CN106520790A - Construction method and application of pig encephalomyocarditis virus BD2 strain full-length infectious clone - Google Patents

Abstract

The invention discloses a construction method and application of a porcine encephalomyocarditis virus BD2 strain full-length infectious clone. Based on the constructed full-length infectious clone of the porcine encephalomyocarditis virus BD2 strain, the ORF2 gene of the porcine circovirus is inserted into the L gene encoding the viral lead protein, and the full-length infectious clone of the porcine circovirus BD2 strain stably carrying the porcine circovirus type 2 ORF2 gene is successfully constructed. The constructed full-length infectious clone of the EMCVBD2 strain and the full-length infectious clone of the encephalomyocarditis virus BD2 strain stably carrying the porcine circovirus type 2 ORF2 gene can rescue infectious recombinant viruses and can be applied to vaccine preparation and virus research.

Description

A kind of construction method of the full-length infectious clone of porcine encephalomyocarditis virus BD2 strains and Using

Technical field

The invention belongs to biological technical field, and in particular to a kind of full-length infectious clone of porcine encephalomyocarditis virus BD2 strains And its construction method and application, further relate to the pig brain heart for stably carrying porcine circovirus 2 type ORF2 genes is built using the clone The full-length infectious clone of myositis virus BD2 strains and the application of recombinant clone.

Background technology

Pig brain myocarditis (Porcine encephalomyocarditis) is by encephalomyocarditis virus One kind that (Encephalomyocarditis virus, EMCV) causes is main with encephalitis, myocarditis and sow breeding difficulty The Arbo infectious disease of feature.Since 1958 since suffer from and be separated to EMCV in myocarditic sick pig, subsequently in the world one A little pig-raising countries all once had the generation of the disease, the report of popular and EMCV infection, caused certain economic damage to pig industry Lose.China has confirmed that in terms of aetology and serology the large-scale pig farm in many Swine Productions area has the sense of EMCV Dye, it is potentially hazardous to Swine Production to receive publicity.

EMCV host ranges extensively, being capable of infected pigs, rodent, ox, elephant, racoon, kangaroo, baboon, monkey, chimpanzee Or even people.According to the data display of clinical data, after different EMCV isolated strains infected pigs, caused clinical symptoms are not Together, some strains only cause a kind of symptom in encephalitis, myocarditis or breeding difficulty, and what is had but can be while cause.EMCV feels After dye muroid, acute forms disorder, diabetes, myocarditis or subclinical infection can be caused.

EMCV belongs to microRNA Viraceae (Picornaviridae) cardiovirus (Cardiovirus) member, belongs to together with which Member also has mengo virus (Mengovirus), Taylor mouse source encephalomyocarditis virus (Theiler ' s murine encephalomyelitis,TMEV).EMCV is single strand plus RNA virus, full-length genome about 7.8kb, big containing one Open reading frame (ORF), coding molecule amount about 2.6 × 10³The polyprotein of kDa.Research shows, positioned at viral genome Poly (C) structure in 5'UTR regions may be related to the pathogenicity of EMCV, by gene delection technology, is artificially shortened The virus of Poly (C) length can make body from the infection of wild poison, and provide long-term as live vaccination susceptible animal Immunity is protected.After this deleted virus infection muroid, virus virulence will not be also returned by force, with good biological safety.Grind Study carefully and also find, after virus continuous passage on the cell is multiple, the pathogenicity of virus can weaken, but strain antigenicity does not occur to become Change.Virus inoculation susceptible animal can be made animal from wild virus infection by the characteristics of substantially being weakened using its virulence.

The basic process of reverse genetics manipulation technology is first to carry out full-length genome extraction to detached strain, by its reverse transcription For cDNA, then by round pcr segmentation amplification full length fragment, selection can accommodate the carrier of full-length genome and be oriented gram It is grand, the total length plasmid for building is obtained into geneome RNA by in-vitro transcription, then is transfected into viral susceptible cell In, and then save out infectious virus, by observing its character mutation, judge these impacts of the operation to virus, so as to for The aspects such as the research and development of viral replication machinery, pathogenesis and new generation vaccine provide platform.Reverse Genetics Technique is wide at present The general every field for being applied to life science, especially in terms of the research of RNA virus, solves rna virus cdna group Unworkable puzzlement, the research for RNA virus provide effective instrument.For the research of EMCV, many plants of quilts are had at present The report of Revive virus is successfully built and obtains, external has PV21, EMC-B, EMC-D, Belgian strain and Mengo strains, the country Have BJC3, NJ08 and HB10 strain, this will be research EMCV gene functions and new generation vaccine etc. there is provided good technology platform.

The content of the invention

Object of the present invention is to provide a kind of porcine encephalomyocarditis virus BD2 strains are full-length infectious cloning and its structure side Method.The full-length infectious clone of porcine encephalomyocarditis virus BD2 strains constructed by the present invention can save the virus for providing infectious, Can be used for preparing porcine encephalomyocarditis virus vaccine, be also used as carrier foreign gene-carrying.Additionally, the clone and its structure Method can be also used for the genome structure for further studying porcine encephalomyocarditis virus, and function and virus are interacted with host Mechanism.

Further object is that providing a kind of stably carrying porcine circovirus 2 type (PCV2) ORF2 genes The full-length infectious clone of EMCV BD2 strains and its construction method.The pig brain of the stable carrying PCV2ORF2 genes constructed by the present invention The full-length infectious clone of myocarditis virus BD2 strains can save the recombinant virus for providing infectious, can be used for preparing multiple-effect Vaccine.Its construction method can be offered reference to build the full-length infectious clone for carrying other foreign genes.

In order to achieve the above object, this invention takes technical scheme below：

The structure of the full-length infectious clone of porcine encephalomyocarditis virus BD2 strains, its step include：

(1) choose low copy carrier pWSK29 as EMCV BD2 wild type full-lengths infectious CDNA clones build it is final Purpose base is entered by carrier with point mutation kit Fast Mutagenesis System and mutant primer SpeI-F/SpeI-R Row mutation, to remove unnecessary I restriction enzyme sites of Spe, mutant primer SpeI-F, SpeI-R gene order such as SEQ ID NO.2, Shown in SEQ ID NO.3, the reaction of tri- steps PCR of Jing is expanded, and is cultivated in being transformed into DMT competent cells, selects Jing PCR Testing result is positive bacterium colony, is carried out expanding numerous, with extraction reagent kit in plasmid after sequencing confirms mutation successPlus Midipreps DNA Purifation System are extracted and plasmid DNA purification；Low after by point mutation is copied Shellfish carrier is named as pWSKs；

(2) EMCV BD2 F5 are extracted for viral genome total serum IgE；

(3) design, three couples of overlapping primers SegA-F, SegA-R, SegB- of synthesis covering EMCV BD2 virus full-length genomes F, SegB-R, SegC-F, SegC-R, each primer sequence such as SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID Shown in NO.7, SEQ ID NO.8, SEQ ID NO.9, SP6RNA polymerase promoters are added in the upstream primer of total length amplification Core sequence, introduces point mutation in primer SegB-R and SegC-F, mutant primer be EcoRI-F, EcoRI-R, SpeI-F, SpeI-R mutant primers sequence is as shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.2, SEQ ID NO.3；

(4) each genomic fragment cDNA synthesis of EMCV and PCR amplifications：Using reverse transcriptaseReverse Transcriptase and three couple of downstream primer SegA-R, SegB-R and SegC-R are inverted to EMCV BD2 geneome RNAs Record, obtains the cDNA of tri- fragments of A, B, C of EMCV BD2 genomes；With reverse transcription obtain cDNA as template, respectively with SegA-F/SegA-R, SegB-F/SegB-R and SegC-F/SegC-R are primer, using high-fidelity enzymeFastPfu Fly DNA Polymerase enter performing PCR amplification to three fragments, and recovery is detected and purified to pcr amplification product, will return PCR primer after receipts is attached respectively with pEASY-Blunt carriers, and connection product is all added to what is just thawed In Trans1-T1 competent cells, Jing process, culture select Jing PCR testing results to be positive bacterium colony, confirm through sequencing Carried out expanding numerous after mutation success, plasmid is extracted with the little extraction reagent kit QIAGEN Plasmid Mini Kit of plasmid, by each The plasmid that section subclone is obtained is respectively designated as pEBSegA, pEBSegB and pEBSegC；

(5) pEBSegC, pEBSegB, pEBSegA are cloned into improved pWSK29s successively, complete total length plasmid Build and gene sequencing is carried out to which, its sequence is shown in SEQ ID NO.1.

The full-length infectious clone of EMCV BD2 strains that the present invention is provided successfully is saved out and parent's strain BD2 growth tendency phases As virus, its step is：

The total length plasmid pWSKBD2 built up with the linearisations of restriction enzyme BamH I, linearization plasmid is used RiboMAXTM Large Scale RNA Production System-SP6 kits carry out in-vitro transcription, and transcription is obtained RNA transfection BHK-21 cell, Jing culture obtain wild type Revive virus, be named as RvEMCV/BD2.

Built using the constructed full-length infectious clone of porcine encephalomyocarditis virus BD2 strains and stably carry 2 porcine circovirus The full-length infectious clone of encephalomyocarditis virus BD2 strains of type ORF2 gene, its step include：

(1) the fusion DNA vaccine amplification of external source Insert Fragment：With the ORF2L of porcine circovirus 2 type gene, fORF2, ORF2R, L-Cap fragments are template, using tetra- primers of up-F/up-R, ORF2-F/ORF2-R, down-F/down-R, up-F/down-R It is right, primer up-F, up-R, ORF2-F, ORF2-R, down-F, down-R sequence as, SEQ ID NO.12, SEQ ID NO.13, Shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, set up according to conventional PCR method and expand Increasing system, independently expands each purpose fragment, and amplification is using high-fidelity enzymeFastPfu Fly DNA Polymerase, purifying reclaim amplified production；In the fusion DNA vaccine system for building, add fragment to be fused each, it is incipient anti- Primer is added without in answering system, each fragment region that coincides is carried out into complementary extension using high-fidelity enzyme, treat the fusions of 8 circulations Step suspends PCR reactions after completing, then primer up-F and down-R are added in system, completes hereafter 25 circulations, to obtain Obtain the fusion product of total length；Reclaim after will be the fusion DNA vaccine product for obtaining identified, connector is set up with pEASY-Blunt carriers System, is transformed in the Trans1-T1 competent cells for just having thawed, Jing cultures, carries out plasmid extraction, the middle interstitial granules that will be obtained It is named as pEBORF2；

(2) structure of recombinant virus infection cloned plasmids and sequencing：Choose fusion fragment L gene insertion site upstream and downstream Unique a pair of restriction enzymes Hind III and Nhe I, using the two specific restriction enzyme sites, will melt The fusion fragment containing external source ORF2 gene for getting togather therefrom is cut on interstitial granules pEBORF2, while by the infectivity for building On Cloning of full length plasmid pWSKBD2, respective regions are cut together with Hind III and Nhe I, and the two are carried cohesive end Digestion products Jing T4 ligases link together, and complete the stable encephalomyocarditis virus for carrying porcine circovirus 2 type ORF2 genes The structure of the full-length infectious cloned plasmids of BD2 strains；Connection product is transformed in Trans1-T1 competent cells and is cultivated, Expand numerous extraction low-copy plasmid, the recombinant plasmid is named as into pWSKBD2-ORF2, with primer up-F/down-R to Insert Fragment It is sequenced, so that it is determined that whether external source ORF2 gene has completely been correctly inserted into the pilot protein region of EMCV genomes, its Sequence is shown in SEQ ID NO.18.

EMCV full-length infectious clones of BD2 strains of the stable carrying PCV2ORF2 genes that the present invention is provided successfully is saved out Stably can breed and can stablize the recombinant virus of foreign gene-carrying on BHK-21 cells, its step is：

Recombinant plasmid pWSKBD2-ORF2 is used into RiboMAXTM Large Jing after restriction enzyme BamH I is linearized Scale RNA Production System-SP6 kits carry out in-vitro transcription, and RNA transfection BHK-21 that transcription is obtained is thin Born of the same parents, Jing cultures obtain restructuring Revive virus, are named as RvEMCV/BD2-ORF2.

The present invention compared with prior art, with advantages below and effect：

1st, the cDNA infection clones of the EMCV BD2 strains that the present invention builds are by by EMCV BD2 strain full-length genomes It is divided into 3 fragment amplifications and is cloned on the low copy carrier pWSK29 for having transformed.The fragment expanded in segmentation amplification is relative It is shorter, not only avoid the operation difficulty of long segment amplification, additionally it is possible to better ensure that the fidelity of institute's amplified fragments sequence.Choosing PWSK29 is taken as carrier, which contains multiple restriction enzyme sites, and recipient cell is safe from harm, and is easily isolated, can self Replicate and stably preserve, while with special marker gene.During this, three couple of Corticovirus full-length genome is have also been devised Overlapping primers, add SP6RNA polymerase promoter core sequences, bacteriophage SP6RNA to gather in the upstream primer of amplification total length Between first nucleic acid of synthase promoter and viral genome, many external sources G, can promote in-vitro transcription efficiency.

2nd, the full-length infectious clone Jing BamH I digestion lines of the EMCV BD2 strains that the present invention builds, in-vitro transcription can be saved Rescue with infective virus, name RvEMCV/BD2.5th generation recovered virus TCID 50 reaches 108/mL.One step growth is bent Line shows that proliferative properties of the Revive virus RvEMCV/BD2 on BHK-21 cells are not notable with parent's strain BD2 differences；

3rd, based on the full-length infectious clone of the EMCV BD2 strains constructed by the present invention, pig brain myocarditis can be prepared Malicious vaccine, can also by mutation, displacement, insertion, disappearance equimolecular biological means encephalomyocarditis virus is carried out gene structure, Functional study, while also providing important research tool with host's interaction mechanism further to study virus, is deeply to open Exhibition EMCV virus replications provide easily platform with pathogenesis.

4th, full-length infectious gram of the EMCV BD2 strains of the stable carrying porcine circovirus 2 type ORF2 genes that the present invention builds Grand is on the EMCV BD2 strain infection clones total length plasmid basics for building, in the L genes of the viral pilot protein of coding The ORF2 genes of insertion porcine circovirus 2 type.Jing in-vitro transcriptions, can save the recombinant virus for providing infectious, be named For RvEMCV/BD2-ORF2.The Revive virus can stably be bred on BHK-21 cells and can be stablized foreign gene-carrying.Surely Surely the cDNA infection clones of the EMCV BD2 strains of PCV2ORF2 genes are carried, can be used to prepare while preventing pig circular ring virus 2 and brain Myocarditic multiple-effect vaccine, the also full-length infectious clone to build the EMCV BD2 strains for carrying other foreign genes are provided and are borrowed Mirror, with highly important theory significance and realistic meaning.

Description of the drawings

Fig. 1 is pWSK29 plasmid maps

Fig. 2 is pWSKBD2 construction strategies

Fig. 3 is expanded for the RT-PCR of EMCV BD2cDNA

Fig. 4 is identified for total length plasmid pWSKBD2 double digestions

Fig. 5 is the cytopathy that wild type Revive virus are produced on BHK-21 cells

Fig. 6 is identified for the genetic marker of Revive virus

Fig. 7 is identified for the IFA of Revive virus

Fig. 8 is one step growth curve of the Revive virus on BHK-21 cells

Fig. 9 is EMCV L genes and exogenous sequences PCR results

Figure 10 is EMCV L genes and foreign gene fusion DNA vaccine result

Figure 11 is the construction strategy of restructuring virus full length infective cloned plasmids

Figure 12 is the cytopathy that restructuring Revive virus are produced on BHK-21 cells

Figure 13 is restructuring one step growth curve of the Revive virus on BHK-21 cells

Specific embodiment

Experimental technique in the present embodiment, if no special instructions, is conventional method.What is be exemplified below is only of the invention Several specific embodiments.It is clear that the invention is not restricted to following examples, can also have many deformations.Therefore the skill of this area Modification or improvement that art personnel are made on the basis of the disclosure of invention, all should belong to the scope of protection of present invention.

Embodiment 1：The structure of the full-length infectious clone of porcine encephalomyocarditis virus BD2 strains

1.1 cells and strain BHK-21：Cell is stored in Agricultural University Of Hebei's veterinary biological product laboratory, cultivates in height In the 10%DMEM culture mediums of sugar.EMCV BD2 are separated and are preserved by Agricultural University Of Hebei's veterinary biological product laboratory, and The whole genome sequence for measuring is uploaded to into NCBI, (GenBank sequence numbers：KF709977), the 5th generation poison is used for this research.

The transformation of 1.2 low copy carrier pWSK29：This test chooses low copy carrier pWSK29 (Fig. 1) as EMCV BD2 The final carrier that wild type infectious cDNA clone builds, for the need that the clone and total length that meet each genetic fragment correctly connect Will, it is an indispensable step that carrier is carried out transforming.By the gene order for analyzing pWSK29 carriers, it is found which there are two I sites of Spe, one be located at MCS in, another be located at MCS outside 3536 bit bases at, due to Spe I site is the necessary restriction enzyme site of fragment connection, so I restriction enzyme sites of Spe outside MCS will affect next step Subclone, therefore, need to be incited somebody to action with point mutation kit Fast Mutagenesis System and mutant primer SpeI-F/SpeI-R Purpose base is mutated, to remove unnecessary I restriction enzyme sites of Spe, mutant primer SpeI-F, SpeI-R gene order such as SEQ Shown in ID NO.2, SEQ ID NO.3.Join it in PCR pipe, with micropipettor inhale blow mixing, Jing 5s it is instantaneous from Being put into after the heart in PCR instrument, the reaction of three steps PCR being carried out according to following amplification condition, the first step is denaturation：94 DEG C of 3min, second Walk as circulation step：94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 3min, totally 30 circulations, the 3rd is extension step Suddenly：72 DEG C of overall elongation 10min.5 μ L PCR primers are detected by 1% agarose gel electrophoresis, add 1 μ L DMT enzymes in In remaining PCR primer, after fully mixed which, be placed in 37 DEG C of water-baths and 1h be incubated with non-mutant matter of degrading Grain template.The DMT competent cells being stored in -80 DEG C of refrigerators are taken out, is put on ice for making which slowly melt.Draw 5 μ L In 50 μ L DMT competent cells (digestion is added to produce when competent cell just thaws by the product of DMT enzymic digestions Thing), it is placed on ice chest after gently mixing, ice bath 30min, after then which to be put into rapidly in 42 DEG C of water-baths accurately thermal shock 45s, Quickly remove and be placed in ice bath 2min in ice chest, be careful not to acutely rock centrifuge tube, its whole is added to after ice bath warmed-up 250 μ L LB fluid nutrient mediums in, by shaking table temperature setting be 37 DEG C, rotating speed be set to 200r/min, through 1h concussion train After supporting, it is placed in a centrifuge, 4000rpm centrifugation 1min are inhaled with the pipettor that range is 100 μ L and abandoned after about 100 μ L of supernatant, then will Precipitation pressure-vaccum is all suctioned out after mixing, and is uniformly coated on LB/Amp with rod⁺On solid plate, after which fully absorbs, will Inverted culture dish is incubated in 37 DEG C of incubators overnight.

The extraction of 1.3 low-copy plasmids：With the aseptic pipette tips picking single bacterium colonies of 10 μ L in LB/Amp⁺In fluid nutrient medium, Select Jing PCR testing results to be positive bacterium colony, and carried out expanding numerous after sequencing confirms mutation success：According to 1/1000 Ratio be inoculated with 5 μ L bacterium solution in 5mL LB/Amp⁺In fluid nutrient medium, it is placed in the shaking table for having set, 28 DEG C, 150r/ Min concussion and cultivates overnight, then again in 1/100 ratio inoculation 1mL bacterium solution to 100mL LB/Amp⁺In fluid nutrient medium, It is placed in the shaking table for having set, 28 DEG C, 150r/min incubated overnights.With extraction reagent kit in plasmidPlus Midipreps DNA Purifation System are extracted and plasmid DNA purification.Concrete grammar is as shown in specification：Will be enlarged by The bacterium solution of culture is averagely collected in two 100mL centrifuge tubes, and centrifuge temperature setting is 4 DEG C, rotating speed is set to 12,000r/ Min, after 10min being centrifuged, is inverted centrifuge tube after discarding supernatant, adds 3mL after exhausting bacterium solution supernatant with blotting paper under the conditions of this Cell resuspension solution, will precipitate repeatedly pressure-vaccum repeatedly with pipettor, until thalline is mixed completely；Thereafter plus Enter 3mL Cell lysis solution, reverse centrifuge tube gently makes which fully crack 3～5 times, and total pyrolysis time is maintained at In 3～5min；5mL Neutralization solution are added, after quickly gently overturning centrifuge tube 6～8 times again, 5min is stored at room temperature, white precipitate now occurs.Centrifuge tube is placed in the centrifuge for having set, 4 DEG C, 12,000rpm Centrifugation 15min, it is careful to suction out supernatant and 15min is centrifuged again after being transferred to another clean centrifuge tube, carefully suction out supernatant；Upper 10mL is added in clearPlus Midipreps DNA Purifation resin, gently rotation are mixed；Previous step In Resin/DNA mixtures all add centrifugal column, centrifugation column outlet is connected with vacuum pump interface, opens vavuum pump and open Close, give 38.1cm Hg pressure.Vavuum pump cuts out after centrifugal column by mixture, in centrifugal column adds 15mL Washing solution, are again turned on vavuum pump, make cleaning solution cross post and washed once after pressurization；Repeat above-mentioned washing step one It is secondary, after cleaning solution all excessively complete centrifugal column, continue pressurization and vacuumize 30s, be dried Resin.Centrifugal column is cut with scissors Head, puts it in a new centrifuge tube, and 12,000rpm are centrifuged 2min to remove residual washing lotion；After drying at room temperature 5min The Nulcease-free water for adding 300 μ L to be preheated to 65～70 DEG C, are stored at room temperature 1～2min.Column is placed in new On 1.5mL centrifuge tubes, 4 DEG C in centrifuge, 12,000rpm centrifugation 1min.The solution of collection is placed again into 4 in centrifuge DEG C, 12,000rpm centrifugation 5min, remove Resin residual.Careful collection supernatant, is placed in -20 DEG C of refrigerators and saves backup.By point Low copy carrier after mutation is named as pWSKs.

The extraction of 1.4 viral genome total serum IgEs：EMCV BD2 F5 is thin in cultured BHK-21 for virus inoculation In born of the same parents' individual layer, 6mL 2%FBS-DMEM are added to maintain culture medium, CO₂Culture is collected after 36h is cultivated in incubator, which is anti- After multiple freeze thawing 3 times, 12, the 000r/min centrifugations 10min in the 4 DEG C of centrifuges for having set is extracted according to RNA after collecting supernatant KitViral DNA/RNA Kit specifications extract virus genome RNA.Which comprises the following steps that：In 1.5mL 200 μ L cell culture supernatants are added in centrifuge tube, is added 20 μ L Proteinase K pressure-vaccums in sample to mix, is added BB5 solution (comprising 5.6ug Carrier RNA) the 200 μ L being configured, are vortexed with turbula shaker and are mixed after 15s, Water-bath 15min in the 56 DEG C of water-baths for having mixed up, adds the 250 μ L of absolute ethyl alcohol in new Kaifeng in the sample after the completion of incubation, Be vortexed again for shaking 15s, fully mix, in brief centrifugation machine brief centrifugation after being stored at room temperature 5min, by whole samples plus Enter in labeled good centrifugal column, 12,000rpm room temperatures centrifugation 1min adds 500 μ L after discarding the waste liquid in collecting pipe WB5 washing lotions, 12,000rpm room temperatures centrifugation 1min, discard efflux, after repeating this washing step once, empty adsorption column are put Enter 12,000rpm room temperatures centrifugation 2min in collecting pipe, thoroughly remove the ethanol remained on adsorbed film, be stored at room temperature several minutes Thoroughly to dry centrifugal column.Centrifugal column is placed in a new marked good 1.5mL centrifuge tube, is dripped in adsorbed film centre Plus 50 in advance preheated RNase-free Water of μ L, after being stored at room temperature 1min, 12, the 000rpm room temperatures centrifugation in centrifuge 1min, the geneome RNA of acquisition is carried out reverse transcription immediately or is stored in standby in -80 DEG C of refrigerators.

1.5 design of primers and synthesis：According to EMCV BD2 whole genome sequences (the GenBank accession number logged in NCBI： KF709977), with three couples of overlapping primers SegA-F of 5 Software for Design Corticovirus full-length genomes of Primer Permier, SegA-R, SegB-F, SegB-R, SegC-F, SegC-R, primer are synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd, each primer Sequence such as SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 It is shown, 10 μM are diluted to the water of nuclease free.In the upstream primer of total length amplification, add SP6RNA polymerases to start Sub- core sequence.Introduce point mutation in primer SegB-R and SegC-F, mutant primer be EcoRI-F, EcoRI-R, SpeI-F, The sequence of SpeI-R, will after amplification as shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.2, SEQ ID NO.3 Base T that genome is the 6256th sports base A, and the codon CGA after mutation is encoded together with the codon CGT before mutation Monoamino-acid (R), belongs to same sense mutation, does not affect the expression of gene, and gene order GAATTC obtained after point mutation is restriction Property restriction endonuclease EcoR I recognition sequence, as the genetic marker of this infection clones test.The three pairs of amplimers and Point mutation primer sequence see the table below.

The each genomic fragment cDNA synthesis of 1.6EMCV and PCR amplifications：Using reverse transcriptaseReverse Geneome RNA is carried out reverse transcription and obtains cDNA by Transcriptase, and step is proceeded as follows to specifications：Take three 1.5mL centrifuge tubes, add 10.5 μ L of EMCV BD2 geneome RNAs to which, then are separately added into the three pairs of downstreams for having diluted and draw 2 μ L of thing SegA-R, SegB-R and SegC-R (10 μM), 2 μ L of dNTPs (2.5mM).Following ingredients are often added to be configured to 20 μ L in pipe Reverse transcription reaction system.The centrifuge tube for having mixed is placed in into water-bath reverse transcription 1h in 42 DEG C of water-baths, EMCV BD2 bases are obtained Because of the cDNA of tri- fragments of A, B, C of group, it is directly used in PCR amplifications or -20 DEG C saves backup.With the cDNA that reverse transcription is obtained For template, respectively with SegA-F/SegA-R, SegB-F/SegB-R and SegC-F/SegC-R is primer, using high-fidelity enzymeFastPfu Fly DNA Polymerase enter performing PCR amplification to three fragments.The amplification condition of three fragments point Wei not A fragments：94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 3min；B fragments：94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 4min；C fragments：94℃ 30s, 50 DEG C of 30s, 72 DEG C of 1min 30s；Period in circulation step is 30 circulations, and denaturation condition is 94 DEG C of 3min, Final extension of time is 72 DEG C of 10min.After amplification, 1% agarose gel electrophoresis of Jing checks pcr amplification product, sees Fig. 3.Each section PCR primer is carried out purifying recovery with SanPrep pillar DNA glue reclaim kits, and method of operating is shown in product description, summary It is as follows：The target DNA band in Ago-Gel is accurately cut with clean blade, one is put it into clean labeled good 1.5mL centrifuge tubes in, add 3 times of volumes Buffer B2 solution, in 50 DEG C of water-baths 5～10min of water-bath (interval 2～ 3min is rocked once), it is ensured that blob of viscose melts completely.Glue can add the isopropanol of 1 times of volume after melting completely, molten by what is melted Liquid stands 1min in all adding adsorption column, if during solution repeatedly can add adsorption column too much, 9 in centrifuge, 000rpm room temperatures are centrifuged 30s, discard efflux in collecting pipe (when the DNA fragmentation for reclaiming is larger or during relatively low concentration, will collect Liquid in pipe is centrifuged in adding same adsorption column again again).In adsorption column, add 500 μ L Wash Solution to wash Wash liquid, 9,000rpm room temperatures centrifugation 30s adds cleaning solution repeated washing once again, discard after waste liquid again by empty adsorption column 9,000rpm room temperatures are centrifuged 2min, to remove the Wash Solution of residual, the lid of adsorption column is opened, number is stored at room temperature Minute thoroughly dries adsorption column, it is to avoid the influence of ethanol follow-up test of residual.Adsorption column is placed in new 1.5mL centrifuge tubes, The Elution Buffer for being added dropwise that 30 μ L are preheated to 65 DEG C are hit exactly in adsorbed film, 9,000rpm room temperatures centrifugation after 1min is stored at room temperature 1min, by the DNA solution being recovered to be placed in -20 DEG C of Refrigerator stores it is standby or be directly used in next step test.After recovery PCR primer is attached respectively with pEASY-Blunt carriers, sets up following 5 μ L linked systems.Inhaled with micropipettor and blow mixing The brief centrifugation 5s in brief centrifugation machine, 37 DEG C of water-baths connection 20min, can change according to the flexible in size of junction fragment and connect afterwards Time, big fragment Connection Time need to be grown a bit.The Trans1-T1 competent cells being stored in -80 DEG C of refrigerators are taken out, is put Put, 5 μ L connection products are all added to the 50 μ L Trans1-T1 competence just thawed thin In born of the same parents, ice bath 30min on ice chest is placed in after gently mixing, in 42 DEG C of water-baths after accurate thermal shock 30s, is immediately placed on ice on ice chest Bath 2min, should not acutely rock centrifuge tube in noting moving process, its whole is added to 250 warmed-up μ L LB after ice bath In fluid nutrient medium, it is placed in the shaking table for setting, 37 DEG C, 200r/min, after concussion and cultivate 1h, is placed in a centrifuge, 4000rpm is centrifuged 1min, after discarding about 100 μ L of supernatant, then all suctions out after precipitation pressure-vaccum is mixed, is uniformly coated with rod On LB/Amp+ solid plates, after which fully absorbs, by inverted culture dish incubated overnight in 37 DEG C of incubators.Use 10 μ The aseptic pipette tips picking single bacterium colonies of L select the bacterium colony that Jing PCR testing results are the positive, and Jing in LB/Amp+ fluid nutrient mediums Cross and carried out expanding numerous after sequencing confirms mutation success, extracted with the little extraction reagent kit QIAGEN Plasmid Mini Kit of plasmid Plasmid, each fragment is subcloned the plasmid for obtaining and is respectively designated as pEBSegA, pEBSegB and pEBSegC.

The structure and sequencing of 1.7pWSKBD2 total length plasmids：As shown in Fig. 2 by pEBSegC, pEBSegB, PEBSegA is cloned into the structure that improved pWSK29s completes total length plasmid successively.First pWSK29s and pEBSegC is used EcoR I and Not I carries out double digestion.Brief centrifugation after digestion system is fully mixed, is placed in after acting on 3h in 37 DEG C of water-baths Digestion products are reclaimed, and step ibid, carries out follow-up test after concentration being determined with ultramicrospectrophotometer, after recovery PWSK29s and pEBSegC digestion products build 10 μ L T4 linked systems, and in constant temperature circulator, 16 DEG C of circulator baths connected Night.Take 5 μ L connection products to be added in the 50 μ L Trans1-T1 competent cells for just having thawed, after gently mixing, be placed in ice chest Upper ice bath 30min, in 42 DEG C of water-baths after accurate thermal shock 30s, is immediately placed on ice bath 2min on ice chest, notes in moving process not Centrifuge tube is acutely rocked, its whole is added in 250 warmed-up μ L LB fluid nutrient mediums, by shaking table temperature after ice bath Be set to 37 DEG C, rotating speed be set to 200r/min, after concussion and cultivate 1h, be placed in a centrifuge, 4000rpm centrifugation 1min, discard After about 100 μ L of supernatant, then all suction out after precipitation pressure-vaccum is mixed, LB/Amp is uniformly coated on rod⁺On solid plate, After which fully absorbs, by inverted culture dish incubated overnight in 37 DEG C of incubators.With the single bacterium of the aseptic pipette tips pickings of 10 μ L Fall within LB/Amp⁺In fluid nutrient medium, the bacterium colony that PCR is accredited as the positive is chosen, expand numerous extraction low-copy plasmid PWSKBD2SegC, extraction step of the method with above-mentioned low-copy plasmid.Again by pEBSegB directed clonings to pWSKBD2SegC, Restriction enzyme used is Sal I and EcoR I, and the digestion system of structure is as follows.It is instantaneous after digestion system is fully mixed Centrifugation, is placed in after acting on 3h in 37 DEG C of water-baths and reclaims digestion products, and step ibid, determines concentration with ultramicrospectrophotometer After carry out follow-up test, by after recovery pWSKBD2SegC and pEBSegB digestion products build 10 μ L T4 linked systems, in perseverance In warm circulator, 16 DEG C of circulator baths connect overnight.5 μ L connection products are transformed in Trans1-T1 competent cells, step Ibid, the bacterium colony that PCR is accredited as the positive is chosen, expands numerous extraction low-copy plasmid pWSKBD2SegBC, method is with above-mentioned low-copy The extraction step of plasmid.Finally by pEBSegA directed clonings to pWSKBD2SegBC, restriction enzyme used is Sal I With Spe I, the volume of the digestion system each component of structure is consistent with during clone's B fragments, in 37 DEG C of water-baths after water-bath digestion 3h Digestion products are reclaimed, pWSKBD2SegBC the and pEBSegA digestion products after recovery are built into 10 μ according to the concentration for determining out L T4 linked systems, in constant temperature circulator, 16 DEG C of circulator baths connect overnight, are transformed in Trans1-T1 competent cells, Picking positive bacterium colony, expands numerous extraction low-copy plasmid pWSKBD2SegABC, completes infection clones total length plasmid pWSKBD2's Build.Fig. 4 is shown in digestion identification to pWSKBD2.The sequencing and splicing of total length plasmid pWSKBD2 is by Beijing six directions Hua Da gene section Skill limited company completes, and its sequence is shown in SEQ ID NO.1.

Embodiment 2：Porcine encephalomyocarditis virus BD2 strain infectious CDNA virus rescues

The in-vitro transcription of 2.1 full length cDNA clones：Plasmid linearization：The 15 μ g of total length plasmid pWSKBD2 for building are taken, The plasmid is linearized with restriction enzyme BamH I.According to restriction endonuclease specification, following 100 μ L reaction systems are set up, 37 DEG C Water-bath digestion 3h.Taking 5 μ L digestion products carries out 1% agarose gel electrophoresis detection, identifies whether the plasmid enzyme restriction is complete.Will be surplus Digestion products after remaining total Linearization are placed in water-bath 20min in 65 DEG C of water-baths, make restriction endonuclease BamH I lose work Property, be subsequently adding 5 μ L 10%SDS make its final concentration of 0.5%, then draw the Proteinase K of 1 μ L 20mg/mL and make its final concentration For 200 μ g/mL, with pipettor pressure-vaccum repeatedly, mixing acts on 30min in 50 DEG C of water-baths；Add isopyknic without RNase Phenol：Chloroform, it has to be noted that phenol：Chloroform should draw lower floor's liquid, and centrifuge tube is placed in a centrifuge 4 DEG C then, and 12, 000rpm is centrifuged 10min, and careful supernatant of drawing repeats extracting once, in the supernatant collected in new 1.5mL centrifuge tubes The absolute ethyl alcohol for adding the 3M NaAc and 2 times of volumes of 1/10 volume pre-cooled, after being placed in -20 DEG C of refrigerator precipitation 1h, 4 DEG C, 12,000rpm centrifugation 15min, careful supernatant discarded wash precipitation with the ethanol of 1mL 70%, and 4 DEG C, 12,000rpm are centrifuged Supernatant is carefully discarded by 5min, after being vacuum dried 20min, draws 10 μ L RNase-free water molten in vacuum drying chamber Solution DNA, determines its concentration with ultramicrospectrophotometer, and -20 DEG C preserve or carry out in-vitro transcription immediately.Plasmid linearization Ultimate density afterwards should reach 0.5～1 μ g/ μ L.In-vitro transcription：By above-mentioned linearization plasmid RiboMAX^TM Large Scale RNA Production System-SP6 kits carry out in-vitro transcription, build 20 μ L reaction systems such as according to product description Under.Water-bath 3h in 37 DEG C of water-baths is placed in after the system is fully mixed, 1 μ L RNase-Free DNase is added, again 15min is incubated in being put into 37 DEG C of water-baths, to digest template plasmid DNA.This process will be operated in clean super-clean bench, be used in combination RNA scavengers wipe desktop and pipettor etc., in order to avoid the RNA after in-vitro transcription is degraded by the RNase in air, affect follow-up Test.The RNA ultramicrospectrophotometers that transcription is obtained are immediately available for transfecting or being placed in -80 DEG C of refrigerators guarantors after determining concentration Deposit standby.

2.2 transfection：Form is normal, in eugonic BHK-21 cell culture to 6 porocyte plates, 2mL is added per hole Cell suspension, in 37 DEG C, CO₂Concentration be 5% incubator in overnight incubation, treat that cell in each hole forms 80% or so cell After individual layer, cell conditioned medium is carefully suctioned out and is discarded, 2mL is slowly added into every hole and is balanced to room temperatureI Reduced Serum Medium cleaning solutions, suction out after softly rocking, and repeat this step once, cleaning solution is inhaled and abandons clean.Take a high pressure to go out The 1.5mL centrifuge tubes without RNase of bacterium, add 1mLI Reduced Serum Medium and 10 μ L liposomes 2000, the concussion that is vortexed is mixed, and adds 5 μ g RNA obtained in the previous step, proceeds to cultured BHK- immediately after soft mixing 21 cell monolayers, while setting upThe control group of I Reduced Serum Medium and liposome 2000.By six holes Plate is placed in CO₂During concentration is 5% incubator, after 37 DEG C of culture 4h, transfection liquid is carefully suctioned out, 2mL 10%FBS- are subsequently adding DMEM cell growth mediums continue to cultivate, observation of cell pathology effect (CPE) after 36h, as a result there is pathology effect, see Fig. 5, will Six orifice plate multigelations three times collect the BHK-21 cell monolayers that culture supernatant is inoculated in 80% or so afterwards, and continuous passage continues , as a result there is pathology effect in observation of cell pathology.The wild type Revive virus of acquisition are named as into RvEMCV/BD2.

The digestion identification of 2.3 Revive virus genetic markers and sequence verification：F5 is collected in the cell culture of Revive virus Clearly, after by its multigelation 3 times, geneome RNA is extracted by step, with the EcoR I-F/EcoR I-R comprising genetic marker area For primer, 5918～7056 base zone of amplicon virus genome.10 μ L PCR primers are entered with restriction enzyme EcoR I Row single endonuclease digestion, while expanding the same area in the wild virus gene groups of EMCV BD2 with EcoR I-F/EcoR I-R primers, also uses Restriction enzyme EcoR I digestions, after being incubated 3h in 37 DEG C of water-baths pcr amplification product and digestion products are led to as control Cross 1% agarose gel electrophoresis to be analyzed, see Fig. 6.The full-length cDNA of Revive virus is segmented using RT-PCR method Amplification, it is spliced connect after obtain Revive virus RvEMCV/BD2 whole genome sequence, by Beijing six directions Hua Da Gene science share Co., Ltd completes sequencing and splicing operation.

Indirect immunofluorescence identification (I FA) of 2.4 Revive virus infection BHK-21 cells：Select form normal, grow prosperous The BHK-21 cell culture of Sheng adds 100 μ L cell suspensions, in 37 DEG C, CO into 96 porocyte plates, per hole₂Concentration is 5% Overnight incubation in incubator, after the cell monolayer that cell in each hole forms 80% or so, cell conditioned medium is carefully suctioned out and is discarded, F5 is inoculated with per hole for 100 μ L of Revive virus RvEMCV/BD2, while setting up negative control.In 37 DEG C, CO₂Adsorb in incubator After 1h, the virus liquid that collecting action is crossed, then draw in 100 μ L, 96 porocyte plates of addition of the cell maintenance medium containing 2%FBS-DMEM Continue culture.Supernatant liquid is gently got rid of after inoculation 12h, cell is fixed with the absolute ethyl alcohol of prior -20 DEG C of refrigerator precoolings 15min, adds appropriate PBS to wash 3 times, and EMCV VP1 monoclonal antibodies are carried out 1 by 3～5min every time：After 100 times of dilutions, in The monoclonal antibody for adding 100 μ L to dilute in per hole, is placed in 37 DEG C of incubators and is incubated 1h, adds appropriate PBS drifts again Wash 3 times, 3～5min, discards cleaning solution every time, 100 μ L 1 are added to every hole：Fluorescence labeling (FITC) sheep anti mouse of 100 dilutions IgG, is placed in 37 DEG C of incubators after lucifuge incubation 45min, is rinsed 3 times with PBS, and 3～5min, is eventually adding 50 μ L every time PBS, in fluorescence microscopy Microscopic observation result and takes a picture, and sees Fig. 7.As a result as Fig. 7 shows that F5 is connect for Revive virus RvEMCV/BD2 After planting BHK-21 cell 12h, in cytoplasm, there is visible specificity fluorescent, cell controls then have no fluorescence.

2.5TCID₅₀Determine：The Revive virus in F5 generations are taken, Revive virus RvEMCV/BD2 is determined on BHK-21 cells Malicious valency, concrete grammar step is as follows：With pancreatin by eugonic BHK-21 cell dissociations into after individual cells, with about 2～3 ×10⁵The cell density of individual/mL proceeds to 96 porocyte culture plates, 100 μ L cell suspensions is added per hole, in 37 DEG C, 5%CO₂, it is full With overnight incubation under humidity, after cell grows to 80% or so individual layer, careful suction abandons supernatant, adds 100 μ L PBS carefully to wash Wash once, cleaning solution is abandoned in suction.The Revive virus in F5 generations are carried out into 10 times of serial dilutions with maintenance culture medium, 10 are taken^-1～10^-10It is dilute The virus inoculation BHK-21 cell monolayers of degree of releasing, each gradient are inoculated with 8 hole of row, and the virus liquid of 100 μ L dilutions is inoculated with per hole, and The control group of normal cell is set, 96 porocyte culture plates are placed in into CO₂37 DEG C of absorption 1h in incubator.Careful suction abandons supernatant Afterwards, after adding 100 μ L PBS washed once, 100 μ L are drawn and maintains culture medium to continue culture in being added to 96 orifice plates.Day by day observe Cytopathy CPE, Continuous Observation 5 days, records the hole count of each gradient CPE.TCID is calculated according to Reed-M ü ench methods₅₀.With The 5th generation Revive virus infection BHK-21 cells of 0.01MOI, collect on cell respectively at 4h, 8h, 12h, 16h, 20h and 24h Clearly, determine the TCID of virus₅₀, and growth curve is drawn, see Fig. 8.As shown in figure 8, the TCID of F5 generation viruses₅₀Reach 10⁸/ mL, With the TCID of parental virus₅₀Without notable difference.

Embodiment 3：The structure and its virus rescue of the restructuring encephalomyocarditis virus of expression Porcine circovirus type 2 Cap

The fusion DNA vaccine amplification of 3.1 external source Insert Fragments：The independent amplification of porcine circovirus 2 type gene fragment to be fused：Profit With the primer pair in following table, corresponding template is used respectively, set up 50 μ L amplification systems according to conventional PCR method, independently expand Increase each purpose fragment, primer up-F, up-R, ORF2-F, ORF2-R, down-F, down-R sequence such as SEQ ID NO.12, SEQ Shown in ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, amplification is using high guarantor True enzymeFastPfu Fly DNA Polymerase, the size of purpose band are shown in Fig. 9.The product Jing obtained after amplification Glue reclaim is cut after the identification of 1% agarose gel electrophoresis, each section of PCR primer is carried out with SanPrep pillar DNA glue reclaim kits Purifying is reclaimed, and method of operating is according to glue reclaim step.The fusion DNA vaccine and subclone of Insert Fragment：In the 50 μ L fusion DNA vaccines for building The each 1 μ L of fragment to be fused are added in system, in incipient reaction system, primer is added without, using high-fidelity enzyme by each fragment phase Overlapping region carries out complementary extension, treats that the fusion steps of 8 circulations suspend PCR reactions after completing, then adds in system and draw The each 1 μ L of thing up-F and down-R, complete hereafter 25 circulations, to obtain the fusion product of total length.The fusion DNA vaccine product that will be obtained The identification of 1% agarose gel electrophoresis of Jing, is shown in Figure 10.After cut glue reclaim, the linked system of 5 μ L is set up with pEASY-Blunt carriers, 37 DEG C of water-baths connect 20min, are transformed in the 50 μ L Trans1-T1 competent cells for just having thawed immediately, are gently mixed rearmounted The ice bath 30min on the ice chest, in 42 DEG C of water-baths after accurate thermal shock 30s, is immediately placed on ice bath 2min on ice chest, notes being moved through Centrifuge tube should not be acutely rocked in journey, its whole is added to after ice bath the 250 μ L LB fluid nutrient mediums for being balanced to room temperature In, 37 DEG C, 200rpm, after concussion and cultivate 1h, are placed in a centrifuge, 4000rpm centrifugation 1min, after discarding about 100 μ L of supernatant, then All suction out after precipitation pressure-vaccum is mixed, LB/Amp is uniformly coated on rod⁺On solid plate, after which fully absorbs, Culture dish is inverted in into overnight incubation in 37 DEG C of incubators.Picking positive bacterium colony expands numerous Jing after sequencing identification is errorless, carries out plasmid Extract, the middle interstitial granules for obtaining is named as into pEBORF2.

The structure of 3.2 recombinant virus infection cloned plasmids and sequencing：Insert in the gene of EMCV pilot proteins PCV2ORF2 genes, choose unique a pair of restriction enzymes Hind, III Hes of fusion fragment L gene insertion site upstream and downstream Nhe I, using the two specific restriction enzyme sites, by the fused good fusion fragment containing external source ORF2 gene Therefrom cut on interstitial granules pEBORF2, while respective regions on the infection clones total length plasmid pWSKBD2 for building are used Hind III and Nhe I are cut together, and the two digestion products Jing T4 ligases with cohesive end are linked together, complete Into the structure for completing the stable full-length infectious cloned plasmids of encephalomyocarditis virus BD2 strains for carrying porcine circovirus 2 type ORF2 genes Build, see Figure 11.Digestion products are reclaimed after 3h is incubated in 37 DEG C of water-baths, Jing ultramicrospectrophotometers determine the dense of recovery product Degree, builds the linked system of 10 μ L, and in 16 DEG C of constant temperature circulating instrument, circulator bath overnight, takes 5 μ L connection products and is transformed into In Trans1-T1 competent cells, after coated plate, picking PCR is accredited as the bacterium colony of the positive, expands numerous extraction low-copy plasmid, and this is heavy Group plasmid is named as pWSKBD2-ORF2.Connection conversion, extraction low-copy plasmid procedure and method refer to chapter 1.By low-copy Plasmid delivers to sequencing company, Insert Fragment is sequenced with primer up-F/down-R, so that it is determined that whether external source ORF2 gene The pilot protein region of EMCV genomes has been correctly inserted into completely.Examining order is had by Beijing six directions Hua Da Gene science share Limit company completes, and its sequence is shown in SEQ ID NO.18.

The rescue of 3.3 recombinant viruses：Recombinant plasmid pWSKBD2-ORF2 Jing restriction enzymes BamH I is linearized After use RiboMAX^TMLarge Scale RNA Production System-SP6 kits carry out in-vitro transcription, according to product It is as follows that specification builds 20 μ L reaction systems.After the system is fully mixed, after 37 DEG C of water-bath effect 3h, 1 μ L RNase- are added The mRNA of acquisition, to digest template plasmid DNA, is transfected into cultured 80% by Free DNase, 37 DEG C of water-bath 15min In the BHK-21 cells of left and right, and blank is set.Six orifice plates are placed in into CO₂It is in incubator, after 37 DEG C of culture 4h, careful to inhale Go out transfection liquid, add 2mL 10%FBS-DMEM cell growth mediums to continue culture, observation of cell pathology effect (CPE) after 36h will Six orifice plate multigelations three times collect the BHK-21 cell monolayers that culture supernatant is inoculated in 80% or so afterwards, and continuous passage continues Observation of cell pathology.The restructuring Revive virus of acquisition are named as into RvEMCV/BD2-ORF2.

PCR identifications, sequencing and the genetic stability analysis of 3.4 Revive virus insertion gene：By the restructuring rescue disease for obtaining Malicious RvEMCV/BD2-ORF2 continuous passages on BHK-21 cells, draw F3, F4 and F5 in the cell culture of Revive virus Clearly, after multigelation 3 times, releasing virus extract restructuring Revive virus geneome RNA, using RT-PCR method, with up-F and Down-R is the insert region that primer expands exogenous sequences, using the amplified production of the virus infection clones total length plasmid as right According to, pcr amplification product is analyzed with 1% agarose gel electrophoresis, as a result find Revive virus RvEMCV/BD2-ORF2 outside Source gene is consistent with the clip size of total length plasmid.PCR primer is sequenced, as a result confirms that PCV2ORF2 genes are completely inserted L gene regions.Recombinant virus after rescue was continuously reached into for the 5th generation on BHK-21 cells, each generation virus is carried Foreign gene carries out sequencing identification, as a result shows that the foreign gene that each generation virus is carried is complete correct, with good Genetic stability.

Multiplication characteristic of 3.5 recombinant viruses on BHK-21 cells：F5 is inoculated with for Revive virus RvEMCV/BD2-ORF2 , there is cytopathic effect (CPE) in BHK-21 cell monolayers, see Figure 12, determine virus 4h, 8h, 12h, 16h, 20h after inoculation With the virus titer of 24h, the one step growth curve of recombinant virus is drawn, Figure 13 is seen.As shown in figure 13, Revive virus of recombinating exist The trend bred on BHK-21 cells is basically identical with non-recombinant Revive virus, shows that the insertion of exogenous sequences does not affect EMCV to exist Propagation on BHK-21 cells.

SEQUENCE LISTING

<110>Agricultural University Of Hebei

<120>A kind of construction method of the full-length infectious clone of porcine encephalomyocarditis virus BD2 strains and application

<130> 2016

<160> 18

<170> PatentIn version 3.3

<210> 1

<211> 13147

<212> DNA

<213>Artificial sequence

<400> 1

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 60

aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 120

tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 180

atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 240

cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 300

gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 360

gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 420

tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 480

tcgtcgtttg gtatggcttc attcagctcc ggttcccagc gatcaaggcg agttacatga 540

tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 600

aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 660

atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 720

tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 780

catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 840

aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 900

tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 960

gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 1020

tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 1080

tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg 1140

taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 1200

accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt 1260

tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 1320

aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa 1380

gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat 1440

ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag 1500

gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg 1560

ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt 1620

gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg 1680

ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga 1740

cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc gggccccccc 1800

tcgaggtcga cggtatcgat aagcttgata tcgaattcct gcagcccggg ggatccacta 1860

gttctagagc ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta 1920

attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 1980

acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 2040

gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 2100

tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 2160

cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 2220

gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 2280

aagaacatgt gagatctcta cgggtcggat ttgaagtcgt cttggtagga ggcagcctga 2340

atggcgaatg ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg 2400

gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca 2460

gggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga 2520

tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca 2580

ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg 2640

acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta 2700

tgattcttct cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc 2760

aggtagatga cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa 2820

cttcgatcat tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga 2880

acgggttggc atggattgta ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc 2940

gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg 3000

attcaccact ccgcagaccc gccataaaac gccctgagaa gcccgtgacg ggcttttctt 3060

gtattatggg tagtttcctt gcatgaatcc ataaaaggcg cctgtagtgc catttacccc 3120

cattcactgc cagagccgtg agcgcagcga actgaatgtc acgaaaaaga cagcgactca 3180

ggtgcctgat ggtcggagac aaaaggaata ttcagcgatt tgcccgagct tgcgagggtg 3240

ctacttaagc ctttagggtt ttaaggtctg ttttgtagag gagcaaacag cgtttgcgac 3300

atccttttgt aatactgcgg aactgactaa agtagtgagt tatacacagg gctgggatct 3360

attcttttta tcttttttta ttctttcttt attctataaa ttataaccac ttgaatataa 3420

acaaaaaaaa cacacaaagg tctagcggaa tttacagagg gtctagcaga atttacaagt 3480

tttccagcaa aggtctagca gaatttacag atacccacaa ctcaaaggaa aaggactagt 3540

aattatcatt gactagccca tctcaattgg tatagtgatt aaaatcacct agaccaattg 3600

agatgtatgt ctgaattagt tgttttcaaa gcaaatgaac tagcgattag tcgctatgac 3660

ttaacggagc atgaaaccaa gctaatttta tgctgtgtgg cactactcaa ccccacgatt 3720

gaaaacccta caaggaaaga acggacggta tcgttcactt ataaccaata cgctcagatg 3780

atgaacatca gtagggaaaa tgcttatggt gtattagcta aagcaaccag agagctgatg 3840

acgagaactg tggaaatcag gaatcctttg gttaaaggct ttgagatttt ccagtggaca 3900

aactatgcca agttctcaag cgaaaaatta gaattagttt ttagtgaaga gatattgcct 3960

tatcttttcc agttaaaaaa attcataaaa tataatctgg aacatgttaa gtcttttgaa 4020

aacaaatact ctatgaggat ttatgagtgg ttattaaaag aactaacaca aaagaaaact 4080

cacaaggcaa atatagagat tagccttgat gaatttaagt tcatgttaat gcttgaaaat 4140

aactaccatg agtttaaaag gcttaaccaa tgggttttga aaccaataag taaagattta 4200

aacacttaca gcaatatgaa attggtggtt gataagcgag gccgcccgac tgatacgttg 4260

attttccaag ttgaactaga tagacaaatg gatctcgtaa ccgaacttga gaacaaccag 4320

ataaaaatga atggtgacaa aataccaaca accattacat cagattccta cctacataac 4380

ggactaagaa aaacactaca cgatgcttta actgcaaaaa ttcagctcac cagttttgag 4440

gcaaaatttt tgagtgacat gcaaagtaag tatgatctca atggttcgtt ctcatggctc 4500

acgcaaaaac aacgaaccac actagagaac atactggcta aatacggaag gatctgaggt 4560

tcttatggct cttgtatcta tcagtgaagc atcaagacta acaaacaaaa gtagaacaac 4620

tgttcaccgt tacatatcaa agggaaaact gtccatatgc acagatgaaa acggtgtaaa 4680

aaagatagat acatcagagc ttttacgagt ttttggtgca ttcaaagctg ttcaccatga 4740

acagatcgac aatgtaacag atgaacagca tgtaacacct aatagaacag gtgaaaccag 4800

taaaacaaag caactagaac atgaaattga acacctgaga caacttgtta cagctcaaca 4860

gtcacacata gacagcctga aacaggcgat gctgcttatc gaatcaaagc tgccgacaac 4920

acgggagcca gtgacgcctc ccgtggggaa aaaatcatgg caattctgga agaaatagcg 4980

ctttcagccg gcaaaccggc tgaagccgga tctgcgattc tgataacaaa ctagcaacac 5040

cagaacagcc cgtttgcggg cagcaaaacc cgtacttttg gacgttccgg cggttttttg 5100

tggcgagtgg tgttcgggcg gtgcgcgcaa gatccattat gttaaacggg cgagtttaca 5160

tctcaaaacc gcccgcttaa caccatcaga aatcctcagc gcgattttaa gcaccaaccc 5220

ccccccgtaa cacccaaatc catactgaaa gtggctttgt tgaataaatc agatttcggg 5280

taagtctccc ccgtagcggg ttgtgttttc aggcaatacg cacgctttca ggcatacctg 5340

ctttcgtcat tttgttcagc gctcgtacca gggccatagc ctccgcaacc tgaccatcgt 5400

agtcacgcag cgtcagtgaa cccccgaaca gagattgaaa gccgggggtg ggagatccgg 5460

attgccagtc tgctcgatat cgcaggctgg gtccgtgact acccactccc cctttcaacg 5520

tgaaggctac gatagtgcca gggcgggtac tgccgtaagt gccaccccaa aacaacaaca 5580

aacccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt 5640

ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg 5700

ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg 5760

tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc 5820

tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca 5880

aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag 5940

ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa 6000

ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt 6060

tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc ctaaccacgg ggacgtggtt 6120

ttcctttgaa aaacacgatg ataatatggc cacaaccatg gaacaagaga cttgcgcgca 6180

ccctctcact tttgaggaat gcccaaaatg ctctgctcta caataccgta atggatttta 6240

cctgctaaag tatgatgaag aatggtaccc agaggagtta ttgattgatg gagaggatga 6300

tgtctttgat cccgaattag acatggaagt cgttttcgag ttacagggca attccacctc 6360

ctcagacaag aataactcct cctcggaagg caatgaaggt gtgatcatca ataactttta 6420

ctccaaccaa tatcaaaact ccattgacct ctctgctaat gcagccgggt ctgacccacc 6480

cagaacctac ggtcaatttt cgaatctcct ttcgggcgca gtgaatgcct tttctaatat 6540

gcttccattg ctagctgatc aaaatacaga agaaatggag aatctgtctg atcgagtgtc 6600

tcaagacact gccggcaata cggtcacaaa cacccagtca acagtgggcc gtcttgtcgg 6660

ttatggtgcc gttcatgatg gagagcatcc ggcatcatgt gctgacactg cttcagaaaa 6720

gatcctggcg gtggaaaggt actacacctt caaggtaaat gattggacat caacacaaaa 6780

gccctttgag tacatccgca ttccccttcc tcacgtcctg tccggtgaag atggtggtgt 6840

ctttggtgcg gccctccgcc ggcactacct ggtgaaaact ggatggcggg tgcaagttca 6900

gtgcaacgcc tctcaattcc acgctggaag tttgctggtg ttcatggcac cagagtatcc 6960

aaccttagat gcttttgtca tggacaaccg ttggtccaag gataacctgc ctaatggaac 7020

cagaactcag acaaacaaaa agggaccatt tgccatggac catcagaact tctggcagtg 7080

gaccttgtat ccccatcaat tcctgaatct gagaactaac accacagtgg atcttgaggt 7140

gccatatgta aacatagccc ccacttcctc ctggacacaa catgcttcct ggactttggt 7200

gattgcagtg gttgctcccc tgacatactc aaccggggct tctaccagtt tggatatcac 7260

cgcttctatt cagccagtaa ggcctgtctt taatggcctc cggcacgaga cactttctag 7320

acagtcgccc attccggtca caattagaga acatgctggt acctggtatt ctactctgcc 7380

agacagcaca gtgcctattt atggcaagac tcctgttgct ccatccaatt acatggtagg 7440

cgaatacaag gacttcctgg agatagctca gattccaacc tttattggga ataagatccc 7500

taatgctgtc ccctacattg aggcatccaa cacagccgtc aagacccaac cgctggccac 7560

ctatcaagtg accttgtcct gctcctgtct ggccaataca ttcttggccg ctttgtctag 7620

aaactttgct cagtaccggg gatcattggt ttataccttt gtgttcactg ggaccgcgat 7680

gatgaagggc aagttcctca ttgcctacac cccacctgga gcgggcaagc ccactagtcg 7740

agaccaagcc atgcaggcga cttatgcgat ttgggatttg gggctaaatt cttcttactc 7800

cttcactgtg ccttttattt ctcccactca cttccgcatg gtaggtactg accaagtcaa 7860

catcactaat gcggatggct gggttaccgt gtggcagctc actcccctca cttacccacc 7920

aggatgcccg acctctgcta agatactaac aatggtgagc gcagggaagg atttctcact 7980

caagatgcct atctcacctg ccccctggag ccctcaggga gtagaaaacg ctgaaaaagg 8040

ggtcactgaa aacgcagacg caactgctga ctttgtggct caaccagttt acttgcctga 8100

gaaccaaacg aaggtggctt tcttctatga taggtccagt cccattggtg ccttcaccgt 8160

gaagtccggc agtctagaat ctggttttgc cccgttctct aatgagactt gcccgaactc 8220

agtgatactg acccctgggc cccaatttga ccccgcctat gaccaactca ggccacagcg 8280

tctgacagaa atttggggca atggaaatga ggagacctca aaagtctttc cgcttaaatc 8340

caaacaggat tattccttct gcctcttctc cccctttgtg tattataaat gtgatttaga 8400

agtgactctt agtcctcaca cttcaggcaa ccatgggctg ttggcgaggt ggtgtcccac 8460

tggtacacca accaagccca ctacccaggt tctccatgaa gtaagttccc tctcagaagg 8520

cagaaccccc caggtttata gtgccggacc tggcatttca aatcagattt cctttgtagt 8580

tccttacaat tctccacttt cagtcctacc agctgtctgg tataatggac acaagagatt 8640

tgacaacact gggagcttgg gcattgcccc taattctgat ttcggcactc tgttctttgc 8700

tggcacaaag cctgacatta aattcacagt ctacttgaga tacaagaata tgagagtttt 8760

ttgcccacgt ccgactgtct ttttcccctg gcccacttcc ggagacaaga ttgatatgac 8820

cccgagagct ggagtcttga tgctagagag tccaaatgcc ctagacattt caagaacata 8880

ccccacgtta catgttctca ttcaattcaa ccatagaggt ttggaggtta gattgtttag 8940

acatggacaa ttttgggctg aaacacgtgc ggacgtgatt ctgagatcga agaccaaaca 9000

ggtctctttc ctgagcaacg ggaactaccc gtcaatggac tctagagctc cctggaatcc 9060

ttggaagaat acctaccagg cggttctaag agcagaacca tgtagagtga ccatggatat 9120

atactataag agagtcaggc cttttagact gcccctggtt cagaaggaat ggcgcgtgcg 9180

agaggagaac gttttcggtt tgtaccggat cttcaatgcc cactacgctg gttactttgc 9240

ggacctactg attcatgaca ttgagacaaa tccagggccc ttcatgttta gaccaaggaa 9300

acaggttttc cagacccaag gagcggcagt gtcatcaatg gctcaaaccc tactgccgaa 9360

cgaccttgcc agcaaagcta tgggatcagc ttttacggct ttgctcgatg ccaacgagga 9420

cgcccaaaaa gcaatgaaga ttataaagac attaagttct ctatcggatg catgggaaaa 9480

tgtaaaagaa acactaaaca acccagagtt ctggaagcag ctcttgagca gatgtgtgca 9540

gctgattgca gggatgacaa tagcagtgat gcatccggac cccttgactc tgctctgctt 9600

aggaacattg acggccgccg agattacaag ccagacaagt ctgtgcgaag aaatagcagc 9660

taagttcaag acaattttca tcactcctcc accacggttt cccacaatct ctcttttcca 9720

acaacaatcc cccttgaaac aggtaaatga tattttctcc ctagccaaga acctggactg 9780

ggccgtcaag actgtggaaa aggtggttga ttggtttggg acatggatag tacaggagga 9840

aaaggaacag accctagatc agctcttgca gcgtttcccc gaacatgcga agcgcatttc 9900

tgatctccgg aatggaatgg ccgcctatgt agagtgcaag gagagttttg atttctttga 9960

aaagctgtac aatcaggcag tgaaagagaa gagaacgggt atcgccgccg tctgtgaaaa 10020

attcagacag aagcatgacc acgccaccgc tcggtgtgag ccagtcgtga ttgtgctccg 10080

cggagacgcg gggcaaggga aatctttatc aagtcaggtt attgcccagg ccgtctccaa 10140

gaccattttc ggtcggcaat ctgtgtattc cctccccccc gattcggatt tctttgatgg 10200

ctatgaaaat cagtttgcag caataatgga tgatctaggg caaaatcctg atggctctga 10260

tttcactacg ttctgtcaga tggtttcgac taccaatttt ctccccaata tggctagtct 10320

agagagaaag ggcaccccct ttacatctca gcttgtggtg gcaactacca atctgcctga 10380

gtttagacct gtcacaatag cccattaccc tgctgttgag agaaggataa cttttgacta 10440

ttcagtgtct gctggtccag tctgctccaa aacagaggcc gggtataagg ttttggatgt 10500

tgaaagagcc tttaggccta ccggtgaggc tcctcttcca tgcttccaga ataactgcct 10560

tttccttgag aaagctgggc tccagttcag agataaccga actaaagaga tcatttccct 10620

ggtagatgtg attgagagag ccgtggctag gattgaaagg aagaagaaag ttctcacaac 10680

cgtgcagacc cttgtggcac aagctccagt agacgaggtc agtttccatt ccgtagtcca 10740

gcagcttaaa acaagacagg aagcgacaga tgaacagctt gaggaattgc aagaggcttt 10800

cgcgaaagta caggagcgta actctgtgtt ttctgattgg ttgaagattt ctgcaatgtt 10860

gtgtgctgcg actctggcac tttcccaagt tgtcaagatg gccaaggcgg tgaagcagat 10920

ggtcaagcct gatctggttc gtgtgcaatt ggatgagcag gagcagggac cttacaatga 10980

gacagcgaga gttaaaccaa aaacactgca gttgttggac attcagggac caaaccctgt 11040

gatggacttt gaaaaatatg tagccaaaca tgtaaccgcc cccattgatt ttgtctaccc 11100

cactggggtg agcacccaga cttgcctcct tgtgagaggc cgcaccttgg cagtaaatag 11160

acacatggcc gagtctgact ggacttccat agtagtgcgt ggagtcacac acgcccgctc 11220

tactgttaaa attttggcca tagctaaagc aggcaaagag actgacgtat ctttcatccg 11280

cctctcttct ggtcctctat tcagagacaa tacatccaaa tttgtcacgg ctggtgatgt 11340

acttcctact ggtgccgctc cagtcacggg gataatgaac acggacatac ccatgatgta 11400

cacaggaacc ttcctgaaag ctggtgtgtc agtcccagtg gaaaccggcc agacctttaa 11460

tcactgtatt cattacaagg ctaacacacg aaagggctgg tgtggatcag ccctactggc 11520

agatcttgga ggaagcaaga aaatccttgg catccattct gctggctcta tgggaatagc 11580

cgccgcctcg attgtgtcac aggagatgat tcgggcggta gtgaatgcct ttgagccaca 11640

gggtgctctc gagagattgc cagatgggcc ccgtattcac gtaccacgta aaacagcact 11700

acgccccacc gttgcccgtc aagtcttcca accagcatat gccccggctg ttctatcgaa 11760

atttgaccct agaacagagg ctgatgtaga tgaagtggct ttctccaaac atacctccaa 11820

ccaggaaagc ctcccaccag tgtttagaat ggtagccaaa gagtatgcca atagagtttt 11880

caccttgctg ggaaaagaca atggccgtct gactgtaaag caggctttgg aaggactgga 11940

ggggatggac cccatggaca ggaacacctc cccggggctt ccatatactg cgctaggaat 12000

gcgcagaaca gatgtcgtag attgggaatc agccaccctg atcccgtttg cggcagaaag 12060

attaagaaaa atgaatgaag gagacttttc cgaagttgtc tatcaaacat tcctcaagga 12120

tgagcttaga ccgatagaga aggttcaagc cgccaagaca cggattgtag atgttccacc 12180

atttgagcat tgcattctgg gtagacaatt gttgggaaag tttgcatcaa agttccagac 12240

ccaaccgggt ctggaactag gatcagccat tggatgtgac ccagatgtac actggactgc 12300

cttcggtgtc gccatgcaag gttttgagcg tgtctacgat gtggactact ccaactttga 12360

ttcgacccat tcggtggcaa tgttccgctt attggctgag gaatttttca ctccagagaa 12420

tggttttgac cccctgacta gagaatatct tgagtcatta gccatttcaa cccatgcgtt 12480

tgaggagaag cgctttctga taaccggtgg tctcccatca ggttgtgcag cgacctcaat 12540

gctaaacact ataatgaata atataataat tagggcgggt ttgtatctca cgtataaaaa 12600

ttttgaattt gatgatgtga aggtgttgtc gtacggagat gatctccttg tggccacaaa 12660

ttaccaattg gattttgata aggtgagagc aagcctcgca aagacaggat ataagataac 12720

tcccgctaac aaaacttcta cctttcctct taattcgacg cttgaagacg ttgtcttctt 12780

aaaaagaaag tttaagaaag agggccctct gtatcggcct gtcatgaaca gagaggcgtt 12840

ggaagcaatg ttgtcatact atcgtccagg gactctatct gagaaactca cttcgatcac 12900

tatgcttgcc gttcattctg gtaagcagga atatgatcgg ctctttgccc cattccgtga 12960

ggtaggggtt gtcgtgccat cattcgagag tgtggagtac agatggagga gtctgttctg 13020

gtagtagtgc agtcactggc acaacgcgtt acccggtaag ccaatcgggt gtacacggtc 13080

gtcatactgc agacagggtt cttctacttt gcaagatagt ctagagtagt aaaataaata 13140

gatagag 13147

<210> 2

<211> 36

<212> DNA

<213>Artificial sequence

<400> 2

ccacaactca aaggaaaagg tctagtaatt atcatt 36

<210> 3

<211> 36

<212> DNA

<213>Artificial sequence

<400> 3

accttttcct ttgagttgtg ggtatctgta aattct 36

<210> 4

<211> 49

<212> DNA

<213>Artificial sequence

<400> 4

ccgtcgacgg atttaggtga cactatagaa ttgaaagccg ggggtggga 49

<210> 5

<211> 26

<212> DNA

<213>Artificial sequence

<400> 5

agggggagaa gaggcagaag gaataa 26

<210> 6

<211> 33

<212> DNA

<213>Artificial sequence

<400> 6

ccgtcgacgg caagttcctc attgcctaca ccc 33

<210> 7

<211> 24

<212> DNA

<213>Artificial sequence

<400> 7

gttttacgtg gtacgtgaat tcgg 24

<210> 8

<211> 20

<212> DNA

<213>Artificial sequence

<400> 8

gccccgaatt cacgtaccac 20

<210> 9

<211> 46

<212> DNA

<213>Artificial sequence

<400> 9

acatgcggcc gcatgtcgcg gatccgcgtt tttttttttt tttttt 46

<210> 10

<211> 22

<212> DNA

<213>Artificial sequence

<400> 10

gatgtacttc ctactggtgc cg 22

<210> 11

<211> 22

<212> DNA

<213>Artificial sequence

<400> 11

tgggttgaaa tggctaatga ct 22

<210> 12

<211> 20

<212> DNA

<213>Artificial sequence

<400> 12

gccctgtctt cttgacgagc 20

<210> 13

<211> 43

<212> DNA

<213>Artificial sequence

<400> 13

cgcctccttg gatacgtcat catggttgtg gccatattat cat 43

<210> 14

<211> 43

<212> DNA

<213>Artificial sequence

<400> 14

atgataatat ggccacaacc atgatgacgt atccaaggag gcg 43

<210> 15

<211> 37

<212> DNA

<213>Artificial sequence

<400> 15

tgcgcgcaag tctcttgttc cttagggtta agtgggg 37

<210> 16

<211> 37

<212> DNA

<213>Artificial sequence

<400> 16

ccccacttaa ccctaaggaa caagagactt gcgcgca 37

<210> 17

<211> 20

<212> DNA

<213>Artificial sequence

<400> 17

gcaccataac cgacaagacg 20

<210> 18

<211> 13852

<212> DNA

<213>Artificial sequence

<400> 18

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 60

aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 120

tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 180

atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 240

cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 300

gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 360

gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 420

tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 480

tcgtcgtttg gtatggcttc attcagctcc ggttcccagc gatcaaggcg agttacatga 540

tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 600

aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 660

atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 720

tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 780

catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 840

aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 900

tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 960

gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 1020

tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 1080

tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg 1140

taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 1200

accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt 1260

tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 1320

aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa 1380

gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat 1440

ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag 1500

gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg 1560

ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt 1620

gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg 1680

ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga 1740

cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc gggccccccc 1800

tcgaggtcga cggtatcgat aagcttgata tcgaattcct gcagcccggg ggatccacta 1860

gttctagagc ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta 1920

attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 1980

acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 2040

gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 2100

tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 2160

cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 2220

gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 2280

aagaacatgt gagatctcta cgggtcggat ttgaagtcgt cttggtagga ggcagcctga 2340

atggcgaatg ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg 2400

gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca 2460

gggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga 2520

tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca 2580

ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg 2640

acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta 2700

tgattcttct cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc 2760

aggtagatga cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa 2820

cttcgatcat tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga 2880

acgggttggc atggattgta ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc 2940

gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg 3000

attcaccact ccgcagaccc gccataaaac gccctgagaa gcccgtgacg ggcttttctt 3060

gtattatggg tagtttcctt gcatgaatcc ataaaaggcg cctgtagtgc catttacccc 3120

cattcactgc cagagccgtg agcgcagcga actgaatgtc acgaaaaaga cagcgactca 3180

ggtgcctgat ggtcggagac aaaaggaata ttcagcgatt tgcccgagct tgcgagggtg 3240

ctacttaagc ctttagggtt ttaaggtctg ttttgtagag gagcaaacag cgtttgcgac 3300

atccttttgt aatactgcgg aactgactaa agtagtgagt tatacacagg gctgggatct 3360

attcttttta tcttttttta ttctttcttt attctataaa ttataaccac ttgaatataa 3420

acaaaaaaaa cacacaaagg tctagcggaa tttacagagg gtctagcaga atttacaagt 3480

tttccagcaa aggtctagca gaatttacag atacccacaa ctcaaaggaa aaggactagt 3540

aattatcatt gactagccca tctcaattgg tatagtgatt aaaatcacct agaccaattg 3600

agatgtatgt ctgaattagt tgttttcaaa gcaaatgaac tagcgattag tcgctatgac 3660

ttaacggagc atgaaaccaa gctaatttta tgctgtgtgg cactactcaa ccccacgatt 3720

gaaaacccta caaggaaaga acggacggta tcgttcactt ataaccaata cgctcagatg 3780

atgaacatca gtagggaaaa tgcttatggt gtattagcta aagcaaccag agagctgatg 3840

acgagaactg tggaaatcag gaatcctttg gttaaaggct ttgagatttt ccagtggaca 3900

aactatgcca agttctcaag cgaaaaatta gaattagttt ttagtgaaga gatattgcct 3960

tatcttttcc agttaaaaaa attcataaaa tataatctgg aacatgttaa gtcttttgaa 4020

aacaaatact ctatgaggat ttatgagtgg ttattaaaag aactaacaca aaagaaaact 4080

cacaaggcaa atatagagat tagccttgat gaatttaagt tcatgttaat gcttgaaaat 4140

aactaccatg agtttaaaag gcttaaccaa tgggttttga aaccaataag taaagattta 4200

aacacttaca gcaatatgaa attggtggtt gataagcgag gccgcccgac tgatacgttg 4260

attttccaag ttgaactaga tagacaaatg gatctcgtaa ccgaacttga gaacaaccag 4320

ataaaaatga atggtgacaa aataccaaca accattacat cagattccta cctacataac 4380

ggactaagaa aaacactaca cgatgcttta actgcaaaaa ttcagctcac cagttttgag 4440

gcaaaatttt tgagtgacat gcaaagtaag tatgatctca atggttcgtt ctcatggctc 4500

acgcaaaaac aacgaaccac actagagaac atactggcta aatacggaag gatctgaggt 4560

tcttatggct cttgtatcta tcagtgaagc atcaagacta acaaacaaaa gtagaacaac 4620

tgttcaccgt tacatatcaa agggaaaact gtccatatgc acagatgaaa acggtgtaaa 4680

aaagatagat acatcagagc ttttacgagt ttttggtgca ttcaaagctg ttcaccatga 4740

acagatcgac aatgtaacag atgaacagca tgtaacacct aatagaacag gtgaaaccag 4800

taaaacaaag caactagaac atgaaattga acacctgaga caacttgtta cagctcaaca 4860

gtcacacata gacagcctga aacaggcgat gctgcttatc gaatcaaagc tgccgacaac 4920

acgggagcca gtgacgcctc ccgtggggaa aaaatcatgg caattctgga agaaatagcg 4980

ctttcagccg gcaaaccggc tgaagccgga tctgcgattc tgataacaaa ctagcaacac 5040

cagaacagcc cgtttgcggg cagcaaaacc cgtacttttg gacgttccgg cggttttttg 5100

tggcgagtgg tgttcgggcg gtgcgcgcaa gatccattat gttaaacggg cgagtttaca 5160

tctcaaaacc gcccgcttaa caccatcaga aatcctcagc gcgattttaa gcaccaaccc 5220

ccccccgtaa cacccaaatc catactgaaa gtggctttgt tgaataaatc agatttcggg 5280

taagtctccc ccgtagcggg ttgtgttttc aggcaatacg cacgctttca ggcatacctg 5340

ctttcgtcat tttgttcagc gctcgtacca gggccatagc ctccgcaacc tgaccatcgt 5400

agtcacgcag cgtcagtgaa cccccgaaca gagattgaaa gccgggggtg ggagatccgg 5460

attgccagtc tgctcgatat cgcaggctgg gtccgtgact acccactccc cctttcaacg 5520

tgaaggctac gatagtgcca gggcgggtac tgccgtaagt gccaccccaa aacaacaaca 5580

aacccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt 5640

ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg 5700

ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg 5760

tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc 5820

tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca 5880

aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag 5940

ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa 6000

ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt 6060

tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc ctaaccacgg ggacgtggtt 6120

ttcctttgaa aaacacgatg ataatatggc cacaaccatg gaacaagaga cttgcgcgca 6180

ccctctcact tttgaggaat gcccaaaatg ctctgctcta caataccgta atggatttta 6240

cctgctaaag tatgatgaag aatggtaccc agaggagtta ttgattgatg gagaggatga 6300

tgtctttgat cccgaattag acatggaagt cgttttcgag ttacagggca attccacctc 6360

ctcagacaag aataactcct cctcggaagg caatgaaggt gtgatcatca ataactttta 6420

ctccaaccaa tatcaaaact ccattgacct ctctgctaat gcagccgggt ctgacccacc 6480

cagaacctac ggtcaatttt cgaatctcct ttcgggcgca gtgaatgcct tttctaatat 6540

gcttccattg ctagctgatc aaaatacaga agaaatggag aatctgtctg atcgagtgtc 6600

tcaagacact gccggcaata cggtcacaaa cacccagtca acagtgggcc gtcttgtcgg 6660

ttatggtgcc gttcatgatg gagagcatcc ggcatcatgt gctgacactg cttcagaaaa 6720

gatcctggcg gtggaaaggt actacacctt caaggtaaat gattggacat caacacaaaa 6780

gccctttgag tacatccgca ttccccttcc tcacgtcctg tccggtgaag atggtggtgt 6840

ctttggtgcg gccctccgcc ggcactacct ggtgaaaact ggatggcggg tgcaagttca 6900

gtgcaacgcc tctcaattcc acgctggaag tttgctggtg ttcatggcac cagagtatcc 6960

aaccttagat gcttttgtca tggacaaccg ttggtccaag gataacctgc ctaatggaac 7020

cagaactcag acaaacaaaa agggaccatt tgccatggac catcagaact tctggcagtg 7080

gaccttgtat ccccatcaat tcctgaatct gagaactaac accacagtgg atcttgaggt 7140

gccatatgta aacatagccc ccacttcctc ctggacacaa catgcttcct ggactttggt 7200

gattgcagtg gttgctcccc tgacatactc aaccggggct tctaccagtt tggatatcac 7260

cgcttctatt cagccagtaa ggcctgtctt taatggcctc cggcacgaga cactttctag 7320

acagtcgccc attccggtca caattagaga acatgctggt acctggtatt ctactctgcc 7380

agacagcaca gtgcctattt atggcaagac tcctgttgct ccatccaatt acatggtagg 7440

cgaatacaag gacttcctgg agatagctca gattccaacc tttattggga ataagatccc 7500

taatgctgtc ccctacattg aggcatccaa cacagccgtc aagacccaac cgctggccac 7560

ctatcaagtg accttgtcct gctcctgtct ggccaataca ttcttggccg ctttgtctag 7620

aaactttgct cagtaccggg gatcattggt ttataccttt gtgttcactg ggaccgcgat 7680

gatgaagggc aagttcctca ttgcctacac cccacctgga gcgggcaagc ccactagtcg 7740

agaccaagcc atgcaggcga cttatgcgat ttgggatttg gggctaaatt cttcttactc 7800

cttcactgtg ccttttattt ctcccactca cttccgcatg gtaggtactg accaagtcaa 7860

catcactaat gcggatggct gggttaccgt gtggcagctc actcccctca cttacccacc 7920

aggatgcccg acctctgcta agatactaac aatggtgagc gcagggaagg atttctcact 7980

caagatgcct atctcacctg ccccctggag ccctcaggga gtagaaaacg ctgaaaaagg 8040

ggtcactgaa aacgcagacg caactgctga ctttgtggct caaccagttt acttgcctga 8100

gaaccaaacg aaggtggctt tcttctatga taggtccagt cccattggtg ccttcaccgt 8160

gaagtccggc agtctagaat ctggttttgc cccgttctct aatgagactt gcccgaactc 8220

agtgatactg acccctgggc cccaatttga ccccgcctat gaccaactca ggccacagcg 8280

tctgacagaa atttggggca atggaaatga ggagacctca aaagtctttc cgcttaaatc 8340

caaacaggat tattccttct gcctcttctc cccctttgtg tattataaat gtgatttaga 8400

agtgactctt agtcctcaca cttcaggcaa ccatgggctg ttggcgaggt ggtgtcccac 8460

tggtacacca accaagccca ctacccaggt tctccatgaa gtaagttccc tctcagaagg 8520

cagaaccccc caggtttata gtgccggacc tggcatttca aatcagattt cctttgtagt 8580

tccttacaat tctccacttt cagtcctacc agctgtctgg tataatggac acaagagatt 8640

tgacaacact gggagcttgg gcattgcccc taattctgat ttcggcactc tgttctttgc 8700

tggcacaaag cctgacatta aattcacagt ctacttgaga tacaagaata tgagagtttt 8760

ttgcccacgt ccgactgtct ttttcccctg gcccacttcc ggagacaaga ttgatatgac 8820

cccgagagct ggagtcttga tgctagagag tccaaatgcc ctagacattt caagaacata 8880

ccccacgtta catgttctca ttcaattcaa ccatagaggt ttggaggtta gattgtttag 8940

acatggacaa ttttgggctg aaacacgtgc ggacgtgatt ctgagatcga agaccaaaca 9000

ggtctctttc ctgagcaacg ggaactaccc gtcaatggac tctagagctc cctggaatcc 9060

ttggaagaat acctaccagg cggttctaag agcagaacca tgtagagtga ccatggatat 9120

atactataag agagtcaggc cttttagact gcccctggtt cagaaggaat ggcgcgtgcg 9180

agaggagaac gttttcggtt tgtaccggat cttcaatgcc cactacgctg gttactttgc 9240

ggacctactg attcatgaca ttgagacaaa tccagggccc ttcatgttta gaccaaggaa 9300

acaggttttc cagacccaag gagcggcagt gtcatcaatg gctcaaaccc tactgccgaa 9360

cgaccttgcc agcaaagcta tgggatcagc ttttacggct ttgctcgatg ccaacgagga 9420

cgcccaaaaa gcaatgaaga ttataaagac attaagttct ctatcggatg catgggaaaa 9480

tgtaaaagaa acactaaaca acccagagtt ctggaagcag ctcttgagca gatgtgtgca 9540

gctgattgca gggatgacaa tagcagtgat gcatccggac cccttgactc tgctctgctt 9600

aggaacattg acggccgccg agattacaag ccagacaagt ctgtgcgaag aaatagcagc 9660

taagttcaag acaattttca tcactcctcc accacggttt cccacaatct ctcttttcca 9720

acaacaatcc cccttgaaac aggtaaatga tattttctcc ctagccaaga acctggactg 9780

ggccgtcaag actgtggaaa aggtggttga ttggtttggg acatggatag tacaggagga 9840

aaaggaacag accctagatc agctcttgca gcgtttcccc gaacatgcga agcgcatttc 9900

tgatctccgg aatggaatgg ccgcctatgt agagtgcaag gagagttttg atttctttga 9960

aaagctgtac aatcaggcag tgaaagagaa gagaacgggt atcgccgccg tctgtgaaaa 10020

attcagacag aagcatgacc acgccaccgc tcggtgtgag ccagtcgtga ttgtgctccg 10080

cggagacgcg gggcaaggga aatctttatc aagtcaggtt attgcccagg ccgtctccaa 10140

gaccattttc ggtcggcaat ctgtgtattc cctccccccc gattcggatt tctttgatgg 10200

ctatgaaaat cagtttgcag caataatgga tgatctaggg caaaatcctg atggctctga 10260

tttcactacg ttctgtcaga tggtttcgac taccaatttt ctccccaata tggctagtct 10320

agagagaaag ggcaccccct ttacatctca gcttgtggtg gcaactacca atctgcctga 10380

gtttagacct gtcacaatag cccattaccc tgctgttgag agaaggataa cttttgacta 10440

ttcagtgtct gctggtccag tctgctccaa aacagaggcc gggtataagg ttttggatgt 10500

tgaaagagcc tttaggccta ccggtgaggc tcctcttcca tgcttccaga ataactgcct 10560

tttccttgag aaagctgggc tccagttcag agataaccga actaaagaga tcatttccct 10620

ggtagatgtg attgagagag ccgtggctag gattgaaagg aagaagaaag ttctcacaac 10680

cgtgcagacc cttgtggcac aagctccagt agacgaggtc agtttccatt ccgtagtcca 10740

gcagcttaaa acaagacagg aagcgacaga tgaacagctt gaggaattgc aagaggcttt 10800

cgcgaaagta caggagcgta actctgtgtt ttctgattgg ttgaagattt ctgcaatgtt 10860

gtgtgctgcg actctggcac tttcccaagt tgtcaagatg gccaaggcgg tgaagcagat 10920

ggtcaagcct gatctggttc gtgtgcaatt ggatgagcag gagcagggac cttacaatga 10980

gacagcgaga gttaaaccaa aaacactgca gttgttggac attcagggac caaaccctgt 11040

gatggacttt gaaaaatatg tagccaaaca tgtaaccgcc cccattgatt ttgtctaccc 11100

cactggggtg agcacccaga cttgcctcct tgtgagaggc cgcaccttgg cagtaaatag 11160

acacatggcc gagtctgact ggacttccat agtagtgcgt ggagtcacac acgcccgctc 11220

tactgttaaa attttggcca tagctaaagc aggcaaagag actgacgtat ctttcatccg 11280

cctctcttct ggtcctctat tcagagacaa tacatccaaa tttgtcacgg ctggtgatgt 11340

acttcctact ggtgccgctc cagtcacggg gataatgaac acggacatac ccatgatgta 11400

cacaggaacc ttcctgaaag ctggtgtgtc agtcccagtg gaaaccggcc agacctttaa 11460

tcactgtatt cattacaagg ctaacacacg aaagggctgg tgtggatcag ccctactggc 11520

agatcttgga ggaagcaaga aaatccttgg catccattct gctggctcta tgggaatagc 11580

cgccgcctcg attgtgtcac aggagatgat tcgggcggta gtgaatgcct ttgagccaca 11640

gggtgctctc gagagattgc cagatgggcc ccgtattcac gtaccacgta aaacagcact 11700

acgccccacc gttgcccgtc aagtcttcca accagcatat gccccggctg ttctatcgaa 11760

atttgaccct agaacagagg ctgatgtaga tgaagtggct ttctccaaac atacctccaa 11820

ccaggaaagc ctcccaccag tgtttagaat ggtagccaaa gagtatgcca atagagtttt 11880

caccttgctg ggaaaagaca atggccgtct gactgtaaag caggctttgg aaggactgga 11940

ggggatggac cccatggaca ggaacacctc cccggggctt ccatatactg cgctaggaat 12000

gcgcagaaca gatgtcgtag attgggaatc agccaccctg atcccgtttg cggcagaaag 12060

attaagaaaa atgaatgaag gagacttttc cgaagttgtc tatcaaacat tcctcaagga 12120

tgagcttaga ccgatagaga aggttcaagc cgccaagaca cggattgtag atgttccacc 12180

atttgagcat tgcattctgg gtagacaatt gttgggaaag tttgcatcaa agttccagac 12240

ccaaccgggt ctggaactag gatcagccat tggatgtgac ccagatgtac actggactgc 12300

cttcggtgtc gccatgcaag gttttgagcg tgtctacgat gtggactact ccaactttga 12360

ttcgacccat tcggtggcaa tgttccgctt attggctgag gaatttttca ctccagagaa 12420

tggttttgac cccctgacta gagaatatct tgagtcatta gccatttcaa cccatgcgtt 12480

tgaggagaag cgctttctga taaccggtgg tctcccatca ggttgtgcag cgacctcaat 12540

gctaaacact ataatgaata atataataat tagggcgggt ttgtatctca cgtataaaaa 12600

ttttgaattt gatgatgtga aggtgttgtc gtacggagat gatctccttg tggccacaaa 12660

ttaccaattg gattttgata aggtgagagc aagcctcgca aagacaggat ataagataac 12720

tcccgctaac aaaacttcta cctttcctct taattcgacg cttgaagacg ttgtcttctt 12780

aaaaagaaag tttaagaaag agggccctct gtatcggcct gtcatgaaca gagaggcgtt 12840

ggaagcaatg ttgtcatact atcgtccagg gactctatct gagaaactca cttcgatcac 12900

tatgcttgcc gttcattctg gtaagcagga atatgatcgg ctctttgccc cattccgtga 12960

ggtaggggtt gtcgtgccat cattcgagag tgtggagtac agatggagga gtctgttctg 13020

gtagtagtgc agtcactggc acaacgcgtt acccggtaag ccaatcgggt gtacacggtc 13080

gtcatactgc agacagggtt cttctacttt gcaagatagt ctagagtagt aaaataaata 13140

gatagagtca cttagggtta agtggggggt ctttaagatt aaattccctg aattgtacat 13200

acatggttat acggatattg tagtcctggt cgtagatact gttttcgaac gcagtgccga 13260

ggcctacatg gtctacattt ccagtagttt gtagtctcag ccagagttga tttcttttgt 13320

tattgggttg gaagtaatcg attgtcctat caaggacagg tttcggggta aagtaccggg 13380

agtggtagga gaagggctgg gttatggtat ggcgggagga gtagtttaca taggggtcat 13440

aggttagggc attggccttt gttacaaagt tatcatctag aataacagca gtggagccca 13500

ctcccctgtc accctgggtg attgggggag caggccagaa ttcaatctta accttcctta 13560

ttctgtagta ttcaaagggc acagtgaggg ggtttgagcc ccctcctggg ggaagaaaat 13620

cattaatatt aaatctcatc atgtccacat tccaggaggg cgttctgact gtggttttct 13680

tgacagtata accgatggtg cgggagaggc gggtgttgaa gatcccattt ttccttctcc 13740

agcggtaacg gtggcggggg tggacgagcc aggggcggcg gcggaggatc tggccaagat 13800

ggctgcgggg gcggtgtctt cgtctgcgga aacgcctcct tggatacgtc at 13852

Claims (6)

1. the full-length infectious clone of a kind of porcine encephalomyocarditis virus BD2 strains, its sequence are shown in SEQ ID NO.1.

2. the construction method of the full-length infectious clone of porcine encephalomyocarditis virus BD2 strains described in claim 1, its step include：

(1) choose the final load that low copy carrier pWSK29 is built as EMCV BD2 wild type full-lengths infectious CDNA clones Purpose base is carried out by body with point mutation kit Fast Mutagenesis System and mutant primer SpeI-F/SpeI-R Mutation, to remove unnecessary I restriction enzyme sites of Spe, mutant primer SpeI-F, SpeI-R gene order such as SEQ ID NO.2, SEQ Shown in ID NO.3, the reaction of tri- steps PCR of Jing is expanded, and is cultivated in being transformed into DMT competent cells, selects Jing PCR detections As a result it is positive bacterium colony, is carried out expanding numerous, with extraction reagent kit in plasmid after sequencing confirms mutation success Plus Midipreps DNA Purifation System are extracted and plasmid DNA purification；By the low copy carrier after point mutation It is named as pWSKs；

(2) EMCV BD2 F5 are extracted for viral genome total serum IgE；

(3) design, synthesis cover three couples of overlapping primers SegA-F of EMCV BD2 virus full-length genome, SegA-R, SegB-F, SegB-R, SegC-F, SegC-R, each primer sequence such as SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID Shown in NO.7, SEQ ID NO.8, SEQ ID NO.9, SP6RNA polymerase promoters are added in the upstream primer of total length amplification Core sequence, introduces point mutation in primer SegB-R and SegC-F, mutant primer be EcoRI-F, EcoRI-R, SpeI-F, SpeI-R mutant primers sequence is as shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.2, SEQ ID NO.3；

(4) each genomic fragment cDNA synthesis of EMCV and PCR amplifications：Using reverse transcriptaseReverse Transcriptase and three couple of downstream primer SegA-R, SegB-R and SegC-R are inverted to EMCV BD2 geneome RNAs Record, obtains the cDNA of tri- fragments of A, B, C of EMCV BD2 genomes；With reverse transcription obtain cDNA as template, respectively with SegA-F/SegA-R, SegB-F/SegB-R and SegC-F/SegC-R are primer, using high-fidelity enzyme FastPfu Fly DNA Polymerase enter performing PCR amplification to three fragments, and pcr amplification product is detected and purified back Receive, the PCR primer after recovery is attached respectively with pEASY-Blunt carriers, connection product is all added to and just thaws Trans1-T1 competent cells in, Jing process, culture select Jing PCR testing results to be positive bacterium colony, through sequencing card Carried out expanding numerous after real mutation success, plasmid is extracted with the little extraction reagent kit QIAGEN Plasmid Mini Kit of plasmid, will be each The plasmid that fragment subclone is obtained is respectively designated as pEBSegA, pEBSegB and pEBSegC；

(5) pEBSegC, pEBSegB, pEBSegA are cloned into improved pWSK29s successively, complete the structure of total length plasmid And gene sequencing is carried out to which.

3. the infection clones described in claim 1 prepare virus, develop vaccine, as viral vector expression alien gene side The application in face.

4. built using a kind of full-length infectious clone of the porcine encephalomyocarditis virus BD2 strains described in claim 1 and stably carry pig The method of the full-length infectious clone of encephalomyocarditis virus BD2 strains of circovurus type 2 ORF2 genes, its step include：

(1) the fusion DNA vaccine amplification of external source Insert Fragment：With ORF2L, fORF2, ORF2R, L-Cap of porcine circovirus 2 type gene Fragment is template, using tetra- primer pairs of up-F/up-R, ORF2-F/ORF2-R, down-F/down-R, up-F/down-R, is drawn Thing up-F, up-R, ORF2-F, ORF2-R, down-F, down-R sequence such as SEQ ID NO.12, SEQ ID NO.13, SEQ ID Shown in NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, amplification system is set up according to conventional PCR method, Each purpose fragment is expanded independently, amplification is using high-fidelity enzymeFastPfu Fly DNA Polymerase, Purifying reclaims amplified production；Fragment to be fused is added in the fusion DNA vaccine system for building, is added without in incipient reaction system Each fragment region that coincides is carried out complementary extension using high-fidelity enzyme by primer, treat the fusion steps of 8 circulations complete after temporarily Stop PCR reactions, then primer up-F and down-R are added in system, complete hereafter 25 circulations, produced with the fusion for obtaining total length Thing；Reclaim after will be the fusion DNA vaccine product for obtaining identified, linked system is set up with pEASY-Blunt carriers, be transformed into and just solve In the Trans1-T1 competent cells of jelly, Jing cultures carry out plasmid extraction, the middle interstitial granules for obtaining are named as pEBORF2；

(2) structure of recombinant virus infection cloned plasmids and sequencing：Choose fusion fragment L gene insertion site upstream and downstream unique A pair of restriction enzyme Hind III and Nhe I, using the two specific restriction enzyme sites, will be fused good Fusion fragment containing external source ORF2 gene is therefrom cut on interstitial granules pEBORF2, while will be the infection clones for building complete On long plasmid pWSKBD2, respective regions are cut together with Hind III and Nhe I, by the two digestion products with cohesive end Jing T4 ligases link together, and complete the stable encephalomyocarditis virus BD2 strain total lengths for carrying porcine circovirus 2 type ORF2 genes The structure of infective cloned plasmids；Connection product is transformed in Trans1-T1 competent cells and is cultivated, expand numerous extraction low The recombinant plasmid is named as pWSKBD2-ORF2, Insert Fragment is sequenced with primer up-F/down-R by copy plasmid, So that it is determined that whether external source ORF2 gene has completely been correctly inserted into the pilot protein region of EMCV genomes.

5. it is a kind of stably carry porcine circovirus 2 type ORF2 genes the full-length infectious clone of encephalomyocarditis virus BD2 strains, its sequence It is classified as shown in SEQ ID NO.18.

6. the infection clones described in claim 5 are preparing the application of virus, the aspect that develops vaccine.