High-sensitivity yellow fever virus humanized monoclonal antibody and application thereof
阅读说明：本技术 一种高灵敏度的黄热病毒人源单克隆抗体及其应用 (High-sensitivity yellow fever virus humanized monoclonal antibody and application thereof ) 是由 严景华 高福 李燕 马素芳 仵丽丽 于 2018-04-04 设计创作，主要内容包括：本发明公开了一种高灵敏度的黄热病毒人源单克隆抗体及其应用,属于医药技术领域。本发明以大肠杆菌表达的黄热病毒E蛋白作为抗原,通过流式分选,从一例康复期患者的PBMCs中筛选到可以特异结合黄热病毒E蛋白的记忆B细胞,然后对筛选的单一B细胞进行RT-PCR及PCR扩增,获得抗体的可变区片段,并进一步与恒定区连接至表达载体中。本发明获得的6株人源抗体均有很强的YFV中和活性,IC50值达0.03～3.5ug/mL,可以完全或部分保护小鼠免受致死剂量的YFVChina的攻击。本发明的6株人源抗体有着临床治疗或预防YFV感染的应用价值。(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.)
1. An antibody, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 7 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 8.
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 heavy chain constant region comprises an amino acid sequence 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 light chain constant region has an amino acid sequence 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 cell expressing the antibody of any one of claims 1 to 3.
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 the ProteinA is purified, the result of antibody SDS-PAGE is obtained;
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 40mL of LB medium and cultured for 6-8 hours. Inoculating to 4L LB medium, and culturing at 37 deg.C to OD600When the concentration was 0.4 to 0.6, IPTG was added to a final concentration of 1mM, and the culture was continued at 37 ℃ for 4 to 6 hours. The inclusion bodies were harvested and renatured by dilution. The renaturation solution is changed into 20mM Tris, 150mM 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 (20mM Tris, 150mM NaCl, pH8.0, 10% glycerol) while monitoring the UV absorbance at 280nm to recover the protein of interest and identify 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+The cells of (4) were collected directly into a 96-well plate at 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 No.33 to SED ID No. 40), and reacted at 55 ℃ for 60 min.
TABLE 1 reverse transcription primers
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: CMVpromoter-EcoR I-Leader sequences-heavy chain variable region-CH-Xho I；
Light chain (κ): CMVpromoter-Sac I-Leader sequences-light chain variable region-CL(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 proteinA pre-column (5mL, 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 10mM HEPES, 150mM 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
(2) Neutralization test
The purified antibody was diluted 3-fold, mixed with YFV (C6/36 amplified) diluted 1:60, and incubated at 37 ℃ for 60 minutes. The mixture was then added to 24-well plates, 300. mu.L/well, which had been plated with Vero cells. After incubation at 37 ℃ for 1 hour, each well was supplemented with 0.7mL of medium (DMEM, 10% FBS), and 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
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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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp
20 25 30
Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile
35 40 45
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