CN113651886B

CN113651886B - High-sensitivity yellow fever virus humanized monoclonal antibody and application thereof

Info

Publication number: CN113651886B
Application number: CN202110864016.7A
Authority: CN
Inventors: 严景华; 高福; 李燕; 马素芳; 仵丽丽
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2018-04-04
Filing date: 2018-04-04
Publication date: 2022-04-15
Anticipated expiration: 2038-04-04
Also published as: CN110343172B; CN110343172A; CN113372441B; CN113651885B; CN113583117B; CN113429479A; CN113651886A; CN113651885A; CN113372441A; CN113583117A; CN113429479B

Abstract

The invention discloses a high-sensitivity yellow fever virus humanized monoclonal antibody and application thereof, belonging to the technical field of medicines. The invention uses yellow fever virus E protein expressed by colon bacillus as antigen, selects memory B cell which can specifically combine with yellow fever virus E protein from PBMCs of a convalescent patient by flow sorting, then carries out RT-PCR and PCR amplification on the selected single B cell to obtain variable region segment of antibody, and further connects the variable region segment and constant region into an expression vector. The 6-strain humanized antibody obtained by the invention has strong YFV neutralization activity, the IC50 value reaches 0.03-3.5 ug/mL, and the mouse can be completely or partially protected from the attack of lethal dose YFVCHina. The 6-strain humanized antibody has the application value of clinically treating or preventing YFV infection.

Description

High-sensitivity yellow fever virus humanized monoclonal antibody and application thereof

The invention relates to a high-sensitivity yellow fever virus humanized monoclonal antibody and divisional application thereof, wherein the original application number is CN201810302091.2, the original application date is 04.04.2018, and the application name is high.

Technical Field

The invention relates to a high-sensitivity yellow fever virus humanized monoclonal antibody and application thereof, belonging to the technical field of medicines.

Background

Yellow Fever Virus (YFV), a single-stranded positive-strand RNA virus, belonging to the flaviviridae family of flaviviridae, is a mosquito-borne pathogen that causes human morbidity, and includes zika virus (ZIKV), dengue virus (dengue virus, DENV), West Nile Virus (WNV), and the like. YFV is an important pathogen causing yellow fever, and in severe cases, it causes hemorrhagic fever with multiple organ failure, especially in the liver, spleen, lymph nodes, heart, and kidney.

In 1996, scientists estimated that yellow fever virus causes 20 million infections and 30 million deaths each year in africa and south america. In recent two years, yellow fever outbreaks occur in brazil, angora and Congo democratic republic, hundreds of people die with a mortality rate of 14%, and 11 cases are imported in 2016 in China. Currently, attenuated vaccines (YFV 17D) are available clinically, but vaccine shortages and inadequate vaccination rates lead to frequent outbreaks of disease, and non-immunized individuals remain at risk. There are no clinically effective specific drugs available to treat this disease after YFV infection.

To date, neutralizing antibodies have proven to be an effective method of treating viral diseases, including Human Immunodeficiency Virus (HIV), influenza, and other flaviviruses, among others. The flavivirus surface E protein (Envelope) recognizes receptors on the cell surface and facilitates membrane fusion of the viral membrane to the cell membrane, a process that is completed by three distinct domains (DI, DII and DIII). Therefore, the E protein is an important epitope for neutralizing antibody action generated by the immune system of the body.

Some human antibodies have been found to have neutralizing activity, and can neutralize some yellow fever virus strains before 2001, such as: 5A, 7A, R3(27) and the like can neutralize Central African Republic (CAR) 1986, Ethiopia 1961, Senegal1990, Nigeria1987, Ghana 1927 (Asibi) and the vaccine strain YFV 17D. However, RNA viruses are characterized by high mutations under antibody pressure, and although some neutralizing antibodies have been identified, more new antibodies directed against different epitopes are essential for therapy. The aim of the invention is to identify specific novel YFV neutralizing antibodies with protective effect.

Disclosure of Invention

In order to solve the problems, the invention firstly uses YFV-E protein expressed by Escherichia coli as antigen, selects memory B cells which can specifically bind to the YFV-E protein from PBMCs of a convalescent YFV patient through flow sorting, then carries out RT-PCR and PCR amplification on the selected single B cells to obtain variable region fragments of 6 strains of antibodies, and further connects the variable region fragments and a constant region into an expression vector. After the sequencing is correct, a series of function detection including the detection of the binding force with YFV-E protein, the in vitro neutralization effect, the in vivo protective capability and the like is carried out through the expression and purification of mammalian cells.

A first object of the present invention is to provide an antibody as set forth in any one of (1) to (6):

(1) the antibody is named as YD2, and the heavy chain variable region of the antibody contains an amino acid sequence shown by SEQ ID NO. 1, and the light chain variable region of the antibody contains an amino acid sequence shown by SEQ ID NO. 2;

(2) the antibody is named as YD25, and the heavy chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 3, and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 4;

(3) the antibody is named as YD62, and the heavy chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 5, and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 6;

(4) the antibody is named as YD86, and the heavy chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 7, and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 8;

(5) the antibody is named as YD97-1, the heavy chain variable region of the antibody contains an amino acid sequence shown by SEQ ID NO. 9, and the light chain variable region of the antibody contains an amino acid sequence shown by SEQ ID NO. 10;

(6) the antibody is named as YD97-2, and the heavy chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 11, and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 12.

In one embodiment of the invention, the heavy chain of the antibody comprises a heavy chain variable region and a heavy chain constant region, wherein the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO. 25, and the nucleotide sequence is set forth in SEQ ID NO. 27.

In one embodiment of the invention, the light chain of the antibody is a kappa chain comprising a light chain variable region and a light chain constant region; the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 26, and the nucleotide sequence is shown as SEQ ID NO. 28.

In one embodiment of the invention, the amino acid sequence of the heavy chain variable region of YD2 is SEQ ID NO 1 and the amino acid sequence of the light chain variable region is SEQ ID NO 2; the amino acid sequence of the heavy chain variable region of YD25 is SEQ ID NO 3 and the amino acid sequence of the light chain variable region is SEQ ID NO 4; the amino acid sequence of the heavy chain variable region of YD62 is SEQ ID NO 5 and the amino acid sequence of the light chain variable region is SEQ ID NO 6; the amino acid sequence of the heavy chain variable region of YD86 is SEQ ID NO. 7 and the amino acid sequence of the light chain variable region is SEQ ID NO. 8; the amino acid sequence of the heavy chain variable region of YD97-1 is SEQ ID NO 9 and the amino acid sequence of the light chain variable region is SEQ ID NO 10; the amino acid sequence of the heavy chain variable region of YD97-2 is SEQ ID NO 11 and the amino acid sequence of the light chain variable region is SEQ ID NO 12.

In one embodiment of the invention, the nucleotide sequence of the heavy chain of YD2 is SEQ ID NO 13 and the nucleotide sequence of the light chain is SEQ ID NO 14.

In one embodiment of the invention, the nucleotide sequence of the heavy chain of YD25 is SEQ ID NO 15 and the nucleotide sequence of the light chain is SEQ ID NO 16.

In one embodiment of the invention, the nucleotide sequence of the heavy chain of YD62 is SEQ ID NO 17 and the nucleotide sequence of the light chain is SEQ ID NO 18.

In one embodiment of the invention, the nucleotide sequence of the heavy chain of YD86 is SEQ ID NO 19 and the nucleotide sequence of the light chain is SEQ ID NO 20.

In one embodiment of the invention, the nucleotide sequence of the heavy chain of YD97-1 is SEQ ID NO 21 and the nucleotide sequence of the light chain is SEQ ID NO 22.

In one embodiment of the invention, the nucleotide sequence of the heavy chain of YD97-2 is SEQ ID NO. 23 and the nucleotide sequence of the light chain is SEQ ID NO. 24.

The 6 antibodies of the invention are derived from the same patient, all target the envelope protein E protein which is specific to yellow fever virus, and inhibit the infection of the virus to cells by inhibiting the receptor combination and/or the membrane fusion process which are mediated by the E protein.

The second purpose of the invention is to provide the application of the antibody in preparing medicines.

In one embodiment of the invention, the medicament is a medicament for the treatment and/or prevention of yellow fever virus.

The third purpose of the invention is to provide a pharmaceutical composition, which contains the human monoclonal antibody YD2, YD25, YD62, YD86, YD97-1 or YD 97-2.

In one embodiment of the present invention, the pharmaceutical composition comprises at least 2 of the human monoclonal antibodies YD2, YD25, YD62, YD86, YD97-1 and YD 97-2.

In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

The fourth purpose of the invention is to provide a kit for immunodiagnosis or detection, wherein the kit contains an antigen of any one of the human monoclonal antibodies YD2, YD25, YD62, YD86, YD97-1 and YD97-2, or a DNA molecule encoding the antigen, or a recombinant vector/an expression cassette/a transgenic cell line/a recombinant bacterium for expressing the antigen.

It is a fifth object of the present invention to provide gene sequences encoding the human monoclonal antibodies YD2, YD25, YD62, YD86, YD97-1 and YD 97-2.

In one embodiment of the invention, the nucleotide sequence of the heavy chain constant region of the antibody is set forth in SEQ ID NO. 27.

In one embodiment of the invention, the nucleotide sequence of the light chain constant region of the antibody is set forth in SEQ ID NO 28.

In one embodiment of the invention, the sequence encoding the heavy chain of the antibody comprises a CMV promoter sequence, an EcoR I cleavage site sequence, a leader sequence, a sequence encoding the heavy chain variable region, a sequence encoding the heavy chain constant region, and an Xho I cleavage site sequence, in that order.

In one embodiment of the invention, the sequence encoding the light chain of the antibody comprises a CMV promoter sequence, a Sac I cleavage site sequence, a leader sequence, a sequence encoding the variable region of the light chain, a sequence encoding the constant region of the light chain, and a cleavage site sequence Xho I in that order.

In one embodiment of the present invention, the first or second cleavage site sequence includes but is not limited to EcoR I, Xho I, Sac I or Xho I cleavage site sequence.

In one embodiment of the invention, the amino acid sequence of the leader sequence is shown in SEQ ID NO. 29 and the nucleotide sequence encoding the leader sequence is shown in SEQ ID NO. 30.

It is a sixth object of the present invention to provide a vector containing the gene sequence of the antibody or a cell expressing the antibody.

The invention has the beneficial effects that:

the invention obtains 6 strains of human YFV antibody with high neutralizing activity: YD2, YD25, YD62, YD86, YD97-1 and YD 97-2. The 6 antibodies are completely different from the reported YFV antibody sequences and are 6 newly discovered antibodies. The 6 humanized antibodies have strong YFV neutralization activity IC50 value reaching 0.03-3.5 ug/mL, and can completely or partially protect mice from being attacked by YFV China with lethal dose. The 6-strain humanized antibody has the application value of clinically treating or preventing YFV infection.

Drawings

FIG. 1: the result of the YFV China-E protein purification molecular sieve and SDS-PAGE;

FIG. 2: after Protein A is purified, an antibody SDS-PAGE result;

FIG. 3: the kinetic curve result of the antibody and YFV China-E;

FIG. 4: results of neutralization curves of antibodies against YFV China;

FIG. 5: protective effect of antibody on YFV China-E infected mice; a is the body weight change of the surviving mice, and B is the survival rate of the mice.

Detailed Description

Example 1: expression and purification of yellow fever virus E protein

The DNA fragment of the extracellular domain of YFV CNYF01/2016(YFV-China) strain membrane protein E protein (the amino acid sequence is shown in SEQ ID NO:31, and the nucleotide sequence is shown in SEQ ID NO: 32) was digested with NdeI and XhoI, and then ligated to pET21a vector. Wherein the 3' end of the YFV E protein coding region is linked to a coding sequence for a 6 histidine-tag (hexa-His-tag) and a translation stop codon. The ligation product was then transformed into BL21 E.coli competent cells. The single clone was inoculated into 40 mL of LB medium and cultured for 6-8 hours. Inoculated into 4L of LB medium, 37^oC culturing to OD₆₀₀=0.4-0.6, add IPTG to final concentration 1mM, 37^oC continued the culture for 4-6 hours. The inclusion bodies were harvested and renatured by dilution. The renaturation solution is changed into 20mM Tris, 150 mM NaCl, pH8.0 buffer solution and 10% glycerol after being concentrated. The concentrated protein solution was further purified by size exclusion chromatography using AKTA-purifier (GE) and superdex200 Hiload 16/60 column (GE) with buffer A (20 mM Tris, 150 mM NaCl, pH8.0, 10% glycerol) while monitoring the UV absorbance at 280 nm, recovering the protein of interest and identifying the protein purity by SDS-PAGE. Through identification, the high-purity E monomer protein with the size of 43kDa can be obtained. The results are shown in FIG. 1.

Example 2: isolation of specific memory B cells binding to YFV China-E protein

With patient informed consent, 20mL of blood was collected and PBMCs were isolated. Isolating the PBMCs at 10⁷Density per mL was combined with incubation of YFV-E protein at a final concentration of 400nM on ice for half an hour, followed by washing 2 times with PBS and incubation with the following antibodies: anti-human CD3/PE-Cy5, anti-human CD16/PE-Cy5, anti-human CD235a/PE-Cy5, anti-human CD19/APC-Cy7, anti-human CD27/Pacific Blue, anti-human CD38/APC, anti-human IgG/FITC, and anti-His/PE. After half an hour incubation on ice, the antibodies were washed 2 times with PBS. PE-Cy5 collected by FACSAria III sorting^- APC^- APC-Cy7⁺ Pacific Blue⁺ FITC⁺ PE⁺Of (2) cellsDirectly collected into 96-well plates, 1 cell/well.

Example 3: single B cell PCR, sequence analysis and design of humanized antibody

The B cells obtained in example 2 were reverse-transcribed by Superscript III reverse transcriptase (Invitrogen) primers shown in Table 1 (sequences shown by SED ID number 33 to SED ID number 40), and reacted at 55 ℃ for 60 min.

TABLE 1 reverse transcription primers

Primer and method for producing the same	5 '-3' sequence
		IgM-RT	ATG GAG TCG GGA AGG AAG TC
IgD-RT	TCA CGG ACG TTG GGT GGT A
		IgE-RT	TCA CGG AGG TGG CAT TGG A
IgA1-RT	CAG GCG ATG ACC ACG TTC C
		IgA2-RT	CAT GCG ACG ACC ACG TTC C
IgG-RT	AGG TGT GCA CGC CGC TGG TC
		Cκ-new RT	GCA GGC ACA CAA CAG AGG CA
Cλ-new-ext	AGG CCA CTG TCA CAG CT

Using this reverse transcription product as a template, PCR was performed using HotStar Tap Plus enzyme (QIAgen) to amplify an antibody variable region sequence (PCRa). Designing corresponding primers, wherein the reaction conditions are as follows: 95 ℃ for 5 min; 95 ℃ 30s, 55 ℃ (heavy chain/kappa chain)/50 ℃ (lambda chain) 30s, 72 ℃ 90s, 35 cycles; 72 ℃ for 7 min. This was used as template for a second round of PCR (PCRb) under the following conditions: 95 ℃ for 5 min; 95 ℃ 30s, 58 ℃ (heavy chain)/60 ℃ (kappa chain)/64 ℃ (lambda chain) 30s, 72 ℃ 90s, 35 cycles; 72 ℃ for 7 min.

1.2% agarose gel electrophoresis, and separating the PCR product. The size of the band is 400-500bp after the gel cutting recovery, and the band is sent to a sequencing company for sequencing. The sequencing results were analyzed using NCBI online software.

Analysis of the correct variable region sequence and the corresponding heavy chain/kappa chain/lambda chain constant region through bridging PCR connection, cloned into the expression vector pCAGGS. Wherein the heavy chain is linked to the lambda chain with EcoRI and XhoI and the kappa chain is linked to XhoI with SacI. B cell sequencing and eukaryotic cell expression plasmid construction strategies are as follows:

the human antibody design strategy is as follows:

heavy chain: CMV promoter-EcoR I-Leader sequences-heavy chain variable region-C_H-Xho I；

Light chain (κ): CMV promoter-Sac I-Leader sequences-light chain variable region-C_L(κ)-Xho I；

Wherein, the amino acid sequence of the Leader sequences is shown as SEQ ID NO. 29 (the nucleotide sequence is shown as SEQ ID NO. 30).

Example 4: expression and purification of antibodies

293T cells were cultured in DMEM with 10% FBS. 293T was co-transfected with plasmids containing the genes encoding the light and heavy chains of the particular antibodies constructed in example 3. After 4-6 hours of transfection, the medium was changed to serum-free DMEM for another 7 days, and the supernatant was collected.

The collected supernatant was centrifuged at 5000rpm for 30min, mixed with an equal volume of buffer containing 20mM sodium phosphate (pH 8.0), filtered through a 0.22 μm filter and bound to a protein A pre-column (5 mL, GE Healthcare). Bound protein was eluted with 0.1M glycine (pH 3.0). The protein is collected, concentrated and then subjected to molecular sieve chromatography. The peak of interest was determined by SDS-PAGE, and the results are shown in FIG. 2, indicating that the target protein was normally expressed.

Finally, 6 antibodies YD2, YD25, YD62, YD86, YD97-1 and YD97-2 which can be combined with YFV China-E protein and have strong neutralizing activity are obtained. The specific gene rearrangement pattern is shown in Table 2 below

TABLE 2 antibody Gene rearrangements

Example 5: performance testing of human antibodies

(1) Surface plasmon resonance technology detection and YFV-E binding capacity

Surface plasmon resonance analysis was performed using Biacore T100 (Biacore Inc.). The method comprises the following specific steps:

first, an antibody against anti-human IgG was immobilized in an amino-coupled manner on a channel (flow cell, Fc) of a CM5 chip. The fixed amount is controlled around a response value (RU) of 10,000. The purified antibody is combined in an antibody capture mode, wherein the liquid flow speed is controlled at 10 mu L/min, the sample injection is carried out for 1min, and the antibody capture amount is about 60 RU. And diluting YFV China-E protein by 10 mM HEPES, 150 mM NaCl and pH 7.4 solution in a multiple ratio, regulating the flow rate to 30 mu L/min, sequentially passing through each channel, and loading the YFV-E protein one by one from low concentration. The curves constitute the kinetic curves shown in figure 3. The results are shown in Table 3. The calculation of binding kinetic constants was performed using BIAevaluation software T100 (Biacore, Inc.). The SPR result shows that 6 strains of antibodies can be combined with YFV-E protein.

TABLE 3 kinetic constants for binding of antibodies to YFV China-E protein

Abs	ka (1/Ms)	kd (1/s)	KD (M)
				YD2	4.85E+05	9.96E-06	2.05E-11
YD25	5.78E+10	4.21E+02	7.28E-09
				YD62	2.02E+05	3.66E-04	1.81E-09
YD86	2.57E+05	2.83E-04	1.10E-09
				YD97-1	1.08E+04	4.44E-03	4.12E-07
YD97-2	5.92E+02	2.89E-03	4.88E-06

(2) Neutralization test

The purified antibody was diluted 3 fold, mixed with YFV (C6/36 amplification) diluted 1:60, 37^oC incubation for 60 min. The mixture was then added to 24-well plates, 300. mu.L/well, which had been plated with Vero cells. 37^oAfter 1 hour incubation for C, each well was supplemented with 0.7mL of media (DMEM, 10% FBS), and the incubation was continued for 40 hours before staining. The cells were collected, treated with 4% paraformaldehyde, 0.05% soponin in PBS, and left on ice in the dark for 30 min. The cells were then washed 2 times with solution (PBS, 1% BSA, 0.01% soponin), incubated with 2. mu.g/mL Z6-FITC antibody on ice for 30min, washed 2 times with solution, and the cell positive ratio was determined using FACSCANTO. The neutralizing capacity of antibodies to YFV was calculated from the positive ratio at different concentrations, as shown in fig. 4, and the statistics are shown in table 4.

TABLE 4 neutralizing Effect of antibodies on YFV China

MAbs	IC50 (ug/ml)
		YD2	0.22
YD25	3.49
		YD62	0.33
YD86	0.031
		YD97-1	0.07517
YD97-2	0.06248

(3) Animal protection test

Female Balb/c mice (Witongli Hua) at 3 weeks of age were divided into 4-5 groups. Each mouse was injected intracranial with 80 PFU virus YFV China. A single dose of 10mg/kg antibody or an equal volume of PBS was injected intraperitoneally 24 hours after infection in infected mice. Survival and weight changes of mice over 14 days were recorded. Mice that changed more than 25% in body weight or developed signs of paralysis were sacrificed. The results are shown in FIG. 5. Mice injected with YD2 antibody began to die by day 11, survival by day 16 was 66.7%, and surviving mice recovered in body weight. Mice injected with YD25 antibody began to die by day 10 with a survival rate of 66.7% by day 16. Mice injected with YD86 antibody began to die by day 11 with a survival rate of 66.7% by day 16. Mice injected with YD97-2 antibody began to die by day 11 with a 80% survival rate by day 16. Mice injected with YD62 and YD97-1 antibodies began to die by day 11 with a 100% survival rate by day 16. The 6 antibody protection rates are summarized in table 5, with 4 mice injected with control antibody beginning to die at day 8 post-infection and all die at day 11 (fig. 5).

TABLE 5 therapeutic Effect of the antibodies on YFV China infected mice

Abs	Survival rate
		YD2	66.7%
YD25	66.7%
		YD62
	100%
		YD86	66.7%
YD97-1	100%
		YD97-2	80%

At present, the human antibodies against yellow fever virus have been found to be 5A, 7A, R3(27), 1A, 2A, R3(9), and the gene rearrangement mode is: VH4-59, VH3-11, VK1-12, VL1-19 (SEQ ID NOS: AY661699, AY661700, AY661701, AY661702, AY661703 and AY661704, respectively, in GenBank): wherein 5A, 7A, R3(27) can neutralize partial strain and vaccine YFV 17D before 2001, and neutralize active IC₅₀About 1-10 ug/ml. No in vivo animal experiment verifies its function. IC of 6 strains of humanized antibody in this experiment₅₀3.5 to 0.03. mu.g/mL. Also, in animal protection experiments, the antibody dose used in the present invention was 10mg/kg, where YD97-1 could completely protect lethal dose challenged mice. The protection rate of YD2, YD25, YD86 and YD97-1 on mice is over 60 percent.

In conclusion, the invention obtains 6 humanized YFV antibodies with high neutralizing activity by screening YFV China-E specific combined memory B cells of rehabilitation patients: YD2, YD25, YD62, YD86, YD97-1 and YD 97-2. The 6 antibodies are completely different from the reported yellow fever antibody sequences, are newly discovered antibodies and have strong yellow fever virus neutralizing activity. Also, mice can be fully or partially protected from a lethal dose of yellow fever virus. This suggests that the 6-strain humanized antibody has clinical application value in treating and preventing yellow fever.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Tyr Asp Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Asp Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Asp Ile Lys

100 105

<210> 9

<211> 126

<212> PRT

<213> Artificial sequence

<400> 9

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ala Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala His Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Val Thr Thr Asp Thr Thr Thr Arg Thr Ala Ser

65 70 75 80

Met Glu Leu Arg Asn Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Pro Trp Glu Tyr Asn Tyr Arg Ser Ser Gly Tyr Tyr Asp

100 105 110

Ser Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 10

<211> 107

<212> PRT

<213> Artificial sequence

<400> 10

Glu Ile Val Leu Thr Gln Ser Pro Pro Leu Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Gly Gln Gly Ile Ser Tyr Ser

20 25 30

Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Asp Leu Leu Val

35 40 45

Tyr Asp Ser Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Lys Thr Phe Pro His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Asp Val Lys

100 105

<210> 11

<211> 126

<212> PRT

<213> Artificial sequence

<400> 11

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ala Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala His Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Val Thr Thr Asp Thr Thr Thr Arg Thr Ala Ser

65 70 75 80

Met Glu Leu Arg Asn Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Pro Trp Glu Tyr Asn Tyr Arg Ser Ser Gly Tyr Tyr Asp

100 105 110

Ser Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 12

<211> 107

<212> PRT

<213> Artificial sequence

<400> 12

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ile Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Glu Ala Ser Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Gly Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 13

<211> 357

<212> DNA

<213> Artificial sequence

<400> 13

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcgtc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc atcagctggg tgcgacaggc ccccggacaa 120

gggcttgagt ggatgggaag gatcatccca atttttggta gcccacacta cgcacacaaa 180

ttccaggaca gagtcacgat cacggcggac aaactcacga acacagccta catggagttg 240

agtagcctga gttctgagga cacggccatg tattactgtg cgagactgtc cgactatgat 300

aatcgtggta ataactttga ctactggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 14

<211> 321

<212> DNA

<213> Artificial sequence

<400> 14

gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

ttcacctgcc gggcaagtca ggacattgga aatcgtttag gctggtatca gcagaaacca 120

ggggaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggctc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240

gcggattttg caacttattt ttgtctacag catgatagtt acccacggac attcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 15

<211> 369

<212> DNA

<213> Artificial sequence

<400> 15

gaccaggtgc agctggtgca gtctggggct gaggtgaaga agcctgggtc ctcggtgagg 60

gtctcctgcg aggcttctgg aggcaccttc agcagttatg ctattagttg gctgcgacag 120

gcccctggac aaggacttga gtggatggga aggatcaccc ctatttttga tatagcagac 180

tattcacaga agttccaggg cagactcacc tttaccgcgg acaaatccac gaacacagcg 240

tacatggaac tgagcagcct gagatctgac gacacggccg tctattactg tgcgcacctt 300

atggtttggg gagttaacgg agagtccttt gatatgtggg gccaagggac catggtcacc 360

gtctcctca 369

<210> 16

<211> 321

<212> DNA

<213> Artificial sequence

<400> 16

gaaattgtgt tgacacagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattgga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctattct gcatccagtt tgcagagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatgaat accctcgaac gttcggccaa 300

gggaccaagg tggaagtcaa a 321

<210> 17

<211> 360

<212> DNA

<213> Artificial sequence

<400> 17

gaggtgcagc tggtggagtc tgggggaggc ttgggccagc cgggggggtc cctgagactc 60

tcctgcgcag cctctggatt cagttttagt agctattgga tgggctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtggccaac ataaagcaag aaggaaatga gaaatactat 180

gtggactcag tgaagggccg attcaccatc tccagagaca acaccaagaa ctcaatgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgttt attactgtgc gagagatatg 300

ggatatggga gtgatgctta tgatatctgg ggccaaggga caatggtcac cgtctcctca 360

<210> 18

<211> 318

<212> DNA

<213> Artificial sequence

<400> 18

gacatcgtga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc aactatttga attggtatca gcaaagacca 120

gggagagccc ctaagctcct gatctactct gcatccactt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag acttacgaaa tctggacgtt cggccaaggg 300

accaaggtgg aaatcaaa 318

<210> 19

<211> 357

<212> DNA

<213> Artificial sequence

<400> 19

caggtgcagc tgcaggagtc ggggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgtag gctcaggatt cagtttcagt gtctatggaa tacactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtgacagta atatcctatg atggcagtaa taaacagtac 180

gcagactccg tgaagggtcg attcgccatc tccagagaca atgacaagaa cacggtgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagtcgagca 300

gtgggtggtg attcggatga ctattggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 20

<211> 321

<212> DNA

<213> Artificial sequence

<400> 20

gaaattgtgt tgacacagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

ggaaaagccc ctaagcgcct gatctatgat gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatgatt acccgtacac ttttggccag 300

gggaccaagc tggacatcaa a 321

<210> 21

<211> 378

<212> DNA

<213> Artificial sequence

<400> 21

gaagtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgtaagg cttctggcta cacctttatc gcctatggta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcagcgctc acaacggtaa cacaaactat 180

gcacagaagt tccagggcag agtcaccgtg accacagaca caaccacgag aacagcctcc 240

atggaactgc ggaacctgag atctgacgac acggccgtgt actactgtgc gcgagctcct 300

tgggagtata attacaggag tagtggttat tacgactcgc cctactgggg ccagggaacc 360

ctggtcaccg tctcctca 378

<210> 22

<211> 321

<212> DNA

<213> Artificial sequence

<400> 22

gaaattgtgt tgacacagtc tccacccctc ctgtctgcat ctgtcggaga cagagtcacc 60

atcacttgcc gggccggtca 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> 23

<211> 378

<212> DNA

<213> Artificial sequence

<400> 23

gaagtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgtaagg cttctggcta cacctttatc gcctatggta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcagcgctc acaacggtaa cacaaactat 180

gcacagaagt tccagggcag agtcaccgtg accacagaca caaccacgag aacagcctcc 240

atggaactgc ggaacctgag atctgacgac acggccgtgt actactgtgc gcgagctcct 300

tgggagtata attacaggag tagtggttat tacgactcgc cctactgggg ccagggaacc 360

ctggtcaccg tctcctca 378

<210> 24

<211> 321

<212> DNA

<213> Artificial sequence

<400> 24

gaaattgtgt tgacacagtc tccatcctct ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc aggcgagtca ggacattagc atctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctacgag gcatccagtt tggaaacagg ggtcccatca 180

aggttcagtg gaggtgggtc tgggacagat ttcactttca ccatcagcag cctgcagcct 240

gaagatattg caacatatta ctgtcaacag tatgataatc tccctctcac tttcggccct 300

gggaccaaag tggatatcaa a 321

<210> 25

<211> 330

<212> PRT

<213> Artificial sequence

<400> 25

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

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

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

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

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 26

<211> 108

<212> PRT

<213> Artificial sequence

<400> 26

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Ser

100 105

<210> 27

<211> 990

<212> DNA

<213> Artificial sequence

<400> 27

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 accaggtcag 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 990

<210> 28

<211> 324

<212> DNA

<213> Artificial sequence

<400> 28

cgaactgtgg ctgcaccaag cgtgtttatc 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 cagc 324

<210> 29

<211> 21

<212> PRT

<213> Artificial sequence

<400> 29

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp

20

<210> 30

<211> 63

<212> DNA

<213> Artificial sequence

<400> 30

atggagacgg atacgctgct cctgtgggtt ttgctgctgt gggttccagg ttccactggt 60

gac 63

<210> 31

<211> 405

<212> PRT

<213> Artificial sequence

<400> 31

Met Ala His Cys Ile Gly Ile Thr Asp Arg Asp Phe Ile Glu Gly Val

1 5 10 15

His Gly Gly Thr Trp Val Ser Ala Thr Leu Glu Gln Asp Lys Cys Val

20 25 30

Thr Val Met Ala Pro Asp Lys Pro Ser Leu Asp Ile Ser Leu Gln Thr

35 40 45

Val Ala Ile Asp Gly Pro Ala Glu Ala Arg Lys Val Cys Tyr Ser Ala

50 55 60

Val Leu Thr His Val Lys Ile Asn Asp Lys Cys Pro Ser Thr Gly Glu

65 70 75 80

Ala His Leu Ala Glu Glu Asn Asp Gly Asp Asn Ala Cys Lys Arg Thr

85 90 95

Tyr Ser Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly

100 105 110

Ser Ile Val Ala Cys Ala Lys Phe Thr Cys Ala Lys Ser Met Ser Leu

115 120 125

Phe Glu Val Asp Gln Thr Lys Ile Gln Tyr Val Ile Arg Ala Gln Leu

130 135 140

His Val Gly Ala Lys Gln Glu Asn Trp Asn Thr Asp Ile Lys Thr Leu

145 150 155 160

Lys Phe Asp Ala Leu Ser Gly Ser Gln Glu Ala Glu Phe Thr Gly Tyr

165 170 175

Gly Lys Ala Thr Leu Glu Cys Gln Val Gln Thr Ala Val Asp Phe Gly

180 185 190

Asn Ser Tyr Ile Ala Glu Met Glu Lys Asp Ser Trp Ile Val Asp Arg

195 200 205

Gln Trp Ala Gln Asp Leu Thr Leu Pro Trp Gln Ser Gly Ser Gly Gly

210 215 220

Ile Trp Arg Glu Met His His Leu Val Glu Phe Glu Pro Pro His Ala

225 230 235 240

Ala Thr Ile Arg Val Leu Ala Leu Gly Asn Gln Glu Gly Ser Leu Lys

245 250 255

Thr Ala Leu Thr Gly Ala Met Arg Val Thr Lys Asp Glu Asn Asp Asn

260 265 270

Asn Leu Tyr Lys Leu His Gly Gly His Val Ser Cys Arg Val Lys Leu

275 280 285

Ser Ala Leu Thr Leu Lys Gly Thr Ser Tyr Lys Met Cys Thr Asp Lys

290 295 300

Met Ser Phe Val Lys Asn Pro Thr Asp Thr Gly His Gly Thr Val Val

305 310 315 320

Met Gln Val Lys Val Pro Lys Gly Ala Pro Cys Lys Ile Pro Val Ile

325 330 335

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

340 345 350

Val Asn Pro Ile Ala Ser Thr Asn Asp Asp Glu Val Leu Ile Glu Val

355 360 365

Asn Pro Pro Phe Gly Asp Ser Tyr Ile Ile Val Gly Thr Gly Asp Ser

370 375 380

Arg Leu Thr Tyr Gln Trp His Lys Glu Gly Ser Ser Ile Gly Lys His

385 390 395 400

His His His His His

405

<210> 32

<211> 1215

<212> DNA

<213> Artificial sequence

<400> 32

atggcacatt gcatcggcat taccgaccgc gatttcatcg agggtgtgca tggtggtaca 60

tgggtgagtg caaccctgga acaggataaa tgcgtgaccg tgatggcccc ggataagcct 120

agtctggata ttagcctgca gaccgtggcc attgatggtc cggcagaagc ccgtaaagtg 180

tgctacagcg ccgttctgac ccacgtgaag atcaacgaca agtgccctag cacaggcgaa 240

gcccatctgg cagaggagaa cgacggtgat aacgcctgta aacgcaccta cagcgaccgt 300

ggctggggta atggctgcgg cctgtttggc aagggtagca ttgtggcctg cgcaaaattc 360

acctgcgcca agagcatgag tctgttcgag gtggaccaga ccaagattca gtatgtgatc 420

cgcgcccagc tgcacgtggg cgcaaagcag gagaactgga acaccgacat caagaccctg 480

aagttcgatg ccctgagcgg cagccaagaa gccgagttta caggttacgg caaggcaacc 540

ctggagtgtc aagtgcagac cgcagtggat ttcggtaata gctatattgc cgagatggag 600

aaagacagct ggatcgtgga tcgccagtgg gcccaagatc tgaccctgcc gtggcagagc 660

ggtagtggtg gcatttggcg cgaaatgcat catctggtgg agtttgagcc gccgcatgcc 720

gcaaccattc gtgtgctggc cctgggcaat caggaaggca gcctgaaaac cgccctgaca 780

ggcgccatgc gcgtgaccaa agacgaaaac gataataatc tgtacaagct gcatggtggc 840

cacgtgagct gccgcgtgaa gctgagcgcc ctgaccctga aaggcaccag ctacaagatg 900

tgtacagaca aaatgagctt cgttaagaat ccgaccgata ccggccacgg caccgtggtg 960

atgcaggtta aggttccgaa aggcgcaccg tgcaaaatcc cggtgattgt tgccgatgac 1020

ctgaccgccg ccgtgaataa gggcattctg gtgaccgtga acccgatcgc aagcaccaac 1080

gatgatgagg tgctgatcga agtgaacccg ccttttggcg acagttacat catcgtgggt 1140

accggcgata gccgcctgac ctatcaatgg cacaaggaag gcagtagcat cggcaaacat 1200

catcaccacc accat 1215

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence

<400> 33

atggagtcgg gaaggaagtc 20

<210> 34

<211> 19

<212> DNA

<213> Artificial sequence

<400> 34

tcacggacgt tgggtggta 19

<210> 35

<211> 19

<212> DNA

<213> Artificial sequence

<400> 35

tcacggaggt ggcattgga 19

<210> 36

<211> 19

<212> DNA

<213> Artificial sequence

<400> 36

caggcgatga ccacgttcc 19

<210> 37

<211> 19

<212> DNA

<213> Artificial sequence

<400> 37

catgcgacga ccacgttcc 19

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence

<400> 38

aggtgtgcac gccgctggtc 20

<210> 39

<211> 20

<212> DNA

<213> Artificial sequence

<400> 39

gcaggcacac aacagaggca 20

<210> 40

<211> 17

<212> DNA

<213> Artificial sequence

<400> 40

aggccactgt cacagct 17

Claims

1. The monoclonal antibody against yellow fever virus E protein is characterized in that the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO. 9, and the amino acid sequence of a light chain variable region is shown as SEQ ID NO. 10.

2. The antibody of claim 1, wherein the heavy chain of said antibody comprises a heavy chain variable region and a heavy chain constant region; the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO. 25.

3. The antibody of claim 1 or 2, wherein the light chain of the antibody is a kappa chain comprising a light chain variable region and a light chain constant region; the amino acid sequence of the light chain constant region is shown in SEQ ID NO 26.

4. A pharmaceutical composition comprising the antibody of any one of claims 1 to 3.

5. Use of an antibody according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment and/or prophylaxis of yellow fever virus.

6. A DNA encoding the antibody according to any one of claims 1 to 3.

7. DNA according to claim 6, characterized in that the nucleotide sequence encoding the heavy chain of the antibody comprises a CMV promoter sequence, a leader sequence, a sequence encoding the variable region of the heavy chain and a sequence encoding the constant region of the heavy chain; sequences encoding the light chain of the antibody include a CMV promoter sequence, a leader sequence, a sequence encoding the variable region of the light chain, and a sequence encoding the constant region of the light chain.

8. A vector comprising the DNA according to claim 6 or 7.

9. A host cell comprising the vector of claim 8.