CN106754982B

CN106754982B - Limited replication West Nile virus system for expressing green fluorescent protein and application thereof

Info

Publication number: CN106754982B
Application number: CN201611154016.3A
Authority: CN
Inventors: 华荣虹; 步志高
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2016-12-14
Filing date: 2016-12-14
Publication date: 2021-03-09
Anticipated expiration: 2036-12-14
Also published as: CN106754982A

Abstract

The invention discloses a restricted replication West Nile virus system for expressing green fluorescent protein and application thereof. The restriction replication West Nile virus system for expressing the green fluorescent protein comprises a West Nile virus restriction replicon plasmid and a recombinant cell line for stably expressing the C-prM-E protein of the West Nile virus. The restrictive replication West Nile virus (delta WNV) obtained by using the system has the characteristics similar to WNV whole virus in a BWNV-CME cell line, can express GFP and has a visualization effect, more importantly, the virus can only replicate in the system, and the progeny virus cannot be generated when other cells are infected, so the system has extremely high safety and can be used for WNV neutralizing antibody detection and antiviral drug screening. The method improves the safety and the simplicity of the WNV related operation, does not depend on the high-grade biological safety conditions of BSL-3, and provides a good technical means for WNV vaccine evaluation, antiviral drug screening and virus pathogenic mechanism research.

Description

Limited replication West Nile virus system for expressing green fluorescent protein and application thereof

Technical Field

The invention relates to a virus replication system and application thereof, in particular to a restricted replication West Nile virus system for expressing green fluorescent protein and application thereof, belonging to the technical field of biology.

Background

West Nile Virus (WNV) belongs to the Flaviviridae family, the flavivirus genus, is an important zoonosis Virus, and can cause serious nervous system diseases of human. WNV virus particles are about 40 nm-60 nm in diameter, have an envelope, and the virus nucleocapsid in the envelope is in icosahedral symmetry and about 30nm in diameter. The WNV genome is a single-stranded positive sense RNA, approximately 11kb in length. The genome consists of a non-coding region (UTR) 96nt long at the 5 'end and 337-649nt long at the 3' end, and a single Open Reading Frame (ORF) of about 10.3 kb. The ORF starts at position 97 at the 5 'end and ends at position 10398 at the 3' end, encodes 1 polyprotein precursor of 3433 amino acids in length, which is cleaved by host and viral proteases to yield 3 structural proteins: c protein (capsid/core protein), prM/M (membrane precursor protein/membrane protein) and E protein (envelope glycoprotein) and 7 non-structural proteins.

West Nile virus disease is infectious disease caused by West Nile virus, and is a zoonosis. The disease is an exotic disease for China, and is not reported in China at present, but the climate and geographic environment of China are complex, mosquitoes are various and have propagation conditions, and peripheral countries such as Russia, India and the like have already prevailed the disease, and with increasingly frequent international communication, the possibility of WNV being introduced into China is not negligible, for example, the WNV is recently reported to be separated from mosquitoes in Xinjiang. Therefore, the risk of WNV epidemic outbreak is very high in China, and no vaccine or effective drug is available at present for preventing and treating the infection of West Nile virus, so that corresponding prevention and control measures are necessary to be researched. However, WNV is a zoonotic pathogen, and the operation of live viruses for relevant researches such as vaccines and the like needs to be carried out in a high-grade biosafety laboratory, which hinders the research of relevant vaccines and drugs to a certain extent.

In order to promote the development of WNV vaccines and screen new antiviral drugs, the invention constructs a whole virus genome with all structural proteins deleted, inserts a WNV replicon plasmid pWNVrepGFP-dCME of a GFP reporter gene in the structural protein region, and constructs a cell line for stably expressing C-prM-E protein; the replicon plasmid, recombinant cell line and packaged Δ WNV virus constitute a restricted replication west nile virus system. The replication-defective west nile virus system of the present invention can provide a safe, simple and convenient method for epidemiological investigation, vaccine evaluation, antiviral drug screening and research of replication and pathogenesis of west nile virus.

Disclosure of Invention

The invention aims to provide a West Nile Virus (WNV) preparation system which expresses green fluorescent protein and only has infectivity and replication capacity in the system, so as to be applied to a safe and visual WNV neutralizing antibody detection system and a drug screening method which are similar to whole virus infection and replication.

In order to achieve the purpose, the invention adopts the following technical means:

the invention relates to a restriction replication West Nile virus system for expressing green fluorescent protein, which comprises:

(1) west nile virus restriction replicon plasmid:

the west nile virus restriction replicon plasmid is obtained by cloning a west nile virus genome to the downstream of a promoter of a eukaryotic expression vector and simultaneously replacing a structural protein C-prM-E sequence in the genome with a green fluorescent protein gene;

(2) recombinant cell line for stably expressing West Nile virus C-prM-E protein

The recombinant cell line is obtained by transfecting a eukaryotic expression vector containing a west nile virus C-prM-E protein expression gene into a mammalian cell.

In the present invention, preferably, in the construction of the west nile virus restriction replicon plasmid, the eukaryotic expression vector used is pCI-neo.

In the present invention, preferably, the nucleotide sequence of the west nile virus restriction replicon plasmid is shown in SEQ ID No. 1.

In the invention, preferably, the recombinant cell line for stably expressing the west nile virus C-prM-E protein is obtained by transfecting a eukaryotic expression vector containing an optimized expression gene of the west nile virus C-prM-E protein into a mammalian cell, more preferably, the eukaryotic expression vector is pCAG-neo, and the mammalian cell is BHK-21 cell.

In the invention, preferably, the nucleotide sequence of the optimized West Nile virus C-prM-E protein expression gene is shown as SEQ ID NO. 2.

In the present invention, preferably, the recombinant cell line stably expressing C-prM-E protein of west nile virus is constructed by the following method:

(1) artificially synthesizing an optimized C-prM-E protein expression gene, wherein the nucleotide sequence of the optimized C-prM-E protein expression gene is shown as SEQ ID NO.2, the optimized sequence is named as opti-WNV-CME, the optimized sequence is amplified by a PCR method after artificial synthesis, a SacI enzyme cutting site is introduced into the 5 'end of a PCR product, an XhoI enzyme cutting site is introduced into the 3' end of the PCR product, the PCR product after double enzyme cutting is connected with a pCAG-neo vector with the same double enzyme cutting, after a connecting product is transformed into DH5 a, recombinant clones are selected, enzyme cutting and sequencing identification are carried out, a large amount of plasmids are prepared by using a kit after positive cloning is obtained, the plasmids are named as pCAG-WNV-CME, and are frozen at-20 ℃ for later use after linearization is carried out by SspI;

(2) BHK-21 cells with good growth state are selected for digestion and passage to a 24-well plate, and when the BHK-21 cells grow to 90% full, the linearized recombinant plasmid pCAG-WNV-CME is used for transfecting the cells to obtain a stably expressed recombinant cell line which stably expresses West Nile virus C-prM-E protein and is named as BWNV-CME.

Furthermore, the invention also provides the application of the system in obtaining the restrictive replication West Nile virus expressing the green fluorescent protein.

Furthermore, the invention also provides a method for obtaining the limited replication West Nile virus expressing the green fluorescent protein, which comprises the following steps:

DMEM medium containing 10% (v/v) FBS was used as growth medium, 1: 4 subculturing the recombinant cell line stably expressing the West Nile virus C-prM-E protein, and culturing the recombinant cell line in the logarithmic growth phase at 5 x 10⁵And (3) inoculating a 6-well cell culture plate per ml, culturing overnight, adding the west nile virus restriction replicon plasmid into Opti-MEM to prepare a DNA diluent, adding 200 mu l of Opti-MEM into every 3 mu g of plasmid, adding 20 mu l X-treme HP transfection reagent, standing at room temperature for 15min, dropwise adding the DNA transfection reagent compound into a culture solution containing recombinant cells stably expressing the west nile virus C-prM-E protein, and observing fluorescence after 72 h. Collecting supernatant, centrifuging, filtering, and storing at-80 deg.C.

Still further, the invention also provides the restrictive replication West Nile virus expressing the green fluorescent protein obtained by the method and application of the restrictive replication West Nile virus expressing the green fluorescent protein in preparing a West Nile virus neutralizing antibody detection reagent and screening anti-West Nile virus medicines.

Compared with the prior art, the invention has the beneficial effects that:

the WNV replication-defective virus developed by the invention utilizes a genome replicon which lacks a structural protein gene to transfect a cell line BWNV-CME which stably expresses WNV C-prM-E protein for delta WNV virus particle packaging, and the structural protein gene in the virus genome is replaced by a gene which expresses green fluorescent protein, so that in the replication process of the virus replicon, the virus cannot continue to generate progeny virus because the virus cannot express the structural protein by itself, but can express a large amount of GFP protein, and has strong indication effect on infected cells. But can only integrate into a complete virion when reassembled with exogenously supplied WNV C (capsid/core), prM/M (membrane precursor/membrane) and E proteins (envelope glycoprotein). In a replication-defective virus neutralization assay, WNV replication-defective viruses can be effectively neutralized by neutralizing antibodies and lost the infectious effect on host cells. Since WNV replication-defective viruses contain a reporter gene and are capable of expressing green fluorescent protein, cells infected with the virus can be visually observed using a fluorescence microscope. When WNV replication-defective virus is used for replacing wild type WNV to detect the neutralizing antibody, a certain amount of replication-defective virus is incubated with a sample to be detected for 1h, the sample is directly infected with susceptible cells BHK-21, and the cells are placed under a fluorescence microscope to observe fluorescence density after 48h to evaluate the neutralizing antibody in the sample to be detected. Meanwhile, since the WNV replication-defective virus is replication-defective, the amount of the added virus is a fixed value, so that the virus cannot be proliferated due to infection of susceptible cells, and qualitative and quantitative detection in antibody detection is facilitated.

Drawings

FIG. 1 is a schematic of the construction of the WNV replicon;

FIG. 2 is the restriction enzyme identification of recombinant plasmid pWNVrep-GFP-dCME;

1: DL 15000DNA Marker; 2: before double enzyme digestion; 3: after double enzyme digestion.

FIG. 3 is the restriction enzyme identification of recombinant plasmid pCAG-WNV-CME;

1: DL 15000DNA Marker; 2. 3: before double enzyme digestion; 4. 5: after double enzyme digestion.

FIG. 4 shows the detection of the recombinant cell line E protein specific monoclonal antibody IFA;

FIG. 5 shows fluorescent and plaque formation by Δ WNV infection of BWNV-CME cells;

FIG. 6 is a graph showing the replication kinetics of Δ WNV virus;

figure 7 is a profile of Δ WNV virus infection;

figure 8 is a WNV-restricted replication virus system used to detect anti-WNV activity of drugs.

Detailed Description

The invention will be further described with reference to specific embodiments and drawings, the advantages and features of which will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 construction of a restrictive replication West Nile Virus System expressing Green fluorescent protein

1 materials and methods

1.1 vectors, cells and Primary reagents

Expression vector plasmids pCAG-neo plasmid (Li paranan et al, China preventive veterinary science bulletin, 2013,35(3): 189-; BHK-21 cells were maintained in this laboratory and the W-92 cell line was constructed in this laboratory. DMEM, premium Fetal Bovine Serum (FBS) and Opti-MEM were purchased from Gibco; x-treme HP transfection reagent was purchased from Roche; FITC-labeled goat anti-mouse IgG was purchased from China fir Jinqiao; infrared fluorescence labeled anti-mouse IgG antibodies were purchased from the public laboratory of the harbin veterinary institute; e protein and NS1 protein monoclonal antibody are prepared in the laboratory; WNV mouse positive sera were stored in this laboratory.

1.2 replicon plasmid pWNVrepGFP-dCME construction

A full-length splicing primer is designed by referring to a WNV virus genome sequence (NY99 strain, Genbank serial number DQ211652NY99), a PAS (PCR-based Accurate Synthesis) based method is adopted, protective bases are respectively designed at two ends of the primer, genome fragments are artificially synthesized, a WNV replicon plasmid is constructed by connecting a cloning site Xho I and an Xba I to the downstream of a CMV promoter of a eukaryotic expression plasmid pCI-neo, a structural protein C-prM-E sequence in the WNV replicon genome is replaced by a Green Fluorescent Protein (GFP) gene, and the obtained recombinant plasmid is named as pWNVrepfGFP-dCME. The major elements of the replicon are shown in FIG. 1. The nucleotide sequence of pWNVrepGFP-dCME is shown in SEQ ID NO. 1.

1.3 construction of plasmid pCAG-WNV-CME

Firstly, codon optimization is carried out on a WNV C-prM-E protein gene by using a bioinformatics method, the optimized C-prM-E protein gene sequence is shown as SEQ ID NO.2, the optimized sequence is named as opti-WNV-CME, the optimized sequence is amplified by a PCR method after artificial synthesis, Sac I enzyme cutting site is introduced into the 5 'end of a PCR product, Xho I enzyme cutting site is introduced into the 3' end of the PCR product, the double-enzyme-cutting PCR product is connected with a pCAG-neo vector which is subjected to double enzyme cutting in the same way, after a connecting product is transformed into DH5 a, recombinant clones are selected, enzyme cutting and sequencing identification are carried out, a large amount of plasmids are prepared by using a kit after positive clones are obtained, the plasmids are named as pCAG-WNV-CME, and are frozen at-20 ℃ for later use after linearization by SspI.

1.4 construction and screening identification of recombinant cell lines

Selecting BHK-21 cells with good growth state, digesting and passaging to a 24-well plate, and when BHK-21 cells grow to 90% full, carrying out the steps of

The HD Transfection Reagent kit operation instruction with linear recombinant plasmid pCAG-WNV-CME transfected cells. After transfection for 48h, G418 (1000. mu.g/mL) containing selective medium was added for pressure culture, and after 4d, the cells were trypsinized, passaged in a 96-well plate by limiting dilution, and cultured again, and after 5d, the number of clones per well was observed under an inverted microscope. The wells containing 1 cell colony (i.e., 1 cell pellet) were selected and expanded in 24-well plates, 6-well plates, and cell culture flasks in sequence, and the cells were subjected to IFA identification using a WNV E protein-specific monoclonal antibody, and cell clones with strong IFA signals were selected. The cell clone stably expressing the C-prM-E protein is obtained and named as BWNV-CME.

1.5 packaging and preservation of the Δ WNV Virus

DMEM medium containing 10% FBS was used as growth medium, 1: 4 subculturing BWNV-CME cells, and culturing the BWNV-CME cells in logarithmic growth phase at 5 × 10⁵Inoculation of 6 wells per mlCell culture plates, overnight culture, plasmid pWNVrep-GFP-dCME was added to Opti-MEM to make DNA dilutions, 200. mu.l Opti-MEM was required for every 3. mu.g plasmid, 20. mu. l X-treme HP transfection reagent was added, and the mixture was allowed to stand at room temperature for 15 min. The DNA transfection reagent complex was added dropwise to the culture medium containing BWNV-CME cells, and fluorescence was observed after 72 hours. Collecting supernatant, centrifuging, filtering, and storing at-80 deg.C.

1.6. DELTA. WNV Virus PFU assay

15ul of virus supernatant packaged by harvested transfected cells was taken, 135ul of 5% FBS DMEM was added, and a series of 10-fold dilutions were performed, with 100ul per six well plate, 37 ℃, 5% CO in each well₂After the culture in the incubator is carried out for 10h, 3% methyl cellulose with the same volume of the culture medium is added into each hole, and the expression of the green fluorescent protein is observed after the culture is carried out for 48 h. Or 6 days after culture, staining with crystal violet to count virus plaques.

1.7 packaging Virus replication kinetics

In order to observe the growth kinetics of the transfected cell packaging virus, the prepared packaging virus is analyzed for infection and replication in BWNV-CME cells, and supernatants are harvested and titer-measured at 24h, 48h, 72h, 96h and 120h after infection.

2 results

2.1 plasmid construction of pWNVrepgFP-dCME

The plasmid pWNVrepGFP-dCME construction is schematically shown in FIG. 1. The plasmid is based on wnv virus genome sequence, CMV and T7 promoters are arranged at 5 ' end, and a hepatitis D nuclease sequence (HDr) is added at 3 ' end to ensure the accuracy of the transcribed 3 ' end sequence. The plasmid is identified by NotI and SalI double enzyme digestion results as shown in figure 2, the sizes of the enzyme digestion fragments are about 8000bp and 5000bp, the sizes are consistent with expected results, and the insertion sequence is confirmed to be correct by forward and reverse sequencing.

2.2 construction of plasmid pCAG-WNV-CME

After the double-restriction enzyme PCR product is connected with a double-restriction enzyme pCAG-neo vector, transformation and plate coating are carried out, monoclonal bacteria are picked up, the plasmid is extracted, double-restriction enzyme digestion verification is carried out by Sac I and Xho I, and the detection is carried out by 1% agarose gel electrophoresis, so that the target fragment is about 2400bp and is consistent with the expectation. And the sequence determination showed that the sequence was consistent with the expected (FIG. 3).

2.3 screening and identification of recombinant cell lines

The linearized plasmid was transfected into BHK-21 cells, which were then screened with G418 and cloned for IFA identification with monoclonal antibodies (FIG. 4). The result obtains a cell line expressing the C-prM-E protein and is named as BWNV-CME. The cell line is subjected to subculture and detection after being cloned and purified again, and shows that the cell line is stable in heredity and can still stably express the E protein after being continuously passaged for 20 generations. The cultured growth characteristics of the recombinant cell line are similar to those of the parent cell BHK-21 cell, and the ratio of 1: and 5, subculturing, wherein the culture medium is DMEM (DMEM) containing 10% fetal calf serum, and subculturing can be performed every other day.

2.4. DELTA. WNV Virus PFU assay

Harvesting 100 mul of supernatant of replication defective virus delta WNV, infecting BWNV-CME cells through a series of 10-fold dilutions, observing the number of GFP-expressing cells in each hole under an inverted microscope after 48 hours, counting, and measuring the virus titer to be 1 × 10^5.5PFU/ml. The fluorescent and plaque formation of Δ WNV infected BWNV-CME cells is shown in FIG. 5.

2.5. DELTA. WNV Virus replication kinetics

After transfection, supernatant is respectively collected at 24h, 48h, 72h, 96h and 120h, the titer is measured, a kinetic curve graph 6 is drawn, the result shows that the titer of the virus is generally in an ascending trend in 24-72 h, the virus is in a plateau period from 72h to 96h, and the virus is slightly reduced after 96h, the result shows that the constructed replication-defective virus has good replication activity, and the optimal time for virus recovery is determined to be between 72h and 96h after transfection.

2.6 Δ WNV has restrictive replication characteristics

Cell culture supernatants transfected with both pWNVrep-GFP-dCME and pCAG-WNV-CME were able to express GFP in first infected BHK-21 cells, whereas cell culture supernatants transfected with either pWNVrep-GFP-dCME or pCAG-WNV-CME plasmid alone were not able to infect BHK-21 cells, with no GFP expression. Plasmid pWNVrep-GFP-dCME transfection BWNV-CME cell line generated virus delta WNV has infectivity on cell line BWNV-CME, can generate progeny virus, generate fluorescent spots and form virus plaques, and maintains virus infection characteristics when the BWNV-CME cell line is infected in the second round and the third round. While the supernatant from infected BHK-21 cells did not have the second round of infectivity, and when the supernatant was re-inoculated into BHK-21 cells, the cells could not express GFP. From this we can conclude that the replication-restricted WNV viruses of the present invention can only have properties similar to whole virus replication in a specific cell line within the system, while other sensitive cells, such as BHK-21 cells, have only a single round of infectivity (FIG. 7).

Example 2 use of a restrictive replication West Nile Virus System expressing Green fluorescent protein

1 method

1.1 restriction replication Virus System for WNV neutralizing antibody detection

Will be 4X 10⁵the/mL BWNV-CME cells are inoculated on a 24-well cell culture plate, cultured overnight at the temperature of 37 ℃ and 5 percent CO2, and then grown into a monolayer; WNV positive and negative sera were inactivated from 1: after the 10 starts gradient dilution, mixing with equal volume of delta WNV, arranging a plurality of holes, and placing in a 5% CO2 incubator for 1h at 37 ℃; then 100 mul of mixed solution of delta WNV virus antibody is inoculated to a monolayer BWNV-CME cell, cultured for 6h at 37 ℃ with 5% CO2, then added with 3% methyl cellulose DMEM solution, cultured for 3 days and then observed for green fluorescence expression. Plaque counts were performed after 6 days. The neutralizing antibody titer of each serum to be tested was calculated by taking the 50% reduction of the fluorescent plaque or plaque as the endpoint.

1.2 restriction replication Virus System for WNV antiviral drug screening

Will be 4X 10⁵cell/mL BWNV-CME cells were seeded in 24-well cell culture plates at 500. mu.L/well, 37 ℃ with 5% CO₂Culturing overnight to allow the cells to grow into a monolayer; preparing medicines such as Tannic acid (Tannic acid) and 6-azauridine (6-azauridine) into 10mM stock solution with DMSO, diluting with DMEM solution to 20 μ M, using 1% DMSO as control, adding each medicine and control into cells, acting for 10 hr, adding 100TCID50/0.1ml delta WNV virus, and treating at 37 deg.C and 5% CO₂The incubator is placed for 10h, the mixed solution of the delta WNV virus and the antibody is inoculated to a monolayer BWNV-CME cell, the cell is cultured for 6h at 37 ℃ with 5% CO2, then 3% methyl cellulose DMEM solution is added, and the green fluorescence expression is observed after the cell is cultured for 3 days. Plaque counts were performed after 6 days. Calculating each drug to be tested by taking DMSO or blank processing hole as a controlThe rate of virus inhibition.

2 results

2.1 detection of WNV neutralizing antibodies in neutralization assay

The limited replication WNV virus system of the invention is used for virus neutralization antibody detection of mouse serum, goose serum and horse serum immunized by WNV subunit vaccine, and negative serum of corresponding non-immunized experimental animals is set as a control. The results show that the serum of the vaccine immunized animals can also detect the neutralization activity through the neutralization test of the WNV restricted replication virus system, the neutralization effect on the WNV replication-defective viruses is gradually reduced along with the increase of the dilution of the serum, the negative control serum has no neutralization effect on the WNV replication-defective viruses, and the virus neutralizing antibodies of all the sera of the test animals are obviously improved after the secondary immunization, so that the immune protection can be provided for the immunized animals (Table 1). The WNV restricted replication virus system of the invention was shown to be useful in assays for the detection of serum neutralizing antibodies.

TABLE 1 detection of WNV neutralizing antibodies Using the Δ WNV Virus System

2.2 screening antiviral drugs Using WNV-restricted replication Virus System

The results of testing known drugs with antiviral activity against WNV using the restricted replication viral system of the present invention indicate that the system can be used to screen drugs against WNV virus. Both drugs in this example were shown to inhibit WNV virus replication, whereas the DMSO control did not (FIG. 8).

Sequence listing

<110> Harbin veterinary institute of Chinese academy of agricultural sciences

<120> restricted replication West Nile Virus System expressing Green fluorescent protein and use thereof

<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

1. A restricted replication west nile virus system expressing green fluorescent protein, comprising:

(1) west nile virus restriction replicon plasmid:

the west nile virus restriction replicon plasmid is obtained by cloning a west nile virus genome to the downstream of a promoter of a eukaryotic expression vector pCI-neo, and simultaneously replacing a structural protein C-prM-E sequence in the genome with a green fluorescent protein gene; the nucleotide sequence of the West Nile virus restriction replicon plasmid is shown in SEQ ID NO. 1;

(2) recombinant cell line for stably expressing West Nile virus C-prM-E protein

The recombinant cell line for stably expressing the West Nile virus C-prM-E protein is constructed by the following method:

1) artificially synthesizing an optimized C-prM-E protein expression gene, wherein the nucleotide sequence of the optimized C-prM-E protein expression gene is shown as SEQ ID NO.2, the optimized sequence is named as opti-WNV-CME, and the optimized C-prM-E protein expression gene is amplified by a PCR method after artificial synthesisSequence, and introducing into the 5' end of the PCR productSacI enzyme cutting site, 3' end introductionXhoI, enzyme cutting sites, connecting PCR products after double enzyme cutting with pCAG-neo vectors of the same double enzyme cutting, after connecting products to transform DH5 alpha, selecting recombinant clones, carrying out enzyme cutting and sequencing identification, preparing a large number of plasmids with a kit after obtaining positive clones, named as pCAG-WNV-CME, carrying out linearization with SspI, and freezing and storing at-20 ℃ for later use;

2) BHK-21 cells with good growth state are selected for digestion and passage to a 24-well plate, and when the BHK-21 cells grow to 90% full, the linearized recombinant plasmid pCAG-WNV-CME is used for transfecting the cells to obtain a stably expressed recombinant cell line which stably expresses West Nile virus C-prM-E protein and is named as BWNV-CME.

2. Use of the system of claim 1 to obtain a restricted replication West Nile Virus expressing Green fluorescent protein.

3. A method for obtaining a restricted replication west nile virus expressing green fluorescent protein, comprising the steps of:

DMEM medium containing 10% (v/v) FBS was used as growth medium, 1: 4 subculturing the recombinant cell line stably expressing West Nile Virus C-prM-E protein according to claim 1, and culturing the recombinant cells in logarithmic growth phase at 5X 10⁵And (2) inoculating a 6-well cell culture plate per ml, culturing overnight, adding the west nile virus restriction replicon plasmid of claim 1 into Opti-MEM to prepare a DNA diluent, adding 200 μ l of Opti-MEM into every 3 μ g of the plasmid, adding 20 μ l X-treme HP transfection reagent, standing at room temperature for 15min, dropwise adding the DNA transfection reagent compound into a culture solution containing recombinant cells stably expressing the west nile virus C-prM-E protein, observing fluorescence after 72h, collecting supernatant, centrifuging, filtering, and storing at-80 ℃ for later use.

4. A restricted replicating West Nile Virus expressing Green fluorescent protein obtained according to the method of claim 3.

5. The use of the limited replication West Nile virus expressing Green fluorescent protein of claim 4 in the preparation of West Nile virus neutralizing antibody detection reagent and in the screening of anti-West Nile virus drugs.