CN113683690B - Antibody for yellow fever virus NS1 protein and application thereof - Google Patents

Abstract

The invention provides an antibody against NS1 protein of yellow fever virus and its application, and the antibody includes YD40 and/or YB36 antibody; the invention provides a highly sensitive yellow fever virus ELISA double-antibody sandwich detection method. This method can be operated in ordinary laboratories, and can be further developed into various kits, such as colloidal gold, ELISA kits, etc., for easy use.

Description

Antibody against yellow fever virus NS1 protein and its application

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to an antibody against NS1 protein of yellow fever virus and application thereof.

Background technique

Yellow fever virus (YFV) is an insect-borne virus transmitted by Aedes mosquitoes, and belongs to the Flavivirus genus of the Flaviviridae family like Zika virus (ZIKV), West Nile virus (WNV) and dengue virus (DENV). The vector of yellow fever virus is mosquitoes, so yellow fever outbreaks are high in mosquito-endemic areas and developing countries. Currently, it is mainly prevalent in tropical and subtropical regions of Africa and South America. Yellow fever virus can cause yellow fever, which seriously endangers human health, manifested as jaundice, hemorrhage, and even multi-system organ failure. Because the symptoms of yellow fever are similar to those caused by viruses of the same family, and yellow fever virus and other flaviviruses may also be circulating in the same area, it is difficult to distinguish clinically based on clinical symptoms. Furthermore, since viruses of the Flaviviridae family have similar antigenic features, antibodies may be cross-reactive. Furthermore, dengue virus can also cause ADE (Antibody-Dependent Enhancement), which complicates the clinical diagnosis. The above problems also pose great challenges to the development of serological diagnostic methods for yellow fever.

Laboratory methods for the diagnosis of yellow fever virus infection mainly include virus isolation, nucleic acid detection, antigen detection, and serological detection. At present, the most reliable diagnostic method is to extract viral RNA and perform qRT-PCR. This method is time-consuming, expensive, and needs to be operated by professionals in a specific laboratory. The instruments used are also complicated and cannot be widely and conveniently applied. Therefore, there is an urgent need to develop a product and method that is simple to operate, can detect infection at an early stage, and can be used in remote areas.

The nonstructural protein 1 (nonstructural protein 1, NS1) of yellow fever virus is a relatively conservative glycoprotein, which has two forms: membrane type and secreted type. good target.

Contents of the invention

In view of the deficiencies in the prior art, one of the objectives of the present invention is to provide an antibody against the NS1 non-structural protein of yellow fever virus and its application in the preparation of products.

The second purpose of the present invention is to develop a highly sensitive yellow fever virus detection kit and detection method based on the antibody disclosed in the present invention. This kit or method can be operated in ordinary laboratories, and can be further developed into various kits, such as colloidal gold, ELISA, etc., for easy use.

To achieve the above object, the specific technical solutions of the present invention are as follows:

In a first aspect, the present invention provides an antibody against the NS1 non-structural protein of yellow fever virus, said antibody comprising YD40 and/or YB36 antibody;

Wherein, the YD40 antibody comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region of the amino acid sequence shown in SEQ ID NO:2;

The YB36 antibody comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:3, and a light chain variable region with the amino acid sequence shown in SEQ ID NO:4.

In some embodiments, the YD40 or YB36 antibody further comprises: a heavy chain constant region with the amino acid sequence shown in SEQ ID NO:5 and a light chain constant region with the amino acid sequence shown in SEQ ID NO:6.

In some embodiments, the above antibody light chains are kappa light chains.

In some embodiments, the YB36 or YD40 antibody further includes an N-terminal signal peptide, the amino acid sequence of the signal peptide is SEQ ID NO:7; the nucleotide sequence of the signal peptide is shown in SEQ ID NO:8 .

In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned antibody.

In some embodiments, the nucleotide sequence is as shown in SEQ ID NO:9-14;

Wherein, for the YD40 antibody, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 9, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 10;

For the YB36 antibody, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:12.

For the YD40 or YB36 antibody, the nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:13 and the nucleotide sequence of the light chain constant region is shown in SEQ ID NO:14.

In a third aspect, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecule.

In a fourth aspect, the present invention provides a yellow fever virus detection kit, which includes any of the antibodies described above.

In some embodiments, the kit includes the YD40 and YB36 antibodies described above, the YD40 being used as a detection antibody with a chromogenic label; the YB36 being immobilized and used as a capture antibody.

In some embodiments, a solid phase carrier for immobilizing the capture antibody, a sample treatment solution, a positive control, a negative control, a chromogenic solution and a stop solution are also included.

In some embodiments, the solid phase support includes: any one or a combination of at least two of nitrocellulose membranes, latex particles, magnetic particles, colloidal gold, glass, fiberglass, polymers, or fiber optic sensors.

In some embodiments, the chromogenic label may be an enzyme, colloidal metal particles, colored latex particles, fluorescent latex particles, luminescent substances, fluorescent substances, and the like. Enzymes can be various enzymes used in enzyme-linked immunoassay (EIA), such as alkaline phosphatase, peroxidase, β-D-galactosidase, etc.; colloidal metal particles such as colloidal gold particles, colloidal Selenium particles, etc. Preferably, horseradish peroxidase.

In some embodiments, the chromogenic solution and stop solution can be purchased commercially or prepared by yourself. The chromogenic solution is, for example, an enzyme substrate, TMB, etc.

In some embodiments, the positive control is yellow fever virus NS1 protein; the negative control is a blank control without yellow fever virus NS1 protein.

In a fifth aspect, the present invention provides a method for non-disease diagnosis and treatment purposes, detection of yellow fever virus or its NS1 protein in a sample, the method comprising the steps of:

The sample to be tested is mixed with the antibody, incubated, and the presence of the yellow fever virus or its corresponding antigen is detected.

In some embodiments, the methods include sandwich and competition methods.

In a preferred embodiment, the sandwich method comprises the steps of:

(1) Binding YB36 as a capture antibody to a solid phase carrier;

(2) Mix the solid phase carrier obtained in step (1) with the sample to be tested, incubate, and compare with a control sample;

(3) After the incubation is completed, wash the solid phase carrier, add a color-labeled detection antibody YD40 and incubate again, and the detection antibody binds to yellow fever virus or its corresponding antigen;

(4) Wash the solid-phase carrier again, then add a chromogenic solution, and use a microplate reader to measure the corresponding absorbance value;

(5) Compare the measured absorbance value with the absorbance value of the control sample to determine the presence and relative amount of the yellow fever virus in the test sample.

Preferably, the sample to be tested is a biological sample, further, the sample includes cell supernatant, plasma, serum and whole blood.

In the sixth aspect, the present invention provides the use of the antibody in the preparation of medicines for treating and/or preventing yellow fever virus.

Preferably, the drug comprises any of the above antibodies.

In the seventh aspect, the present invention provides the use of the antibody in the preparation of products for detecting and/or diagnosing yellow fever virus.

In some embodiments, the products may be used in methods including, but not limited to, enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, and the like.

Any detection method using the YB36 and/or YD40 antibody disclosed in the present invention and the products thereof are within the protection scope of the present invention.

Preferably, the products include (but not limited to) detection reagents, detection test strips and detection kits.

In some embodiments, the detection test paper includes a base, a sample area, a binding area and a reaction area, wherein along the flow direction of the sample to be tested, the sample area, the binding area and the reaction area are sequentially arranged on the base; The binding area is loaded with chromogenic labeled YD40 antibody; the reaction area is provided with a detection area, and the detection area is loaded with YB36 antibody, which is immobilized on the detection area and used as a capture antibody. The reaction area is also provided with a quality control area.

In some embodiments, the detection test paper further includes an absorption zone on the side of the reaction zone away from the sample zone.

In some embodiments, the chromogenic label is a colloidal gold label or a fluorescent label.

In some embodiments, the detection test paper can be prepared according to the prior art, such as the method described in the examples of Chinese patent application CN102747040A.

Based on the above technical scheme, the present invention has the following beneficial effects:

1. The monoclonal antibodies YB36 and YD40 of the present invention can specifically recognize the YFV-NS1 protein, have high affinity, and have the advantages of good sensitivity and specificity; and they are used to prepare products for diagnosing yellow fever virus.

2. The present invention provides a highly sensitive and reliable double-antibody sandwich ELISA method, which can be used to detect the YFV-NS1 protein antigen in different types of samples including animal or human serum, and is used for the early diagnosis of flavivirus infection. To achieve the purpose of early detection, early isolation, and avoid transmission.

3. The products prepared based on double antibodies of the present invention include YFV-NS1 virus detection kit and detection test paper; because the monoclonal antibody used has high affinity and specificity, the obtained detection results are relatively accurate; and it is easy to carry and store, and the operation is simple , the method is stable, and the test can be completed in a short time. Compared with nucleic acid detection, it has the characteristics of shorter detection time and lower cost, and is especially suitable for screening of a large number of samples and remote areas.

Description of drawings

Figure 1: Molecular sieve (A) and SDS-PAGE results (B) for YFV-NS1 protein purification.

Figure 2: Antibody SDS-PAGE results after antibody purification.

Figure 3: The results of antibody binding to yellow fever virus and NS1 protein of the same family virus.

Figure 4: The surface plasmon resonance technique detects the affinity of the antibody to the YFV-NS1 protein.

Figure 5: Octet RED 96 instrument detects non-competitive binding of YD40 and YB36 to YFV-NS1 protein.

Figure 6: The result curve of the double-antibody sandwich ELISA test of the capture antibody YB36 and the labeled antibody YD40; (A) explore the amount of coated antibody YB36 used; (B) detect the amount of antibody YD40 used; (C) explore the detectable antigen YFV - Lower bound for NS1.

Figure 7: Comparison results between the sandwich ELISA method (B) established by the present invention and the existing nucleic acid detection method (A).

Figure 8: The verification results of the sandwich ELISA method established by the present invention to detect clinical samples.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

All materials and reagents in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1 Expression and purification of yellow fever virus nonstructural protein 1 (YFV-NS1)

The extracellular region gene fragment of YFV-NS1 was cloned into the pFastBac I vector by Nde I and Xho I, and transformed into Escherichia coli DH10 to obtain the recombinant plasmid Bacmid-NS1. Then, sf9 cells were transfected to obtain baculovirus containing NS1 gene. After baculovirus infects Hi5 cells, it can secrete and express NS1 protein into the culture supernatant. The protein was purified by affinity chromatography and molecular sieve chromatography, and the protein purity was identified by SDS-PAGE. The results showed that the NS1 protein existed in monomer and dimer forms, and the monomer size was 43kDa, as shown in Figure 1 . Concentrate YFV-NS1 to 1 mg/mL and store it frozen at -80°C for future use.

For the construction, expression and purification of yellow fever virus YFV-NS1 C terminal and other flaviviruses WNV-NS1, ZIKV-NS1, DENV1-NS1, DENV2-NS1, DENV3-NS1, DENV4-NS1 refer to the YFV-NS1 above.

The amino acid sequence of yellow fever virus YFV-NS1 is shown in SEQ ID NO: 15, and the amino acid sequence of YFV-NS1 C terminal is shown in SEQ ID NO: 15 at AA at position 171-351; other flaviviruses: West Nile virus WNV-NS1 (size is 45kDa) amino acid sequence such as SEQ ID NO: 16, Zika virus ZIKV-NS1 (size is about 45kDa) amino acid sequence such as SEQ ID NO: 17, four serotype dengue fever virus DENV-NS1 (DV1- The amino acid sequences of NS1/DV2-NS1/DV3-NS1/DV4-NS1 proteins are 45kDa/75kDa/43kDa/43kDa) such as SEQ ID NO: 18-21.

Example 2 Preparation of Human Monoclonal Antibody

1. Single cell sorting and clone construction

With the informed consent of the recovered patients, peripheral blood samples were collected from two recovered patients infected with YFV for 6 months, peripheral blood mononuclear cells (PBMCs) were separated, and various blood cell-specific marker antibodies and specific YFV-NS1 antigen were added Incubated together. After sorting by FACSAria II (BD Biosciences), the YFV-NS1 positive memory B cells were collected into a 96-well plate, with 1 cell per well.

The inventors amplified antibody variable region gene sequences (VH and VL) from a single B cell by means of RT-PCR and nested PCR. First, the obtained B cells were reverse-transcribed using the SuperScript III reverse transcriptase kit. After obtaining the cDNA, nested PCR was performed using HotStar plus enzyme (QIAGEN) to amplify the antibody variable region sequence. The first round of PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 55°C (VH and Vκ) or 50°C (Vλ) for 60 s, 72°C for 90 s, 35 cycles; 72°C for 7 min. The obtained product was used as a template for the second round of PCR under the following conditions: 95°C for 5 min; 95°C for 30 s, 58°C (heavy chain)/60°C (K chain)/64°C (λ chain) for 30 s, 72°C for 90 s, 35°C cycle; 72°C for 7min.

The PCR products were separated by agarose gel electrophoresis, the PCR band with a molecular weight of about 400 bp was extracted with a gel extraction kit (CWBIO), and the purified products were sequenced. The obtained target sequence was analyzed by IMGT (international ImMunoGeneTics) information system and IgBlast. The variable region sequence and the corresponding constant region were connected by bridging PCR, and cloned into the expression vector pCAGGS, in which EcoRI and XhoI restriction sites were used for the heavy chain, and SacI and XhoI restriction sites were used for the κ chain (light chain).

2. Protein expression and purification

The heavy chain and light chain construct plasmids were transfected into HEK-293T cells. Six hours after transfection, cultures were replaced with fresh serum-free medium. On days 3 and 7, cell culture supernatants were collected. Mix the collected supernatant with an equal volume of buffer solution containing 20mM sodium phosphate (pH7.0), centrifuge at 6500rpm for 30min to remove debris, filter through a 0.22um filter membrane, and then pass the supernatant through the HiTrap Protein A affinity chromatography column , and the bound antibody was then eluted using 0.1M glycine (pH 3.0). The collected eluate was concentrated and subjected to molecular sieve chromatography. The target peak was identified by SDS-PAGE. The molecular weight of the heavy chain was about 50kDa, and the molecular weight of the light chain was about 25kDa. The results are shown in Figure 2, indicating that the target protein was expressed normally.

Eleven kinds of antibodies were isolated, and the antibody binding affinity was determined on the basis of preliminary ELISA, and the monoclonal antibodies YB36 and YD40 with strong binding activity to YFV-NS1 protein were screened out.

Embodiment 3 YB36 and YD40 antibody functional identification

1. ELISA detection of antibody specific binding to YFV-NS1

1) Dilute antigen 2μg/mL (YFV-NS1, YFV-NS1 C terminal, WNV-NS1, ZIKV-NS1, DENV1-NS1, DENV2-NS1, DENV3-NS1, DENV4-NS1) with coating solution, add to enzyme In the target plate, 100 μL/well, overnight at 4°C.

2) The next day, add 200 μL/well of PBST, wash the plate 5 times with a plate washer (BIO-TEK, 405_LS), add 5% skimmed milk powder blocking solution, 100 μL/well, and incubate in a 37°C incubator for 1 hour.

3) Rinse 5 times with PBST, add antibody YB36 or YD40, 400ng/well, incubate at 37°C for 1 hour.

4) Rinse 5 times with PBST, HPR-labeled goat anti-human IgG, 100 μL/well, and incubate at 37° C. for 45 minutes.

5) Rinse with PBST for 5 times, add TMB chromogenic solution, 100 μL/well, react at room temperature in the dark for 2 minutes, then add 2M H ₂ SO ₄ to terminate the reaction, 100 μL/well.

6) Detect the OD value at the wavelength of 450nm, save and record the original data.

The results are shown in FIG. 3 , both antibodies YB36 and YD40 are YFV-specific monoclonal antibodies; they have no binding to other Flaviviridae NS1 proteins.

2. Surface plasmon resonance (SPR) was used to detect the binding ability of antibodies to YFV-NS1

This experiment was completed with a BIAcore ^TM 8K machine. Specific steps are as follows:

First, the anti-human secondary antibody was immobilized on the channel (flow cell, Fc) of the CM5 chip by amino coupling. The fixed amount is controlled at around 10,000 response units (RU). Immobilize YB36 or YD40 in the way of antibody capture, and the capture amount is about 60RU. Buffer (10mM HEPES, 150mM NaCl, pH 7.4) was used to dilute the YFV-NS1 protein by a factor of two, and flowed through each channel in turn at a flow rate of 30μL/min. Binding kinetic constants were calculated using BIAevaluation software (Biacore, Inc.).

The results of SPR showed that all antibodies could bind to YFV-NS1 protein (Figure 4). The obtained association constant (ka), dissociation constant (kd) and equilibrium dissociation constant (kD) of each interaction are shown in Table 1.

The kinetic constants of table 1 antibody binding to YFV-NS1 protein

3. YB36 and YD40 bind to different epitopes of YFV-NS1

The Octet RED96 biomolecular interaction analyzer was used to detect whether the antibodies YB36 and YD40 competitively bound to the YFV-NS1 protein.

)，然后将生物传感器浸泡于含YD40的缓冲液中，反应300s以达到饱和。然后再浸泡于YD40与YB36的混合液中，反应300s以达到饱和。抗体均以相同浓度(1μM)测定。比较结合曲线的变化来判定两种抗体结合抗原的表位。First, YFV-NS1 was immobilized on the AR2G biosensor (Pall

), and then soak the biosensor in the buffer solution containing YD40, and react for 300s to reach saturation. Then soak in the mixture of YD40 and YB36, react for 300s to reach saturation. Antibodies were all assayed at the same concentration (1 μM). The changes in the binding curves were compared to determine the epitopes of the antigens bound by the two antibodies.

The results are shown in Figure 5. After the biosensor was soaked in the solution containing YD40, the curve rose, and when it reached the plateau and then soaked in the solution containing the YB36 antibody, the curve still rose, indicating that YFV-NS1 binds YD40 and can still bind after saturation. YB36, the two antibodies bind to different epitopes of YFV-NS1, and there is no competitive relationship.

Example 4 Establishment of Double Antibody Sandwich ELISA Method for Detection of YFV-NS1 Antigen Using YB36 and YD40 Antibodies

Considering the specificity and affinity of the monoclonal antibody and the antigen YFV-NS1, the present invention selects YD40 with stronger affinity as the detection antibody, and uses HRP labeling for subsequent determination, and YB36 is used as the capture antibody to establish the detection of yellow fever virus YFV-NS1. Double-antibody sandwich ELISA assay for NS1 antigen.

The ELISA method refers to the ELISA method in Example 3, and is modified according to experimental needs. Among them, the YB36 antibody is coated on the microtiter plate, after rinsing and blocking, the antigen is added for incubation, after rinsing again, YD40 is added for reaction, and then the color is developed after rinsing.

YB36 was serially diluted with coating solution (as shown in Figure 6A) and then coated on the microtiter plate. After blocking and rinsing, YFV-NS1 (10 μg/mL, 100 μL/well) and YD40-HRP (2 μg/mL, 100 μL/well). The results show that when YB36 is greater than 200ng/well, the OD value does not increase any more, as shown in Figure 6A.

After coating 200ng/well of YB36 on the ELISA plate, YFV-NS1 (10 μg/mL, 100 μL/well) and YD40-HRP serially diluted in PBST were added sequentially (as shown in Figure 6B). The results showed that the antigen could be detected when the concentration of YD40-HRP was 74.07ng/mL, and when it exceeded 667ng/mL, the OD value reached the plateau stage, as shown in Figure 6B.

In summary, YB36 can be used as a capture antibody at 200 ng/well, and YD40 as a detection antibody can be used at a concentration of 2 μg/mL.

In order to evaluate the detection level of antigen by sandwich ELISA, YFV-NS1 was diluted from 30 μg/mL to 1.52 ng/mL, and the OD value was measured. The results are shown in Figure 6C, the minimum detection concentration of YFV-NS1 is about 40ng/mL; when the concentration exceeds 370ng/mL, the OD value reaches the plateau stage.

The performance of embodiment 5ELISA method and kit thereof

In the present invention, YD40 is used as a detection antibody and YB36 is used as a capture antibody, and the established double-antibody sandwich ELISA method and kit for measuring the protein content of yellow fever virus YFV-NS1 are evaluated for performance.

1. The double-antibody sandwich ELISA method was used to detect the content of YFV-NS1 in the mouse serum after virus infection, and compared with the Q-PCR method.

Nine IFN-αβR-KO mice (6 weeks old) were infected by intraperitoneal injection of 1×10 ⁵ PFU YFV-17D virus. Orbital blood was collected before infection and on days 3, 5, 7, 10, and 14 after infection. Blood samples were placed on ice for 60 minutes and then centrifuged for 12 minutes at 6000 rpm/min. Serum was collected and stored at -80°C. YFV-NS1 was quantified by Q-PCR and the double-antibody sandwich ELISA method established in the present invention. YFV-17D virus preparation refers to Lu X, Xiao H, Li S, et al. Double lock of a humanneutralizing and protective monoclonal antibody targeting the yellow fevervirus envelope [J]. Cell Reports, 2019, 26(2): 438-446. e5.

The results are shown in Figure 7, the Q-PCR method (Figure 7A) can detect infection in the 1d and 10d part of the mice, and the virus in the serum of all mice from the 3rd day to the 7th day; the double-antibody sandwich ELISA method ( Fig. 7B) YFV-NS1 can be detected in the sera of all mice on the 3d, 5d and part of the mice on the 7d.

2. The double-antibody sandwich ELISA method established by the present invention detects clinical samples

In 2016, the inventor collected the serum of 4 cases of YFV infection during hospitalization in Beijing Ditan Hospital and the serum of 5 healthy people for NS1 detection. The detection of NS1 in patient serum by the double-antibody sandwich ELISA method of the present invention ranges from 1.6 μg/mL to 59 μg/mL, while NS1 in healthy human serum is lower than 0.1 μg/mL ( FIG. 8 ). The results showed that the sandwich ELISA could detect NS1 sensitively and specifically.

The invention provides a highly sensitive and reliable double-antibody sandwich ELISA method, which can be used to detect the YFV-NS1 protein antigen in different types of samples including YFV-infected animals or human serum, and is used for the early diagnosis of yellow fever. To achieve the purpose of early detection, early isolation, and avoid transmission.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

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<213> Artificial sequence (Artificial sequence)

<400> 8

atggagacgg atacgctgct cctgtgggtt ttgctgctgt gggttccagg ttccactggt 60

gac 63

<210> 9

<211> 360

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

gaggtgcagc tggtggagtc tgggggaggc tttgttctgc ctggggggtc cctgagactc 60

tcctgtggag gctctggatt catctttaag agctatgtca tgacctgggt ccgccaggct 120

ccagggaagg gtctggagtg ggtctcaact attackggtc gtggagatac acactacgca 180

gactccgtga tgggccggtt caccatctcc agagacaatt ctaagaatat ggtgtttctg 240

caaatgaagg gcctgagagc cgacgacacg gccgtctatt actgtgcgaa agaagtggcg 300

tttagtggcg acgattacat tgactcttgg ggccaggggga ccctggtcgc cgtctcctca 360

<210> 10

<211> 321

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

gacatccagt tgacccagtc tccttccacc ctgtctgcat ctattggaga cagagtcacc 60

atcacttgcc gggccagtca gagtattaat agctggttgg cctggtatca gcagagacca 120

gggaaagccc ctaacctcct gatgtatgag gcgtctcgtt tagaaagtgg cgtcccatca 180

aggttcagcg gcagtggatc tgggacagag ttcactctca ccatcagcag cctgcagcct 240

aatgattttg caacttatta ctgccaacag tacaatactt attcccggac gttcggccaa 300

gggaccaagg tggaaatcag a 321

<210> 11

<211> 366

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

gaggtgcagc tggtggagtc tgggggaggc ttgatccagc cgggggggtc cctgagactc 60

tcctgtgcag cctctgggtt cagcgtcaga agttactaca tgacctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcaatt atttatagcg ggggtagtag atactacgca 180

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctctatctt 240

caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agttggtgac 300

ggtgacgact acttctacta ctacatggac gtctggggca aagggaccac ggtcaccgtc 360

tcctca 366

<210> 12

<211> 327

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttacc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccagggt cctcatctat ggtgcatcca ggagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtgtggtt tctcacctcc cttcactttc 300

ggccctggga ccaaagtgga tatcaaa 327

<210> 13

<211> 993

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

gccagcacca aaggcccgag cgtgtttccg ctggcgccga gcagcaaaag caccagcggc 60

ggcaccgcgg cgctgggctg cctggtgaaa gattattttc cggaaccggt gaccgtgagc 120

tggaacagcg gcgcgctgac cagcggcgtg catacctttc cggcggtgct gcagagcagc 180

ggcctgtata gcctgagcag cgtggtgacc gtgccgagca gcagcctggg cacccagacc 240

tatatttgca acgtgaacca taaaccgagc aacaccaaag tggataaacg cgtggagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720

ctgaccaaga accagtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagaagagcc tctccctgtc tccgggtaaa tga 993

<210> 14

<211> 327

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

cgaactgtgg ctgcaccatc tgtgttcatc ttccctccca gcgacgagca gctgaagagc 60

ggcaccgcca gcgtggtctg tctcctgaac aacttctatc ccagggaggc caaggtccag 120

tggaaagtgg acaacgccct gcaaagcggc aatagccagg agtccgtcac agagcaggac 180

agcaaggaca gcacctacag cctgtccagc accctgaccc tcagcaaggc cgactacgag 240

aagcacaagg tgtacgcttg cgaggtgacc catcagggcc tgtccagccc cgtgaccaag 300

tccttcaaca ggggcgaatg cagctaa 327

<210> 15

<211> 351

<212> PRT

<213> YFV-NS1

<400> 15

Gln Gly Cys Ala Val Asn Phe Gly Lys Arg Glu Leu Lys Cys Gly Asp

1 5 10 15

Gly Ile Phe Val Phe Arg Asp Ser Asp Asp Trp Leu Thr Lys Tyr Ser

20 25 30

Tyr Tyr Pro Glu Asp Pro Val Lys Leu Ala Ser Ile Ile Lys Ala Ser

35 40 45

His Glu Glu Gly Lys Cys Gly Leu Asn Ser Val Asp Ser Leu Glu His

50 55 60

Glu Met Trp Arg Ser Arg Ala Asp Glu Ile Asn Ala Ile Phe Glu Glu

65 70 75 80

Asn Glu Val Asp Ile Ser Val Val Val Gln Asp Pro Lys Asn Ile Tyr

85 90 95

Gln Arg Gly Thr His Pro Phe Ser Arg Ile Arg Asp Gly Leu Gln Tyr

100 105 110

Gly Trp Lys Thr Trp Gly Lys Asn Leu Val Phe Ser Pro Gly Arg Lys

115 120 125

Asn Gly Ser Phe Ile Ile Asp Gly Lys Ser Arg Lys Glu Cys Pro Phe

130 135 140

Ser Asn Arg Val Trp Asn Ser Phe Gln Ile Glu Glu Phe Gly Met Gly

145 150 155 160

Val Phe Thr Thr Arg Val Phe Met Asp Ala Thr Phe Asp Tyr Ser Val

165 170 175

Asp Cys Asp Gly Ala Ile Leu Gly Ala Ala Val Asn Gly Lys Lys Ser

180 185 190

Ala His Gly Ser Pro Thr Phe Trp Met Gly Ser His Glu Val Asn Gly

195 200 205

Thr Trp Met Ile His Thr Leu Glu Thr Leu Asp Tyr Lys Glu Cys Glu

210 215 220

Trp Pro Leu Thr His Thr Ile Gly Thr Ser Val Glu Glu Ser Asp Met

225 230 235 240

Phe Met Pro Arg Ser Ile Gly Gly Pro Val Ser Ser His Asn Arg Ile

245 250 255

Pro Gly Tyr Lys Val Gln Thr Asn Gly Pro Trp Met Gln Val Pro Leu

260 265 270

Glu Val Lys Arg Glu Val Cys Pro Gly Thr Ser Val Val Val Asp Ser

275 280 285

Asn Cys Asp Gly Arg Gly Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly

290 295 300

Lys Ile Ile Pro Glu Trp Cys Cys Arg Ser Cys Thr Met Pro Pro Val

305 310 315 320

Ser Phe His Gly Ser Asp Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro

325 330 335

Met Lys Thr Ser Asp Ser His Leu Val Arg Ser Trp Val Thr Ala

340 345 350

<210> 16

<211> 351

<212> PRT

<213> WNV-NS1

<400> 16

Thr Gly Cys Ala Ile Asp Ile Ser Arg Gln Glu Leu Arg Cys Gly Ser

1 5 10 15

Gly Val Phe Ile His Asn Asp Val Glu Ala Trp Met Asp Arg Tyr Lys

20 25 30

Tyr Tyr Pro Glu Thr Pro Gln Gly Leu Ala Lys Ile Ile Gln Lys Ala

35 40 45

His Lys Glu Gly Val Cys Gly Leu Arg Ser Val Ser Arg Leu Glu His

50 55 60

Gln Met Trp Glu Ala Val Lys Asp Glu Leu Asn Thr Leu Leu Lys Glu

65 70 75 80

Asn Gly Val Asp Leu Ser Val Val Val Glu Lys Gln Glu Gly Met Tyr

85 90 95

Lys Ser Ala Pro Lys Arg Leu Thr Ala Thr Thr Glu Lys Leu Glu Ile

100 105 110

Gly Trp Lys Ala Trp Gly Lys Ser Ile Leu Phe Ala Pro Glu Leu Ala

115 120 125

Asn Asn Thr Phe Val Val Asp Gly Pro Glu Thr Lys Glu Cys Pro Thr

130 135 140

Gln Asn Arg Ala Trp Asn Ser Leu Glu Val Glu Asp Phe Gly Phe Gly

145 150 155 160

Leu Thr Ser Thr Arg Met Phe Leu Lys Val Arg Glu Ser Asn Thr Thr

165 170 175

Glu Cys Asp Ser Lys Ile Ile Gly Thr Ala Val Lys Asn Asn Leu Ala

180 185 190

Ile His Ser Asp Leu Ser Tyr Trp Ile Glu Ser Arg Leu Asn Asp Thr

195 200 205

Trp Lys Leu Glu Arg Ala Val Leu Gly Glu Val Lys Ser Cys Thr Trp

210 215 220

Pro Glu Thr His Thr Leu Trp Gly Asp Gly Ile Leu Glu Ser Asp Leu

225 230 235 240

Ile Ile Pro Val Thr Leu Ala Gly Pro Arg Ser Asn His Asn Arg Arg

245 250 255

Pro Gly Tyr Lys Thr Gln Asn Gln Gly Pro Trp Asp Glu Gly Arg Val

260 265 270

Glu Ile Asp Phe Asp Tyr Cys Pro Gly Thr Thr Val Thr Leu Ser Glu

275 280 285

Ser Cys Gly His Arg Gly Pro Ala Thr Arg Thr Thr Thr Thr Glu Ser Gly

290 295 300

Lys Leu Ile Thr Asp Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu

305 310 315 320

Arg Tyr Gln Thr Asp Ser Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro

325 330 335

Gln Arg His Asp Glu Lys Thr Leu Val Gln Ser Gln Val Asn Ala

340 345 350

<210> 17

<211> 351

<212> PRT

<213> Zika

<400> 17

Val Gly Cys Ser Val Asp Phe Ser Lys Lys Glu Thr Arg Cys Gly Thr

1 5 10 15

Gly Val Phe Val Tyr Asn Asp Val Glu Ala Trp Arg Asp Arg Tyr Lys

20 25 30

Tyr His Pro Asp Ser Pro Arg Arg Leu Ala Ala Ala Val Lys Gln Ala

35 40 45

Trp Glu Asp Gly Ile Cys Gly Ile Ser Ser Val Ser Arg Met Glu Asn

50 55 60

Ile Met Trp Arg Ser Val Glu Gly Glu Leu Asn Ala Ile Leu Glu Glu

65 70 75 80

Asn Gly Val Gln Leu Thr Val Val Val Gly Ser Val Lys Asn Pro Met

85 90 95

Trp Arg Gly Pro Gln Arg Leu Pro Val Pro Val Asn Glu Leu Pro His

100 105 110

Gly Trp Lys Ala Trp Gly Lys Ser Tyr Phe Val Arg Ala Ala Lys Thr

115 120 125

Asn Asn Ser Phe Val Val Asp Gly Asp Thr Leu Lys Glu Cys Pro Leu

130 135 140

Lys His Arg Ala Trp Asn Ser Phe Leu Val Glu Asp His Gly Phe Gly

145 150 155 160

Val Phe His Thr Ser Val Trp Leu Lys Val Arg Glu Asp Tyr Ser Leu

165 170 175

Glu Cys Asp Pro Ala Val Ile Gly Thr Ala Val Lys Gly Lys Glu Ala

180 185 190

Val His Ser Asp Leu Gly Tyr Trp Ile Glu Ser Glu Lys Asn Asp Thr

195 200 205

Trp Arg Leu Lys Arg Ala His Leu Ile Glu Met Lys Thr Cys Glu Trp

210 215 220

Pro Lys Ser His Thr Leu Trp Thr Asp Gly Ile Glu Glu Ser Asp Leu

225 230 235 240

Ile Ile Pro Lys Ser Leu Ala Gly Pro Leu Ser His His Asn Thr Arg

245 250 255

Glu Gly Tyr Arg Thr Gln Met Lys Gly Pro Trp His Ser Glu Glu Leu

260 265 270

Glu Ile Arg Phe Glu Glu Cys Pro Gly Thr Lys Val His Val Glu Glu

275 280 285

Thr Cys Gly Thr Arg Gly Pro Ser Leu Arg Ser Thr Thr Ala Ser Gly

290 295 300

Arg Val Ile Glu Glu Trp Cys Cys Arg Glu Cys Thr Met Pro Pro Leu

305 310 315 320

Ser Phe Arg Ala Lys Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro

325 330 335

Arg Lys Glu Pro Glu Ser Asn Leu Val Arg Ser Met Val Thr Ala

340 345 350

<210> 18

<211> 351

<212> PRT

<213> DENV1

<400> 18

Ser Gly Cys Val Ile Asn Trp Lys Gly Arg Glu Leu Lys Cys Gly Ser

1 5 10 15

Gly Ile Phe Val Thr Asn Glu Val His Thr Trp Thr Glu Gln Tyr Lys

20 25 30

Phe Gln Ala Asp Ser Pro Lys Arg Leu Ser Ala Ala Ile Gly Lys Ala

35 40 45

Trp Glu Glu Gly Val Cys Gly Ile Arg Ser Ala Thr Arg Leu Glu Asn

50 55 60

Ile Met Trp Lys Gln Ile Ser Asn Glu Leu Asn His Ile Leu Leu Glu

65 70 75 80

Asn Asp Met Lys Leu Thr Val Val Val Gly Asp Val Thr Gly Ile Leu

85 90 95

Ala Gln Gly Lys Lys Met Ile Arg Pro Gln Pro Met Glu His Lys Tyr

100 105 110

Ser Trp Lys Ser Trp Gly Lys Ala Lys Ile Thr Gly Ala Asp Val Gln

115 120 125

Asn Thr Thr Phe Ile Ile Asp Gly Pro Asn Thr Pro Glu Cys Pro Asp

130 135 140

Asn Gln Arg Ala Trp Asn Ile Trp Glu Val Glu Asp Tyr Gly Phe Gly

145 150 155 160

Val Phe Thr Thr Asn Ile Trp Leu Lys Leu Arg Asp Ser Tyr Thr Gln

165 170 175

Val Cys Asp His Arg Leu Met Ser Ala Ala Ile Lys Asp Ser Lys Ala

180 185 190

Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser Glu Lys Asn Glu Thr

195 200 205

Trp Lys Leu Glu Arg Ala Ser Phe Ile Glu Val Lys Thr Cys Ile Trp

210 215 220

Pro Arg Ser His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Glu Met

225 230 235 240

Ile Ile Pro Lys Ile Tyr Gly Gly Pro Val Ser Gln His Asn Tyr Arg

245 250 255

Pro Gly Tyr Phe Thr Gln Thr Ala Gly Pro Trp His Leu Gly Lys Leu

260 265 270

Glu Leu Asp Phe Asp Leu Cys Glu Gly Thr Thr Val Val Val Asp Glu

275 280 285

Asn Cys Gly Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Val Thr Gly

290 295 300

Lys Thr Ile His Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu

305 310 315 320

Arg Phe Lys Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro

325 330 335

Val Lys Glu Lys Glu Glu Asn Leu Val Lys Ser Met Val Ser Ala

340 345 350

<210> 19

<211> 351

<212> PRT

<213> DENV2-NS1

<400> 19

Ser Gly Cys Val Val Ser Trp Lys Asn Lys Glu Leu Lys Cys Gly Ser

1 5 10 15

Gly Ile Phe Ile Thr Asp Asn Val His Thr Trp Thr Glu Gln Tyr Lys

20 25 30

Phe Gln Pro Glu Ser Pro Ser Lys Leu Ala Ser Ala Ile Gln Lys Ala

35 40 45

His Glu Glu Gly Ile Cys Gly Ile Arg Ser Val Thr Arg Leu Glu Asn

50 55 60

Leu Met Trp Lys Gln Ile Thr Pro Glu Leu Asn His Ile Leu Ser Glu

65 70 75 80

Asn Glu Val Lys Leu Thr Ile Met Thr Gly Asp Ile Lys Gly Ile Met

85 90 95

Gln Ala Gly Lys Arg Ser Leu Arg Pro Gln Pro Thr Glu Leu Lys Tyr

100 105 110

Ser Trp Lys Thr Trp Gly Lys Ala Lys Met Leu Ser Thr Glu Ser His

115 120 125

Asn Gln Thr Phe Leu Ile Asp Gly Pro Glu Thr Ala Glu Cys Pro Asn

130 135 140

Thr Asn Arg Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr Gly Phe Gly

145 150 155 160

Val Phe Thr Thr Asn Ile Trp Leu Lys Leu Lys Glu Lys Gln Asp Val

165 170 175

Phe Cys Asp Ser Lys Leu Met Ser Ala Ala Ile Lys Asp Asn Arg Ala

180 185 190

Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser Ala Leu Asn Asp Thr

195 200 205

Trp Lys Ile Glu Lys Ala Ser Phe Ile Glu Val Lys Asn Cys His Trp

210 215 220

Pro Lys Ser His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Glu Met

225 230 235 240

Ile Ile Pro Lys Asn Leu Ala Gly Pro Val Ser Gln His Asn Tyr Arg

245 250 255

Pro Gly Tyr His Thr Gln Ile Thr Gly Pro Trp His Leu Gly Lys Leu

260 265 270

Glu Met Asp Phe Asp Phe Cys Asp Gly Thr Thr Val Val Val Thr Glu

275 280 285

Asp Cys Gly Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly

290 295 300

Lys Leu Ile Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu

305 310 315 320

Arg Tyr Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro

325 330 335

Leu Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala

340 345 350

<210> 20

<211> 351

<212> PRT

<213> DENV3-NS1

<400> 20

Met Gly Cys Val Ile Asn Trp Lys Gly Lys Glu Leu Lys Cys Gly Asn

1 5 10 15

Gly Ile Phe Val Thr Asn Glu Val His Thr Trp Thr Glu Gln Tyr Lys

20 25 30

Phe Gln Ala Asp Ser Pro Lys Arg Leu Ala Thr Ala Ile Ala Gly Ala

35 40 45

Trp Glu Asn Gly Val Cys Gly Ile Arg Ser Thr Thr Arg Met Glu Asn

50 55 60

Leu Leu Trp Lys Gln Ile Ala Asn Glu Leu Asn Tyr Ile Leu Trp Glu

65 70 75 80

Asn Asn Ile Lys Leu Thr Val Val Val Gly Asp Ile Thr Gly Val Leu

85 90 95

Glu Gln Gly Lys Arg Thr Leu Thr Pro Gln Pro Met Glu Leu Lys Tyr

100 105 110

Ser Trp Lys Thr Trp Gly Lys Ala Lys Ile Val Thr Ala Glu Thr Gln

115 120 125

Asn Ser Ser Phe Ile Ile Asp Gly Pro Asn Thr Pro Glu Cys Pro Ser

130 135 140

Ala Ser Arg Ala Trp Asn Val Trp Glu Val Glu Asp Tyr Gly Phe Gly

145 150 155 160

Val Phe Thr Thr Asn Ile Trp Leu Lys Leu Arg Glu Met Tyr Thr Gln

165 170 175

Leu Cys Asp His Arg Leu Met Ser Ala Ala Val Lys Asp Glu Arg Ala

180 185 190

Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser Gln Lys Asn Gly Ser

195 200 205

Trp Lys Leu Glu Lys Ala Ser Leu Ile Glu Val Lys Thr Cys Thr Trp

210 215 220

Pro Lys Ser His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Asp Met

225 230 235 240

Ile Ile Pro Lys Ser Leu Ala Gly Pro Ile Ser Gln His Asn Tyr Arg

245 250 255

Pro Gly Tyr His Thr Gln Thr Ala Gly Pro Trp His Leu Gly Lys Leu

260 265 270

Glu Leu Asp Phe Asn Tyr Cys Glu Gly Thr Thr Val Val Ile Thr Glu

275 280 285

Asn Cys Gly Thr Arg Gly Pro Ser Leu Arg Thr Thr Thr Val Ser Gly

290 295 300

Lys Leu Ile His Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu

305 310 315 320

Arg Tyr Met Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro

325 330 335

Ile Asn Glu Lys Glu Glu Asn Met Val Lys Ser Leu Val Ser Ala

340 345 350

<210> 21

<211> 351

<212> PRT

<213> DENV4-NS1

<400> 21

Met Gly Cys Val Val Ser Trp Asn Gly Lys Glu Leu Lys Cys Gly Ser

1 5 10 15

Gly Ile Phe Val Ile Asp Asn Val His Thr Arg Thr Glu Gln Tyr Lys

20 25 30

Phe Gln Pro Glu Ser Pro Ala Arg Leu Ala Ser Ala Ile Leu Asn Ala

35 40 45

His Lys Asp Gly Val Cys Gly Val Arg Ser Thr Thr Arg Leu Glu Asn

50 55 60

Val Met Trp Lys Gln Ile Thr Asn Glu Leu Asn Tyr Val Leu Trp Glu

65 70 75 80

Gly Gly His Asp Leu Thr Val Val Ala Gly Asp Val Lys Gly Val Leu

85 90 95

Thr Glu Gly Lys Arg Ala Leu Thr Pro Pro Val Asn Asp Leu Lys Tyr

100 105 110

Ser Trp Lys Thr Trp Gly Lys Ala Lys Ile Phe Thr Leu Glu Ala Arg

115 120 125

Asn Ser Thr Phe Leu Ile Asp Gly Pro Asp Thr Ser Glu Cys Pro Asn

130 135 140

Glu Arg Arg Ala Trp Asn Phe Leu Glu Val Glu Asp Tyr Gly Phe Gly

145 150 155 160

Met Phe Thr Thr Asn Ile Trp Met Lys Phe Arg Glu Gly Ser Ser Ser Glu

165 170 175

Val Cys Asp His Arg Leu Met Ser Ala Ala Ile Lys Asp Gln Lys Ala

180 185 190

Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser Ser Lys Asn Gln Thr

195 200 205

Trp Gln Ile Glu Lys Ala Ser Leu Ile Glu Val Lys Thr Cys Leu Trp

210 215 220

Pro Lys Thr His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Gln Met

225 230 235 240

Leu Ile Pro Arg Ser Tyr Ala Gly Pro Phe Ser Gln His Asn Tyr Arg

245 250 255

Gln Gly Tyr Ala Thr Gln Thr Met Gly Pro Trp His Leu Gly Lys Leu

260 265 270

Glu Ile Asn Phe Gly Glu Cys Pro Gly Thr Thr Val Ala Ile Gln Glu

275 280 285

Asp Cys Gly His Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly

290 295 300

Lys Leu Val Thr Gln Trp Cys Cys Arg Ser Cys Ala Met Pro Pro Leu

305 310 315 320

Arg Phe Leu Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro

325 330 335

Leu Ser Glu Lys Glu Glu Asn Met Val Lys Ser Gln Val Thr Ala

340 345 350

Claims (10)

1. Antibodies against yellow fever virus NS1 non-structural proteins, characterized in that the antibodies include YD40 and/or YB36 antibodies; Wherein, the YD40 antibody comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region of the amino acid sequence shown in SEQ ID NO:2; The YB36 antibody comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:3, and a light chain variable region with the amino acid sequence shown in SEQ ID NO:4. 2. The antibody according to claim 1, wherein the YD40 or YB36 antibody further comprises: the heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 5 and the amino acid sequence shown in SEQ ID NO: 6 light amino acid sequence Chain constant region. 3. The antibody according to claim 1, wherein the antibody light chains are all κ-type light chains. 4. A nucleic acid molecule, characterized in that it encodes the antibody according to any one of claims 1-3. 5. An expression vector, characterized in that it comprises the expression vector of the nucleic acid molecule according to claim 4. 6. A yellow fever virus detection kit, characterized in that the kit includes any one of claims 1-3 Antibody. 7. The yellow fever virus detection kit according to claim 6, wherein the kit includes the YD40 and YB36 antibodies of any one of claims 1-3, wherein the YD40 is used as a detection antibody with a significant Color label; the YB36 was immobilized and used as capture antibody. 8. The yellow fever virus detection kit according to claim 7, further comprising Solid phase carrier for antibody, sample treatment solution, positive control, negative control, chromogenic solution and stop solution. 9. A method for non-disease diagnosis and treatment purpose, detection of yellow fever virus or its NS1 protein in a sample, characterized in that the method comprises the following steps: Mix the sample to be tested with the antibody of any one of claims 1-3, incubate, and detect yellow fever virus or its NS1 protein existence. 10. Application of the antibody according to any one of claims 1-3 and/or the kit according to any one of claims 6-8 in the preparation of products for detection and diagnosis of yellow fever virus.

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