CN113683691B

CN113683691B - Flavivirus antibodies and uses thereof

Info

Publication number: CN113683691B
Application number: CN202110955389.5A
Authority: CN
Inventors: 高福; 严景华; 李燕; 仵丽丽; 王奇慧; 马素芳
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2021-08-19
Filing date: 2021-08-19
Publication date: 2023-05-23
Anticipated expiration: 2041-08-19
Also published as: CN113683691A

Abstract

A flavivirus antibody and uses thereof. The invention discloses a flavivirus antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in a sequence table. Also disclosed are nucleotide sequences encoding the heavy and light chain variable regions, the use of the antibodies of the invention in the preparation of medicaments or reagents for detecting, treating and/or preventing flaviviruses, and pharmaceutical compositions or reagents comprising the antibodies of the invention. The antibodies have high affinity for flaviviruses.

Description

Flavivirus antibodies and uses thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to a flavivirus antibody and application thereof.

Background

Flaviviruses fall into three general categories based on the transmission vector: mosquito-borne flaviviruses, tick-borne flaviviruses, and flaviviruses without known transmission vectors. Yellow fever virus, zika virus, west Nile virus, japanese encephalitis virus and dengue virus all belong to mosquito-borne flavivirus, and tick-borne encephalitis virus and jumping disease virus all belong to tick-borne flavivirus. Yellow fever virus infection mainly causes fever, muscle pain, headache, chills, inappetence, nausea and vomiting, severe fever, huang Qi abdominal pain and vomiting accompanied by death; dengue fever infection causes clinical manifestations such as hyperpyrexia, headache, muscle bone joint pain and the like, and is one of main causes of death of children in southeast Asia: west Nile virus infection can cause aseptic meningitis and even death when severe.

The current laboratory methods for diagnosing flavivirus infection mainly comprise virus isolation, nucleic acid detection, antigen detection, serological detection and the like. The most reliable diagnosis method at present mainly comprises the steps of extracting viral RNA and then carrying out qRT-PCR, and the method is time-consuming, high in cost and needs to be operated by professionals in a specific laboratory, and the used instrument is complex and cannot be widely and conveniently applied. Thus, there is a great need to develop a product and method that is simple to operate, can be detected early in infection, and can be used in remote areas.

Recently, it has been reported that non-structural protein 1 (nonstructural protein, NS1) of flavivirus is a relatively conserved glycoprotein, has two forms of membrane type and secretion, has high expression and long time in infected body, and is a good target for detecting virus infection.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a flavivirus antibody which can be combined with a flavivirus NS1 protein and has higher affinity and better stability.

A flavivirus antibody comprising an amino acid sequence of any one of (1) - (4) below:

(1) As set forth in amino acid sequence SEQ ID NO:1, such as the amino acid sequence of SEQ ID NO:2, and a light chain variable region of an antibody shown in seq id no;

(2) As set forth in amino acid sequence SEQ ID NO:3, such as the amino acid sequence of SEQ ID NO:4, an antibody light chain variable region shown in figure 4;

(3) As set forth in amino acid sequence SEQ ID NO:5, such as the amino acid sequence of SEQ ID NO:6, an antibody light chain variable region shown in figure 6;

(4) As set forth in amino acid sequence SEQ ID NO:7, such as the amino acid sequence of SEQ ID NO: 8.

The antibody described above, wherein the amino acid sequence of SEQ ID NO:9, such as the amino acid sequence of SEQ ID NO:10, and a light chain constant region of an antibody.

The antibody, wherein the coding nucleotide sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO:11, the coding nucleotide sequence of the antibody light chain constant region is shown as SEQ ID NO: shown at 12.

The antibody, wherein the antibody further comprises a signal peptide sequence, and the amino acid sequence of the signal peptide is shown in SEQ ID NO:13, the coding nucleotide sequence of the signal peptide sequence is shown as SEQ ID NO: 14.

The antibody of above, wherein the amino acid light chains are all K-type light chains.

A nucleic acid molecule of the invention, wherein the nucleic acid molecule comprises a nucleotide sequence encoding an antibody as described above, said nucleotide sequence being as follows:

(1) As set forth in nucleotide sequence SEQ ID NO:15, such as the amino acid heavy chain variable region shown in nucleotide sequence SEQ ID NO:16, an amino acid light chain variable region shown in seq id no;

(2) As set forth in nucleotide sequence SEQ ID NO:17, such as the amino acid heavy chain variable region shown in nucleotide sequence SEQ ID NO:18, an amino acid light chain variable region shown in seq id no;

(3) As set forth in nucleotide sequence SEQ ID NO:19, such as the amino acid heavy chain variable region shown in nucleotide sequence SEQ ID NO:20, an amino acid light chain variable region shown in seq id no;

(4) As set forth in nucleotide sequence SEQ ID NO:21, such as the amino acid heavy chain variable region shown in nucleotide sequence SEQ ID NO:22, and an amino acid light chain variable region shown in seq id no.

The invention also comprises expression vectors for the nucleic acid molecules described above.

The invention also comprises a host cell of the expression vector described above.

A pharmaceutical composition of the invention comprising an antibody as described in any of the above.

A detection reagent according to the present invention, comprising an antibody according to any one of the preceding claims.

A test kit according to the invention, comprising an antibody according to any one of the preceding claims.

The use of any of the above antibodies in the preparation of a medicament or agent for detecting, treating and/or preventing a flavivirus.

The use as described above wherein the flavivirus comprises four serotypes of yellow fever virus, zika virus, west nile virus and dengue virus.

Advantageous effects

The flavivirus antibody provided by the invention has high affinity with the flavivirus, has high application value for the detection of the flavivirus and the preparation of detection products, provides a novel method for detecting the flavivirus, and can rapidly and accurately finish the detection.

Drawings

FIG. 1 is a diagram showing the result of affinity chromatography and SDS-PAGE identification of yellow fever virus YFV-NS1 protein;

FIG. 2 is a diagram showing the result of affinity chromatography and SDS-PAGE identification of ZIKV-NS1 protein of Zika virus;

FIG. 3 is a diagram showing the result of affinity chromatography and SDS-PAGE identification of the serotype dengue virus DV1-NS1/DV2-NS1/DV3-NS1/DV4-NS1 proteins;

FIG. 4 is a diagram showing the result of affinity chromatography and SDS-PAGE identification of WN-NS1 protein of West Nile virus;

FIG. 5 is a graph showing the results of Western Blotting and SDS-PAGE identification of antibody proteins;

FIG. 6 is a graph showing the results of measurement of binding activity of a flavivirus antibody;

FIG. 7 is a graph of the results of determining the affinity of the flavivirus antibodies.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.

Example 1 preparation of human antibodies

1. Expression and purification of yellow fever virus nonstructural protein 1 (YFV-NS 1)

The extracellular region gene fragment of YFV-NS1 is cloned into pFastBac I vector through Nde I and Xho I, and transformed into E.coli DH10 to obtain recombinant plasmid Bacmid-NS1. Then, sf9 cells were transfected to obtain baculovirus containing NS1 gene. After infection of Hi5 cells by baculovirus, the NS1 protein can be expressed secreted into the culture supernatant. Purification was performed by means of affinity chromatography and molecular sieve chromatography, and protein purity was identified by SDS-PAGE. The results show that the NS1 protein exists in monomeric and dimeric form, the monomer size is 43kDa (as shown in SEQ ID NO: 23), and the results are shown in FIG. 1. The YFV-NS1 is concentrated to 1mg/mL and frozen for storage at-80 ℃.

Other flaviviridae viruses WNV-NS1, ZIKV-NS1, DENV-NS1 (DV 1-NS1/DV2-NS1/DV3-NS1/DV4-NS 1) were constructed, expressed and purified with reference to YFV-NS1 described above.

2. Expression and purification of other flavivirus NS1 proteins

Other flaviviridae viruses: ziKV-NS1 (about 45kDa in size, shown in SEQ ID NO:24, shown in FIG. 2), four serotypes of dengue virus DENV-NS1 (DV 1-NS1/DV2-NS1/DV3-NS1/DV4-NS1 proteins, 45kDa/75kDa/43kDa/43kDa in size, shown in FIG. 3, shown in SEQ ID NO: 25-28), west Nile virus WNV-NS1 (45 kDa in size, shown in SEQ ID NO:29, shown in FIG. 4), and protein expression purification procedures refer to the YFV-NS1 protein expression purification method described above.

3. Preparation of humanized monoclonal antibodies

1 Single cell sorting and cloning construction

Peripheral Blood Mononuclear Cells (PBMCs) of two persons infected with YFV for 6 months are isolated, and various blood cell-specific labeled antibodies and specific YFV-NS1 antigens are added for incubation under informed consent. The YFV-NS1 positive memory B cells were collected into 96-well plates, 1 cell per well, by FACSaria II (BD Biosciences) sorting.

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

The PCR products were separated by agarose gel electrophoresis, PCR bands having a molecular weight of about 400bp were extracted using a gel extraction kit (CWBIO), and the purified products were sequenced. The obtained target sequence was analyzed using IMGT (international ImMunoGeneTics) information system and IgBlast. The variable region sequence and the corresponding constant region are connected by bypass PCR, and cloned into an expression vector pCAGGS, wherein the heavy chain and the lambda chain adopt EcoRI and XhoI cleavage sites, and the kappa chain adopts SacI and XhoI cleavage sites.

2 protein expression and purification

HEK-293T cells were transfected with heavy and light chain construction plasmids. The culture was performed 6 hours after transfection with medium replaced with fresh serum-free medium. Cell culture supernatants were collected on days 3 and 7. The collected supernatant was mixed with an equal volume of buffer containing 20mM sodium phosphate (pH 7.0), centrifuged at 6500rpm for 30min to remove fragments, filtered through a 0.22um filter, and passed through a HiTrap Protein A affinity chromatography column, followed by elution of bound antibody using 0.1M glycine (pH 3.0). Collecting the eluent, concentrating, and performing molecular sieve chromatography. The peak of interest was determined by SDS-PAGE, as shown in FIG. 5, to isolate 11 antibodies, heavy chain of about 45kDa and light chain of about 25kDa, indicating normal expression of the target protein.

Example 2 functional identification of antibodies

1. ELISA for detecting binding Activity of antibodies

1) Antigen was diluted 2. Mu.g/mL (YFV-NS 1, YFV-NS 1C terminal, WNV-NS1, ZIKV-NS1, DENV1-NS1, DENV2-NS1, DENV3-NS1, DENV4-NS 1) with coating solution, added to the ELISA plate at 100. Mu.L/well, and allowed to stand at 4℃overnight.

2) The next day, 200. Mu.L/well of PBST was added, and after 5 plate washes with a plate washer (BIO-TEK, 405 LS), 5% nonfat dry milk blocking solution was added, 100. Mu.L/well, and incubated in a incubator at 37℃for 1 hour.

3) PBST was rinsed 5 times, and incubated with antibodies YB31, YB44, YD4 or YD33, 400 ng/well, at 37℃for 1 hour.

4) PBST was rinsed 5 times, and HPR-labeled goat anti-human IgG was incubated at 100. Mu.L/well for 45min at 37 ℃.

5) PBST is rinsed 5 times, TMB color development solution is added, 100 mu L/hole is added, the reaction is carried out for 2 minutes at room temperature in dark place, and then 2M H2SO is added ₄ The reaction was stopped at 100. Mu.L/well.

6) OD values at 450nm wavelength were detected and the original data was saved.

Results: the results of the results shown in FIG. 6 indicate that the flavivirus antibodies YB31, YB44, YD4 or YD33 of the invention can be combined with YFV-NS1/DV1-NS1/DV2-NS1/DV3-NS1/DV4-NS1/WN-NS1 proteins, and have higher combination activity. Does not bind the ZIKV-NS1 protein.

2. Binding affinity assay of antibodies YB31, YB44, YD4, YD33 to anti-YFV-NS 1

The experiment uses BIAcore ^TM The 8K machine is complete. The method comprises the following specific steps:

first, an anti-human secondary antibody was immobilized in an amino-coupled manner on a channel (Fc) of a CM5 chip. The fixed amount is controlled around 10,000 Response Units (RU). The YB31, YB44, YD4 or YD33 is immobilized in a mode of capturing the antibody, and the capturing amount is about 60 RU. The YFV-NS1 protein was diluted in a buffer (10mM HEPES,150mMNaCl,pH 7.4) to a multiple ratio and flowed through each channel sequentially at a flow rate of 30. Mu.L/min. Binding kinetic constants were calculated using BIAeva1 station software (Biacore, inc.).

The results of SPR showed that both antibodies could bind to the YFV-NS1 protein (FIG. 7). The binding constant (ka), dissociation constant (kD) and equilibrium dissociation constant (kD) of each interaction obtained are shown in table 1.

TABLE 1

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

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ccagggaagg ggctggagtg cgtctcacat attactggta atggagttac acattacgca 180

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac actgtatctg 240

caaatgaaca gcctgagagt cgaggacacg gccgtatatt actgtgcgaa agaagtggca 300

tatagtgact acgattactc ggacgtctgg ggccaaggga cgacggtcac cgtctcctca 360

<210> 18

<211> 324

<212> DNA

<213> Artificial sequence (Synthetic sequences)

<400> 18

gacatccagt tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gagtattagt gcctggttgg cctggtatca gtataaacca 120

gggaaagccc cgaagctcct gatctatgag gcttctaatt tagaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240

gatgattttg caacttatta ctgccaacag tgttacactt attctcagtg ggcgttcggc 300

caagggacca aggtggacat caaa 324

<210> 19

<211> 375

<212> DNA

<213> Artificial sequence (Synthetic sequences)

<400> 19

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttccggata catcttcgat agccacgctg tgcattgggt gcgccaggcc 120

cccggtcaaa ggccagagtg gatgggatgg gtcaacgttg ccaatggtca gaggaagtat 180

tcagagaact tgcggggcag actcaccata attagggaca tatccgcgac cacagcctac 240

atggagctga ccagcctgag acctgaagat acggctatat attactgtgc gagagataac 300

ttagacggtg actacaacgc ctactactac ggcatggacg tctggggcca ggggaccacg 360

gtcaccgtct cctca 375

<210> 20

<211> 321

<212> DNA

<213> Artificial sequence (Synthetic sequences)

<400> 20

gaaattgtgt tgacgcagtc tccatccctc ctgtctgcat ctgtcggaga cagagtcacc 60

atcacttgcc gggccagtca gggcattagc tattctttag cctggtatcg gcaaaaacca 120

gggaaagccc ctgatctcct ggtctatgat tcatccactt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacaa cttaaaactt tccctcacac ttttggccag 300

gggaccaagc tggacgtcaa a 321

<210> 21

<211> 363

<212> DNA

<213> Artificial sequence (Synthetic sequences)

<400> 21

gaggttcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc actcagactc 60

tcctgtgtag cctctggatt cacctttagc agttatgcca tgacctgggt ccgccaggct 120

ccagggaggg ggctggagtg ggtctcatca attagtggaa atggtgttag cacatattac 180

gcagactccg tgcagggccg cttcaccatc tccagagaca aatccaagaa gacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgttt attactgtgc gaaggatgta 300

cactggaacg acgctggata ctgtgactcc tggggccagg gaactcttgt gaccgtctcc 360

tca 363

<210> 22

<211> 321

<212> DNA

<213> Artificial sequence (Synthetic sequences)

<400> 22

gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gagctttagt acctggttgg cctggtatca gcagaaacca 120

gggaaagcac ctaaggtcct gatctatgag gcgtctagat tagaaagtgg ggtcccatcg 180

aggttcagcg gcagtgaatc tgggacagaa ttcactctca ccatcagtag cttgcagcct 240

gatgattttg caacttatta ctgccaacaa tataatagtt atccactcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 23

<211> 351

<212> PRT

<213> Artificial sequence (Synthetic sequences)

<400> 23

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> 24

<211> 351

<212> PRT

<213> Artificial sequence (Synthetic sequences)

<400> 24

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> 25

<211> 351

<212> PRT

<213> Artificial sequence (Synthetic sequences)

<400> 25

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> 26

<211> 351

<212> PRT

<213> Artificial sequence (Synthetic sequences)

<400> 26

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> 27

<211> 351

<212> PRT

<213> Artificial sequence (Synthetic sequences)

<400> 27

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> 28

<211> 351

<212> PRT

<213> Artificial sequence (Synthetic sequences)

<400> 28

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 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

<210> 29

<211> 351

<212> PRT

<213> Artificial sequence (Synthetic sequences)

<400> 29

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 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

Claims

1. A flavivirus antibody, said antibody comprising:

as set forth in amino acid sequence SEQ ID NO: 1. the antibody heavy chain variable region shown, e.g., amino acid sequence SEQ ID NO: 2. the antibody light chain variable region is shown.

2. The antibody of claim 1, comprising an amino acid sequence as set forth in SEQ ID NO: 9. by a means of

The antibody heavy chain constant region is shown as SEQ ID NO: 10. the antibody light chain constant regions are shown.

3. The antibody of claim 1, wherein the amino acid light chains are all K-type light chains.

4. A nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding an antibody as described above.

5. An expression vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the expression vector of claim 5.

7. A detection reagent comprising the antibody of any one of claims 1 to 3.

8. A test kit comprising the antibody of any one of claims 1 to 3.

9. Use of an antibody according to any one of claims 1 to 3 for the preparation of a detection reagent.