a BAC system to obtain an intermediate vector pBAC-M-PDCoV;

(5) 5 gene segments are subjected to homologous recombination in proportion and are cloned into an intermediate vector pBAC-M-PDCoV in one step to obtain a recombinant plasmid pBAC-CHN-HN-2014;

(6) transforming the recombinant product into DH10B chemically competent cells, screening positive clones by PCR, carrying out amplification culture on positive clones with correct sequencing, and extracting plasmids.

The 5 'end gene sequence, the 3' end gene sequence and the sequences of the 5 gene segments are respectively as follows:

5' end gene sequence: as shown in SEQ ID NO. 1;

3' end gene sequence: as shown in SEQ ID NO. 2;

gene fragment A: as shown in SEQ ID NO. 3;

gene fragment B: as shown in SEQ ID NO. 4;

gene fragment C: as shown in SEQ ID NO. 5;

gene fragment D: as shown in SEQ ID NO. 6;

gene fragment E: shown as SEQ ID NO. 7.

The primers for PCR amplification of 5 'end gene sequence, 3' end gene sequence and 5 gene segments are respectively as follows:

PDCoV-5’-F：SEQ ID NO:11

PDCoV-5’-R：SEQ ID NO:12

PDCoV-3’-F：SEQ ID NO:13

PDCoV-3’-R：SEQ ID NO:14

A-F：SEQ ID NO:15

A-R：SEQ ID NO:16

B-F：SEQ ID NO:17

B-R：SEQ ID NO:18

C-F：SEQ ID NO:19

C-R：SEQ ID NO:20

D-F：SEQ ID NO:21

D-R：SEQ ID NO:22

E-F：SEQ ID NO:23

E-R：SEQ ID NO:24。

the sequence of the CMV promoter is shown as SEQ ID NO. 8, and the sequence of the HDV-BGH gene is shown as SEQ ID NO. 9.

The primers for PCR amplification of the CMV promoter and the HDV-BGH gene are respectively as follows:

CMV-F：SEQ ID NO:25

CMV-R：SEQ ID NO:26

HDV-F：SEQ ID NO:27

BGH-R：SEQ ID NO:28。

in the step (5), the molar ratio of the 5 gene segments to the intermediate vector pBAC-M-PDCoV is 1:2:2:1:1: 1.

The method comprises the following specific steps:

(1) extracting RNA of a PDCoV CHN-HN-2014 strain and preparing cDNA;

(2) amplification and cloning of related genes of the PDCoV CHN-HN-2014 strain: the cDNA is taken as a template, and PCR amplification is carried out by utilizing primer pairs PDCoV-5 '-F/R (SEQ ID NO:11, SEQ ID NO:12), PDCoV-3' -F/R (SEQ ID NO:13, SEQ ID NO:14), A-F/R (SEQ ID NO:15, SEQ ID NO:16), B-F/R (SEQ ID NO:17, SEQ ID NO:18), C-F/R (SEQ ID NO:19, SEQ ID NO:20), D-F/R (SEQ ID NO:21, SEQ ID NO:22), E-F/R (SEQ ID NO:23, SEQ ID NO:24) to obtain corresponding gene segments. Wherein, A (348) and 4538 and 4191bp), B (4519 and 9652 and 5134bp), C (9633 and 15132 and 5500bp), D (15113 and 19455 and 4343bp) and E (19436 and 23403 and 3968bp) gene fragments contain 20bp homologous sequences, and in addition, 15121 site C is silenced and mutated into A by primer design, a ClaI endonuclease sequence (ATCGAT) is eliminated, and the ClaI endonuclease sequence is used as a genetic marker to distinguish the parental virus from the recombinant virus. PCR amplification is respectively carried out by taking pBAC-AJ1102(PEDV AJ1102 strain cDNA clone plasmid, stored in a laboratory) constructed in a laboratory as a template and CMV-F/R (SEQ ID NO:25, SEQ ID NO:26) and HDV-F/BGH-R (SEQ ID NO:27, SEQ ID NO:28) as primer pairs to obtain a CMV promoter and an HDV-BGH gene sequence. These gene fragments were cloned into pJET1.2/blunt vector for sequencing analysis, and the plasmids were named pJET1.2-CMV, pJET1.2-PDCoV-5 '(1-375), pJET1.2-PDCoV-3' (23365-25421bp), pJET1.2-A, pJET1.2-B, pJET1.2-C, pJET1.2-D, pJET1.2-E, and pJET1.2-HDV-BGH, respectively.

(3) Construction of intermediate vector (pBAC-M-PDCoV): cloning CMV promoter, 5 'end gene sequence (1-375bp, 3' end containing AvrII restriction endonuclease), 3 'end gene sequence (23365-25421bp, 5' end containing BamHI restriction endonuclease), poly (A) structure of 27 adenine deoxynucleotides (A), hepatitis C virus (HDV) ribozyme self-cutting site and bovine growth hormone termination sequence (BGH) into a low-copy vector pBeloBAC11 by using ApaLI and HindIII restriction endonuclease and a multi-step fusion PCR method to obtain an intermediate vector pBAC-M-PDCoV;

(4) construction of a full-length cDNA cloning plasmid (pBAC-CHN-HN-2014) of the PDCoV CHN-HN-2014 strain: PCR amplification of 5 gene fragments, A (SEQ ID NO:3), B (SEQ ID NO:4), C (SEQ ID NO:5), D (SEQ ID NO:6), E (SEQ ID NO:7), each containing 20bp of homologous sequence between each fragment, using the correctly sequenced pJET1.2-A, pJET1.2-B, pJET1.2-C, pJET1.2-D, pJET1.2-E plasmids as templates, A-F/R (SEQ ID NO:15, SEQ ID NO:16), B-F/R (SEQ ID NO:17, SEQ ID NO:18), C-F/R (SEQ ID NO:19, SEQ ID NO:20), D-F/R (SEQ ID NO:21, SEQ ID NO:22), E-F/R (SEQ ID NO:23, SEQ ID NO:24) as primer pairs, wherein, 20bp homologous arms exist at the 5 'end of the A fragment and the 3' end of the E fragment and the two ends of pBAC-M-PDCoV linearized by AvrII and BamHI respectively. Then, 5 fragments are cloned into pBAC-M-PDCoV in one step through homologous recombination according to a certain proportion to obtain the recombinant plasmid pBAC-CHN-HN-2014.

(5) Transforming the recombinant product into DH10B chemically competent cells, and screening positive clones by using PCR;

(6) carrying out amplification culture on positive clones with correct sequencing, and extracting plasmids;

(7) the extracted plasmid was transfected into LLC-PK1 cells using Lipofectamine 3000 for 4h, and then replaced with serum-free DMEM medium containing 10. mu.g/mL of pancreatin, and the cytopathic effect was observed day by day.

The invention has the beneficial effects that:

the invention provides a PDCoV CHN-HN-2014 strain full-length cDNA infectious clone plasmid and a recombinant virus rCHN-HN-2014 method which are quickly obtained based on a BAC system and a homologous recombination one-step cloning method, and provides a powerful technical platform for deeply researching genetic variation of PDCoV, a potential cross-species propagation mechanism, a virulence gene, a virus infection and pathogenesis, vaccine development and the like. The invention also has the advantages of high homologous recombination efficiency, high positive colony content, short period and the like.

Drawings

FIG. 1: schematic diagram of construction of infectious clone of porcine delta coronavirus CHN-HN-2014 strain.

FIG. 2: pictures of the rescued virus rchnn-HN-2014 were identified by indirect Immunofluorescence (IFA). Description of the drawings: a, picture A: pictures under an inverted microscope of normal virus-uninfected LLC-PK1 cells; and B, drawing: a picture of LLC-PK1 cells infected by a PDCoV wild-type virus CHN-HN-2014 strain under an inverted microscope; and (C) diagram: images of LLC-PK1 cells infected with the PDCoV rescue virus rCHN-HN-2014 strain under an inverted microscope.

FIG. 3: RT-PCR analysis identifies pictures of genetic markers.

FIG. 4: DNA sequencing analyzes pictures of genetic markers.

FIG. 5: the PDCoV rCHN-HN-2014 strain is compared with the CHN-HN-2014 strain in vitro with one-step growth curves.

FIG. 6: rCHN-HN-2014 was compared to CHN-HN-2014 for the plaque phenotype formed on LLC-PK1 cells.

Detailed Description

The present invention is described in detail below with reference to specific examples, but the scope of the present invention is not limited thereto.

Example 1:

1. the formula of the main test materials, carriers, plasmids, reagents and related culture media or solutions:

PDCoV CHN-HN-2014 strain: the virus is separated, identified and stored by the national emphasis laboratory of agricultural microbiology of university of agriculture in Huazhong in different rooms (Dong N, Fan L, Yang H, Liu H, Du T, Fan P, Wang D, Chen H, Xiao S.isolation, genomic characterization, and clinical importance of a Chinese pesticide deltacononavir strain CHN-HN-2014.Vet Microbiol.2016,196: 98-106). Whole genome sequences such as GenBank accession No.: KT 336560.

pBAC-AJ1102 is an infectious clone containing the full-length cDNA of PEDV AJ1102 strain: the virus is constructed and stored in different rooms for the national key laboratory of agricultural microbiology of Huazhong university of agriculture.

LLC-PK1 cell: purchased from American Type Culture Collection (ATCC).

Chloramphenicol (cat # BS 049B): purchased from Biosharp corporation.

Various restriction enzymes: purchased from New England Biolabs.

DMEM medium, T4 DNA ligase (cat # 15224025), Lipofectamine 3000 (cat # L3000015), pJET1.2/blunt vector were purchased from Thermofisiher Scientific, USA.

FastPfu DNA Polymerase was purchased from TransGen Biotech. Transcriptor Reverse Transcriptase (cat # 39083420).

Fetal bovine serum (FBS, cat # P30-3302): purchased from PAN Biotech, usa.

Cycle Pure Kit (cat # D6492-02), Gel Extraction Kit (cat # D2500-02): purchased from Omega Bio-tek, USA.

Infusion Clone Kit (cat # 639648), Plasmid DNA purification Kit (cat # 740410.50): purchased from TAKARA corporation.

DH10B chemically competent cells: purchased from Shanghai super research Biotechnology, Inc.

The pBeloBAC11 vector (cat # P1028) was purchased from Shanghai grain, noon Biotech, Inc.

Preparing an LB culture medium: weighing 5g peptone, 2.5g yeast extract, 5g NaCl in 1L Erlenmeyer flask, adding ddH₂And O is added to 500mL, and the mixture is autoclaved at 121 ℃ for 20 min.

2. Construction of infectious clone plasmid of porcine delta coronavirus CHN-HN-2014 strain

2.1 extraction of PDCoV CHN-HN-2014 strain RNA and preparation of cDNA

The genome RNA of the PDCoV CHN-HN-2014 strain is extracted by using a TRIzol reagent. Taking the extracted RNA as a template, and carrying out reverse transcription by using a Transcriptor First strand cDNA synthesis Kit to obtain cDNA, wherein the reverse transcription conditions are as follows: 1 μ 1 oligo (dT)18 or fragment specific downstream primer, 12 μ L of extracted RNA, supplemented with ddH₂O to the total volume of 13 mu L, uniformly mixing, incubating for 10min at 65 ℃, incubating for 5min on ice, adding 4 mu L of 5 × RT reaction buffer, 2 mu L of dNTP mixture, 0.5 mu L of RNase inhibitor and 0.5 mu L of Transcriptor Reverse Transcriptase, incubating for 30min at 55 ℃, and incubating for 10min at 85 ℃ to obtain cDNA for subsequent experiments.

2.2 amplification and cloning of related genes of PDCoV CHN-HN-2014 strain

PDCoV CHN-HN-2014 strain cDNA is taken as a template, and PCR amplification is respectively carried out on PDCoV-5 '-F/R (SEQ ID NO:11, SEQ ID NO:12), PDCoV-3' -F/R (SEQ ID NO:13, SEQ ID NO:14), A-F/R (SEQ ID NO:15, SEQ ID NO:16), B-F/R (SEQ ID NO:17, SEQ ID NO:18), C-F/R (SEQ ID NO:19, SEQ ID NO:20), D-F/R (SEQ ID NO:21, SEQ ID NO:22), E-F/R (SEQ ID NO:23, SEQ ID NO:24) by utilizing primer pairs to obtain corresponding gene fragments. Wherein, the A (SEQ ID NO:3), B (SEQ ID NO:4), C (SEQ ID NO:5), D (SEQ ID NO:6) and E (SEQ ID NO:7) gene segments contain 20bp homologous sequences, and in addition, the 15121 site C is artificially silenced and mutated into A by primer design, so that a ClaI endonuclease sequence (ATCGAT → ATAGAT) is eliminated as a genetic marker. The PCR reaction system is as follows: 10 μ L of 5 XPCR reaction buffer, 4 μ L of dNTP mix, 10 μ M of upstream and downstream primers, 2 μ L of cDNA template,

FastPfu DNA Polymerase 1. mu.L, plus ddH₂O to a total volume of 50. mu.L. The reaction conditions were as follows: 2min at 95 ℃; 20s at 95 ℃; 20s at 95 ℃,20 s at 58 ℃ and 2min at 72 ℃; after 34 cycles, extension was carried out for 5min at 72 ℃. After the reaction is finished, the amplification product is subjected to 1% agarose Gel electrophoresis, and then is recovered by using Gel Extraction Kit according to the instruction, and the recovered product is the gene segment related to the virus.

PCR amplification is respectively carried out by taking pBAC-AJ1102(PEDV AJ1102 strain cDNA clone plasmid) constructed in a laboratory as a template and CMV-F/R (SEQ ID NO:25, SEQ ID NO:26) and HDV-F/BGH-R (SEQ ID NO:27, SEQ ID NO:28) as primer pairs to obtain a CMV promoter and an HDV-BGH gene sequence.

The obtained gene fragments are respectively cloned into pJET1.2/blunt vector (Thermo scientific) for sequencing analysis, the authenticity of the sequence is ensured, and the fragment cloning reaction system is as follows: 5 mu.L of 2 × Reaction buffer, 4 mu.L of PCR product, 0.5 mu.L of pJET1.2/blunt vector and 0.5 mu. L T4 DNA ligase, the total volume is 10 mu.L, the temperature is 25 ℃ for 30min, then DH5 alpha is transformed, positive clones are picked and sent to Wuhan Pongson biotechnology Limited for sequencing, and positive plasmids are extracted and named as pJET1.2-CMV, pJET1.2-PDCoV-5 '(1-375), pJET 1.2-PDCoV-3' (23365 + 25421bp), pJET1.2-A, pJET1.2-B, pJET1.2-C, pJET1.2-D, pJET1.2-E, pHDT 1.2-V-BGH.

2.3 construction of the intermediate vector pBAC-M-PDCoV:

2.3.1 amplification of the intermediate fragment (M-PDCoV): the fusion fragment 1 is obtained by overlapping extension PCR method of CMV gene fragment obtained by amplification of the CMV-F/R (SEQ ID NO:25, SEQ ID NO:26) primer pair and PDCoV CHN-HN-2014 strain PDCoV 5 'end (1-375bp) gene fragment obtained by amplification of PDCoV-5' -F/R (SEQ ID NO:11, SEQ ID NO:12) primer pair. Overlap extension PCR system: 10 μ L of 5 XPCR reaction buffer, 4 μ L of dNTP mix, 10 μ M of upstream and downstream primers (CMV-F/PDCoV-5 '-R), 2 μ L of DNA template (equimolar amounts of CMV and PDCoV-5'),

fastpfu DNA Polymerase 1. mu.L, plus ddH₂O to a total volume of 50. mu.L. The reaction conditions were as follows:2min at 95 ℃; 20s at 95 ℃; 20s at 95 ℃,20 s at 58 ℃ and 2min at 72 ℃; after 34 cycles, extension was carried out for 5min at 72 ℃. After the reaction is finished, the amplification product is subjected to 1% agarose Gel electrophoresis, and then is recovered by using Gel Extraction Kit according to the instruction, and the recovered product is the fusion fragment 1.

Obtaining a fusion fragment 2 by overlapping extension PCR method of a PDCoV CHN-HN-2014 strain 3 'end (23365-plus 25421bp) gene fragment obtained by amplifying a PDCoV-3' -F/R (SEQ ID NO:13, SEQ ID NO:14) primer pair and an HDV-BGH gene fragment obtained by amplifying an HDV-F/BGH-R (SEQ ID NO:27, SEQ ID NO:28) primer pair; finally, the fusion fragments 1 and 2 are subjected to an overlap extension PCR method to obtain an intermediate fragment M-PDCoV (SEQ ID NO:10), and both ends of the intermediate fragment respectively contain ApaLI and HindIII restriction endonuclease sequences.

2.3.2 cloning of the intermediate fragment M-PDCoV into the pBeloBAC11 vector: the pBeloBAC11 vector and the intermediate fragment M-PDCoV were subjected to ApaLI and HindIII double digestion for 2h, subjected to 1% agarose Gel electrophoresis, and then recovered with Gel Extraction Kit according to the instructions. T4 DNA ligase is used for ligation, and the ligation reaction system is as follows: M-PDCoV 200ng, linearized pBeloBAC11 vector 200ng, 1 mu LT4 DNA Ligase, 1 mu L10 XLigase buffer with the total volume of 10 mu L and the temperature of 22 ℃ for 5h, then DH10B competence is transformed, positive clone is picked up, plasmid is extracted, and the obtained plasmid is named pBAC-M-PDCoV.

2.4 digestion and recovery of pBAC-M-PDCoV: pBAC-M-PDCoV was subjected to double digestion with AvrII and BamHI from NEB, and the digestion reaction system and conditions were as follows: 2.5. mu.g of pBAC-M-PDCoV, 2. mu.L each of AvrII and BamHI, 3. mu.L of 10 × reaction buffer, supplemented with ddH₂O to a total volume of 30. mu.L, mixing, and incubating at 37 ℃ for 2 h. The recovery was performed with the Cycle Pure Kit according to the instructions. Wherein, 20bp homologous arms exist at the two ends of the pBAC-M-PDCoV linearized by AvrII and BamHI and at the 5 'end of the A fragment and the 3' end of the E fragment respectively.

2.5 linearized pBAC-M-PDCoV was subjected to homologous recombination with 5 fragments to clone in one step:

2.5.1 taking pJET1.2-A, pJET1.2-B, pJET1.2-C, pJET1.2-D and pJET1.2-E plasmids with correct sequencing as templates and taking A-F/R, B-F/R, C-F/R, D-F/R and E-F/R as primersPCR amplification is carried out to obtain corresponding fragments. The reaction system is as follows: 10 μ L of 5 XPCR reaction buffer, 4 μ L of dNTP mix, 10 μ M of upstream and downstream primers, 2 μ L of cDNA template,

fastpfu DNA Polymerase 1. mu.L, plus ddH₂O to a total volume of 50. mu.L. The reaction conditions were as follows: 2min at 95 ℃; 20s at 95 ℃; 20s at 95 ℃,20 s at 58 ℃ and 2min at 72 ℃; after 34 cycles, extension was carried out for 5min at 72 ℃. After the reaction is finished, the amplification product is subjected to 1% agarose Gel electrophoresis, and then is recovered by using Gel Extraction Kit according to the instruction, and the recovered product is used for subsequent homologous recombination experiments.

2.5.2 Infusion reaction System was constructed using In-Fusion PCR Cloning Kits: mixing 5 gene fragments (A, B, C, D, E) with different proportions and the pBAC-M-PDCoV vector (F fragment) linearized In the step (4) uniformly, adding 4 mu L of 5 XIn-Fusion Enzyme Premix with the total volume of 20 mu L and the temperature of 50 ℃ for 30min, then incubating for 2min on ice, taking 5 mu L of the transformant DH10B competence, and carrying out positive clone screening. We tried recombination with different molar ratios of fragments (A, B, C, D, E, F) and found that homologous recombination with the most commonly used ratio (1:1:1:1:1:1) produced very few positive colonies, only about 2%; for this purpose, we further adjust the ratio, including (1:1:1:1:1:2), (1:1:1:1:2:1), (1:1:1:2:1:1), (1:2:1:1: 1), (2:1:1:1:1:1), and find that increasing the ratio of the B fragment or the C fragment significantly increases the positive rate of the colony, reaching about 30% and 36%, respectively; meanwhile, the proportion of B and C is increased (1:2:2:1:1:1), the positive rate of the colony can reach 65%, which shows that the increase of the proportion of the B fragment and the C fragment is favorable for improving the efficiency of one-step homologous recombination; we further tried to increase the proportion of other fragments on the basis of this, and found that the rate of positive colonies was not increased but decreased. In addition, the ratio of the B fragment to the C fragment is simultaneously increased on the basis of the ratio (1:2:2:1:1:1) (1:4:4:1: 1), the positive rate of colonies is not increased again, but is reduced, which is probably the unique property of the PDCoV gene fragment, and the previous research does not report.

2.6 conversion of ligation products: mu.L of the ligation product from step (2.5.2) was added slowly to 100. mu.L of DH10B chemocompetent cells, gently mixed and left to stand in an ice-water bath for 30 min. After heat shock at 42 ℃ for 60s, the cells were immediately placed in an ice-water bath for 2min, 600. mu.L of LB medium was added, and the cells were thawed at 37 ℃ and 180rpm for 1 h. After completion of the recovery, centrifugation was carried out at 5000rpm for 5min, 500. mu.L of LB medium was discarded, and competent cells were suspended with the remaining LB medium and plated on LB solid plates containing 25. mu.g/mL of chloramphenicol. After 10min, the plate was placed upside down in an incubator at 37 ℃ and cultured for another 15 h.

2.7 screening of Positive clones: a plurality of colonies were picked up and cultured in a bacterial flask containing 1mL of LB at 37 ℃ for 8 hours at 225rpm, and then the gene sequences containing the genetic markers were PCR-amplified using the primers Clone-F and Clone-R for identification. The PCR conditions were as follows: 2 XTaq Master Mix 12.5. mu.L, upstream and downstream primers 10. mu.M each, 1. mu.L of bacterial solution, and ddH₂O to a total volume of 25. mu.L. The reaction conditions were as follows: pre-denaturation at 95 ℃ for 3 min; 30s at 95 ℃, 30s at 58 ℃ and 1min at 72 ℃; after 25 cycles, extension was carried out for 5min at 72 ℃. After the reaction is finished, 8 mu L of the mixture is subjected to electrophoresis by using 1% agarose gel to identify positive clones, the bacteria identified as the positive clones are sent to Wuhan Pongzi biotechnology limited company for sequencing, and the plasmids of the positive clones are named as pBAC-CHN-HN-2014.

2.8 extraction of pBAC-CHN-HN-2014 plasmid: after the colony identified as positive in step (2.7) was expanded and cultured, the Plasmid pBAC-CHN-HN-2014 was extracted in large quantities with the Plasmid DNA purification Kit according to the instructions. The last step was performed with 100. mu.L ddH₂Eluting with O, and storing at-20 deg.C for use.

2.9 rescue of recombinant virus rCHN-HN-2014: mu.g of recombinant plasmid pBAC-CHN-HN-2014 was transfected to 80% confluency LLC-PK1 cells with Lipofectamine 3000 according to the instructions. After 4h of transfection, the culture medium is changed into serum-free DMEM containing 10 mug/mL pancreatin, the culture is continued until more than 80% of cells are diseased, the cell culture is harvested, the cells are frozen and thawed for 2 times at minus 70 ℃, the cells are centrifuged for 5min at 2000rpm, and the supernatant is taken and inoculated with LLC-PK1 cells for subculture.

As a result: 48 hours after the cDNA clone pBAC-CHN-HN-2014 transfects LLC-PK1 cells, the cells have obvious lesions which are mainly manifested as cell rounding, aggregation and shedding and are consistent with the cell lesions caused by wild PDCoV CHN-HN-2014 strains, which indicates that the virus rescue is successful, and the cDNA clone pBAC-CHN-HN-2014 is named as rCHN-HN-2014. Indirect immunofluorescence experiments show that specific green fluorescence can be observed in cells infected by both the recombinant virus and the wild type virus, the control cells have NO specific fluorescence (figure 2), virus RNA is further extracted to identify the genetic marker (SEQ ID NO:29), the RT-PCR product from the wild type virus is cut into two bands (364bp and 716bp) by ClaI enzyme, but the RT-PCR product from the recombinant virus cannot be cut (figure 3), the results are further verified by DNA sequencing, and the 15121 site C is mutated into A to eliminate the ClaI enzyme cutting site in the RT-PCR product from the recombinant virus (figure 4). These results confirm the success of the virus rescue. The obtained recombinant virus is continuously transmitted for 5 generations on LLC-PK1 cells, cytopathic effect can be stably observed, RT-PCR analysis proves that genetic markers stably exist, and the recombinant virus rCHN-HN-2014 has stable proliferation capacity. At the same time, further analysis of the in vitro biological properties of the recombinant and parental viruses revealed that rCHN-HN-2014 had a similar growth curve (fig. 5) and plaque size and morphology (fig. 6) as the parental virus.

Wherein, the primers used for obtaining the 1080bp genetic marker-containing gene fragment by amplification are as follows:

the upstream primer Clone-F: 5'-CCAAGTTGGTGATTACATTC-3'

The downstream primer Clone-R: 5'-GCTGAGTCAGTGGTCACGCA-3' are provided.

The invention provides a method for quickly obtaining a PDCoV CHN-HN-2014 strain full-length cDNA infectious clone plasmid and a recombinant virus rCHN-HN-2014 based on a BAC system and a homologous recombination one-step cloning method. Provides a powerful technical platform for deeply researching the genetic variation of the PDCoV, a potential cross-species propagation mechanism, a virulence gene, a virus infection and pathogenesis mechanism, vaccine development and the like.

Description of sequence listing:

1, SEQ ID NO: PDCoV CHN-HN-2014 strain 5' end gene sequence.

2, SEQ ID NO: PDCoV CHN-HN-2014 strain 3' end gene sequence.

3, SEQ ID NO: fragment A gene sequence.

4, SEQ ID NO: b fragment gene sequence.

5, SEQ ID NO: c fragment gene sequence.

6 of SEQ ID NO: d fragment gene sequence.

7, SEQ ID NO: e fragment gene sequence.

8, SEQ ID NO: CMV promoter gene sequence.

9 of SEQ ID NO: HDV-BGH gene sequence.

10, SEQ ID NO: an intermediate fragment M-PDCoV gene sequence comprising: the CMV promoter, a PDCoV CHN-HN-2014 strain 5 'end gene sequence, a PDCoV CHN-HN-2014 strain 3' end gene sequence, 27A poly (A) structures, an HDV gene sequence and a BGH gene sequence.

11, SEQ ID NO: and amplifying a forward primer (PDCoV-5 '-F) sequence of the 5' end gene.

12, SEQ ID NO: and amplifying a downstream primer (PDCoV-5 '-R) sequence of the 5' end gene.

13 in SEQ ID NO: and amplifying a forward primer (PDCoV-3 '-F) sequence of the 3' end gene.

14, SEQ ID NO: and amplifying a downstream primer (PDCoV-3 '-R) sequence of the 3' end gene.

15, SEQ ID NO: the upstream primer (A-F) sequence of the A fragment gene was amplified.

16 in SEQ ID NO: amplifying the downstream primer (A-R) sequence of the A fragment gene.

17 in SEQ ID NO: and amplifying the upstream primer (B-F) sequence of the B fragment gene.

18, SEQ ID NO: and amplifying the downstream primer (B-R) sequence of the B fragment gene.

19, SEQ ID NO: and amplifying the upstream primer (C-F) sequence of the C fragment gene.

20, SEQ ID NO: and amplifying a downstream primer (C-R) sequence of the C fragment gene.

21, SEQ ID NO: and amplifying an upstream primer (D-F) sequence of the D fragment gene.

22, SEQ ID NO: amplifying the downstream primer (D-R) sequence of the D fragment gene.

23, SEQ ID NO: and amplifying the upstream primer (E-F) sequence of the E fragment gene.

24, SEQ ID NO: and amplifying the sequence of a downstream primer (E-R) of the E fragment gene.

25 in SEQ ID NO: the upstream primer (CMV-F) sequence of the CMV promoter was amplified.

26, SEQ ID NO: the downstream primer (CMV-R) sequence of the CMV promoter was amplified.

27 of SEQ ID NO: the sequence of the upstream primer (HDV-F) of the HDV-BGH gene is amplified.

28, SEQ ID NO: and amplifying a downstream primer (BGH-R) sequence of the HDV-BGH gene.

29 in SEQ ID NO: a gene sequence comprising a genetic marker.

Sequence listing

<110> university of agriculture in Huazhong

<120> method for constructing infectious clone plasmid of porcine delta coronavirus

<160> 29

<170> SIPOSequenceListing 1.0

<210> 1

<211> 375

<212> DNA

<213> Porcine delta coronavirus (Porcine deltacoronavirus)

<400> 1

acatggggac taaagataaa aattatagca ttagtctata attttatctc cctagcttcg 60

ctagttctct accgacacca atccaggtgc gtctgccacc aagttggcta ccctttctag 120

gggcgctttt gcgcttgctc accattagat tacctggaaa ccagccattc aggttggagt 180

ttccccaggc acttttgcgt gggcattagc ggcttgtggt ttttgcacaa aatctaagct 240

acttaccgtt cctctgacca ttcaccactt ctatagacag cactgactac cgtagggttc 300

aagtcacacc ggtctgcacc gcccgtcagc ggacacatta cccagcatag cactccttgc 360

accgagccta ggtag 375

<210> 2

<211> 2057

<212> DNA

<213> Porcine delta coronavirus (Porcine deltacoronavirus)

<400> 2

cttgcaggga ttatggatcc aatgggtaca tggaggtgca ttcccatcga ccacatggct 60

ccaattctca caccagtcgt taagcatggc aagctcaagc tacatgggca agagctggcc 120

aatggcatat cagtcagaaa tccgccacag gatatggtga tagtgtcacc aagtgacacc 180

tttcactaca cttttaagaa acctgtggaa tcaaacaacg atccagaatt cgctgttctg 240

atataccagg gtgaccgcgc ttcaaacgct ggacttcaca ccataaccac ttcaaaggcc 300

ggtgacgctc gcctgtataa gtatatgtaa tgtgcaactg ccacctgcag ctgcgagatt 360

tatatagatt gtgcaataag cggcacatca gaagagagga tgttcctgag cttattgacc 420

ctctcgttaa aactcgctgt tttgcttaca gtctcgtggt tcttgctaat gctaatccaa 480

ttgcatttag catactacct cggaaacttc ttatcaatgg tgagccttta ctgcttgaat 540

atggtagcat atatggtaaa gactttatca ttcgaccatc gctccaagtc attcttgaag 600

atgaattaaa ttaaagtttt gacaccaatc tatcatggct gcaccagtag tccctactac 660

tgacgcgtct tggtttcagg tgctcaaagc tcaaaacaaa aaagccattc atcctcagtt 720

tcgtggcaat ggagttccgc ttaactccgc catcaaaccc gttgaaaatc atggctactg 780

gctgcgttac accagacaaa agccaggtgg tactccgatt cctccatcct atgcctttta 840

ttatactggc acaggtccca gaggaaatct taagtatggt gaactccctc ctaatgatac 900

cccagcaacc actcgtgtta cttgggttaa gggttcggga gctgacactt ctattaagcc 960

tcatgttgcc aaacgcaacc ccaacaatcc taaacatcag ctgctacctc tccgattccc 1020

aaccggagat ggcccagctc aaggtttcag agttgacccc ttcaacgcta gaggaagacc 1080

tcaggagcgt ggaggtggcc caagatctca atctgttaac tccagaggca caggcaatca 1140

gcctaggaaa cgcgaccaat ctgcacccgc tgcggtacgt cgtaagaccc aacatcaagc 1200

tcccaagcgg actttaccta agggtaaaac catttctcag gtatttggca accggtctcg 1260

tactggtgcc aatgtcggct ctgcagacac tgagaagacg ggtatggctg atcctcgcat 1320

catggctcta gccagacatg tgcctggtgt tcaggaaatg cttttcgctg gtcacctcga 1380

gagcaacttt caggcggggg caattaccct taccttctct tactcaatca cagtcaagga 1440

gggttctcct gactatgaga gacttaagga tgcgctcaac acggtcgtta accagaccta 1500

tgagccaccc accaaaccaa ctaaggacaa gaagcctgac aaacaagacc agtctgctaa 1560

atccaaacag cagaagaaac ctaaaaaggt aactctgcca gcagacaaac aggattggga 1620

gtgggatgat gcttttgaga taaagcagga atcagcagcg tagacatcaa tctatgtctg 1680

ttaaacccac ccaactccac tcaaatatct ctttggttcc agagagtcgt agtgtatagc 1740

cagagagcca gtcagagggc gctatcatgc aaactagggc tggctactct agcacagaat 1800

cacatcccga taatcaacag tgctagaagg ttgattatac catttaatat gccgaggcca 1860

cgcggagtac gatcgagggt acagcataat ctcaactttt gttgagccac aattttaatc 1920

ctaattggag aaggccaaag gactgtacta cttctgtagg tgtagcagtc gcccagtggg 1980

aaagcgccaa ctaggttaca attgtggtgg ggacaaatta ggggaaatta aattggctta 2040

taggggggat ggagcaa 2057

<210> 3

<211> 4191

<212> DNA

<213> Porcine delta coronavirus (Porcine deltacoronavirus)

<400> 3

tagcactcct tgcaccgagc ctaggtagga taaaaccccc taccgggtga ctcttaaggc 60

ctttcctcca cgggatagct actagtcact aggtgtaagt gatctgatct gggcgtattg 120

tgttgcgcaa gtgtgatacc cataggagcg tggaatccta ttctgcggct cagtgcctga 180

tatagctgtg aaatggccaa gaacaagtcc aagcgcgacg ctatcgcgtt gcctgaaaat 240

gtaccaccac ctctgcaact tttcattcac gttgcagctg ctgaagaggg tcaccctaag 300

gttactactt accttggcaa ctataacctc tatgccacca aggctccgcc tggcgtgcag 360

gttcttagtg ctaaaacctc tcttactgac tttgagaatg tctttggagc ccaacccacc 420

ttgcgatcaa ttcgtaatct ggtttgtgag gctcgctcgg ctgaatggac aacttccaag 480

aatgcttttg cactcaaagc cactcaactt gactactctg atgccgtttt gagggcaatg 540

attcgtttct gccctccaaa ggtgtccaca ctcgctgcct ttgctctttt tggcagattg 600

gttaaaattg aggacaagga acttgctgag ttagctcgtg acactgccct tgagttggcg 660

tacacggcta aaattggtac atctcttgct gacacgagat ctgtctcact tattcataag 720

gacgcttatc taactctcag taatgaggtt gttggcgtaa cttttactgc cgcacttatg 780

gcaaaggcta ccactgttaa tggagcaatg caatactcaa acttttacct ctaccctcgt 840

gccactatta aggtgaccga tggtaaggtt gaagcaattg caaccaagcc tctttctgct 900

gctactaaag gcaagcaaat cacagaggat gtcaaccttc tccctgacta tcagcagctg 960

cttgttgatc aagtgactgg cactgaggtt aaggttggag ctttaaccta tgttaagacc 1020

actgattcac caccccttta ctttcccaaa gtcaagggtg gtgttattgg tattgcactt 1080

aagcagcagg gcactgcggc taagaagctc aatgtagtct tccatgctca acctgatgat 1140

gttctgctag ccttcataca acttcagcaa ttcttgaacc gtacttcgga ttcaagtgtt 1200

gaaattactg gttgccagag ttatgaagta tctccaactg tgactgtcaa aattggcccg 1260

tctaaacctg gggatgtcat cgtggctact gatgaggaat accttaaatg ctttgaaacc 1320

cctgaggtag gtaggctcta taaggttttc caaactcaat cttgggctat cattgagcgt 1380

tccttctcca gtttgaagat ccgcgtgtcc aaagctttat cagcatttat aagttttctg 1440

caaaaccttg cagataactt tactgcaata agtggtgttg tcactgcact cattcgtgaa 1500

ctccaggatc ttaccctgga tgtggcgaca cgtatcacta acatacaatt tgtttaccgt 1560

gccggtaagc ttattgtcga cactacaagt gtcatagcta aacttttcca gccattctgt 1620

gattttatat cacctttcct tcggaaagtt gctggttttg caatttacac tgttggtaat 1680

cgcatgctta tgtttaccag caccggcacc tttcttctta caagggcaac tactaagata 1740

ctcaataagg caaagtacat ctttgatgtg gagcctgagt acccagtaga tgtaacaaca 1800

tccaaagttg tagtacatga agcactccag caaaccgaca ctaagcctac tagagctcta 1860

gaggctgttg atgtcgttgt tggtaatact gtactgcaaa tggctactga tggcactgcg 1920

ttctacccat cggatggtac gcacgcctct cttccaggat tcaaagcagg ctcggatgag 1980

ctttttataa gcttcaactg cgacctcttt gatgatgaga ctaatgctca aatcaacgaa 2040

acactcgctg catatgagct taaccaacta gtggctccag gtgattctac accgcgtcaa 2100

attgcgacgt tggttgtcga tacacttgta gatgctataa cagaccactt tccggagaaa 2160

accattgatc tacctgaaga ctatcaagtc ttttctgacc atgatgatct cccactcgca 2220

caataccaca tccctgatca cctgagcctg tatattcagg ctatggaagg tgaagatgat 2280

agtggtgatg aaatatgtat tgaggacgat gattacgact gtcctcaagc cgacgaagac 2340

acagaaggag taattcccca acagtgggaa cttcctgatg ttgataaatt tttactcaag 2400

atccaggaac ggaagaccag cagcgacgaa gtacttagcg tcgacgtcta tcctaaacca 2460

gagccagtcg gcaatgttgg gattgacgac agcgcgtcgg aaaagaagcc aaatggggac 2520

tcagtaccgg atcctgaggt ccatccaaca ctagagagtg tggatgttga acgaccaacc 2580

gaaacagcaa accaggctgt tgaagacaaa ccttctgata ccacctttgt ggttgatgag 2640

gaacaattac aagaatcaac accagaacat gaactccgct cctatgaggg ggagtttgat 2700

tctgatgatg aaattattat tcctatagta ccagtaacac ctgcggattt aaaaccacag 2760

actattacta taaaggagta ctttaagtct gaaaaacttg agactattaa cgaaggatcc 2820

acagagtcag ttacacaatc tgacgattcg tttgacgagt catttgttga tgctgagtct 2880

gatgatccac aagatcctgc tgtatatgat gatacaacaa ttataacgga cagtactgat 2940

gtaggcgatg agcctgagac aactctagct accatcgtta acacacctct gacactcgat 3000

aataacttgc cacctgaagc cattaaacaa cccagcccga ctaaggttga gttagttgtt 3060

ggcgaattgg caagtattaa atttgacaat tctgtcctag tcaaccctgc taatgcgcaa 3120

ttaacaaatg gcggtggagc tgcccgtgca attgcaaagt tagctggtcc aaaataccaa 3180

gagtactgta atagtgtggc tcctatctca ggaccgctta ccacggactc ttttgatgcc 3240

aagaaacttg gtgtagcctg catcttgcat gtagtgccac ccaaaggttc tgaccctaat 3300

gtacaagaac tcctgtatca agcttacaag agtatcctta ctgaaccagc acactatgtt 3360

atacctatac taggtgctgg tatctttgga tgcaacccag tccactctct ggatgcgttc 3420

aggaaagcat gtccaagtga cataggtcgt gtcacccttg tcactatgaa caaaaaccat 3480

ttgcaggtgt gggatgctct caataggacc attgtacgca ccactactga ctatgatcaa 3540

gttaccacca aggcccttac accccaggga gtgttagaag ccaatctctt tgatggtgag 3600

gactttgttc aagaaccaaa acccggtcaa atttaccttg aggttactga agaagttcag 3660

aaccaagcca aggaacttga ccttaacctt cagcaatact gcgtctacct gaagacttgc 3720

caccataaat gggttgtgag tcgtacgaac gggttgatgc atctaaaaca aaaagataac 3780

aattgttttg ttagtgcagg tgtaaacttg tttcaaaaca ctgcttatca atttagacct 3840

gctattgatg ctctctatag ggagtatctc aatggtaatc caaacagatt tgttgcttgg 3900

atctacgcat ccactaaccg tcgtgttggt gagatgggtt gtccacagca agttatttct 3960

ttgctcgtta gtaactctga cgcagcattt tcagcaacta cagcctgttg taacacctac 4020

tttaaccaca caggtgttat ttcagtagct cgtgaatatg acccaataca accaaaggtc 4080

tactgcatga agtgcgatgt gtggactccc tttacacccc agagtggaaa aggtgcagtt 4140

gcaattggta cttctgcaga tgaacctacc ggtcctgcca ttaaatttgc c 4191

<210> 4

<211> 5134

<212> DNA

<213> Porcine delta coronavirus (Porcine deltacoronavirus)

<400> 4

gtcctgccat taaatttgcc gcagctcact gctggtacac taatggcaag aaaacagtta 60

atggctatga cactaaagct aatgttgtag ctacttatca taggtttgac gtgcctaagc 120

ctcaacttgt cgaggacgtg gttgcgctgc ctactaaaaa tgactttgaa gttctcaatg 180

ttgaagaact gccacaggat agtgtgctcc atttggaccc acctcctgta caggccttac 240

aacctaaggc taaccaacac attgagattt tagaaaaccc agattatctg gacattttgg 300

atctttggat tcgtaaaccc aaattcatcc tcgtaaagtc gtggagtgtt ttgggtagag 360

cactatgtaa ggcaggtaaa gttgtctttg tcagtgcttc gcttttgacc cgtttctaca 420

attaccttgt agagattggt gctcttgact caacaataag gttgtcagtc gatcttacct 480

gtaaatttgt tagaacggta ctcccatcgt ctaacactgt acacaaaact tgtcttggtc 540

tgtattattc agcccagaca ctttttgttt ctttagcacc attccttatg ttaccagctg 600

tagttagtct gcttaattca ggctatacaa ttggcacata tttgtatgca aaaactggct 660

ggccttgtaa ttacaatgcc acgcaacacc ttgattataa ttcttactgt gcaggtgact 720

tggtttgtca agcctgtttt gacggtcaag actccctaca tttgtatccg catttacgtg 780

ttaatcagca gcctcttcag accactgact acactgttta tgcgctttca ctaatactac 840

tattagctaa catgactctt gtcatgggta cgctaatagt tactttcttt gtgaacttct 900

atggtgtgca aataccattt tatggtacac ttctgataga ttatcagtcc gcgctgatga 960

tgactttctc agtgtactac ttttataagg taatgaagtt tttccgccat ctcacacatg 1020

gatgtaaaat tccaacgtgt atggtatgtg ctaaacttcg taccccacct actataacag 1080

ttgagactgt cgttcaaggc aggaaatacc catctgttat tgaaacaaat ggcgggttta 1140

caatttgtaa agaacacaac ttctattgca aagactgctc tctacaaaca cccggcactt 1200

tcatcccgac agaagctatt gagtcgctct cacgagctac caggcttagt gtcaaaccaa 1260

cagcaccagc attcttactt gctagagatg ttgagtgcca aactgatgtt gtcgttgctc 1320

gcgcaatgca taaccaaaat gcgcatgtgt gcatttcaaa atactctgat atccgtaccg 1380

ttgaccaact acttaagcct actccactgt tttcatacac tcccgatgtt atcatcgcgg 1440

cagactttga caacagaggt agtcttaaga cagctaaaga attagctgtg gttttgtcaa 1500

tggaccttaa acgtactata attatcattg atcaggctta ttctagaccc attgataatt 1560

atcaggaagt tgcttctcgt attgagaagt attacccagt tgcaaagatt acacccacag 1620

gtgacatctt tacagacatt aagcaagcga ccaatggcca agctagtgac tctgctatta 1680

atgcagctgt tctggctgtc cagcgcggtc ttgattttac aatcgacaac cctaacaata 1740

tattaccaca ttacgccttt gacttttcaa cccttaatgc agaagaccag tctaccattt 1800

tggagagtgg ttgtgctaaa ggcaatctca agggcactaa tgttggtgtt gttctttcag 1860

ctagccttgt tacacgtctt agtcagcagg ctatatgtgt gattgctaat gctgcttcac 1920

gtaatggtgt aacatgcgct gttactccat ctacacttgt tatgcgtggg aatattgcaa 1980

cacagccctt gactcgcatc aaagctggtg cacctcccat gcgtcaaaaa attttatgtg 2040

ttatcctggc acttgctatt gtgtactttg ctgctatggc ttttggcttt tttgcaagtc 2100

aaattacgct taatacagtg cctacgatta aatctgatat ccgcgcctct accttctacg 2160

ttgttagaga tggagtcttg gatactgttc gctcaaatga caagtgcttt gcaaataagt 2220

ttttggcatt tgatagcttc attcaagcac cttacactaa ttcacctgac tgtccagtcg 2280

ttgtgggagt tgttgaagta acgacgcact ctattcctgg aattccagca ggtgtcattc 2340

atagagacgg tctcatactt aacatttatg aacagtctct ttatgaaatc catcagcgtc 2400

agtctatggt tagggatgcg ttgtcactca agacagcaaa tctctttaac ctaggcaagc 2460

gtgttgtagt aggatacact caacatgaag ttgttgtggg tacctcctat tttaattctc 2520

ctgcactttt taacgcaaag tgcaccttct tacagtacca ggacactaga caactctatt 2580

gctatgatac tgttcctact gaacataagc tttactctga tgtgcttccg cacgtcgagt 2640

ataaggctat tgacattaat ggtgatcttg ttcctttcaa gataccggag cagataatgt 2700

tctatccaca tattgtgcgc tatactagca attcctattg ccgtatgggg cattgtttta 2760

atactaaccc tggtatttgc atttcattta cggacgaatt tccgtatagt gaaaatgtta 2820

aacccggtgt atactgtgct gatacctctt tgcagttgtt ttcaaacctc gttttgggca 2880

ctgtatctgg tattcacatc tttacatcaa cagctgcatt gcttggatct actattgtga 2940

tcatactatg cgttgttgct gttcttgcag ttcagcgatt cttcaaggag tacacaactt 3000

ttgttatgta cacttgtggt cttgctcttg tcaacattgt gggcattgca cttatataca 3060

agtgccttgt cttcgcgatc ttctattatg caatctacct ttactttgtc cttactttcc 3120

cctcctttaa gaggaatgtg gcattgtttt acttcgctgt agtgatcgtg ccgcacgtga 3180

gtaacatgca attgcttgcg ctcattgtgt gtagcattat ctactttctc tacacctatg 3240

ttcatactgt agctaagaca gctgggaaat tctcttcctt cttagacgca gctaaagcta 3300

cttttgtcat tgacaatgaa aagtacgtgt tgcttaaaga cctcgctggt gctgaatttg 3360

accagtatct ggcctcttac aacaagtaca aatatttttc tggtactgct tctgataagg 3420

attatgataa ggtctgtatg gcatttcttg ccaaggcttt gtcatctttt cgtgaaggag 3480

gcggttcaca gttgtacaca ccacctaaat ttgcagttgt tcagagtctt aagaccaagc 3540

tgcaagcagg tatcaaaatc ctcctgcacc cttcaggtgt agttgagcga tgtatggtct 3600

cagttgtcta caatggatct gcattgaatg gcatctggct taagaatgtt gtctactgcc 3660

cacgccatgt aattggaaaa ttccgtggtg accagtggac tcacatggtc tcaattgctg 3720

attgccgcga ctttatagtc aagtgtccaa tacagggtat tcagctaaat gtccaatcag 3780

ttaagatggt aggagctctc ctccagttaa ctgttcatac caacaacaca gccactccag 3840

actataagtt tgaaaggctc caaccaggat catcgatgac aattgcttgt gcttatgatg 3900

gcattgtacg gcatgtctat cacgtggtcc tccaacttaa taatcttatt tatgcaagct 3960

tccttaacgg agcttgtggt agtgtgggtt acactcttaa gggtaaaaca ctctacttac 4020

attacatgca ccacattgag tttaacaaca aaactcatag tggtacagat cttgaaggta 4080

acttctatgg cccctatgtg gatgaggaag ttattcagca acaaacagca ttccagtatt 4140

acactgataa tgttgttgct caattatatg cacacttact gactgttgat gctagaccaa 4200

aatggctggc acaatctcag ataagtatcg aggattttaa ctcatgggct gctaacaatt 4260

cctttgctaa cttcccatgt gaacaaacta atatgtccta cattatggga ctctcgcaaa 4320

ctgctcgagt ccctgtagaa cgtatcctca ataccattat acagctaacc accaatagag 4380

atggtgcttg tattatggga tcttatgatt tcgagtgtga ttggacgcca gagatggtat 4440

acaatcaggc tccaatttca ttgcagtcag gagtagttaa gaaaacttgt acgtggttct 4500

tccacttctt gtttatggct attaccatgc tactcgctgc catgcatgtt ttccctgtac 4560

acctgtaccc aatagtactg ccatgcttca ttgtcgtggc attcctgttg actttaacca 4620

ttaaacacac tgttgtgttt accactacat atttgcttcc gtcacttttg atgatggttg 4680

taaatgctaa cactttttgg ataccgaaca catttctgcg cacctgctac gaaactatat 4740

ttggttcccc aattgctcag cgactgtatg gttacactgt tgctttttat atgctgatct 4800

atgctggact tgcaatcaac tatacgttga aaacactccg gtatagagca acttcattct 4860

tatctttttg catgcagtgg tttcaatatg gttatgttgc acacattgcg tacaaactgc 4920

ttaataaacc ctggacagaa tcactactct tcacagcctt cacaatgcta accagtcatc 4980

ctttgttggc tgctcttagc tggtggctag ctggtcgcgt aactctgccc attatcatgc 5040

ctgacttagc tattcgtgtt ttggcgtata acgtcattgg ctatgtcata tgtgttcgat 5100

ttggccttat gtggcttgca aatcgattca caac 5134

<210> 5

<211> 5500

<212> DNA

<213> Porcine delta coronavirus (Porcine deltacoronavirus)

<400> 5

cttgcaaatc gattcacaac tgtacctatg ggcacatacc agtatatggt gtctgtagag 60

caacttaagt acatgatggc agttaagatg tccccaccgc gtaatgcgtt tgaggtgctt 120

atagccaaca ttagacttct tggtttgggt ggaaaccgta acattgctgt ttctactgtc 180

caaaacaaaa ttcttgatgc aaaagctact gctgttgttg ttgctaacct tcttgaaaag 240

gctggcgtca caaacaagca cgctatttgc aaaaagattg tgaaactcca caatgatacc 300

cttaaagcca ccacttatga ggaggttgag gtagcacttg tgaaacttct ttctcacata 360

attgagttct tgccaactga tcaggtagat gcttatctag ctgatgcggc caatgctcaa 420

catgttaata cctatttcga caacttgctt gagaacaaag ctgttgttca ggctgttgcc 480

gatatcaaca ttaatctgga ttcttataga atttataagg aggcagatgc tatttacaaa 540

cgatctgttg agatgaacga atctccacag gagcaaaaga aaaagcttaa agctgttaac 600

attgcaaagg cggaatggga gcgtgaggct gcttctcagc gtaagcttga aaagcttgct 660

gatgctgcta tgaagtctat gtatcttgca gaacgtgctg aggatcgtcg cattaagcta 720

acctctggac ttactgcaat gctttaccat atgcttagac gtcttgactc agatagggta 780

aaagctctgt ttgagtgcgc taaggcacaa atcttgccaa tacatgctgt agttggaatt 840

tctaatgaca accttaaagt tatttttaac gataaggata gctactctca ttatgtagag 900

ggcaacacac ttatacataa gggagttcgc tacactattg tgaagaaact ctccttagat 960

aatgcaccta ttgaaggcgt accagaagaa ttccctgtgg tcgttgagac tgttagggaa 1020

ggtgtgcccc agttgcaaaa caatgagcta tgtttgcgca atgttttcac tgctcagaac 1080

acagctcagg acttcaatgg caatgaatcc actgtaaaat ctttttatgt tactagagcc 1140

ggtaagaaga ttttggttgc cattacatca actaaagaca atcttaagac tgtgacctgc 1200

cttactgaga ccggtaagac agtccttaac ttggaccctc ctatgcgctt cgcacatacc 1260

gtaggtggaa aacagtctgt tgtctatctc tattttattc agaatattag ttcactcaac 1320

agaggtatgg ttattggcca catctctgaa actactatcc ttcaggcaag tggcactcaa 1380

attgagtacc agcaaaatgc ctctcttttg acctatttgg ctttcgctgt agaccctaag 1440

acagcctacc ttaagcatct tgctgatggt gggtctccta tacagggttg tattcagatg 1500

attgctacta tgggtcctgg atttgcagtt actactaaac cacaacctaa tgagcatcag 1560

tattcttatg gtggtgcttc aatttgtctt tattgccgtg ctcatatacc acatcctggt 1620

gttgatggac ggtgccccta caaaggccgc tttgttcaca tcgacaaaga taaggaacct 1680

gtttccttcg ccttgactca tgagccatgc agttcttgtc aacggtgggt caattatgac 1740

tgcacctgtg gatctagtct gcagaattcg gcttatttaa acgagtaacg ggttctagtg 1800

acgcccggct agaacccctg cagcctggaa ctcaaccaga tgctgtaaaa agggccttcc 1860

atgtgcataa tgataccacc tctggtatat tcttaagcac aaaatctaac tgcgctcggt 1920

ttaaaaccac acgcagtgcc ctgcctttac ctaacaaggg agaggttgaa ttgtactttg 1980

ttactaagca gtgtgcagct aaagtcttcg aaatcgagga ggaatgctac aacgctctta 2040

gtacagagtt ttatactact gatgatacat ttggtgtcct tgccaaaact gagttcttca 2100

agtttgacaa gatacctaat gtcaatcgcc agtatctgac taaatataca ctcctggact 2160

tggcttatgc tctacgccat ttgtcaacat ctaaggatgt tattcaagaa attttgatca 2220

ccatgtgcgg aacccctgaa gattggtttg gagaaaattg gtttgatcca attgagaatc 2280

catcctttta caaggagttc cataaacttg gagatattct taaccgttgt gttcttaatg 2340

ccaataagtt tgctagtgcc tgtatagacg ctggtcttgt tggcatatta acacccgaca 2400

atcaagacct cctgggtcag atctatgact ttggagattt tattattaca caaccaggta 2460

atggatgtgt ggacttagca tcctattatt cttatttaat gcccattatg tccatgactc 2520

acatgttaaa gtgtgagtgt atggatagtg atggcaaccc acttgagtat gatggatttc 2580

agtatgactt cacggacttc aagcttggct tgttcgagaa gtattttaag tactgggacc 2640

gtccttacca tcctaacact gttgaatgtc cagatgaccg ttgcgtattg cactgtgcga 2700

acttcaatgt gttgtttgct atgtgtatac ctaatacggc atttggcaat ctttgttcaa 2760

gagctactgt tgatggccac cttgtggtcc agacagtggg tgtacacttg aaagaacttg 2820

gtatagtcct taacgaggac gttaccacac acatggcaaa tattaatcta aacactctat 2880

tgcgattggt tggtgatccc accaccattg caagtgtctc agacaagtgt gtagatttaa 2940

gaactccttg tcagaccttg gctactatgt ctagcggaat tgctaaacag tcagtcaagc 3000

ccgggcattt taatcaacac ttctacaagt atttgcttga tagtaaccta ttagaccaac 3060

ttggaataga cattcgccac ttctactata tgcaggatgg tgaagcggct atcacagact 3120

acagctacta caggtataat acccccacga tggtagatat caagatgttc ttattttgcc 3180

ttgaggtggc agataagtat cttgagccct acgaaggtgg atgtattaat gcacagtcag 3240

ttgtggtctc taatttggac aagtcagcgg gctacccctt taacaagcta ggtaaggctc 3300

gtaactatta cgacatgact catgccgagc aaaatcaact gtttgagtat acaaaacgca 3360

atgttttgcc tacactcact cagatgaacc ttaagtatgc aatttcagcc aaggatcgtg 3420

ctcgcactgt ggcaggagtg tctataatta gcaccatgac taacaggcag taccatcaaa 3480

agatgctgaa atctatttca cttgcacgca atcagaccat cgtgattgga acaaccaaat 3540

tctatggtgg ttgggacaac atgttacgac gactgatgtg taatatcaac aatcccattt 3600

tagtgggttg ggattaccct aagtgtgatc gttctatgcc aaacatgctg cgcattgctg 3660

cttcgtgctt gctagcacga aaacacactt gttgtaacca aagccagcga ttctaccgtt 3720

tggctaatga atgttgccaa gtactatctg aagtggtagt ctctggtaac aacctctatg 3780

taaaaccagg tggcactagc agtggtgatg cgaccacagc ttatgccaat tcggtattta 3840

acatcttaca ggtggtttct gctaatgtag ccaccttctt atcaacttcc accacgacac 3900

atcttaataa ggacattgct gacttgcatc gtagtcttta tgaagatatt tatcgtggtg 3960

actctaatga tatcaccgtc atcaatagat tctaccagca tctccaaagt tactttggac 4020

ttatgatatt gtctgatgat ggtgttgcat gcatagactc agccgttgca aaggctggag 4080

ctgttgctga tcttgatggt ttccgagaca ttttgtttta ccaaaacaat gtttacatgg 4140

cagactcaaa gtgttggaca gaaactgaca tgaatgttgg ccctcatgaa ttttgctcac 4200

agcatactgt gttagcagag catgatggta aaccttacta cttaccttac ccagatgtct 4260

cgcgcattct gggtgcatgc atctttgtgg atgacgttaa caaggctgac cccgttcaga 4320

accttgaacg ttacatctca cttgcaattg atgcatatcc cctcaccaag gttgacccta 4380

ttaagggtaa agtcttttat ttgttactag actacatacg tgttcttgct caggagttac 4440

aggatggtat ccttgatgct ttccaatcac tcactgacat gtcgtatgta aataacttta 4500

tgaatgaggc cttttatgct cagatgtatg agcaaagtcc tacactacag gccagcggtg 4560

tttgtgtggt gtgtaactca cccactatac tgcgctgtgg tgattgcatt cgtcgaccac 4620

tactttgttg cgtctgtgcc taccagcatg ttacgcagac tacacataaa cgtatcattg 4680

ctatcaacaa ctacatttgt agtgttgaga attgcaatga ggacaatgtt gaaaaacttt 4740

tcatttctgg cactgcaatt tattgtgaga atcacaaacc cacgctgtgc atacccattg 4800

tagctaatgg ttctgttttt ggtatctatc gccacactgc ccgtggtagt gatgacatag 4860

acctctttaa cgagcttgct acatctaact atgacactat tgaaccttat cagaaggcca 4920

atcgtgcacc tttatcactt atgctcttcg ctgctgaaac cattaaggca ctcgaggagt 4980

ctatcaagaa gtcatatgct accgcaaccg tcaaggatgt gtatgaccaa cgcttcatta 5040

aacttctatg ggaacagggt aaaaagccgc cacccataac gaagaaccac attttcactg 5100

gctaccattt taacaagaat ggaaaaaccc aagttggtga ttacattctt gctaaaacag 5160

atggcagtga cacttatact tacagaggaa catctaccta caaactccaa acaggtgatg 5220

ttctagtctt aatggcacat gttgttacac cgctctcagc accccctgtg ctaacgcaga 5280

caacatatgt cagaaaatca cttttacccg actctgttgg tgcgtcttat tatgtgcaac 5340

attttaagtc atataatgag atagctatgc agagggttac aacagtatta ggtccgccag 5400

gcacaggtaa gtcaaccttt gctattggtt tggctaagta ctttcctagt gcacgtattt 5460

gctacactgc gtcttcgcat gcagcaatag atgcactctg 5500

<210> 6

<211> 4343

<212> DNA

<213> Porcine delta coronavirus (Porcine deltacoronavirus)

<400> 6

gcagcaatag atgcactctg tgaaaaagct ttcaagacaa tacctgtagg ccaatgcagt 60

cgtatcgtac ccacacgtac aactgttgag tgctttcagg agtttgtcgt aaataacaca 120

actgcacagt atatcttctc gactatcaat gccttacctg acattaagtg tgacattgta 180

gttgtagatg aggtttctat gttgaccaat tatgagcttt cctctgtgaa tgctcgtttg 240

gtttacaatc acattgtgta tgttggtgat ccttatcagt taccttcacc tagaactatg 300

cttacgtctg gccagctttc gccagctgac tataacgtag ttactgatat aatggtacat 360

gcaggagcgg acgttatgct cgacatgtgc tacagatgcc cacgtgaaat cgttgagaca 420

gtgtctaaac ttgtctacga taacaaacta aaagcggcga aaccgaactc aagacagtgt 480

tacaagacca ttgtgaactt tggtcctgga gacgttgctc atgagggaca atctgcctac 540

aacgaagcac agttgcgttt cgcactcgca tttagacaac aaaagcggtg ggataacgtg 600

actttcatat ctccatataa tgctatgaat gtgaaagcat ccttagcagg tttctctact 660

cagaccgttg actcttctca aggttctgag tatgattatg ttatcttttg cgtgaccact 720

gactcagcac acgcacttaa catggctcgt ttgaacgttg cccttacacg cgcaaagata 780

ggtatccttg tggtgtttag gcaggcaaac gaactttaca atagtttgca gtttgaatct 840

attgattcac agcttcagtc gagtgctgag aaaaacctca caccactgtt taagcgctgc 900

ggctatgagt ataatggcgt ccatccagct catgctttga cctggcatga ttgtggtgca 960

gagtaccgct gtgaggagcc acttgctaaa ttagtaggag ttgccgatgg cactcttata 1020

tcatataaaa ccctagtatc cacacttggg tttcttccat cacttaaaat tgatgcatat 1080

cataatatgt tcctaacacg tgacgcgtgt cgcacctatg ttcagagttg gatcggcata 1140

gacgttgaag cagcacacgc cataaaacct aacaccggga ctaacctgcc attgcaaata 1200

ggttttagta ccggaaagaa tttttcagtt actccagagg gaatttgggt aaacgagcac 1260

ggatcttgca ctgagcccgt ccctgccaaa atacctcctg gagaacaatt tcgtcacctt 1320

aagaaggaca tgcgccaggc gcgtccttgg aaggttgttc gacgtgagat tgctactcac 1380

attgctgagg tagctcctca cactgattat atatgctttg tcacttgggc tcaccagctt 1440

gagctagcga caatgcgcta ctttgtcaaa ctaggtatgg aagagaaatg cttttgtggc 1500

aggcgggctt gtttcactaa tggaactgag ttcgcttgca aagcacacca ttctctcacc 1560

attccacaat gtgattatgt gtacaatcca ttcctcatcg acgtggctac gtggggattc 1620

tcgggacggc tttccaccaa ccatgacgcg gtatgcacat atcatgctaa tgcccatgtt 1680

gcatcagctg atgcaatcat gacggtatgt ttagctatcc atgaactgtt cagtactgtt 1740

gactggaacc ttgaatttcc agtaactgct gagcaatcac aacttaacaa ggcctgtcgc 1800

ttagtacagg caaattattt aaatatacta ctcactacaa ccaaagccac ggtggttcat 1860

gatattggta acccaaaagg tatccctatc gtgcgcaaac ctggtgttaa atatcacttc 1920

tatgatcaag cacccattgt caaacacgtt caaaaactaa agtacaagcc agagatggag 1980

gcccgtttca ccgatggttt gactatgttt tggaattgta atgttgacac ataccctgct 2040

aatgcccttg tgtgccgcta cgatactcat cggcagaagc atttaattgg acctaatggt 2100

tcagcattat atgttaataa gcatgctttt ctcacccctg agatgcatac ttatgctaca 2160

cataaactca acttggctcc actcatctac tactccacca cagattgtag tagtgaacag 2220

cctattgttg ttacctacag agattgtgtc acccggtgca atactggaaa aactctctgt 2280

ccaaatcatg ctcttgaata ccaagagttt atcaatgcat acaatctcat ggctcgccat 2340

ggatttaatg tttacatacc acgcaatgtc aacgtctata actgttggct tactttcact 2400

aatctccaaa accttgaaaa cttagcttac aactgttatt ataagaactg caatgctcac 2460

gttgatgggc agcttgatgt agttattaat aataacgctg tatatgctaa ggtcgacaat 2520

aatcttgtca aactctttga caatcgcact aacttacctg tctcagtggc ctttgaacat 2580

tacactaaca ggcatacccg ttcactgcca actacacagc tgttatctgg tttaggcgta 2640

actgccacca gaaatttcac tgtgtggttc gacaatgata caattttcca atacactatt 2700

aatgtatcta cttatactga catcgaccct agtacccatg ttgtcctctg tgatgatagg 2760

tacggaacag attggagtca gtttaaccaa cttcctaatg cagtgttcct caccaaaact 2820

aaggtgaaga aaacagaacc gtttgtttgt acagcactga ccctaaatgg cctcgccatt 2880

gacggtgaag agctatacat ctatgtacgc tataacaatc aactgaccac atttgctact 2940

acttgtacac agggtagaaa tgttgagcag tttataccta aaacacctat ggaaagagac 3000

ttccttgaga tgtctcaaca gtccttcatt gaacaatatc aattgcagga actgggtgtt 3060

gaacacatta tctatggtga tgattccagt ccagtcattg gcggaactca cacacttatc 3120

tcactagtta aaaacaagtt tgaacatcag cttgtcaacc atgtttacaa cccagtccag 3180

aactgtgttg ttacctcacc taacgcaagc tccaagaacg tttgcactgt tcttgatgtt 3240

cttcttgatg actacattga catcataaga caagcacatg ccagttacac aagtaaatct 3300

aaagtattca ctgtgtcaat tgacaaccaa caaattagat tcatgctctg gcatgatgag 3360

caagtcaaga cttgctaccc aatcttacag tcacttacca atggttacca gatgccatct 3420

gtgtacaaaa cattggttac tgacttacaa ccagctgaca tccctaatta tcattcctac 3480

accccccggg tgcctggagt agttaagaat gttatcaagt accgccaact tttcaactac 3540

atagttaaaa aggataggtt ggcagtacca cacaatatga ctgtattaca ccttggagct 3600

gcatctgcac taggtacagc accaggttct tcagtcataa aacaaatgtt tcctgaagga 3660

actgttctta ttgaccttga tataagagag ttcacttcag atgctaacca aataatagtt 3720

acagactaca gaacttacat accaccacac cacgtagacg tcatattttc tgacctctac 3780

tgttgtgatg acatacactt ctttgacaat ctaataagga tagttaagga gaggctcgcc 3840

ctcggtggtt ctatctttgt taagataact gaacattcat tctcacccga actctactca 3900

cttgcggggt ggttcgatga ttatcaacta ttttgcacag cagtcaatgc ctcgtcttca 3960

gaagcatttt tatgctgttt taattatttg gggcaagcta aggaaaacat taatggtttt 4020

aacttacatg cttcctacat tcaatggcgc aatgaaatag cattgacacc aacctattct 4080

cctttagcgg ataacccggc tacggcctgt aagctaaaag caacgcctat tatctcggct 4140

cgtgagttag agaagaagcc tattcttcgc tatctcgttg catcaggacg ccttcttgtg 4200

aggccaccag aatgcagaga gctctattga ttatgacctt actttgtctc gttcgagcaa 4260

agtttgctga tgatctactc gatttactca cctttccggg tgcacatcgc ttcttacata 4320

aacccacgag gaattccagc agt 4343

<210> 7

<211> 3968

<212> DNA

<213> Porcine delta coronavirus (Porcine deltacoronavirus)

<400> 7

ccacgaggaa ttccagcagt ctctactcgc gggctaataa ttttgatgtt ggcgttcttc 60

ctggctaccc cactaagaac gttaacctct tctcaccact tactaactcc actttgccca 120

ttaatggcct tcatcggagt tatcaaccac tcatgctgaa ttgtcttact aaaataacta 180

accacactct cagcatgtat ctccaaccta gtgatataca aacctatagc tgcggcggtg 240

ccatggttaa acaccagaca catgatgcag ttcgtatcat tttagacctc actgccactg 300

accacatctc tgttgaagtc gttggccagc atggtgaaaa ttatgtgttt gtttgtagtg 360

agcagtttaa ctataccact gcattacaca actctaccgt cttctcactt aattctgagc 420

tttattgctt tactaataac acctacttag gtattcttcc acctgattta actgacttta 480

cggtctatcg tactggtcag ttctatgcta atggttacct tttaggtact ttacctatta 540

cggttaacta tgttaggttg tatcggggtc acttggcggc caatagtgcc cactttgccc 600

ttgcaaactt aaccgataca cttataacac ttaccaatac tactatatcg caaatcactt 660

attgtgataa gtcagtagtt gattcaatag catgccagcg ctcttctcac gaagtggagg 720

atgggtttta ctctgaccct aaatctgccg ttagagctag gcaacgtact attgttacac 780

tacctaagct ccctgagctt gaagtagtgc agttaaatat ttctgcacac atggattttg 840

gcgaagccag acttgacagc gttaccatta atggtaacac atcctactgt gtcactaagc 900

cttacttcag gcttgaaact aactttatgt gtacaggttg cactatgaat ctgcgcactg 960

atacctgtag ttttgacctg tcagcagtaa acaatggcat gtcattctct caattctgtc 1020

taagcactga atctggtgct tgtgagatga aaattattgt tacctacgta tggaattact 1080

tgctaaggca gcgtttgtat gttacagctg tagagggtca gactcacact ggaaccactt 1140

cagtacatgc aacagacact tctagtgtaa tcactgatgt ctgcactgac tacactatct 1200

atggagtctc tggcactggc attattaagc catcagatct cttattgcac aatggcatag 1260

cattcacctc tccaacaggt gagctttatg catttaaaaa tataaccact ggcaaaaccc 1320

ttcaggtctt accgtgtaaa accccttctc tactgattgt gataaacaac accgttgtcg 1380

gtgctatcac atccagtaat tcaactgaaa ataataggtt tactactact attgtcacac 1440

ctactttctt ttattccaca aatgccacca ccttcaactg caccaagcct gttttgtcct 1500

atggacccat cagcgtgtgt agtgatggtg caattgcggg aacatccaca ttacagaata 1560

ctcgaccatc catagtttca ctatacgatg gcgaagttga aataccatct gcattttctc 1620

tttctgttca gacggagtat ttgcaagttc aatcagagca agttatagtt gattgtcctc 1680

agtatgtatg caatggcaac agccgttgtc tacaattact ggcacaatac acctcagctt 1740

gctctaacat tgaagcagct ctgcattcct ctgtacagtt ggatagcaga gagattataa 1800

atatgtttaa aacatcaaca cagtccttgc agttggctaa tattaccaac ttcaagggtg 1860

actacaattt tagcagcata ctaaccacca gacttggtgg cagatctgct attgaagacc 1920

ttctttttaa taaagttgtt actagtggcc ttggcactgt tgatcaggac tacaaagcct 1980

gctctagaga catggccatc gctgacttag tttgttccca gtattacaat ggcatcatgg 2040

ttctacctgg tgttgttgat gctgagaaaa tggcaatgta tactggctct cttactggag 2100

ctatggtatt tgggggactg actgctgcag cggcaatacc attcgccacg gcagtacaag 2160

cccgccttaa ttatgtcgca ctgcaaacaa atgtactaca agaaaaccag aaaattcttg 2220

cagaatcatt taaccaagca gttggcaata tatcacttgc actatcctct gttaatgatg 2280

ccatcaagca aacttctgag gctcttaaca ccgtagctat tgctattaaa aagattcaaa 2340

cagttgttaa ccagcagggt gaggcattat cacacctgac tgcacagctg tcaaacaatt 2400

ttcaggcaat ttcgacttct attcaagaca tttacaaccg tcttgaggaa gtagaggcta 2460

accagcaagt tgaccgtctc atcacaggac ggttggctgc acttaatgca tatgttactc 2520

agttactcaa tcagatgtct cagattagac aatctcgatt gctagctcag caaaagatta 2580

atgagtgtgt caaatctcag tcgtccagat acggtttctg tggaaatggc acacacatct 2640

tctcacttac acagactgca ccaaatggca tatttttcat gcatgcagtg cttgtaccca 2700

acaaattcac acgtgtcaac gcttctgccg gcatttgtgt ggataatacc agaggctact 2760

cattgcagcc tcaacttata ctctaccagt ttaataactc ctggagagtt acacctagaa 2820

atatgtatga acccagactg ccccggcaag ctgattttat acaattaact gattgcagcg 2880

ttacttttta caacaccaca gctgctaatc ttcccaatat tattcctgac gttatagatg 2940

ttaatcaaac agtcagtgat attattgaca atctacctac agcaacacct cctcagtggg 3000

atgttggtat ctataataat actattctca accttaccgt tgagattaat gatctacaag 3060

agcggtctaa aaacctctct cagattgcag atcgtttaca aaattatatt gataatctta 3120

ataatactct agttgacctt gattggctca acagagtgga aacttacctt aaatggccgt 3180

ggtatatatg gcttgccatt gccctggctc ttattgcatt tgtgacaatc ctcataacaa 3240

tctttctttg tactggttgt tgtggtggtt gctttggttg ttgtggcggt tgttttggcc 3300

ttttctctaa gaagaaaagg tataccgacg accaaccaac accgtccttt aagtttaagg 3360

aatggtagtc gatgactggg ccgttaccat ccctggacaa tatattattg ctatactagt 3420

tgtcacctgc attggtgtgg cactactttt tattaacact tgcttagctt gtgttaaatt 3480

attttacaag tgctacctag gggcagcata tcttgttagg cctattatag tgtactactc 3540

caagccgaac cccgtacctg aggatgagtt tgtaaaagta caccaatttc ctagaaacac 3600

tcactatgtc tgacgcagaa gagtggcaaa ttattgtttt cattgcgatc atatgggcac 3660

ttggcgtcat cctccaagga ggctatgcca cgcgtaatcg tgtgatctat gttattaaac 3720

ttattctgct ttggctgctc caacccttca ccctagtggt gaccatttgg accgcagttg 3780

acagatcatc taagaaggac gcagttttca ttgtgtccat aatttttgcc gtactgacct 3840

tcatatcctg ggccaagtac tggtatgact caattcgttt attaatgaaa accagatctg 3900

catgggcact ctcacctgag agtagactcc ttgcagggat tatggatcca atgggtacat 3960

ggaggtgc 3968

<210> 8

<211> 635

<212> DNA

<213> Porcine delta coronavirus (Porcine deltacoronavirus)

<400> 8

ggcgtgcact tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt 60

agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 120

ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 180

gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 240

ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 300

atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 360

catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 420

gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 480

gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 540

attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt 600

agtgaaccgt acatggggac taaagataaa aatta 635

<210> 9

<211> 366

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

atagggggga tggagcaaaa aaaaaaaaaa aaaaaaaaaa aaagggtcgg catggcatct 60

ccacctcctc gcggtccgac ctgggcatcc gaaggaggac gcacgtccac tcggatggct 120

aagggagagc cactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 180

cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 240

catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 300

agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggaag 360

cttctc 366

<210> 10

<211> 3390

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

ggcgtgcact tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt 60

agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 120

ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 180

gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 240

ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 300

atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 360

catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 420

gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 480

gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 540

attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt 600

agtgaaccgt acatggggac taaagataaa aattatagca ttagtctata attttatctc 660

cctagcttcg ctagttctct accgacacca atccaggtgc gtctgccacc aagttggcta 720

ccctttctag gggcgctttt gcgcttgctc accattagat tacctggaaa ccagccattc 780

aggttggagt ttccccaggc acttttgcgt gggcattagc ggcttgtggt ttttgcacaa 840

aatctaagct acttaccgtt cctctgacca ttcaccactt ctatagacag cactgactac 900

cgtagggttc aagtcacacc ggtctgcacc gcccgtcagc ggacacatta cccagcatag 960

cactccttgc accgagccta ggtagcttgc agggattatg gatccaatgg gtacatggag 1020

gtgcattccc atcgaccaca tggctccaat tctcacacca gtcgttaagc atggcaagct 1080

caagctacat gggcaagagc tggccaatgg catatcagtc agaaatccgc cacaggatat 1140

ggtgatagtg tcaccaagtg acacctttca ctacactttt aagaaacctg tggaatcaaa 1200

caacgatcca gaattcgctg ttctgatata ccagggtgac cgcgcttcaa acgctggact 1260

tcacaccata accacttcaa aggccggtga cgctcgcctg tataagtata tgtaatgtgc 1320

aactgccacc tgcagctgcg agatttatat agattgtgca ataagcggca catcagaaga 1380

gaggatgttc ctgagcttat tgaccctctc gttaaaactc gctgttttgc ttacagtctc 1440

gtggttcttg ctaatgctaa tccaattgca tttagcatac tacctcggaa acttcttatc 1500

aatggtgagc ctttactgct tgaatatggt agcatatatg gtaaagactt tatcattcga 1560

ccatcgctcc aagtcattct tgaagatgaa ttaaattaaa gttttgacac caatctatca 1620

tggctgcacc agtagtccct actactgacg cgtcttggtt tcaggtgctc aaagctcaaa 1680

acaaaaaagc cattcatcct cagtttcgtg gcaatggagt tccgcttaac tccgccatca 1740

aacccgttga aaatcatggc tactggctgc gttacaccag acaaaagcca ggtggtactc 1800

cgattcctcc atcctatgcc ttttattata ctggcacagg tcccagagga aatcttaagt 1860

atggtgaact ccctcctaat gataccccag caaccactcg tgttacttgg gttaagggtt 1920

cgggagctga cacttctatt aagcctcatg ttgccaaacg caaccccaac aatcctaaac 1980

atcagctgct acctctccga ttcccaaccg gagatggccc agctcaaggt ttcagagttg 2040

accccttcaa cgctagagga agacctcagg agcgtggagg tggcccaaga tctcaatctg 2100

ttaactccag aggcacaggc aatcagccta ggaaacgcga ccaatctgca cccgctgcgg 2160

tacgtcgtaa gacccaacat caagctccca agcggacttt acctaagggt aaaaccattt 2220

ctcaggtatt tggcaaccgg tctcgtactg gtgccaatgt cggctctgca gacactgaga 2280

agacgggtat ggctgatcct cgcatcatgg ctctagccag acatgtgcct ggtgttcagg 2340

aaatgctttt cgctggtcac ctcgagagca actttcaggc gggggcaatt acccttacct 2400

tctcttactc aatcacagtc aaggagggtt ctcctgacta tgagagactt aaggatgcgc 2460

tcaacacggt cgttaaccag acctatgagc cacccaccaa accaactaag gacaagaagc 2520

ctgacaaaca agaccagtct gctaaatcca aacagcagaa gaaacctaaa aaggtaactc 2580

tgccagcaga caaacaggat tgggagtggg atgatgcttt tgagataaag caggaatcag 2640

cagcgtagac atcaatctat gtctgttaaa cccacccaac tccactcaaa tatctctttg 2700

gttccagaga gtcgtagtgt atagccagag agccagtcag agggcgctat catgcaaact 2760

agggctggct actctagcac agaatcacat cccgataatc aacagtgcta gaaggttgat 2820

tataccattt aatatgccga ggccacgcgg agtacgatcg agggtacagc ataatctcaa 2880

cttttgttga gccacaattt taatcctaat tggagaaggc caaaggactg tactacttct 2940

gtaggtgtag cagtcgccca gtgggaaagc gccaactagg ttacaattgt ggtggggaca 3000

aattagggga aattaaattg gcttataggg gggatggagc aaaaaaaaaa aaaaaaaaaa 3060

aaaaaaaggg tcggcatggc atctccacct cctcgcggtc cgacctgggc atccgaagga 3120

ggacgcacgt ccactcggat ggctaaggga gagccactgt gccttctagt tgccagccat 3180

ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 3240

tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg 3300

ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg 3360

gggatgcggt gggctctatg gaagcttctc 3390

<210> 11

<211> 37

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

acatggggac taaagataaa aattatagca ttagtct 37

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

ctacctaggc tcggtgcaag g 21

<210> 13

<211> 52

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

cttgcaccga gcctaggtag cttgcaggga ttatggatcc aatgggtaca tg 52

<210> 14

<211> 37

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

ttgctccatc ccccctataa gccaatttaa tttcccc 37

<210> 15

<211> 43

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

tagcactcct tgcaccgagc ctaggtagga taaaaccccc tac 43

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

ggcaaattta atggcaggac 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

gtcctgccat taaatttgcc 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

gttgtgaatc gatttgcaag 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

cttgcaaatc gattcacaac 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

cagagtgcat ctattgctgc 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

gcagcaatag atgcactctg 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

actgctggaa ttcctcgtgg 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

ccacgaggaa ttccagcagt 20

<210> 24

<211> 42

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

gcacctccat gtacccattg gatccataat ccctgcaagg ag 42

<210> 25

<211> 34

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

ggcgtgcact tgacattgat tattgactag ttat 34

<210> 26

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

taatttttat ctttagtccc catgtacggt tcactaaacg agctctgctt atatagacc 59

<210> 27

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

atagggggga tggagcaaaa aaaaaaaaaa aaaaaaaaaa aaagggtcgg catggcatc 59

<210> 28

<211> 34

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

gagaagcttc catagagccc accgcatccc cagc 34

<210> 29

<211> 1080

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

ccaagttggt gattacattc ttgctaaaac agatggcagt gacacttata cttacagagg 60

aacatctacc tacaaactcc aaacaggtga tgttctagtc ttaatggcac atgttgttac 120

accgctctca gcaccccctg tgctaacgca gacaacatat gtcagaaaat cacttttacc 180

cgactctgtt ggtgcgtctt attatgtgca acattttaag tcatataatg agatagctat 240

gcagagggtt acaacagtat taggtccgcc aggcacaggt aagtcaacct ttgctattgg 300

tttggctaag tactttccta gtgcacgtat ttgctacact gcgtcttcgc atgcagcaat 360

agatgcactc tgtgaaaaag ctttcaagac aatacctgta ggccaatgca gtcgtatcgt 420

acccacacgt acaactgttg agtgctttca ggagtttgtc gtaaataaca caactgcaca 480

gtatatcttc tcgactatca atgccttacc tgacattaag tgtgacattg tagttgtaga 540

tgaggtttct atgttgacca attatgagct ttcctctgtg aatgctcgtt tggtttacaa 600

tcacattgtg tatgttggtg atccttatca gttaccttca cctagaacta tgcttacgtc 660

tggccagctt tcgccagctg actataacgt agttactgat ataatggtac atgcaggagc 720

ggacgttatg ctcgacatgt gctacagatg cccacgtgaa atcgttgaga cagtgtctaa 780

acttgtctac gataacaaac taaaagcggc gaaaccgaac tcaagacagt gttacaagac 840

cattgtgaac tttggtcctg gagacgttgc tcatgaggga caatctgcct acaacgaagc 900

acagttgcgt ttcgcactcg catttagaca acaaaagcgg tgggataacg tgactttcat 960

atctccatat aatgctatga atgtgaaagc atccttagca ggtttctcta ctcagaccgt 1020

tgactcttct caaggttctg agtatgatta tgttatcttt tgcgtgacca ctgactcagc 1080

Claims (7)

1. A construction method of infectious clone plasmid of porcine delta coronavirus is characterized by comprising the following steps:

(1) extracting RNA of the porcine delta coronavirus and performing reverse transcription to obtain cDNA;

(2) using the cDNA as a template, designing primers to carry out PCR amplification respectively to obtain a 5 'end gene sequence, a 3' end gene sequence and 5 gene fragments of the porcine delta coronavirus, wherein the 5 gene fragments contain a homologous sequence of 20bp, and in addition, a 15121 site C silencing mutation is changed into A, so that a ClaI endonuclease sequence is eliminated and is used as a genetic marker;

(3) taking pBAC-AJ1102 plasmid as a template, designing primers, and respectively carrying out PCR amplification to obtain a CMV promoter and an HDV-BGH gene sequence;

(4) cloning a CMV promoter, a 5 'end gene sequence, a 3' end gene sequence, a poly (A) structure of 27 adenine deoxynucleotides (A), a hepatitis C virus (HDV) ribozyme self-cleavage site and a bovine growth hormone termination sequence (BGH) into a low-copy vector pBeloBAC11 by using an ApaLI and HindIII restriction endonuclease and a multi-step fusion PCR method by using a BAC system to obtain an intermediate vector pBAC-M-PDCoV;

(5) 5 gene segments are subjected to homologous recombination in proportion and are cloned into an intermediate vector pBAC-M-PDCoV in one step to obtain a recombinant plasmid pBAC-CHN-HN-2014;

(6) transforming the recombinant product into DH10B chemically competent cells, screening positive clones by PCR, carrying out amplification culture on positive clones with correct sequencing, and extracting plasmids.

2. The method of claim 1, wherein the method comprises the steps of: the porcine delta coronavirus is CHN-HN-2014 strain.

3. The method for constructing infectious cloned plasmid of porcine delta coronavirus according to claim 1, wherein the 5 'end gene sequence, the 3' end gene sequence and the 5 gene segments have the sequences of:

5' end gene sequence: 1, SEQ ID NO;

3' end gene sequence: 2, SEQ ID NO;

gene fragment A: 3, SEQ ID NO;

gene fragment B: 4, SEQ ID NO;

gene fragment C: 5, SEQ ID NO;

gene fragment D: 6, SEQ ID NO;

gene fragment E: SEQ ID NO 7.

4. The method for constructing infectious cloned plasmid of porcine delta coronavirus according to claim 3, wherein the primers for PCR amplification of 5 'end gene sequence, 3' end gene sequence and 5 gene fragments are respectively:

PDCoV-5’-F：SEQ ID NO:11

PDCoV-5’-R：SEQ ID NO:12

PDCoV-3’-F：SEQ ID NO:13

PDCoV-3’-R：SEQ ID NO:14

A-F：SEQ ID NO:15

A-R：SEQ ID NO:16

B-F：SEQ ID NO:17

B-R：SEQ ID NO:18

C-F：SEQ ID NO:19

C-R：SEQ ID NO:20

D-F：SEQ ID NO:21

D-R：SEQ ID NO:22

E-F：SEQ ID NO:23

E-R：SEQ ID NO:24。

5. the method of claim 1, wherein the method comprises the steps of: the sequence of the CMV promoter is shown as SEQ ID NO. 8, and the sequence of the HDV-BGH gene is shown as SEQ ID NO. 9.

6. The method of claim 5, wherein the method comprises the steps of: the primers for PCR amplification of the CMV promoter and the HDV-BGH gene are respectively as follows:

CMV-F：SEQ ID NO:25

CMV-R：SEQ ID NO:26

HDV-F：SEQ ID NO:27

BGH-R：SEQ ID NO:28。

7. the method of claim 1, wherein the method comprises the steps of: in the step (5), the molar ratio of the 5 gene segments to the intermediate vector pBAC-M-PDCoV is 1:2:2:1:1: 1.

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