CN114437232A - 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 and a preparation method and application thereof. The phase transition peptide segment is fused on the transmembrane protein of the ligand quantitative presenting cell, the receptor expression cell is a synNotch synthetic receptor expression cell, and the extracellular region of the synNotch synthetic receptor is fused with a region which can be specifically recognized and combined with the ligand on the surface of the ligand presenting cell. When the ligand quantitative presenting cells and the receptor expressing cells are cultured together, 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 batch acquisition of high-affinity antibodies, so that batch high-affinity antibodies can be quickly obtained, and subsequent tedious verification work of technologies such as phage display and the like 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 and a preparation method and application thereof.

Background

How mammalian cells sense and differentiate the magnitude of cell surface receptors and their corresponding ligand affinities when the cells are in contact with each other, and convert them into specific quantitative biological events, remains a scientific puzzle. For example, the mechanism of how lymphoid B cells 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 hallmark of the immune response of the adaptive immune system, during which high affinity antibodies to a particular antigen will be produced. Whereas antibody affinity maturation of Germinal Centers (GCs) mainly involves high frequency mutation (SHM) of antibody variable regions and affinity-based B cell selection. Among them, high frequency mutation induces a large number of diverse B Cell Receptors (BCRs) with different affinities, and in the germinal center, B cells with a higher affinity BCR can better compete to bind follicular helper T cells (Tfh) located in the bright zone of the germinal center, thereby allowing a small number of high affinity B cells to finally dominate. This affinity-based selection depends on the amount of antigen that is extracted from the surface of the Antigen Presenting Cell (APC) by the B cell and re-presented at the plasma membrane of the autologous cell. Activation of B cell downstream signaling pathway responses by specific antigens is largely determined by the affinity between the antigen and the BCR. It has been reported that high affinity antigen-antibody interactions may lead to stronger mechanical forces, and that the particular micro-cluster structure of BCR formation in germinal centers may also be associated with this mechanism. However, the molecular mechanism of how the high affinity BCR captures more antigenic molecules is still unclear. The synNotch synthesis system reported as an antibody expression system does not discriminate well between antibody-antigen affinity differences.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention is directed to a quantitative display system for cell surface macromolecules, and a method for preparing the same and use thereof, which are used to solve the problems of the prior art.

In order to achieve the above objects and other related objects, the present invention provides a recombinant transmembrane protein fused with a phase transition peptide fragment.

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

The invention also provides a nucleic acid construct, which comprises the polynucleotide.

The invention also provides a cell on which the transmembrane protein is fused.

The invention also provides a cell surface macromolecule quantitative display system, which comprises the ligand quantitative presenting cell and the receptor expressing cell, wherein the receptor of the receptor expressing cell can be specifically and quantitatively activated when the ligand quantitative presenting cell and the receptor expressing 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 the target receptor on the cell membrane of the receptor expression cell;

2) cloning the target ligand to 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 method for detecting ligand-receptor affinity, which comprises the steps of co-culturing the ligand quantitative presenting cells and the receptor expression cells obtained in the preparation method, and then detecting the binding condition of the target ligand and the target receptor.

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

1) constructing receptor expression cells 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 a cell surface macromolecule quantitative display system;

3) collecting cells in a co-culture system, and primarily enriching receptor expression cells with positive report groups;

4) culturing the receptor expression cells primarily enriched in the step 3) for 5-10 days, and enriching again by using the new ligand quantitative presenting cells to obtain the high-affinity antibody carried by the receptor expression cells.

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

1) the cell surface macromolecule quantitative display technology based on protein phase transition can convert the affinity of a ligand-receptor, such as an antigen-antibody, into the strength of the expression of a reporter gene in cells by combining with a synNotch system;

2) the cell surface macromolecule display technology is proved to be related to the phase transition of the intracellular part of the membrane protein, and a plurality of tool molecules capable of supporting quantitative display are found, and the molecules can generate protein liquid-liquid phase transition 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 of coronavirus neutralizing antibody or receptor protein ACE2 and coronavirus spike protein;

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

Drawings

FIG. 1 shows an example of the cell surface macromolecule quantitative display system and the detection of ligand-receptor binding affinity of the present invention, wherein the following figures are illustrated: schematic system diagram of Ag-mem and Ag-m-LC cell interacting with anti-GFP synNotch receptor cell respectively; schematic representation of expression vectors for Ag-mem, Ag-m-LC and synNotch receptors; GFP-mem and GFP-mem-FUS_LCReporting the activation ratio of the system after the cell interacts with the anti-GFP/mCherry receptor cell; reporting the activation ratio of the system after interaction of the mCherry-mem cell and the anti-GFP/mCherry receptor cell; GFP-mem and GFP-m-FUS_LCReporting the activation ratio of the system after the cell interacts with two high/low anti-GFP receptor cells according to different ratios; F. the information of the anti-GFP nano antibody and the expression quantity on the cell membrane after corresponding syncnotch receptors are fused; GFP-mem and GFP-m-FUS_LCThe corresponding BFP of the cells reports the proportion of systemic activation.

FIG. 2 shows the principle of quantitative presentation of a system for quantitative display of macromolecules on the surface of cells, in which the figures are illustrated as follows: GFP-mem and GFP-m-FUS_LCConfocal fluorescence imaging of GFP in cells; GFP-mem and GFP-m-FUS_LCPerforming GFP (green fluorescent protein) localization statistics on the cells; C. GFP-mem and GFP-m-FUS with different expression amounts_LCFlow quantification of cell surface GFP protein; D. GFP-mem and GFP-m-FUS with different expression amounts_LCThe ratio of reporter system activation to cell; E. interaction schematic diagram of red fluorescence labeling synNotch receptor expression cell (modified in HEK293T cell line of human embryonic kidney cell) and ligand quantitative presenting cell (modified in K562 cell line of human myeloid leukemia cell); F. the interface where the ligand quantitative presenting cells interact with synNotch receptor cells; qDis system mode of action diagram; HER2-mem and HER2-m-FUS_LCSchematic representation of the system for interaction with anti-HER2 synNotch receptor cells; I. cell surface HER2 extracellular domain expression level; J. the expression level of cell surface anti-HER2-scfv synNotch receptor; HER2-mem and HER2-m-FUS_LCThe corresponding reporting system activation ratio.

FIG. 3 shows the principle of phase transition for quantification of a system for quantitatively displaying macromolecules on a cell surface, wherein the diagrams are illustrated as follows: FUS_LCDistribution of Q, G, Y amino termini in proteins, and FUS_LCThe protein membrane surface expression quantity of different mutant cells and the corresponding activation ratio of a report system; GFP-m-FUS_LC-478 cell corresponding reporter activation ratio; GFP-m-TAF15_LCAnd GFP-m-DDX4_LCThe ratio of reporter system activation to cell; the corresponding reporter system activation ratio of the GFP-foldon-m cells and the GFP-Fc-m cells;

FIG. 4 shows the principle of quantification and mutation analysis for a quantitative display system for macromolecules on the cell surface, wherein the figures are illustrated as follows: A. different GFP-m-FUS_LCMutants form a statistical map of the rate of protein aggregation in the opto-droplet system, with the corresponding statistical cell numbers noted in the legend; GFP-m-FUS_LC Q>gPG confocal fluorescence imaging of G cells, flow assay and reporting of system activation ratio results；C.GFP-m-FUS_LC G>GFP confocal fluorescence imaging of cells A, flow detection and reporting of system activation ratio results; GFP-m-FUS_LCGFP confocal fluorescence imaging of Y27S cells, flow assay and reporting of the system activation ratio results; GFP-m-FUS_LCGFP confocal fluorescence imaging of Y7A cells, flow detection and reporting of the system activation ratio results; GFP-m-FUS_LCGFP confocal fluorescence imaging of 27R cells, flow detection and reporting of system activation ratio results;

FIG. 5 shows the ratio of reporter activation for different GFP-m-LC cells.

FIG. 6 shows the interaction between the surface spinous process protein S-RBD of various coronaviruses and ACE2 or its corresponding antibody, wherein the illustration of each figure is as follows: A. synNotch cells expressing hACE2 and various antibodies and S-RBD-m-FUS fusing coronavirus surface spinous process proteins of various species_LCThe reporter ratio for protein interaction activation; B. S-RBD-m-FUS on the surface of chimeric receptor expressing cells of ACE2 of different species and three coronaviruses_LCReporter ratio of protein interaction activation. C. Three-dimensional structure diagram of interaction of human ACE2 protein (green) and SARS-CoV-2S-RBD protein (blue), wherein the peptide fragment directly acting on ACE2 is marked orange.

FIG. 7 shows the batch isolation of high affinity antibodies for a cell surface macromolecular quantitative display system, wherein the figures are illustrated as follows: schematic diagram of hcd8 reporting system; GFP-mem and GFP-m-FUS_LCThe hCD8 report system activation proportion is obtained after the cells interact with high, medium and low affinity anti-GFP receptor cells respectively; C. the high affinity antibody expression cell proportion after each round of qDis-MACS enrichment; qDis-MACS flow diagram; E. after three rounds of qDis-MACS screening, the antibody is enriched in proportion, and the red color is the final selected expression antibody; bli method determines the corresponding affinity of the antibody.

FIG. 8 shows the binding dissociation curve for the antibody measured by the BLI method.

Detailed Description

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

The phase-transition peptide fragment refers to a peptide fragment capable of undergoing phase transition under physiological conditions. The phase transition peptide fragment can be any one or more peptide fragments in LLPSDB database (http:// bio-comp. ucas. ac. cn/LLPSDB), PhaSePro database (https:// PhaSePro. elte. hu) or PhaSepDB database (http:// db. phasep. pro), or peptide fragments mutated based on peptide fragments in the database. The peptide segment mutated based on the peptide segment in the database has a similarity of more than 80%, more than 90%, more than 95% or more than 99% with the phase-change peptide segment in the database. The low complexity sequence (also called LC tag) is one of the phase-change peptide fragments.

The low-complexity sequence refers to a protein sequence which is enriched with a specific amino acid component and/or repeated appearance of a specific amino acid sequence. 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 regions of intrinsic disorder, and proteins or peptide fragments containing low complexity sequences can aggregate under physiological conditions and drive liquid-liquid phase separation to occur. 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 protein to be concentrated in a localized area with a significant increase in concentration.

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

Specifically, the low-complexity sequence source comprises amino acids 1-214 (shown in SEQ ID NO. 1) of the human FUS protein, amino acids 1-478 (shown in SEQ ID NO. 2) of the FUS protein, amino acids 1-208 (shown in SEQ ID NO. 3) of the human TAF15 protein, and amino acids 1-236 (shown in SEQ ID NO. 4) of the human DDX4 protein.

The low-complexity sequence can also be applied to the ligand quantitative presenting cells of the invention by modifying some protein sequences of natural LC tags (such as amino acids 415-218 of human TDP-43 protein, amino acids 46-266 of human EWSR1 protein, amino acids 341-190-186 of human HNRNPA1 protein, amino acids 320-186 of human HNRNPA2 protein, and twenty, thirty, forty, fifty polymers of repeated arrangement of two amino acids proline and arginine). For example by adjusting the amount of protein expression to approach the threshold required for phase separation in a 2D system, or by altering the rate at which proteins form multivalent binding, etc.

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

In one embodiment, the phase change peptide segment is fused to an intracellular domain 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 (ligand-PDGFR) transmembrane domain fusion protein (Ag-mem) is abundantly expressed in the plasma membrane of antigen-presenting cells (corresponding to the ligand quantitative-presenting cells of the present application). The recombinant transmembrane protein fused with the LC label can be called Ag-m-LC or Ag-mem-LC for short. The Ag-m-LC recombinant transmembrane protein is obtained by combining any target ligand with a PDGFR transmembrane domain by a molecular biology method well known in the field, then fusing an intracellular peptide segment of a PDGFR receptor, and finally fusing different LC tags at the C end. In one embodiment, the ligand of interest is selected from an antigen. 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 of the ligand-PDGFR recombinant transmembrane protein fused to the low-complexity sequence at the C-terminus not including the ligand moiety (i.e., the amino acid sequence including only the transmembrane region, intracellular region and LC tag of PDGFR) is as set forth in SEQ ID No. 5:

(the PDGFR transmembrane region is underlined; the PDGFR intracellular short peptide is underlined; and FUS is the remainder)_LCPeptide fragment)

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

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

In one embodiment, the polynucleotide has the nucleotide sequence shown as SEQ ID No. 6.

The invention also relates to variants of the above polynucleotides which encode fragments, analogues and derivatives of the enzymes or proteins having the same amino acid sequence as the present invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is 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 protein encoded thereby.

The invention also provides a nucleic acid construct, which comprises the polynucleotide.

In certain embodiments of the invention, the nucleic acid construct is constructed by inserting the isolated polynucleotide into a multiple cloning site of an expression vector. The expression vector of the present invention is generally referred to various commercially available expression vectors well known in the art, and may be, for example, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.

The invention provides a cell expressing the recombinant transmembrane protein.

In one embodiment, the cell is a quantitative ligand-presenting cell; preferably, the ligand-quantitatively-presenting cells include antigen-quantitatively-presenting cells. The ligand quantitative presenting cells are selected from K562 human chronic myelogenous leukemia cell lines. In one embodiment, the quantitative ligand presenting cell is a stable transformable cell line stably expressing Ag-m-LC.

In another embodiment, the cell is a host cell for producing the transmembrane protein.

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

The macromolecule refers to a macromolecule existing in organisms such as protein, nucleic acid, polysaccharide and the like or artificially synthesized.

Specifically, the extracellular domain of the receptor-expressing cells is fused to a region capable of specifically recognizing and binding to the ligand presented by the ligand-presenting cells, and upon contact with each other, specifically activates synNotch synthesis receptors.

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 synthetic receptors. The synNotch synthetic receptor is fused with a receptor of interest.

synNotch synthetic receptors are located on cell membranes, extracellular domains of the synNotch synthetic receptors are fused with purposeful receptors, such as nano antibodies, single chain antibodies (scFv), antibody analogs or cell membrane surface protein receptors and extracellular protein peptide fragments, and the purposeful receptors in the extracellular domains can be specifically combined after being contacted with antigens on the surface of ligand quantitative presenting cells; the intracellular domain inside the cytoplasmic membrane of synNotch synthetic receptors is fused with a transcriptional activator, which is released and initiates the expression of the intracellular blue fluorescent protein when the receptor of interest and the ligand of interest are bound (fig. 1A, 1B). Activation of synNotch synthetic receptors in receptor-expressing cells will be affected slightly when the affinity between ligand-receptor is altered. The synNotch synthetic receptor of the present invention is obtained by fusing only the intended receptor to an existing synNotch receptor, and methods for fusing either an existing synNotch receptor or an intended receptor are well known to those skilled in the art.

In one embodiment, the receptor expressing cell is a stable transgenic cell line expressing both synNotch synthetic receptor and reporter.

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

In general, the fluorescent reporter 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 is only required to be capable of promoting protein expression and positioning expression on a cytoplasmic membrane when the antigen is combined with the antibody, and sorting out cells expressing the protein by utilizing a commercially available magnetic bead sorting technology. For example, the non-fluorescent reporter is selected from the group consisting of a CD8 reporter or a CD4 reporter. The CD8 and CD4 reporter genes are derived from mammals. Preferably hCD8 and hCD4 reporter genes, namely human CD8 and 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 synthetic 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, the quantitative display system is a mammalian cell surface macromolecule quantitative display system.

The cell surface macromolecule quantitative display system can detect the molecular interaction of the ligand and the receptor with the affinity ranging from micromolar to nanomolar, but the activation of the receptor and the intermolecular affinity are not well correlated in the affinity range.

The liquid-liquid phase separation of membrane proteins inside the cytoplasmic membrane rapidly increases the concentration of receptor proteins in local regions, thereby increasing the affinity (avidity) between ligand-receptors in the extracellular region, i.e., the cumulative strength of multiple ligand-receptor molecular interactions, which in turn amplifies the differences in individual ligand-receptor affinities (affinity). The inventors have found that fusion of a variety of low complexity sequences to ligand (antigen) presenting transmembrane proteins can result in the production of large aggregates of antigen molecules on the membrane. This aggregated ligand (antigen) presentation pattern, when combined with the synNotch synthetic receptor system, enables the ligand-receptor system to be converted into an affinity-proportional quantitative mammalian cell membrane display system.

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

1) cloning the target receptor on the cell membrane of the receptor expression cell;

2) cloning a ligand of interest onto said ligand quantitative presenting cells;

the method of cloning the receptor of interest into receptor expressing cells and the ligand of interest into the ligand-presenting cells is a molecular biological method well known to those skilled in the art.

The receptor expressing cells carry a fluorescent reporter group.

The ligand and the receptor can be antigen and antibody.

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 method for detecting ligand-receptor affinity, which comprises the steps of culturing the ligand quantitative presenting cells cloned with the target ligand and the receptor expression cells together, and detecting the binding condition of the target ligand and the target receptor.

In particular, 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, comprising the following steps:

1) constructing receptor expression cells 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 a cell surface macromolecule quantitative display system;

3) collecting cells in a co-culture system, and primarily enriching cells with positive reporter groups;

4) and (3) culturing the cells primarily enriched in the step 3) for 5-10 days, and enriching again by using the new ligand quantitative presenting cells, wherein the receptor expression cells obtained by enrichment 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. In general, the synNotch antibody library can be obtained by the existing antibody library construction method. For example by phage display technology.

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

In one embodiment, in step 2), the following method is used to activate the cell surface macromolecule quantitative display system: separately taking receptor expression cells and antigen expression cells, resuspending the cells, and placing the cells in CO₂And performing shake culture in an incubator for 36-60 hours.

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

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

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

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

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention. Unless otherwise specified, the experimental procedures used in the present invention are well known to those skilled in the art.

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

the synNotch synthetic receptor is formed by fusing a plurality of GFP nano antibodies (including G1-G9), mCherry nano antibodies, HER2 single-chain antibodies, SARS-CoV S protein antibodies, SARS-CoV-2S protein antibodies, ACE2 chimeras (shown as SEQ ID NO. 7-SEQ ID NO. 26) of different species and ACE2 chimeras of different species (raccoon, dog, ferret, raccoon, bear, cat, paguma, horse, Chinese chrysanthemum head bat, cow, sheep, pig, camel, mouse, rat, squirrel, hedgehog, pangolin and monkey) with a commercial mouse Notch1 partial domain and Gal4-VP64 transcriptional activator respectively, and carrying a myc tag (purchased from addge, cat number: 79127) at the front end of the receptor.

The blue fluorescence reporter system is a fusion of a copy of the Gal4 DNA binding domain sequence to a portion of the CMV promoter and cloned in front of the BFP gene, as described in L.Morshut et al, Engineering custom 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 constructed by fusing a copy of the Gal4 DNA binding domain sequence to the 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 in SEQ ID NO. 28), SARS-CoV S-RBD protein (nucleotide sequence shown in SEQ ID NO. 29), SARS-CoV-2S-RBD protein (nucleotide sequence shown in SEQ ID NO. 30), TGP protein (derived from D.W.Close et al, Thermal green protein, an extreme state, non-amplifying fluorescing fluorescent protein secreted by structure-regulated surface engineering, proteins 83,1225-1237(2015)) with PDGFR domain (sequence shown in SEQ ID NO. 5), and fusing myc tag at N-terminal.

The Ag-m-LC expression vector is formed by combining a plurality of proteins including GFP (including G1-G9), mChery protein, HER2 extracellular domain, SARS-CoV S-RBD protein, SARS-CoV-2S-RBD protein and TGP protein with PDGFR transmembrane domain (namely a sequence shown in SEQ ID NO. 5), fusing an intracellular peptide segment (aa562-599) of PDGFR receptor, and finally fusing different LC tags at the C end.

The nano antibody of GFP and the mCherry nano antibody in the expression vector are derived from the following sources: fridy et al, A robust pipeline for rapid production of very small antibodies 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 inhibition an incorporated Therapeutic Index against turbines in Rice cancer Res 75, 3596-.

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

SARS-CoV S protein antibody S230 is derived from: A.C. walls et al, connected receiver function minor semiconductors Activation of Coronavir fusion.cell 176, 1026-.

SARS-CoV-2S protein antibody m396 is derived from: Z.Zhu et al, content cross-reactive conjugation of SARS coronavirs isolates by human monoclonal antibodies. Proc Natl Acad Sci U S A104, 12123-.

The SARS-CoV-2S protein antibody CV30 is derived from: seydoux et al, Analysis of a SARS-CoV-2-fed induced visual developments of Point neural fibres with Limited textual information Immunity 53,98-105e105 (2020).

The LC tag comprises amino acids 1-214 of the human FUS protein, amino acids 1-478 of the FUS protein, amino acids 1-208 of the human TAF15 protein, amino acids 1-236 of the human DDX4 protein, 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 and fifty polymers with repeated arrangement of two amino acids of proline and arginine.

Example 1 construction and activation of a cell line quantitatively displaying the desired stably transformed Strain

Construction: GFP-mem fusion proteins were expressed on the surface of ligand-presenting cells (Ag-mem APC) and GFP-M-LC fusion proteins were expressed on the surface of ligand-quantitative-presenting cells (Ag-M-LC APC) and fused GFP and mCherry nanobody (anti-GFP: KD ═ 0.31. mu.M, anti-mCherry: KD ═ 0.18nM, antibody derived from Fridy, P.C.et. A robust pipeline for rapid production of a vertical negative antibody synthesized receptor, 1253. 1260, doi:10.1038/nmeth.3170(2014), respectively, on the surface of K562 cells, called receptor expressing cells or receptor expressing cells (ABC) (FIGS. 1A, 1B.) the method is as follows: obtaining a virus transfected transiently with the corresponding T293 in the art using the corresponding liposome vectors, and simultaneously infecting in the desired cells, and a myc label or a fluorescent gene is used for sorting to obtain a cell line for stably expressing the target gene. synNotch synthetic receptor expression cells stably express synNotch fusion expression vectors and reporter system expression vectors in K562 cells. The cell lines constructed were stable populations of transformed cells, not corresponding monoclonal cells.

Activating: the synNotch synthetic receptor expressing cells and the antigen expressing cells were counted separately using a hemocytometer, and the two cells were mixed at a ratio of 1:1, wherein 0.025million cells were added to each 96-well plate and suspended in 200. mu.l of medium, 0.1million cells were added to each 24-well plate and suspended in 1ml of medium, and 1million cell was added to each 10ml of medium during shaking culture in a culture flask. Mixing well, at 37 deg.C and 5% CO₂After culturing for 48h in the incubator, the cells were detected by a fluorescence reporter system using a flow cytometer.

The results showed that the BFP reporter of anti-GFP synNotch synthetic receptor expressing cells was successfully activated within 48 hours of co-culture, whereas the reporter failed to be activated when mixed in equal proportion to anti-mCherry synNotch synthetic receptor expressing cells (fig. 1C, 1D). 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, 1D). The experimental results show that the mammalian surface antibody display system effectively reports the existence of the combination between the molecules, and the synNotch synthesis receptor is activated with a certain threshold.

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

To examine the effect of intermolecular affinity exerted during synNotch synthetic receptor activation, GFP-APC and two anti-GFP synNotch ABC cell lines were run at 2: 1:1 for 48 hours. Although the affinity of the two anti-GFP nanobodies differed by two orders of magnitude ("Hi": KD ═ 3.5 nM; "Lo": KD ═ 600nM), a similar proportion of BFP positive cells was observed in both anti-GFP synNotch cells (fig. 1E). And in addition, the ratio of 1: 3 or 1: 9, the ABC content is reduced by reducing the number of APC cells in the system, so that the ABC of the high-affinity antibody can obtain competitive advantage under extreme conditions.

The results show that 1: 9-time anti-GFP^HiThe proportion of BFP positive cells in SynNotch ABC was slightly increased (fig. 1E). Thus, molecular interactions with affinities in the micromolar to nanomolar range can activate synNotch synthetic receptors when stimulated by Ag-mem cells, but the activation of this receptor is not well correlated with intermolecular affinities.

Example 3 intracellular Low complexity sequence (LC) tags to achieve ligand-receptor affinity discrimination

Fusion of the intracellular domain of ligand-presenting cells with LC tags (FIG. 1D, called Ag-m-LC to distinguish from conventional Ag-mem) can significantly reduce anti-GFP^LoActivation of blue fluorescent protein in synNotch cells (FIG. 1E), with a significant increase in anti-GFP^HiAnd anti-GFP^LoDifferences in fluorescence activation in synNotch cells (fig. 1E). For anti-GFP^HisynNotch synthetic receptor expressing cells showed higher activation capacity of Ag-m-LC expressing APC compared to commonly used Ag-mem APC (FIG. 1E).

To further explore the affinity differentiation of Ag-m-LC, a panel of anti-GFP nanobodies with different affinities was cloned into synNotch synthetic receptor expressing cells. These anti-GFP nanobodies recognize two non-overlapping epitopes (interface of antibody-antigen interaction) on the GFP protein with affinities (KD) ranging from micromolar to sub-nanomolar (fig. 1F). Through expression quantity analysis of synNotch synthetic receptors located on the surface of antibody-expressing cells, the receptors fused with various anti-GFP nanobodies were found to have differences in quantity (FIG. 1F). When a panel of anti-GFP synNotch ABC cells were separately co-cultured with GFP-mem APC, the level of synNotch synthetic receptor activation was similar in cells of different affinities (FIG. 1G). Also in this case, the level of synNotch synthetic receptor activation is determined primarily by its number corresponding to the plasma membrane surface of the cell (FIG. 1F). When synNotch synthetic receptors are expressed in consistent amounts, affinity has less effect on the level of activation of the fluorescent reporter system in the recipient cells. However, when co-cultured with GFP-m-LC APC, activation of anti-GFP synNotch synthetic receptor was highly correlated with avidity, regardless of identity of the antibody-binding epitope or high or low expression level of ABC cell surface receptor (FIG. 1G). Thus, the addition of an LC tag to the intracellular region of an antigen allows for affinity-dependent activation of synNotch synthetic receptors, a system referred to as the 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 affinity-dependent activation of synNotch synthetic receptor-displaying cells, the following experiments were performed.

First, subcellular localization of the two different forms of membrane-bound GFP antigen described above was observed. Although the overall GFP fluorescence signal in the cells was at a similar level, GFP-mem was predominantly expressed in the cytoplasmic membrane, including the microvilli structure at the plasma membrane surface, while most of the GFP-m-LC fluorescence signal came from the intracellular membrane (FIGS. 2A, 2B). The expression level of GFP localized on the plasma membrane surface of the cell is detected by using a fluorescently-labeled anti-GFP antibody in combination with a flow cell surface protein staining technique. The experimental results showed that the GFP-m-LC plasma membrane surface displayed significantly reduced content of GFP protein (fig. 2C), confirming that the fusion of the LC tag reduced the amount of protein expressed on the plasma membrane.

Secondly, based on the above experimental results, the effect of the decrease in the expression level of the antigen on the activation of synNotch synthetic receptors was investigated by decreasing the expression level of the antigen on the surface in GFP-mem APC cells without LC tags. The SFFV promoter on the 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 a significant reduction of plasma membrane surface GFP expression (fig. 2C). At this time, the GFP-mem cells with a low expression level showed the same level of surface GFP as the GFP-m-LC cells (FIG. 2C). When the corresponding APC was co-cultured with synNotch ABC cells of different affinities in a panel, the activation level of the fluorescent reporter system was greatly reduced in all synNotch synthetic receptor expressing cells by GFP-mem at low expression levels, but this reduction was not clearly correlated with affinity (FIG. 2D).

Third, the interaction between APC-ABC was visualized. To visualize the anti-GFP synNotch synthetic receptors in real time, the 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), an interface of GFP-anti-GFP molecular interaction was observed (FIG. 2E, 2F). Surprisingly, despite the low expression level of GFP-m-LC on the cell surface, significant aggregation was observed at the APC-ABC interaction interface when co-cultured with anti-GFP-mCherry-synNotch (FIG. 2F).

The three observations above reveal the role of intracellular LC tags in the quantitative display system (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 synNotch synthetic receptor-activated cellular response. In this case, phase separation driven aggregation between ligand-receptor will significantly increase receptor activation, and this aggregation will be influenced by intermolecular forces, i.e., affinity, such that activation of signaling in synNotch synthetic receptor expressing cells is proportional to the ligand-receptor affinity.

Based on the above conclusion, the activation condition of blue fluorescent protein after the mixed culture of ABC cells expressing anti-Her2 scFv synNotch synthetic receptors and APC cells with different surface antigen display modes is detected. Extracellular domain of human HER2 and PDGFR or PDGFR-FUS_LCFusions, abbreviated as Her2-mem or Her2-m-LC (FIG. 2H). The results show that high affinity anti-Her2 synNotch synthetic receptor ABC cells show a higher proportion of fluorescent reporter activation when co-cultured with Her2-mem APC (FIG. 2K). However, when the antigen is Her2-The affinity distinction is more pronounced in the presence of the m-LC form (FIG. 2K). For anti-Her2 synNotch synthetic receptor expressing cells with nanomolar affinity, the expression level of each fusion receptor is consistent (fig. 2J), while the expression level of Her2-m-LC fusion antigen on the cytoplasmic membrane is lower, but the activation ratio of the fluorescence reporter gene in the whole cells can be obviously improved (fig. 2I). Therefore, the reduction of the expression amount of the Ag-m-LC cell surface antigen protein and the increase of the total affinity highlight the capability of quantitative display.

Example 5 affinity differentiation requires proper aggregation of low complexity sequences within cells

The 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 antigenic monomers and increasing avidity (avidity). A number of proteins have been shown to phase separate, and FUS proteins are typical examples of such proteins, and therefore in the initial experiments FUS was selected_LCPeptide fragments were studied. Various FUS mutants were designed to examine 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 mutants, which have an influence on the hardness and flowability, respectively, of the protein agglutination complex. In an opto-Droplet system, FUS_LC Q>The G mutant tag produced protein aggregation more rapidly (fig. 4A), while, when expressed as a fusion in the plasma membrane of the cell, the expression of antigen on the membrane was reduced (fig. 3A,4B), and thus synNotch synthesis receptor-expressing cells could not be activated (fig. 3A, 4B). FUS_LC G>The a mutation tag can activate synNotch synthetic receptor expressing cells, but cannot distinguish the difference between different levels of affinity (fig. 3A, 4C). In addition, two mutants were designed to reduce the formation of aggregation, FUS_LCThe Y27S mutant significantly reduced phase transition capacity and was not able to efficiently support quantitative display (fig. 3A,4D), whereas FUS_LCThe 7A mutant slightly reduced phase transition capacity (fig. 4A), but still supported quantitative display (fig. 3A, 4E).

Long FUS peptide fragments (aa 1-478) and FUS_LCThe 27R mutants could all be induced to phase separate at lower protein levels, but the interaction between sub-nanomolar GFP-anti-GFP failed to activate synNotch synthetic receptors (fig. 3A,3B, 4F). 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 indicate that affinity discrimination requires the LC tag to have the appropriate ability to phase separate.

Example 6 multiple low complexity sequences can support quantitative display

Continued exploration into whether there are other proteins or fragments that form phase separation can also achieve affinity discrimination in the qDis system. A group of low complexity tags is designed, which comprises PrL structural domains of a plurality of FUS protein family members, LC regions of DDX4 genes and poly PR synthetic peptide fragments. Antigens fused to the LC domain of hnRNPA1 or hnRNPA2 lose the ability to activate synNotch synthetic receptors when the various PrL domains of the FUS protein family are overexpressed (fig. 5), and when antigens are fused to the LC tag from TDP43 or EWSR1, activation of synNotch synthetic receptors can be supported, but this activation is not clearly related to affinity (fig. 5). While fusing the LC tag from TAF15 (TAF 15)_LC) Can also be mixed with FUS_LCAs such, synNotch synthetic receptor activation was made proportional to affinity (fig. 3C). Likewise, the LC tag from DDX4 also supported quantitative display (fig. 3C). But the synthetic polyPR peptide did not 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 affinity discrimination in quantitative display systems. Although the current experimental results show that many LC tags cannot support quantitative display, modification of the protein sequences of the LC tags, such as adjusting the protein expression level to be close to the threshold required for phase separation in a 2D system, or changing the rate of multivalent binding of the protein, can make the LC tags reusable in a quantitative display system. Meanwhile, other polymer labels including a trimer Foldon label and a dimer antibody Fc (antibody crystallizable fragment) label are tested, and the experimental results show that the polymer labels are allCannot support affinity-dependent activation of synNotch synthetic receptors (FIG. 3D). In summary, more LC tags can be applied in a quantitative presentation system, whereas multimerization tags cannot be applied in a system. At the same time, the different sequence patterns of these LC tags further indicate that in this system, the receptor molecules are aggregated by phase separation to differentiate the affinities.

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, the interaction between the spike protein RBD domain (S-RBD) of various SARS coronavirus types and ACE2 or its corresponding antibody was taken as an example.

First, the affinity between S-RBD and human ACE2 or several antibodies was examined. S-RBD was cloned into 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), which is consistent with the results of protein interaction in biochemical experiments reported previously. Three neutralizing antibodies against SARS-CoV were able to bind to 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 (CV30) binds to SARS-CoV-2S-RBD only with high affinity (FIG. 6A).

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

Example 8 quantitative display System for Rapid batch isolation of high affinity antibodies

The quantitative display system can directly separate the high-affinity antibody without subsequent large-scale screening and verification experiments. To develop the quantitative display system into a screening system for high affinity antibodies, the fluorescent reporter in synNotch synthetic receptor expressing cells was replaced with the hCD8 reporter (fig. 7A). In this system, hCD8 gene initiates expression and localizes expression 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 was also proportional after binding of receptors of different affinities to the corresponding Ag-m-LC cells (fig. 7B). ABC cells of two high (nM) (red fluorescent protein labeled) and low (μ M) affinities were separately plated at 1: 1000,1: the qDis-MACS system was tested for discriminatory ability by mixing at 100 or 1:10 ratios. In each round, two ABCs were mixed in the above ratio, co-cultured with an equal amount of Ag-m-LC APC cells, and synNotch synthetic receptor activated ABC cells were enriched with magnetic beads carrying 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 antibody (0.1% of total ABC) in the original cell population can be significantly enriched to about 10%.

To test whether the quantitative display system could be applied to screen for high affinity antibodies, a phage display and mammalian quantitative display system were combined to screen for high affinity antibodies against the thermostable gfp (tgp) protein (fig. 7D). Although phage display libraries can reach 10¹¹Orders of magnitude, but usually requires that hundreds of clones be expressed individually in subsequent validation steps and further tested for affinity by ELISA or similar methods after two or three rounds of enrichment. Antibody libraries from prior art phage display were cloned into a quantitative display system and potential high affinity antibodies were 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 qDisTo verify binding affinity and to compare with randomly selected clones directly from phage display libraries (FIG. 7E). The results showed that 18 of the 19 antibodies from the quantitative display system were able to detect binding to TGP by BLI, while only 4 of the 188 antibodies randomly selected were able to detect binding to TGP. Of these 18 antibodies, 16 bound TGP in the nanomolar range, and two additional antibodies bound TGP with picomolar affinity (fig. 7F, 8). Therefore, the quantitative display system can be used as a method for separating high-affinity antibodies from a medium-sized antibody library, and the high-affinity antibodies can be separated in batches by combining a conventional phage display technology 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 synNotch vector and used as a new vector backbone. Primers containing synNotch vector backbone linker sequences were designed to specifically amplify the antibodies in the library of interest. And recombining the recovered product with the vector skeleton after enzyme digestion by using a Gibson assembly method, and constructing a new recombinant library in escherichia coli by using a bacterial electrotransfer method.

2. Construction of stably transfected cell lines for qDIS-MACS screening

Retroviruses corresponding to expression vectors were obtained by transient transfection of liposomes in 293T. Overexpression of Ag-mem-FUS in K562 cell line_LCAnd obtaining the cell line stably expressing the fusion antigen by flow sorting. The hCD8 reporter system was overexpressed in the K562 cell line and a stable transgenic cell line was selected using the Blastidin resistance gene. The synNotch antibody library is over-expressed in a K562-hCD8 cell line, and after a cell population expressing the antibody library is screened for 7 days by using puromycin resistance genes to obtain a stable cell population, the next experiment is carried out.

3. Activating qDIS system

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

MACS enriched hCD8 reporter positive cells

The co-cultured cells were counted and collected, centrifuged at 300g for 5min and resuspended in 10ml MACS buffer, centrifuged at 300g for 5min and the cells were plated at 80. mu.l/10⁷The proportion of cells was resuspended. According to 20. mu.l/10⁷Adding anti-hCD8 microbeads according to the proportion of the cells, uniformly mixing, and incubating at 4 ℃ for 15 min. After the incubation was complete, 5ml of 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 prepared in advance to be placed in a magnetic field. After the cells completely passed through the sorting column, unbound hCD8 negative cells were removed by adding 1ml of MACS buffer and repeating the procedure twice more. And finally, removing the sorting column from the magnetic field, adding 1ml of MACS buffer solution, quickly beating out the cells, and finally collecting the cells which are positive to the hCD8 reporter gene. And taking a small amount of obtained positive cells, staining the positive cells, and detecting the expression level of hCD8 in the enriched cells by using flow cytometry.

5. Multi-round qDIS-MACS enrichment

And (3) centrifuging the cells obtained by enrichment, re-suspending, taking a part of the cells, extracting genomic DNA, and remaining most of the cells for continuous culture. After the expression level of the hCD8 in the round of cells is remarkably reduced, the 10 million-enriched cells and new Ag-m-FUS are continuously taken_LCThe cells were mixed and the experiment was terminated after three rounds of enrichment.

6. Cell genome DNA enrichment and library construction

The extracted genomic DNA was dissolved in TE solution at a ratio of 20. mu.l/million cell, and the amount of cells was 100 times the volume of the antibody library as the initial cell amount for the library construction. The coding sequence of the antibody gene is amplified by using Q5 high fidelity polymerase, and in one round of amplification, 5 mul of DNA is added into each 50 mul of reaction system to be used as a template, and 20 cycles of amplification are carried out. In the two-round amplification, 4 reactions were performed for each sample, and 3. mu.l of one-round product was added to 50. mu.l of the reaction system as a template for 20 cycles of amplification. And (3) performing gel electrophoresis on the 2 nd round PCR product, recovering a target band, adding 50ng of the target band serving as a template for three rounds of amplification, and amplifying for 8 cycles. Finally, the third round of amplification product is purified and recovered, and high-throughput sequencing is carried out.

7. Analysis of antibody enrichment Using bioinformatics

And respectively extracting the sequences of the CDR1, the CDR2 and the 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. And comparing the sequence with the statistical frequency of 1 with other sequences, and classifying the sequences as the same sequence when the CDR sequences only differ by 1 base. And combining the sequencing results of multiple screens into the same file, carrying out homogenization treatment on the results according to the total data measured in each experiment, and filtering sequences which appear only once in the plasmid library and sequences which appear less than 10 times in the previous three enrichments. Finally, selecting 15 obviously enriched antibodies through the multiple difference of the single antibodies after the first round and the third round of screening, and selecting 4 obviously enriched antibodies through the multiple difference of the single antibodies in the initial cells after the third round of screening.

8. Antibody purification

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

9. Antibody affinity validation

Affinity between the purified antibody and TGP antigen was analyzed using an oct Red 96 protein interactor. Biotin labeling was fused to TGP, and the protein was diluted to 2. mu.g/ml with buffer (1xPBS, 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), further diluted to 100nM and finally diluted in a 2-fold gradient to 50nM,25nM,12.5nM,6.25nM,3.7nM, etc. During the experiment, antigen and antibody were allowed to bind for 240s, dissociate for 240s, and the corresponding binding and dissociation constants were calculated on the analytical software using a 1:1 binding mode. After completion of one set of experiments, the sensors were regenerated using a regeneration solution (10mM glycine, PH 2.0) to remove bound antibody, corresponding to the procedure of 5s regeneration, 5s equilibration, and the above procedure was repeated three times. And (4) repeatedly using the regenerated sensor to measure other antibodies, and when the signal of the sensor is obviously weakened, reusing a new sensor to measure.

The invention fuses a series of proteins which can be separated in cells with antigens, and realizes a quantitative display system based on affinity in mammalian cells. Among other things, the low complexity domain of the protein can help to differentiate the affinity of protein interactions outside the plasma membrane of the cell. Receptor aggregation and associated coagulation of downstream effector proteins contribute to signal transduction. The synNotch synthetic receptors and antigen presentation systems of the present invention indicate that aggregation of antigenic molecules inside the plasma membrane of cells may affect extracellular binding. Whether lymphocyte antigen receptors such as BCR and TCR also distinguish antigens with different affinities by a similar strategy is worthy of continued investigation in the future.

From the experimental results of the present invention, the qDis system provides a new method for the existing surface display technology, and high affinity antibodies can be found in batches. The size of the mammalian display library is limited by plasmid transfection efficiency and cell number, and these limitations can be overcome by combining multiple display systems. Antibodies selected from the initial or synthetic display libraries are generally of lower affinity, in which case the qDis system can help find high affinity antibodies in the library that are less abundant. Furthermore, by combining in vitro mutagenesis and qDis systems, we provided the possibility to mimic the antibody affinity process in germinal centers in vitro. In addition to searching for antibodies, affinity-based displays can also be used to reveal ligand-receptor binding, providing a quantitative approach to finding and/or validating potential invading receptors of viruses and potential ligand-receptor pairs in cellular interactions.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons 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 above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Sequence listing

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Tyr Ser Gln Ser Thr Asp Thr Ser Gly Tyr Gly Gln Ser Ser Tyr Ser

130 135 140

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

145 150 155 160

Gln Gly Tyr Gly Ser Thr Gly Gly Tyr Gly Ser Ser Gln Ser Ser Gln

165 170 175

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

180 185 190

Ala Pro Ser Ser Thr Ser Gly Ser Tyr Gly Ser Ser Ser Gln Ser Ser

195 200 205

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

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 (20)

1. A recombinant transmembrane protein, which is fused with a phase-change peptide fragment.

2. The recombinant transmembrane protein according to claim 1, wherein the phase transition peptide fragment is a low complexity sequence.

3. The recombinant transmembrane protein according to claim 2, wherein the low-complexity sequence is derived from a TAF15 protein, a FUS protein or a DDX4 protein; preferably, the low complexity sequence is selected from one or more of the following:

1) an amino acid sequence shown as SEQ ID NO. 1;

2) an amino acid sequence shown as SEQ ID NO. 2;

3) an amino acid sequence shown as SEQ ID NO. 3;

4) the amino acid sequence shown as SEQ ID NO. 4.

4. The recombinant transmembrane protein according to claim 1, wherein the phase transition peptide fragment is fused to an intracellular domain of the transmembrane protein.

5. The recombinant transmembrane protein according to claim 1, wherein the recombinant transmembrane protein is a ligand-PDGFR recombinant transmembrane protein.

6. The recombinant transmembrane protein according to claim 5, wherein the C-terminal of the ligand-PDGFR recombinant transmembrane protein is fused to a low-complexity sequence; preferably, the amino acid sequence of the ligand-PDGFR recombinant transmembrane protein fused with the C-terminal low-complexity sequence is shown as SEQ ID NO. 5.

7. An isolated polynucleotide encoding a recombinant transmembrane protein according to any one of claims 1 to 6.

8. The polynucleotide of claim 7, wherein the polynucleotide has the nucleotide sequence shown in SEQ ID No. 6.

9. A nucleic acid construct comprising the polynucleotide of claim 7 or 8.

10. A cell expressing a recombinant transmembrane protein according to any one of claims 1 to 6.

11. The cell of claim 10, wherein the cell is a ligand-presenting cell; preferably, the ligand quantitative presenting cell is an antigen quantitative presenting cell.

12. A system for quantitatively displaying macromolecules on the surface of cells, the system comprising the cells of claim 11 and receptor-expressing cells, wherein the receptors of the receptor-expressing cells are quantitatively activated specifically when the ligand-quantitatively-presenting cells and the receptor-expressing cells are co-cultured.

13. The system for quantitatively displaying macromolecules on the surface of a cell according to claim 12, wherein the receptor-expressing cells are synNotch synthetic receptor-expressing cells, and preferably, extracellular regions of synNotch synthetic receptors on the synNotch synthetic receptor-expressing cells are fused with regions capable of specifically recognizing and binding to ligands on the surface of the ligand quantitative-presenting cells.

14. The system for quantitatively displaying macromolecules on the surface of a cell as claimed in claim 12, wherein the receptor-expressing cell is a homeostatic cell line which expresses synNotch synthetic receptor and reporter group simultaneously.

15. The quantitative cell surface macromolecule display system of claim 14, wherein the reporter is selected from the group consisting of a fluorescent reporter and a non-fluorescent reporter.

16. A preparation method of a cell surface macromolecule quantitative display system is characterized by comprising the following steps:

1) constructing a receptor expression cell expressing a target receptor on a cell membrane;

2) constructing a ligand quantitative presenting cell expressing a ligand of interest, said ligand quantitative presenting cell expressing a recombinant transmembrane protein according to any one of claims 1 to 6.

17. The method of claim 16, further comprising one or more of the following features:

1) the receptor expressing cells carry a reporter group;

2) the receptor expression cells are synNotch synthetic receptor expression cells; preferably, the extracellular region of synNotch synthetic receptor on the synNotch synthetic receptor-expressing cell is fused to the receptor of interest.

18. Use of the cell surface macromolecule quantitative display system of any one of claims 12-15 for ligand-receptor affinity detection and/or bulk acquisition of high affinity antibodies.

19. A method for detecting ligand-receptor affinity, comprising co-culturing the receptor-expressing cells of step 1) and the ligand-quantitatively presenting cells of step 2) in the preparation method of claim 16, and then detecting the binding between the target ligand and the target receptor.

20. A method for obtaining high affinity antibodies in bulk, comprising the steps of:

1) constructing a synNotch antibody library and a receptor expression cell library of a reporter group;

2) co-culturing the pool of receptor-expressing cells of step 1) with a quantitative ligand-presenting cell expressing the recombinant transmembrane protein of any one of claims 1-6 to activate a cell surface macromolecular quantitative display system;

3) collecting cells in a co-culture system, and primarily enriching receptor expression cells with positive report groups;

4) culturing the receptor expression cells primarily enriched in the step 3) for 5-10 days, and enriching the cells again by using the ligand quantitative presenting cells to obtain synNotch antibodies carried by the receptor expression cells, namely the high-affinity antibodies.

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