CN114805577B

CN114805577B - Antibody for IL-17RA protein, preparation method and application thereof

Info

Publication number: CN114805577B
Application number: CN202210438597.2A
Authority: CN
Inventors: 苏志鹏; 张云; 韩顶
Original assignee: Nanjing Rongjiekang Biotechnology Co ltd
Current assignee: Nanjing Rongjiekang Biotechnology Co ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2023-11-21
Anticipated expiration: 2039-12-31
Also published as: CN114805577A; CN111040035B; CN111040035A

Abstract

The application discloses an antibody aiming at IL-17RA protein, a preparation method and application thereof, and relates to the technical field of immunity, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of a heavy chain variable region of the antibody are selected from any one of (1) - (14), (16) - (19) or (21) - (24), so that a new way is provided for the research of autoimmune diseases or the drug development thereof, and the development of a new related therapeutic means of autoimmune diseases is facilitated.

Description

Antibody for IL-17RA protein, preparation method and application thereof

Technical Field

The application relates to the technical field of immunity, in particular to an antibody aiming at IL-17RA protein, a preparation method and application thereof.

Background

Interleukin 17receptor A (IL-17 receptor A, IL-17 RA) was first discovered in 1995 and was identified as Interleukin 17A (IL-17A) and Interleukin 17F (IL-17F) receptors. Subsequently, a series of other interleukin 17receptor family members were found, IL-17RB, IL-17RC, IL-17RD and IL-17RE, respectively, all of which 5 family members contained one and the same domain in sequence, but were quite different from other known, e.g., tumor Necrosis Factor (TNF) receptors, toll-like receptors (TLRs) and the type I/II Interferon (IFN) receptor family.

IL-17 receptor family members may be specifically recognized by specific ligands of IL-17 family members. The IL-17 family comprises 6 members, IL-17A, B, C, F, E and F. Typically, IL-17A and IL-17F will bind to a receptor complex consisting of IL-17RA and IL-17RC, IL-17B will bind to IL-17RB, IL-17C will bind to IL-17RE, and IL-17E (also known as IL-25) will bind to a receptor complex consisting of IL-17RA and IL-17RB (also known as IL-25R). One study in 2012 showed that IL-17RD may also be involved in IL-17A-mediated signaling pathways, but little is currently known about IL-17D.

The signaling pathway mediated by IL-17RA has two main pathways: IL-17A (F) signaling and IL-17E (IL-25) signaling. As known from the prior studies, IL-17A and IL-17F have the highest homology in sequence and can form IL-17A/A, IL17-F/F homodimers or IL1-7A/F heterodimers, which then bind to the IL-17RA/RC heterodimer receptor complex to activate downstream signaling. IL-17A and IL-17F have been recognized as important pro-inflammatory factors and trigger inflammatory signals such as NF-. Kappa. B, MAPKs and C/EBPs.

Currently, studies have been made to distinguish between the different biological activities of mice deficient in IL-17A/F, respectively. IL-17A showed an indirect pro-inflammatory effect in the mouse inflammation model. Whereas O' Connor et al studies showed that IL-17A deficient T cells could trigger an exacerbated condition in the mouse CD45RBhi metastatic enteritis model.

IL-17RA may activate multiple downstream inflammatory signaling pathways, including the NF-. Kappa.B pathway.

NF- κB is a typical inflammation-associated transcription factor, IκBα is an inhibitor of NF- κB, which can be phosphorylated and degraded by the p50 and p56 subunits. Interestingly, even et al found by microaray analysis that IL-17A/IL-17RA promoted expression of IκBζ, which was required for IL-6 expression, demonstrating that IL-17A synergistically promotes inflammatory responses with TNFα. In addition, IL-17RA activates MAPKs signaling and C/EBP signaling.

Still other studies suggest that the PI3K and JAK/STAT pathways are also involved in IL-17RA signaling.

At present, therapies targeting IL-17 signaling molecules are an important tool for treating patients with autoimmune diseases, which can alleviate very exaggerated inflammatory responses in patients, and multiple molecules in the IL-17 signaling pathway become targeting molecules for monoclonal antibodies, such as Ustekinumab and Briakiumab targeting IL-12/23p40, clazakizumab, olokizumab, sirukumab targeting IL-6, tocilizumab, sarilumab targeting IL-6R, secukinumab, ixekizumab targeting IL-17A.

Whereas only the monoclonal antibody Brodalumab, which targets IL-17RA, is currently in clinical study. Brodalumab is a fully humanized monoclonal antibody that binds to IL-17R, blocks the binding of various IL-17 cytokines (A, F, A/F and C) to receptors, inhibits inflammatory signaling, and the IL-17 pathway is a key role in the initiation and promotion of inflammatory processes, which was marketed by Valean pharmaceutical company, FDA, USA, at 15, 2, 2017 for use in the treatment of psoriasis (atopic dermatitis). However, the medicine is sunk into the wind wave which can cause the suicidal thoughts of patients at the clinical test stage once, and is only used for treating psoriasis as a second-line medicine at present. Clearly, this drug has problems as a therapeutic drug.

In view of this, the present application has been made.

Disclosure of Invention

The application aims at providing an antibody aiming at IL-17RA protein, a preparation method thereof, an isolated nucleic acid containing the antibody, a recombinant vector, a host cell and a kit for detecting IL-17 RA.

The application is realized in the following way:

in a first aspect, embodiments of the application provide antibodies to IL-17RA proteins,

the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody are selected from any one of (1) to (37):

(1) Sequences shown as SEQ ID No. 1-3;

(2) As shown in SEQ ID No. 4-6;

(3) As shown in SEQ ID No. 7-9;

(4) As shown in SEQ ID No. 10-12;

(5) As shown in SEQ ID No. 13-15;

(6) As shown in SEQ ID No. 16-18;

(7) As shown in SEQ ID No. 19-21;

(8) As shown in SEQ ID No. 22-24;

(9) As shown in SEQ ID No. 25-27;

(10) As shown in SEQ ID No. 28-30;

(11) Sequences shown as SEQ ID No. 31-33;

(12) As shown in SEQ ID No. 34-36;

(13) As shown in SEQ ID No. 37-39;

(14) As shown in SEQ ID No. 40-42;

(15) As shown in SEQ ID No. 43-45;

(16) As shown in SEQ ID Nos. 46 to 48;

(17) As shown in SEQ ID No. 49-51;

(18) As shown in SEQ ID No. 52-54;

(19) As shown in SEQ ID No. 55-57;

(20) As shown in SEQ ID No. 58-60;

(21) Sequences shown as SEQ ID No. 61-63;

(22) As shown in SEQ ID No. 64-66;

(23) As shown in SEQ ID No. 67-69;

(24) As shown in SEQ ID Nos. 70-72.

In a second aspect, embodiments of the application provide an isolated nucleic acid encoding an antibody against an IL-17RA protein as described in the previous embodiments.

In a third aspect, embodiments of the present application provide a recombinant vector comprising the isolated nucleic acid of the previous embodiments.

In a fourth aspect, embodiments of the present application provide a host cell comprising the recombinant vector of the previous embodiments.

In a fifth aspect, embodiments of the application provide a method of producing an antibody against an IL-17RA protein, comprising culturing a host cell as described in the previous embodiments to obtain an antibody against an IL-17RA protein.

In a sixth aspect, embodiments of the application provide a kit for detecting IL-17RA comprising an antibody directed against an IL-17RA protein as described in the previous embodiments.

In a seventh aspect, embodiments of the present application provide the use of an antibody against IL-17RA protein as described in the previous embodiments for the preparation of a medicament for inhibiting or preventing an autoimmune disease.

The application has the following beneficial effects:

the application provides a new anti-IL-17 RA antibody, provides a new way for the research of autoimmune diseases or the drug development thereof, and is helpful for developing new related therapeutic means of autoimmune diseases.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the SDS-PAGE analysis of the human recombinant IL-17RA protein of example 1;

FIG. 2 shows the results of a VHH fragment correct insertion analysis of a single domain antibody library against IL-17RA in example 1;

FIG. 3 is the result of target enrichment of specific antibodies against IL-17RA in a solid phase panning based on the constructed nanobody library against IL-17RA in example 1;

FIG. 4 is a SDS-PAGE analysis of the results of purification of a portion of the single domain antibodies screened in example 1 in a prokaryotic expression system;

FIG. 5 is a schematic diagram of a single domain antibody Fc fusion expression vector in example 1;

FIG. 6 shows the results of a primary affinity ELISA assay for a single domain antibody purified in example 2 and a target protein;

FIG. 7 is a graph showing the affinity of the single domain antibodies purified in example 3 to a target protein;

FIG. 8 shows the results of in vitro neutralizing cytokine release experiments with single domain antibodies.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Noun definition

The term "single domain antibody" as used herein refers to an antibody naturally lacking a light chain, which comprises only one heavy chain variable region (VHH), also known as nanobody.

"CDR" as referred to in this specification is the complementarity determining region of an antibody, which typically comprises two variable regions, a heavy chain variable region and a light chain variable region, which typically comprises 3 CDRs.

The present embodiments provide antibodies to IL-17RA proteins,

the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody are selected from any one of (1) to (24):

(1) Sequences shown as SEQ ID No. 1-3;

(2) As shown in SEQ ID No. 4-6;

(3) As shown in SEQ ID No. 7-9;

(4) As shown in SEQ ID No. 10-12;

(5) As shown in SEQ ID No. 13-15;

(6) As shown in SEQ ID No. 16-18;

(7) As shown in SEQ ID No. 19-21;

(8) As shown in SEQ ID No. 22-24;

(9) As shown in SEQ ID No. 25-27;

(10) As shown in SEQ ID No. 28-30;

(11) Sequences shown as SEQ ID No. 31-33;

(12) As shown in SEQ ID No. 34-36;

(13) As shown in SEQ ID No. 37-39;

(14) As shown in SEQ ID No. 40-42;

(15) As shown in SEQ ID No. 43-45;

(16) As shown in SEQ ID Nos. 46 to 48;

(17) As shown in SEQ ID No. 49-51;

(18) As shown in SEQ ID No. 52-54;

(19) As shown in SEQ ID No. 55-57;

(20) As shown in SEQ ID No. 58-60;

(21) Sequences shown as SEQ ID No. 61-63;

(22) As shown in SEQ ID No. 64-66;

(23) As shown in SEQ ID No. 67-69;

(24) As shown in SEQ ID Nos. 70-72.

In alternative embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody are selected from the group consisting of: (2) Any one of (10), (11), (12), (13), (14), (15), (18), (20) and (22).

In an alternative embodiment, the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody are selected from any one of (15), (20), and (22).

In an alternative embodiment, the heavy chain variable region further comprises a framework region, in particular comprising FR1, FR2, FR3 and FR4, the heavy chain variable region having the structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。

when the amino acid sequences of CDRs 1 to 3 of the heavy chain variable region of the antibody are shown in (1) to (24) in sequence, the amino acid sequences of the heavy chain variable region are shown in SEQ ID Nos. 73 to 96 in sequence. In alternative embodiments, the antibody is selected from the group consisting of single domain antibodies, heavy chain antibodies, fc fragment fusion proteins, igGI, igG2, igG4, igA, igE, igM, igD, fab ', fab, F (ab') ₂ At least one of Fv and scFv fragments.

Specifically, a single domain antibody (VHH) is a heavy chain variable region as shown in any one of the previous embodiments. It should be noted that, when the "antibody against IL-17RA protein" as claimed in the examples of the present specification is a single domain antibody, the sequence of the antibody is equivalent to the sequence of the heavy chain variable region of the antibody.

Heavy chain antibodies (hcabs) are antibodies comprising the single domain antibodies described above and two constant regions (CH 2 and CH3 regions); the Fc fragment fusion protein is an antibody obtained by fusing the heavy chain variable region and the Fc fragment of an immunoglobulin.

The said "IgGI, igG2, igG4, igA, igE, igM, igD, fab ', fab, F (ab') ₂ Fv and scFv fragments "are antibodies comprising the heavy chain variable regions described above, where" IgGI, igG2, igG4, igA, igE, igM, igD, fab ', fab, F (ab') ₂ Fv and scFv fragments "are merely illustrative of possible types of antibodies, and are within the scope of the present application as long as they comprise CDR1, CDR2 and CDR3 of the heavy chain variable region described above.

In alternative embodiments, the antibody is a single domain antibody or an Fc-fragment fusion protein comprising the heavy chain variable region and an Fc-fragment of an immunoglobulin.

In an alternative embodiment, the antibody is a single domain antibody.

The single domain antibody may be synthesized artificially or may be obtained by first synthesizing a gene encoding the single domain antibody and then biologically expressing the single domain antibody.

The present examples provide an isolated nucleic acid encoding an antibody against an IL-17RA protein as described in any one of the previous embodiments;

in alternative embodiments, the nucleic acid comprises any one of the sequences set forth in SEQ ID Nos. 97 to 120.

It should be noted that, when the antibody is a single domain antibody and its sequence is shown in SEQ ID No. 73-96, the sequence of the nucleic acid is shown in SEQ ID No. 97-120.

The present examples provide a recombinant vector comprising an isolated nucleic acid as described in any one of the previous embodiments.

In alternative embodiments, the recombinant vector may be a plasmid, phage, or viral vector.

The present application provides a host cell comprising the recombinant vector as described in the previous examples.

In alternative embodiments, the host cell may be a prokaryotic cell or a eukaryotic cell.

The present application provides a method for producing an antibody against an IL-17RA protein, comprising culturing a host cell as described in the previous examples to obtain an antibody against an IL-17RA protein.

The present examples also provide a kit for detecting IL-17RA comprising an antibody directed against an IL-17RA protein as described in any of the preceding embodiments.

In addition, the embodiment of the application also provides application of the antibody aiming at the IL-17RA protein in preparing a medicament for inhibiting or preventing autoimmune diseases.

In alternative embodiments, the autoimmune disease comprises at least one of crohn's disease, psoriasis, hidradenitis suppurativa, systemic inflammation, and multiple sclerosis.

In alternative embodiments, the antibody inhibits or prevents autoimmune disease by inhibiting the binding of IL-17 receptor to IL-17 cytokine.

The features and capabilities of the present application are described in further detail below in connection with the examples.

Example 1

Preparation of a single domain antibody directed against a human recombinant IL-17RA protein.

1. Constructing an expression vector of a human recombinant IL-17RA protein:

the coding sequence for IL-17RA, which is identified as NM-014339.6, was retrieved from NCBI and encoded to produce the amino acid sequence accession No. NP-055154.3. The amino acid sequence corresponding to np_055154.3 was then analyzed for the transmembrane region and extracellular end of the protein via TMHMM and SMART websites, respectively.

The analysis result shows that the extracellular end of the IL-17RA protein is the 1 st to 320 th amino acid of the above sequence, wherein, the 1 st to 32 nd amino acid is the signal peptide of the protein.

The nucleotide sequences encoding amino acids 1 to 320 of the IL-17RA protein were cloned into the vector pcDNA3.4 by means of the restriction enzymes XbaI and AgeI using sequence-specific primers. And (3) carrying out Sanger sequencing on the constructed vector, comparing the original sequences, carrying out batch extraction on the recombinant plasmid after confirming no errors, removing endotoxin, carrying out expression and purification of a target protein (IL-17 RA protein) by transfecting suspension 293F, and carrying out SDS-PAGE analysis after purification of the humanized recombinant IL-17RA protein, wherein the SDS-PAGE analysis is shown in figure 1.

As can be seen from FIG. 1, the purity of the purified protein is up to 90%, and the animal immunity requirement is met.

2. Construction of a Single-Domain antibody library of IL-17RA proteins

Mixing 1mg of the human recombinant IL-17RA protein obtained by purification in the step 1 with an equal volume of Freund's complete adjuvant, immunizing an inner Mongolian Alexander alpacad, immunizing once a week, continuously immunizing 7 times, and carrying out animal immunization by mixing 1mg of IL-17RA protein with the Freund's incomplete adjuvant in equal volume for six times except the first immunization, wherein the immunization process aims at intensively stimulating the camel to generate antibodies against the IL-17RA protein.

After the animal immunization is finished, 150mL of camel peripheral blood lymphocytes are extracted, and RNA of the cells is extracted. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified by a nested PCR reaction using the cDNA as a template.

Then, the pMECS vector and the VHH fragment were digested separately using restriction enzymes, and the digested fragments and vector were ligated. Transforming the ligated fragment spots into competent cells TG1, constructing a phage display library of MMP9 protein and measuring the library capacity, wherein the library capacity is about 1X 10 ⁹ At the same time, the correct insertion rate of the library into the target fragment was detected by colony PCR identification, and the results are shown in FIG. 2.

The results showed that 28 clones amplified a band of 600bp (predicted size) and 2 clones amplified an incorrect band after PCR amplification of 30 randomly selected colonies from the library, so the correct insertion rate was 28.times.30%. Apprxeq.93.3%.

3. Screening of Single-Domain antibodies against IL-17RA proteins

200. Mu.L of the recombinant TG1 cells in step 2 were cultured in 2 XTY medium, during which 40. Mu.L of helper phage VCSM13 was added to infect TG1 cells, and cultured overnight to amplify phages, the phages were precipitated the next day with PEG/NaCl, and the amplified phages were collected by centrifugation.

NaHCO diluted at 100mM pH 8.3 ₃ 500 mug of IL-17RA protein is coupled on an ELISA plate, and is placed at 4 ℃ overnight, and a negative control hole is formed; the next day 200 μl of 3% skim milk was added and blocked at room temperature for 2h; after blocking was completed, 100. Mu.l of amplified phage library (approximately 2X 10 ¹¹ Individual phage particles), 1h at room temperature; after 1 hour of action, wash 5 times with PBS+0.05% Tween-20 to wash away unbound phage.

The phage specifically combined with IL-17RA protein is dissociated by trypsin with a final concentration of 25mg/mL, and E.coli TG1 cells in logarithmic phase are infected, cultured for 1h at 37 ℃, phage are generated and collected for the next round of screening, the same screening process is repeated for 1 round, enrichment is gradually obtained, and when the enrichment multiple reaches more than 10 times, the enrichment effect is shown in figure 3.

In fig. 3, P/N = number of monoclonal bacteria grown after infection of TG1 bacteria by phage with positive Kong Xi removal by biopanning/number of monoclonal bacteria grown after infection of TG1 bacteria by phage with positive Kong Xi removal, this parameter gradually increases after enrichment has occurred; I/E = total phage added to positive wells per round of biopanning/total phage removed from positive Kong Xi per round of biopanning, which parameter gradually approaches 1 after enrichment has occurred.

4. Screening of specific positive clones against IL-17RA by ELISA

Screening the single domain antibody against IL-17RA protein for 3 rounds according to the screening method in the step 3, wherein the phage enrichment factor of the anti-IL-17 RA protein is more than 10, after screening, selecting 400 single colonies from positive clones obtained by screening, respectively inoculating the single colonies into 96 deep well plates containing 100 mug/mL of ampicillin TB culture medium, setting blank control, culturing at 37 ℃ to logarithmic phase, adding IPTG with a final concentration of 1mM, and culturing at 28 ℃ for overnight.

Obtaining a crude extract antibody by using a permeation swelling method; the IL-17RA recombinant protein was released to 100mM NaHCO pH 8.3, respectively ₃ 100. Mu.g of protein was coated in an ELISA plate (ELISA plate) at 4℃overnight. Transferring 100uL of the obtained crude antibody extract to an ELISA plate added with antigen, and incubating for 1h at room temperature; washing the unbound antibody with PBST, adding 100 μl of Mouse anti-HA tag antibody (Mouse anti-HA antibody, thermo Fisher) diluted 1:2000, and incubating for 1h at room temperature; unbound antibody was washed off with PBST, 100. Mu.l of Anti-Rabbit HRP conjugate (goat Anti-rabbit horseradish peroxidase-labeled antibody, available from Thermo Fisher) diluted 1:20000 was added and incubated for 1h at room temperature; washing unbound antibody with PBST, adding horseradish peroxidase chromogenic solution, reacting at 37deg.C for 15min, adding stop solution, and reading absorption value at 450nm wavelength on a microplate reader.

When the OD value of the sample hole is more than 5 times that of the control hole, judging that the sample hole is a positive cloning hole; the positive clone well was transferred to LB medium containing 100. Mu.g/mL ampicillin to extract plasmids and sequenced.

And analyzing the gene sequences of all clone strains according to sequence comparison software Vector NTI, and regarding the strains with the same CDR1, CDR2 and CDR3 sequences as the same clone strain and the strains with different sequences as different clone strains to finally obtain the single-domain antibody specific to the IL-17RA protein.

The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure, which forms the whole VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally the single-domain antibody protein is obtained.

5. Purification and expression of anti-IL-17 RA protein single domain antibodies in host E.coli.

The plasmids of the different clones obtained by sequencing analysis in step 4 (pMECS-VHH) were electrotransformed into E.coli HB2151 and spread on LB+amp+glucose-containing culture plates, which were incubated overnight at 37 ℃; single colonies were selected and inoculated in 5mL of LB medium containing shoreside penicillin, and shake cultured overnight at 37 ℃.

Inoculating 1mL of overnight culture strain into 330mL of TB culture solution, shake culturing at 37deg.C, adding 1M IPTG when OD600nm value reaches 0.6-0.9, shake culturing at 28deg.C overnight; centrifuging, collecting escherichia coli, and obtaining an antibody crude extract by using a osmotic bursting method;

the antibodies were purified by nickel column affinity chromatography and the purified single domain antibodies, shown in FIG. 4, included VHHs 1-18.

6. Construction of Fc segment fusion protein eukaryotic expression vector containing anti-IL-17 RA protein single domain antibody.

Subcloning the target sequence obtained in step 4 into a eukaryotic expression vector: and (3) sequencing the single domain antibody screened in the step (4) by Sanger to obtain the nucleotide sequence of the single domain antibody. The nucleotide sequence after codon optimization is synthesized into a target vector RJK-V4-hFC which is common to the designed and modified nano antibody in a sequence synthesis mode.

RJK-V4-hFC was engineered on the basis of the Fc segment in the heavy chain coding sequence (NCBI Accession No:AB 776838.1) of human IgG fused to the live-heavy chain coding sequence (NCBI Accession No:AB 776838.1) of an human IgG, i.e.the vector contains the Hinge (Hinge) CH2 and CH3 regions of the IgG heavy chain, on the basis of the vector data link https:// packages. The concrete improvement scheme is as follows:

selecting restriction enzyme cutting sites XbaI and AgeI on pcDNA3.4; multiple cloning sites (MCS, multiple Cloning Site) and a 6 xhis tag are introduced at the 5 'and 3' ends of the Fc fragment coding sequence, respectively, by means of overlap PCR, as shown in fig. 5; amplifying the fragments by PCR by using a pair of primers with XbaI and AgeI enzyme cutting sites respectively to obtain recombinant DNA fragments; respectively carrying out enzyme digestion on pcDNA3.4 and the obtained recombinant DNA fragment by using restriction enzymes XbaI and AgeI; and (3) connecting the digested vector and the inserted fragment under the action of T4 ligase, then converting the connection product into escherichia coli, amplifying, and checking by sequencing to obtain the recombinant plasmid.

Transforming the constructed recombinant eukaryotic expression vector into DH5 alpha escherichia coli, culturing to carry out plasmid large extraction, and removing endotoxin; sequencing and identifying the sequence of the plasmid after large extraction; and preparing the recombinant vector after the determination of no error for subsequent eukaryotic cell transfection and expression.

7. Fc fragment fusion proteins containing single domain antibodies against IL-17RA protein were expressed in suspension Expi CHO-S cells.

3 days before transfection at 2.5X10 ⁵ Expi CHO-S cells passaged/expanded culture ^TM The cells, calculated desired cell volume, were transferred to an ExpiCHO containing fresh pre-warmed 120ml (final volume) ^TM 500ml shake flask of expression medium; to achieve a cell concentration of about 4X 10 ⁶ ～6×10 ⁶ Living cells/mL.

One day prior to transfection, expiCHO-S was used ^TM Cell dilution concentration to 3.5X10 ⁶ Living cells/mL, allowing the cells to incubate overnight; the day of transfection, cell density and percent viable cells were determined. The cell density should reach about 7X 10 before transfection ⁶ ～10×10 ⁶ Living cells/mL.

Fresh Expi CHO preheated to 37 ℃ ^TM Dilution of cells to 6X 10 in expression Medium ⁶ Each living cell/mL. The calculated desired cell volume was transferred to an ExpiCHO containing fresh pre-warmed 100ml (final volume) ^TM 500ml shake flask of expression medium: gently mixing the mixture with the mixture of the Expifectamine in a reverse manner ^TM CHO reagent with 3.7ml OptiPRO ^TM Dilution of Expifectamine in Medium ^TM CHO reagent, whipping or mixing; with refrigerated 4ml OptiPRO ^TM Diluting plasmid DNA with culture medium, and mixing; the ExpiFectamine CHO/plasmid DNA complex was incubated for 1-5 minutes at room temperature and then gently added to the prepared cell suspension, during which time the flask was gently swirled.

The cells were incubated at 37℃with 8% CO ₂ Shake culturing in humidified air; mu.l of Expifectamine was added on day 1 (18-22 hours post transfection) ^TM CHO enhancement and 24ml of expi CHO feed. Supernatants were collected about 8 days after transfection (cell viability below 70%).

8. Expression of Fc-segment fusion proteins containing single domain antibodies against IL-17RA protein in suspension 293F cells.

Recombinant single domain antibody expression experimental procedure (500 ml shake flask for example):

3 days before transfection at 2.5X10 ⁵ The cells/ml were passaged and expanded 293F cells and the calculated desired cell volume was transferred to 500ml shake flasks with fresh pre-warmed 120ml (final volume) OPM-293CD05 Medium. The cell concentration was brought to about 2X 106-3X 106 viable cells/mL.

The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 2X 10 before transfection ⁶ ～3×10 ⁶ Living cells/mL. Dilution of cells to 1X 10 with pre-warmed OPM-293CD05Medium ⁶ Each living cell/mL. The calculated cell volume was transferred to a 500ml shake flask containing fresh pre-warmed 100ml (final volume) of medium.

Diluting PEI (1 mg/ml) reagent with 4ml Opti-MEM culture medium, and stirring or blowing to mix uniformly; the plasmid DNA was diluted with 4ml of Opt-MEM medium, mixed back and forth, and filtered with a 0.22um filter. Incubate at room temperature for 5min.

Diluted PEI reagent was added to the diluted DNA and mixed upside down. PEI/plasmid DNA complexes were incubated for 15-20 minutes at room temperature and then gently added to the prepared cell suspension, during which time the shake flask was gently swirled.

The cells were incubated at 37℃with 5% CO ₂ Shake culturing at 120 rpm. 5ml OPM-CHO PFF05 feed was added 24h, 72h post transfection. Supernatants were collected about 7 days after transfection (cell viability below 70%).

9. Purification of Fc-segment fusion proteins containing single domain antibodies against IL-17RA proteins

Filtering the protein expression supernatant obtained in the step 6 or 7 by using a disposable filter head with the diameter of 0.45 mu m to remove insoluble impurities; purifying the filtrate by affinity chromatography using a Protein purifier, and purifying by using agarose filler coupled with Protein A by utilizing the binding capacity of human Fc and Protein A;

passing the filtrate through a Protein A pre-packed column at a flow rate of 1 mL/min, wherein the target Protein in the filtrate is combined with the packing; washing the column-bound impurity proteins with a low-salt and high-salt buffer; eluting the target protein bound on the column with a low pH buffer; the eluate was rapidly added to Tris-HCl solution at pH9.0 for neutralization.

And (3) dialyzing the neutralized protein solution, performing SDS-PAGE analysis to determine that the protein purity is above 95%, and preserving the protein at a low temperature for later use after the concentration is above 0.5 mg/mL.

Example 2

ELISA detection of the affinity of the Single Domain antibodies provided in example 1 to IL-17 RA.

Using an anti-IL-6 VHH as a isotype control, the binding capacity of the anti-IL-17 RA protein VHH to the target was tested as follows:

coating 50 mu L of a human recombinant IL-17RA sample with the concentration of 1 ng/. Mu.L on an ELISA plate, and coating at 4 ℃ overnight; sealing the coated plate by using 5% skimmed milk powder for 1 hour, wherein 200 mu L of skimmed milk powder is filled in each hole; adding the single domain antibody obtained in the step 4 in the example 1, wherein the single domain antibody is provided with HA or a human Fc tag, and incubating for 1 hour; adding a detection antibody (HRP label) specific to the HA tag protein or the human Fc, and incubating for 0.5 hour; adding a chromogenic substrate TMB, and developing; the reaction was terminated by adding a stop solution. OD450 values were measured as shown in figure 6. The results showed that only a portion of the 37 single domain antibodies (24 antibodies) showed significant specific binding to IL17RA in the ELISA assay, and the remaining 13 antibodies were judged to be non-specific binding.

The amino acid sequences of 24 single domain antibodies are shown in SEQ ID Nos. 73-96, please refer to Table 1. TABLE 1 Single Domain antibodies

Example 3

The single domain antibodies provided in example 1 were assayed for affinity to IL-17RA by dose-response curve.

50. Mu.L of a sample of human recombinant IL-17RA at a concentration of 1 ng/. Mu.L was coated onto ELISA plates at 4℃overnight; washing the plate; 200 mu L of 5% milk is added and the mixture is sealed for 1h at 37 ℃; the 24 single domain antibodies with higher P/N values in the example 2 are respectively diluted to 2 mug/ml, and then 8 concentration gradients are added to the 5-time gradient diluted antibodies; washing the plate; adding 50 mu L of antibody, and incubating for 1h at 37 ℃ in two duplicate wells; washing the plate; adding 50 mu L of mouse anti-HA tag HRP secondary antibody, and incubating for 30min at 37 ℃; washing the plate; adding 50 μl of TMB which is recovered in advance at normal temperature, and reacting for 15min at normal temperature in dark place; adding 50 μl of stop solution (1N HCl), and preserving the reading of the enzyme label instrument; the EC50 was calculated by plotting the curves and the results are shown in fig. 7.

As can be seen from fig. 7, in the 24 antibodies obtained by the analysis in example 2, the EC50 value of the dose-response curve of binding of 9 single domain antibodies to IL17RA was low, and the curve window was large, which proves that the affinity constant of the 9 single domain antibodies to IL17RA was small and the affinity was high.

The 9 single domain antibodies were: 1H10, 3B1, 4A8, 1B6, 4A9, 1G12, 3B8, 1C11 and 3A10.

The affinity constant of the antibody at immature has reached the nM level. Among them, clone 1B12 showed better biological functional activity in the functional results of example 5, although the EC50 curve window was smaller in this experiment.

Example 4

Single domain antibody neutralizing cytokine release experiments for IL-17RA in example 1.

IL-17 induces the release of IL-6 by HeLa cells, and neutralizing antibodies to IL-17R may block this response. The experimental procedure was as follows:

spreading Hela cells in 96-well plates, ten thousand cells per well; the highest concentration of 10 μg/ml, 5-fold gradient of anti-IL-17 RA VHH (single domain antibody with lower EC50 in example 3), and control set 3 control, lxekizumab, brodalumab and blank hIgG, respectively, were mixed with 220ng/ml IL-17 at 1:1; mixing the mixed mixture with cells according to a ratio of 1:1, culturing for 24 hours, and collecting cell supernatants; IL-6 expression in cell supernatants was detected using the human IL-6ELSIA kit, ELISA assay conditions were as described in Thermo, cat#88-7066-88, and the results are shown in FIG. 8.

The results show that the 9 single domain antibodies obtained in example 3 with lower EC50 (high affinity) perform better in functional experiments, clone 3B8, 4A9, but their function can be optimized by amino acid mutation, whereas clone 1B12, although with a small dose-response curve window in example 3, performs better in this example with lower EC50 and a larger curve window.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Sequence listing

<110> Nanjing Rongjiekang biotechnology Co., ltd

<120> antibody against IL-17RA protein, method for producing the same and use thereof

<160> 120

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Gly Tyr Thr Ser Ser Ser Tyr Cys

1 5

<210> 2

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Ile Asp Ser Lys Gly Ser Thr

1 5

<210> 3

<211> 22

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Ala Ala Gly Asp Lys Tyr Asp Cys Tyr Ser Gly Ser Trp Ser Asn Ala

1 5 10 15

Glu Ile Val Gly Tyr Trp

20

<210> 4

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Lys Tyr Thr Asn Arg Ser Tyr Cys

1 5

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Ile Asp Ser Asp Gly Ser Thr

1 5

<210> 6

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Val Ala Asp Ala Gly Tyr Asp Cys Tyr Ser Gly Ser Trp Phe Glu Thr

1 5 10 15

Val Pro Ala Leu Gly Val Gly Tyr Trp

20 25

<210> 7

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Gly Tyr Thr His Arg Ser Tyr Tyr

1 5

<210> 8

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Ile Tyr Thr Gly Asp Gly Ser Thr

1 5

<210> 9

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Ala Ala Asp Thr Gln Asn Ser Phe Thr Ala Pro Tyr Trp

1 5 10

<210> 10

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Gly Phe Thr Phe Ser Ser Tyr Gly

1 5

<210> 11

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Ile Asn Ser Gly Val Gly Ser Thr Thr

1 5

<210> 12

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

Ala Lys Gly Ser Ile Glu Tyr Asp Ser Asp Tyr Arg Val Asn Tyr Val

1 5 10 15

Glu Ala Lys

<210> 13

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 13

Gly Phe Ile Phe Ser Asn Tyr Gly

1 5

<210> 14

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 14

Ile Asn Ile Asp Gly Ser Arg Thr

1 5

<210> 15

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 15

Ala Lys Gly Ser Ile Thr Tyr Asp Met Asp Tyr Arg Val Thr Thr Ile

1 5 10 15

Glu Glu Lys

<210> 16

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 16

Gly Tyr Thr Val Arg Lys Ser Asp

1 5

<210> 17

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 17

Ile Asp Lys Asp Gly Asn Thr

1 5

<210> 18

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 18

Arg Ser Arg Tyr Tyr Ser Ser Asp Tyr Arg Val Leu Asn Tyr Tyr Trp

1 5 10 15

<210> 19

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 19

Gly Asn Thr Tyr Ser Ser Asn Trp

1 5

<210> 20

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 20

Phe Phe Thr Gly Gly Gly Ala Pro

1 5

<210> 21

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 21

Ala Arg Cys Ala Val Ala Ser Trp Tyr Gly Ser Arg Ser Cys Arg Asp

1 5 10 15

Thr Tyr Trp

<210> 22

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 22

Gly Asn Thr Tyr Ser Ser Asn Trp

1 5

<210> 23

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 23

Phe Phe Thr Gly Gly Gly Ala Pro

1 5

<210> 24

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 24

Ala Arg Cys Ala Ala Gly Ser Trp Phe Gly Ser Arg Ser Cys Arg Asp

1 5 10 15

Thr Tyr Trp

<210> 25

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 25

Gly Asn Thr Tyr Ser Ser Asn Trp

1 5

<210> 26

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 26

Phe Phe Thr Gly Gly Gly Ala Pro

1 5

<210> 27

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 27

Ala Arg Cys Ala Ala Gly Ser Trp Tyr Gly Ser Arg Ser Cys Arg Asp

1 5 10 15

Ser Tyr Trp

<210> 28

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 28

Gly Tyr Thr Phe Lys Thr Tyr His

1 5

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 29

Ile Asn Ser Asp Gly Ile Thr

1 5

<210> 30

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 30

Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg His

1 5 10 15

His Tyr Asn Ala Trp

20

<210> 31

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 31

Gly Tyr Thr Phe Lys Thr Tyr His

1 5

<210> 32

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 32

Ile Asn Ser Asp Gly Ile Thr

1 5

<210> 33

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 33

Ala Ala Gly Ser Gly Asn Leu Phe Asp Phe Leu Leu Leu Ser Arg His

1 5 10 15

His Tyr Asn Ala Trp

20

<210> 34

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 34

Gly Tyr Thr Phe Lys Thr Tyr His

1 5

<210> 35

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 35

Ile Asn Ser Glu Gly Ser Thr

1 5

<210> 36

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 36

Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg His

1 5 10 15

His Tyr Asn Ala Trp

20

<210> 37

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 37

Gly Tyr Thr Phe Lys Thr Tyr His

1 5

<210> 38

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 38

Ile Asn Ser Asp Gly Ala Thr

1 5

<210> 39

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 39

Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg His

1 5 10 15

His Tyr Asn Ala Trp

20

<210> 40

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 40

Gly Tyr Thr Phe Lys Thr Tyr His

1 5

<210> 41

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 41

Ile Asn Ser Asp Gly Val Thr

1 5

<210> 42

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 42

Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg His

1 5 10 15

His Tyr Asn Ala Trp

20

<210> 43

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 43

Gly Tyr Thr Phe Lys Thr Tyr His

1 5

<210> 44

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 44

Ile Asn Ser Asp Gly Val Thr

1 5

<210> 45

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 45

Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg His

1 5 10 15

His Tyr Asn Ala Trp

20

<210> 46

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 46

Gly Tyr Thr Phe Lys Thr Tyr His

1 5

<210> 47

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 47

Ile Asn Ser Asp Gly Val Thr

1 5

<210> 48

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 48

Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg His

1 5 10 15

His Tyr Asn Ala Trp

20

<210> 49

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 49

Gly Tyr Thr Ser Lys Thr Tyr His

1 5

<210> 50

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 50

Ile Asn Ser Asp Gly Val Thr

1 5

<210> 51

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 51

Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg His

1 5 10 15

His Tyr Asn Ala Trp

20

<210> 52

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 52

Gly Tyr Thr Phe Lys Thr Tyr His

1 5

<210> 53

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 53

Ile Asn Ser Asp Gly Val Thr

1 5

<210> 54

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 54

Ala Ala Gly Ser Gly Tyr Met Phe Asp Phe Leu Leu Leu Ser Arg His

1 5 10 15

His Tyr Ser Ala Trp

20

<210> 55

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 55

Ile Tyr Thr Tyr Lys Thr Tyr His

1 5

<210> 56

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 56

Ile Asn Ser Asn Ala Arg Thr

1 5

<210> 57

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 57

Ala Ala Gly Val Gly Asn Val Phe His Leu Leu Ser Arg Asn Asn Tyr

1 5 10 15

Asn Ala Trp

<210> 58

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 58

Gly Tyr Thr Phe Lys Thr Tyr Arg

1 5

<210> 59

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 59

Phe Phe Thr Gly Gly Gly Ala

1 5

<210> 60

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 60

Gly Ala Gly Ser Gly Lys Leu Phe Asp Phe Leu Leu Leu Arg Ile His

1 5 10 15

His Tyr Asn Ala Trp

20

<210> 61

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 61

Gly Phe Thr Pro Ser Lys Tyr Cys

1 5

<210> 62

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 62

Ile Ser Thr Arg Gly Thr Thr Thr

1 5

<210> 63

<211> 20

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 63

Ala Ala Asp Pro Ala Pro Cys Thr Met Gly Gly Ser Thr Ala Val Asn

1 5 10 15

Tyr Asn Tyr Trp

20

<210> 64

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 64

Gly Ile Tyr Tyr Ser Gly Arg Cys

1 5

<210> 65

<211> 6

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 65

Ile Asp Lys Ser Asn Thr

1 5

<210> 66

<211> 22

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 66

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

1 5 10 15

Arg Glu Leu Arg Tyr Trp

20

<210> 67

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 67

Lys Phe Pro Val Ser Gly Tyr Cys

1 5

<210> 68

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 68

Ile Asn Ser Asp Gly Ser Thr

1 5

<210> 69

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 69

Ala Ala Ser Thr Trp Tyr Asn Asn Cys Tyr Ile Gly Arg Thr Ala Phe

1 5 10 15

Ser Tyr Trp

<210> 70

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 70

Gly Tyr Thr Tyr Ser Asn Tyr Cys

1 5

<210> 71

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 71

Ile Ile Ser Leu Gly Gly Ser Thr

1 5

<210> 72

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 72

Ala Ala Ser Pro Ala Met Gly Trp Ala Cys Leu Gly Gly Arg Asp Phe

1 5 10 15

Arg Tyr Trp

<210> 73

<211> 129

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 73

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Ser

20 25 30

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

35 40 45

Gly Val Ala Ala Ile Asp Ser Lys Gly Ser Thr Met Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Ser Thr Leu

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Ala Gly Asp Lys Tyr Asp Cys Tyr Ser Gly Ser Trp Ser Asn

100 105 110

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

115 120 125

Ser

<210> 74

<211> 132

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 74

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Lys Tyr Thr Asn Arg

20 25 30

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

35 40 45

Gly Val Ala Ala Ile Asp Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser

50 55 60

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

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Val Ala Asp Ala Gly Tyr Asp Cys Tyr Ser Gly Ser Trp Phe Glu

100 105 110

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

115 120 125

Thr Val Ser Ser

130

<210> 75

<211> 121

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 75

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Thr His Arg

20 25 30

Ser Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Lys Arg Glu

35 40 45

Gly Val Ala Ser Ile Tyr Thr Gly Asp Gly Ser Thr His Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Gln Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr

85 90 95

Tyr Cys Ala Ala Asp Thr Gln Asn Ser Phe Thr Ala Pro Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 76

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 76

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

1 5 10 15

Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

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

35 40 45

Trp Val Ser Thr Ile Asn Ser Gly Val Gly Ser Thr Thr Tyr Tyr Ala

50 55 60

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

65 70 75 80

Thr Leu Tyr Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met

85 90 95

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

100 105 110

Asn Tyr Val Glu Ala Lys Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 77

<211> 127

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 77

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

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser

20 25 30

Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Asn Ile Asn Ile Asp Gly Ser Arg Thr Phe Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr

65 70 75 80

Leu Tyr Leu Gln Leu Asn Ser Leu Arg Thr Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Ala Lys Gly Ser Ile Thr Tyr Asp Met Asp Tyr Arg Val Thr

100 105 110

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

115 120 125

<210> 78

<211> 123

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 78

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Leu Ala Ser Gly Tyr Thr Val Arg

20 25 30

Lys Ser Asp Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Phe Val Ser Thr Ile Asp Lys Asp Gly Asn Thr Asn Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Arg Ser Arg Tyr Tyr Ser Ser Asp Tyr Arg Val Leu Asn Tyr Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 79

<211> 127

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 79

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Asn Thr Tyr Ser

20 25 30

Ser Asn Trp Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu

35 40 45

Arg Val Ala Thr Phe Phe Thr Gly Gly Gly Ala Pro Ala Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asp Ile Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Gly Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Ala Arg Cys Ala Val Ala Ser Trp Tyr Gly Ser Arg Ser Cys

100 105 110

Arg Asp Thr Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 80

<211> 127

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 80

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Asn Thr Tyr Ser

20 25 30

Ser Asn Trp Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu

35 40 45

Arg Val Ala Thr Phe Phe Thr Gly Gly Gly Ala Pro Ser Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Gly Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Ala Arg Cys Ala Ala Gly Ser Trp Phe Gly Ser Arg Ser Cys

100 105 110

Arg Asp Thr Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 81

<211> 127

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 81

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Asn Thr Tyr Ser

20 25 30

Ser Asn Trp Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu

35 40 45

Arg Val Ala Thr Phe Phe Thr Gly Gly Gly Ala Pro Thr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Gly Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Ala Arg Cys Ala Ala Gly Ser Trp Tyr Gly Ser Arg Ser Cys

100 105 110

Arg Asp Ser Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 82

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 82

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Lys

20 25 30

Thr Tyr His Met Ala Trp Phe Arg Gln Ala Pro Gly Met Glu Arg Glu

35 40 45

Gly Val Ala Gly Ile Asn Ser Asp Gly Ile Thr Lys Tyr Ala Asp Ser

50 55 60

Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg

100 105 110

His His Tyr Asn Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 83

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 83

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

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Phe Lys

20 25 30

Thr Tyr His Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Gly Val Ala Gly Ile Asn Ser Asp Gly Ile Thr Lys Tyr Ala Asp Ser

50 55 60

Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu

65 70 75 80

Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Ala Gly Ser Gly Asn Leu Phe Asp Phe Leu Leu Leu Ser Arg

100 105 110

His His Tyr Asn Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 84

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 84

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Lys

20 25 30

Thr Tyr His Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Gly Val Ala Gly Ile Asn Ser Glu Gly Ser Thr Lys Tyr Ala Asp Ser

50 55 60

Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Ala Ala Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg

100 105 110

His His Tyr Asn Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 85

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 85

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Lys

20 25 30

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

35 40 45

Gly Val Ala Gly Ile Asn Ser Asp Gly Ala Thr Lys Tyr Ala Asp Ser

50 55 60

Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg

100 105 110

His His Tyr Asn Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 86

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 86

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Lys

20 25 30

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

35 40 45

Gly Val Ala Gly Ile Asn Ser Asp Gly Val Thr Lys Tyr Ala Asp Ser

50 55 60

Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg

100 105 110

His His Tyr Asn Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 87

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 87

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Arg Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Lys

20 25 30

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

35 40 45

Gly Val Ala Gly Ile Asn Ser Asp Gly Val Thr Lys Tyr Ala Asp Ser

50 55 60

Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg

100 105 110

His His Tyr Asn Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 88

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 88

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Lys

20 25 30

Thr Tyr His Met Ser Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg Glu

35 40 45

Gly Val Ala Gly Ile Asn Ser Asp Gly Val Thr Lys Tyr Ala Asp Ser

50 55 60

Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg

100 105 110

His His Tyr Asn Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 89

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 89

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Lys

20 25 30

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

35 40 45

Gly Val Ala Gly Ile Asn Ser Asp Gly Val Thr Lys Tyr Ala Asp Ser

50 55 60

Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Ala Gly Ser Gly Asn Met Phe Asp Phe Leu Leu Leu Ser Arg

100 105 110

His His Tyr Asn Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 90

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 90

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Lys

20 25 30

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

35 40 45

Gly Val Ala Gly Ile Asn Ser Asp Gly Val Thr Lys Tyr Ala Asp Ser

50 55 60

Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Ala Gly Ser Gly Tyr Met Phe Asp Phe Leu Leu Leu Ser Arg

100 105 110

His His Tyr Ser Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 91

<211> 126

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 91

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Ile Tyr Thr Tyr Lys

20 25 30

Thr Tyr His Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Gly Val Ala Gly Ile Asn Ser Asn Ala Arg Thr Glu Tyr Ala Asp Ser

50 55 60

Val Gln Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Ala Gly Val Gly Asn Val Phe His Leu Leu Ser Arg Asn Asn

100 105 110

Tyr Asn Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 92

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 92

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Lys

20 25 30

Thr Tyr Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Gly Val Ala Ser Phe Phe Thr Gly Gly Gly Ala Lys Tyr Ala Asp Ser

50 55 60

Ala Gln Gly Arg Phe Phe Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu

65 70 75 80

Leu Leu Leu Met Glu Ser Leu Met His Asp Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Gly Ala Gly Ser Gly Lys Leu Phe Asp Phe Leu Leu Leu Arg Ile

100 105 110

His His Tyr Asn Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 93

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 93

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

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

20 25 30

Lys Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

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

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

65 70 75 80

Leu Asn Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Ala Ala Asp Pro Ala Pro Cys Thr Met Gly Gly Ser Thr Ala

100 105 110

Val Asn Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 94

<211> 128

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 94

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Val Pro Thr Gly Ile Tyr Tyr Ser

20 25 30

Gly Arg Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Arg Val Ala Ser Ile Asp Lys Ser Asn Thr Thr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

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

100 105 110

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

115 120 125

<210> 95

<211> 126

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 95

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

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

20 25 30

Gly Tyr Cys Arg Ala Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu

35 40 45

Gly Val Ala Thr Ile Asn Ser Asp Gly Ser Thr Thr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Leu Asp Asn Ala Lys Asn Thr Leu

65 70 75 80

Tyr Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr

85 90 95

Cys Ala Ala Ser Thr Trp Tyr Asn Asn Cys Tyr Ile Gly Arg Thr Ala

100 105 110

Phe Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 96

<211> 127

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 96

Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser

20 25 30

Asn Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

35 40 45

Gly Val Ala Arg Ile Ile Ser Leu Gly Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Ala Ala Ser Pro Ala Met Gly Trp Ala Cys Leu Gly Gly Arg

100 105 110

Asp Phe Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 97

<211> 387

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 97

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccgccag cggctacacc agcagcagct actgcatggg ctggttcagg 120

caggcccccg gcaaggagta cgagggcgtg gccgccatcg acagcaaggg cagcaccatg 180

tacgccgaca gcgtgaaggg caggttcacc atcagccagg acaacgccaa gagcaccctg 240

tacctgcaga tgaacagcct gaagcccgag gacaccgcca tgtactactg cgccgccggc 300

gacaagtacg actgctacag cggcagctgg agcaacgccg agatcgtggg ctactggggc 360

cagggcaccc aggtgaccgt gagcagc 387

<210> 98

<211> 396

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 98

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccgccag caagtacacc aacaggagct actgcatggg ctggttcagg 120

agggcccccg gcaaggagag ggagggcgtg gccgccatcg acagcgacgg cagcaccagc 180

tacgccgaca gcgtgaaggg caggttcacc gtgagccagg gcaacgccaa gaacaccctg 240

tacctgcaga tgaacagcct gaagcccgag gacaccgcca tgtactactg cgtggccgac 300

gccggctacg actgctacag cggcagctgg ttcgagaccg tgcccgccct gggcgtgggc 360

tactggggcc agggcaccca ggtgaccgtg agcagc 396

<210> 99

<211> 363

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 99

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccgtgag cggctacacc cacaggagct actacatggg ctggttcagg 120

caggcccccg gcaagaagag ggagggcgtg gccagcatct acaccggcga cggcagcacc 180

cactacgccg acagcgtgaa gggcaggttc accatcagcc aggacaacgc ccagaacacc 240

ctgtacctgc agatgaacag cctgaagccc gaggacagcg ccatgtacta ctgcgccgcc 300

gacacccaga acagcttcac cgccccctac tggggccagg gcacccaggt gaccgtgagc 360

agc 363

<210> 100

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 100

atggcccagg tgcagctgca ggagagcggc ggcggcctgg tgcagcccgg cggcagcctg 60

aagctgagct gcgccgccag cggcttcacc ttcagcagct acggcatgac ctgggtgagg 120

caggcccccg gcaagggcct ggagtgggtg agcaccatca acagcggcgt gggcagcacc 180

acctactacg ccgacagcgt gaagggcagg ttcaccatca gcagggacaa cgccaagaac 240

accctgtacc tgcagctgaa cagcctgaag accgaggaca ccgccatgta ctactgcgcc 300

aagggcagca tcgagtacga cagcgactac agggtgaact acgtggaggc caagggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 101

<211> 381

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 101

atggcccagg tgcagctgca ggagagcggc ggcggcctgg tgcagcccgg cggcagcctg 60

aggctgagct gcgccgccag cggcttcatc ttcagcaact acggcatgaa ctgggtgagg 120

caggcccccg gcaagggcct ggagtgggtg agcaacatca acatcgacgg cagcaggacc 180

ttctacgccg acagcgtgaa gggcaggttc accatcagca gggacaacgc caggaacacc 240

ctgtacctgc agctgaacag cctgaggacc gaggacaccg ccatgtacta ctgcgccaag 300

ggcagcatca cctacgacat ggactacagg gtgaccacca tcgaggagaa gggccagggc 360

acccaggtga ccgtgagcag c 381

<210> 102

<211> 369

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 102

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcctggccag cggctacacc gtgaggaaga gcgacatgag ctggtacagg 120

caggcccccg gcaaggagag ggagttcgtg agcaccatcg acaaggacgg caacaccaac 180

tacgccgaca gcgtgaaggg caggttcacc atcagccagg acaacgccaa gaacaccgtg 240

tacctgcaga tgaacagcct gaagcccgag gacaccgcca tgtactactg caggagcagg 300

tactacagca gcgactacag ggtgctgaac tactactggg gccagggcac ccaggtgacc 360

gtgagcagc 369

<210> 103

<211> 381

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 103

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcaccgccag cggcaacacc tacagcagca actggatggg ctggttcagg 120

cagccccccg gcaaggagag ggagagggtg gccaccttct tcaccggcgg cggcgccccc 180

gcctacgccg acagcgtgaa gggcaggttc accatcagcc aggacgacat caagaacacc 240

ctgtacctgc agatgggcag cctgaagccc gaggacaccg ccatgtacta ctgcgccagg 300

tgcgccgtgg ccagctggta cggcagcagg agctgcaggg acacctactg gggcaggggc 360

acccaggtga ccgtgagcag c 381

<210> 104

<211> 381

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 104

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcaccgccag cggcaacacc tacagcagca actggatggg ctggttcagg 120

cagccccccg gcaaggagag ggagagggtg gccaccttct tcaccggcgg cggcgccccc 180

agctacgccg acagcgtgaa gggcaggttc accatcagcc aggacaacgc caagaacacc 240

ctgtacctgc agatgggcag cctgaagccc gaggacaccg ccatgtacta ctgcgccagg 300

tgcgccgccg gcagctggtt cggcagcagg agctgcaggg acacctactg gggcaggggc 360

acccaggtga ccgtgagcag c 381

<210> 105

<211> 381

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 105

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcaccgccag cggcaacacc tacagcagca actggatggg ctggttcagg 120

cagccccccg gcaaggagag ggagagggtg gccaccttct tcaccggcgg cggcgccccc 180

acctacgccg acagcgtgaa gggcaggttc accatcagcc aggacaacgc caagaacacc 240

ctgtacctgc agatgggcag cctgaagccc gaggacaccg ccatgtacta ctgcgccagg 300

tgcgccgccg gcagctggta cggcagcagg agctgcaggg acagctactg gggcaggggc 360

acccaggtga ccgtgagcag c 381

<210> 106

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 106

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccgccag cggctacacc ttcaagacct accacatggc ctggttcagg 120

caggcccccg gcatggagag ggagggcgtg gccggcatca acagcgacgg catcaccaag 180

tacgccgaca gcgtgcaggg caggttcacc atcagcaggg acaacgccaa gaacaccctg 240

tacctgcaga tggacagcct gaagcccgag gacaccgcca tgtactactg cgccgccggc 300

agcggcaaca tgttcgactt cctgctgctg agcaggcacc actacaacgc ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 107

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 107

atggcccagg tgcagctgca ggagagcggc ggcggcctgg tgaggcccgg cggcagcctg 60

aggctgagct gcaccgccag cggctacacc ttcaagacct accacatggc ctggttcagg 120

caggcccccg gcaaggagag ggagggcgtg gccggcatca acagcgacgg catcaccaag 180

tacgccgaca gcgtgcaggg caggttcacc atcagcaggg acaacgccaa gaacatcctg 240

tacctgcaga tggacagcct gaagcccgag gacaccgcca tgtactactg cgccgccggc 300

agcggcaacc tgttcgactt cctgctgctg agcaggcacc actacaacgc ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 108

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 108

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccgccag cggctacacc ttcaagacct accacatggc ctggttcagg 120

caggcccccg gcaaggagag ggagggcgtg gccggcatca acagcgaggg cagcaccaag 180

tacgccgaca gcgtgcaggg caggttcacc atcagcaggg acgccgccaa gaacaccctg 240

tacctgcaga tggacagcct gaagcccgag gacaccgcca tgtactactg cgccgccggc 300

agcggcaaca tgttcgactt cctgctgctg agcaggcacc actacaacgc ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 109

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 109

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccgccag cggctacacc ttcaagacct accacatgag ctggttcagg 120

caggcccccg gcaaggagag ggagggcgtg gccggcatca acagcgacgg cgccaccaag 180

tacgccgaca gcgtgcaggg caggttcacc atcagcaggg acaacgccaa gaacaccctg 240

tacctgcaga tggacagcct gaagcccgag gacaccgcca tgtactactg cgccgccggc 300

agcggcaaca tgttcgactt cctgctgctg agcaggcacc actacaacgc ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 110

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 110

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aagctgagct gcgccgccag cggctacacc ttcaagacct accacatgag ctggttcagg 120

caggcccccg gcaaggagag ggagggcgtg gccggcatca acagcgacgg cgtgaccaag 180

tacgccgaca gcgtgcaggg caggttcacc atcagcaggg acaacgccaa gaacaccctg 240

tacctgcaga tggacagcct gaagcccgag gacaccgcca tgtactactg cgccgccggc 300

agcggcaaca tgttcgactt cctgctgctg agcaggcacc actacaacgc ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 111

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 111

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgagggccgg cggcagcctg 60

aggctgagct gcgccgccag cggctacacc ttcaagacct accacatgag ctggttcagg 120

caggcccccg gcaaggagag ggagggcgtg gccggcatca acagcgacgg cgtgaccaag 180

tacgccgaca gcgtgcaggg caggttcacc atcagcaggg acaacgccaa gaacaccctg 240

tacctgcaga tggacagcct gaagcccgag gacaccgcca tgtactactg cgccgccggc 300

agcggcaaca tgttcgactt cctgctgctg agcaggcacc actacaacgc ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 112

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 112

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccgccag cggctacacc ttcaagacct accacatgag ctggttcagg 120

caggcccccg gcagggagag ggagggcgtg gccggcatca acagcgacgg cgtgaccaag 180

tacgccgaca gcgtgcaggg caggttcacc atcagcaggg acaacgccaa gaacaccctg 240

tacctgcaga tggacagcct gaagcccgag gacaccgcca cctactactg cgccgccggc 300

agcggcaaca tgttcgactt cctgctgctg agcaggcacc actacaacgc ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 113

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 113

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccgccag cggctacacc agcaagacct accacatgag ctggttcagg 120

caggcccccg gcaaggagag ggagggcgtg gccggcatca acagcgacgg cgtgaccaag 180

tacgccgaca gcgtgcaggg caggttcacc atcagcaggg acaacgccaa gaacaccctg 240

tacctgcaga tggacagcct gaggcccgag gacaccgcca tgtactactg cgccgccggc 300

agcggcaaca tgttcgactt cctgctgctg agcaggcacc actacaacgc ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 114

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 114

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccgccag cggctacacc ttcaagacct accacatgag ctggttcagg 120

caggcccccg gcaaggagag ggagggcgtg gccggcatca acagcgacgg cgtgaccaag 180

tacgccgaca gcgtgcaggg caggttcacc atcagcaggg acaacgccaa gaacaccctg 240

tacctgcaga tggacagcct gaagcccgag gacaccgcca tgtactactg cgccgccggc 300

agcggctaca tgttcgactt cctgctgctg agcaggcacc actacagcgc ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 115

<211> 378

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 115

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccagcag catctacacc tacaagacct accacatggc ctggttcagg 120

caggcccccg gcaaggagag ggagggcgtg gccggcatca acagcaacgc caggaccgag 180

tacgccgaca gcgtgcaggg caggttcacc atcagccagg acaacgccaa gaacaccctg 240

tacctgcaga tggacagcct gaagcccgag gacaccgcca tgtactactg cgccgccggc 300

gtgggcaacg tgttccacct gctgagcagg aacaactaca acgcctgggg ccagggcacc 360

caggtgaccg tgagcagc 378

<210> 116

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 116

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccgccag cggctacacc ttcaagacct acaggatggg ctggttcagg 120

caggcccccg gcaaggagag ggagggcgtg gccagcttct tcaccggcgg cggcgccaag 180

tacgccgaca gcgcccaggg caggttcttc atcagcaggg acaacgccga gaacaccctg 240

ctgctgctga tggagagcct gatgcacgac gacaccgccg tgtactactg cggcgccggc 300

agcggcaagc tgttcgactt cctgctgctg aggatccacc actacaacgc ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 117

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 117

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccaccag cggcttcacc cccagcaagt actgcatggg ctggttcagg 120

caggcccccg gcaaggagag ggagggcgtg gccagcatca gcaccagggg caccaccacc 180

tactacgccg acagcgtgaa gggcaggttc accatcagca gggacaacgc caagaacacc 240

ctgaacctgc agatgaacag cctgaagccc gaggacaccg ccatgtacta ctgcgccgcc 300

gaccccgccc cctgcaccat gggcggcagc accgccgtga actacaacta ctggggccag 360

ggcacccagg tgaccgtgag cagc 384

<210> 118

<211> 384

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 118

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgtgcccac cggcatctac tacagcggca ggtgcatggc ctggttcagg 120

caggcccccg gcaaggagag ggagagggtg gccagcatcg acaagagcaa caccacctac 180

gccgacagcg tgaagggcag gttcaccatc agccaggaca acgccaagaa caccctgtac 240

ctgctgatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccagcagc 300

tggggcaact actgcccccc caacgacagg agcggcaggg agctgaggta ctggggcccc 360

ggcacccagg tgaccgtgag cagc 384

<210> 119

<211> 378

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 119

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccaccag caagttcccc gtgagcggct actgcagggc ctggttcagg 120

cagacccccg gcaaggagag ggagggcgtg gccaccatca acagcgacgg cagcaccacc 180

tacgccgaca gcgtgaaggg caggttcacc atcagcctgg acaacgccaa gaacaccctg 240

tacctggaga tgaacagcct gaagcccgag gacagcgcca tgtactactg cgccgccagc 300

acctggtaca acaactgcta catcggcagg accgccttca gctactgggg ccagggcacc 360

caggtgaccg tgagcagc 378

<210> 120

<211> 381

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 120

atggcccagg tgcagctgca ggagagcggc ggcggcagcg tgcaggccgg cggcagcctg 60

aggctgagct gcgccgccag cggctacacc tacagcaact actgcatggg ctggttcagg 120

caggcccccg gcaaggagag ggagggcgtg gccaggatca tcagcctggg cggcagcacc 180

tactacgccg acagcgtgaa gggcaggttc accatcagca gggacaacgc caagaacacc 240

ctgtacctgc agatgaacag cctgaagccc gaggacaccg ccatgtacta ctgcgccgcc 300

agccccgcca tgggctgggc ctgcctgggc ggcagggact tcaggtactg gggccagggc 360

acccaggtga ccgtgagcag c 381

Claims

1. The single domain antibody for the IL-17RA protein is characterized in that the amino acid sequence of CDR 1-3 of the heavy chain variable region of the single domain antibody is shown as SEQ ID No. 64-66.

2. The single domain antibody directed against IL-17RA protein of claim 1, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 94.

3. An Fc-fragment fusion protein comprising the single domain antibody to IL-17RA protein of claim 1 or 2 and an Fc-fragment of an immunoglobulin.

4. An isolated nucleic acid encoding a single domain antibody against an IL-17RA protein according to claim 1 or 2.

5. An isolated nucleic acid according to claim 4, wherein said nucleic acid sequence is set forth in SEQ ID No. 118.

6. A recombinant vector comprising the isolated nucleic acid of claim 4 or 5.

7. A host cell comprising the recombinant vector of claim 6.

8. A method for producing a single domain antibody against IL-17RA protein, comprising culturing the host cell of claim 7 to obtain a single domain antibody against IL-17RA protein.

9. A kit for detecting IL-17RA, comprising a single domain antibody directed against IL-17RA protein according to claim 1 or 2.

10. Use of a single domain antibody directed against IL-17RA protein according to claim 1 or 2 for the manufacture of a medicament for inhibiting or preventing an autoimmune disease, which is psoriasis.