CN114437232B - Cell surface macromolecule quantitative display system and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a cell surface macromolecule quantitative display system, a preparation method and application thereof. Wherein the transmembrane protein of the ligand quantitative presenting cell is fused with a phase change peptide fragment, the receptor expression cell is synNotch synthetic receptor expression cell, and the extracellular region of the synNotch synthetic receptor is fused with a region capable of specifically recognizing and binding to the ligand on the surface of the ligand presenting cell. Ligand quantitative presenting cells and receptor expressing cells are co-cultured, and the receptor of the receptor expressing cells can be specifically and quantitatively activated. The cell surface macromolecule quantitative display system can be applied to ligand-receptor (including antigen-antibody) affinity detection and/or high-affinity antibody batch acquisition, so that batch high-affinity antibodies can be rapidly obtained, and the subsequent tedious verification work of phage display and other technologies is avoided.

Description

Cell surface macromolecule quantitative display system and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a cell surface macromolecule quantitative display system, a preparation method and application thereof.

Background

When cells are in contact with each other, it remains a scientific puzzle on how mammalian cells sense and differentiate the size of cell surface receptors and their corresponding ligand affinities and convert them into specific quantitative biological events. For example, the mechanism of how lymphocytes expressing high affinity antibodies (i.e., B cell receptors) compete with low affinity B cells for antigen molecules displayed on the surface of antigen presenting cells remains unclear.

Antibody affinity maturation is a marker of the immune response of the adaptive immune system, during which high affinity antibodies against specific antigens will be produced. Whereas antibody affinity maturation of Germinal Centers (GC) mainly involves high frequency mutation (SHM) of the antibody variable regions and affinity-based B cell selection. Wherein, the high frequency mutation can induce a large number of B Cell Receptors (BCRs) with different affinities, and B cells with higher affinity BCRs can better competitively bind with follicular helper T cells (Tfh) positioned in a bright area of the germinal center in the germinal center, so that a small number of B cells with high affinity can finally dominate. This affinity-based selection depends on the amount of antigen that B cells extract from the surface of Antigen Presenting Cells (APCs) and re-present on their own cytoplasmic membranes. The downstream signaling pathway response of a particular antigen activated B cell is primarily determined by the affinity between the antigen and BCR. It has been reported that high affinity antigen-antibody interactions may form stronger mechanical forces, and that the specific micro-cluster structure formed by BCR in the hair-growing center may also be related to this mechanism. However, the molecular mechanism of how high affinity BCR captures more antigen molecules is currently unclear. The synNotch synthesis system reported as an antibody expression system does not distinguish well between antibody-antigen affinity differences.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a quantitative display system for cell surface macromolecules, and a preparation method and application thereof, which are used for solving the problems in the prior art.

To achieve the above and other related objects, the present invention first provides a recombinant transmembrane protein having a phase-change peptide fused thereto.

The invention also provides an isolated polynucleotide encoding the recombinant transmembrane protein.

The invention also provides a nucleic acid construct comprising the polynucleotide.

The invention also provides a cell, wherein the cell is fused with the transmembrane protein.

The invention also provides a cell surface macromolecule quantitative display system, which comprises the ligand quantitative presenting cell and a receptor expression cell, wherein the receptor of the receptor expression cell can be specifically and quantitatively activated when the ligand quantitative presenting cell and the receptor expression cell are cultured together.

The invention also provides a preparation method of the cell surface macromolecule quantitative display system, which comprises the following steps:

1) Cloning a receptor of interest onto a cell membrane of a receptor expressing cell;

2) Cloning the ligand of interest onto the ligand-quantitative presenting cell.

The invention also provides application of the cell surface macromolecule quantitative display system in ligand-receptor affinity detection and/or batch acquisition of high-affinity antibodies.

The invention also provides a ligand-receptor affinity detection method, which comprises the step of detecting the binding condition of a target ligand and a target receptor after the ligand quantitative presenting cell and the receptor expression cell obtained in the preparation method are co-cultured.

The invention also provides a method for obtaining high-affinity antibodies in batches, which is characterized by comprising the following steps:

1) Constructing a receptor expression cell carrying a synNotch antibody library and a reporter group;

2) Co-culturing the receptor expressing cells of step 1) with said ligand quantitative presenting cells to activate the cell surface macromolecule quantitative display system;

3) Collecting cells in the co-culture system, and primarily enriching receptor expression cells positive to the reporter group;

4) Culturing the receptor expression cells preliminarily enriched in the step 3) for 5-10 days, and carrying out secondary enrichment by using the new ligand quantitative presenting cells, wherein the enriched receptor expression cells carry the high-affinity antibody.

As described above, the cell surface macromolecule quantitative display system and the preparation method and the application thereof have the following beneficial effects:

1) Cell surface macromolecule quantitative display technology based on protein phase transition, binding to synNotch system can convert ligand-receptor, e.g., antigen-antibody affinity, to intensity of intracellular reporter gene expression;

2) The cell surface macromolecule display technology is related to membrane protein intracellular phase transformation, and a plurality of tool molecules capable of supporting quantitative display are discovered, and protein liquid-liquid phase transformation can be carried out on the molecules under specific physiological conditions;

3) The cell surface macromolecule display technology can be used for cell surface ligand-receptor affinity identification, such as quantitative verification of interaction between coronavirus neutralizing antibody or receptor protein ACE2 and coronavirus spinous process protein;

4) By combining phage display technology or other antibody library construction technology, high-affinity antibodies in batches can be obtained rapidly, and the subsequent tedious verification work of phage display technology can be avoided.

Drawings

FIG. 1 shows an example of a quantitative display system for cell surface macromolecules and detection of ligand-receptor binding affinity according to the present invention, wherein the following is illustrated in the figures: schematic system of interaction of Ag-mem and Ag-m-LC cells with anti-GFP synNotch receptor cells, respectively; schematic representation of expression vectors for Ag-mem and Ag-m-LC and synNotch receptor; GFP-mem and GFP-mem-FUS _LC Reporting the system activation ratio after interaction of the cells with anti-GFP/mCherry receptor cells; reporting the system activation ratio after interaction of mCherry-mem cells with anti-GFP/mCherry receptor cells; E.GFP-mem and GFP-m-FUS _LC Reporter lines after interaction of cells with two high/low anti-GFP receptor cells in different ratiosThe proportion of the system activation; F. information of the anti-GFP nano antibody used and the expression quantity of the anti-GFP nano antibody on a cell membrane after corresponding fusion of a synNotch receptor; G.GFP-mem and GFP-m-FUS _LC The BFP report system activation ratio corresponding to the cells.

FIG. 2 shows the principle of quantitative presentation for a cell surface macromolecular quantitative display system, wherein the figures are described as follows: GFP-mem and GFP-m-FUS _LC Confocal fluorescence imaging of GFP in cells; GFP-mem and GFP-m-FUS _LC GFP protein localization statistics of cells; C. GFP-mem and GFP-m-FUS with different expression levels _LC Cell surface GFP protein flow quantification results; D. GFP-mem and GFP-m-FUS with different expression levels _LC The cell-corresponding reporting system activation ratio; E. schematic of the interaction of red fluorescent labeled synNotch receptor expressing cells (engineered to human embryonic kidney cell HEK293T cell line) with ligand quantitative presenting cells (engineered to human myeloid leukemia cell K562 cell line); F. ligand quantifies the interface at which the presenting cell interacts with synNotch receptor cells; a G.qDis system action mode diagram; HER2-mem and HER2-m-FUS _LC Schematic of the system for interaction with anti-HER2 synNotch receptor cells; I. cell surface HER2 extracellular domain expression level; J. cell surface anti-HER2-scfv synNotch receptor expression level; HER2-mem and HER2-m-FUS _LC The corresponding reporting system activates the ratio.

Fig. 3 shows the quantitative phase change principle for a cell surface macromolecule quantitative display system, wherein the figures are described as follows: FUS _LC Q, G, Y amino terminal distribution in proteins and FUS _LC Protein membrane surface expression amounts of different mutant cells and corresponding report system activation ratios; GFP-m-FUS _LC -a reporter system activation ratio corresponding to 478 cells; GFP-m-TAF15 _LC And GFP-m-DDX4 _LC The cell-corresponding reporting system activation ratio; the reporter system activation ratio for GFP-foldon-m cells and GFP-Fc-m cells;

FIG. 4 shows the principle of quantification and mutation analysis for a cell surface macromolecule quantitative display system, wherein the figures are described below: A. different GFP-m-FUS _LC Rate statistics of protein aggregation of mutants in an opto-droplet system, corresponding to the systemCell count is noted in the legend; GFP-m-FUS _LC Q>GFP confocal fluorescence imaging of G cells, and activating proportion results of a flow detection and reporting system; GFP-m-FUS _LC G>GFP confocal fluorescence imaging of A cells, and activating a proportional result by a flow detection and reporting system; GFP-m-FUS _LC GFP confocal fluorescence imaging of Y27S cells, flow detection and reporting system activation proportion results; E.GFP-m-FUS _LC GFP confocal fluorescence imaging of Y7A cells, flow detection and reporting system activation proportion results; F.GFP-m-FUS _LC GFP confocal fluorescence imaging of 27R cells, flow detection and reporting system activation ratio results;

FIG. 5 shows the corresponding reporter system activation ratios for different GFP-m-LC cells.

FIG. 6 shows interactions between various coronavirus surface spinous process proteins S-RBD and ACE2 or its corresponding antibodies, wherein the following is a description of the figures: A. synNotch cells expressing hACE2 and multiple antibodies with S-RBD-m-FUS fused to coronavirus surface spinous process proteins of multiple different species _LC The proportion of reporter systems activated by protein interactions; B. S-RBD-m-FUS of ACE2 chimeric receptor expression cells of different species and three coronavirus surfaces _LC The proportion of reporter system activated by protein interactions. C. Three-dimensional structure of interaction of human ACE2 protein (green) with SARS-CoV-2S-RBD protein (blue), wherein direct acting peptide fragments on ACE2 are marked orange.

FIG. 7 shows the bulk isolation of high affinity antibodies for a cell surface macromolecular quantitative display system, wherein the illustrations of the figures are as follows: schematic diagram of hcd8 reporting system; GFP-mem and GFP-m-FUS _LC The activation proportion of the hCD8 report system after the cells respectively interact with the anti-GFP receptor cells with high, medium and low affinities; C. ratio of high affinity antibody expressing cells after each round of qDis-MACS enrichment; schematic flow chart of qddis-MACS; E. after three rounds of qDis-MACS screening, the antibody enrichment ratio, red is the final choice of expressed antibody; the BLI method determines the affinity of the antibody.

FIG. 8 shows the binding dissociation curves corresponding to the antibodies determined for the BLI method.

Detailed Description

The invention firstly provides a recombinant transmembrane protein, wherein a phase change peptide segment is fused on the recombinant transmembrane protein.

The phase-change peptide segment refers to a peptide segment capable of undergoing phase change under physiological conditions. The phase change peptide may be any one or several peptide fragments of LLPSDB database (http:// bio-comp. Ucas. Ac. Cn/LLPSDB), phaSePro database (https:// Phasepro. Elte. Hu), or PhaSepDB database (http:// db. Phasep. Pro), or a peptide fragment mutated based on the peptide fragment in the database. The peptide fragments mutated based on the peptide fragments in the database have a similarity of 80% or more, 90% or more, 95% or more, or 99% or more with the phase-change peptide fragments in the database. The low complexity sequence (also called LC tag) is one of the phase change peptide fragments.

By low complexity sequence is meant that the protein sequence is enriched for a particular amino acid component and/or that a particular amino acid arrangement is repeated. Such as repeated expression of specific amino acid residues, repeated arrangement of single amino acid chains or short amino acid chains. Low Complexity Sequences (LCs) exist in intrinsically disordered regions and proteins or peptide fragments containing low complexity sequences can aggregate under physiological conditions and drive the onset of liquid-liquid phase separation. The liquid-liquid phase separation occurs as a general property of proteins and nucleic acids in a particular situation, and the phase separation allows the proteins to increase their concentration significantly in localized areas.

The low complexity sequence is from one or more of TAF15 protein, prL domain of FUS protein and DDX4 protein.

Specifically, the low-complexity sequence sources comprise amino acids 1-214 (the amino acid sequence is shown as SEQ ID NO. 1) of the human FUS protein, amino acids 1-478 (the amino acid sequence is shown as SEQ ID NO. 2) of the FUS protein, amino acids 1-208 (the amino acid sequence is shown as SEQ ID NO. 3) of the human TAF15 protein, and amino acids 1-236 (the amino acid sequence is shown as SEQ ID NO. 4) of the human DDX4 protein.

The low complexity sequence may also be used in ligand quantitative presenting cells of the present application by modifying some of the native LC tags (e.g., amino acids 218-415 of the human TDP-43 protein, amino acids 46-266 of the human EWSR1 protein, amino acids 190-341 of the human HNRNPA1 protein, amino acids 186-320 of the human HNRNPA2 protein, and twenty, thirty, forty, fifty polymers of repeated arrangements of two amino acids proline and arginine). For example by adjusting the amount of protein expression to approach a threshold value required for phase separation in a 2D system, or by changing the rate at which the protein forms multivalent binding, etc.

The low complexity sequences enable quantitative detection of ligand-receptor binding by reducing cell surface antigen expression and increasing the overall affinity between ligand-receptors.

In one embodiment, the phase change peptide is fused to an intracellular region of a transmembrane protein.

In a preferred embodiment, the low complexity sequence is fused to a ligand-PDGFR (platelet derived growth factor receptor) transmembrane protein, i.e., the recombinant transmembrane protein is a ligand-PDGFR recombinant transmembrane protein. Under physiological conditions, antigen-PDGFR (corresponding to ligand-PDGFR of the present application) transmembrane domain fusion proteins (Ag-mem) are expressed in large amounts on the plasma membrane of antigen presenting cells (corresponding to ligand-quantitative presenting cells of the present application). The recombinant transmembrane protein fused with the LC tag in the application can be simply called Ag-m-LC or Ag-mem-LC. The Ag-m-LC recombinant transmembrane protein is obtained by combining any target ligand with a PDGFR transmembrane domain through a molecular biological method well known in the art, fusing a section of intracellular peptide of a PDGFR receptor, and finally fusing different LC labels at the C end. In one embodiment, the ligand of interest is selected from antigens. Such as GFP protein, mCherry protein, HER2 extracellular domain, SARS-CoV S protein, SARS-CoV-2S protein, TGP protein.

In one embodiment, the amino acid sequence excluding the ligand portion (i.e., the amino acid sequence including only the transmembrane region, intracellular region, and LC tag of PDGFR) after fusing the low complexity sequence to the C-terminus of the ligand-PDGFR recombinant transmembrane protein is shown in SEQ ID No. 5:

(Linear underline PDGFR transmembrane region; curved underline PDGFR intracellular short peptide; the remainder is FUS) _LC Peptide segment

The invention also provides an isolated polynucleotide encoding the recombinant transmembrane protein.

The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

In one embodiment, the polynucleotide has a nucleotide sequence as set forth in SEQ ID No. 6.

The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the enzymes or proteins having the same amino acid sequence as the invention. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded protein.

The invention also provides a nucleic acid construct comprising the polynucleotide.

In certain embodiments of the invention, the nucleic acid construct is constructed from the isolated polynucleotide inserted into a multiple cloning site of an expression vector. Expression vectors in the present invention generally refer to a variety of commercially available expression vectors well known in the art, and may be, for example, bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

The present invention provides a cell that expresses the recombinant transmembrane protein.

In one embodiment, the cell is a ligand quantitative presenting cell; preferably, the ligand quantitative presenting cell comprises an antigen quantitative presenting cell. The ligand quantitative presenting cell is selected from a K562 human chronic myelogenous leukemia cell line. In one embodiment, the ligand quantitative presenting cell is a stably transfected cell line stably expressing Ag-m-LC.

In another embodiment, the cell is a host cell that produces the transmembrane protein.

The invention also provides a cell surface macromolecule quantitative display system: the system includes the ligand-presenting cell and the receptor-expressing cell, wherein the receptor of the receptor-expressing cell is specifically activated when the ligand-presenting cell and the receptor-expressing cell are co-cultured.

The macromolecules refer to macromolecules existing in organisms such as proteins, nucleic acids, polysaccharides and the like or synthesized artificially.

Specifically, the extracellular region of the receptor-expressing cell receptor is fused to a region capable of specifically recognizing and binding to a ligand presented by a ligand quantitative presenting cell, and the two regions are contacted with each other to specifically activate synNotch synthesis receptor.

The receptor-expressing cells are also known as antibody-expressing cells (ABC). In one embodiment, the receptor of the receptor expressing cell is selected from synNotch synthesis receptors. The synNotch synthetic receptor is fused to a receptor of interest.

The synNotch synthesis receptor is positioned on a cell membrane, an extracellular domain of the synNotch synthesis receptor is fused with a target receptor, and the target receptor is, for example, a nano antibody, a single chain antibody (scFv), an antibody analogue or a cell membrane surface protein receptor and an extracellular protein peptide fragment, and the target receptor in the extracellular domain can be specifically combined after contacting with an antigen on the surface of a ligand quantitative presentation cell; the intracellular domain inside the cytoplasmic membrane of synNotch synthetic receptor is fused to a transcriptional activator which, when the receptor of interest and ligand of interest are bound, will be released and initiate the expression of blue fluorescent protein in the cell (FIGS. 1A, 1B). Activation of synNotch synthetic receptors in receptor expressing cells will be slightly affected when the affinity between ligand-receptor is altered. The synNotch synthetic receptor of the present invention is a synNotch receptor fused to a receptor of interest, and both the existing synNotch receptor and the method of fusing the receptor of interest are well known to those skilled in the art.

In one embodiment, the receptor expressing cell is a stably transfected cell line that expresses both a synNotch synthesis receptor and a reporter group.

In one embodiment, the receptor expressing cell carries a fluorescent reporter group. The fluorescent reporter group is used to determine whether the receptor expressing cell is activated.

In general, the fluorescent reporter group may be selected from fluorescent protein encoding genes such as GFP, mCherry, BFP, and the like. In one embodiment, the fluorescent reporter group is selected from BFP.

In another embodiment, the reporter is a non-fluorescent reporter. The non-fluorescent reporter group may be one that initiates protein expression and localized expression in the cytoplasmic membrane when the antigen and antibody are bound, and that is capable of sorting out cells expressing the protein using commercially available magnetic bead sorting techniques. For example, the non-fluorescent reporter is selected from a CD8 reporter or a CD4 reporter. The CD8 and CD4 reporter genes are derived from mammals. Preferred are hCD8, hCD4 reporter genes, i.e. human CD8, human CD4 reporter genes. The non-fluorescent reporter group may also be selected from drug resistance genes. For example, common drug resistance genes such as puromycin resistance, blasticidin resistance, and the like.

In general, any cell line that expresses a receptor can be used as the receptor-expressing cell. In one embodiment, the receptor expressing cells are prepared by expressing synNotch synthesis receptors in K562 myeloid leukemia cells.

The cell surface macromolecule quantitative display system is a mammalian cell or non-mammalian cell surface macromolecule quantitative display system. Preferably, a mammalian cell surface macromolecule quantitative display system.

The cell surface macromolecular quantitative display system is capable of detecting ligand-receptor molecular interactions with affinities in the micromolar to nanomolar range, but the activation of the receptor is not well correlated with intermolecular affinities in this affinity range.

The liquid-liquid phase separation of membrane proteins inside the cytoplasmic membrane can rapidly increase the receptor protein concentration in localized areas, thereby increasing the affinity (avidity) between ligand-receptors in extracellular areas, i.e., the cumulative strength of multiple ligand-receptor molecule interactions, which in turn can amplify the differences in individual ligand-receptor affinities (affinities). The inventors have found that fusion of a variety of low complexity sequences to ligand (antigen) presenting transmembrane proteins can produce aggregates of antigen macromolecules on the membrane. This aggregated ligand (antigen) presentation, when bound to the synNotch synthetic receptor system, enables the ligand-receptor system to be converted into a mammalian cell membrane quantitative display system proportional to affinity.

The invention also provides a preparation method of the cell surface macromolecule quantitative display system, which comprises the following steps:

1) Cloning a receptor of interest onto a cell membrane of a receptor expressing cell;

2) Cloning a ligand of interest onto said ligand quantitative presenting cell;

methods for cloning the receptor of interest into receptor expressing cells and the ligand of interest into the ligand-presenting cell in quantitative amounts are well known to those skilled in the art of molecular biology.

The receptor expressing cells carry a fluorescent reporter group.

The ligand and receptor in the invention can be antigen and antibody.

The invention also provides the use of the cell surface macromolecule quantitative display system in ligand-receptor affinity detection and/or bulk acquisition of high affinity antibodies.

The present invention also provides a method for detecting affinity of a ligand-receptor, which comprises co-culturing a ligand-quantitatively presenting cell cloned with a target ligand and a cell expressing the receptor, and detecting binding between the target ligand and the target receptor.

Specifically, binding is detected by a reporter group. The greater the number of cells expressing the reporter group, the greater the affinity between the ligand of interest and the receptor of interest, and vice versa.

The invention also provides a method for obtaining high affinity antibodies in batches, which comprises the following steps:

1) Constructing a receptor expression cell carrying a synNotch antibody library and a reporter group;

2) Co-culturing the receptor expressing cells of step 1) with the ligand quantitative presenting cells to activate the cell surface macromolecule quantitative display system;

3) Collecting cells in the co-culture system, and primarily enriching cells positive to the reporter group;

4) Culturing the cells initially enriched in step 3) for 5-10 days, and re-enriching the cells with new ligand quantitative presenting cells, wherein the enriched receptor expressing cells carry a large amount of high affinity antibodies.

The synNotch antibody library includes a plurality of synNotch synthetic receptors fused to a receptor of interest. Typically, the synNotch antibody library can be obtained by existing methods of antibody library construction. Obtained, for example, by phage display technology.

Specifically, the reporter group in step 1) is selected from a fluorescent reporter group or a non-fluorescent reporter group. Preferably selected from non-fluorescent reporter groups. More preferably, it is selected from hCD8 reporter genes.

In one embodiment, in step 2), the cell surface macromolecule quantitative display system is activated using the following method: taking receptor expression cells and antigen expression cells, respectively, and re-suspending them, and placing them in CO ₂ Shake culturing in incubator for 36-60 h.

In one embodiment, step 3) is performed with a preliminary enrichment in a magnetic field using magnetic beads that specifically bind to the reporter group expressed protein. The magnetic beads capable of binding specifically to the protein expressed by the reporter group are, for example, anti-hCD8 magnetic beads.

In one embodiment, the initially enriched cells of step 3) are cultured for about 1 week, and after the expression level of hCD8 is significantly reduced, the initially enriched cells are mixed with new ligand-quantitative presenting cells for re-enrichment. The re-enrichment may be performed in one or more rounds. For example: the second enrichment can be performed in two, three, four or more rounds.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention. Unless otherwise indicated, all experimental methods used in the present invention are well known to those skilled in the art.

The plasmids required for the quantitative display system are as follows:

the synNotch synthesis receptor is prepared by fusing a plurality of GFP nanobodies (comprising G1 to G9), mCherry nanobodies, HER2 single chain antibodies, SARS-CoV S protein antibodies, SARS-CoV-2S protein antibodies, different species ACE2 chimeras (raccoon, dog, ferret, raccoon, bear, cat, civet, horse, chinese chrysanthemum baton, cow, sheep, pig, camel, mouse, rat, squirrel, guinea pig, hedgehog, pangolin and monkey) with a commercially available mouse Notch1 partial domain and Gal4-VP64 transcriptional activator, and carrying a myc tag (available from adedge, cat: 79127) at the front end of the receptor.

The blue fluorescence reporter system is prepared by fusing a copy of the Gal4 DNA binding domain sequence to a partial CMV promoter and cloning it in front of the BFP gene, see L.Morput et al, engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch receptors.cell 164,780-791 (2016).

The hCD8 reporter system (nucleotide sequence shown in SEQ ID No. 27) was obtained by fusing a copy of the Gal4 DNA binding domain sequence to a partial CMV promoter and cloning it in front of the hCD8 gene.

The Ag-mem expression vector is prepared by combining a plurality of proteins including GFP protein, mCherry protein, HER2 extracellular domain (nucleotide sequence shown as SEQ ID NO. 28), SARS-CoV S-RBD protein (nucleotide sequence shown as SEQ ID NO. 29), SARS-CoV-2S-RBD protein (nucleotide sequence shown as SEQ ID NO. 30), TGP protein (from D.W. close et al, thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering protein 83,1225-1237 (2015)) with PDGFR transmembrane domain (i.e. sequence shown as SEQ ID NO. 5) respectively, and fusing myc tag at N-terminal.

The Ag-m-LC expression vector is prepared by combining multiple proteins, including GFP protein (including G1-G9), mCherry protein, HER2 extracellular domain, SARS-CoV S-RBD protein, SARS-CoV-2S-RBD protein, TGP protein and PDGFR transmembrane domain (i.e. sequence shown in SEQ ID NO. 5), fusing a section of intracellular peptide (aa 562-599) of PDGFR receptor, and finally fusing different LC labels at C terminal.

The above nano-antibodies and mCherry nano-antibodies for GFP in expression vectors were derived from: fridy et al A robust pipeline for rapid production of versatile nanobody repertiries Nat. Methods 11,1253-1260 (2014).

HER2 single chain antibody is derived from: liu et al, affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice.cancer Res 75,3596-3607 (2015).

SARS-CoV S protein antibody CR3022 is derived from: ter Meulen et al Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape variants. PLoS Med 3, e237 (2006).

SARS-CoV S protein antibody S230 is derived from: ac. walls et al Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus fusion.cell 176,1026-1039e1015 (2019).

SARS-CoV-2S protein antibody m396 is derived from: zhu et al, post cross-reactive neutralization of SARS coronavirus isolates by human monoclonal anti-bodies, proc Natl Acad Sci U S A104,12123-12128 (2007).

The SARS-CoV-2S protein antibody CV30 is derived from: seydoux et al Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic mutation.immunity 53,98-105e105 (2020).

The LC tag comprises amino acids 1-214 of human FUS protein, amino acids 1-478 of FUS protein, amino acids 1-208 of human TAF15 protein, amino acids 1-236 of human DDX4 protein, amino acids 218-415 of human TDP-43 protein, amino acids 46-266 of human EWSR1 protein, amino acids 190-341 of human HNRNPA1 protein, amino acids 186-320 of human HNRNPA2 protein, and twenty, thirty, forty and fifty polymers of repeated arrangement of two amino acids of proline and arginine.

EXAMPLE 1 quantitative display of the construction and activation of the desired stably transformed cell lines

And (3) construction: GFP-mem fusion protein is expressed on the surface of ligand presenting cells (Ag-mem APCs) and GFP-M-LC fusion protein is expressed on the surface of ligand quantitative presenting cells (Ag-M-LC APCs), and fusion GFP and mCherry nanobodies (anti-GFP: KD=0.31. Mu.M, anti-mCherry: KD=0.18 nM) are expressed on the surface of K562 cells, respectively, the antibodies are derived from Fridy, P.C.et al.A robust pipeline for rapid production of versatile nanobody repoiries.Nature methods 11,1253-1260, doi:10.1038/nmeth.3170 (2014), and the K562 cells are called receptor expressing cells or antibody expressing cells (ABC) (FIG. 1A, 1B) are obtained by a method conventional in the art by liposome transient transfection in 293T to obtain a retrovirus corresponding to the expression vector, and infection in cells with a virus required for the use of a c-tag or fluorescent gene sorting to obtain a stable gene, and a stable cell line is constructed in the cell line expressing a stable reporter cell line, whereas the receptor expressing cell line expressing a receptor is a stable reporter cell line is not a fusion vector in a cell expressing a cell of interest.

Activating: the synNotch synthetic receptor-expressing cells and antigen-expressing cells were counted separately using a hemocytometer, and the two cells were mixed at 1:1, wherein 0.025million cells were added to each 96-well plate and resuspended in 200. Mu.l medium, 0.1million cells were added to each 24-well plate and resuspended in 1ml medium, and each 1million cell was added to each 10ml medium when shaking culture was performed in a flask. Fully and uniformly mixing at 37 ℃ with 5 percent CO ₂ After culturing in the incubator for 48 hours, the cells were detected by a fluorescence reporting system using a flow cytometer.

The results showed that the BFP reporter gene of anti-GFP synNotch synthetic receptor expressing cells was successfully activated within 48 hours of co-culture, whereas the reporter system failed to be activated when mixed with anti-mCherry synNotch synthetic receptor expressing cells in equal proportions (fig. 1c,1 d). Vice versa, ligand presenting cells expressing mCherry-mem fusion proteins successfully activated anti-mCherry synNotch antibody expressing cells, but not anti-GFP synNotch antibody expressing cells (fig. 1c,1 d). Experimental results show that this mammalian surface antibody display system effectively reports the presence/absence of binding between molecules, while the activation of synNotch synthetic receptors has a threshold.

Example 2 quantitative detection of ligand-receptor binding force by mammalian cell surface antibody display System

To examine the effect of intermolecular affinities in synNotch synthesis receptor activation, GFP-APCs were combined with two anti-GFP synNotch ABC cell lines at 2:1:1 for 48 hours. Although the affinities of the two anti-GFP nanobodies differed by two orders of magnitude ("Hi": kd=3.5 nM; "Lo": kd=600 nM), similar BFP positive cell ratios were observed in both anti-GFP synNotch cells (fig. 1E). In addition, 1:3 or 1:9, reducing the antigen content by reducing the number of APC cells in the system, thereby enabling ABC of high affinity antibodies to gain a competitive advantage under extreme conditions.

The results showed that 1: anti-GFP at 9 ^Hi There was a slight increase in the proportion of BFP positive cells in SynNotch ABC (figure 1E). Thus, molecular interactions with affinities in the micromolar to nanomolar range can activate synNotch synthesis receptors when stimulated by Ag-mem cells, but activation of the receptor has no good correlation with intermolecular affinities.

Example 3 intracellular low complexity sequence (LC) tags can achieve ligand-receptor affinity discrimination

Fusing the intracellular domain of ligand-presenting cells with an LC tag (FIG. 1D, called Ag-m-LC to distinguish it from conventional Ag-mem) can significantly reduce anti-GFP ^Lo Activation of blue fluorescent protein in synNotch cells (FIG. 1E) with significant increase in anti-GFP ^Hi And anti-GFP ^Lo Differences in fluorescence activation in synNotch cells (fig. 1E). For anti-GFP ^Hi The Ag-m-LC expressing APC exhibited a higher activation capacity than the conventional Ag-mem APC for synNotch synthetic receptor expressing cells (FIG. 1E).

To further explore the affinity discrimination of Ag-m-LC, a set of anti-GFP nanobodies with different affinities were cloned into synNotch synthetic receptor expressing cells. These anti-GFP nanobodies recognize two non-overlapping epitopes (interfaces of antibody-antigen interactions) on GFP proteins with affinities (KD) ranging from micromolar to subnanomolar (fig. 1F). By performing expression level analysis of synNotch synthesis receptors on the surface of antibody-expressing cells, it was found that the number of receptors was different after fusing a plurality of anti-GFP nanobodies (FIG. 1F). When a panel of anti-GFP synNotch ABC cells were co-cultured with GFP-mem APCs, respectively, the level of activation of synNotch synthetic receptors was similar in cells of different affinities (FIG. 1G). Also in this case, the level of activation of synNotch synthesis receptor is primarily determined by its corresponding number on the cytoplasmic membrane surface (fig. 1F). When the expression level of synNotch synthetic receptor is consistent, affinity has less effect on the level of activation of the fluorescent reporter system in the receptor cells. However, when co-cultured with GFP-m-LC APCs, activation of the anti-GFP synNotch synthetic receptor was highly correlated with affinity, whether or not the antibody binding epitope was identical or whether the ABC cell surface receptor expression level was low (FIG. 1G). Thus, the addition of LC tags to the intracellular region of the antigen can achieve affinity-dependent activation of synNotch synthetic receptors, a system called quantitative display (qDis) system.

Example 4 reduction of cell surface antigen expression and increase of Total affinity are key points for achieving quantitative display

To explain how intracellular LC tags allow synNotch synthetic receptors to display affinity-dependent activation of cells, the following experiments were performed.

First, subcellular localization of the two different forms of membrane-bound GFP antigen was observed. While the overall GFP fluorescence signal was at a similar level in cells, GFP-mem was predominantly expressed in the plasma membrane, including the microvilli structure of the plasma membrane surface, whereas most GFP-m-LC fluorescence signal was from the intracellular membrane (fig. 2a,2 b). The GFP protein expression content localized on the cytoplasmic membrane surface was detected by using a fluorescent-labeled anti-GFP antibody in combination with a flow cell surface protein staining technique. The experimental results showed that GFP protein content displayed on the surface of GFP-m-LC plasma membrane was significantly reduced (fig. 2C), which demonstrates that fusion of LC tags reduced the expression of proteins on plasma membrane.

Second, based on the above experimental results, it was examined what effect the decrease in the antigen expression level would have on the activation of synNotch synthesis receptor by decreasing the antigen expression level on the surface of GFP-mem APC cells without the LC tag. The SFFV promoter on GFP-mem expression vector was replaced with CMV promoter without kozak sequence or with SV40 weak promoter, while flow cytometry experiments demonstrated that GFP-mem cells after promoter replacement had lower overall GFP expression levels and significant reduction in cytoplasmic membrane surface GFP expression (fig. 2C). At this time, the GFP-mem cells with low expression level were consistent with the GFP-m-LC cells in terms of surface GFP level (FIG. 2C). When the corresponding APC was co-cultured with synNotch ABC cells of different affinities in a panel, low expression levels of GFP-mem resulted in a significant decrease in activation levels of the fluorescence reporter system in all synNotch synthetic receptor expressing cells, but this decrease was not significantly associated with affinity (fig. 2D).

Third, interactions between APC-ABC are visualized. To observe anti-GFP synNotch synthetic receptor in real time, mCherry fluorescent tag was added to the extracellular domain of anti-GFP synNotch and stably expressed in HEK293T cells. When anti-GFP-mCherry-synNotch HEK293T cells were co-cultured with GFP-mem APC (K562 cells), the interface of GFP-anti-GFP molecular interactions was observed (FIGS. 2E, 2F). Surprisingly, despite the low expression of GFP-m-LC on the cell surface, significant aggregation was observed at the APC-ABC action interface when co-cultured with anti-GFP-mCherry-synNotch (FIG. 2F).

The three observations reveal the role of intracellular LC tags in quantitative display systems (fig. 2G). Its low complexity allows a significant reduction in the expression of antigen on the cell surface of Ag-m-LC APC, thereby reducing the cellular response to activation of synNotch synthesis receptors. In this case, aggregation between ligand-receptor produced by phase separation driving will significantly increase receptor activation, which in turn will be affected by intermolecular forces, i.e., affinities, such that activation of signals in synNotch-expressing receptor-expressing cells is proportional to the affinity between ligand-receptor.

Based on the above conclusion, blue fluorescent protein activation of ABC cells expressing anti-Her2 scFv synNotch synthetic receptor after mixed culture with APC cells having different surface antigen display modes was examined. Extracellular domain of human HER2 and PDGFR or PDGFR-FUS _LC Fusion, abbreviated as Her2-mem or Her2-m-LC (fig. 2H). The results show that when the same as Her2-meWhen mAPC was co-cultured, high affinity anti-Her2 synNotch synthetic receptor ABC cells showed a higher proportion of fluorescent reporter activation (FIG. 2K). However, when the antigen is present in Her2-m-LC form (fig. 2K), the affinity discrimination is more pronounced. For anti-Her2 synNotch synthetic receptor expressing cells with nanomolar affinity, the expression level of each fusion receptor was consistent (fig. 2J), while Her2-m-LC fusion antigen expressed on cytoplasmic membrane was lower, but the fluorescent reporter activation ratio in whole cells could be significantly improved (fig. 2I). Thus, the reduction in the expression level of the Ag-m-LC cell surface antigen protein and the increase in the total affinity highlight the ability to quantitatively display it.

Example 5 affinity discrimination requires that low complexity sequences within cells undergo proper aggregation

Protein phase separation/aggregation can significantly increase its concentration in localized areas. In Ag-m-LC cells, multivalent interactions are formed between LC tags inside the cytoplasmic membrane, thereby promoting aggregation of antigen monomers and increasing avidity. Many proteins have been shown to phase separate, and FUS proteins are typical examples thereof, so in the initial experiments FUS was selected _LC Peptide fragments were studied. Various FUS mutants were designed to detect the effect of phase separation on affinity discrimination in the qDis system. Construction of FUS using methods well known to those skilled in the art _LC Q>G and G>A mutant, which affects the hardness and fluidity of the protein aggregation complex, respectively. In an opto-Droplet system, FUS _LC Q>The G mutant tag produced aggregation of the protein faster (fig. 4A), and at the same time, when it was fusion expressed in the cytoplasmic membrane, the expression of the antigen on the membrane was reduced (fig. 3a,4 b), and thus synNotch synthetic receptor expressing cells could not be activated (fig. 3a,4 b). While FUS _LC G>The a mutation tag activated synNotch synthetic receptor expressing cells but failed to distinguish differences between different levels of affinities (fig. 3a,4 c). In addition, two mutants were designed to reduce aggregation and FUS formation _LC The Y27S mutant significantly reduced the phase change ability and was unable to efficiently support quantitative display (fig. 3a,4 d), whereas FUS _LC 7A mutantThe phase change ability is slightly reduced (fig. 4A) but still quantitative display can be supported (fig. 3a,4 e).

Long FUS peptide (aa 1-478) and FUS _LC Both 27R mutants induced phase separation at lower protein levels, but interaction between subnanomolar GFP-anti-GFP failed to activate synNotch synthesis receptor (fig. 3a,3b,4 f). Observations indicate that the threshold concentration required for phase separation of proteins in three-dimensional (cytoplasmic) and two-dimensional (cell membrane) systems varies. These FUS mutants demonstrate that affinity discrimination requires LC tags with appropriate phase separation capability.

Example 6 multiple low complexity sequences can support quantitative display

It was continued to investigate whether other proteins or fragments that form phase separation could also achieve affinity discrimination in qDis systems. A set of low complexity tags was designed, comprising PrL domains of multiple FUS protein family members, LC region of DDX4 gene and poly PR synthetic peptide. When overexpressing the various PrL domains of the FUS protein family, antigens fused to the LC domains of hnRNPA1 or hnRNPA2 lose the ability to activate synNotch synthesis receptors, which can be supported when fused to LC tags from TDP43 or EWSR1 (fig. 5), but with no apparent relationship to affinity (fig. 5). And fusion of LC tag from TAF15 (TAF 15 _LC ) Can also and FUS _LC As such, activation of synNotch synthetic receptor was made proportional to affinity (fig. 3C). Likewise, the LC tag from DDX4 also supports quantitative display (fig. 3C). However, the synthesized polyPR peptide cannot support quantitative display (fig. 5). Notably, DDX4 has a different amino acid composition and overall sequence pattern compared to the PrL domain of the FUS protein family, suggesting that the phase separation process plays an important role in the affinity discrimination of the quantitative display system. Although many LC tags do not support quantitative display, it is possible to re-use these LC tags in quantitative display systems by modifying the protein sequence, for example, by adjusting the protein expression to a level close to the threshold required for phase separation in 2D systems, or by changing the rate at which the protein forms multivalent bonds. At the same time, also test Several other multimeric tags were included, including trimeric Foldon tags and dimeric antibody Fc (antibody crystallizable fragment) tags, and experimental results showed that none of these multimeric tags was able to support synNotch synthesis receptor formation affinity-dependent activation (fig. 3D). In summary, more LC tags can be used in a quantitative display system, while multimerization tags cannot be used in a system. At the same time, the different sequence patterns of these LC tags also further indicate that in this system, receptor molecules are distinguished by phase separation by aggregation.

Example 7 quantitative display of ligand-receptor binding

Quantitative display systems can also be used as a quantitative method to reveal ligand-receptor interactions. To demonstrate this, interactions between the spike protein RBD domain (S-RBD) of the various classes of SARS coronaviruses and ACE2 or its corresponding antibodies are taken as examples.

First, the affinity between S-RBD and human ACE2 or several antibodies was examined. S-RBD was cloned into the Ag-m-LC construct and hACE 2/antibody (scFv) was cloned into the cytoplasmic membrane extracellular domain of synNotch synthetic receptor. The binding of hACE2 to SARS-CoV-2S-RBD was much higher than that of hACE2 to SARS-CoV or SARSr-CoV S-RBD (FIG. 6A), consistent with the results of protein interactions in the biochemical experiments reported previously. All three SARS-CoV neutralizing antibodies were able to bind SARS-CoV S-RBD with high affinity (FIG. 6A), while only CR3022 showed cross-binding between SARS-CoV and SARS-CoV-2 (FIG. 6A). Vice versa, the SARS-CoV-2 neutralizing antibody (CV 30) binds SARS-CoV-2S-RBD with only high affinity (FIG. 6A).

Second, the interaction between S-RBD and ACE2 of different species was examined. Based on the spatial structure of S-RBD/ACE2 interaction, the key peptide segment of hACE2 and S-RBD interaction was replaced by the corresponding protein sequence of other species (FIG. 6C). After co-culturing the S-RBD-m-LC cells with ACE2 chimera-synNotch cells, the binding force between S-RBD and different species of ACE2 protein was quantitatively shown by a fluorescence reporting system (FIG. 6B). Compared to SARS-CoV S-RBD, SARS-CoV-2S-RBD can bind to hACE2 with high affinity and interact with chimeric ACE2 from cats, dogs, etc. with low affinity (FIG. 6B).

Example 8 quantitative display System high affinity antibodies can be rapidly mass isolated

The quantitative display system can directly separate and obtain the high-affinity antibody without subsequent mass screening verification experiments. To develop a quantitative display system into a screening system for high affinity antibodies, the fluorescent reporter gene in synNotch synthetic receptor expressing cells was replaced with hCD8 reporter gene (fig. 7A). In this system, hCD8 gene expression is initiated and localized to the cytoplasmic membrane when synNotch synthetic receptor expressing cells are specifically activated, at which time hCD8 positive cells can be enriched by magnetic cell sorting (MACS). As with blue fluorescent protein activation, the hCD8 protein expression on the surface of the receptor with different affinities was also proportional after binding to the corresponding Ag-m-LC cells (fig. 7B). Two high (nM) (red fluorescent protein labeled) and low (μm) affinity ABC cells were each labeled with 1:1000,1:100 or 1:10 ratio to test the discriminatory power of the qDis-MACS system. In each round, two ABC were mixed in the above ratio, co-cultured with equal amounts of Ag-m-LC APC cells, and enriched for synNotch synthesis receptor activated ABC cells using magnetic beads with anti-hCD8 antibody. By examining the ratio of two high/low affinity ABC cells after each round of qDis-MACS enrichment, it was found that high affinity ABC cells could be enriched 3 to 14 fold (fig. 7C). Meanwhile, after two rounds of screening, the rare high affinity antibodies (0.1% of the total ABC) in the original cell population can be significantly enriched to about 10%.

To test whether the quantitative display system can be applied to screen for high affinity antibodies, phage display and mammalian quantitative display systems were combined to screen for high affinity antibodies against Thermostable GFP (TGP) proteins (fig. 7D). Although phage display libraries can reach 10 ¹¹ On the order of magnitude, but often several hundred clones are expressed separately in a subsequent validation step and the affinity is further tested by ELISA or similar methods after two rounds of enrichment. Cloning of phage display-derived antibody libraries of the prior art to quantitative displayPotential high affinity antibodies were shown in the system and selected by three rounds of qDis-MCAS. Based on the fold enrichment of different antibodies in each round of display, 19 clones were selected from qDis to verify binding affinity and compared to randomly selected clones directly from phage display library (fig. 7E). The results indicate that 18 of the 19 antibodies from the quantitative display system were able to detect binding to TGP by BLI, whereas of the 188 antibodies randomly selected, only 4 were able to detect binding to TGP. Of these 18 antibodies, 16 were able to bind TGP in the nanomolar range, the other two bound TGP with picomolar affinity (fig. 7f, 8). Thus, the quantitative display system can be used as a method for separating high affinity antibodies from a medium-sized antibody library, and high affinity antibodies can be isolated in bulk by combining conventional phage display techniques with the quantitative display system.

The specific experimental method is as follows:

1. construction of synNotch antibody library

The puromycin resistance gene was added to the synNotch vector and used as a novel vector backbone. Primers containing synNotch vector backbone linker sequences were designed to specifically amplify antibodies in the library of interest. And (3) recombining the recovered product with the digested carrier skeleton by using a Gibson assembly method, and constructing a new recombinant library in escherichia coli by using a bacterial electrotransformation method.

2. Construction of qDIS-MACS selection of the desired stably transformed cell lines

Retrovirus corresponding to the expression vector was obtained by liposome transient transfection in 293T. Overexpression of Ag-mem-FUS in K562 cell line _LC Cell lines stably expressing the fusion antigen were obtained by flow sorting. The hCD8 reporter system is over-expressed in the K562 cell line, and the stable transgenic cell line is obtained by screening with the Blastidin resistance gene. The synNotch antibody library is overexpressed in a K562-hCD8 cell line, and after 7 days of screening of the cell population expressing the antibody library by using a puromycin resistance gene, a stable transgenic cell population is obtained, and then the next experiment is carried out.

3. Activating qDIS system

The cell number is always ensured to be more than 100 times of the number of the antibody library during cell passage. Cell populations were counted on day 8, 10million each of synNotch synthetic receptor-expressing cells and antigen-expressing cells was resuspended in 100ml of medium and shake-cultured in a 5% CO2 incubator at 37℃for 48h at 50 rpm/min.

MACS enriched hCD8 reporter positive cells

Co-cultured cells were counted and collected, centrifuged at 300g for 5min, resuspended in 10ml MACS buffer, centrifuged at 300g for 5min, and the cells were counted at 80. Mu.l/10 ⁷ The proportion of cells was resuspended. According to 20 μl/10 ⁷ The anti-hCD8 microbeads are added into the cells according to the proportion, and the cells are incubated for 15min at 4 ℃ after being evenly mixed. After incubation was complete, 5ml MACS buffer was added and centrifuged at 300g for 5min to remove excess unbound microbeads. After centrifugation, the cells were resuspended in 500. Mu.l buffer and added to an MS sorting column ready for placement in the magnetic field. After the cells were completely passed through the sorting column, 1ml MACS buffer was added to remove unbound hCD8 negative cells and the procedure was repeated two more times. Finally, the sorting column is taken down from the magnetic field, 1ml of MACS buffer solution is added, the cells are rapidly beaten out, and finally the cells positive to the hCD8 reporter gene are obtained. And taking out a small amount of obtained positive cells for staining, and detecting the expression quantity of hCD8 in the enriched cells by using a flow cytometry.

5. Multiple rounds of qDIS-MACS enrichment

And centrifuging the cells obtained by enrichment, and taking part of the cells to extract genomic DNA after resuspension, so that most of the cells remain to be cultured. After the expression level of hCD8 in the cells of the previous round is obviously reduced, the cells enriched by 10million and the new Ag-m-FUS are continuously taken for about 1 week _LC The cells were mixed and the experiment was ended after three rounds of enrichment.

6. Enrichment of genomic DNA from cells

The extracted genomic DNA was dissolved in TE solution at a rate of 20. Mu.l/million cell, and the amount of the initial cell mass was 100 times the antibody library capacity. The coding sequence of the antibody gene is amplified by using Q5 high-fidelity polymerase, and 5 mu l of DNA is added into each 50 mu l of reaction system as a template for 20 cycles of amplification during one round of amplification. In two rounds of amplification, 4 reactions were performed for each sample, and 3. Mu.l of one round of product was added to 50. Mu.l of the reaction system as a template for 20 cycles of amplification. The PCR product of round 2 was subjected to gel electrophoresis, the target band was recovered, and 50ng was added as a template for three rounds of amplification, and amplified for 8 cycles. Finally, the amplified product of the third round is purified and recovered, and high-throughput sequencing is performed.

7. Analysis of antibody enrichment using bioinformatics

And respectively extracting the sequences of CDR1, CDR2 and CDR3 in each sequence from the original sequencing file according to the fixed sequences at the two sides of the CDR, and counting the corresponding occurrence times of the sequences. The sequence with the statistics of 1 is compared with other sequences, and the sequence is classified as the same sequence when the CDR sequences differ by only 1 base. Combining the sequencing results of the multiple screening into the same file, carrying out homogenization treatment on the results according to the total data amount measured by each experiment, filtering out sequences which only appear once in the plasmid library, and accumulating the sequences which appear less than 10 times in the previous three enrichment. Finally, 15 obviously enriched antibodies are selected by the difference multiple of the single antibodies after the first round and the third round of screening, and 4 obviously enriched antibodies are selected by the difference multiple of the single antibodies in the initial cells after the third round of screening.

8. Antibody purification

Primers were designed and expression vectors for 19 antibodies were constructed and verified by sequencing. The constructed plasmid was transformed into E.coli MC1061 strain, and protein expression was induced overnight with 0.02% (w/v) arabinose. Bacterial cultures were collected, resuspended in TES buffer (20% (w/v) Sucrose,0.5mM EDTA,200mM Tris pH 8.0), spun at 4 degrees for 30min, and spun for 1h with twice the volume of pre-chilled ice water. Centrifuge at 20,000g at 4℃for 30min. And (3) purifying the proteins of the supernatant after centrifugation by using Ni-NTA, and finally adding an eluent containing 300mM imidazole to elute the target proteins. Protein concentration was measured using Bradford while the purified protein band size was verified by SDS-PAGE.

9. Antibody affinity validation

Affinity between purified antibodies and TGP antigen was analyzed using an ott Red 96 protein interactometer. The biotin-labeled protein was fused to TGP, and the protein was diluted to 2. Mu.g/ml with a buffer (1 xPBS,0.02% Tween-20) and immobilized on an SA sensor with a control signal of about 0.6. The antibody was diluted to 0.2mg/ml (12.6. Mu.M), re-diluted to 100nM, and finally diluted to 50nM,25nM,12.5nM,6.25nM,3.7nM, etc. in a 2-fold gradient. During the experiment, antigen and antibody were allowed to bind for 240s, dissociated for 240s, and the corresponding binding and dissociation constants were calculated on the analysis software using a 1:1 binding pattern. After completion of a set of experiments, the sensor was regenerated using a regeneration solution (10mM glycine,PH 2.0) to remove bound antibodies, corresponding to a 5s regeneration, 5s equilibration, and the above procedure was repeated three times. The regenerated sensor is reused for measuring other antibodies, and when the sensor signal is obviously weakened, a new sensor is reused for measuring.

The invention fuses a series of proteins which can be separated in cells with antigens, and realizes an affinity-based quantitative display system in mammalian cells. Among other things, the low complexity domain of proteins can help to distinguish between affinities of cytoplasmic membrane outside protein interactions. Receptor aggregation and associated coagulation of downstream effector proteins contribute to signaling. The synNotch synthesis receptor and antigen presentation system of the invention demonstrate that aggregation of antigen molecules inside the plasma membrane may affect extracellular binding. Whether lymphocyte antigen receptors such as BCR and TCR also distinguish antigens with different affinities by similar strategies would be worth continuing research in the future.

From the experimental results of the invention, the qDis system provides a new method for the existing surface display technology, and high-affinity antibodies can be found in batches. The library size for mammalian display is limited by plasmid transfection efficiency and cell number, which can be overcome by combining multiple display systems. The affinity of the antibodies selected from the initial or synthetic display libraries is typically low, in which case the qDis system can help to find higher affinity antibodies that account for a lower library. Furthermore, by combining in vitro mutations with the qdisc system, we have made it possible to mimic the antibody affinity process in germinal centers in vitro. In addition to finding antibodies, affinity-based displays can also be used to reveal ligand-receptor binding, providing a quantitative approach to find and/or verify possible invading receptors for viruses and possible ligand-receptor pairs in cellular interactions.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

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210 215 220

Gly Gly Gln Gln Gln Ser Tyr Gly Gln Gln Gln Ser Tyr Asn Pro Pro

225 230 235 240

Gln Gly Tyr Gly Gln Gln Asn Gln Tyr Asn Ser Ser Ser Gly Gly Gly

245 250 255

Gly Gly Gly Gly Gly Gly Gly Asn Tyr Gly Gln Asp Gln Ser Ser Met

260 265 270

Ser Ser Gly Gly Gly Ser Gly Gly Gly Tyr Gly Asn Gln Asp Gln Ser

275 280 285

Gly Gly Gly Gly Ser Gly Gly Tyr Gly Gln Gln Asp Arg Gly

290 295 300

<210> 6

<211> 906

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

aacgccgtgg gccaggatac gcaggaggtg atcgtcgtgc cccacagctt gcccttcaag 60

gtcgtggtta ttagtgctat cctggcgttg gtggtgctga ctattatctc ccttatcata 120

cttatcatgc tgtggcaaaa aaaaccccga tacgagatcc gatggaaggt gattgagtct 180

gtgagctctg acggccatga gtacatctac gtggacccca tgcagctgcc ctatgactcc 240

acgtgggagc tgccgcggga ccagatggcc tcaaacgatt atacccaaca agcaacccaa 300

agctatgggg cctaccccac ccagcccggg cagggctatt cccagcagag cagtcagccc 360

tacggacagc agagttacag tggttatagc cagtccacgg acacttcagg ctatggccag 420

agcagctatt cttcttatgg ccagagccag aacacaggct atggaactca gtcaactccc 480

cagggatatg gctcgactgg cggctatggc agtagccaga gctcccaatc gtcttacggg 540

cagcagtcct cctatcctgg ctatggccag cagccagctc ccagcagcac ctcgggaagt 600

tacggtagca gttctcagag cagcagctat gggcagcccc agagtgggag ctacagccag 660

cagcctagct atggtggaca gcagcaaagc tatggacagc agcaaagcta taatccccct 720

cagggctatg gacagcagaa ccagtacaac agcagcagtg gtggtggagg tggaggtgga 780

ggtggaggta actatggcca agatcaatcc tccatgagta gtggtggtgg cagtggtggc 840

ggttatggca atcaagacca gagtggtgga ggtggcagcg gtggctatgg acagcaggac 900

cgtgga 906

<210> 7

<211> 595

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Ser Thr Glu Asp Leu Val Asn Thr Phe Leu Glu Lys Phe Asn Tyr Glu

1 5 10 15

Ala Glu Glu Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr Asn

20 25 30

Thr Asn Ile Thr Asp Glu Asn Leu Gln Lys Met Asn Asn Ala Gly Ala

35 40 45

Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Lys Leu Ala Lys Thr Tyr

50 55 60

Pro Leu Glu Glu Ile Gln Asp Ser Thr Val Lys Arg Gln Leu Arg Ala

65 70 75 80

Leu Gln His Ser Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys Arg

85 90 95

Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly Lys

100 105 110

Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro Gly

115 120 125

Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu Trp

130 135 140

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

145 150 155 160

Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn His

165 170 175

Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn Gly

180 185 190

Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val Glu

195 200 205

His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala Tyr

210 215 220

Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro Ile

225 230 235 240

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

245 250 255

Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn Ile

260 265 270

Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg Ile

275 280 285

Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met

290 295 300

Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Glu Pro Ser Asp Ser

305 310 315 320

Trp Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Arg Gly Asp

325 330 335

Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr

340 345 350

Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala

355 360 365

Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu Ala

370 375 380

Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu Lys

385 390 395 400

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

405 410 415

Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu Pro

420 425 430

Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly Glu

435 440 445

Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg Glu

450 455 460

Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys Asp

465 470 475 480

Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg Tyr

485 490 495

Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys Gln

500 505 510

Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn Ser

515 520 525

Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys Ser

530 535 540

Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala Lys Asn Met

545 550 555 560

Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp Leu

565 570 575

Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp Ser

580 585 590

Pro Tyr Ala

595

<210> 8

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Ser Thr Glu Asp Leu Val Lys Thr Phe Leu Glu Lys Phe Asn Tyr Glu

1 5 10 15

Ala Glu Glu Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr Asn

20 25 30

Ile Asn Ile Thr Asp Glu Asn Val Gln Lys Met Asn Asn Ala Gly Ala

35 40 45

Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Lys Leu Ala Lys Thr Tyr

50 55 60

Pro Leu Glu Glu Ile Gln Asp Ser Thr Val Lys Arg Gln Leu Arg Ala

65 70 75 80

Leu Gln His Ser Gly Ser Ser Val Leu Ser Ala Asp Lys Asn Gln Arg

85 90 95

Leu Asn Thr Ile Leu Asn Ser Met Ser Thr Val Tyr Ser Thr Gly Lys

100 105 110

Ala Cys Asn Pro Ser Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro Gly

115 120 125

Leu Asp Asp Ile Met Glu Asn Ser Lys Asp Tyr Asn Glu Arg Leu Trp

130 135 140

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

145 150 155 160

Tyr Glu Glu Tyr Val Ala Leu Lys Asn Glu Met Ala Arg Ala Asn Asn

165 170 175

Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Glu Glu Trp

180 185 190

Glu Asn Gly Tyr Asn Tyr Ser Arg Asn Gln Leu Ile Asp Asp Val Glu

195 200 205

Leu Thr Phe Thr Gln Ile Met Pro Leu Tyr Gln His Leu His Ala Tyr

210 215 220

Val Arg Thr Lys Leu Met Asp Thr Tyr Pro Ser Tyr Ile Ser Pro Thr

225 230 235 240

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

245 250 255

Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Gln Lys Pro Asn Ile

260 265 270

Asp Val Thr Asn Ala Met Val Asn Gln Ser Trp Asp Ala Arg Lys Ile

275 280 285

Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn Met

290 295 300

Thr Gln Glu Phe Trp Gly Asn Ser Met Leu Thr Glu Pro Ser Asp Ser

305 310 315 320

Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly Asp

325 330 335

Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu Thr

340 345 350

Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala Ala

355 360 365

Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu Ala

370 375 380

Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Asn His Leu Lys

385 390 395 400

Asn Ile Gly Leu Leu Pro Pro Ser Phe Phe Glu Asp Ser Glu Thr Glu

405 410 415

Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu Pro

420 425 430

Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly Glu

435 440 445

Ile Pro Lys Asp Gln Trp Met Lys Thr Trp Trp Glu Met Lys Arg Asn

450 455 460

Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys Asp

465 470 475 480

Pro Ala Ser Leu Phe His Val Ala Asn Asp Tyr Ser Phe Ile Arg Tyr

485 490 495

Tyr Thr Arg Thr Ile Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys Gln

500 505 510

Ile Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn Ser

515 520 525

Ser Glu Ala Gly Gln Lys Leu Leu Glu Met Leu Lys Leu Gly Lys Ser

530 535 540

Lys Pro Trp Thr Tyr Ala Leu Glu Ile Val Val Gly Ala Lys Asn Met

545 550 555 560

Asp Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp Leu

565 570 575

Lys Glu Gln Asn Arg Asn Ser Phe Val Gly Trp Asn Thr Asp Trp Ser

580 585 590

Pro Tyr Ala Asp

595

<210> 9

<211> 595

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

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

1 5 10 15

Glu Ala Glu Glu Leu Ser Tyr Gln Asn Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Asp Glu Asn Ile Gln Lys Met Asn Ile Ala Gly

35 40 45

Ala Lys Trp Ser Ala Phe Tyr Glu Glu Glu Ser Gln His Ala Lys Thr

50 55 60

Tyr Pro Leu Glu Glu Ile Gln Asp Pro Ile Ile Lys Arg Gln Leu Arg

65 70 75 80

Ala Leu Gln Gln Ser Gly Ser Ser Val Leu Ser Ala Asp Lys Arg Glu

85 90 95

Arg Leu Asn Thr Ile Leu Asn Ala Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Ala Cys Asn Pro Asn Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asp Asp Ile Met Glu Asn Ser Lys Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Ala Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

Asn Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Glu Glu

180 185 190

Trp Ala Asp Gly Tyr Ser Tyr Ser Arg Asn Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Thr Gln Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asp Ala Tyr Pro Ser Arg Ile Ser Pro

225 230 235 240

Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Pro Leu Met Val Pro Phe Arg Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asn Gln Ser Trp Asp Ala Arg Arg

275 280 285

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

290 295 300

Met Thr Glu Gly Phe Trp Gln Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Asn Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Arg

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Glu Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Asn His Leu

385 390 395 400

Lys Asn Ile Gly Leu Leu Pro Pro Asp Phe Ser Glu Asp Ser Glu Thr

405 410 415

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

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Glu Gln Trp Met Gln Lys Trp Trp Glu Met Lys Arg

450 455 460

Asp Ile Val Gly Val Val Glu Pro Leu Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ala Leu Phe His Val Ala Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Ile Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ile Ala Lys His Glu Gly Pro Leu Tyr Lys Cys Asp Ile Ser Asn

515 520 525

Ser Ser Glu Ala Gly Gln Lys Leu His Glu Met Leu Ser Leu Gly Arg

530 535 540

Ser Lys Pro Trp Thr Phe Ala Leu Glu Arg Val Val Gly Ala Lys Thr

545 550 555 560

Met Asp Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Glu Gln Asn Arg Asn Ser Phe Val Gly Trp Asn Thr Asp Trp

580 585 590

Ser Pro Tyr

595

<210> 10

<211> 595

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Ser Thr Thr Glu Asp Leu Ala Asn Thr Phe Leu Glu Asn Phe Asn Asn

1 5 10 15

Glu Thr Glu Glu Leu Ser Tyr Gln Asn Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Asp Glu Asn Ile Gln Lys Met Asn Asp Ala Ala

35 40 45

Ala Lys Trp Ser Ala Phe Tyr Asp Glu Gln Ser Lys Gln Ala Lys Thr

50 55 60

Tyr Pro Leu Glu Glu Ile Gln Asp Pro Thr Asn Lys Arg Gln Leu Gln

65 70 75 80

Ala Leu Gln His Ser Gly Ser Ser Val Leu Ser Ala Asp Lys Arg Glu

85 90 95

Arg Leu Asn Thr Ile Leu Asn Ala Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Thr Cys Asn Pro Asn Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asp Asp Ile Met Glu Asn Ser Lys Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Thr Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

Asn Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Glu Glu

180 185 190

Trp Ala Asp Gly Tyr Asn Tyr Ser Arg Ser Gln Leu Ile Asp Asp Val

195 200 205

Glu His Thr Phe Lys Gln Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asp Thr Tyr Pro Ser His Met Ser Pro

225 230 235 240

Thr Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Pro Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asn Gln Ser Trp Asp Ala Arg Arg

275 280 285

Ile Phe Glu Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Asn Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Ser His Leu

385 390 395 400

Lys Asn Ile Gly Leu Leu Pro Pro Gly Phe Ser Glu Asp Asn Glu Thr

405 410 415

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

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Glu Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Leu Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ala Leu Phe His Val Ala Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Ile Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ile Ala Lys His Glu Gly Pro Leu Tyr Lys Cys Asp Ile Ser Asn

515 520 525

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

530 535 540

Ser Lys Pro Trp Thr Leu Ala Leu Glu Thr Val Val Gly Ala Lys Thr

545 550 555 560

Met Asp Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Gln Glu Arg Asn Arg Asn Ser Phe Val Gly Trp Asn Thr Asp Trp

580 585 590

Ser Pro Tyr

595

<210> 11

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

Glu Ala Glu Asp Leu Tyr Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Asn Glu Asn Ile Gln Lys Met Asn Asp Ala Gly

35 40 45

Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Lys His Ala Lys Thr

50 55 60

Tyr Pro Leu Glu Glu Ile His Asn Ser Thr Val Lys Arg Gln Leu Gln

65 70 75 80

Ala Leu Gln His Ser Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Glu Phe Trp Glu Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Gly Gln Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 12

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

Ser Thr Thr Glu Glu Leu Ala Lys Thr Phe Leu Glu Lys Phe Asn His

1 5 10 15

Glu Ala Glu Glu Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Asp Glu Asn Val Gln Lys Met Asn Glu Ala Gly

35 40 45

Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Lys Leu Ala Lys Thr

50 55 60

Tyr Pro Leu Ala Glu Ile His Asn Thr Thr Val Lys Arg Gln Leu Gln

65 70 75 80

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

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Ser Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 13

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 13

Ser Thr Thr Glu Glu Leu Ala Lys Thr Phe Leu Glu Thr Phe Asn Tyr

1 5 10 15

Glu Ala Gln Glu Leu Ser Tyr Gln Ser Ser Val Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Asp Glu Asn Ala Lys Asn Met Asn Glu Ala Gly

35 40 45

Ala Lys Trp Ser Ala Tyr Tyr Glu Glu Gln Ser Lys Leu Ala Gln Thr

50 55 60

Tyr Pro Leu Ala Glu Ile Gln Asp Ala Lys Ile Lys Arg Gln Leu Gln

65 70 75 80

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

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 14

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 14

Ser Thr Thr Glu Asp Leu Ala Lys Thr Phe Leu Glu Lys Phe Asn Ser

1 5 10 15

Glu Ala Glu Glu Leu Ser His Gln Ser Ser Leu Ala Ser Trp Ser Tyr

20 25 30

Asn Thr Asn Ile Thr Asp Glu Asn Val Gln Lys Met Asn Glu Ala Gly

35 40 45

Ala Arg Trp Ser Ala Phe Tyr Glu Glu Gln Cys Lys Leu Ala Lys Thr

50 55 60

Tyr Pro Leu Glu Glu Ile Gln Asn Leu Thr Val Lys Arg Gln Leu Gln

65 70 75 80

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

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 15

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 15

Ser Thr Thr Glu Asp Glu Ala Lys Met Phe Leu Asp Lys Phe Asn Thr

1 5 10 15

Lys Ala Glu Asp Leu Ser His Gln Ser Ser Leu Ala Ser Trp Asp Tyr

20 25 30

Asn Thr Asn Ile Asn Asp Glu Asn Val Gln Lys Met Asp Glu Ala Gly

35 40 45

Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Lys Leu Ala Lys Asn

50 55 60

Tyr Ser Leu Glu Gln Ile Gln Asn Val Thr Val Lys Leu Gln Leu Gln

65 70 75 80

Ile Leu Gln Gln Ser Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Glu Gly Phe Trp Asn Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Glu Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 16

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 16

Ser Thr Thr Glu Glu Gln Ala Lys Thr Phe Leu Glu Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Asp Glu Asn Val Gln Lys Met Asn Glu Ala Arg

35 40 45

Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Arg Met Ala Lys Thr

50 55 60

Tyr Ser Leu Glu Glu Ile Gln Asn Leu Thr Leu Lys Arg Gln Leu Lys

65 70 75 80

Ala Leu Gln His Ser Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Asp Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 17

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 17

Ser Thr Thr Glu Glu Gln Ala Lys Thr Phe Leu Glu Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Asp Glu Asn Val Gln Lys Met Asn Glu Ala Arg

35 40 45

Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Arg Met Ala Arg Thr

50 55 60

Tyr Ser Leu Glu Glu Ile Gln Asn Leu Thr Leu Lys Arg Gln Leu Lys

65 70 75 80

Ala Leu Gln His Ser Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Asn Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 18

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 18

Ser Thr Thr Glu Glu Leu Ala Lys Thr Phe Leu Glu Lys Phe Asn Leu

1 5 10 15

Glu Ala Glu Asp Leu Ala Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Asp Glu Asn Ile Gln Lys Met Asn Asp Ala Arg

35 40 45

Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Arg Ile Ala Lys Thr

50 55 60

Tyr Pro Leu Asp Glu Ile Gln Thr Leu Ile Leu Lys Arg Gln Leu Gln

65 70 75 80

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

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Asn Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 19

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 19

Ser Thr Thr Glu Glu Leu Ala Lys Thr Phe Leu Glu Glu Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Asp Glu Asn Val Gln Lys Met Asn Asp Ala Arg

35 40 45

Ala Lys Trp Ser Thr Phe Tyr Glu Glu Lys Ser Lys Thr Ala Lys Thr

50 55 60

Tyr Pro Leu Glu Glu Ile Gln Asn Val Thr Leu Lys Arg Gln Leu Gln

65 70 75 80

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

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Asp Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 20

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 20

Ser Leu Thr Glu Glu Asn Ala Lys Thr Phe Leu Asn Asn Phe Asn Gln

1 5 10 15

Glu Ala Glu Asp Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Ala Gln Lys Met Ser Glu Ala Ala

35 40 45

Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Lys Thr Ala Gln Ser

50 55 60

Phe Ser Leu Gln Glu Ile Gln Thr Pro Ile Ile Lys Arg Gln Leu Gln

65 70 75 80

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

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Ala Asn Ser Met Leu Thr Glu Pro Ala Asp

305 310 315 320

Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly His Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asn Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 21

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 21

Ser Leu Ile Glu Glu Lys Ala Glu Ser Phe Leu Asn Lys Phe Asn Gln

1 5 10 15

Glu Ala Glu Asp Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Ala Gln Lys Met Asn Glu Ala Ala

35 40 45

Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Lys Ile Ala Gln Asn

50 55 60

Phe Ser Leu Gln Glu Ile Gln Asn Ala Thr Ile Lys Arg Gln Leu Lys

65 70 75 80

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

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Pro Gly Phe Trp Thr Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Asp Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly His Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asn Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 22

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 22

Ser Thr Ile Glu Glu Leu Ala Lys Thr Phe Leu Asp Lys Phe Asn Gln

1 5 10 15

Glu Ala Glu Asp Leu Asp Tyr Gln Arg Ser Leu Ala Ala Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Thr Gln Lys Met Asn Glu Ala Glu

35 40 45

Ala Lys Trp Ser Ala Phe Tyr Glu Glu Gln Ser Lys Leu Ala Thr Ala

50 55 60

Tyr Pro Leu Gln Glu Ile Gln Asn Phe Thr Leu Lys Arg Gln Leu Gln

65 70 75 80

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

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Glu Pro Thr Asp

305 310 315 320

Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gln Lys Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asn Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 23

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 23

Phe Asn Leu Glu Glu Gln Ala Lys Thr Phe Leu Asp Glu Phe Asn Leu

1 5 10 15

Lys Ala Glu Asp Leu Tyr Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Asp Glu Asn Val Gln Lys Met Ser Glu Ala Gly

35 40 45

Gly Ile Leu Ser Ala Phe Tyr Glu Glu Gln Ser Asn Leu Ala Lys Ala

50 55 60

Tyr Pro Leu Gln Asp Ile Gln Asn Leu Thr Val Lys Arg Gln Leu Arg

65 70 75 80

Ile Leu Gln Gln Ser Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Lys Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Gly Gln Lys Val Val Cys His Pro Thr Ala Trp Asp Met Gly Lys Asn

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp His Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 24

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 24

Thr Asn Ile Glu Glu Glu Ala Lys Lys Phe Leu Asp Asp Phe Asn Arg

1 5 10 15

Gln Ala Glu Asn Val Ser Tyr Glu Ser Ala Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Ile Asn Ile Thr Glu Glu Asn Ile Gln Lys Met Asn Asp Ala Gly

35 40 45

Ala Lys Trp Ser Glu Phe Tyr Glu Glu Gln Ser Lys Thr Ala Arg Asn

50 55 60

Tyr Pro Leu Gln Asp Ile Gln Asn Pro Thr Val Arg Arg Gln Leu Gln

65 70 75 80

Ile Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Glu Gly Phe Trp Asn Asn Ser Met Leu Thr Glu Pro Gln Asp

305 310 315 320

Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Asn Gly

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 25

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 25

Ser Thr Ser Asp Glu Glu Ala Lys Thr Phe Leu Glu Lys Phe Asn Ser

1 5 10 15

Glu Ala Glu Glu Leu Ser Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Asp Glu Asn Val Gln Lys Met Asn Val Ala Gly

35 40 45

Ala Lys Trp Ser Thr Phe Tyr Glu Glu Gln Ser Lys Ile Ala Lys Asn

50 55 60

Tyr Gln Leu Gln Asn Ile Gln Asn Asp Thr Ile Lys Arg Gln Leu Gln

65 70 75 80

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

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Thr Phe Trp Glu Asn Ser Met Leu Thr Glu Pro Gly Asp

305 310 315 320

Gly Arg Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys His

325 330 335

Asp Phe Arg Ile Lys Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 26

<211> 596

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 26

Ser Thr Ile Glu Glu Gln Ala Arg Thr Phe Leu Asp Lys Phe Asn His

1 5 10 15

Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp Asn Tyr

20 25 30

Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn Ala Gly

35 40 45

Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala Gln Met

50 55 60

Tyr Pro Pro Gln Glu Ile Gln Asn Leu Thr Ile Lys Leu Gln Leu Gln

65 70 75 80

Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys Ser Lys

85 90 95

Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser Thr Gly

100 105 110

Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu Glu Pro

115 120 125

Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg Leu

130 135 140

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

145 150 155 160

Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala Asn

165 170 175

His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val Asn

180 185 190

Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp Val

195 200 205

Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu His Ala

210 215 220

Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser Pro

225 230 235 240

Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg Phe

245 250 255

Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro Asn

260 265 270

Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln Arg

275 280 285

Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro Asn

290 295 300

Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly Asn

305 310 315 320

Val Gln Lys Val Val Cys His Pro Thr Ala Trp Asp Leu Gly Lys Gly

325 330 335

Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe Leu

340 345 350

Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr Ala

355 360 365

Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His Glu

370 375 380

Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His Leu

385 390 395 400

Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu Thr

405 410 415

Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly Thr Leu

420 425 430

Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe Lys Gly

435 440 445

Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys Arg

450 455 460

Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr Tyr Cys

465 470 475 480

Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile Arg

485 490 495

Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu Cys

500 505 510

Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser Asn

515 520 525

Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly Lys

530 535 540

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

545 550 555 560

Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe Thr Trp

565 570 575

Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp Trp

580 585 590

Ser Pro Tyr Ala

595

<210> 27

<211> 905

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 27

Cys Gly Gly Ala Gly Cys Ala Cys Thr Gly Thr Cys Cys Thr Cys Cys

1 5 10 15

Gly Ala Ala Cys Gly Thr Cys Gly Gly Ala Gly Cys Ala Cys Thr Gly

20 25 30

Thr Cys Cys Thr Cys Cys Gly Ala Ala Cys Gly Thr Cys Gly Gly Ala

35 40 45

Gly Cys Ala Cys Thr Gly Thr Cys Cys Thr Cys Cys Gly Ala Ala Cys

50 55 60

Gly Thr Cys Gly Gly Ala Gly Cys Ala Cys Thr Gly Thr Cys Cys Thr

65 70 75 80

Cys Cys Gly Ala Ala Cys Gly Gly Ala Gly Cys Ala Thr Gly Thr Cys

85 90 95

Cys Thr Cys Cys Gly Ala Ala Cys Gly Thr Cys Gly Gly Ala Gly Cys

100 105 110

Ala Cys Thr Gly Thr Cys Cys Thr Cys Cys Gly Ala Ala Cys Gly Ala

115 120 125

Cys Thr Ala Gly Thr Thr Ala Gly Gly Cys Gly Thr Gly Thr Ala Cys

130 135 140

Gly Gly Thr Gly Gly Gly Ala Gly Gly Cys Cys Thr Ala Thr Ala Thr

145 150 155 160

Ala Ala Gly Cys Ala Gly Ala Gly Cys Thr Cys Gly Thr Thr Thr Ala

165 170 175

Gly Thr Gly Ala Ala Cys Cys Gly Thr Cys Ala Gly Ala Thr Cys Gly

180 185 190

Cys Cys Thr Gly Gly Ala Gly Ala Cys Gly Cys Cys Ala Thr Cys Cys

195 200 205

Ala Cys Gly Cys Thr Gly Thr Thr Thr Thr Gly Ala Cys Cys Thr Cys

210 215 220

Cys Ala Thr Ala Gly Ala Ala Gly Ala Cys Ala Cys Cys Gly Gly Gly

225 230 235 240

Ala Cys Cys Gly Ala Thr Cys Cys Ala Gly Cys Cys Thr Cys Thr Cys

245 250 255

Gly Ala Cys Ala Thr Thr Cys Gly Thr Thr Gly Gly Ala Thr Cys Cys

260 265 270

Gly Cys Cys Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Thr Ala Cys

275 280 285

Cys Gly Gly Thr Gly Ala Cys Cys Gly Cys Cys Thr Thr Gly Cys Thr

290 295 300

Cys Cys Thr Gly Cys Cys Gly Cys Thr Gly Gly Cys Cys Thr Thr Gly

305 310 315 320

Cys Thr Gly Cys Thr Cys Cys Ala Cys Gly Cys Cys Gly Cys Cys Ala

325 330 335

Gly Gly Cys Cys Gly Ala Gly Cys Cys Ala Gly Thr Thr Cys Cys Gly

340 345 350

Gly Gly Thr Gly Thr Cys Gly Cys Cys Gly Cys Thr Gly Gly Ala Thr

355 360 365

Cys Gly Gly Ala Cys Cys Thr Gly Gly Ala Ala Cys Cys Thr Gly Gly

370 375 380

Gly Cys Gly Ala Gly Ala Cys Ala Gly Thr Gly Gly Ala Gly Cys Thr

385 390 395 400

Gly Ala Ala Gly Thr Gly Cys Cys Ala Gly Gly Thr Gly Cys Thr Gly

405 410 415

Cys Thr Gly Thr Cys Cys Ala Ala Cys Cys Cys Gly Ala Cys Gly Thr

420 425 430

Cys Gly Gly Gly Cys Thr Gly Cys Thr Cys Gly Thr Gly Gly Cys Thr

435 440 445

Cys Thr Thr Cys Cys Ala Gly Cys Cys Gly Cys Gly Cys Gly Gly Cys

450 455 460

Gly Cys Cys Gly Cys Cys Gly Cys Cys Ala Gly Thr Cys Cys Cys Ala

465 470 475 480

Cys Cys Thr Thr Cys Cys Thr Cys Cys Thr Ala Thr Ala Cys Cys Thr

485 490 495

Cys Thr Cys Cys Cys Ala Ala Ala Ala Cys Ala Ala Gly Cys Cys Cys

500 505 510

Ala Ala Gly Gly Cys Gly Gly Cys Cys Gly Ala Gly Gly Gly Gly Cys

515 520 525

Thr Gly Gly Ala Cys Ala Cys Cys Cys Ala Gly Cys Gly Gly Thr Thr

530 535 540

Cys Thr Cys Gly Gly Gly Cys Ala Ala Gly Ala Gly Gly Thr Thr Gly

545 550 555 560

Gly Gly Gly Gly Ala Cys Ala Cys Cys Thr Thr Cys Gly Thr Cys Cys

565 570 575

Thr Cys Ala Cys Cys Cys Thr Gly Ala Gly Cys Gly Ala Cys Thr Thr

580 585 590

Cys Cys Gly Cys Cys Gly Ala Gly Ala Gly Ala Ala Cys Gly Ala Gly

595 600 605

Gly Gly Cys Thr Ala Cys Thr Ala Thr Thr Thr Cys Thr Gly Cys Thr

610 615 620

Cys Gly Gly Cys Cys Cys Thr Gly Ala Gly Cys Ala Ala Cys Thr Cys

625 630 635 640

Cys Ala Thr Cys Ala Thr Gly Thr Ala Cys Thr Thr Cys Ala Gly Cys

645 650 655

Cys Ala Cys Thr Thr Cys Gly Thr Gly Cys Cys Gly Gly Thr Cys Thr

660 665 670

Thr Cys Cys Thr Gly Cys Cys Ala Gly Cys Gly Ala Ala Gly Cys Cys

675 680 685

Cys Ala Cys Cys Ala Cys Gly Ala Cys Gly Cys Cys Ala Gly Cys Gly

690 695 700

Cys Cys Gly Cys Gly Ala Cys Cys Ala Cys Cys Ala Ala Cys Ala Cys

705 710 715 720

Cys Gly Gly Cys Gly Cys Cys Cys Ala Cys Cys Ala Thr Cys Gly Cys

725 730 735

Gly Thr Cys Gly Cys Ala Gly Cys Cys Cys Cys Thr Gly Thr Cys Cys

740 745 750

Cys Thr Gly Cys Gly Cys Cys Cys Ala Gly Ala Gly Gly Cys Gly Thr

755 760 765

Gly Cys Cys Gly Gly Cys Cys Ala Gly Cys Gly Gly Cys Gly Gly Gly

770 775 780

Gly Gly Gly Cys Gly Cys Ala Gly Thr Gly Cys Ala Cys Ala Cys Gly

785 790 795 800

Ala Gly Gly Gly Gly Gly Cys Thr Gly Gly Ala Cys Thr Thr Cys Gly

805 810 815

Cys Cys Thr Gly Thr Gly Ala Thr Ala Thr Cys Thr Ala Cys Ala Thr

820 825 830

Cys Thr Gly Gly Gly Cys Gly Cys Cys Cys Thr Thr Gly Gly Cys Cys

835 840 845

Gly Gly Gly Ala Cys Thr Thr Gly Thr Gly Gly Gly Gly Thr Cys Cys

850 855 860

Thr Thr Cys Thr Cys Cys Thr Gly Thr Cys Ala Cys Thr Gly Gly Thr

865 870 875 880

Thr Ala Thr Cys Ala Cys Cys Cys Thr Thr Thr Ala Cys Thr Gly Cys

885 890 895

Ala Ala Cys Cys Ala Cys Thr Gly Ala

900 905

<210> 28

<211> 731

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 28

Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu

1 5 10 15

Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys

20 25 30

Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His

35 40 45

Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr

50 55 60

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

65 70 75 80

Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu

85 90 95

Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr

100 105 110

Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro

115 120 125

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

130 135 140

Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln

145 150 155 160

Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn

165 170 175

Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys

180 185 190

His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser

195 200 205

Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys

210 215 220

Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys

225 230 235 240

Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu

245 250 255

His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val

260 265 270

Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg

275 280 285

Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu

290 295 300

Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln

305 310 315 320

Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys

325 330 335

Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu

340 345 350

Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys

355 360 365

Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp

370 375 380

Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe

385 390 395 400

Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro

405 410 415

Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg

420 425 430

Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu

435 440 445

Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly

450 455 460

Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val

465 470 475 480

Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr

485 490 495

Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His

500 505 510

Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys

515 520 525

Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys

530 535 540

Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys

545 550 555 560

Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys

565 570 575

Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser

645 650 655

Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly

660 665 670

Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg

675 680 685

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

690 695 700

Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu

705 710 715 720

Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe

725 730

<210> 29

<211> 193

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 29

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys

1 5 10 15

Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val

20 25 30

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys

35 40 45

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

50 55 60

Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile

65 70 75 80

Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

85 90 95

Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp

100 105 110

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

115 120 125

Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser

130 135 140

Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro

145 150 155 160

Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro

165 170 175

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

180 185 190

Val

<210> 30

<211> 273

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 30

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

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

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn

210 215 220

Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys

225 230 235 240

Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp

245 250 255

Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys

260 265 270

Ser

Claims (18)

1. The recombinant transmembrane protein is characterized in that a phase change peptide segment is fused on the recombinant transmembrane protein, the recombinant transmembrane protein is ligand-PDGFR recombinant transmembrane protein, the phase change peptide segment is fused in an intracellular region of the transmembrane protein, and a low-complexity sequence is fused at the C end of the ligand-PDGFR recombinant transmembrane protein; the phase change peptide is a low-complexity sequence derived from TAF15 protein, FUS protein or DDX4 protein; the low complexity sequence is selected from one or more of the following:

1) An amino acid sequence as shown in SEQ ID NO. 1;

2) An amino acid sequence as shown in SEQ ID NO. 2;

3) An amino acid sequence as shown in SEQ ID NO. 3;

4) An amino acid sequence as shown in SEQ ID NO. 4;

when the low complexity sequence is SEQ ID NO.1, the amino acid sequence of the ligand-PDGFR recombinant transmembrane protein which does not comprise a ligand part after the C end of the ligand-PDGFR recombinant transmembrane protein is fused with the low complexity sequence is shown as SEQ ID NO. 5;

when the low complexity sequence is SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, the amino acid sequence of the ligand-PDGFR recombinant transmembrane protein which does not comprise a ligand part after the C-terminal fusion of the low complexity sequence is obtained by replacing SEQ ID NO.1 in SEQ ID NO.5 with SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO. 4.

2. An isolated polynucleotide encoding the recombinant transmembrane protein of claim 1.

3. The polynucleotide according to claim 2, wherein the polynucleotide has a nucleotide sequence as shown in SEQ ID No. 6.

4. A nucleic acid construct comprising the polynucleotide of claim 2 or 3.

5. A cell expressing the recombinant transmembrane protein of claim 1.

6. The cell of claim 5, wherein the cell is a ligand quantitative presenting cell.

7. The cell of claim 6, wherein the ligand-presenting cell is an antigen-presenting cell.

8. A cell surface macromolecular quantitative display system comprising the cell of claim 7 and a receptor-expressing cell, wherein the receptor of the receptor-expressing cell is specifically quantitatively activated when the ligand quantitative presenting cell and the receptor-expressing cell are co-cultured.

9. The cell surface macromolecule quantitative display system of claim 8, wherein the receptor-expressing cells are synNotch synthesis receptor-expressing cells.

10. The cell surface macromolecular quantitative display system according to claim 9, wherein the extracellular region of the synNotch synthetic receptor on the synNotch synthetic receptor-expressing cell is fused with a region capable of specifically recognizing and binding to the ligand on the surface of the ligand quantitative presenting cell.

11. The cell surface macromolecule quantitative display system of claim 8, wherein the receptor expressing cell is a stably transfected cell line that expresses both a synNotch synthesis receptor and a reporter group.

12. The cell surface macromolecule quantitative display system of claim 11, wherein the reporter is selected from a fluorescent reporter or a non-fluorescent reporter.

13. A method for preparing a cell surface macromolecule quantitative display system, which is characterized by comprising the following steps:

1) Constructing a receptor expression cell expressing a receptor of interest on a cell membrane;

2) Constructing ligand-quantitative presenting cells expressing a ligand of interest, said ligand-quantitative presenting cells expressing the recombinant transmembrane protein of claim 1.

14. The method of claim 13, further comprising one or more of the following features:

1) The receptor expressing cells carry a reporter group;

2) The receptor-expressing cell is a synNotch synthetic receptor-expressing cell.

15. The method of claim 14, wherein the extracellular region of the synNotch synthetic receptor on the synNotch synthetic receptor-expressing cell fuses the receptor of interest.

16. Use of the cell surface macromolecule quantitative display system of any one of claims 8-12 for ligand-receptor affinity detection and/or bulk acquisition of high affinity antibodies.

17. A method for ligand-receptor affinity assay, comprising co-culturing the receptor expressing cells of step 1) and the ligand-presenting cells of step 2) of the method of preparation of claim 13, and detecting binding of the target ligand to the target receptor.

18. A method for obtaining high affinity antibodies in bulk, comprising the steps of:

1) Constructing a receptor expression cell library carrying a synNotch antibody library and a reporter group;

2) Co-culturing the pool of receptor expressing cells of step 1) with ligand-presenting cells expressing the recombinant transmembrane protein of claim 1 to activate a cell surface macromolecular quantitative display system;

3) Collecting cells in the co-culture system, and primarily enriching receptor expression cells positive to the reporter group;

4) Culturing the receptor expression cells preliminarily enriched in the step 3) for 5-10 days, and carrying out secondary enrichment by using the ligand quantitative presenting cells, wherein the synNotch antibody carried by the receptor expression cells obtained by enrichment is the high-affinity antibody.