CN106754982A - The restricted duplication west nile virus system of expressing green fluorescent protein and its application - Google Patents

The restricted duplication west nile virus system of expressing green fluorescent protein and its application Download PDF

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CN106754982A
CN106754982A CN201611154016.3A CN201611154016A CN106754982A CN 106754982 A CN106754982 A CN 106754982A CN 201611154016 A CN201611154016 A CN 201611154016A CN 106754982 A CN106754982 A CN 106754982A
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华荣虹
步志高
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Harbin Veterinary Research Institute of CAAS
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Abstract

The invention discloses a kind of restricted duplication west nile virus system of expressing green fluorescent protein and its application.The restricted duplication west nile virus system of expressing green fluorescent protein of the present invention includes the recombinant cell lines of the restricted replicon plasmid of west nile virus and stabilization expression west nile virus C prM E proteins.The restricted duplication west nile virus (△ WNV) obtained using system of the invention, the virus has the characteristic similar to WNV totivirus in BWNV CME cell lines, and GFP can be expressed, with effect of visualization, importantly, the virus can only be replicated in the system, young generation virus can not be then produced when infecting other cells, therefore there is high security, can be used for the detection of WNV neutralizing antibodies and antiviral drugs screening.The security and simplicity that improve WNV associative operations of the invention, does not rely on the high-grade bio-safety conditions of BSL 3, for WNV vaccine evaluations, antiviral drugs screening and viral pathogenesis Mechanism Study provide good technological means.

Description

The restricted duplication west nile virus system of expressing green fluorescent protein and its application
Technical field
The present invention relates to a kind of viral replication system and its application, more particularly to a kind of limitation of expressing green fluorescent protein Property replicate west nile virus system and its application, the invention belongs to biological technical field.
Background technology
West nile virus (West Nile Virus, WNV) belongs to flaviviridae, and Flavivirus is that a kind of important people beast is total to Suffer from infectious disease virus, the nervous system disease that people can be caused serious.WNV virions diameter about 40nm~60nm, there is cyst membrane, film Interior virus nucleocapsid is in icosahedral symmetry, diameter about 30nm.WNV genomes are a single stranded positive-sense RNA, are about 11kb. Noncoding region (UTR) and the single opening that is about 10.3kb of the genome by 5 ' the end ends of 96nt long and 3 ' 337-649nt long Reading frame (open reading frame, ORF) is constituted.ORF, to the termination of 10398,3 ' end, encodes 1 from 5 ' 97 startings in end Individual total length is 3433 polyprotein precursors of amino acid, and 3 kinds of structures are produced after the cutting processing of host and virus protease Albumen：C protein (capsid/core protein), prM/M (film precursor protein/memebrane protein) and E protein (membrane glycoprotein) and 7 kinds Non-structural protein.
West nile virus disease is the infectious disease caused by west nile virus, is a kind of Amphixenosis.The disease is come to China Say it is exotic disease, had not been reported in inland of China at present, but the weather of China, geographical environment are complicated, and mosquito specie is various, tool Standby propagation conditions, and the existing disease of the surrounding countries such as Russia, India prevalence, with international exchange increasingly frequently, WNV biographies The possibility for entering within Chinese territory can not be ignored, and such as isolate the report of WNV from the mosquito in Xinjiang in recent years.So to me For state, the risk of the popular outbursts of WNV is very big, and western to prevent and treat infection there is presently no vaccine or effective medicine Nile virus, it is therefore necessary to study corresponding Control Measure.But WNV is former Zoonosis venereal disease, the correlation such as vaccine is ground The live virus operation studied carefully needs to be carried out in high-level biosafety laboratory, and this hinders related vaccines and medicine to a certain extent Thing is studied.
In order to promote WNV vaccine developments and screening new anti-virus medicine, the present invention constructs missing entire infrastructure albumen Totivirus genome, and its structural protein region insert GFP reporter genes WNV replicon plasmids pWNVrepGFP- DCME, and construct the cell line of stabilization expression C-prM-E albumen；Replicon plasmid, recombinant cell lines and the △ WNV for packing Virus constitutes a west nile virus system for restricted duplication.Replication defect type west nile virus system of the invention can be The epidemiology survey of west nile virus, vaccine evaluation, antiviral drugs are screened and West Nile Virus duplication and pathogenesis Research safe and simple method is provided.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of expressing green fluorescent protein and can only have in system Infectious west nile virus (WNV) preparation system with replication capacity, be applied to it is safe, visual, similar to totivirus Infection and the WNV neutralizing antibodies detecting system and drug screening method that replicate.
In order to achieve the above object, present invention employs following technological means：
A kind of restricted duplication west nile virus system of expressing green fluorescent protein of the invention, including：
(1) the restricted replicon plasmid of west nile virus：
The restricted replicon plasmid of described west nile virus is to be cloned into eucaryon table by by wnv cdna group Up to the promoter downstream of carrier, and the structural proteins C-prM-E sequences in genome are replaced with into green fluorescent protein base simultaneously Obtained because after；
(2) recombinant cell lines of stabilization expression west nile virus C-prM-E albumen
Described recombinant cell lines are carried by by the eukaryotic expression containing west nile virus C-prM-E protein-encoding genes Obtained after body transfection mammalian cell.
In the present invention, it is preferred to, in the structure of the restricted replicon plasmid of described west nile virus, what is used is true Nuclear expression carrier is pCI-neo.
In the present invention, it is preferred to, the nucleotide sequence such as SEQ of the restricted replicon plasmid of described west nile virus Shown in ID NO.1.
In the present invention, it is preferred to, the recombinant cell lines of described stabilization expression west nile virus C-prM-E albumen be by Obtained after eukaryotic expression vector transfection mammalian cell containing the west nile virus C-prM-E protein-encoding genes after optimization , it is furthermore preferred that described carrier for expression of eukaryon is pCAG-neo, described mammalian cell is BHK-21 cells.
In the present invention, it is preferred to, the nucleotides of the west nile virus C-prM-E protein-encoding genes after described optimization Sequence is as shown in SEQ ID NO.2.
In the present invention, it is preferred to, the recombinant cell lines of described stabilization expression west nile virus C-prM-E albumen are logical Cross following methods and build what is obtained：
(1) the C-prM-E protein-encoding genes after artificial synthesized optimization, its nucleotide sequence as shown in SEQ ID NO.2, Sequence designations after optimization are opti-WNV-CME, carry out it is artificial synthesized after the sequence after optimizing is expanded by PCR method, and 5 ' ends of PCR primer introduce the restriction enzyme sites of Sac I, and 3 ' ends introduce the restriction enzyme sites of Xho I, PCR primer after double digestion with it is same The pCAG-neo carriers of double digestion are connected, and after connection product conversion DH5 а, select recombinant clone, through digestion and sequencing identification, obtain Largely prepare plasmid with kit after positive colony, be named as pCAG-WNV-CME, frozen after being linearized with SspI in- 20 DEG C standby；
(2) the BHK-21 cell dissociations for selecting growth conditions good are passaged in 24 orifice plates, treat that BHK-21 cells are long to 90% Man Shi, with the recombinant plasmid pCAG-WNV-CME transfectional cells after linearisation, obtains the stabilization expression Xi Niluo diseases of stabilization expression The recombinant cell lines of malicious C-prM-E albumen, are named as BWNV-CME.
Further, the invention allows for described system in the restricted duplication west for obtaining expressing green fluorescent protein Purposes in Nile virus.
Further, the invention allows for a kind of restricted duplication Xi Niluo diseases for obtaining expressing green fluorescent protein The method of poison, comprises the following steps：
It is growth medium, 1 with the DMEM culture mediums containing 10% (v/v) FBS：4 Secondary Cultures are of the present invention steady Surely the recombinant cell lines of west nile virus C-prM-E albumen are expressed, by the recombinant cell of exponential phase with 5 × 105/ ml is inoculated with 6 porocyte culture plates, incubated overnight, by the restricted replicon plasmid addition Opti-MEM of west nile virus of the present invention DNA dilutions are made, every 3 μ g plasmids add 200 μ l opti-MEM, add 20 μ l X-treme HP transfection reagents, and room temperature is quiet 15min is put, adds the restructuring containing stabilization expression west nile virus C-prM-E albumen thin DNA transfection reagents complexes drop-wise In the nutrient solution of born of the same parents, fluorescence is observed after 72h.Collect supernatant, be stored in after centrifugal filtration -80 DEG C it is standby.
Further, the invention allows for the restricted of the expressing green fluorescent protein obtained according to described method The restricted duplication west nile virus for replicating west nile virus and described expressing green fluorescent protein is preparing Xi Niluo diseases Purposes in malicious neutralizing antibody detection reagent and the anti-west nile virus medicine of screening.
Compared to prior art, the beneficial effects of the present invention are：
The WNV replication-defective viruses that the present invention is developed into are turned using the subgenomic replicons of deletion construct GFP The cell line BWNV-CME of dye stabilization expression WNV C-prM-E albumen carries out △ WNV virus particle packages, in the viral genome Structural protein gene be expressed the gene of green fluorescent protein and replaced, therefore in Viral Replicon reproduction process, due to It is unable to oneself expression structural proteins and cannot continue to produce progeny virus, but can be with great expression GFP albumen, to infection cell With extremely strong indicative function.And only when the WNV C proteins (capsid/core protein), prM/M (the film precursors that are provided with external source Albumen/memebrane protein) and complete virion can be integrated into after E protein (membrane glycoprotein) is ressembled.Lack replicating In swaged virus neutralization tests, WNV replication-defective viruses can be neutralized antibody and effectively neutralize and lose the sense to host cell Dye is acted on.Because WNV replication-defective viruses contain reporter gene, can expressing green fluorescent protein, therefore can utilize glimmering Light microscope intuitively observes the cell being infected.In being carried out instead of wild type WNV using WNV replication-defective viruses During detection with antibody, after only the replication-defective virus of doses need to being incubated into 1h jointly with measuring samples, direct infection , be placed in cell under fluorescence microscope after 48h observe fluorescence intensity by permissive cell BHK-21, in evaluating in measuring samples And antibody, the operation is operated compared to live virus has high security and simplicity, does not rely on the high-grade biologies of BSL-3 Safety condition, it is time-consuming, improve efficiency.Simultaneously as WNV replication-defective viruses are replication defect types, therefore added Virus quantity be fixed numerical value, the propagation of virus will not be produced because infecting permissive cell, advantageously in carrying out antibody Qualitative and quantitative detection during detection.
Brief description of the drawings
Fig. 1 is that WNV replicons build schematic diagram；
Fig. 2 is identified for the digestion of recombinant plasmid pWNVrep-GFP-dCME；
1：DL 15000DNA Marker；2：Before double digestion；3：After double digestion.
Fig. 3 is identified for the digestion of recombinant plasmid pCAG-WNV-CME；
1：DL 15000DNA Marker；2、3：Before double digestion；4、5：After double digestion.
Fig. 4 is detected for recombinant cell lines E protein monoclonal antibody specific IFA；
Fig. 5 is that △ WNV infect the plastidogenetic fluorescent spots of BWNV-CME and plaque；
Fig. 6 is the duplicating dynamics curve of △ WNV viruses；
Fig. 7 is △ WNV virus infection characterizations；
Fig. 8 is to detect the anti-WNV activity of medicine with the restricted virus systems that replicate of WNV.
Specific embodiment
The present invention is further described with reference to specific embodiments and the drawings, advantages of the present invention and feature will be with Description and it is apparent.But these embodiments are only exemplary, do not constitute any limitation to the scope of the present invention.This area Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and Form is modified or is replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
The structure of the restricted duplication west nile virus system of the expressing green fluorescent protein of embodiment 1
1 material and method
1.1 carriers, cell and main agents
Expression vector plasmid pCAG-neo plasmids (Li Yenan etc., Chinese Preventive Veterinary Medicine report, 2013,35 (3)::189- 192；Hua RH,et al,BMC Biotechnology,2014,14:62) (the flat stabilizations of Guo Li are expressed with PCAG-WNV plasmids Structure [D] the Chinese Academy of Agricultural Sciences of west nile virus prM-E albuminous cells system, 2015) built by literature procedure； BHK-21 cells are preserved by this laboratory, and W-92 cell lines are built by this laboratory.DMEM, excellent hyclone (FBS) and Opti-MEM is purchased from Gibco companies；X-treme HP transfection reagents are purchased from Roche companies；FITC mark mountain sheep anti-mouse iggs are purchased from Company of Zhong Shan Golden Bridge；The dynamics of infrared fluorescent labels are purchased from the public laboratory of Harbin veterinary institute；E protein and NS1 protein monoclonal antibodies are prepared by this laboratory；WNV mouse positive serum is preserved by this laboratory.
1.2 replicon plasmid pWNVrepGFP-dCME build
By reference to WNV virus genome sequences (NY99 plants, Genbank sequence number DQ211652NY99), using being based on The method of PAS (PCR-based Accurate Synthesis), design total length splicing primer, respectively devises at the two ends of primer Protectiveness base, synthetic gene pack section, eukaryon expression plasmid pCI- is connected to by cloning site Xho I and Xba I The CMV promoter downstream of neo builds WNV replicon plasmids, and structural proteins C-prM-E sequences are replaced during WNV replicates subgenom It is green fluorescent protein (green fluorescent protein, GFP) gene, the recombinant plasmid of acquisition is named as pWNVrepGFP-dCME.Each main element of replicon is as shown in Figure 1.The nucleotide sequence of pWNVrepGFP-dCME such as SEQ ID Shown in NO.1.
1.3 plasmid pCAG-WNV-CME build
Codon optimization is carried out to coding WNV C-prM-E GFPs with bioinformatics method first, after optimization C-prM-E protein gene sequences as shown in SEQ ID NO.2, sequence designations after optimization are opti-WNV-CME, enter pedestrian Sequence after work synthesis after PCR method expands optimization, and the restriction enzyme sites of Sac I are introduced at 5 ' ends of PCR primer, 3 ' ends are drawn Enter the restriction enzyme sites of Xho I, double digestion PCR primer is connected with the pCAG-neo carriers of same double digestion, connection product conversion DH5 а Afterwards, select recombinant clone, through digestion and sequencing identification, largely prepare plasmid with kit after obtaining positive colony, be named as PCAG-WNV-CME, freezes standby in -20 DEG C after being linearized with SspI.
The structure and Screening and Identification of 1.4 recombinant cell lines
The BHK-21 cell dissociations for selecting growth conditions good are passaged in 24 orifice plates, treat that BHK-21 cells are long to 90% full When, according toRestructuring matter after the linearisation of HD Transfection Reagent transfection reagent boxes operating instruction Grain pCAG-WNV-CME transfectional cells.Added after transfection 48h carries out pressurization training containing G418 (1000 μ g/mL) selective medium Support, cell is digested with pancreatin after 4d, passed on limiting dilution assay and continue to cultivate in 96 orifice plates, after 5d under inverted microscope Colony counts of the observation per hole cell.The hole containing 1 cell colony (i.e. 1 cell mass) is selected in succession in 24 orifice plates, 6 holes Amplification Culture in plate, Tissue Culture Flask, while carrying out IFA identifications with WNV E proteins monoclonal antibody specific to each cell, sieves The cell clone for selecting IFA signals stronger.The cell clone of stabilization expression C-prM-E albumen is obtained, BWNV-CME is named as.
The packaging of 1.5 △ WNV viruses and preservation
It is growth medium, 1 with the DMEM culture mediums containing 10%FBS：4 Secondary Culture BWNV-CME cells, by logarithm The BWNV-CME cells in growth period are with 5 × 105/ ml is inoculated with 6 porocyte culture plates, incubated overnight, by plasmid pWNVrep-GFP- DCME is made DNA dilutions in adding Opti-MEM, and every 3 μ g plasmids need 200 μ l opti-MEM, add 20 μ l X-treme HP transfection reagents, are stored at room temperature 15min.DNA transfection reagents complexes drop-wise is added into the nutrient solution containing BWNV-CME cells In, fluorescence is observed after 72h.Collect supernatant, be stored in after centrifugal filtration -80 DEG C it is standby.
1.6 △ WNV viruses PFU are determined
The viral supernatants 15ul packaged by results transfectional cell is taken, 135ul 5%FBS DMEM is added, by a series of 10 Dilute again, six orifice plates add 100ul, 37 DEG C, 5%CO per hole2Incubator culture 10h adds culture medium isometric in backward every hole 3% methylcellulose, egfp expression is observed after culture 48h.Or with crystal violet liquid dyeing counting virus after cultivating 6 days Plaque.
1.7 packaging virus duplicating dynamics
In order to observe the growth kinetics process of transfectional cell packaging virus, to prepared packaging virus in BWNV-CME Infection is analyzed with duplication situation in cell, and 24h, 48h, 72h, 96h, 120h collect supernatant and survey after infection respectively Its titre.
2 results
2.1 plasmid pWNVrepGFP-dCME build
Plasmid pWNVrepGFP-dCME builds schematic diagram and sees Fig. 1.The plasmid based on wnv virus genome sequences, 5 ' It is CMV and T7 promoters, to ensure the accuracy of 3 ' terminal sequences after transcribing, 3 ' ends are added with fourth liver nucleotide sequence (HDr).Plasmid Through Not I and SalI double digestions qualification result such as Fig. 2, endonuclease bamhi size is 8000bp and 5000bp or so, with expected results one Cause, forward and reverse sequencing confirms that insetion sequence is correct.
2.2 plasmid pCAG-WNV-CME build
After double digestion PCR primer is connected with the pCAG-neo carriers of double digestion, coated plate is converted, choose monoclonal bacterium, extract matter Verified with Sac I and the double digestions of Xho I after grain, detected through 1% agarose gel electrophoresis, purpose fragment 2400bp or so is and pre- Phase is consistent.And sequencing results show sequence with expected consistent (Fig. 3).
The Screening and Identification of 2.3 recombinant cell lines
After the plasmid transfection BHK-21 cells that will be linearized, screened with G418, cell is anti-with monoclonal after clone Body carries out IFA identifications (Fig. 4).Result obtains one plant of cell line of expression C-prM-E proteins, is named as BWNV-CME.Should Cell line carries out Secondary Culture after clone purification again and shows the cell line inheritance stability with detection, after the generation of continuous passage 20 Expression E protein can still be stablized.The culture growth characteristics of recombinant cell lines are similar to parental cell BHK-21 cells, with 1：5 Ratio carries out Secondary Culture, and culture medium is the DMEM liquid containing 10% hyclone, can pass on again every other day.
2.4 △ WNV viruses PFU are determined
Harvest the μ l of replication-defective virus △ WNV supernatants 100 and infect BWNV-CME cells, 48h through a series of 10 times dilutions Afterwards, observed under inverted microscope and GFP cell numbers are expressed per hole, and counted, it is 1 × 10 to measure virus titer5.5PFU/ml。△ The WNV infection plastidogenetic fluorescent spots of BWNV-CME are as shown in Figure 5 with plaque.
2.5 △ WNV Virus replication kinetics
In 24h, 48h, 72h, 96h, 120h supernatant is collected after transfection respectively and survey its potency and draw kinetic curve Fig. 6, as a result shows 24~72h, and virus titer is totally in rising trend, and 72h to 96h is in plateau, is slightly decreased after 96h, Result shows that the replication-defective virus of structure has good replication activity, and it is transfection that the malicious Best Times of receipts are determined Afterwards between 72h-96h.
The 2.6 restrictive duplication characteristics of △ WNV
The cells and supernatant for transfecting pWNVrep-GFP-dCME and pCAG-WNV-CME simultaneously can be in the first subinfection BHK-21 cells in express GFP, and transfect the cell culture of pWNVrep-GFP-dCME or pCAG-WNV-CME plasmids merely Supernatant can not then infect BHK-21 cells, without GFP expression.Plasmid pWNVrep-GFP-dCME transfection BWNV-CME cell lines are produced Viral △ WNV have infectivity on cell line BWNV-CME, can produce young generation virus, produce fluorescent spot to be bitten with virus is formed Spot, and viral infection characterization is kept when the second wheel infects BWNV-CME cell lines with third round.And infect BHK-21 cells Supernatant it is then infectious without the second wheel, when its supernatant inoculates BHK-21 cells, cell can not express GFP.Thus I Can draw by it is of the invention replicate in the specific cell line that restricted WNV virus can only be in the system have similar to The characteristic that totivirus is replicated, and it is total to his sensitive cells and only has single-wheel infectivity (Fig. 7) if in BHK-21 cells.
The restricted duplication west nile virus systematic difference of the expressing green fluorescent protein of embodiment 2
1 method
The 1.1 restricted virus systems that replicate are detected for WNV neutralizing antibodies
By 4 × 105/ mL BWNV-CME cells are inoculated in 24 porocyte culture plates, 500 μ L/ holes, 37 DEG C, 5%CO2 cultures Overnight, cell is made to grow up to individual layer；From 1 after WNV positive serums and negative serum are inactivated：10 start gradient dilutions after with it is isometric △ WNV mixing, set multiple holes, in 37 DEG C, 5%CO2 incubators place 1h；Afterwards by the μ l of △ WNV antiviral antibodies mixed liquor 100 It is inoculated in individual layer BWNV-CME cells, 37 DEG C, add 3% methylcellulose DMEM liquid after 5%CO2 cultures 6h, culture is seen after 3 days Examine green fluorescence expression.Plaque counting is carried out after 6 days.50% is reduced to judge terminal with fluorescent spot or plaque, is calculated each to be measured The NAT of serum.
1.2 restricted replicability virus systems are screened for WNV antiviral drugs
By 4 × 105Cell/mL BWNV-CME cells are inoculated in 24 porocyte culture plates, 500 μ L/ holes, 37 DEG C, 5%CO2 Overnight incubation, makes cell grow up to individual layer；By tannic acid (Tannic acid), the medicine such as 6- aza uridines (6-azauridine) 10mM reservoirs are formulated as with DMSO, then 20 μM are diluted to DMEM liquid, 1%DMSO is control, by each medicine and and control addition Into cell, the △ WNV viruses of 100TCID50/0.1ml are added after effect 10h, in 37 DEG C, 5%CO2After incubator places 10h Add and △ WNV antiviral antibody mixed liquors are inoculated in individual layer BWNV-CME cells, 37 DEG C, add 3% first after 5%CO2 cultures 6h Green fluorescence expression is observed in base cellulose DMEM liquid, culture after 3 days.Plaque counting is carried out after 6 days.With DMSO or space management hole It is control, calculates the viral suppression of each medicine to be measured.
2 results
With experiment detection WNV neutralizing antibodies in 2.1
Using restricted duplication WNV virus systems of the invention to WNV subunit vaccines be immunized mice serum, goose blood Clearly, and horse serum has carried out virucidin's detection, while setting up the negative serum conduct that corresponding experimental animal is not immunized Control.Result shows, vaccine immunity animal blood serum is equally detected by the restricted neutralization tests for replicating virus system of WNV Neutralization activity, and with the increase of serum dilution, its neutralization to WNV replication-defective viruses is gradually reduced, and Negative control sera has no neutralization to WNV replication-defective viruses, and all experimental animal serum are exempted from restrovirus and neutralized two Antibody is significantly improved, and can provide immunoprotection (table 1) for immune animal.Show the restricted duplication virus stocks of WNV of the invention System can be used to detect the analysis of serum neutralizing antibody.
Table 1 is using △ WNV virus systems detection WNV neutralizing antibodies
2.2 screen antiviral drugs with the restricted virus systems that replicate of WNV
Through with the restricted detection knot for replicating virus system to the known medicine for having antiviral activity to WNV of the invention Fruit shows that the system can be used for screening anti-WNV virus drugs.Two kinds of medicines show and can suppress WNV viruses in this example Duplication, and DMSO control then it is this activity (Fig. 8).
Sequence table
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture
<120>The restricted duplication west nile virus system of expressing green fluorescent protein and its application
<160> 2
<210> 1
<211> 11713bp
<212> DNA
<213> pWNVrep-GFP-dCME
<400> 1
agtagttcgc ctgtgtgagc tgacaaactt agtagtgttt gtgaggatta acaacaatta 60
acacagtgcg agctgtttct tagcacgaag atctcgatgt ctaagaaacc aggagggccc 120
ggcaagagcc gggctgtcaa tatgctaaaa cgcggaatgg tgagcaaggg cgaggagctg 180
ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 240
agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 300
tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 360
gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 420
atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 480
acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 540
atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 600
cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 660
cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 720
atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 780
agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 840
gggatcactc tcggcatgga cgagctgtac aagctgctga actttgacct gctcaagttg 900
gctggagatg tggagtccaa ccctggaccc tttaagaagg aactagggac cttgaccagt 960
gctatcaatc ggcggagctc aaaacaaaag aaaagaggag gaaagaccgg aattgcagtc 1020
atgattggcc tgatcgccag cgtaggagca gttaccctct ctaacttcca agggaaggtg 1080
atgatgacgg taaatgctac tgacgtcaca gatgtcatca cgattccaac agctgctgga 1140
aagaacctat gcattgtcag agcaatggat gtgggataca tgtgcgatga tactatcact 1200
tatgaatgcc cagtgctgtc ggctggtaat gatccagaag acatcgactg ttggtgcaca 1260
aagtcagcag tctacgtcag gtatggaaga tgcaccaaga cacgccactc aagacgcagt 1320
cggaggtcac tgacagtgca gacacacgga gaaagcactc tagcgaacaa gaagggggct 1380
tggatggaca gcaccaaggc cacaaggtat ttggtaaaaa cagaatcatg gatcttgagg 1440
aaccctggat atgccctggt ggcagccgtc attggttgga tgcttgggag caacaccatg 1500
cagagagttg tgtttgtcgt gctattgctt ttggtggccc cagcttacag cttcaactgc 1560
cttggaatga gcaacagaga cttcttggaa ggagtgtctg gagcaacatg ggtggatttg 1620
gttctcgaag gcgacagctg cgtgactatc atgtctaagg acaagcctac catcgatgtg 1680
aagatgatga atatggaggc ggccaacctg gcagaggtcc gcagttattg ctatttggct 1740
accgtcagcg atctctccac caaagctgcg tgcccgacca tgggagaagc tcacaatgac 1800
aaacgtgctg acccagcttt tgtgtgcaga caaggagtgg tggacagggg ctggggcaac 1860
ggctgcggac tatttggcaa aggaagcatt gacacatgcg ccaaatttgc ctgctctacc 1920
aaggcaatag gaagaaccat cttgaaagag aatatcaagt acgaagtggc catttttgtc 1980
catggaccaa ctactgtgga gtcgcacgga aactactcca cacaggttgg agccactcag 2040
gcagggagat tcagcatcac tcctgcggcg ccttcataca cactaaagct tggagaatat 2100
ggagaggtga cagtggactg tgaaccacgg tcagggattg acaccaatgc atactacgtg 2160
atgactgttg gaacaaagac gttcttggtc catcgtgagt ggttcatgga cctcaacctc 2220
ccttggagca gtgctggaag tactgtgtgg aggaacagag agacgttaat ggagtttgag 2280
gaaccacacg ccacgaagca gtctgtgata gcattgggct cacaagaggg agctctgcat 2340
caagctttgg ctggagccat tcctgtggaa ttttcaagca acactgtcaa gttgacgtcg 2400
ggtcatttga agtgtagagt gaagatggaa aaattgcagt tgaagggaac aacctatggc 2460
gtctgttcaa aggctttcaa gtttcttggg actcccgcag acacaggtca cggcactgtg 2520
gtgttggaat tgcagtacac tggcacggat ggaccttgca aagttcctat ctcgtcagtg 2580
gcttcattga acgacctaac gccagtgggc agattggtca ctgtcaaccc ttttgtttca 2640
gtggccacgg ccaacgctaa ggtcctgatt gaattggaac caccctttgg agactcatac 2700
atagtggtgg gcagaggaga acaacagatc aatcaccatt ggcacaagtc tggaagcagc 2760
attggcaaag cctttacaac caccctcaaa ggagcgcaga gactagccgc tctaggagac 2820
acagcttggg actttggatc agttggaggg gtgttcacct cagttgggaa ggctgtccat 2880
caagtgttcg gaggagcatt ccgctcactg ttcggaggca tgtcctggat aacgcaagga 2940
ttgctggggg ctctcctgtt gtggatgggc atcaatgctc gtgataggtc catagctctc 3000
acgtttctcg cagttggagg agttctgctc ttcctctccg tgaacgtgca cgctgacact 3060
gggtgtgcca tagacatcag ccggcaagag ctgagatgtg gaagtggagt gttcatacac 3120
aatgatgtgg aggcttggat ggaccggtac aagtattacc ctgaaacgcc acaaggccta 3180
gccaagatca ttcagaaagc tcataaggaa ggagtgtgcg gtctacgatc agtttccaga 3240
ctggagcatc aaatgtggga agcagtgaag gacgagctga acactctttt gaaggagaat 3300
ggtgtggacc ttagtgtcgt ggttgagaaa caggagggaa tgtacaagtc agcacctaaa 3360
cgcctcaccg ccaccacgga aaaattggaa attggctgga aggcctgggg aaagagtatt 3420
ttatttgcac cagaactcgc caacaacacc tttgtggttg atggtccgga gaccaaggaa 3480
tgtccgactc agaatcgcgc ttggaatagc ttagaagtgg aggattttgg atttggtctc 3540
accagcactc ggatgttcct gaaggtcaga gagagcaaca caactgaatg tgactcgaag 3600
atcattggaa cggctgtcaa gaacaacttg gcgatccaca gtgacctgtc ctattggatt 3660
gaaagcaggc tcaatgatac gtggaagctt gaaagggcag ttctgggtga agtcaaatca 3720
tgtacgtggc ctgagacgca taccttgtgg ggcgatggaa tccttgagag tgacttgata 3780
ataccagtca cactggcggg accacgaagc aatcacaatc ggagacctgg gtacaagaca 3840
caaaaccagg gcccatggga cgaaggccgg gtagagattg acttcgatta ctgcccagga 3900
actacggtca ccctgagtga gagctgcgga caccgtggac ctgccactcg caccaccaca 3960
gagagcggaa agttgataac agattggtgc tgcaggagct gcaccttacc accactgcgc 4020
taccaaactg acagcggctg ttggtatggt atggagatca gaccacagag acatgatgaa 4080
aagaccctcg tgcagtcaca agtgaatgct tataatgctg atatgattga cccttttcag 4140
ttgggccttc tggtcgtgtt cttggccacc caggaggtcc ttcgcaagag gtggacagcc 4200
aagatcagca tgccagctat actgattgct ctgctagtcc tggtgtttgg gggcattact 4260
tacactgatg tgttacgcta tgtcatcttg gtgggggcag ctttcgcaga atctaattcg 4320
ggaggagacg tggtacactt ggcgctcatg gcgaccttca agatacaacc agtgtttatg 4380
gtggcatcgt ttctcaaagc gagatggacc aaccaggaga acattttgtt gatgttggcg 4440
gctgttttct ttcaaatggc ttatcacgat gcccgccaaa ttctgctctg ggagatccct 4500
gatgtgttga attcactggc ggtagcttgg atgatactga gagccataac attcacaacg 4560
acatcaaacg tggttgttcc gctgctagcc ctgctaacac ccgggctgag atgcttgaat 4620
ctggatgtgt acaggatact gctgttgatg gtcggaatag gcagcttgat cagggagaag 4680
aggagtgcag ctgcaaaaaa gaaaggagca agtctgctat gcttggctct agcctcaaca 4740
ggacttttca accccatgat ccttgctgct ggactgattg catgtgatcc caaccgtaaa 4800
cgcggatggc ccgcaactga agtgatgaca gctgtcggcc taatgtttgc catcgtcgga 4860
gggctggcag agcttgacat tgactccatg gccattccaa tgactatcgc ggggctcatg 4920
tttgctgctt tcgtgatttc tgggaaatca acagatatgt ggattgagag aacggcggac 4980
atttcctggg aaagtgatgc agaaattaca ggctcgagcg aaagagttga tgtgcggctt 5040
gatgatgatg gaaacttcca gctcatgaat gatccaggag caccttggaa gatatggatg 5100
ctcagaatgg tctgtctcgc gattagtgcg tacaccccct gggcaatctt gccctcagta 5160
gttggatttt ggataactct ccaatacaca aagagaggag gcgtgttgtg ggacactccc 5220
tcaccaaagg agtacaaaaa gggggacacg accaccggcg tctacaggat catgactcgt 5280
gggctgctcg gcagttatca agcaggagcg ggcgtgatgg ttgaaggtgt tttccacacc 5340
ctttggcata caacaaaagg agccgctttg atgagcggag agggccgcct ggacccatac 5400
tggggcagtg tcaaggagga tcgactttgt tacggaggac cctggaaatt gcagcacaag 5460
tggaacgggc aggatgaggt gcagatgatt gtggtggaac ctggcaagaa cgttaagaac 5520
gtccagacga aaccaggggt gttcaaaaca cctgaaggag aaatcggggc cgtgactttg 5580
gacttcccca ctggaacatc aggctcacca atagtggaca aaaacggtga tgtgattggg 5640
ctttatggca atggagtcat aatgcccaac ggctcataca taagcgcgat agtgcagggt 5700
gaaaggatgg atgagccaat cccagccgga ttcgaacctg agatgctgag gaaaaaacag 5760
atcactgtac tggatctcca tcccggcgcc ggtaaaacaa ggaggattct gccacagatc 5820
atcaaagagg ccataaacag aagactgaga acagccgtgc tagcgccaac cagggttgtg 5880
gctgctgaga tggctgaagc actgagagga ctgcccatcc ggtaccagac atccgcagtg 5940
cccagagaac ataatggaaa tgagattgtt gatgtcatgt gtcatgctac cctcacccac 6000
aggctgatgt ctcctcacag ggtgccgaac tacaacctgt tcgtgatgga tgaggctcat 6060
ttcaccgacc cagctagcat tgcagcaaga ggttacattt ccacaaaggt cgagctaggg 6120
gaggcggcgg caatattcat gacagccacc ccaccaggca cttcagatcc attcccagag 6180
tccaattcac caatttccga cttacagact gagatcccgg atcgagcttg gaactctgga 6240
tacgaatgga tcacagaata caccgggaag acggtttggt ttgtgcctag tgtcaagatg 6300
gggaatgaga ttgccctttg cctacaacgt gctggaaaga aagtagtcca attgaacaga 6360
aagtcgtacg agacggagta cccaaaatgt aagaacgatg attgggactt tgttatcaca 6420
acagacatat ctgaaatggg ggctaacttc aaggcgagca gggtgattga cagccggaag 6480
agtgtgaaac caaccatcat aacagaagga gaagggagag tgatcctggg agaaccatct 6540
gcagtgacag cagctagtgc cgcccagaga cgtggacgta tcggtagaaa tccgtcgcaa 6600
gttggtgatg agtactgtta tggggggcac acgaatgaag acgactcgaa cttcgcccat 6660
tggactgagg cacgaatcat gctggacaac atcaacatgc caaacggact gatcgctcaa 6720
ttctaccaac cagagcgtga gaaggtatat accatggatg gggaataccg gctcagagga 6780
gaagagagaa aaaactttct ggaactgttg aggactgcag atctgccagt ttggctggct 6840
tacaaggttg cagcggctgg agtgtcatac cacgaccgga ggtggtgctt tgatggtcct 6900
aggacaaaca caattttaga agacaacaac gaagtggaag tcatcacgaa gcttggtgaa 6960
aggaagattc tgaggccgcg ctggattgac gccagggtgt actcggatca ccaggcacta 7020
aaggcgttca aggacttcgc ctcgggaaaa cgttctcaga tagggctcat tgaggttctg 7080
ggaaagatgc ctgagcactt catggggaag acatgggaag cacttgacac catgtacgtt 7140
gtggccactg cagagaaagg aggaagagct cacagaatgg ccctggagga actgccagat 7200
gctcttcaga caattgcctt gattgcctta ttgagtgtga tgaccatggg agtattcttc 7260
ctcctcatgc agcggaaggg cattggaaag ataggtttgg gaggcgctgt cttgggagtc 7320
gcgacctttt tctgttggat ggctgaagtt ccaggaacga agatcgccgg aatgttgctg 7380
ctctcccttc tcttgatgat tgtgctaatt cctgagccag agaagcaacg ttcgcagaca 7440
gacaaccagc tagccgtgtt cctgatttgt gtcatgaccc ttgtgagcgc agtggcagcc 7500
aacgagatgg gttggctaga taagaccaag agtgacataa gcagtttgtt tgggcaaaga 7560
attgaggtca aggagaattt cagcatggga gagtttcttc tggacttgag gccggcaaca 7620
gcctggtcac tgtacgctgt gacaacagcg gtcctcactc cactgctaaa gcatttgatc 7680
acgtcagatt acatcaacac ctcattgacc tcaataaacg ttcaggcaag tgcactattc 7740
acactcgcgc gaggcttccc cttcgtcgat gttggagtgt cggctctcct gctagcagcc 7800
ggatgctggg gacaagtcac cctcaccgtt acggtaacag cggcaacact ccttttttgc 7860
cactatgcct acatggttcc cggttggcaa gctgaggcaa tgcgctcagc ccagcggcgg 7920
acagcggccg gaatcatgaa gaacgctgta gtggatggca tcgtggccac ggacgtccca 7980
gaattagagc gcaccacacc catcatgcag aagaaagttg gacagatcat gctgatcttg 8040
gtgtctctag ctgcagtagt agtgaacccg tctgtgaaga cagtacgaga agccggaatt 8100
ttgatcacgg ccgcagcggt gacgctttgg gagaatggag caagctctgt ttggaacgca 8160
acaactgcca tcggactctg ccacatcatg cgtgggggtt ggttgtcatg tctatccata 8220
acatggacac tcataaagaa catggaaaaa ccaggactaa aaagaggtgg ggcaaaagga 8280
cgcaccttgg gagaggtttg gaaagaaaga ctcaaccaga tgacaaaaga agagttcact 8340
aggtaccgca aagaggccat catcgaagtc gatcgctcag cggcaaaaca cgccaggaaa 8400
gaaggcaatg tcactggagg gcatccagtc tctaggggca cagcaaaact gagatggctg 8460
gtcgaacgga ggtttctcga accggtcgga aaagtgattg accttggatg tggaagaggc 8520
ggttggtgtt actatatggc aacccaaaaa agagtccaag aagtcagagg gtacacaaag 8580
ggcggtcccg gacatgaaga gccccaacta gtgcaaagtt atggatggaa cattgtcacc 8640
atgaagagtg gagtggatgt gttctacaga ccttctgagt gttgtgacac cctcctttgt 8700
gacatcggag agtcctcgtc aagtgctgag gttgaagagc ataggacgat tcgggtcctt 8760
gaaatggttg aggactggct gcaccgaggg ccaagggaat tttgcgtgaa ggtgctctgc 8820
ccctacatgc cgaaagtcat agagaagatg gagctgctcc aacgccggta tgggggggga 8880
ctggtcagaa acccactctc acggaattcc acgcacgaga tgtattgggt gagtcgagct 8940
tcaggcaatg tggtacattc agtgaatatg accagccagg tgctcctagg aagaatggaa 9000
aaaaggacct ggaagggacc ccaatacgag gaagatgtaa acttgggaag tggaaccagg 9060
gcggtgggaa aacccctgct caactcagac accagtaaaa tcaagaacag gattgaacga 9120
ctcaggcgtg agtacagttc gacgtggcac cacgatgaga accacccata tagaacctgg 9180
aactatcacg gcagttatga tgtgaagccc acaggctccg ccagttcgct ggtcaatgga 9240
gtggtcaggc tcctctcaaa accatgggac accatcacga atgttaccac catggccatg 9300
actgacacta ctcccttcgg gcagcagcga gtgttcaaag agaaggtgga cacgaaagct 9360
cctgaaccgc cagaaggagt gaagtacgtg ctcaacgaga ccaccaactg gttgtgggcg 9420
tttttggcca gagaaaaacg tcccagaatg tgctctcgag aggaattcat aagaaaggtc 9480
aacagcaatg cagctttggg tgccatgttt gaagagcaga atcaatggag gagcgccaga 9540
gaagcagttg aagatccaaa attttgggag atggtggatg aggagcgcga ggcacatctg 9600
cggggggaat gtcacacttg catttacaac atgatgggaa agagagagaa aaaacccgga 9660
gagttcggaa aggccaaggg aagcagagcc atttggttca tgtggctcgg agctcgcttt 9720
ctggagttcg aggctctggg ttttctcaat gaagaccact ggcttggaag aaagaactca 9780
ggaggaggtg tcgagggctt gggcctccaa aaactgggtt acatcctgcg tgaagttggc 9840
acccggcctg ggggcaagat ctatgctgat gacacagctg gctgggacac ccgcatcacg 9900
agagctgact tggaaaatga agctaaggtg cttgagctgc ttgatgggga acatcggcgt 9960
cttgccaggg ccatcattga gctcacctat cgtcacaaag ttgtgaaagt gatgcgcccg 10020
gctgctgatg gaagaaccgt catggatgtt atctccagag aagatcagag ggggagtgga 10080
caagttgtca cctacgccct aaacactttc accaacctgg ccgtccagct ggtgaggatg 10140
atggaagggg aaggagtgat tggcccagat gatgtggaga aactcacaaa agggaaagga 10200
cccaaagtca ggacctggct gtttgagaat ggggaagaaa gactcagccg catggctgtc 10260
agtggagatg actgtgtggt aaagcccctg gacgatcgct ttgccacctc gctccacttc 10320
ctcaatgcta tgtcaaaggt tcgcaaagac atccaagagt ggaaaccgtc aactggatgg 10380
tatgattggc agcaggttcc attttgctca aaccatttca ctgaattgat catgaaagat 10440
ggaagaacac tggtggttcc atgccgagga caggatgaat tggtaggcag agctcgcata 10500
tctccagggg ccggatggaa cgtccgcgac actgcttgtc tggctaagtc ttatgcccag 10560
atgtggctgc ttctgtactt ccacagaaga gacctgcggc tcatggccaa cgccatttgc 10620
tccgctgtcc ctgtgaattg ggtccctacc ggaagaacca cgtggtccat ccatgcagga 10680
ggagagtgga tgacaacaga ggacatgttg gaggtctgga accgtgtttg gatagaggag 10740
aatgaatgga tggaagacaa aaccccagtg gagaaatgga gtgacgtccc atattcagga 10800
aaacgagagg acatctggtg tggcagcctg attggcacaa gagcccgagc cacgtgggca 10860
gaaaacatcc aggtggctat caaccaagtc agagcaatca tcggagatga gaagtatgtg 10920
gattacatga gttcactaaa gagatatgaa gacacaactt tggttgagga cacagtactg 10980
tagatattta atcaattgta aatagacaat ataagtatgc ataaaagtgt agttttatag 11040
tagtatttag tggtgttagt gtaaatagtt aagaaaattt tgaggagaaa gtcaggccgg 11100
gaagttcccg ccaccggaag ttgagtagac ggtgctgcct gcgactcaac cccaggagga 11160
ctgggtgaac aaagccgcga agtgatccat gtaagccctc agaaccgtct cggaaggagg 11220
accccacatg ttgtaacttc aaagcccaat gtcagaccac gctacggcgt gctactctgc 11280
ggagagtgca gtctgcgata gtgccccagg aggactgggt taacaaaggc aaaccaacgc 11340
cccacgcggc cctagccccg gtaatggtgt taaccagggc gaaaggacta gaggttagag 11400
gagaccccgc ggtttaaagt gcacggccca gcctggctga agctgtaggt caggggaagg 11460
actagaggtt agtggagacc ccgtgccaca aaacaccaca acaaaacagc atattgacac 11520
ctgggataga ctaggagatc ttctgctctg cacaaccagc cacacggcac agtgcgccga 11580
caatggtggc tggtggtgcg agaacacagg atctgggtcg gcatggcatc tccacctcct 11640
cgcggtccga cctgggcatc cgaaggagga cgcacgtcca ctcggatggc taagggaggg 11700
cggcggccgc tat 11713
<210> 2
<211> 2376bp
<212> DNA
<213> opti-WNV-CME
<400> 2
atgtccaaga agcccggcgg ccccggcaag tcccgcgccg tgaacatgct gaagcgcggc 60
atgccccgcg tgctgtccct gatcggcctg aagcgcgcca tgctgtccct gatcgacggc 120
aagggcccca tccgcttcgt gctggccctg ctggccttct tccgcttcac cgccatcgcc 180
cccacccgcg ccgtgctgga ccgctggcgc ggcgtgaaca agcagaccgc catgaagcac 240
ctgctgtcct tcaagaagga gctgggcacc ctgacctccg ccatcaaccg ccgctcctcc 300
aagcagaaga agcgcggcgg caagaccggc atcgccgtga tgatcggcct gatcgcctcc 360
gtgggcgccg tgaccctgtc caacttccag ggcaaggtga tgatgaccgt gaacgccacc 420
gacgtgaccg acgtgatcac catccccacc gccgccggca agaacctgtg catcgtgcgc 480
gccatggacg tgggctacat gtgcgacgac accatcacct acgagtgccc cgtgctgtcc 540
gccggcaacg accccgagga catcgactgc tggtgcacca agtccgccgt gtacgtgcgc 600
tacggccgct gcaccaagac ccgccactcc cgccgctccc gccgctccct gaccgtgcag 660
acccacggcg agtccaccct ggccaacaag aagggcgcct ggatggacag caccaaggcc 720
acccgctacc tggtgaagac cgagagctgg atcctgcgca accccggcta cgccctggtg 780
gccgccgtga tcggctggat gctgggcagc aacaccatgc agcgcgtggt gttcgtggtg 840
ctgctgctgc tggtggcccc cgcctacagc ttcaactgcc tgggcatgag caaccgcgac 900
ttcctggagg gcgtgagcgg cgccacctgg gtggacctgg tgctggaggg cgacagctgc 960
gtgaccatca tgagcaagga caagcccacc atcgacgtga agatgatgaa catggaggcc 1020
gccaacctgg ccgaggtgcg cagctactgc tacctggcca ccgtgagcga cctgagcacc 1080
aaggccgcct gccccaccat gggcgaggcc cacaacgaca agcgcgccga ccccgccttc 1140
gtgtgccgcc agggcgtggt ggaccgcggc tggggcaacg gctgcggcct gttcggcaag 1200
ggcagcatcg acacctgcgc caagttcgcc tgcagcacca aggccatcgg ccgcaccatc 1260
ctgaaggaga acatcaagta cgaggtggcc atcttcgtgc acggccccac caccgtggag 1320
agccacggca actacagcac ccaggtgggc gccacccagg ccggccgcct gagcatcacc 1380
cccgccgccc ccagctacac cctgaagctg ggcgagtacg gcgaggtgac cgtggactgc 1440
gagccccgca gcggcatcga caccaacgcc tactacgtga tgaccgtggg caccaagacc 1500
ttcctggtgc accgcgagtg gttcatggac ctgaacctgc cctggagcag cgccggcagc 1560
accgtgtggc gcaaccgcga gaccctgatg gagttcgagg agccccacgc caccaagcag 1620
agcgtgatcg ccctgggcag ccaggagggc gccctgcacc aggccctggc cggcgccatc 1680
cccgtggagt tcagcagcaa caccgtgaag ctgaccagcg gccacctgaa gtgccgcgtg 1740
aagatggaga agctgcagct gaagggcacc acctacggcg tgtgcagcaa ggccttcaag 1800
ttcctgggca cccccgccga caccggccac ggcaccgtgg tgctggagct gcagtacacc 1860
ggcaccgacg gcccctgcaa ggtgcccatc agcagcgtgg ccagcctgaa cgacctgacc 1920
cccgtgggcc gcctggtgac cgtgaacccc ttcgtgagcg tggccaccgc caacgccaag 1980
gtgctgatcg agctggagcc ccccttcggc gacagctaca tcgtggtggg ccgcggcgag 2040
cagcagatca accaccactg gcacaagagc ggcagcagca tcggcaaggc cttcaccacc 2100
accctgaagg gcgcccagcg cctggccgcc ctgggcgaca ccgcctggga cttcggcagc 2160
gtgggcggcg tgttcaccag cgtgggcaag gccgtgcacc aggtgttcgg cggcgccttc 2220
cgcagcctgt tcggcggcat gagctggatc acccagggcc tgctgggcgc cctgctgctg 2280
tggatgggca tcaacgcccg cgaccgcagc atcgccctga ccttcctggc cgtgggcggc 2340
gtgctgctgt tcctgagcgt gaacgtgcac gcctga 2376

Claims (10)

1. the restricted duplication west nile virus system of a kind of expressing green fluorescent protein, it is characterised in that including：
(1) the restricted replicon plasmid of west nile virus：
The restricted replicon plasmid of described west nile virus is carried by the way that wnv cdna group is cloned into eukaryotic expression The promoter downstream of body, and after the structural proteins C-prM-E sequences in genome are replaced with into green fluorescence protein gene simultaneously Obtain；
(2) recombinant cell lines of stabilization expression west nile virus C-prM-E albumen
Described recombinant cell lines are turned by by the carrier for expression of eukaryon containing west nile virus C-prM-E protein-encoding genes Obtained after dye mammalian cell.
2. the system as claimed in claim 1, it is characterised in that the structure of the restricted replicon plasmid of described west nile virus In, the carrier for expression of eukaryon for being used is pCI-neo.
3. the system as claimed in claim 1, it is characterised in that the nucleosides of the restricted replicon plasmid of described west nile virus Acid sequence is as shown in SEQ ID NO.1.
4. the system as claimed in claim 1, it is characterised in that the weight of described stabilization expression west nile virus C-prM-E albumen Group cell line is to move the eukaryotic expression vector transfection lactation containing the west nile virus C-prM-E protein-encoding genes after optimization Obtained after thing cell, it is preferred that described carrier for expression of eukaryon is pCAG-neo, described mammalian cell is BHK-21 Cell.
5. system as claimed in claim 4, it is characterised in that the west nile virus C-prM-E protein expressions after described optimization The nucleotide sequence of gene is as shown in SEQ ID NO.2.
6. the system as claimed in claim 1, it is characterised in that the weight of described stabilization expression west nile virus C-prM-E albumen Group cell line builds obtain by the following method：
(1) the C-prM-E protein-encoding genes after artificial synthesized optimization, its nucleotide sequence optimizes as shown in SEQ ID NO.2 Sequence designations afterwards are opti-WNV-CME, carry out the artificial synthesized rear sequence after PCR method expands optimization, and in PCR 5 ' ends of product introduce the restriction enzyme sites of Sac I, and 3 ' ends introduce the restriction enzyme sites of Xho I, PCR primer and same pair of enzymes after double digestion The pCAG-neo carriers cut are connected, and after connection product conversion DH5 а, select recombinant clone, through digestion and sequencing identification, obtain sun Property clone after plasmid is largely prepared with kit, be named as pCAG-WNV-CME, frozen in -20 DEG C after being linearized with SspI It is standby；
(2) the BHK-21 cell dissociations for selecting growth conditions good are passaged in 24 orifice plates, when BHK-21 cells are long full to 90%, With the recombinant plasmid pCAG-WNV-CME transfectional cells after linearisation, the stabilization expression west nile virus C- of stabilization expression is obtained The recombinant cell lines of prM-E albumen, are named as BWNV-CME.
7. the system described in any one of claim 1-6 is obtaining the restricted duplication west nile virus of expressing green fluorescent protein In purposes.
8. it is a kind of obtain expressing green fluorescent protein restricted duplication west nile virus method, it is characterised in that including following Step：
It is growth medium, 1 with the DMEM culture mediums containing 10% (v/v) FBS：4 Secondary Culture any one of claim 1-6 institutes The recombinant cell lines of the stabilization expression west nile virus C-prM-E albumen stated, by the recombinant cell of exponential phase with 5 × 105/ Ml is inoculated with 6 porocyte culture plates, incubated overnight, by the restricted replicon of west nile virus described in claim any one of 1-6 Plasmid is made DNA dilutions in adding Opti-MEM, and every 3 μ g plasmids add 200 μ l opti-MEM, add 20 μ l X-treme HP transfection reagents, are stored at room temperature 15min, DNA transfection reagents complexes drop-wise is added and contains stabilization expression west nile virus C- In the nutrient solution of the recombinant cell of prM-E albumen, fluorescence is observed after 72h.Collect supernatant, be stored in after centrifugal filtration -80 DEG C it is standby With.
9. the restricted duplication west nile virus of the expressing green fluorescent protein for obtaining in accordance with the method for claim 8.
10. the restricted duplication west nile virus of the expressing green fluorescent protein described in claim 9 is preparing west nile virus Purposes in neutralizing antibody detection reagent and the anti-west nile virus medicine of screening.
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CN113917139A (en) * 2021-10-18 2022-01-11 扬州大学 Detection method of serum 4 type avian adenovirus neutralizing antibody based on recombinant fluorescent virus

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