Yellow fever virus antibody and application thereof
阅读说明：本技术 黄热病毒抗体及其应用 (Yellow fever virus antibody and application thereof ) 是由 高福 严景华 李燕 仵丽丽 王奇慧 马素芳 于 2021-08-19 设计创作，主要内容包括：本发明公开了黄热病毒抗体,包括重链可变区和轻链可变区,其中所述重链可变区及轻链可变区的氨基酸序列如序列表中所示。还公开了编码所述重链可变区和轻链可变区的核苷酸酸序列以及本发明抗体在制备用于检测、治疗和/或预防黄热病毒药物或试剂上的应用、及包含本发明抗体的药物组合物或试剂。该抗体对于黄热病毒具有高亲和力和特异性。(The invention discloses a yellow fever virus 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 discloses a nucleotide sequence for encoding the heavy chain variable region and the light chain variable region, application of the antibody in preparing a medicament or reagent for detecting, treating and/or preventing yellow fever virus, and a medicinal composition or reagent containing the antibody. The antibody has high affinity and specificity for yellow fever virus.)
1. An antibody against yellow fever virus, comprising the amino acid sequence of any one of (1) to (5) below:
(1) as shown in the amino acid sequence of SEQ ID NO: 1, such as the amino acid sequence of SEQ ID NO: 2;
(2) as shown in the amino acid sequence of SEQ ID NO: 3, such as the amino acid sequence of SEQ ID NO: 4, or a light chain variable region of an amino acid;
(3) as shown in the amino acid sequence of SEQ ID NO: 5, such as the amino acid sequence of SEQ ID NO: 6;
(4) as shown in the amino acid sequence of SEQ ID NO: 7, such as the amino acid sequence of SEQ ID NO: 8, or a light chain variable region of an amino acid shown in figure 8;
(5) as shown in the amino acid sequence of SEQ ID NO: 9, such as the amino acid sequence of SEQ ID NO: 10, or a light chain variable region of an amino acid sequence shown in seq id no.
2. The antibody of claim 1, wherein the amino acid sequence of SEQ ID NO: 11, as shown in amino acid sequence SEQ ID NO: 12, or a light chain constant region of an antibody.
3. The antibody of claim 1, wherein the amino acid light chains are all K-type light chains.
4. A nucleic acid molecule, wherein said nucleic acid molecule comprises a nucleotide sequence encoding said antibody.
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 pharmaceutical composition comprising the antibody of any one of claims 1 to 3.
8. A detection reagent comprising the antibody of any one of claims 1 to 3.
9. A test kit comprising the antibody of any one of claims 1 to 3.
10. Use of the antibody of any one of claims 1 to 3 in the preparation of a medicament, reagent or kit for detecting, treating and/or preventing yellow fever virus.
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to a yellow fever virus antibody and application thereof.
Background
Yellow Fever Virus (YFV) is an insect-borne infectious virus mediated by Aedes, and belongs to the Flaviviridae family of flaviviridae, equivalent to Zika virus (ZIKV), West Nile Virus (WNV), and dengue virus (DENV). The transmission medium of yellow fever virus is mosquitoes, and therefore, the incidence of yellow fever is high in areas where mosquitoes are prevalent and in developing countries. Currently, the prevalence is mainly in tropical and subtropical regions of africa and south america. Yellow fever virus can cause yellow fever which seriously harms human health, and is manifested as jaundice, bleeding, and even multiple systems of organ failure. Because the yellow fever symptoms are similar to those caused by the same family of viruses, and the yellow fever virus and other flaviviruses may be epidemic at the same time in the same area, the yellow fever symptoms are difficult to be clinically distinguished according to clinical symptoms. Furthermore, antibodies may be cross-reactive due to antigenically similar characteristics of viruses of the flaviviridae family. Furthermore, dengue virus also causes ADE (antibody-dependent enhancement), which further complicates clinical diagnosis. The above problems also present a great challenge to develop serological diagnostic methods for yellow fever.
The laboratory method for diagnosing yellow fever virus infection mainly comprises virus separation, nucleic acid detection, antigen detection, serology detection and the like. At present, the most reliable diagnosis method is mainly to perform qRT-PCR after viral RNA is extracted, the method is time-consuming and high in cost, operation needs to be performed by professional personnel in a specific laboratory, and used instruments are complex and cannot be widely and conveniently applied. Therefore, there is an urgent need to develop a product and method that is easy to operate, can detect early infection, and can be used in remote areas.
The nonstructural protein 1 (NS 1) of yellow fever virus is a relatively conserved glycoprotein, has two forms of membrane type and secretory type, has high expression and long time in an infected organism, and is a good target point for detecting yellow fever virus infection.
Disclosure of Invention
In order to solve the above technical problems, the present invention aims to provide a yellow fever virus antibody, which can bind to the NS1 protein of yellow fever virus, and has high affinity, specificity and stability.
An antibody against yellow fever virus, comprising an amino acid sequence of any one of (1) to (5) below:
(1) as shown in the amino acid sequence of SEQ ID NO: 1, such as the amino acid sequence of SEQ ID NO: 2;
(2) as shown in the amino acid sequence of SEQ ID NO: 3, such as the amino acid sequence of SEQ ID NO: 4, or a light chain variable region of an amino acid;
(3) as shown in the amino acid sequence of SEQ ID NO: 5, such as the amino acid sequence of SEQ ID NO: 6;
(4) as shown in the amino acid sequence of SEQ ID NO: 7, such as the amino acid sequence of SEQ ID NO: 8, or a light chain variable region of an amino acid shown in figure 8;
(5) as shown in the amino acid sequence of SEQ ID NO: 9, such as the amino acid sequence of SEQ ID NO: 10, or a light chain variable region of an amino acid sequence shown in seq id no.
The antibody of above, wherein the amino acid sequence of SEQ ID NO: 11, as shown in amino acid sequence SEQ ID NO: 12, or a light chain constant region of an antibody.
The antibody of above, wherein the coding nucleotide sequence of the antibody heavy chain constant region is as shown in SEQ ID NO: 13, and the coding nucleotide sequence of the antibody light chain constant region is shown as SEQ ID NO: as shown at 14.
The antibody of above, wherein the antibody further comprises a signal peptide sequence, and the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 15, and the coding nucleotide sequence of the signal peptide sequence is shown as SEQ ID NO: shown at 16.
The antibody of above, wherein the amino acid light chains are all K-type light chains.
The nucleic acid molecule of the invention comprises a nucleotide sequence encoding the antibody, wherein the nucleotide sequence is as follows:
(1) as shown in the nucleotide sequence of SEQ ID NO: 17, such as the amino acid heavy chain variable region shown in the nucleotide sequence SEQ ID NO: 18, or a light chain variable region of amino acids;
(2) as shown in the nucleotide sequence of SEQ ID NO: 19, such as the amino acid heavy chain variable region shown in a nucleotide sequence SEQ ID NO: 20, the light chain variable region of amino acids;
(3) as shown in the nucleotide sequence of SEQ ID NO: 21, such as the amino acid heavy chain variable region shown in a nucleotide sequence SEQ ID NO: 22;
(4) as shown in the nucleotide sequence of SEQ ID NO: 23, such as the amino acid heavy chain variable region shown in the nucleotide sequence SEQ ID NO: 24, the amino acid light chain variable region shown in seq id no;
(5) as shown in the nucleotide sequence of SEQ ID NO: 25, such as the amino acid heavy chain variable region shown in the nucleotide sequence SEQ ID NO: 26, or a light chain variable region of an amino acid shown in seq id no.
The invention also comprises an expression vector of the nucleic acid molecule.
The present invention also includes host cells of the above-described expression vectors.
The invention provides a pharmaceutical composition, which comprises the antibody.
The detection reagent of the present invention comprises the antibody according to any one of the preceding claims.
The detection kit of the present invention comprises the antibody of any one of the preceding claims.
The application of any one of the antibodies in preparation of drugs, reagents or kits for detecting, treating and/or preventing yellow fever viruses.
Advantageous effects
The yellow fever virus antibody provided by the invention can specifically recognize yellow fever virus, has no cross reaction with other viruses of the Flaviviridae family, has high affinity with the yellow fever virus, and has higher application value for detection of the yellow fever virus and preparation of a detection product.
Drawings
FIG. 1 is a diagram showing the results of affinity chromatography and SDS-PAGE identification of YFV-NS1 protein;
FIG. 2 is a view showing the results of affinity chromatography and SDS-PAGE identification of ZiKV-NS1 protein of Zika virus;
FIG. 3 is a diagram showing the results of affinity chromatography and SDS-PAGE identification of dengue virus serotype DV1-NS1/DV2-NS1/DV3-NS1/DV4-NS1 proteins;
FIG. 4 is a diagram showing the results of affinity chromatography and SDS-PAGE identification of WN-NS1 protein of West Nile virus;
FIG. 5 is a graph showing the results of the identification of antibody proteins by Western Blotting and SDS-PAGE;
FIG. 6 is a graph showing the results of measuring the binding activity of yellow fever virus antibodies;
FIG. 7 is a graph showing the results of measuring the affinity of yellow fever virus antibodies.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
EXAMPLE 1 preparation of human antibodies
Expression and purification of yellow fever virus nonstructural protein l (YFV-NS1)
The extracellular region gene fragment of YFV-NS1 was cloned into pFastBac I vector by Nde I and Xho I, transformed into E.coli DH10, and recombinant plasmid Bacmid-NS1 was obtained. Then, sf9 cells were transfected to obtain baculovirus containing the NS1 gene. After baculovirus infects Hi5 cells, NS1 protein can be secreted and expressed to culture supernatant. Purifying by affinity chromatography and molecular sieve chromatography, and identifying protein purity by SDS-PAGE. The results showed that NS1 protein exists in monomeric and dimeric form, with a monomer size of 43kDa, as shown in SEQ ID NO: 27, the results are shown in fig. 1. YFV-NS1 was concentrated to 1mg/mL and frozen for storage at-80 ℃ until use.
Expression and purification of NS1 protein of other flaviviridae
Other flaviviruses: west Nile virus WNV-NS1(WNV-NS1 monomeric protein, size 92kDa, shown in SEQ ID NO: 28, shown in FIG. 2), Zika virus ZIKV-NS1(ZIKV-NS1 monomeric protein, 90kDa, shown in SEQ ID NO: 29, shown in FIG. 3), four serotypes of dengue virus DENV-NS1(DV1-NS 1/DV2-NS1/DV3-NS1/DV4-NS1 monomeric proteins, size 92kDa/86kDa/43kDa/43kDa, shown in SEQ ID NO: 30-33, shown in FIG. 4) protein expression and purification process refer to the above YFV-NS1 protein expression and purification method.
Preparation of human monoclonal antibody
1 Single cell sorting and cloning construction
Under the condition of obtaining the informed consent of the convalescent, two convalescent persons infected with YFV for 6 months are sampled from the peripheral blood, the Peripheral Blood Mononuclear Cells (PBMCs) are separated, and various blood cell specific marker antibodies and specific YFV-NS1 antigens are added for incubation. YFV-NS1 positive memory B cells were collected into 96-well plates, 1 cell per well, by FACSAria II (BD Biosciences) sorting.
We amplified antibody variable region gene sequences (VH and VL) from single B cells by RT-PCR and nested PCR methods. First, the obtained B cells were subjected to reverse transcription by 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: 5min at 95 ℃; 95 ℃ 30s, 55 ℃ (VH and V)K) Or 50 ℃ (V lambda) 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: 5min at 95 ℃; 95 ℃ 30s, 58 ℃ (heavy chain)/60 ℃ (K chain)/64 ℃ (lambda chain) 30s, 72 ℃ 90s, 35 cycles; 7min at 72 ℃.
The PCR product was separated by agarose gel electrophoresis, and the PCR band of about 400bp molecular weight was extracted with a gel extraction kit (CWBIO), and the purified product was sequenced. The obtained target sequences were analyzed using the IMGT (international ImmunoGeneTiCs) information system and Igblast. The variable region sequence and the corresponding constant region are connected through bridging PCR and cloned into an expression vector pCAGGS, wherein EcoRI and XhoI cleavage sites are adopted for a heavy chain and a lambda chain, and SacI and XhoI cleavage sites are adopted for a kappa chain.
2 protein expression and purification
The heavy and light chain construction plasmids were transfected into HEK-293T cells. After 6 hours of transfection, the cells were cultured in a serum-free medium. On days 3 and 7, cell culture supernatants were collected. The collected supernatant was mixed with a buffer containing 20mM sodium phosphate (pH 7.0) in equal volume, centrifuged at 6500rpm for 30min to remove debris, filtered through a 0.22um filter, and the supernatant was passed through a HiTrap Protein A affinity chromatography column, followed by elution of bound antibody using 0.1M glycine (pH 3.0). Collecting eluate, concentrating, and performing molecular sieve chromatography. The target peak was subjected to SDS-PAGE to separate 11 antibodies, each having a heavy chain of about 45kDa and a light chain of about 25kDa, indicating that the target protein was normally expressed.
Example 2 functional characterization of antibodies
First, ELISA detects antibody specific binding YFV-NS11) antigen dilution 2 μ g/mL (YFV-NS1, YFV-NS 1C terminal, WNV-NS1, ZIKV-NS1, DENV1-NS1, DENV2-NS1, DENV3-NS1, DENV4-NS1) using coating solution, accelerated to ELISA plate, 100 μ L/well, 4 ℃ overnight.
2) The next day, PBST 200. mu.L/well was added, and after washing the plate 5 times with a plate washer (BIO-TEK, 405LS), 5% nonfat dry milk blocking solution was added, 100. mu.L/well was incubated at 37 ℃ for L hours.
3) PBST was rinsed 5 times, antibodies YB5, YB17, YB19, YB33 or YD30 were added at 400 ng/well, and incubated at 37 ℃ for 1 hour in an incubator.
4) PBST was rinsed 5 times and HPR-labeled goat anti-human IgG, 100. mu.L/well, incubated at 37 ℃ in an incubator for 45 min.
5) PBST was rinsed 5 times, TMB developing solution was added thereto, 100. mu.L/well was left dark at room temperature, reaction was carried out for 2 minutes, and then 2M H was added2SO4The reaction was stopped, 100. mu.L/well.
6) And detecting the OD value at the wavelength of 450nm, and storing and recording the original data.
The results are shown in fig. 6, and antibodies YB5, YB17, YB19, YB33 and YD30 are specific monoclonal antibodies of YFV; no binding to other flaviviridae NS1 proteins.
Secondly, the Surface Plasmon Resonance (SPR) technology is used for detecting the binding capacity of the antibody to YFV-NS1
BIAcore was used in this experimentTMAnd 8K machine completion. The method comprises the following specific steps:
first, an anti-human secondary antibody was immobilized in a channel (flow cell, Fc) of a CM5 chip by amino coupling. The fixed amount is controlled around a response value (RU) of 10,000. YB5, YB17, YB19, YB33 or YD30 were immobilized by antibody capture in an amount of about 60 RU. The YFV-NS1 protein was diluted in a buffer (10mM HEPES, 150mM NaCl, pH7.4) in two times and flowed through each channel in sequence at a flow rate of 30. mu.L/min. Binding kinetic constants were calculated using BIAevaluation software (Biacore, Inc.).
The results of SPR showed that the antibodies all bound to YFV-NS1 protein (FIG. 7). The resulting binding constant (ka), dissociation constant (kD), and equilibrium dissociation constant (kD) for each interaction are shown in table 1.
TABLE 1
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> institute of microbiology of Chinese academy of sciences
<120> yellow fever virus antibody and application thereof
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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 tga 993
<210> 14
<211> 327
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<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> 21
<212> PRT
<213> Artificial sequences (Synthetic sequences)
<400> 15
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> 16
<211> 63
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 16
atggagacgg atacgctgct cctgtgggtt ttgctgctgt gggttccagg ttccactggt 60
gac 63
<210> 17
<211> 372
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 17
gaggtgcagc tggtgcagtc tggaggtgag gtgaagaagc ctggggcctc agtgaaggtc 60
tcctgcaagg cttctggtta cacctttagc agttatggaa tcagctgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggatgg atcagcgttt ataaaaaagt taccacaaaa 180
tatgcacaga agttccaggg cagagtcacc atgaccgcag acacatccac gagcacagcg 240
tacatggaac tgaggagcct gagatctgac gacacggccg tgtattattg tgcgagagca 300
gaatttgtcc cacttttagc ccccggggac tttgactact ggggccaggg aaccctggtc 360
accgtctcct ca 372
<210> 18
<211> 336
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 18
gaaattgtgt tgacgcagtc tccactctct ctgtccgtcg cccctggaca gccggcctcc 60
atctcctgca agtctagtca gagcctcctg catagtgatg gaaaggccta tttgtattgg 120
ttcttgcaga agccaggcca gtctccacaa ctcctaatct atgaagcttc gagccggttc 180
tctggagtgc cagataggtt cactggcagc gggtcaggga cagatttcac actgaaaatc 240
agccgggtgg aggctgacga tgttggagtt tattattgca tgcaatgtat agaccttcct 300
ctcactttcg gcggagggac caaggtggag atcaaa 336
<210> 19
<211> 354
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 19
gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60
tcctgcaagg tttccggata caccctcact gaattttcca tacactgggt gcgacaggct 120
cctggaaaag ggcttgagtg gatgggaagt tttgatcctg aagatggtga aacaatctac 180
gtacagaagt tccagggcag agttaccatg accgcggaca catctacaga cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attattgtgc aagaagacgt 300
ggctacgatg ggctggacta ctggggccag ggaaccctgg tcaccgtctc ctca 354
<210> 20
<211> 321
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 20
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgttggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc aacaacttaa attggtatca gcagaaacca 120
gggagagccc ctaagttgct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gccctggatc tgggacatat ttcactctca ccatcaccgg tctgcaacct 240
gaagattttg caacttacta ctgtcaacag acttacagta ccccgtacac ttttggccag 300
gggaccaagc tggagatcaa a 321
<210> 21
<211> 375
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 21
gaggttcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60
tcctgcaagg cttctggaga caccttcagc agctatgcta tcagctgggt gcggcaggcc 120
cctggacaag ggcttgagtg gatgggaagg atcatccctg tccttgttac accaaactac 180
gcacagaagt tccagggcag agtcacgata accgcggaca aatccacgag cacagcctac 240
atggagctga gcagcctgac atctgaggac acggccgtgt attattgtgc gactaagata 300
ccagtggctg gcagaccact ttactatgat gcttttgata tctggggcca agggacaatg 360
gtcaccgtct cctca 375
<210> 22
<211> 321
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 22
gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcgagtca gggcattagg aattatttag cctggtatca gcagaaacca 120
gggaaagttc ctaaactcct gatctctgct gcatccactt tgcaatcagg ggtcccatct 180
cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagatgttg caacttatta ctgtcaaaag tataacagtg ccccgaacac tttcggccct 300
gggaccaaag tggatatcaa a 321
<210> 23
<211> 357
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 23
caggtgcagc tgcaggagtc gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60
acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120
ccagggaagg ggctggagtg gattggggaa atcaatcata ctggaagcac caactacaac 180
ccgtccctca agagtcgact caccatatca gtagacaggt ccaagaacca attctccctg 240
aagctgagct ctgtgaccgc cgcggacacg gctgtgtttt actgtgcgag atgggggtat 300
accagtggct ggtacgctga ctcctggggc cagggaaccc tggtcaccgt ctcctca 357
<210> 24
<211> 321
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 24
gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60
atcacttgtc gggcgagtca gggtattagc aggtggttag cctggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaaatgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttacta ttgtcaacag ggtgccggtt tccctcgggc gttcggccaa 300
gggaccaagg tggaaatcaa a 321
<210> 25
<211> 366
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 25
gaggttcagc tggtggagtc tgggggaggc gtggtccagc ctgggacgtc cctgagactc 60
tcctgtgcag cctctggatt caccttccgt agctatggca tgcactgggt ccgccaggct 120
cccggcaagg ggctggagtg ggtggcaatt atatcatatg atggaagaaa tacacactac 180
gcagattcag tgaagggccg attcaccatc tccagagaca atcccaagaa caagttgtat 240
ctgcaaatga acagcctgag aagtgaggac acggcttttt attactgtgc gagagatact 300
gcagcagctg gcgattgggg ggtctttgac tcctggggcc agggaaccct ggtcatcgtc 360
tcctca 366
<210> 26
<211> 324
<212> DNA
<213> Artificial sequences (Synthetic sequences)
<400> 26
gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc aggcgagtca ggacattacc tactatttga attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctttgat tcatccaatt tggaaccagg ggtcccatca 180
aggttcagtg gaagtggatc tggaacagat tttactttca ccatcagcag cctgcagcct 240
gaagatattg caacatatta ctgtcaacag tatgctaatg tccctccgta cacttttggc 300
caggggacca aactggagat caaa 324
<210> 27
<211> 351
<212> PRT
<213> Artificial sequences (Synthetic sequences)
<400> 27
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> 28
<211> 351
<212> PRT
<213> Artificial sequences (Synthetic sequences)
<400> 28
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
<210> 29
<211> 351
<212> PRT
<213> Artificial sequences (Synthetic sequences)
<400> 29
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> 30
<211> 351
<212> PRT
<213> Artificial sequences (Synthetic sequences)
<400> 30
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> 31
<211> 351
<212> PRT
<213> Artificial sequences (Synthetic sequences)
<400> 31
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> 32
<211> 351
<212> PRT
<213> Artificial sequences (Synthetic sequences)
<400> 32
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> 33
<211> 351
<212> PRT
<213> Artificial sequences (Synthetic sequences)
<400> 33
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
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