CN111040035B - Antibody aiming at IL-17RA protein and preparation method and application thereof - Google Patents

Antibody aiming at IL-17RA protein and preparation method and application thereof Download PDF

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CN111040035B
CN111040035B CN201911424799.6A CN201911424799A CN111040035B CN 111040035 B CN111040035 B CN 111040035B CN 201911424799 A CN201911424799 A CN 201911424799A CN 111040035 B CN111040035 B CN 111040035B
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CN111040035A (en
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苏志鹏
张云
韩顶
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Nanjing Rongjiekang Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7155—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]

Abstract

The invention discloses an antibody aiming at IL-17RA protein, a preparation method and application thereof, relating to the technical field of immunity, wherein the amino acid sequences of CDR1, CDR2 and CDR3 in a heavy chain variable region of the antibody are selected from any one of (1) to (37), which provides a new way for the research of autoimmune diseases or the development of medicaments thereof and is beneficial to developing a new related treatment means of autoimmune diseases.

Description

Antibody aiming at IL-17RA protein and preparation method and application thereof
Technical Field
The invention relates to the technical field of immunization, in particular to an antibody aiming at IL-17RA protein, a preparation method and application thereof.
Background
Interleukin 17 receptor A (IL-17 RA) was first discovered in 1995 and identified as receptors for Interleukin 17A (IL-17A) and Interleukin 17F (IL-17F). Subsequently, a series of other interleukin 17 receptor family members were discovered, IL-17RB, IL-17RC, IL-17RD, and IL-17RE, respectively, these 5 family members all comprising a single domain in sequence, but with great differences from other known families of receptors such as Tumor Necrosis Factor (TNF), Toll-like receptors (TLRs), and type I/II Interferon (IFN) receptors.
IL-17 receptor family members are specifically recognized by IL-17 family member specific ligands. The IL-17 family contains 6 members, IL-17A, B, C, F, E and F. In general, 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 referred to as IL-25) will bind to a receptor complex consisting of IL-17RA and IL-17RB (also referred to as IL-25R). A study in 2012 indicated that IL-17RD also participates in IL-17A-mediated signaling, but IL-17D is currently poorly understood.
There are two main signaling pathways mediated by IL-17 RA: IL-17A (F) signal and IL-17E (IL-25) signal. As is known from the results of the prior art, 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 long recognized as important proinflammatory factors and trigger inflammatory signals such as NF-. kappa. B, MAPKs and C/EBPs.
At present, it has been studied to distinguish between different biological activities in mice deficient in IL-17A/F, respectively. In the mouse model of inflammation, IL-17A showed indirect proinflammatory effects. Studies by O' Connor et al have shown that IL-17A deficient T cells cause an exacerbated disease in a mouse model of CD45RBhi metastatic enteritis.
IL-17RA may activate a number of downstream inflammatory signaling pathways, including the NF-. kappa.B pathway.
NF-. kappa.B is a typical inflammation-associated transcription factor, I.kappa.B.alpha.is an inhibitor of NF-. kappa.B, which phosphorylates and degrades I.kappa.B.alpha.via the p50 and p56 subunits. Interestingly, Shen et al found by MicroArray analysis that IL-17A/IL-17RA promoted expression of I.kappa.B.. zeta.required for IL-6 expression, demonstrating that IL-17A synergistically promotes inflammatory responses with TNF. alpha. In addition, IL-17RA also activates MAPKs signaling as well as C/EBP signaling.
There are also studies that suggest that PI3K and the JAK/STAT pathway are also involved in IL-17RA signaling.
Currently, therapies that target IL-17 signaling molecules are becoming an important tool for treating patients with autoimmune diseases, which can alleviate the very exaggerated inflammatory response in patients, and multiple molecules in the IL-17 signaling pathway are targeting molecules for monoclonal antibodies, such as Ustekinumab and Briakinumab targeting IL-12/23p40, Clazakizumab, Olokizumab, Sirukumab targeting IL-6, Tocilizumab, Sarilumab targeting IL-6R, Secukinumab, Ixekizumab targeting IL-17A.
While the only monoclonal antibody currently under clinical investigation that targets IL-17RA is Brodalumab. 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 the receptor, inhibits inflammatory signaling, and the IL-17 pathway is the key role in initiating and promoting the inflammatory process, and is marketed as monoclonal antibody drug Siliq (bromolumab) developed by Valerant pharmaceuticals, approved by the FDA USA, on 15/2 2017 for the treatment of psoriasis (atopic demattitis). However, this drug is first to be trapped in clinical trials to trigger a suicidal ideative wave in patients and is currently only used as a second-line drug for the treatment of psoriasis. Clearly, the drug presents problems as a therapeutic agent.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The present invention aims to provide an antibody against IL-17RA protein, a method for preparing the same, an isolated nucleic acid comprising the antibody, a recombinant vector, a host cell, and a kit for detecting IL-17 RA.
The invention is realized in the following way:
in a first aspect, embodiments of the invention provide antibodies to IL-17RA protein,
the amino acid sequences of the 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) shown as SEQ ID No. 4-6;
(3) shown as SEQ ID No. 7-9;
(4) shown as SEQ ID No. 10-12;
(5) shown as SEQ ID No. 13-15;
(6) shown as SEQ ID No. 16-18;
(7) shown as SEQ ID Nos. 19-21;
(8) shown as SEQ ID No. 22-24;
(9) shown as SEQ ID No. 25-27;
(10) shown as SEQ ID No. 28-30;
(11) sequences shown as SEQ ID No. 31-33;
(12) shown as SEQ ID No. 34-36;
(13) shown as SEQ ID No. 37-39;
(14) shown as SEQ ID No. 40-42;
(15) shown as SEQ ID No. 43-45;
(16) shown as SEQ ID No. 46-48;
(17) shown as SEQ ID Nos. 49-51;
(18) shown as SEQ ID Nos. 52-54;
(19) Shown as SEQ ID No. 55-57;
(20) shown as SEQ ID No. 58-60;
(21) the sequence shown as SEQ ID No. 61-63;
(22) shown as SEQ ID No. 64-66;
(23) shown as SEQ ID Nos. 67-69;
(24) shown as SEQ ID No. 70-72.
In a second aspect, embodiments of the invention provide an isolated nucleic acid encoding an antibody against IL-17RA protein as described in previous embodiments.
In a third aspect, embodiments of the present invention provide a recombinant vector comprising the isolated nucleic acid described in the previous embodiments.
In a fourth aspect, embodiments of the present invention provide a host cell comprising a recombinant vector as described in the previous embodiments.
In a fifth aspect, embodiments of the invention provide methods for producing antibodies against IL-17RA protein, comprising culturing a host cell as described in the preceding embodiments to obtain antibodies against IL-17RA protein.
In a sixth aspect, embodiments of the invention provide a kit for detecting IL-17RA, comprising an antibody to IL-17RA protein as described in previous embodiments.
In a seventh aspect, embodiments of the present invention provide the use of an antibody against IL-17RA protein as described in the previous embodiments in the manufacture of a medicament for inhibiting or preventing autoimmune disease.
The invention has the following beneficial effects:
the invention provides a novel anti-IL-17 RA antibody, provides a novel approach for the research of autoimmune diseases or the development of medicaments thereof, and is beneficial to developing a novel treatment means related to autoimmune diseases.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is the SDS-PAGE analysis of the recombinant IL-17RA protein of human origin in example 1;
FIG. 2 is the results of analysis of the correct insertion rate of VHH fragments for the single domain antibody library against IL-17RA in example 1;
FIG. 3 shows the results of target enrichment of specific antibodies against IL-17RA by solid phase panning in example 1 based on the constructed nanobody library against IL-17 RA;
FIG. 4 is an SDS-PAGE analysis of the purification of a portion of the single domain antibodies selected 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 result of primary affinity ELISA detection of the single domain antibody and the target protein after purification in example 2;
FIG. 7 is an affinity dose-effect curve of the single domain antibody and the target protein after purification in example 3;
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 invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Noun definitions
A "single domain antibody" as referred to in the present specification is an antibody that naturally lacks a light chain and comprises only one heavy chain variable region (VHH), also known as a nanobody.
Reference to a "CDR" in this specification is a 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 includes 3 CDRs.
The embodiments of the present invention provide antibodies against IL-17RA protein,
the amino acid sequences of the 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) shown as SEQ ID No. 4-6;
(3) shown as SEQ ID No. 7-9;
(4) shown as SEQ ID No. 10-12;
(5) shown as SEQ ID No. 13-15;
(6) shown as SEQ ID No. 16-18;
(7) shown as SEQ ID Nos. 19-21;
(8) shown as SEQ ID No. 22-24;
(9) shown as SEQ ID No. 25-27;
(10) shown as SEQ ID No. 28-30;
(11) sequences shown as SEQ ID No. 31-33;
(12) shown as SEQ ID No. 34-36;
(13) shown as SEQ ID No. 37-39;
(14) shown as SEQ ID No. 40-42;
(15) shown as SEQ ID No. 43-45;
(16) shown as SEQ ID No. 46-48;
(17) shown as SEQ ID Nos. 49-51;
(18) shown as SEQ ID Nos. 52-54;
(19) shown as SEQ ID No. 55-57;
(20) shown as SEQ ID No. 58-60;
(21) the sequence shown as SEQ ID No. 61-63;
(22) shown as SEQ ID No. 64-66;
(23) shown as SEQ ID Nos. 67-69;
(24) shown as SEQ ID No. 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 alternative embodiments, 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, specifically, the framework region comprises FR1, FR2, FR3 and FR4, and the structure of the heavy chain variable region is:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
when the amino acid sequences of the CDRs 1-3 of the heavy chain variable region of the antibody are sequentially shown in (1) - (24), the amino acid sequences of the heavy chain variable region are sequentially shown in SEQ ID Nos. 73-96. In alternative embodiments, the antibody is selected from the group consisting of a single domain antibody, a heavy chain antibody, an Fc fragment fusion protein, IgGI, IgG2, IgG4, IgA, IgE, IgM, IgD, Fab ', Fab, F (ab')2Fv and scFv fragments.
In particular, a single domain antibody (VHH) is a heavy chain variable region as set out in any one of the preceding examples. It should be noted that, when the "antibody against IL-17RA protein" 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.
The heavy chain antibody (HcAb) is an antibody comprising the above-described single domain antibody and two constant regions (CH2 region and CH3 region); the Fc fragment fusion protein is an antibody obtained by fusing the heavy chain variable region with the Fc fragment of immunoglobulin.
The "IgGI, IgG2, IgG4, IgA, IgE, IgM, IgD, Fab ', Fab, F (ab')2Fv and scFv fragments "are antibodies comprising the heavy chain variable region as defined above, where" IgGI, IgG2, IgG4, IgA, IgE, IgM, IgD, Fab ', Fab, F (ab')2Fv and scFv fragments "are only illustrative of the possible types of antibodies, and are within the scope of the present invention, provided that they contain the 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 alternative embodiments, the antibody is a single domain antibody.
The single domain antibody may be artificially synthesized, or may be obtained by synthesizing a gene encoding the antibody and expressing the antibody biologically.
The embodiments provide an isolated nucleic acid encoding an antibody against IL-17RA protein according to any one of the preceding embodiments;
in alternative embodiments, the nucleic acid comprises any one of the sequences set forth in SEQ ID Nos. 97-120.
It is noted that when the antibody is a single domain antibody and the sequence thereof is shown in SEQ ID Nos. 73-96 in sequence, the sequence of the nucleic acid is shown in SEQ ID Nos. 97-120 in sequence.
The embodiments provide a recombinant vector comprising an isolated nucleic acid as described in any of the preceding embodiments.
In alternative embodiments, the recombinant vector may be a plasmid, a phage, or a viral vector.
Embodiments of the present invention provide a host cell containing a recombinant vector as described in the preceding embodiments.
In alternative embodiments, the host cell may be a prokaryotic cell or a eukaryotic cell.
The embodiment of the invention provides a preparation method of an antibody aiming at IL-17RA protein, which comprises the steps of culturing the host cell as described in the previous embodiment to obtain the antibody aiming at the IL-17RA protein.
The embodiment of the invention also provides a kit for detecting IL-17RA, which comprises the antibody aiming at the IL-17RA protein according to any one of the previous embodiments.
In addition, the embodiment of the invention also provides application of the antibody aiming at the IL-17RA protein in preparing a medicament for inhibiting or preventing autoimmune diseases.
In an alternative embodiment, the autoimmune disease comprises at least one of crohn's disease, psoriasis, hidradenitis suppurativa, systemic inflammation, and multiple sclerosis of tissue.
In alternative embodiments, the antibodies inhibit or prevent autoimmune diseases by inhibiting the binding of IL-17 receptors to IL-17 cytokines.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Preparation of single domain antibody against human recombinant IL-17RA protein.
1. Constructing an expression vector of the human recombinant IL-17RA protein:
the coding sequence for IL-17RA was retrieved at NCBI and included under NM-014339.6, and the amino acid sequence generated by this sequence was accession NP-055154.3. Then, the amino acid sequence corresponding to NP-055154.3 was analyzed for the transmembrane region and extracellular end of the protein by TMHMM and SMART websites, respectively.
The analysis result shows that the extracellular end of the IL-17RA protein is amino acids 1 to 320 th of the sequence, wherein, the 1 st to 32 th sites are signal peptides of the protein.
The nucleotide sequence of the 1 st to 320 th amino acid of the coding IL-17RA protein is cloned into a vector pcDNA3.4 by restriction enzymes XbaI and AgeI by using a sequence specific primer. Sanger sequencing is carried out on the constructed vector, the original sequence is compared, after no error is confirmed, batch extraction is carried out on the recombinant plasmid, endotoxin is removed, expression and purification of target protein (IL-17RA protein) are carried out by transfection and suspension 293F, and SDS-PAGE analysis after purification of the human recombinant IL-17RA protein is shown in figure 1.
As can be seen from figure 1, the purity of the purified protein is as high as 90%, and the animal immunity requirement is met.
2. Construction of Single Domain antibody library of IL-17RA protein
1mg of the human recombinant IL-17RA protein obtained by purification in the step 1 is mixed with Freund complete adjuvant with the same volume, one inner Mongolia alashan bactrian camel is immunized once a week for 7 times in a continuous way, the rest six times except the first immunization are animal immunization by mixing 1mg of the IL-17RA protein with Freund incomplete adjuvant with the same volume, and the immunization process is to intensively stimulate the camel to generate antibodies aiming at the IL-17RA protein.
After 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 nested PCR reaction using the cDNA as a template.
Then, respectively carrying out enzyme digestion on the pMECS vector and the VHH fragment by using restriction enzymes, and then linking the enzyme-digested fragment with the vector. The ligated fragments were spotted into competent cells TG1, a phage display library of MMP9 protein was constructed and the library size was determined to be about 1X 109Meanwhile, the correct insertion rate of the test library in the target fragment is identified by colony PCR, and the result is shown in FIG. 2.
The results showed that after PCR amplification of 30 randomly selected colonies from the library, 28 clones amplified a band of 600bp (predicted size) and 2 clones amplified a band of incorrect size, so the correct insertion rate was 28 ÷ 30X 100% ≈ 93.3%.
3. Screening of Single-Domain antibodies against IL-17RA protein
200 μ L of recombinant TG1 cells from step 2 were cultured in 2 XTY medium during which 40 μ L of helper phage VCSM13 was added to infect TG1 cells and cultured overnight to amplify phages, which were precipitated with PEG/NaCl the next day and collected by centrifugation.
NaHCO diluted at 100mM pH 8.33 Coupling 500 mu g of IL-17RA protein on an enzyme label plate, standing overnight at 4 ℃, and simultaneously setting a negative control hole; adding 200 μ L of 3% skimmed milk the next day, sealing at room temperature for 2 hr; after the end of blocking, 100. mu.l of the amplified phage library (approx.2X 10) was added11Individual phage particles), and reacting for 1h at room temperature; after 1 hour of action, the unbound phage were washed away with PBS + 0.05% Tween-20 5 times.
The phage specifically bound with the IL-17RA protein is dissociated by trypsin with the final concentration of 25mg/mL, escherichia coli TG1 cells in the logarithmic growth phase are infected, the cells are cultured for 1h at 37 ℃, the 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 is the number of monoclonal bacteria that grow after phage eluted from positive well was infected with TG1 bacteria in biopanning/the number of monoclonal bacteria that grow after phage eluted from positive well was infected with TG1 bacteria, which parameter gradually increases after enrichment has occurred; I/E ═ total phage added to positive wells per biopanning round/total phage eluted from positive wells per biopanning round, this parameter will gradually approach 1 after enrichment has occurred.
4. Enzyme-linked method for screening specific positive clone resisting IL-17RA
And (3) performing 3 rounds of screening on the single domain antibody against the IL-17RA protein according to the screening method in the step 3, wherein the phage enrichment factor against the IL-17RA protein reaches more than 10, after screening is finished, selecting 400 single colonies from positive clones obtained by screening, respectively inoculating the single colonies into a 96-deep-well plate containing 100 mu g/mL ampicillin TB culture medium, setting a blank control, culturing at 37 ℃ until the logarithm phase, adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM, and culturing at 28 ℃ overnight.
Obtaining a crude antibody by using a permeation cracking method; the IL-17RA recombinant protein was released separately to 100mM NaHCO, pH 8.33And 100. mu.g of protein was coated overnight at 4 ℃ in an ELISA plate (ELISA plate). Transferring 100uL of the obtained antibody crude extract to an ELISA plate added with an antigen, and incubating for 1h at room temperature; unbound antibody was washed away with PBST, 100. mu.l of Mouse anti-HA tag antibody (Mouse anti-HA antibody, Thermo Fisher) diluted at 1:2000 was added, and incubated at room temperature for 1 h; unbound antibody was washed away with PBST, 100. mu.l of Anti-Rabbit HRP conjugate (goat Anti-Rabbit horseradish peroxidase labeled antibody, purchased from Thermo Fisher) diluted at 1:20000 was added, and incubated at room temperature for 1 h; washing away unbound antibody with PBST, adding horseradish peroxidase developing solution, reacting at 37 deg.C for 15min, adding stop solution, and reading the absorption value at 450nm wavelength on enzyme-labeling instrument.
When the OD value of the sample hole is more than 5 times of that of the control hole, judging the sample hole as a positive cloning hole; the positive colony well was transferred into LB medium containing 100. mu.g/mL ampicillin to extract plasmids and sequenced.
Analyzing the gene sequences of the clones according to the Vector NTI of the sequence alignment software, regarding the strains with the same CDR1, CDR2 and CDR3 sequences as the same clones, and regarding the strains with different sequences as different clones, and finally obtaining the single domain antibody specific to the IL-17RA protein.
The amino acid sequence of the antibody is the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the whole VHH is formed. The obtained single domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally, single domain antibody protein is obtained.
5. And (3) purifying and expressing the single-domain antibody of the anti-IL-17 RA protein in host Escherichia coli.
The plasmids (pMECS-VHH) of the different clones obtained by sequencing analysis in step 4 were electrically transformed into E.coli HB2151 and spread on LB + amp + glucose, i.e., a culture plate containing ampicillin and glucose, and cultured overnight at 37 ℃; individual colonies were picked and inoculated into 5mL of LB medium containing shoal penicillin and shake-cultured overnight at 37 ℃.
Inoculating 1mL of overnight cultured strain into 330mL of TB culture solution, performing shake culture at 37 ℃ until OD600nm value reaches 0.6-0.9, adding 1M IPTG, and performing shake culture at 28 ℃ overnight; centrifuging, collecting Escherichia coli, and obtaining crude antibody extractive solution by use of osmotic bursting method;
The antibody is purified by nickel column affinity chromatography, and the purified single-domain antibody comprises VHH 1-18 as shown in figure 4.
6. And (3) constructing an Fc fragment fusion protein eukaryotic expression vector containing a single-domain antibody of the anti-IL-17 RA protein.
Subcloning the target sequence obtained in step 4 into a eukaryotic expression vector: and (5) carrying out Sanger sequencing on the single-domain antibody screened out in the step 4 to obtain the nucleotide sequence of the single-domain antibody. Synthesizing the nucleotide sequence after codon optimization into a target vector RJK-V4-hFC which is universal for the designed and modified nano antibody by a sequence synthesis mode.
RJK-V4-hFC is modified by fusing Fc segment in heavy chain coding sequence of human IgG (NCBI Accession No: AB776838.1) on the basis of Invitrogen commercial vector pCDNA3.4 (vector data link: https:// Assets. thermofisher. com/TFS-Assets/LSG/manuals/pcdna 3-4-topo-ta-cloning-man. pdf), i.e., the vector contains the Hinge region (Hinge) CH2 and CH3 regions of IgG heavy chain. The specific modification scheme is as follows:
selecting restriction sites XbaI and AgeI on pcDNA3.4; introducing a Multiple Cloning Site (MCS) and a 6 XHis tag at the 5 'end and the 3' end of the Fc fragment coding sequence, respectively, by means of overlapping PCR, as shown in FIG. 5; amplifying the fragments by using a pair of primers with XbaI and AgeI enzyme cutting sites respectively in a PCR (polymerase chain reaction) mode to obtain recombinant DNA fragments; using restriction enzymes XbaI and AgeI to respectively enzyme-cut pcDNA3.4 and the obtained recombinant DNA fragment; and (3) connecting the vector and the insert after enzyme digestion under the action of T4 ligase, then transforming the connection product into escherichia coli, amplifying, sequencing and verifying to obtain the recombinant plasmid.
Transforming the constructed recombinant eukaryotic expression vector into DH5 alpha escherichia coli, culturing, carrying out plasmid large extraction, and removing endotoxin; carrying out sequencing identification on the plasmid subjected to the large extraction; and preparing the recombinant vector without errors for subsequent eukaryotic cell transfection 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 2.5X 105/ml cell passage and expanded culture Expi CHO-STMCells, calculated required cell volume transferred to ExpCHO filled with fresh preheated 120ml (final volume)TMIn a 500ml shake flask of expression medium; to achieve a cell concentration of about 4X 106～6×106Viable cells/mL.
One day before transfection, ExpicHO-STMCell dilution to 3.5X 106Viable cells/mL, cells were cultured overnight; on the day of transfection, cell density and percentage of viable cells were determined. The cell density before transfection should reach about 7X 106～10×106Viable cells/mL.
With fresh Expi CHO preheated to 37 ℃TMExpression media cells were diluted to 6X 106Viable cells/mL. The calculated required cell volume was transferred to ExpicHO containing fresh preheated 100ml (final volume)TMIn a 500ml shake flask of expression medium: expifeacmine was mixed by gentle inversion TMCHO reagent, 3.7ml OptiPROTMDilution of Expifeacylamine in culture MediumTMCHO reagent, swirling or mixing; with refrigerated 4ml OptiPROTMDiluting plasmid DNA with a culture medium, and mixing uniformly; expifeacylamine CHO/plasmid DNA complex was incubated at room temperature for 1-5 minutes, then added gently to the prepared cell suspension, with gentle swirling of the flask during the addition.
Cells were incubated at 37 ℃ with 8% CO2Carrying out shake culture in humidified air; on day 1 post-transfection (18-22 hours later) 600. mu.l Expifeacylamine was addedTMCHO Enhancer and 24ml ExpicHO feed. Supernatants were collected approximately 8 days after transfection (cell viability below 70%).
8. Expression of Fc fragment fusion proteins containing single domain antibodies against IL-17RA protein in suspension 293F cells.
Recombinant single domain antibody expression experimental protocol (taking 500ml shake flask as an example):
3 days before transfection at 2.5X 105The 293F cells were passaged and expanded in culture and the calculated required cell volume was transferred to a 500ml shake flask containing fresh pre-warmed 120ml (final volume) OPM-293CD05 Medium. The cell concentration was brought to about 2X 106 to 3X 106 viable cells/mL.
On the day of transfection, cell density and percentage of viable cells were determined. The cell density before transfection should reach about 2X 10 6～3×106Viable cells/mL. Cells were diluted to 1X 10 with preheated OPM-293 CD05 Medium6Viable cells/mL. The required cell volume was calculated and transferred to a 500ml shake flask containing fresh pre-warmed 100ml (final volume) of medium.
Diluting PEI (1mg/ml) reagent with 4ml of Opti-MEM medium, and swirling or blowing to mix evenly; the plasmid DNA was diluted with 4ml Opt-MEM medium, vortexed, mixed well, and filtered through a 0.22um filter tip. Incubate at room temperature for 5 min.
Diluted PEI reagent was added to the diluted DNA and mixed by inversion. The PEI/plasmid DNA complex was incubated for 15-20 minutes at room temperature and then gently added to the prepared cell suspension, with gentle swirling of the flask during the addition.
Cells were incubated at 37 ℃ with 5% CO2And shake culturing at 120 rpm. 5ml OPM-CHO PFF05 feed was added at 24h, 72h post transfection. Supernatants were collected approximately 7 days after transfection (cell viability below 70%).
9. Purification of Fc fragment fusion proteins containing single domain antibodies against IL-17RA protein
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-derived Fc and Protein A (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 bound to the packing material; washing the impurity protein bound on the column by low-salt and high-salt buffer solutions; eluting the target protein bound on the column by using a low pH buffer solution; the eluate was rapidly added to a Tris-HCl solution of pH9.0 to neutralize it.
Dialyzing the neutralized protein solution, performing SDS-PAGE analysis to determine that the protein has a purity of 95% or more and a concentration of 0.5mg/mL or more, and storing at low temperature for later use.
Example 2
The ELISA assay for affinity of the single domain antibodies provided in example 1 to IL-17 RA.
The VHH of anti-IL-6 is used as an isotype control to detect the binding capacity of the VHH of the anti-IL-17 RA protein and a target spot, and the operation steps are as follows:
50 mu L of human recombinant IL-17RA sample with the concentration of 1 ng/mu L is coated on an ELISA plate and is coated overnight at 4 ℃; sealing the coated plate by using 5% of skimmed milk powder for 1 hour, wherein each hole is 200 mu L of skimmed milk powder; adding the single domain antibody obtained in step 4 of example 1, which carries HA or a human Fc tag, and incubating for 1 hour; adding a detection antibody (HRP mark) specific to HA-labeled protein or human Fc, and incubating for 0.5 hour; adding a chromogenic substrate TMB for color development; stop solution was added to terminate the reaction. OD450 values were measured as shown in FIG. 6. The results showed that only a portion (24 antibodies) of the 37 single-domain antibodies biopanning had significant specific binding to IL17RA in the ELISA assay, and the remaining 13 antibodies were judged to be non-specific binding.
The amino acid sequence of the 24-strain single-domain antibody is shown in SEQ ID No. 73-96, please refer to Table 1. TABLE 1 Single Domain antibodies
Single domain antibody numbering Cloning SEQ ID No.
1 1A12 73
2 3A10 74
3 3C8 75
4 2B9 76
5 3E11 77
6 3D4 78
7 1F6 79
8 1F9 80
9 2F7 81
10 1B6 82
11 3B1 83
12 1H10 84
13 1C11 85
14 1G12 86
15 3B8 87
16 3F7 88
17 3D3 89
18 4A8 90
19 2A7 91
20 4A9 92
21 2F4 93
22 1B12 94
23 2H8 95
24 2H11 96
Example 3
The single domain antibody provided in example 1 was assayed for affinity to IL-17RA using a dose-response curve.
50 mu L of human recombinant IL-17RA sample with the concentration of 1 ng/mu L is coated on an ELISA plate and stays overnight at 4 ℃; washing the plate; adding 200 μ L of 5% milk, sealing at 37 deg.C for 1 h; respectively diluting the 24 single-domain antibodies with higher P/N values in example 2 to 2 mu g/ml, and then diluting the antibodies by 5 times of gradient to obtain 8 concentration gradients; washing the plate; adding 50 mu L of antibody, carrying out two-hole reaction, and incubating for 1h at 37 ℃; washing the plate; adding 50 mu L of mouse anti-HA label HRP secondary antibody, and incubating for 30min at 37 ℃; washing the plate; adding 50 μ l of TMB recovered to normal temperature in advance, and reacting for 15min at normal temperature in the dark; adding 50 mul of stop solution (1N HCl), and reading and storing by an enzyme-linked immunosorbent assay; curves were drawn and EC50 was calculated and the results are shown in fig. 7.
As can be seen from fig. 7, among the 24 antibodies analyzed and obtained in example 2, 9 single-domain antibodies bound to IL17RA with a lower dose-response curve EC50 value and a larger curve window, which demonstrates that the 9 single-domain antibodies have a lower affinity constant and a higher affinity for IL17 RA.
The 9 single-domain antibodies are respectively: 1H10, 3B1, 4a8, 1B6, 4a9, 1G12, 3B8, 1C11, and 3a 10.
Antibodies have achieved nM in their immature affinity constants. Among them, clone 1B12 showed better biofunctional activity in the functional results of example 5, although the EC50 curve window was smaller in this experiment.
Example 4
IL-17RA in example 1 was tested for cytokine release by neutralizing single domain antibodies.
IL-17 induces the release of IL-6 from HeLa cells, and neutralizing antibodies to IL-17R can block this response. The experimental procedure was as follows:
spreading Hela cells on a 96-well plate, wherein each well contains ten thousand cells; a maximum concentration of 10. mu.g/ml of VHH (single domain antibody with lower EC50 in example 3) diluted in a 5-fold gradient against IL-17RA was mixed 1:1 with 220ng/ml IL-17, and 3 control sets of controls, lxekizumab, Brodalumab and blank control hIgG, respectively; mixing the mixed mixture with cells according to a ratio of 1:1, culturing for 24 hours, and collecting cell supernatant; the expression of IL-6 in the cell supernatant was determined using a human IL-6ELSIA kit, and the ELISA assay conditions were as described in Thermo, Cat #88-7066-88, with the results shown in FIG. 8.
The results show that the 9-strain single-domain antibody with lower EC50 (high affinity) obtained in example 3 is better in functional experiments with clones 3B8 and 4a9, but the functions thereof can be optimized by amino acid mutation, while clone 1B12 is excellent in this example although the dose-response curve window is small in example 3, and EC50 is lower and the curve window is larger.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Nanjing Rodgeikang Biotech Ltd
<120> antibody for IL-17RA protein, preparation method and application thereof
<160> 120
<170> PatentIn version 3.5
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Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala
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Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Ser
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Ser Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Tyr Glu
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Gly Val Ala Ala Ile Asp Ser Lys Gly Ser Thr Met Tyr Ala Asp Ser
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Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Ser Thr Leu
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Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr
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Cys Ala Ala Gly Asp Lys Tyr Asp Cys Tyr Ser Gly Ser Trp Ser Asn
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Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala
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Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Lys Tyr Thr Asn Arg
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Ser Tyr Cys Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu
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Gly Val Ala Ala Ile Asp Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser
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Val Lys Gly Arg Phe Thr Val Ser Gln Gly Asn Ala Lys Asn Thr Leu
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Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr
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Cys Val Ala Asp Ala Gly Tyr Asp Cys Tyr Ser Gly Ser Trp Phe Glu
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Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala
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Ser Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Lys Arg Glu
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Gly Val Ala Ser Ile Tyr Thr Gly Asp Gly Ser Thr His Tyr Ala Asp
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Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Gln Asn Thr
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Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr
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Tyr Cys Ala Ala Asp Thr Gln Asn Ser Phe Thr Ala Pro Tyr Trp Gly
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Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro
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Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
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Ser Tyr Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
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Trp Val Ser Thr Ile Asn Ser Gly Val Gly Ser Thr Thr Tyr Tyr Ala
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Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
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Thr Leu Tyr Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met
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Tyr Tyr Cys Ala Lys Gly Ser Ile Glu Tyr Asp Ser Asp Tyr Arg Val
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Asn Tyr Val Glu Ala Lys Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro
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Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser
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Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
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Trp Val Ser Asn Ile Asn Ile Asp Gly Ser Arg Thr Phe Tyr Ala Asp
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Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr
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Leu Tyr Leu Gln Leu Asn Ser Leu Arg Thr Glu Asp Thr Ala Met Tyr
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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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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 (11)

1. The nano antibody for the IL-17RA protein is characterized in that the amino acid sequence of CDR 1-3 of a heavy chain variable region of the antibody is shown as SEQ ID No. 43-45 or SEQ ID No. 58-60.
2. The nanobody against IL-17RA protein of claim 1, wherein the heavy chain variable region further comprises a framework region.
3. The nanobody against IL-17RA protein of claim 2, wherein when the amino acid sequences of CDRs 1-3 of the heavy chain variable region of the nanobody are sequentially represented as SEQ ID nos. 43-45, the amino acid sequence of the heavy chain variable region is represented as SEQ ID No. 87;
When the amino acid sequences of the CDRs 1-3 of the heavy chain variable region of the nano antibody are sequentially shown as SEQ ID Nos. 58-60, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 92.
4. An isolated nucleic acid encoding the nanobody for IL-17RA protein of any one of claims 1 to 3.
5. The isolated nucleic acid of claim 4, wherein the nucleic acid comprises a sequence set forth as SEQ ID No.111 or 116.
6. An Fc fragment fusion protein comprising the nanobody against the IL-17RA protein according to any one of claims 1 to 3 and an Fc fragment of an immunoglobulin.
7. A recombinant vector comprising the isolated nucleic acid of claim 4 or 5.
8. A host cell comprising the recombinant vector of claim 7.
9. A method for producing a nanobody against IL-17RA protein, which comprises culturing the host cell according to claim 8 to obtain a nanobody against IL-17RA protein.
10. A kit for detecting IL-17RA, comprising the nanobody of any one of claims 1 to 3, which is directed against the IL-17RA protein.
11. Use of a nanobody against an IL-17RA protein as claimed in any one of claims 1 to 3 for the preparation of a medicament for the inhibition or prevention of an autoimmune disease, psoriasis.
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