CN111334528B - Mink enteritis parvovirus whole genome infectious clone and construction method and application thereof - Google Patents

Mink enteritis parvovirus whole genome infectious clone and construction method and application thereof Download PDF

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CN111334528B
CN111334528B CN202010203738.3A CN202010203738A CN111334528B CN 111334528 B CN111334528 B CN 111334528B CN 202010203738 A CN202010203738 A CN 202010203738A CN 111334528 B CN111334528 B CN 111334528B
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CN111334528A (en
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谢之景
朱倩
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Shandong Agricultural University
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Abstract

The invention provides a mink enteritis parvovirus whole genome infectious clone and a construction method and application thereof. The method is to carry out enzyme digestion on the complete genome in the MEV replication form, and directionally clone the complete genome to a vector pUC18-M in a segmented form in sequence to obtain a recombinant plasmid. The recombinant plasmid and the transfection reagent are mixed and then transfect CRFK cells, and the rescued virus can be obtained. The invention utilizes the mink enteritis parvovirus infectious clone constructed by the reverse genetic technology to transfect CRFK cells in vitro, and can induce the cells to generate cytopathy and growth tendency which are the same as those of parental viruses. The method is simple, rapid, time-saving and labor-saving, has higher accuracy compared with the traditional PCR method, and can rapidly and conveniently carry out base mutation on any position of the MEV genome by applying the cloning system, thereby providing an effective way and means for the subsequent research and development of molecular biology of MEV and the development of vaccines.

Description

Mink enteritis parvovirus whole genome infectious clone and construction method and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to a mink enteritis parvovirus whole genome infectious clone using a reverse genetic manipulation technology, and a construction method and application thereof.
Background
Mink enteritis parvovirus (MEV) can cause Mink viral enteritis, a highly contagious disease characterized by gastrointestinal mucosal hemorrhage and severe diarrhea, and has high morbidity and mortality, with young minks being more susceptible. The disease is fulminantly epidemic in China beginning in the eighties of the last century, causes great economic loss to the mink breeding industry in China, and is one of three diseases which are recognized to damage the mink breeding industry in the world. At present, although vaccines are used for preventing mink viral enteritis, the disease is still more frequent in production practice, and the health of minks is seriously harmed.
The reverse genetic technology is an effective molecular biology means for developing virology research, and plays an important role in the elucidation of virus pathogenic mechanisms and the development of vaccines. The infectious clone of mink enteritis parvovirus is constructed by utilizing a reverse genetic technology, so that mutation is introduced into any site of the virus, and the pathogenic mechanism of the virus can be conveniently and more finely and accurately researched, so that the virus is more deeply researched and understood on a gene level, and meanwhile, the infectious clone of mink enteritis parvovirus is an effective way for researching mink enteritis virus vaccine, and has great veterinary public health significance.
According to the MEV whole genome sequence reported by GenBank, the hairpin structure at the 3 '-end is 205nt long and forms a Y-shaped structure, while the hairpin structure at the 5' -end is 62nt long and forms a U-shaped structure. The existence of the special structure seriously influences the cloning of the parvovirus whole genome and the establishment of a reverse genetics operation platform. The prior art mainly adopts a PCR method for constructing infectious clones of mink enteritis parvovirus, but the PCR method has the problem of mismatching when a virus sequence is amplified. Meanwhile, the conventional plasmid is adopted, so that the problems of small capacity and difficulty in bearing long fragment sequences exist.
At present, the research on the replication process of the whole parvovirus family is very little, especially on the aspect of mink enteritis parvovirus molecules. Although GenBank has reported MEV whole genome sequences, few examples of full-length clones have been studied, particularly full-length infectious clones. Because the genome structure of the autonomously replicating parvovirus is special, the full-length genome is difficult to clone, and the establishment of a reverse genetics operation platform is seriously influenced.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a novel construction method of infectious clone plasmids of Mink enterovirus parvovirus (MEV), and a reverse genetics operating system is established, so that technical support is provided for virus virulence research and novel vaccine development in the future.
In order to achieve the above object, the present invention provides, in one aspect, a mink enteritis parvovirus whole genome infectious clone obtained by digesting a mink enteritis parvovirus whole genome, and sequentially cloning the mink enteritis parvovirus whole genome to a vector pUC18-M in a segmented manner. The nucleotide sequence is shown in SEQ ID NO. 1.
On the other hand, the invention also provides a construction method of the mink enteritis parvovirus whole genome infectious clone, which mainly comprises the following steps: carrying out enzyme digestion on the complete genome of the mink enteritis parvovirus replication form; and (3) directionally cloning two sections of gene fragments obtained after enzyme digestion to a vector pUC18-M in a segmented form in sequence to obtain a recombinant plasmid.
The vector pUC18-M is formed by enzyme digestion transformation of a vector pUC 18; the transformation process is as follows: the circular plasmid was first linearized by digestion with Hind III and then blunt-ended by Blunting enzyme treatment to blunt-ended cohesive ends at both ends of the plasmid DNA, designated the vector pUC 18-M.
The complete genome of the mink enteritis parvovirus replication form is obtained by the following method: inoculating the mink enteritis parvovirus to CRFK cells, collecting the cells for genome extraction when the cells grow vigorously and most of swelling becomes round, and obtaining a complete genome of the mink enteritis parvovirus replication form; the genome was digested with Pst I to generate two DNA fragments.
Specifically, the construction method of the mink enteritis parvovirus whole genome infectious clone comprises the following steps,
(1) transformation of vector pUC18
Firstly, using Hind III to linearize pUC18, and then using Blunting blunt-ended enzyme to treat two ends of a vector fragment from a viscous end to a blunt end, wherein the vector fragment is named as pUC 18-M; carrying out PstI enzyme digestion on the pUC18-M vector to change one end of the vector fragment into a viscous tail end;
(2) MEV replication form genome extraction
Inoculating MEV virus into CRFK cells at a cell density of about 106The titer of MEV is 10 per mL7TCID50The method comprises inoculating 0.5mL of MEV virus solution into 10mL of LCRFK cells, infecting for about 36h, collecting cells when the cells grow vigorously and most of swelling becomes round, and extracting genome to obtain complete genome of MEV replication form, wherein the length of the complete genome is about 5.1 kb.
(3) MEV replication form genome segmentation cloning
Carrying out enzyme digestion on the MEV replication form double-stranded genome obtained in the step (2) by using Pst I, and cutting two DNA fragments MEV-5 '(about 3.1kb) and MEV-3' (about 2kb), wherein the sequences are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3; connecting and transforming pUC18-M, MEV-3 'to obtain pM-MEV-3' recombinant plasmid; then carrying out PstI enzyme digestion on the pM-MEV-3' recombinant plasmid, and then carrying out SmaI enzyme digestion; and connecting and transforming the enzyme-cut pM-MEV-3 'and MEV-5' to obtain a pM-MEV recombinant plasmid.
In still another aspect, the invention provides a reverse genetics operation system of mink parvovirus, which comprises the mink enteritis parvovirus whole genome infectious clone and a host cell. The host cell is a CRFK cell.
The reverse genetics operating system is applied to mink parvovirus rescue, research on pathogenesis and pathogenicity of mink parvovirus and development of mink parvovirus vaccines.
In another aspect of the invention, a method for rescuing mink parvovirus is provided: adding a Lipofectamine2000 transfection reagent into a centrifugal tube, adding an Opti-DMEM culture solution, uniformly mixing, and standing; adding pM-MEV recombinant plasmid, mixing, standing at room temperature for 20min to obtain transfection mixture; removing culture medium from full monolayer CRFK cells, washing with PBS or serum-free medium for 2-3 times, adding the transfection mixture, and placing the cells in 5% CO at 37 deg.C2Culturing for 1h in an incubator, adding a complete culture medium with 7% of serum content, and continuously culturing for 24-48h for cell passage; collecting cell supernatant with suspected cytopathic effect to obtain the rescued virus.
Compared with the prior art, the invention has the following beneficial effects:
(1) the mink enteritis parvovirus whole genome infectious clone constructed by the invention covers the mink enteritis parvovirus full-length genome for the first time. Due to the adoption of the DNA in a replication form, the mismatching problem when a PCR method is used for amplifying a virus sequence is avoided, the problems of small capacity and difficulty in bearing a long fragment sequence of a conventional plasmid are solved, and an important molecular tool is provided for the subsequent modification of an MEV whole gene and the research of the influence of different amino acid sites on biological characteristics.
(2) The invention adopts the methods of ampicillin resistance and enzyme digestion verification of plasmids to screen infectious clones of mink enteritis parvovirus, and is convenient and quick.
(3) The reverse genetics operating system of the mink parvovirus established by the invention can induce the cells to generate cytopathy and growth tendency the same as those of parental viruses by infecting and cloning the mink enteritis parvovirus in vitro to transfect CRFK cells. The method is simple, rapid, time-saving and labor-saving, has higher accuracy compared with the traditional PCR method, and can rapidly and conveniently carry out base mutation on any position of the MEV genome by applying the cloning system, thereby providing an effective way and means for the molecular biology research of the MEV and the vaccine development in the future.
Drawings
FIG. 1 agarose gel electrophoresis of extraction of genomic DNA of MEVSD1 strain. M: DNA Marker DL 5000; 1: MEV copies around 5kb in form of double stranded DNA molecules.
FIG. 2 restriction identification of pM-MEV-3' plasmid. M: DNA Marker DL 5000; 1: a DNA fragment with the size of 4748bp generated by the enzyme digestion of Pst I; 2: DNA fragments with the sizes of 2355bp, 1575bp and 727bp are generated by PvuII enzyme digestion; 3: the Ssp I enzyme digestion generates DNA fragments with the sizes of 2568bp, 1549bp, 444bp and 187 bp.
FIG. 3 construction strategy of infectious clone pM-MEV of MEV strain.
FIG. 4 enzyme digestion identification of pM-MEV plasmid. M: DNA Marker DL 5000; 1: a DNA fragment of 7823bp generated by enzyme digestion with Pst I; 2: DNA fragments with the sizes of 6384bp and 1439bp generated by EcoRI enzyme digestion; 3: DNA fragments with the sizes of 7161bp and 662bp generated by HindIII enzyme digestion; 4: DNA fragments with the sizes of 3893bp, 2355bp and 1575bp generated by PvuII enzyme digestion; 5: the SspI digestion produces DNA fragments of 5824bp, 1368bp, 444bp and 187 bp.
FIG. 5 agarose gel electrophoresis to verify successful transfection of pM-MEV plasmid. M: DNA Marker DL 2000; 1: the supernatant after pM-MEV transfection is cut by DNase and PCR amplification is carried out to obtain a VP2 gene fragment with the size of 1755 bp.
Detailed Description
For a better understanding of the present invention, the present invention is further described below in conjunction with specific biological materials and parameters, but not further limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; the reagents involved are commercially available.
1. Transformation of vector pUC18
Firstly, linearizing pUC18 by using Hind III, performing 1% agarose gel electrophoresis after acting for 1h at 37 ℃, cutting DNA of a target band, recovering and purifying; then treating the purified vector by using Blunting blunt-ended enzyme, performing 1% agarose gel electrophoresis after acting for 10min at 37 ℃ and 5min at 70 ℃, cutting DNA of a target band, recovering and purifying, wherein the two ends of a vector fragment are changed from sticky ends to flat ends in the step and named as pUC 18-M; and finally carrying out PstI enzyme digestion on the vector purified in the last step, carrying out 1% agarose gel electrophoresis after acting for 1h at 37 ℃, cutting off DNA of a target band, recovering and purifying, wherein one end of a vector fragment is changed into a viscous tail end in the step, so that subsequent cloning is facilitated.
MEV replicative genome extraction
The MEV virus is inoculated on CRFK cells, and the inoculation dose and the virus inoculation time are groped according to the virus titer. When the cell density is about 106The titer of MEV is 10 per mL7TCID50And (4) inoculating 0.5mL of MEV virus solution into 10mL of CRFK cells, and collecting cells for genome extraction when the cells grow vigorously and most of cells swell and become round after about 36 hours of infection to obtain the complete genome of the MEV replication form. The specific method for extracting the genome is as follows: sucking out the nutrient solution in the cell culture bottle, and washing for 2-3 times by using PBS; pouring out PBS, adding 1mL of lysate, and acting for 1h at 37 ℃; adding 250 mu L of 5M NaCl solution, and simultaneously shaking the cell culture bottle, and carrying out ice bath for 1 h; transferring the viscous lysate into a 1.5mL centrifuge tube, and centrifuging at 12000rpm at 4 ℃ for 30-45 min; sucking out the supernatant, transferring into a clean centrifuge tube, adding equal volume of phenol/chloroform (1: 1), inverting and standing for a while, centrifuging at 12000rpm for 15min, and repeating the step if the intermediate protein part is too much after centrifugal separation; adding 0.25M sodium acetate and 2 times volume of anhydrous ethanol, acting for 10-15min, centrifuging at 12000rpm for 15min, and pouring off supernatant; eluting with 70% ethanol, slowly adding ethanol and mixingDrying; adding 25-50 μ L TE, adding 1-2 μ L RNase, and reacting at 37 deg.C for 20-30 min; finally, 1% agarose gel electrophoresis is carried out, and a target band of about 5.1kb is recovered and purified by incision in an ultraviolet gel cutter (see figure 1).
MEV replication form genome segmentation cloning (see FIG. 3)
The purified double-stranded genome of the MEV replication form is subjected to enzyme digestion by using Pst I, 1% agarose gel electrophoresis is carried out after 1 hour of action at 37 ℃, two DNA fragments MEV-5'(3098bp) and MEV-3' (2064bp) are respectively cut, and the sequences are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3. And respectively recovering and purifying. Mu.l of pUC18-M, 4. mu.L of MEV-3' and 6. mu.L of Ligation solution I were blown and mixed well and left overnight at 16 ℃. Adding the ligation product into DH5 alpha competent cells, gently and uniformly mixing, carrying out ice bath for 30min, carrying out heat shock for 90s at 42 ℃, carrying out ice bath for 2-3min, adding 800 mu L of nonresistant LB liquid culture medium, and then placing on a shaking table at 37 ℃ for shaking for 45 min; sucking 200 μ L of the conversion product, uniformly coating on an ampicillin plate, and inverting at 37 deg.C for 8-12 h; picking single colony on the ampicillin plate, putting the single colony into LB liquid culture medium with ampicillin resistance, placing the single colony in a shaker at 37 ℃ for shaking for 8-12h, then carrying out plasmid extraction, and screening pM-MEV-3' recombinant plasmid by a method of enzyme digestion identification (see figure 2).
Carrying out PstI enzyme digestion on the purified pM-MEV-3', carrying out 1% agarose gel electrophoresis after 1h action at 37 ℃, recovering and purifying the obtained recombinant plasmid, carrying out SmaI enzyme digestion again, carrying out 1% agarose gel electrophoresis after 1h action at 30 ℃, blowing and uniformly mixing 2 mu L of enzyme digestion purified pM-MEV-3', 4 mu L LMEV-5' and 6 mu L Ligation solution I, placing the mixture at 16 ℃ overnight, repeating the transformation process, selecting a single colony for amplification culture, and screening the pM-MEV recombinant plasmid by an enzyme digestion identification method (see figure 4).
4. CRFK cell rescue by recombinant plasmid transfection
4.1. Preparation of transfection reagents
The transfection mixture was prepared according to the Lipofectamine2000 transfection reagent (Invitrogen, Carlsbad, USA) instructions. The vector and transfection reagent were performed at 1:2.5 (. mu.g/. mu.L). Mu.g of the purified plasmid solution was taken into 1 sterilized 1.5mL centrifuge tube, diluted to 1mL with DMEM medium (Invitrogen), and allowed to stand for 5 min. Another sterilized 1.5mL centrifuge tube was added 40. mu.L Lipofectamine2000 transfection reagent, and 960. mu.L DMEM was added to mix well and allowed to stand for 5 min. After standing, the diluted plasmid and transfection reagent are gently mixed, and the mixture is stood at room temperature for 20min for transfection.
4.2. Transfection of CRFK cells
Removing culture medium from full monolayer cells, washing with PBS or serum-free culture medium for 2-3 times, adding the prepared transfection reagent, and placing the cells in 5% CO at 37 deg.C2Culturing for 1h in an incubator, adding complete culture medium with 7% serum content, continuously culturing for 24-48h, carrying out cell passage, observing whether the cells have lesions, and determining whether secondary transfection is needed according to the cell state. Collecting cell supernatant with suspected cytopathy, randomly cutting off recombinant plasmids which are not transfected into cells by using DNase, and carrying out PCR amplification of VP2 fragments on the cut supernatant, wherein the primer sequence: VP2-F:5'-CTTTGCCTCAATCTGAAGGAGT-3' and VP2-R:5'-GAATTGGATTCCAAGTATGAGAT-3' under the reaction condition of 96 ℃ for 3 min; denaturation at 95 ℃ for 30 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 140 s; 32 cycles, extension at 72 ℃ for 10min, storage at 4 ℃. Successful transfection was confirmed by agarose gel electrophoresis of the band of interest at around 1.8kb (see FIG. 5).
VP2 gene PCR reaction system
Figure BDA0002420235580000051
Sequence listing
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<120> mink enteritis parvovirus whole genome infectious clone and construction method and application thereof
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cgccaccttt tcccgcccaa gtttaaacac acaaaccgcc tatcattctt tagaaccaac 60
tgaccaagtt cacgtacgta tgacgtgatg acgcgcgctg cgcgcgctgc ctacggcagt 120
cacacgtcat acgtacgctc cttggtcagt tggttctaaa gaatgatagg cggtttgtgt 180
gtttaaactt gggcgggaaa aggtggcggg cattgtgggc gtggttaaag gtataaaaga 240
caaaccatag accgttactg acattcgctt cttgtctttg acagagtgaa cctctcttac 300
tttgactaac catgtctggc aaccagtata ctgaggaagt tatggaggga gtaaattggt 360
taaaaaaaca tgcagaaaat gaagcatttt cgtttgtttt taaatgtgac aacgtccaac 420
taaatggaaa ggatgttcgc tggaacaact ataccaaacc aattcaaaat gaagagctaa 480
catctttaat tagaggagca caaacagcaa tggatcaaac cgaagaagaa gaaatggact 540
gggaatcgga agttgatagt ctcgccaaaa agcaagtaca aacttttgat gcattaatta 600
aaaaatgtct ttttgaagtc tttgtttcta aaaatataga accaaatgaa tgtgtttggt 660
ttattcaaca tgaatgggga aaagatcaag gctggcattg tcatgtttta cttcatagta 720
agaacttaca acaagcaact ggtaaatggc tacgcagaca aatgaatatg tattggagta 780
gatggttggt gactctttgt tcggtaaatt taacaccaac tgaaaagatt aagctcagag 840
aaattgcaga agatagtgaa tgggtgacta tattaacata cagacataag caaacaaaaa 900
aagactatgt taaaatggtt cattttggaa atatgatagc atattacttt ttaacaaaga 960
aaaaaattgt ccacatgaca aaagaaagtg gctatttttt aagtactgat tctggttgga 1020
aatttaactt tatgaagtat caagacagac aaactgtcag cacactttac actgaacaaa 1080
tgaaaccaga aaccgttgaa accacagtga cgacagcaca ggaaacaaag cgcgggagaa 1140
ttcaaactaa aaaggaagtt tcaatcaaat gtactttgcg ggacttggtt agtaaaagag 1200
taacatcacc tgaagattgg atgatgttac aaccagatag ttatattgaa atgatggcac 1260
aaccaggagg tgaaaatctt ttaaaaaata cacttgaaat ttgtactttg actttagcaa 1320
gaacaaaaac agcatttgaa ttaatacttg aaaaagcaga taatactaaa ctaactaact 1380
ttgatcttgc aaattctaga acatgtcaaa tttttagaat gcacggatgg aattggatta 1440
aagtttgtca cgctatagca tgtgttttaa atagacaagg tggtaaaaga aatacagttc 1500
tttttcatgg accagcaagt acaggaaaat ctattattgc tcaagccata gcacaagctg 1560
tggggaatgt tggttgttat aatgcagcaa atgtaaattt tccatttaat gactgtacca 1620
ataaaaattt aatttggatt gaagaagctg gtaactttgg tcaacaagtt aatcaattta 1680
aagcaatttg ttctggacaa acaattagaa ttgatcaaaa aggtaaagga agtaagcaaa 1740
ttgaaccaac tccagtaatt atgacaacta atgaaaatat aacaattgta agaattggat 1800
gtgaagaaag acctgaacat acacaaccaa taagagacag aatgttgaac attaagttag 1860
tatgtaagct tccaggagac tttggtttgg ttgataaaga agaatggcct ttaatatgtg 1920
catggttagt taaacatggt tatgaaccaa ccatggctaa ctatacacat cattggggaa 1980
aagtaccaga atgggatgaa aactgggcgg agcctaaaat acaagaaggt ataaattcac 2040
caggttgcaa agacttagag acacaagcgg caagcaatcc tcagagtcaa gaccaagttc 2100
taactcctct gactccggac gtagtggacc ttgcactgga accgtggagt actccagata 2160
cgcctattgc agaaactgca aatcaacaat caaaccaact tggcgttact cacaaagacg 2220
tgcaagcgag tccgacgtgg tccgaaatag aggcagacct gagagccatc tttacttctg 2280
aacaattgga ggaagatttt cgagacgact tggattaagg tacgatggca cctccggcaa 2340
agagagccag gagaggtaag ggtgtgttag taaagtgggg ggaggggaaa gatttaataa 2400
cttaactaag tatgtgtttt cttataggac ttgtgcctcc aggttataaa tatcttgggc 2460
ctgggaacag tcttgaccaa ggagaaccaa ctaacccttc tgacgccgct gcaaaagaac 2520
acgacgaagc ttacgctgct tatcttcgct ctggtaaaaa cccatactta tatttctcgc 2580
cagcagatca acgctttata gatcaaacta aggacgctaa agattggggg gggaaaatag 2640
gacattattt ttttagagct aaaaaggcaa ttgctccagt attaactgat acaccagatc 2700
atccatcaac atcaagacca acaaaaccaa ctaaaagaag taaaccacca cctcatattt 2760
tcatcaatct tgcaaaaaaa aaaaaagccg gtgcaggaca agtaaaaaga gacaatcttg 2820
caccaatgag tgatggagca gttcaaccag acggtggtca acctgctgtc agaaatgaaa 2880
gagctacagg atctgggaac gggtctggag gcgggggtgg tggtggttct gggggtgtgg 2940
ggatttctac gggtactttc aataatcaga cggaatttaa atttttggaa aacggatggg 3000
tggaaatcac agcaaactca agcagacttg tacatttaaa tatgccagaa agtgaaaatt 3060
ataaaagagt agttgtaaat aatatggata aaactgcagt taaaggaaac atggctttag 3120
atgatactca tgtacaaatt gtaacacctt ggtcattggt tgatgcaaat gcttggggag 3180
tttggtttaa tccaggagat tggcaactaa ttgttaatac tatgagtgag ttgcatttag 3240
ttagttttga acaagaaatt tttaatgttg ttttaaagac tgtttcagaa tctgctactc 3300
aaccaccaac taaagtttat aataatgatt taactgcatc attgatggtt gcattagata 3360
gtaataatac tatgccattt actccagcag ctatgagatc tgagacattg ggtttttatc 3420
catggaaacc aaccatacca actccatgga gatattattt tcaatgggat agaacattaa 3480
taccatctca tactggaact agtggcacac caacaaatgt atattatggt acagatccag 3540
atgatgttca attttatact attgaaaatt ctgtgccagt acacttacta agaacaggtg 3600
atgaatttgc tacaggaaca tttttttttg attgtaaacc atgtagacta acacatacat 3660
ggcaaacaaa tagagcattg ggcttaccac catttctaaa ttctttgcct caatctgaag 3720
gagctactaa ctttggtgat ataggagttc aacaagataa aagacgtggt gtaactcaaa 3780
tgggaaatac agactatatt actgaagcta ctattatgag accagctgag gttggttata 3840
gtgcaccata ttattctttt gaagcatcta cacaagggcc atttaaaaca cctattgcag 3900
caggacgggg gggagcgcaa acagatgaaa atcaagcagc agatggtgat ccaagatatg 3960
catttggtag acaacatggt caaaaaacta ctacaacagg agaaacaccc gagagattta 4020
catatatagc acatcaagat acaggaagat atccagaagg agattggatt caaaatatta 4080
actttaacct tcctgtaaca aatgataatg tattgctacc aacagatcca attggaggta 4140
aaacaggaat taactatact aatatattta atacttatgg tcctttaact gcattaaata 4200
atgtaccacc agtttatcca aatggtcaaa tttgggataa agaatttgat actgacttaa 4260
aaccaagact tcatgtaaat gcaccatttg tttgtcaaaa taattgtcct ggtcaattat 4320
ttgtaaaagt tgcgcctaat ttaacaaatg aatatgatcc tgatgcatct gctaatatgt 4380
caagaattgt gacttactca gatttttggt ggaaaggtaa attagtattt aaagctaaac 4440
taagagcatc tcatacttgg aatccaattc aacaaataag tattaatgta gataaccaat 4500
ttaactatgt accaaataat attggagcta tgaaaattgt atatgaaaaa tctcaactag 4560
cacctagaaa attatattaa tatacttact atgtttttat gtttattaca tatcaactag 4620
cacctagaaa attatattaa tatacttact atgtttttat gtttattaca tattatttta 4680
agattaatta aattacagca tagaaatatt gtacttgtat ttgatatagg atttagaagg 4740
tttgttatat ggtatacaat aactgtaaga aatagaagaa catttagatc atagttagta 4800
gtttgtttta taaaatgtat tgtaaactat taatgtatgt tgttatggtg tgggtggttg 4860
gttggtttgc ccttagaata tgttaaggac caaaaaaatc aataaaagac atttaaaact 4920
aaatggtctc gtatactgtc tataaggtga actaacctta ccataagtat caatctgtct 4980
ttaagggggg ggtgggtggg agatacacaa catcagtaga ctgactggcc tggttggttg 5040
ctctgcttaa tcaaccagac cgcgtagcgg tctggttgat taagcgcaac caaccaggcc 5100
agtcagtcta ctgatgttgt gtatctccca cccacccccc ccttaaagac agattgatac 5160
tt 5162
<210> 2
<211> 3098
<212> DNA
<213> Mink enteritis parvovirus
<400> 2
cgccaccttt tcccgcccaa gtttaaacac acaaaccgcc tatcattctt tagaaccaac 60
tgaccaagtt cacgtacgta tgacgtgatg acgcgcgctg cgcgcgctgc ctacggcagt 120
cacacgtcat acgtacgctc cttggtcagt tggttctaaa gaatgatagg cggtttgtgt 180
gtttaaactt gggcgggaaa aggtggcggg cattgtgggc gtggttaaag gtataaaaga 240
caaaccatag accgttactg acattcgctt cttgtctttg acagagtgaa cctctcttac 300
tttgactaac catgtctggc aaccagtata ctgaggaagt tatggaggga gtaaattggt 360
taaaaaaaca tgcagaaaat gaagcatttt cgtttgtttt taaatgtgac aacgtccaac 420
taaatggaaa ggatgttcgc tggaacaact ataccaaacc aattcaaaat gaagagctaa 480
catctttaat tagaggagca caaacagcaa tggatcaaac cgaagaagaa gaaatggact 540
gggaatcgga agttgatagt ctcgccaaaa agcaagtaca aacttttgat gcattaatta 600
aaaaatgtct ttttgaagtc tttgtttcta aaaatataga accaaatgaa tgtgtttggt 660
ttattcaaca tgaatgggga aaagatcaag gctggcattg tcatgtttta cttcatagta 720
agaacttaca acaagcaact ggtaaatggc tacgcagaca aatgaatatg tattggagta 780
gatggttggt gactctttgt tcggtaaatt taacaccaac tgaaaagatt aagctcagag 840
aaattgcaga agatagtgaa tgggtgacta tattaacata cagacataag caaacaaaaa 900
aagactatgt taaaatggtt cattttggaa atatgatagc atattacttt ttaacaaaga 960
aaaaaattgt ccacatgaca aaagaaagtg gctatttttt aagtactgat tctggttgga 1020
aatttaactt tatgaagtat caagacagac aaactgtcag cacactttac actgaacaaa 1080
tgaaaccaga aaccgttgaa accacagtga cgacagcaca ggaaacaaag cgcgggagaa 1140
ttcaaactaa aaaggaagtt tcaatcaaat gtactttgcg ggacttggtt agtaaaagag 1200
taacatcacc tgaagattgg atgatgttac aaccagatag ttatattgaa atgatggcac 1260
aaccaggagg tgaaaatctt ttaaaaaata cacttgaaat ttgtactttg actttagcaa 1320
gaacaaaaac agcatttgaa ttaatacttg aaaaagcaga taatactaaa ctaactaact 1380
ttgatcttgc aaattctaga acatgtcaaa tttttagaat gcacggatgg aattggatta 1440
aagtttgtca cgctatagca tgtgttttaa atagacaagg tggtaaaaga aatacagttc 1500
tttttcatgg accagcaagt acaggaaaat ctattattgc tcaagccata gcacaagctg 1560
tggggaatgt tggttgttat aatgcagcaa atgtaaattt tccatttaat gactgtacca 1620
ataaaaattt aatttggatt gaagaagctg gtaactttgg tcaacaagtt aatcaattta 1680
aagcaatttg ttctggacaa acaattagaa ttgatcaaaa aggtaaagga agtaagcaaa 1740
ttgaaccaac tccagtaatt atgacaacta atgaaaatat aacaattgta agaattggat 1800
gtgaagaaag acctgaacat acacaaccaa taagagacag aatgttgaac attaagttag 1860
tatgtaagct tccaggagac tttggtttgg ttgataaaga agaatggcct ttaatatgtg 1920
catggttagt taaacatggt tatgaaccaa ccatggctaa ctatacacat cattggggaa 1980
aagtaccaga atgggatgaa aactgggcgg agcctaaaat acaagaaggt ataaattcac 2040
caggttgcaa agacttagag acacaagcgg caagcaatcc tcagagtcaa gaccaagttc 2100
taactcctct gactccggac gtagtggacc ttgcactgga accgtggagt actccagata 2160
cgcctattgc agaaactgca aatcaacaat caaaccaact tggcgttact cacaaagacg 2220
tgcaagcgag tccgacgtgg tccgaaatag aggcagacct gagagccatc tttacttctg 2280
aacaattgga ggaagatttt cgagacgact tggattaagg tacgatggca cctccggcaa 2340
agagagccag gagaggtaag ggtgtgttag taaagtgggg ggaggggaaa gatttaataa 2400
cttaactaag tatgtgtttt cttataggac ttgtgcctcc aggttataaa tatcttgggc 2460
ctgggaacag tcttgaccaa ggagaaccaa ctaacccttc tgacgccgct gcaaaagaac 2520
acgacgaagc ttacgctgct tatcttcgct ctggtaaaaa cccatactta tatttctcgc 2580
cagcagatca acgctttata gatcaaacta aggacgctaa agattggggg gggaaaatag 2640
gacattattt ttttagagct aaaaaggcaa ttgctccagt attaactgat acaccagatc 2700
atccatcaac atcaagacca acaaaaccaa ctaaaagaag taaaccacca cctcatattt 2760
tcatcaatct tgcaaaaaaa aaaaaagccg gtgcaggaca agtaaaaaga gacaatcttg 2820
caccaatgag tgatggagca gttcaaccag acggtggtca acctgctgtc agaaatgaaa 2880
gagctacagg atctgggaac gggtctggag gcgggggtgg tggtggttct gggggtgtgg 2940
ggatttctac gggtactttc aataatcaga cggaatttaa atttttggaa aacggatggg 3000
tggaaatcac agcaaactca agcagacttg tacatttaaa tatgccagaa agtgaaaatt 3060
ataaaagagt agttgtaaat aatatggata aaactgca 3098
<210> 3
<211> 2064
<212> DNA
<213> Mink enteritis parvovirus
<400> 3
gttaaaggaa acatggcttt agatgatact catgtacaaa ttgtaacacc ttggtcattg 60
gttgatgcaa atgcttgggg agtttggttt aatccaggag attggcaact aattgttaat 120
actatgagtg agttgcattt agttagtttt gaacaagaaa tttttaatgt tgttttaaag 180
actgtttcag aatctgctac tcaaccacca actaaagttt ataataatga tttaactgca 240
tcattgatgg ttgcattaga tagtaataat actatgccat ttactccagc agctatgaga 300
tctgagacat tgggttttta tccatggaaa ccaaccatac caactccatg gagatattat 360
tttcaatggg atagaacatt aataccatct catactggaa ctagtggcac accaacaaat 420
gtatattatg gtacagatcc agatgatgtt caattttata ctattgaaaa ttctgtgcca 480
gtacacttac taagaacagg tgatgaattt gctacaggaa catttttttt tgattgtaaa 540
ccatgtagac taacacatac atggcaaaca aatagagcat tgggcttacc accatttcta 600
aattctttgc ctcaatctga aggagctact aactttggtg atataggagt tcaacaagat 660
aaaagacgtg gtgtaactca aatgggaaat acagactata ttactgaagc tactattatg 720
agaccagctg aggttggtta tagtgcacca tattattctt ttgaagcatc tacacaaggg 780
ccatttaaaa cacctattgc agcaggacgg gggggagcgc aaacagatga aaatcaagca 840
gcagatggtg atccaagata tgcatttggt agacaacatg gtcaaaaaac tactacaaca 900
ggagaaacac ccgagagatt tacatatata gcacatcaag atacaggaag atatccagaa 960
ggagattgga ttcaaaatat taactttaac cttcctgtaa caaatgataa tgtattgcta 1020
ccaacagatc caattggagg taaaacagga attaactata ctaatatatt taatacttat 1080
ggtcctttaa ctgcattaaa taatgtacca ccagtttatc caaatggtca aatttgggat 1140
aaagaatttg atactgactt aaaaccaaga cttcatgtaa atgcaccatt tgtttgtcaa 1200
aataattgtc ctggtcaatt atttgtaaaa gttgcgccta atttaacaaa tgaatatgat 1260
cctgatgcat ctgctaatat gtcaagaatt gtgacttact cagatttttg gtggaaaggt 1320
aaattagtat ttaaagctaa actaagagca tctcatactt ggaatccaat tcaacaaata 1380
agtattaatg tagataacca atttaactat gtaccaaata atattggagc tatgaaaatt 1440
gtatatgaaa aatctcaact agcacctaga aaattatatt aatatactta ctatgttttt 1500
atgtttatta catatcaact agcacctaga aaattatatt aatatactta ctatgttttt 1560
atgtttatta catattattt taagattaat taaattacag catagaaata ttgtacttgt 1620
atttgatata ggatttagaa ggtttgttat atggtataca ataactgtaa gaaatagaag 1680
aacatttaga tcatagttag tagtttgttt tataaaatgt attgtaaact attaatgtat 1740
gttgttatgg tgtgggtggt tggttggttt gcccttagaa tatgttaagg accaaaaaaa 1800
tcaataaaag acatttaaaa ctaaatggtc tcgtatactg tctataaggt gaactaacct 1860
taccataagt atcaatctgt ctttaagggg ggggtgggtg ggagatacac aacatcagta 1920
gactgactgg cctggttggt tgctctgctt aatcaaccag accgcgtagc ggtctggttg 1980
attaagcgca accaaccagg ccagtcagtc tactgatgtt gtgtatctcc cacccacccc 2040
ccccttaaag acagattgat actt 2064
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
ctttgcctca atctgaagga gt 22
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
gaattggatt ccaagtatga gat 23

Claims (5)

1. A construction method of mink enteritis parvovirus whole genome infectious clone is characterized by mainly comprising the following steps: carrying out enzyme digestion on the complete genome of the mink enteritis parvovirus replication form; sequentially and directionally cloning two sections of gene fragments obtained after enzyme digestion to a vector pUC18-M in a segmented form to obtain a recombinant plasmid;
the vector pUC18-M is formed by enzyme digestion transformation of a vector pUC 18: firstly, carrying out enzyme digestion by using Hind III to linearize a circular plasmid, and then treating by using Blunting blunt-ended enzyme to change sticky ends at two ends of plasmid DNA into blunt ends;
the two segments of gene segments are obtained by the following method: inoculating the mink enteritis parvovirus to CRFK cells, collecting the cells for genome extraction when the cells grow vigorously and most of swelling becomes round, and obtaining a complete genome of the mink enteritis parvovirus replication form; the genome was digested with Pst I to generate two DNA fragments.
2. The method of claim 1, wherein the specific steps are as follows,
(1) transformation of vector pUC18
Firstly, using Hind III to linearize pUC18, and then using Blunting blunt-ended enzyme to treat two ends of a vector fragment from a viscous end to a blunt end, wherein the vector fragment is named as pUC 18-M; carrying out PstI enzyme digestion on the pUC18-M vector to change one end of the vector fragment into a viscous tail end;
(2) MEV replication form genome extraction
Inoculating the MEV virus to CRFK cells, and extracting a genome when the cells are in a vigorous growth state and most of the cells are swollen and rounded;
(3) MEV replication form genome segmentation cloning
Carrying out enzyme digestion on the MEV replication form double-stranded genome obtained in the step (2) by using Pst I, and cutting two DNA fragments MEV-5 'and MEV-3'; firstly, pUC18-M, MEV-3 'is connected and transformed to obtain pM-MEV-3' recombinant plasmid; then carrying out Pst I enzyme digestion on the pM-MEV-3' recombinant plasmid, and then carrying out Sma I enzyme digestion; and connecting and transforming the enzyme-cut pM-MEV-3 'and MEV-5' to obtain a pM-MEV recombinant plasmid.
3. The mink enteritis parvovirus whole genome infectious clone constructed by the method of claim 1 or 2.
4. The mink enteritis parvovirus whole genome infectious clone of claim 3 is applied to research on pathogenesis and pathogenicity of mink enteritis parvovirus for the purposes of mink enteritis parvovirus rescue and non-disease treatment and preparation of mink enteritis parvovirus vaccines.
5. A method for rescuing mink enteritis parvovirus is characterized by comprising the following steps: adding a Lipofectamine2000 transfection reagent into a centrifugal tube, adding DMEM culture solution, uniformly mixing, and standing; adding pM-MEV recombinant plasmid obtained by the method of claim 2, mixing, standing at room temperature for 20min to obtain transfection mixture; removing culture medium from full monolayer CRFK cells, washing with PBS or serum-free medium for 2-3 times, adding the transfection mixture, placing the cells at 37 deg.C and 5% CO2Culturing for 1h in an incubator, adding a complete culture medium with 7% of serum content, and continuously culturing for 24-48h for cell passage; collecting cell supernatant with suspected cytopathic effect to obtain the rescued virus.
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