Altered activin A

Abstract

The object of the present invention is to provide modified activin A. The present invention provides an activin A which comprises a modified pro-region.

Description

Technical Field

[0001] The present invention relates to activin A comprising an altered amino acid sequence. Background Art

[0002] Activin A is a cytokine belonging to the transforming growth factor β (TGFβ) superfamily. It participates in development and differentiation as an erythroid differentiation factor and a mesoderm-inducing factor. It is also a physiologically and industrially useful protein that regulates various functions in various cells.

[0003] On the other hand, methods for producing proteins include transient expression of the protein in plants (Non-Patent Document 1: Sainsbury et al., Curr. Opin. Plant Biol., 19, 1-7, 2014), and expression of the target protein in the apoplast (Patent Document 1: Japanese Patent Publication No. 2005-501558).

[0004] However, the following problem has been pointed out: when expressing proteins in plants, the expressed target protein is degraded by the endogenous proteases of the plant, and its yield is reduced (non-patent literature 2: Mandal MK. et al., Front Plant Sci., 7, 267, 2015). In particular, the apoplast is rich in broad-spectrum (wide range) specific proteases (non-patent literature 3: Pillary P. et al., Bioengineered, 5: 1, 15-20, 2014). In addition, the protease recognition site in the target protein is different depending on the type of protein, so it is difficult to predict it. In view of this, it is known to use protein localization signals to change the localization or co-express protease inhibitors in plants (non-patent literature 2, non-patent literature 3).

[0005] The three-dimensional structure of activin A has been reported many times (Non-Patent Document 4: Wang X. et al., Nat. Commun. 2016 Jul 4; 7: 12052). However, the structural information of the site recognized by proteases other than proprotein convertase (furin) is still unknown.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application No. 2005-501558

[0009] Non-patent literature

[0010] Non-patent literature 1: Sainsbury et al., Curr. Opin. Plant Biol., 19, 1-7, 2014

[0011] Non-patent document 2: Mandal MK. et al., Front Plant Sci., 7, 267, 2015

[0012] Non-patent literature 3: Pillary P. et al., Bioengineered, 5:1, 15-20, 2014

[0013] Non-patent literature 4: Wang X. et al., Nat. Commun. 2016 Jul 4; 7: 12052

[0014] Non-patent document 5: Koretz K. et al., Histochemistry, 86: 5,471-8, 1987 Summary of the Invention

[0015] Problems to be solved by the invention

[0016] Conventional methods have not been able to address the problem of activin A being degraded by proteases (hereinafter sometimes referred to as "proteases" in this specification) other than proprotein convertase (furin). Therefore, there is a need for novel activin A that is less susceptible to degradation by (is resistant to degradation by) such proteases.

[0017] Solutions for solving problems

[0018] To address the above-mentioned issues, the present inventors conducted intensive research and, as a result, discovered a previously unknown protease recognition site in activin A. Furthermore, by altering the amino acid sequence of the proregion of activin A, they succeeded in producing a modified form of activin A that is less susceptible to protease degradation, thus completing the present invention.

[0019] That is, the present invention is as follows.

[0020] (1) Activin A that is resistant to degradation by proteases other than proprotein convertase (furin).

[0021] (2) Activin A according to (1) above, wherein the protease other than proprotein convertase (furin) is an endogenous protease of a plant.

[0022] (3) The activin A according to (1) or (2) above, comprising an altered proregion.

[0023] (4) The activin A according to (3) above, wherein the amino acid sequence of the aforementioned altered front region comprises an amino acid sequence in which at least one amino acid is deleted, substituted and / or added in the amino acid sequence from positions 180 to 201 of the amino acid sequence shown in any one of sequence numbers 4 to 6.

[0024] (5) An activin A comprising an altered proregion, wherein the amino acid sequence of the altered proregion comprises an amino acid sequence in which at least one amino acid is deleted, substituted, and / or added in the amino acid sequence from positions 180 to 201 of the amino acid sequence shown in any one of SEQ ID NOs. 4 to 6.

[0025] (6) The activin A according to (5) above, wherein the amino acid sequence of the modified proregion comprises any amino acid sequence selected from the group consisting of (a) to (d):

[0026] (a) an amino acid sequence in which amino acids 182 to 199 of the amino acid sequence set forth in any one of SEQ ID NOs: 4 to 6 are deleted,

[0027] (b) an amino acid sequence in which amino acids 182 to 199 of the amino acid sequence represented by any one of SEQ ID NOs: 4 to 6 are substituted with a spacer sequence consisting of 1 to 10 amino acids,

[0028] (c) an amino acid sequence in which at least one of the amino acids 182 to 199 of the amino acid sequence of any one of SEQ ID NOs: 4 to 6 is substituted with another amino acid,

[0029] (d) An amino acid sequence in which at least one amino acid is inserted into the amino acid sequence at positions 180 to 201 of the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6.

[0030] (7) The activin A according to (6) above, wherein in (b), the amino acids constituting the spacer sequence are at least one amino acid selected from the group consisting of glycine, alanine, and serine.

[0031] (8) Activin A according to (6) above, wherein in (c), the other amino acid is selected from alanine, serine, glycine, valine, leucine, and isoleucine.

[0032] (9) Activin A according to any one of (5) to (8) above, which is resistant to degradation by proteases other than proprotein convertase (furin). Examples of proteases other than proprotein convertase (furin) include endogenous proteases of plants.

[0033] (10) The activin A according to (5) above, wherein the amino acid sequence of the modified proregion comprises any amino acid sequence selected from the group consisting of (e) to (g) below:

[0034] (e) the amino acid sequence represented by any one of SEQ ID NOs: 9 to 20,

[0035] (f) an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence of any one of SEQ ID NOs: 9 to 20, and activin A comprising the amino acid sequence is resistant to degradation by proteases other than proprotein convertase (furin);

[0036] (g) An amino acid sequence having 80% or more identity with the amino acid sequence shown in any one of SEQ ID NOs: 9 to 20, wherein activin A comprising the amino acid sequence is resistant to degradation by proteases other than proprotein convertase (furin).

[0037] (11) A polynucleotide encoding activin A according to any one of (1) to (10) above.

[0038] (12) A vector comprising the polynucleotide described in (11) above.

[0039] (13) A transformant expressing activin A according to any one of (1) to (10) above.

[0040] (14) The transformant according to (13) above, wherein the transformant is a plant.

[0041] (15) The transformant according to (14) above, wherein activin A is expressed in the apoplast.

[0042] (16) A method for producing activin A, comprising the step of recovering activin A expressed by the transformant according to any one of (13) to (15) above.

[0043] (17) A method for producing activin A, comprising the following steps:

[0044] (a) a step of recovering activin A expressed by the transformant according to any one of (13) to (15); and

[0045] (b) A step of treating the obtained activin A with proprotein convertase.

[0046] Effects of the Invention

[0047] By using the modified activin A of the present invention, when producing activin A in the presence of a protease other than proprotein convertase (furin) (for example, in plants), the yield of activin A can be improved compared to unmodified wild-type activin A. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a schematic diagram showing the structure of human activin A.

[0049] Figure 2 This is a graph showing that degradation of modified activin A by proteases is suppressed. DETAILED DESCRIPTION

[0050] Hereinafter, the present invention will be described in detail. The following embodiments are examples for illustrating the present invention, and the present invention is not limited to these embodiments. As long as it does not deviate from its main purpose, the present invention can be implemented in various ways. In addition, this specification includes the contents described in the specification and drawings of the Japanese patent application (Special Application No. 2018-214630) filed on November 15, 2018, which is the basis for the priority claim of this application.

[0051] 1. summary

[0052] Activin A is a cytokine belonging to the TGFβ superfamily. It participates in development and differentiation as an erythroid differentiation factor and a mesoderm-inducing factor. It is also a physiologically and industrially useful protein that regulates various functions in various cells.

[0053] On the other hand, as a method for producing a target protein, a method for producing by transiently expressing the protein in a plant can be enumerated. However, the following problem has been pointed out: when expressing a protein in a plant, the expressed target protein is degraded by proteases (e.g., endogenous proteases of the plant) other than the precursor protein convertase (furin), resulting in a reduction in yield. In particular, the fact that the apoplast is rich in endogenous proteases is a huge obstacle to expressing the target protein in the apoplast. Furthermore, the protease recognition site in the target protein is different depending on the type of protein, making it difficult to predict.

[0054] To address this issue, existing technologies utilize methods such as using protein localization signals to change the localization of protein expression or co-expressing protease inhibitors in plants, but the problem of activin A being degraded by proteases remains unsolved.

[0055] Under these circumstances, the present inventors discovered a previously unknown protease recognition site in activin A. By altering the amino acid sequence of the pro-region of activin A (also referred to herein as the "propeptide domain"), they successfully produced a modified activin A that is less susceptible to degradation by proteases other than proprotein convertase (furin) (and is resistant to degradation by such proteases), thereby completing the present invention. The present invention provides a modified activin A that is less susceptible to degradation by such proteases by altering the amino acid sequence of the pro-region of activin A. The present inventors discovered that by altering the amino acid sequence of the pro-region without altering the amino acid sequence of the physiologically active mature region, activin A becomes less susceptible to degradation by such proteases.

[0056] The modified activin A of the present invention is extremely useful in that when activin A is produced in the presence of a protease other than proprotein convertase (furin) (e.g., in a plant body), its yield can be improved compared to the unmodified wild-type activin A.

[0057] 2. Activin A and its variants

[0058] Activin A is a cytokine belonging to the TGFβ (transforming growth factor-β) superfamily. It participates in development and differentiation as an erythroid differentiation factor and a mesoderm-inducing factor. It is also a protein that regulates various functions in various cells.

[0059] The present invention provides: activin A that is not easily degraded by proteases other than proprotein convertase (furin) (is resistant to degradation by the aforementioned protease) by changing the amino acid sequence of the pro-region of activin A, and when activin A is produced in the presence of the aforementioned protease, the yield of activin A can be improved compared to unaltered wild-type activin A.

[0060] In the present invention, "activin A" refers to either or both of preactivin A and mature activin A. When referred to as "activin A" in the present invention, activin A may be a mixture of preactivin A and mature activin A, or may be any one protein. Preactivin A is a precursor of mature activin A and is a protein comprising a pro-region, a proprotein convertase recognition region, and a mature region ( Figure 1 ).

[0061] In the present invention, as the amino acid sequence of preactivin A, for example, the amino acid sequence shown in sequence number 1 (human), sequence number 2 (mouse) or sequence number 3 (rat) can be listed, but it is not limited to these. In addition, in the present invention, as the amino acid sequence of the proregion, there is no limitation, for example, the amino acid sequence shown in sequence number 4 (human), sequence number 5 (mouse), or sequence number 6 (rat), the proprotein convertase recognition region is the amino acid sequence shown in sequence number 7, and the amino acid sequence of the mature region (mature activin A) is shown in sequence number 8. These amino acid sequences and three-dimensional structural information can be easily obtained by those skilled in the art from well-known databases such as GenBank and UniProt Protein Data Bank (PDB). For example, the accession number of human preactivin A in GenBank is NP_002183.1, and the accession number in PDB is 5HLY.

[0062] Mature activin A is a protein containing the mature domain, produced by cleavage of preactivin A by the proprotein convertase (furin). Mature activin A is a homodimer composed of 116 amino acid residue βA chains linked by disulfide bonds.

[0063] In the present invention, the animal species from which activin A is derived is not limited; examples include humans, mice, rats, guinea pigs, rabbits, cats, dogs, pigs, monkeys, and cattle, with humans being preferred. The amino acid sequence of the mature region of activin A has 100% homology among humans, mice, rats, cats, pigs, and cattle.

[0064] The present invention provides activin A that is resistant to degradation by proteases other than proprotein convertase (furin). In the present invention, "proteases other than proprotein convertase (furin)" are not limited as long as they have the activity of degrading (e.g., cutting) activin A. The protease in the present invention can be an endogenous protease in plants or non-human mammals, an endogenous protease derived from these and contained in a transformant with cells, bacteria, yeast, fungi, etc. as hosts, or a protease present outside the aforementioned transformant. It is preferably an endogenous protease of a plant, or a protease present outside a plant, more preferably an endogenous protease of a plant. In particular, the plant body (e.g., apoplast) is rich in proteases with a broad spectrum (wide range) of specificity (Pillary P. et al., Bioengineered, 5: 1, 15-20, 2014), so the endogenous protease of the plant is not limited as long as it has the activity of degrading activin A.

[0065] On the other hand, those skilled in the art can evaluate whether a specific protease has the activity to degrade activin A using known methods such as SDS-PAGE and Western blotting.

[0066] In addition, in the present invention, "resistance to degradation caused by proteases other than proprotein convertase (furin)" specifically means: proactivin A is expressed in a transformant and extracted, purified as needed, and when the resulting protein is analyzed, it contains generally more than 70%, preferably more than 80%, and more preferably more than 84% proactivin A relative to the entire protein obtained. This does not necessarily mean that it must contain 100% proactivin A (degradation must be 100% inhibited). The content or content of this proactivin A can be measured using protein blotting as described in the Examples. That is, whether the modified activin A is resistant to degradation caused by proteases other than proprotein convertase (furin) can be evaluated by measuring the content or content of proactivin A relative to the entire protein using protein blotting.

[0067] In the present invention, "protease other than proprotein convertase (furin)" may also be referred to as "protease (excluding proprotein convertase (furin))".

[0068] The present invention relates to activin A comprising an altered proregion. The altered proregion is a region in which the sequence of the expected loop region present in the proregion is altered. Preferably, at least one sequence expected to be a loop region is deleted from the loop region present in the proregion. Here, the loop region refers to the portion having a loop structure, more specifically, the portion from positions 182 to 199 of the amino acid sequence set forth in any of SEQ ID NOs: 4 to 6.

[0069] In the present invention, the modified amino acid sequence of the proregion comprises an amino acid sequence in which at least one amino acid is deleted, substituted and / or added in the amino acid sequence from positions 180 to 201 of the amino acid sequence shown in any one of sequence numbers 4 to 6 (also referred to as "modified activin A" in this specification).

[0070] In addition, the present invention provides an activin A comprising an altered pro-region, wherein the amino acid sequence of the altered pro-region comprises: an amino acid sequence in which at least one amino acid is deleted, substituted and / or added in the amino acid sequence from positions 180 to 201 of the amino acid sequence shown in any one of sequence numbers 4 to 6, and activin A is resistant to degradation caused by proteases other than proprotein convertase (furin).

[0071] Examples of the modified proregion amino acid sequence include, but are not limited to, the following amino acid sequences (a) to (d).

[0072] (a) an amino acid sequence in which amino acids 182 to 199 of the amino acid sequence set forth in any one of SEQ ID NOs: 4 to 6 are deleted,

[0073] (b) an amino acid sequence in which amino acids 182 to 199 of the amino acid sequence represented by any one of SEQ ID NOs: 4 to 6 are substituted with a spacer sequence consisting of 1 to 10 amino acids,

[0074] (c) an amino acid sequence in which at least one of the amino acids 182 to 199 of the amino acid sequence of any one of SEQ ID NOs: 4 to 6 is substituted with another amino acid,

[0075] (d) An amino acid sequence in which at least one amino acid is inserted into the amino acid sequence at positions 180 to 201 of the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6.

[0076] In addition, as the amino acid sequence of the modified pro-region, the following amino acid sequences (a) to (d) can be listed, and activin A containing the amino acid sequence is resistant to degradation by proteases other than proprotein convertase (furin), but is not limited to these.

[0077] (a) an amino acid sequence in which amino acids 182 to 199 of the amino acid sequence set forth in any one of SEQ ID NOs: 4 to 6 are deleted,

[0078] (b) an amino acid sequence in which amino acids 182 to 199 of the amino acid sequence represented by any one of SEQ ID NOs: 4 to 6 are substituted with a spacer sequence consisting of 1 to 10 amino acids,

[0079] (c) an amino acid sequence in which at least one of the amino acids 182 to 199 of the amino acid sequence of any one of SEQ ID NOs: 4 to 6 is substituted with another amino acid,

[0080] (d) An amino acid sequence in which at least one amino acid is inserted into the amino acid sequence at positions 180 to 201 of the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6.

[0081] The amino acid sequence of (a) above is the amino acid sequence shown in any one of SEQ ID NO: 9 (human), SEQ ID NO: 10 (mouse), and SEQ ID NO: 11 (rat).

[0082] In the amino acid sequence of (b) above, a "spacer sequence" refers to an amino acid sequence consisting of 1 to 10 amino acids, wherein the constituent amino acids are at least one selected from glycine, alanine, and serine. That is, the spacer sequence may be an amino acid sequence consisting of only one amino acid, such as only glycine or only alanine, or an amino acid sequence consisting of two or more amino acids, such as glycine and alanine, or glycine, alanine, and leucine. The number of amino acids in the spacer sequence is 1 to 10, preferably 1 to 5, more preferably 1 to 4, and even more preferably 1 to 3. More specifically, examples of spacer sequences include -G-, -GG-, -GGG-, and -GGGG-. It should be noted that, in the present invention, when the spacer consists of one amino acid residue, the "spacer sequence" may also be referred to as a "spacer residue."

[0083] Those skilled in the art can use molecular visualization software, described below, to investigate the types and number of candidate amino acids constituting the spacer sequence, as well as candidate amino acid sequences. Furthermore, in the present invention, a partial structure registered in the PDB can be inserted in place of the spacer sequence, as long as the relative coordinates of the ends of the partial structure are consistent with the coordinates of the insertion site of activin A, the partial structure can form a peptide bond, and the insertion does not affect the spatial structure.

[0084] As the amino acid sequence of the above-mentioned (b), for example, the amino acid sequence shown in any one of sequence number 12 (human), sequence number 13 (mouse) and sequence number 14 (rat) containing one glycine as a spacer sequence; the amino acid sequence shown in any one of sequence number 15 (human), sequence number 16 (mouse) and sequence number 17 (rat) containing two glycines as a spacer sequence; the amino acid sequence shown in any one of sequence number 18 (human), sequence number 19 (mouse) and sequence number 20 (rat) containing three glycines as a spacer sequence, but are not limited to these.

[0085] In the amino acid sequence of (c) above, the "other amino acids" are not limited as long as they do not cause steric hindrance in activin A, and examples thereof include alanine, serine, glycine, valine, leucine, and isoleucine.

[0086] In the amino acid sequence (d) above, the inserted amino acid is not limited as long as it does not cause steric hindrance in activin A, and examples thereof include alanine, serine, glycine, valine, leucine, and isoleucine.

[0087] In the present invention, as the amino acid sequence of the modified proregion, in addition to the amino acid sequence represented by any one of SEQ ID NOs: 9 to 20, the following amino acid sequence can be used:

[0088] (f) an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added to the amino acid sequence of any one of SEQ ID NOs: 9 to 20, and activin A comprising the amino acid sequence is resistant to degradation by proteases other than proprotein convertase (furin);

[0089] (g) An amino acid sequence having 80% or more identity with the amino acid sequence shown in any one of SEQ ID NOs: 9 to 20, wherein activin A comprising the amino acid sequence is resistant to degradation by proteases other than proprotein convertase (furin).

[0090] Among the amino acid sequences of (f) above, examples of amino acid sequences in which one or more amino acids are deleted, substituted, and / or added to the amino acid sequence represented by any one of SEQ ID NOs: 9 to 20 include the following amino acid sequences.

[0091] (i) an amino acid sequence in which 1 to 10 (e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) amino acids are deleted from the amino acid sequence of any one of SEQ ID NOs: 9 to 20,

[0092] (ii) an amino acid sequence in which 1 to 10 (e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) amino acids in the amino acid sequence of any one of SEQ ID NOs: 9 to 20 are substituted with other amino acids,

[0093] (iii) an amino acid sequence in which 1 to 10 (e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) amino acids are added to the amino acid sequence of any one of SEQ ID NOs: 9 to 20,

[0094] (iv) Amino acid sequence in which a combination of mutations (i) to (iii) above has occurred

[0095] In the above-mentioned amino acid sequence (g), as an amino acid sequence having 80% or more identity with the amino acid sequence shown in any one of sequence numbers 9 to 20, an amino acid sequence having 80% or more identity, 90% or more, 95% or more, 98% or more, or 99% or more identity with the amino acid sequence shown in any one of sequence numbers 9 to 20 can be listed.

[0096] Those skilled in the art can easily investigate the homology of amino acid sequences using known databases such as FASTA and BLAST.

[0097] In the present invention, "resistance to degradation by proteases other than proprotein convertase (furin)" refers to the property that activin A is not easily degraded by proteases other than proprotein convertase (furin). Furthermore, in the present invention, as described above, "resistance to degradation by proteases other than proprotein convertase (furin)" specifically means that when proactivin A is expressed in a transformant and extracted, purified as needed, and the resulting protein is analyzed, it generally contains 70% or more, preferably 80% or more, and more preferably 84% or more of proactivin A relative to the total protein obtained. This does not necessarily mean that 100% proactivin A must be contained (degradation must be 100% inhibited). Whether the modified activin A is resistant to degradation by proteases other than proprotein convertase (furin) can be evaluated by measuring the content or ratio of proactivin A relative to the total protein using Western blotting, as described in the Examples.

[0098] In addition, the present invention provides activin A, which comprises an altered pro-region, wherein the amino acid sequence of the altered pro-region comprises: an amino acid sequence in which at least one amino acid is deleted, substituted and / or added in the amino acid sequence from positions 180 to 201 of the amino acid sequence shown in any one of sequence numbers 4 to 6, and has the activity of inducing differentiation into erythroblasts when cleaved by a proprotein convertase.

[0099] Examples of activin A include, but are not limited to, activin A containing the following amino acid sequences (a) to (g) as the modified proregion amino acid sequence and having an activity of inducing differentiation into erythroblasts when cleaved by proprotein convertase.

[0100] (a) an amino acid sequence in which amino acids 182 to 199 of the amino acid sequence set forth in any one of SEQ ID NOs: 4 to 6 are deleted,

[0101] (b) an amino acid sequence in which amino acids 182 to 199 of the amino acid sequence represented by any one of SEQ ID NOs: 4 to 6 are substituted with a spacer sequence consisting of 1 to 10 amino acids,

[0102] (c) an amino acid sequence in which at least one of the amino acids 182 to 199 of the amino acid sequence of any one of SEQ ID NOs: 4 to 6 is substituted with another amino acid,

[0103] (d) an amino acid sequence in which at least one amino acid is inserted into the amino acid sequence at positions 180 to 201 of the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6,

[0104] (e) the amino acid sequence represented by any one of SEQ ID NOs: 9 to 20,

[0105] (f) an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added to the amino acid sequence of any one of SEQ ID NOs: 9 to 20,

[0106] (g) An amino acid sequence having 80% or more identity with the amino acid sequence represented by any one of SEQ ID NOs: 9 to 20.

[0107] In the present invention, "having the activity of inducing differentiation into erythroblasts when cleaved by proprotein convertase" means that activin A (a mixture of proactivin A and mature activin A, or mature activin A) obtained by cleavage by proprotein convertase has the activity of inducing differentiation of any erythroblast-lineage cultured cells (for example, F5-5 cells, K562 cells, etc.) into erythroblasts.

[0108] The activity of inducing differentiation into erythroblasts can be evaluated by a known method, for example, by adding a test polypeptide (test activin A) to the culture medium of erythroblast-line cultured cells, staining the cultured cells with aminoethylcarbazole (AEC) or the like, and measuring the ED of the test activin A. 50 value, thereby evaluating the differentiation rate.

[0109] In the present invention, examples of the amino acid sequence of activin A containing the modified proregion include, but are not limited to, amino acid sequences of activin A containing the amino acid sequences shown in SEQ ID NOs. 9 to 20, respectively (amino acid sequences shown in SEQ ID NOs. 21 to 32).

[0110] For the analysis and simulation of the three-dimensional structure when activin A is subjected to changes (deletion, substitution, addition or a combination thereof), molecular visualization software that displays the structure of the protein in 3D images based on data such as PDB can be used. As such software, Chimera, PyMOL, etc. can be listed. For example, by utilizing Chimera's in silico analysis, whether steric hindrance is generated when the amino acid sequence of the 182nd to 199th is deleted and when no spacer sequence is inserted in the amino acid sequence of the above-mentioned (a) is simulated. In addition, for the amino acid sequence of the above-mentioned (b), the type and amount of the amino acids constituting the spacer sequence or the amino acid sequence candidate can be retrieved using the above-mentioned software. Similarly, for the amino acid sequences of the above-mentioned (c) and (d), it is also possible to simulate whether steric hindrance is generated when the amino acids of the 182nd to 199th are replaced by other amino acids or when other amino acids are inserted. That is, by using such molecular visualization software, those skilled in the art can appropriately select amino acid residues that will not generate steric hindrance even if deletion, substitution and / or addition occur in activin A.

[0111] Furthermore, the chemical and physical properties of amino acids (polarity / non-polarity, acidity / basicity, hydrophilicity / hydrophobicity, side chain size, etc.) are well known, and those skilled in the art can appropriately select amino acids to be deleted, substituted, and / or added based on these properties. It is speculated that substitution of amino acids with similar structures or properties is less likely to substantially inhibit the biological activity of the polypeptide.

[0112] 1) Polar amino acids: lysine, arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tyrosine, cysteine

[0113] 2) Non-polar amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, proline

[0114] 3) Acidic amino acids: aspartic acid, glutamic acid

[0115] 4) Basic amino acids: lysine, arginine, histidine

[0116] 5) Neutral amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, glutamine, methionine, cysteine, proline

[0117] 6) Aliphatic amino acids: glycine, alanine, valine, leucine, isoleucine

[0118] 7) Aromatic amino acids: phenylalanine, tyrosine, tryptophan

[0119] 8) Amino acids with hydroxyl groups: serine, threonine

[0120] 9) Amino acids with amide groups: asparagine, glutamine

[0121] 10) Sulfur-containing amino acids: methionine, cysteine

[0122] 11) Hydrophobic amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, methionine, cysteine, proline (glycine, tyrosine, tryptophan, and cysteine ​​are sometimes also classified as hydrophilic amino acids)

[0123] 12) Hydrophilic amino acids: lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine

[0124] 3. Polynucleotides, vectors, and transformants

[0125] (1) Polynucleotide

[0126] As long as the polynucleotide of the present invention is a DNA or RNA encoding the modified activin A of the present invention, its base sequence is not limited. Those skilled in the art can optimize the codons of the polynucleotide according to the type of host expressing activin A. Through this optimization, the expression level of the modified activin A in the host can be improved. As polynucleotides encoding the modified activin A of the present invention, for example, polynucleotides comprising the base sequence shown in any one of SEQ ID NOs. 33 to 36 (all of which are human base sequences) or polynucleotides consisting of such base sequences can be listed, but are not limited to these.

[0127] As the polynucleotide of the present invention, in addition to a polynucleotide comprising or consisting of the base sequence set forth in any of SEQ ID NOs: 33 to 36, a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence set forth in any of SEQ ID NOs: 33 to 36 and that encodes activin A that is resistant to degradation by proteases other than proprotein convertase (furin) can also be used. In the present invention, "stringent conditions" can be any of low stringency conditions, moderate stringency conditions, and high stringency conditions. "Low stringency conditions" include, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Moderate stringency conditions" include, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C. “High stringency conditions” include, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, and 50° C. For detailed procedures of hybridization, see “Molecular Cloning, A Laboratory Manual (4th edition)” (Cold Spring Harbor Laboratory Press (2012)).

[0128] In addition, as the polynucleotide of the present invention, the following polynucleotide can be used, which has a homology of more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98% or more with the base sequence shown in any one of sequence numbers 33 to 36, and encodes activin A that is resistant to degradation by proteases other than proprotein convertase (furin).

[0129] Methods for introducing mutations into polynucleotides are well known. For example, those skilled in the art can introduce mutations into the polynucleotides of the present invention using known methods such as the Kunkel method, the gapped duplex method, site-directed mutagenesis, overlap extension PCR, and the QuikChange method. By introducing mutations into the polynucleotides of the present invention, the amino acid sequence of activin A can be altered.

[0130] In the present invention, in order to affinity purify activin A, an affinity tag can be added to activin A. As an affinity tag, for example, a histidine tag (an amino acid sequence consisting of consecutive histidine residues), a GST tag, a FLAG tag, a c-myc tag, etc. can be used, but are not limited to these. The method of adding a histidine tag is well known, and those skilled in the art can easily add a histidine tag to the N-terminus of activin A of the present invention based on a well-known method. An example of an amino acid sequence with a histidine tag added to the N-terminus of activin A is shown in SEQ ID NO: 37.

[0131] In the present invention, a signal sequence may be added to activin A. Examples of the signal sequence include the KDEL signal, etc. Methods for adding a signal sequence are well known, and sequences optimized for expression in plant cells can be used.

[0132] (2) Carrier

[0133] In the present invention, the "vector" is not limited as long as it contains the polynucleotide of the present invention described in (1) above, and for example, a plasmid vector, a viral vector, Agrobacterium, etc. can be used. As a vector when a plant is used as a host, for example, a plant virus vector, Agrobacterium, etc. can be used, and more specifically, for example, a TMV vector, a PVX vector, a CPMV vector, a CMV vector, a PPV vector, an AIMV vector, a ZYMV vector, etc. can be used. The vector used in the present invention can be a vector for transient expression or a vector for stable expression. Those skilled in the art can appropriately select the vector to be used according to the type and purpose of the host into which the vector is to be introduced.

[0134] In addition to the polynucleotide of the present invention, the vector of the present invention may also be connected to cis-elements such as enhancers, splicing signals, polyadenylation addition signals, ribosome binding sequences (SD sequences), selective marker genes, reporter genes, etc. as desired. It should be noted that, as selective marker genes, for example, dihydrofolate reductase genes, ampicillin resistance genes, neomycin resistance genes, etc. can be listed. As reporter genes, genes such as green fluorescent protein (GFP) or its mutants (fluorescent proteins such as EGFP, BFP, and YFP), luciferase, alkaline phosphatase, and LacZ can be listed.

[0135] (3) Transformants

[0136] In the present invention, a transformant can be obtained by introducing the vector described in (2) above into a host. In the present invention, a "transformant" is a non-human transformant. In the present invention, a "host" refers to an organism that becomes the object of the introduction of the vector of the present invention, and is a person who expresses the target modified activin A. As a host, there is no limitation as long as the modified activin A of the present invention is expressed. For example, plants, non-human mammals and cells derived therefrom, bacteria, yeast, fungi, etc. can be used, but plants are preferred. Examples of plants include Nicotiana plants, mosses (Physcolycium patens, etc.), potatoes (S. tuberosum, etc.), grasses (Asian cultivated rice (O. sativa), etc.), crucifers, lettuce, and preferably Nicotiana plants. Examples of Nicotiana plants include N. benthamiana, N. tabacum, N. excelsior, etc., but are not limited to these. Examples of non-human mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, cats, dogs, pigs, monkeys, and cows, as well as cells derived therefrom. Examples of bacteria include, but are not limited to, Escherichia coli.

[0137] The introduction of a vector into the host can be carried out using a known method. As known gene introduction methods, for example, methods using viral vectors, Agrobacterium infection, calcium phosphate method, microinjection, particle gun method, DEAE-dextran method, electroporation method, cationic lipid method, etc. can be listed. When the host is a plant, a method using a plant viral vector or Agrobacterium infection method is preferably used. The vectors that can be used in these methods are such as the vectors described as "vectors when plants are used as hosts" in (2) above.

[0138] In the present invention, when a plant is used as a host, the plant is cultivated before transformation. The plant cultivation method is as follows.

[0139] First, seeds are sown in a seedling tray containing fertilizer. Using a weather meter to adjust the light cycle, the plants are allowed to grow for several days. If using liquid fertilizer, this can be soaked in a hydroponic polyurethane mat and placed in the seedling tray.

[0140] Next, the plant bodies obtained by raising seedlings are transplanted into cultivation (early stage) plates, and the transplanted plates are mounted on an artificial weather apparatus and cultivated for several days using, for example, a deep flow technique (DFT method).

[0141] The plant bodies were then removed from the cultivation (early stage) plates and planted on cultivation (late stage) plates. The transplanted cultivation (late stage) plates were mounted on an artificial weather meter and cultivated for several days using the DFT method to obtain plant bodies.

[0142] In the above method, liquid fertilizer can be used as fertilizer, but it is not limited to this. When using liquid fertilizer, the liquid fertilizer can be infiltrated into a polyurethane mat for hydroponic cultivation and placed in a seedling tray.

[0143] In the present invention, the liquid fertilizer can be used in combination with any commercially available liquid fertilizer, without limitation. The liquid fertilizer can be dissolved in dechlorinated water. In addition, the liquid fertilizer can be used after adjusting the conductivity and pH, which can be adjusted by a person skilled in the art using known methods.

[0144] In the present invention, the environmental conditions can be set as follows, for example: temperature of 10 to 40°C (for example, 28°C), relative humidity of 60 to 80%, CO2 concentration of 300 to 5000 ppm (for example, 400 ppm, 500 ppm), cultivation days of 0 to 35 days (for example, 9 days) in the early stage of cultivation, and 0 to 35 days (for example, 7 days) in the late stage of cultivation, but are not limited to these. Those skilled in the art can appropriately adjust these conditions according to the growth conditions of the plants, etc.

[0145] In the present invention, as a hydroponic method, a deep flow method (DFT method) and a nutrient film technique (NFT method) can be mainly used.

[0146] As described above, when the host is a plant, methods using plant virus vectors and Agrobacterium infection can be used. These methods are well known to those skilled in the art, and the Agrobacterium infection method is briefly described below as an example.

[0147] First, the vector of the present invention described in (2) above is introduced into Agrobacterium to transform the Agrobacterium by electroporation, etc. Agrobacterium that can be used in the present invention is not limited, and examples thereof include GV3101, LBA4404, EHA101, EHA105, and AGL1.

[0148] Next, the transformed Agrobacterium is infected with the leaves of the plant. As a method for infecting plants with Agrobacterium, for example, vacuum infiltration method, infiltration injection method, leaf disc method, leaf surface spreading method, etc. can be listed. As a step when using the vacuum infiltration method, for example, first, the cultivated plant is inverted and immersed in the Agrobacterium liquid in the beaker in a manner such that all the leaves are completely immersed in the liquid. Then, the beaker is placed in a vacuum desiccator and allowed to stand for several minutes (for example, 1 minute) to decompress. Then, the valve is immediately opened to restore the pressure. After the pressure is restored, the plant is restored to an upright position and planted in an artificial weather meter. After infection, the plant is cultivated for 1 to 14 days (for example, 6 days) using an artificial weather meter, for example, by DFT method. The environmental conditions are the same as those described above, and those skilled in the art can appropriately adjust these conditions according to the growth conditions of the plant.

[0149] This allows the production of plant transformants. When infecting plants with Agrobacterium, multiple Agrobacterium containing different vectors can be simultaneously infected. In this case, Agrobacterium containing vectors other than the vectors of the present invention can also be used in combination. Examples of such vectors include vectors containing the signal peptide of barley or rice α-amylase and PhiC31 integrase expression vectors.

[0150] Those skilled in the art can also easily produce transformants derived from other hosts based on known methods such as "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)).

[0151] 4. Method for producing activin A

[0152] In the present invention, activin A can be produced by recovering activin A from the transformant of the present invention described in 3. above. In addition, in the method of the present invention, the recovered activin A (proactivin A) is purified and the purified proactivin A is treated with a proprotein convertase (e.g., furin) to obtain activin A (mature activin A) from which the mature region of the proregion has been removed.

[0153] The type of transformant used in the method for producing activin A is not limited. For example, the method for producing activin A when the transformant is a plant is as follows.

[0154] First, the leaves of the transformed plant body that have been cultivated for 1 to 14 days (for example, 6 days) after infection with Agrobacterium are collected, and activin A (pre-activin A) is extracted using an extraction buffer. The amount of plant leaves collected varies depending on the type of plant body. In addition, it can be frozen and stored at -80°C before extraction. As extraction buffers, phosphate buffer, Tris buffer, acetate buffer, etc. can be listed, but are not limited to these. The pH is adjusted to a normal pH between 2 and 11, including the range in which the above-mentioned buffers are suitable for functioning.

[0155] Next, activin A contained in the extract is purified. Purification can be performed by conventional methods such as aqueous two-phase partitioning, ammonium sulfate fractionation, affinity chromatography, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and reverse phase chromatography, alone or in combination.

[0156] Confirmation that the purified substance is the target protein, activin A, can be performed using conventional methods such as SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis, Western blotting, enzyme immunoassay (ELISA), and mass spectrometry.

[0157] In this way, purified activin A (proactivin A) can be obtained.

[0158] Furthermore, by treating the purified proactivin A with a proprotein convertase (Furin), activin A with the mature region of the proregion removed (mature activin A) can be obtained. Alternatively, a mixture of the proregion and mature activin A can be used in the experiment.

[0159] The activity of the obtained mature activin A (or a mixture of the pro-region and mature activin A) can be evaluated, for example, by culturing F5-5 cell line (Friend erythroblastic leukemia cell line) in a medium supplemented with mature activin A, and then performing AEC (aminoethylcarbazole) staining to measure the ED value of mature activin A for the differentiation rate. 50 Evaluation was performed by measuring the value (indicating the concentration of mature activin A at which the differentiation rate becomes 50%).

[0160] 5. Compositions containing activin A

[0161] The present invention can provide a composition comprising the modified activin A of the present invention. The composition of the present invention can contain, in addition to the modified activin A, known additives such as physiological saline, buffer, and excipients.

[0162] Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

[0163] Example 1

[0164] 1. Design of modified activin A

[0165] In this example, a sequence was designed in which a specific sequence of the proregion was deleted.

[0166] After extracting the preactivin A expressed transiently by plants, it was subjected to Western blotting, and the results confirmed a variety of unexpected degradation products. Therefore, multiple site-specific mutation introduction sequences were designed, and QuikChange (Agilent) was used to introduce mutations. The expressed protein after overexpression was evaluated in the same manner as the above-mentioned initial sequence. As a result, degradation was confirmed to be reduced for multiple sequences. The sequence is a sequence that removes Pro182-Gly199 in the loop region expected to be the propeptide domain. When the dihedral angles of H181 and E200 were adjusted using PDB 5HLY and the two residues were bonded by a peptide bond, it was found that no steric hindrance occurred, and it was designed as Mut3-1. In addition, in order to introduce a suitable linker sequence between the above-mentioned H181 and E200 and implement expression screening, a sequence into which (Gly)n (n=1~4) was introduced was designed. According to SuperLooper (http: / / bioinf-applied.charite.de / superlooper / description.php), since the above-mentioned linker (Gly)n (n=2,3) (3DZM, 2VK3, 5AA5 with GG, 4GNO with GGG) with the relative position coordinates of the linker-imported residues H181, E200 can be extracted from the registered PDB structure information, the sequences were designed as Mut3-2: n=1, Mut3-3: n=2, and Mut3-4: n=3.

[0167] Example 2

[0168] Production of modified activin A

[0169] Based on the information on the modified activin A designed in Example 1, the modified activin A was produced by the following method.

[0170] 1. Preparation of vector

[0171] A proactivin A expression vector with a histidine tag (HHHHHH) added to the N-terminus was constructed as follows. The preactivin A gene with a histidine tag added to the N-terminus was inserted into the Bsa I site of the tobacco mosaic virus (TMV) 3'-pro vector (pICH31070, NOMAD) using Golden Gate cloning (Engler et al., 2008) to create pN-His-proactivin.

[0172] Next, a TMV 3'-pro vector expressing the modified proactivin A gene was prepared as follows: pMut3-1 was prepared by PCR using pN-His-proactivin as a template and the QuikChange Lightning Kit (Agilent) with the following mutation introduction primers.

[0173] 5'-CCAACAGCAGAAGCACGAGAGATCTGAGTTGC-3' (SEQ ID NO: 38)

[0174] 5'-GCAACTCAGATCTCTCGTGCTTCTGCTGTTGG-3 (SEQ ID NO: 39)

[0175] 2. Preparation of transformants

[0176] (1) Cultivation of host plants

[0177] In this example, Nicotiana benthamiana, a plant of the genus Nicotiana, was used as a host plant.

[0178] (1-1) Sowing

[0179] Liquid fertilizer for sowing (OTSUKA Haas S1 (OAT Agrio Co., Ltd.) 0.78 g / L, OTSUKA Haas 2 (OAT Agrio Co., Ltd.) 0.25 g / L, pH 5.0) was infiltrated into a hydroponic polyurethane mat (Esong Chemical W587.5 mm × D282 mm × H28 mm: 12 × 2 pieces (Japanese: マス), pore diameter 9 mm) and placed in a seedling tray (W600 mm × D300 mm × H300 mm), and tobacco (Nictiana benthamiana) seeds were sown.

[0180] (1-2) Seedling cultivation

[0181] The sown plants were grown for 12 days at room temperature of 28°C under a photoperiod of 16 hours day and 8 hours night using an artificial weather meter (NC-410HC) (Japan Ikakigi Mfg. Co., Ltd.).

[0182] (1-3) Cultivation (early stage)

[0183] The polyurethane mats used for seedling cultivation were separated one by one and transplanted into cultivation (early stage) plates (600 mm W x 300 mm D, 30 wells). The transplanted cultivation (early stage) plates were mounted on an artificial weather meter (LH-410SP) (Japan Ikagi Seisakusho Co., Ltd.) and cultivated for 9 days using the deep flow technique (DFT). Environmental conditions and liquid fertilizer conditions were controlled as follows.

[0184] Environmental Conditions

[0185] - Temperature: 28℃

[0186] -Relative humidity: 40-60%

[0187] -CO2 concentration: 400ppm

[0188] -Lighting: Average photosynthetic photon flux density (PPFD): 140 μmol / m 2 Seconds, 24-hour continuous illumination, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation)

[0189] Liquid Fertilizer Conditions

[0190] Liquid fertilizers were prepared by dissolving Fertilizer A (150 g / L OTSUKA Haas S1, 2.5 g / L OTSUKA Haas 5 (OAT Agrio Co., Ltd.)) and Fertilizer B (100 g / L OTSUKA Haas 2) in dechlorinated water and mixing them in equal amounts. pH was adjusted using a pH adjuster (OAT Agrio Co., Ltd.) and a 4% KOH aqueous solution. The electrical conductivity (EC) and pH of the liquid fertilizers were adjusted to 2.3 mS / cm and 6.0 using the "Easy Fertilizer Management Machine 3" (CEM Corporation Co., Ltd.).

[0191] (1-4) Cultivation (late stage)

[0192] Plants were removed from the cultivation (early stage) plates and transplanted into cultivation (late stage) plates (6 holes, W600 mm x D300 mm). The transplanted cultivation (late stage) plates were mounted on an artificial weather meter (LH-410SP) (Japan Ikagi Seisakusho) and cultivated using the DFT method for 7 days (28 days after sowing). Environmental conditions were controlled as follows.

[0193] Environmental Conditions

[0194] - Temperature: 28℃

[0195] -Relative humidity: 60-80%

[0196] -CO2 concentration: 500ppm

[0197] -Lighting: Average photosynthetic photon flux density (PPFD): 140 μmol / m 2 Seconds, 24-hour continuous illumination, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation)

[0198] (2) Preparation of transformants

[0199] (2-1) Infection based on vacuum infiltration

[0200] The vectors of the present invention prepared in the above "1." were introduced into the Agrobacterium GV3101 strain by electroporation, and together with the GV3101 strain containing the 5'-provector (pICH20155, NOMAD) containing the signal peptide of rice α-amylase and the PhiC31 integrase expression vector (pICH14011, NOMAD), Nicotiana benthamiana was infected by the Agrobacterium infiltration method.

[0201] Specifically, the Nicotiana benthamiana leaves obtained in the above "2. (1)" on the 28th day after sowing were inverted and immersed in the Agrobacterium bacterial solution in a beaker so that all leaves were completely immersed in the liquid.

[0202] The beaker was then placed in a vacuum desiccator (FV-3P) (Tokyo Glass Instruments Co., Ltd.) and allowed to stand for 1 minute to reduce the pressure to -0.09 MPa. The valve was then suddenly opened to restore the pressure.

[0203] After the stress recovery was completed, the plants were restored to an upright position and planted in an artificial weather machine (LH-410SP) (Japan Ikagi Seisakusho Co., Ltd.).

[0204] (2-2) Cultivation of infected leaves (expression process)

[0205] After infection, cultivation was performed using an artificial weather meter (LH-410SP) (Japan Ikakigi Co., Ltd.) and was carried out by DFT for 6 days. Environmental conditions were controlled as follows.

[0206] Environmental Conditions

[0207] - Temperature: 28℃

[0208] -Relative humidity: 60-80%

[0209] -CO2 concentration: 500ppm

[0210] -Lighting: Average photosynthetic photon flux density (PPFD): 140 μmol / m 2 Seconds, 24-hour continuous illumination, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation)

[0211] 3. Production of modified proactivin A

[0212] (1) Extraction

[0213] Tobacco leaves were obtained from transformed Nicotiana benthamiana 6 days after infection and stored frozen at -80°C until extraction.

[0214] As the extraction buffer, 0.1 M sodium phosphate, 0.5 M arginine, 5 mM sodium sulfite, pH 8.0 (or phosphate buffer) was used.

[0215] (2) Purification

[0216] (2-1) Purification by ammonium sulfate fractionation

[0217] Ammonium sulfate was added to the supernatant recovered by the above method to obtain a 35% saturated ammonium sulfate solution. After stirring at room temperature for 1 hour, the mixture was centrifuged at 15,000 × g for 15 minutes at room temperature to recover a 35% ammonium sulfate fraction supernatant. Next, ammonium sulfate was added to obtain a 60% saturated ammonium sulfate solution, and the mixture was stirred at room temperature for 1 hour. This solution was centrifuged at 15,000 × g for 15 minutes to recover a 60-90% ammonium sulfate fraction precipitate.

[0218] (2-2) Purification based on affinity chromatography

[0219] To 50 g of activin A-expressing tobacco leaves, 60% to 90% ammonium sulfate fractionated precipitation was added 10 mL of histidine (His) tag affinity purification equilibration solution (20 mM HEPES, 150 mM NaCl, 10% (w / v) glycerol, pH 8.0) to dissolve the precipitate. This solution was then passed through a HisTrap HP 1 mL column (GE Healthcare) equilibrated with the His-tag affinity purification equilibration solution at a retention time of 3 minutes. The column was then washed with His-tag affinity purification wash solution (20 mM HEPES, 150 mM NaCl, 20 mM imidazole, 10% (w / v) glycerol, pH 8.0). Finally, the column was passed through His-tag affinity purification elution solution (20 mM HEPES, 150 mM NaCl, 200 mM imidazole, 10% (w / v) glycerol, pH 8.0), and the eluted peak was recovered to obtain the modified pro-activin A.

[0220] 4. Production of mature activin A

[0221] Relative to the modified pro-activin A (Mut3-1 to Mut3-4 (containing the amino acid sequences of sequence numbers 9, 12, 15 and 18, respectively)) obtained by the above method, mature activin A was obtained by adding precursor protein convertase (Furin) under the conditions of 25mM Tris-HCl, pH 8.0, 10% glycerol, and 1mM CaCl2.

[0222] Through this example, activin A with a modified amino acid sequence in the proregion (modified proactivin A and mature activin A) can be produced.

[0223] Example 3

[0224] 1. Functional analysis of altered proactivin A

[0225] The purified modified proactivin A obtained in "3. Production of modified proactivin A" of Example 2 was analyzed by protein blotting to confirm the degradation behavior caused by the endogenous protease of the plant. Specifically, Penta-His Antibody HRP Conjugate (QIAGEN) was used as an antibody, and protein blotting of each sample containing modified proactivin A was performed. LumiGLO Reagent and Peroxide (Cell Signaling Technology) was used as a luminescent reagent, and detection was performed using ImageQuant LAS 500 (GE Healthcare). The obtained protein blot image is shown in Figure 2As a control, wild-type activin A (N-His-activin A in the figure) (SEQ ID NO: 37) was used. In addition, the results of optical density analysis using the analysis software ImageJ are shown in Table 1. In Table 1, other polypeptides are peptides produced by degradation of the expressed protein by endogenous proteases of the plant.

[0226] [Table 1]

[0227]

[0228] That is, the degradation of the modified activin A of the present invention by plant endogenous proteases is significantly suppressed compared to the unmodified activin A (N-His-activin A as wild-type activin A) ( Figure 2 ).

[0229] This example demonstrates that the modified activin A of the present invention is less susceptible to protease degradation (resistance to protease degradation). Furthermore, this example demonstrates that when activin A is produced in the presence of proteases present in the expression host, in purification processes, in reagents / culture media, etc., the modified activin A of the present invention can improve expression levels and / or yields compared to unmodified activin A, demonstrating its significant utility in this regard.

[0230] 2. Activity evaluation of mature activin A

[0231] The activity of the mature activin A obtained in "4. Production of mature activin A" of Example 2 was evaluated. F5-5 cells cultured in an incubator (37°C, 5% CO2) were seeded into a 96-well plate and stained with AEC (aminoethylcarbazole, Tokyo Chemical Industry) based on the method described in non-patent document 5 (Koretz K. et al., Histochemistry, 86: 5, 471-8, 1987). The cells were then stained with AEC (aminoethylcarbazole, Tokyo Chemical Industry ... 50 The results of the values ​​are shown in Table 2.

[0232] [Table 2]

[0233]

[0234] As shown in Table 2, the ED values ​​of mature activin A derived from modified activin A ("Mut3-1" to "Mut3-4" in Table 2) are 50 The values ​​were all lower than 5 ng / mL, which were equivalent to those of mature activin A derived from wild-type activin A ("N-His-activin A" in Table 2).

[0235] This result indicates that the mature activin A obtained by treating the modified activin A with furin has the same ability to induce differentiation of F5-5 cells into erythroblasts as the wild-type activin A; that is, it has the same activity as the wild-type activin A as a mature activin A.

[0236] Industrial applicability

[0237] By using the modified activin A of the present invention, the yield of activin A can be improved when it is produced in the presence of a protease.

[0238] SEQ ID NOs. 9 to 32 and 37: Synthetic peptides

[0239] Sequence numbers 33 to 36, 38, and 39: Synthetic DNA Sequence Listing <110> Mitsubishi Chemical Corporation UniBio Co., Ltd. (UniBio Corporation) <120> Altered activin A <130> G2018WO <150> JP2018-214630 <151> 2018-11-15 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 426 <212> PRT <213> Homo sapiens <400> 1 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Pro Gln Gly Ser Leu Asp Thr Gly Glu Glu Ala 180 185 190 Glu Glu Val Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 195 200 205 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser 210 215 220 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 225 230 235 240 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 245 250 255 Leu Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys 260 265 270 Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser 275 280 285 His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro 290 295 300 His Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile 305 310 315 320 Cys Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn 325 330 335 Asp Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly 340 345 350 Glu Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe 355 360 365 His Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe 370 375 380 Ala Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser 385 390 395 400 Met Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln 405 410 415 Asn Met Ile Val Glu Glu Cys Gly Cys Ser 420 425 <210> 2 <211> 424 <212> PRT <213> Mouse (Mus musculus) <400> 2 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile '1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Pro Gln Gly Ser Leu Asp Thr Gly Asp Glu Ala 180 185 190 Glu Glu Met Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 195 200 205 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 210 215 220 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 225 230 235 240 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 245 250 255 Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 260 265 270 Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg 275 280 285 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg 290 295 300 Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys 305 310 315 320 Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp 325 330 335 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 340 345 350 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser 355 360 365 Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn 370 375 380 Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu 385 390 395 400 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met 405 410 415 Ile Val Glu Glu Cys Gly Cys Ser 420 <210> 3 <211> 424 <212> PRT <213> Rat (Rattus norvegicus) <400> 3 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Pro Gln Gly Ser Leu Asp Met Gly Asp Glu Ala 180 185 190 Glu Glu Met Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 195 200 205 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 210 215 220 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 225 230 235 240 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 245 250 255 Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 260 265 270 Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg 275 280 285 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg 290 295 300 Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys 305 310 315 320 Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp 325 330 335 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 340 345 350 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser 355 360 365 Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn 370 375 380 Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu 385 390 395 400 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met 405 410 415 Ile Val Glu Glu Cys Gly Cys Ser 420 <210> 4 <211> 305 <212> PRT <213> Homo sapiens <400> 4 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 7]Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Pro Gln Gly Ser Leu Asp Thr Gly Glu Glu Ala 180 185 190 Glu Glu Val Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 195 200 205 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser 210 215 220 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 225 230 235 240 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 245 250 255 Leu Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys 260 265 270 Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser 275 280 285 His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro 290 295 300 His 305 <210> 5 <211> 303 <212> PRT <213> mice (Mus musculus) <400> 5 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Pro Gln Gly Ser Leu Asp Thr Gly Asp Glu Ala 180 185 190 Glu Glu Met Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 195 200 205 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 210 215 220 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 225 230 235 240 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 245 250 255 Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 260 265 270 Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg 275 280 285 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 290 295 300 <210> 6 <211> 303 <212> PRT <213> Rattus norvegicus <400> 6 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Pro Gln Gly Ser Leu Asp Met Gly Asp Glu Ala 180 185 190 Glu Glu Met Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 195 200 205 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 210 215 220 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 225 230 235 240 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 245 250 255 Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 260 265 270 Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg 275 280 285 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 290 295 300 <210> 7 <211> 5 <212> PRT <213> Artificial <220> <223> synthetic peptides <400> 7 Arg Arg Arg Arg Arg 1 5 <210> 8 <211> 116 <212> PRT <213> Artificial <220> <223> synthetic peptides <400> 8 Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys Lys Gln Phe 1 5 10 15 Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro 20 25 30 Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile 35 40 45 Ala Gly Thr Ser Gly Ser Ser Ser Leu Ser Phe His Ser Thr Val Ile Asn 50 55 60 His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys 65 70 75 80 Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp 85 90 95 Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val Glu Glu 100 105 110 Cys Gly Cys Ser 115 <210> 9 <211> 287 <212> PRT <213> artificial sequence <220> <223> peptide synthesis <400> 9 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val 180 185 190 Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser Ser Ser 195 200 205 Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg Ile 210 215 220 Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu Gly 225 230 235 240 Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys Gly Gly 245 250 255 Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser His Arg 260 265 270 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 <210> 10 <211> 285 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 10 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val 180 185 190 Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser Ser Ser 195 200 205 Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg Ile 210 215 220 Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu Gly 225 230 235 240 Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp Gly 245 250 255 Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro Phe 260 265 270 Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 <210> 11 <211> 285 <212> PRT <213> artificial sequence <220> <223> peptide synthesis <400> 11 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val 180 185 190 Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser Ser Ser 195 200 205 Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg Ile 210 215 220 Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu Gly 225 230 235 240 Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp Gly 245 250 255 Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro Phe 260 265 270 Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 <210> 12 <211> 288 <212> PRT <213> artificial sequence <220> <223> peptide synthesis <400> 12 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys 180 185 190 Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser Ser 195 200 205 Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg 210 215 220 Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu 225 230 235 240 Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys Gly 245 250 255 Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser His 260 265 270 Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 <210> 13 <211> 286 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 13 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80<​​​​Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys 180 185 190 Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser Ser 195 200 205 Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg 210 215 220 Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu 225 230 235 240 Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp 245 250 255 Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro 260 265 270 Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 <210> 14 <211> 286 <212> PRT <213> artificial sequence <220> <223> peptide synthesis <400> 14 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys 180 185 190 Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser Ser 195 200 205 Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg 210 215 220 Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu 225 230 235 240 Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp 245 250 255 Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro 260 265 270 Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 <210> 15 <211> 289 <212> PRT <213> artificial sequence <220> <223> peptide synthesis <400> 15 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 180 185 190 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser 195 200 205 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 210 215 220 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 225 230 235 240 Leu Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys 245 250 255 Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser 260 265 270 His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro 275 280 285 His <210> 16 <211> 287 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 16 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 180 185 190 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 195 200 205 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 210 215 220 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 225 230 235 240 Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 245 250 255 Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg 260 265 270 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 <210> 17 <211> 287 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 17 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ala Ala<000l213>20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 180 185 190 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 195 200 205 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 210 215 220 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 225 230 235 240 Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 245 250 255 Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg 260 265 270 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 <210> 18 <211> 290 <212> PRT <213> artificial sequence <220> <223> peptide synthesis <400> 18 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser 180 185 190 Glu Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val 195 200 205 Ser Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp 210 215 220 Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val 225 230 235 240 Leu Leu Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys 245 250 255 Lys Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln 260 265 270 Ser His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His 275 280 285 Pro His 290 <210> 19 <211> 288 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 19 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser 180 185 190 Glu Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val 195 200 205 Ser Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp 210 215 220 Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val 225 230 235 240 Leu Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys 245 250 255 Lys Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His 260 265 270 Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 <210> 20 <211> 288 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 20 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser 180 185 190 Glu Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val 195 200 205 Ser Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp 210 215 220 Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val 225 230 235 240 Leu Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys 245 250 255 Lys Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His 260 265 270 Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 <210> 21 <211> 408 <212> PRT <213> artificial sequence <220> <223> peptide synthesis <400> 21 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val 180 185 190 Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser Ser Ser 195 200 205 Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg Ile 210 215 220 Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu Gly 225 230 235 240 Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys Gly Gly 245 250 255 Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser His Arg 260 265 270 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg 275 280 285 Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys 290 295 300 Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp 305 310 315 320 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 325 330 335 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser 340 345 350 Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn 355 360 365 Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu 370 375 380 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met 385 390 395 400 Ile Val Glu Glu Cys Gly Cys Ser 405 <210> 22 <211> 406 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 22 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val 180 185 190 Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser Ser Ser 195 200 205 Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg Ile 210 215 220 Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu Gly 225 230 235 240 Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp Gly 245 250 255 Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro Phe 260 265 270 Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg Arg Arg 275 280 285 Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys Lys 290 295 300 Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile 305 310 315 320 Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser 325 330 335 His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val 340 345 350 Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu Lys 355 360 365 Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr 370 375 380 Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val 385 390 395 400 Glu Glu Cys Gly Cys Ser 405 <210> 23 <211> 406 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 23 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val 180 185 190 Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser Ser Ser 195 200 205 Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg Ile 210 215 220 Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu Gly 225 230 235 240 Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp Gly 245 250 255 Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro Phe 260 265 270 Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg Arg Arg 275 280 285 Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys Lys 290 295 300 Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile 305 310 315 320 Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser 325 330 335 His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val 340 345 350 Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu Lys 355 360 365 Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr 370 375 380 Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val 385 390 395 400 Glu Glu Cys Gly Cys Ser 405 <210> 24 <(211)> 409 <(212)> PRT <(213)> Artificial <220> <(223)> Synthetic peptide <400> 24 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn Note: The tags like <(211)> and <(212)> etc. in the original seem to have some incorrect formatting. I've translated them as best as possible while keeping the original structure. If these are meant to be something specific, the translation might need adjustment.50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys 180 185 190 Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser Ser 195 200 205 Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg 210 215 220 Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu 225 230 235 240 Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys Gly 245 250 255 Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser His 260 265 270 Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys 290 295 300 Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp 305 310 315 320 Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu 325 330 335 Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His 340 345 350 Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala 355 360 365 Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met 370 375 380 Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn 385 390 395 400 Met Ile Val Glu Glu Cys Gly Cys Ser 405 <210> 25 <211> 407 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 25 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys 180 185 190 Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser Ser 195 200 205 Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg 210 215 220 Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu 225 230 235 240 Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp 245 250 255 Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro 260 265 270 Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg Arg 275 280 285 Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys 290 295 300 Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile 305 310 315 320 Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro 325 330 335 Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr 340 345 350 Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu 355 360 365 Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr 370 375 380 Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile 385 390 395 400 Val Glu Glu Cys Gly Cys Ser 405 <210> 26 <211> 407 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 26 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys 180 185 190 Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser Ser 195 200 205 Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val Arg 210 215 220 Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu Leu 225 230 235 240 Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys Asp 245 250 255 Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg Pro 260 265 270 Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg Arg 275 280 285 Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys 290 295 300 Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile 305 310 315 320 Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro 325 330 335 Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr 340 345 350 Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu 355 360 365 Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr 370 375 380 Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile 385 390 395 400 Val Glu Glu Cys Gly Cys Ser 405 <210> 27 <211> 410 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 27 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 180 185 190 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser 195 200 205 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 210 215 220 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 225 230 235 240 Leu Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys 245 250 255 Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser 260 265 270 His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro 275 280 285 His Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile 290 295 300 Cys Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn 305 310 315 320 Asp Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly 325 330 335 Glu Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe 340 345 350 His Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe 355 360 365 Ala Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser 370 375 380 Met Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln 385 390 395 400 Asn Met Ile Val Glu Glu Cys Gly Cys Ser 405 410 <210> 28 <211> 408 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 28 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 180 185 190 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 195 200 205 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 210 215 220 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 225 230 235 240 Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 245 250 255 Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg 260 265 270 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg 275 280 285 Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys 290 295 300 Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp 305 310 315 320 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 325 330 335 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser 340 345 350 Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn 355 360 365 Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu 370 375 380 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met 385 390 395 400 Ile Val Glu Glu Cys Gly Cys Ser 405 <210> 29 <211> 408 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 29 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu 180 185 190 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val Ser 195 200 205 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 210 215 220 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val Leu 225 230 235 240 Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys Lys 245 250 255 Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His Arg 260 265 270 Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His Arg 275 280 285 Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys 290 295 300 Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp 305 310 315 320 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 325 330 335 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser 340 345 350 Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn 355 360 365 Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu 370 375 380 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met 385 390 395 400 Ile Val Glu Glu Cys Gly Cys Ser 405 <210> 30 <211> 411 <212> PRT <213> artificial sequence <220> <223> peptide synthesis <400> 30 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser 180 185 190 Glu Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val 195 200 205 Ser Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp 210 215 220 Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val 225 230 235 240 Leu Leu Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys Lys 245 250 255 Lys Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln 260 265 270 Ser His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His 275 280 285 Pro His Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn 290 295 300 Ile Cys Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp 305 310 315 320 Asn Asp Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu 325 330 335 Gly Glu Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser 340 345 350 Phe His Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro 355 360 365 Phe Ala Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met 370 375 380 Ser Met Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile 385 390 395 400 Gln Asn Met Ile Val Glu Glu Cys Gly Cys Ser 405 410 <210> 31 <211> 409 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 31 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ser Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser 180 185 190 Glu Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val 195 200 205 Ser Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp 210 215 220 Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val 225 230 235 240 Leu Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys 245 250 255 Lys Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His 260 265 270 Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys 290 295 300 Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp 305 310 315 320 Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu 325 330 335 Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His 340 345 350 Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala 355 360 365 Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met 370 375 380 Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn 385 390 395 400 Met Ile Val Glu Glu Cys Gly Cys Ser 405 <210> 32 <211> 409 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 32 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Gly Ala Ala 20 25 30 Pro Asp Cys Pro Ser Cys Ala Leu Ala Thr Leu Pro Lys Asp Gly Pro 35 40 45 Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 Gln Gln Gln Lys His Gly Gly Gly Glu Arg Ser Glu Leu Leu Leu Ser 180 185 190 Glu Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Ile Phe Pro Val 195 200 205 Ser Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp 210 215 220 Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val 225 230 235 240 Leu Leu Gly Lys Lys Lys Lys Lys Glu Val Asp Gly Asp Gly Lys Lys 245 250 255 Lys Asp Gly Ser Asp Gly Gly Leu Glu Glu Glu Lys Glu Gln Ser His 260 265 270 Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro His 275 280 285 Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys 290 295 300 Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp 305 310 315 320 Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu 325 330 335 Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His 340 345 350 Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala 355 360 365 Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met 370 375 380 Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn 385 390 395 400 Met Ile Val Glu Glu Cys Gly Cys Ser 405 <210> 33 <211> 1167 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 33 agtcctactc ctggtagcga aggacattct gctgctccag attgtccttc ttgtgctctt 60 gctgctctgc ctaaggatgt gcctaattct cagcctgaga tggttgaggc tgtgaagaaa 120 cacatcctga acatgctgca cctgaagaaa aggcctgatg tgactcagcc tgttcctaag 180 gctgctttgc tgaacgctat taggaagctg cacgttggta aggtgggaga gaatggttac 240 gttgagatcg aggatgatat cggtagaagg gctgagatga acgagctgat ggaacagacc 300 tctgagatca tcaccttcgc tgagtctgga accgctagaa agactctgca cttcgagatc 360 agcaaagagg gtagcgatct gtctgttgtt gagagggctg aggtgtggct tttcttgaag 420 gtgccaaagg ctaataggac caggaccaag gtgaccatta ggcttttcca acagcagaag 480 cacgagagat ctgagttgct gctgtctgag aaggttgtgg atgctagaaa gtccacctgg 540 cacgttttcc ctgtgtcctc ttcaattcag aggctgctgg atcagggtaa gagcagcctt 600 gatgttagga ttgcttgcga gcagtgccaa gagtctggtg cttctcttgt gcttctgggt 660 aagaagaaaa agaaagagga agagggagaa ggtaagaaaa agggtggtgg tgaaggtggt 720 gctggtgctg atgaagagaa agagcagtct cacaggcctt tcttgatgct tcaggctagg 780 cagtctgagg atcaccctca cagaaggaga agaaggggtc ttgagtgtga tggaaaggtg 840 aacatctgct gcaagaagca gttcttcgtt agcttcaagg atatcggttg gaacgattgg 900 atcattgctc caagcggtta ccacgctaat tactgtgagg gagagtgccc ttctcacatt 960 gctggtacta gcggaagctc tctgtctttc catagcaccg tgatcaacca ctacaggatg 1020 aggggacata gccctttcgc taacctgaag tcttgctgcg tgccaactaa gctgaggcct 1080 atgtctatgc tgtactacga tgatggtcag aacatcatca aaaaggatat ccagaacatg 1140 atcgtggaag agtgcggttg ctcttag 1167 <210> 34 <211> 1170 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 34 agtcctactc ctggtagcga aggacattct gctgctccag attgtccttc ttgtgctctt 60 gctgctctgc ctaaggatgt gcctaattct cagcctgaga tggttgaggc tgtgaagaaa 120 cacatcctga acatgctgca cctgaagaaa aggcctgatg tgactcagcc tgttcctaag 180 gctgctttgc tgaacgctat taggaagctg cacgttggta aggtgggaga gaatggttac 240 gttgagatcg aggatgatat cggtagaagg gctgagatga acgagctgat ggaacagacc 300 tctgagatca tcaccttcgc tgagtctgga accgctagaa agactctgca cttcgagatc 360 agcaaagagg gtagcgatct gtctgttgtt gagagggctg aggtgtggct tttcttgaag 420 gtgccaaagg ctaataggac caggaccaag gtgaccatta ggcttttcca acagcagaag 480 cacggtgaga gatctgagtt gctgctgtct gagaaggttg tggatgctag aaagtccacc 540 tggcacgttt tccctgtgtc ctcttcaatt cagaggctgc tggatcaggg taagagcagc 600 cttgatgtta ggattgcttg cgagcagtgc caagagtctg gtgcttctct tgtgcttctg 660 ggtaagaaga aaaagaaaga ggaagaggga gaaggtaaga aaaagggtgg tggtgaaggt 720 ggtgctggtg ctgatgaaga gaaagagcag tctcacaggc cttctttgat gcttcaggct 780 aggcagtctg aggatcaccc tcacagaagg agaagaaggg gtcttgagtg tgatggaaag 840 gtgaacatct gctgcaagaa gcagttcttc gttagcttca aggatatcgg ttggaacgat 900 tggatcattg ctccaagcgg ttaccacgct aattactgtg agggagagtg cccttctcac 960 attgctggta ctagcggaag ctctctgtct ttccatagca ccgtgatcaa ccactacagg 1020 atgaggggac atagcccttt cgctaacctg aagtcttgct gcgtgccaac taagctgagg 1080 cctatgcta tgctgtacta cgatgatggt cagaacatca tcaaaaagga tatccagaac 1140 atgatcgtgg aagagtgcgg ttgctcttag 1170 <210> 35 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 35 agtcctactc ctggtagcga aggacattct gctgctccag attgtccttc ttgtgctctt 60 gctgctctgc ctaaggatgt gcctaattct cagcctgaga tggttgaggc tgtgaagaaa 120 cacatcctga acatgctgca cctgaagaaa aggcctgatg tgactcagcc tgttcctaag 180 gctgctttgc tgaacgctat taggaagctg cacgttggta aggtgggaga gaatggttac 240 gttgagatcg aggatgatat cggtagaagg gctgagatga acgagctgat ggaacagacc 300 tctgagatca tcaccttcgc tgagtctgga accgctagaa agactctgca cttcgagatc 360 agcaaagagg gtagcgatct gtctgttgtt gagagggctg aggtgtggct tttcttgaag 420 gtgccaaagg ctaataggac caggaccaag gtgaccatta ggcttttcca acagcagaag 480 cacggtggtg agagatctga gttgctgctg tctgagaagg ttgtggatgc tagaaagtcc 540 acctggcacg ttttccctgt gtcctcttca attcagaggc tgctggatca gggtaagagc 600 agccttgatg ttaggattgc ttgcgagcag tgccaagagt ctggtgcttc tcttgtgctt 660 ctgggtaaga agaaaaagaa agaggaagag ggagaaggta agaaaaaggg tggtggtgaa 720 ggtggtgctg gtgctgatga agagaaagag cagtctcaca ggcctttctt gatgcttcag 780 gctaggcagt ctgaggatca ccctcacaga aggagaagaa ggggtcttga gtgtgatgga 840 aaggtgaaca tctgctgcaa gaagcagttc ttcgttagct tcaaggatat cggttggaac 900 gattggatca ttgctccaag cggttaccac gctaattact gtgagggaga gtgcccttct 960 cacattgctg gtactagcgg aagctctctg tctttccata gcaccgtgat caaccactac 1020 aggatgaggg gacatagccc tttcgctaac ctgaagtctt gctgcgtgcc aactaagctg 1080 aggcctatgt ctatgctgta ctacgatgat ggtcagaaca tcatcaaaaa ggatatccag 1140 aacatgatcg tggaagagtg cggttgctct tag 1173 <210> 36 <211> 1176​​​​​​​<223> Synthetic DNA <400> 36 agtcctactc ctggtagcga aggacattct gctgctccag attgtccttc ttgtgctctt 60 gctgctctgc ctaaggatgt gcctaattct cagcctgaga tggttgaggc tgtgaagaaa 120 cacatcctga acatgctgca cctgaagaaa aggcctgatg tgactcagcc tgttcctaag 180 gctgctttgc tgaacgctat taggaagctg cacgttggta aggtgggaga gaatggttac 240 gttgagatcg aggatgatat cggtagaagg gctgagatga acgagctgat ggaacagacc 300 tctgagatca tcaccttcgc tgagtctgga accgctagaa agactctgca cttcgagatc 360 agcaaagagg gtagcgatct gtctgttgtt gagagggctg aggtgtggct tttcttgaag 420 gtgccaaagg ctaataggac caggaccaag gtgaccatta ggcttttcca acagcagaag 480 cacggtggtg gtgagagatc tgagttgctg ctgtctgaga aggttgtgga tgctagaaag 540 tccacctggc acgttttccc tgtgtcctct tcaattcaga ggctgctgga tcagggtaag 600 agcagccttg atgttaggat tgcttgcgag cagtgccaag agtctggtgc ttctcttgtg 660 cttctgggta agaagaaaaa gaaagaggaa gagggagaag gtaagaaaaa gggtggtggt 720 gaaggtggtg ctggtgctga tgaagagaaa gagcagtctc acaggccttt cttgatgctt 780 caggctaggc agtctgagga tcaccctcac agaaggagaa gaaggggtct tgagtgtgat 840 ggaaaggtga acatctgctg caagaagcag ttcttcgtta gcttcaagga tatcggttgg 900 aacgattgga tcattgctcc aagcggttac cacgctaatt actgtgaggg agagtgccct 960 tctcacattg ctggtactag cggaagctct ctgtctttcc atagcaccgt gatcaaccac 1020 tacaggatga ggggacatag ccctttcgct aacctgaagt cttgctgcgt gccaactaag 1080 ctgaggccta tgtctatgct gtactacgat gatggtcaga acatcatcaa aaaggatatc 1140 cagaacatga tcgtggaaga gtgcggttgc tcttag 1176 <210> 37 <211> 432 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 37 His His His His His His Met Pro Leu Leu Trp Leu Arg Gly Phe Leu 1 5 10 15 Leu Ala Ser Cys Trp Ile Ile Val Arg Ser Ser Pro Thr Pro Gly Ser 20 25 30 Glu Gly His Ser Ala Ala Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala 35 40 45 Leu Pro Lys Asp Val Pro Asn Ser Gln Pro Glu Met Val Glu Ala Val 50 55 60 Lys Lys His Ile Leu Asn Met Leu His Leu Lys Lys Arg Pro Asp Val 65 70 75 80 Thr Gln Pro Val Pro Lys Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu 85 90 95 His Val Gly Lys Val Gly Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp 100 105 110 Ile Gly Arg Arg Ala Glu Met Asn Glu Leu Met Glu Gln Thr Ser Glu 115 120 125 Ile Ile Thr Phe Ala Glu Ser Gly Thr Ala Arg Lys Thr Leu His Phe 130 135 140 Glu Ile Ser Lys Glu Gly Ser Asp Leu Ser Val Val Glu Arg Ala Glu 145 150 155 160 Val Trp Leu Phe Leu Lys Val Pro Lys Ala Asn Arg Thr Arg Thr Lys 165 170 175 Val Thr Ile Arg Leu Phe Gln Gln Gln Lys His Pro Gln Gly Ser Leu 180 185 190 Asp Thr Gly Glu Glu Ala Glu Glu Val Gly Leu Lys Gly Glu Arg Ser 195 200 205 Glu Leu Leu Leu Ser Glu Lys Val Val Asp Ala Arg Lys Ser Thr Trp 210 215 220 His Val Phe Pro Val Ser Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly 225 230 235 240 Lys Ser Ser Leu Asp Val Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser 245 250 255 Gly Ala Ser Leu Val Leu Leu Gly Lys Lys Lys Lys Lys Glu Glu Glu 260 265 270 Gly Glu Gly Lys Lys Lys Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp 275 280 285 Glu Glu Lys Glu Gln Ser His Arg Pro Phe Leu Met Leu Gln Ala Arg 290 295 300 Gln Ser Glu Asp His Pro His Arg Arg Arg Arg Arg Gly Leu Glu Cys 305 310 315 320 Asp Gly Lys Val Asn Ile Cys Cys Lys Lys Gln Phe Phe Val Ser Phe 325 330 335 Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro Ser Gly Tyr His 340 345 350 Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile Ala Gly Thr Ser 355 360 365 Gly Ser Ser Leu Ser Phe His Ser Thr Val Ile Asn His Tyr Arg Met 370 375 380 Arg Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys Cys Val Pro Thr 385 390 395 400 Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp Gly Gln Asn Ile 405 410 415 Ile Lys Lys Asp Ile Gln Asn Met Ile Val Glu Glu Cys Gly Cys Ser 420 425 430 <210> 38 <211> 32 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 38 ccaacagcag aagcacgaga gatctgagtt gc 32 <210> 39 <211> 32 <212> DNA <213> Artificial <220> <223> Synthetic DNA <400> 39 gcaactcaga tctctcgtgc ttctgctgtt gg 32

Claims

1. A modified activin A, wherein the pro-region of activin A having an amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 is modified, and the amino acid sequence of the modified pro-region is the amino acid sequence of (a) or (b) below: (a) An amino acid sequence lacking the amino acid sequence from position 182 to position 199 of the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6; (b) An amino acid sequence in which the amino acid sequence from position 182 to position 199 of the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6 is replaced with a spacer sequence consisting of 1 to 3 amino acids, and the amino acids are at least 1 kind of amino acid selected from glycine, alanine, and serine.

2. The modified activin A according to claim 1, wherein, The amino acid sequence of the modified pro-region is the amino acid sequence shown in any one of SEQ ID NOs: 9 to 20.

3. A polynucleotide encoding the modified activin A according to claim 1 or 2.

4. A vector comprising the polynucleotide according to claim 3.

5. A method for manufacturing modified activin A, comprising: A process for recovering the modified activin A expressed by a transformant expressing the modified activin A according to claim 1 or 2.

6. A method for producing a modified activin A, comprising the following steps: (a) A process for recovering the modified activin A expressed by a transformant expressing the modified activin A according to claim 1 or 2; and (b) A process for treating the obtained modified activin A with a proprotein convertase.

7. A mixture, which is a mixture of a modified activin A in which the pro-region of activin A having an amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 is modified and mature activin A, and the amino acid sequence of the modified pro-region is the amino acid sequence of (a) or (b) below: (a) An amino acid sequence lacking the amino acid sequence from position 182 to position 199 of the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6; (b) An amino acid sequence in which the amino acid sequence from position 182 to position 199 of the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6 is replaced with a spacer sequence consisting of 1 to 3 amino acids, and the amino acids are at least 1 kind of amino acid selected from glycine, alanine, and serine.

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