CN112028972A

CN112028972A - Signal peptide for improving secretion expression of recombinant protein of mammalian cell and application thereof

Info

Publication number: CN112028972A
Application number: CN202010876341.0A
Authority: CN
Inventors: 秦丽丽; 范华; 李明月
Original assignee: Beijing Baipusai Biotechnology Co Ltd
Current assignee: Beijing Baipusai Biotechnology Co Ltd
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2020-12-04
Anticipated expiration: 2040-08-27
Also published as: CN112028972B

Abstract

The invention provides a signal peptide for improving secretion expression of recombinant protein of mammalian cells and application thereof. The present invention first provides a polypeptide of the following (a) or (b): (a) polypeptide consisting of an amino acid sequence shown in SEQ ID No. 1; (b) the polypeptide which is formed by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID No.1 and has the same function with the polypeptide formed by the amino acid sequence shown in SEQ ID No. 1. The polypeptide can be used as a signal peptide to secrete and express a target protein in mammalian cells, is favorable for improving the secretion and expression level of the target protein, screens a high-expression monoclonal cell strain, and greatly reduces the time and cost for screening the high-expression signal peptide of the secreted protein.

Description

Signal peptide for improving secretion expression of recombinant protein of mammalian cell and application thereof

Technical Field

The present invention relates to a signal peptide and applications thereof, and more particularly, to a signal peptide for enhancing secretory expression of recombinant proteins in mammalian cells and applications thereof.

Background

Recombinant protein drugs are bright pearl in biological drugs, and with the progress of drug production technology and the increase of drug demand, the types of recombinant protein drugs in China are continuously abundant.

Systems for producing recombinant proteins using mammalian cells have been established for a long time. Compared with lower eukaryotic cell expression systems or prokaryotic expression systems, mammalian cell expression systems can guide the correct folding of proteins, provide various post-translational processing modification functions such as complex N-type glycosylation and accurate O-type glycosylation, so that the expressed product is closest to natural higher biological Protein molecules in terms of molecular structure, physicochemical properties and biological functions, and becomes the best choice for expressing recombinant proteins in the field of biopharmaceuticals (Chen P, Hutter D, Liu P, et al, Protein Expr Purif, 2002; 24 (3); 481-488).

One prominent feature of mammalian cell expression systems is the low level of protein expression. In recent years, with the development of molecular biology and Cell biology, a series of studies on the improvement of protein expression level have been carried out, including Cell strain modification (Davy AM, et al, Cell Syst.2017Mar 22; 4(3): 262-. Many researchers have done a lot of work on vector engineering, including selecting appropriate promoters, enhancers, signal peptides, all of which can increase protein expression levels in mammalian cells to some extent. Different signal peptides direct different levels of secretory protein expression, and selection of an appropriate signal peptide can increase the expression level of the protein (Steve Barash, et al, Biochem Biophys Res Commun.2002Jun 21; 294(4): 835-42).

Signal peptides are short peptide chains that direct the transfer of newly synthesized proteins to the secretory pathway, are located at the N-terminus of secreted proteins, can be cleaved, and typically consist of a 15-30 amino acid leader sequence, including three regions: a positively charged N-terminus of 2-3 polar amino acids, termed the basic amino terminus; an intermediate hydrophobic sequence, mainly neutral amino acids, capable of forming an alpha helical structure, which is the main functional region of the signal peptide; a longer, negatively charged C-terminus is the signal sequence cleavage site, also called the processing region. When the signal peptide sequence is synthesized, it is recognized by a Signal Recognition Particle (SRP), protein synthesis is suspended or slowed, the signal recognition particle carries the ribosome to the endoplasmic reticulum, protein synthesis is restarted, the newly synthesized protein enters the endoplasmic reticulum cavity under the guidance of the signal peptide, and the signal peptide sequence is cleaved by the action of a signal peptidase. The signal peptide is sufficient to transport any additional polypeptide to the target membrane. Different proteins are expressed in mammalian cells, different signal peptides are needed, and the secretion efficiency of the same protein under the action of different signal peptides is far from the same even if the protein can be secreted and expressed. For example, Jia-Ye Wang researches on the influence of mutating Pro at position 22 of a human tissue plasminogen activator signal peptide to Ala/Gly on the secretion effect of influenza virus H3N2-HA1, and the results show that compared with the non-mutated human tissue plasminogen activator signal peptide, the mutation of Pro at position 22 to Ala or Gly can effectively improve the secretion efficiency and expression level of H3N2-HA1 protein, wherein the mutation is the best Ala (Jia-Ye Wang et al, apple Microbiol Biotechnol.2011Aug; 91(3): 731-40). For secretory protein, the expression level can be improved by times by selecting proper signal peptide, and the method has a very profound significance for industrial production and scientific research.

In the prior art, if a higher expression level is required to be obtained, a plurality of signal peptides need to be screened separately for each secreted protein, and a proper signal peptide is selected from the signal peptides, so that the defects of long screening time, high cost and the like exist.

Disclosure of Invention

It is an object of the present invention to provide a novel signal peptide.

It is another object of the present invention to provide related uses of the signal peptide.

In one aspect, the present invention provides a polypeptide of (a) or (b):

(a) polypeptide consisting of an amino acid sequence shown in SEQ ID No. 1;

(b) the polypeptide which is formed by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID No.1 and has the same function with the polypeptide formed by the amino acid sequence shown in SEQ ID No. 1.

The polypeptide of the invention is a signal peptide which can effectively improve the secretory expression of target proteins (including antibodies) in mammalian cells. In some specific embodiments of the invention, 9 verification proteins are selected to verify the function of the polypeptide as a signal peptide, and experimental results show that the expression level of the selected verification proteins is greatly improved by using the polypeptide consisting of the amino acid sequence shown in SEQ ID No.1 as the signal peptide, except that the expression level of the Human PD-1 is improved by about 4 times, the expression level of other verification proteins is improved by 5 times or more, and the expression level of more than 50% of the verification proteins is improved by 10 times or more. The HumanIL-17RC and the Human Decorin protein which are hardly expressed by the self signal peptide can successfully obtain the target protein under the condition of using the signal peptide, and the signal peptide can be widely used for the high-efficiency secretory expression of the target protein in mammalian cells and can effectively solve the production problem of the protein which is difficult to express.

According to a specific embodiment of the present invention, the polypeptide consisting of the amino acid sequence shown in SEQ ID No.1 with one or more amino acid substitutions, deletions and/or additions and having the same function as the polypeptide shown in SEQ ID No.1 at least comprises a sequence of at least 9 consecutive amino acids, preferably at least 10 consecutive amino acids, more preferably at least 11 consecutive amino acids at the C-terminal of the amino acid sequence shown in SEQ ID No. 1.

According to a specific embodiment of the present invention, the polypeptide having the same function as the polypeptide consisting of the amino acid sequence shown in SEQ ID No.1 refers to the polypeptide having the same function of increasing the secretory expression level of the target protein in the mammalian cells (compared to the use of the self-signal peptide, the secretory expression level of the target protein in the mammalian cells) as the polypeptide consisting of the amino acid sequence shown in SEQ ID No.1, preferably, having the same function of increasing the secretory expression level of the target protein in the mammalian cells by at least 4 times.

In another aspect, the invention also provides polynucleotides encoding the polypeptides of the invention.

The polynucleotide encoding the polypeptide of the present invention may be designed and prepared according to a conventional method.

In another aspect, the present invention also provides an expression vector comprising the polynucleotide of the present invention.

According to a specific embodiment of the present invention, the promoter of the expression vector of the present invention may be EF-1. alpha. promoter, hCMV (human cytokine viral) promoter, SV40(Simian vacuolating virus40) late promoter, or the like. In some embodiments of the invention, the expression vector is pcdan3.1 (+).

In another aspect, the present invention also provides a mammalian cell comprising the polynucleotide of the present invention or the expression vector of the present invention. The mammalian cell is selected from one or more of Chinese Hamster Ovary (CHO), mouse plasmacytoma (A non-Ig cloning, non-light chain-synthesizing of NS-1, NSO), Baby Hamster Kidney (BHK), human embryo kidney293 (HEK 293), etc.

In another aspect, the invention also provides the application of the polypeptide as a signal peptide in secretory expression of a target protein in mammalian cells.

According to a specific embodiment of the invention, the protein of interest can be selected from one or more of exogenous proteins such as Human IL-17RC, Human CD28, Human PD-1, Human ICOS, Human Decorin, Human Siglec-15, Human IL-7, Human IL-17F, and Human CD 73.

In another aspect, the present invention provides a method for secretory expression of a target protein in a mammalian cell, the method comprising:

the target protein is secreted and expressed by mammalian cells containing the polynucleotide or the expression vector.

According to a specific embodiment of the present invention, the method for secretory expression of a protein of interest in a mammalian cell of the present invention comprises:

linking a polynucleotide encoding the polypeptide of the present invention and a polynucleotide encoding a protein of interest, which is positioned at the front end of the polynucleotide encoding the protein of interest, and cloning the linked polynucleotide into a mammalian cell expression vector;

and (3) transfecting the expression vector into a mammalian cell to express the target protein.

According to a specific embodiment of the present invention, the method for secretory expression of a target protein in a mammalian cell of the present invention further comprises culturing the transfected mammalian cell to secretory expression of the target protein, and isolating the target protein from the culture.

In some specific embodiments of the present invention, 9 tumor immune drug development key target proteins or cytokines are selected as verification proteins of the application effect of the signal peptide of the present invention, and the amino acid sequences of the verification proteins are shown as SEQ ID nos. 11 to 19 in table 1. The amino acid sequence of the self signal peptide of the verification protein is shown as SEQ ID No. 2-SEQ ID No.10 in the table 1. In the invention, a section of poly His tag is introduced into the C terminal of the verification protein for separating and purifying the protein, and the amino acid sequence is shown as SEQ ID No.20 in Table 1. The amino acid sequences comprising the signal peptide, the verification protein and the poly His tag of the present invention are shown in SEQ ID No.21 to SEQ ID No.29 of Table 1. The amino acid sequences of the self-signal peptide, the verification protein and the poly His tag are shown in SEQ ID No. 30-SEQ ID No.38 in Table 1. The 9 selected tumor immune drugs are used for developing key target proteins or cytokines as foreign proteins to be expressed in HEK293 cells. Compared with the signal peptide of the protein self, the signal peptide of the invention is greatly improved in the expression level of the foreign protein in the HEK293 cell, except that the expression level of the Human PD-1 is improved by about 4 times, the expression level of other 8 verification proteins is improved by 5 times or more on average, and the expression level of more than 50% verification proteins is improved by 10 times or more.

In conclusion, the invention provides a novel signal peptide, which is applied to the improvement of the secretory expression level of secretory protein, screening of high-expression monoclonal cell strains and reduction of the production cost; on the other hand, the method greatly reduces the time and cost for screening the high-expression signal peptide of the secreted protein, and has wide application prospect.

Drawings

FIG. 1A is a map of expression vector pcDNA3.1, and FIG. 1B is a map of expression vector pEE14.4.

FIG. 2 is a schematic diagram of recombinant plasmid construction.

FIG. 3 shows the comparison of the effect of the signal peptide of the present invention and that of the self signal peptide.

FIG. 4 shows the results of comparison of the expression effect of Human protein in CHO cells using the signal peptide of the present invention and self signal peptide.

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. The experimental methods in the examples, in which specific conditions are not noted, are conventional methods and conventional conditions well known in the art, or conditions as recommended by the manufacturer.

Example 1

In this example, 9 proteins were selected as verification proteins for the effect of the signal peptide of the present invention, and the signal peptide of the present invention and the self signal peptide of the verification protein were used to compare the secretion expression level of the verification protein in HEK293 cells under the same conditions. The specific operation is as follows:

1. construction of recombinant expression vectors

First, a recombinant expression vector using a signal peptide of the present invention (SEQ ID No.1, see Table 1 for specific sequence) was constructed, named pHEK1.0: synthesizing polynucleotides with amino acid sequences shown as SEQ ID No. 21-SEQ ID No.29 (the specific sequences are shown in Table 1) with sequences shown as SEQ ID No. 39-SEQ ID No.47 (wherein the polynucleotide sequence for encoding the signal peptide is shown as nucleotide sequences from 1 st to 51 st in SEQ ID No. 39-SEQ ID No. 47), and adding restriction enzyme BamH I cutting sites and Kozak sequences at the 5' tail end; the 3' end is added with restriction enzyme XhoI cutting site. The synthesized product is subjected to double enzyme digestion by restriction enzymes BamH I and Xho I, then agarose gel electrophoresis is carried out, and the gel is cut to recover the target fragment. The target fragment was ligated to pcNDA3.1(+) (purchased from Invitrogen) which was also double-digested with restriction enzymes BamHI and XhoI (expression vector pcDNA3.1 map is shown in FIG. 1A), recombinant eukaryotic expression vector pHEK1.0-IL-17RC/pHEK1.0-CD28/pHEK1.0-PD-1/pHEK1.0-ICOS/pHEK1.0-Decorin/pHEK1.0-Siglec-15/pHEK1.0-IL-7/pHEK1.0-IL-17F/pHEK1.0-CD73 (recombinant plasmid construction is shown in FIG. 2), E.coli DH5 alpha was transformed, cultured at 37 ℃ for 16h, single colony was picked up for plasmid extraction, the extracted plasmid was identified by double-restriction enzyme BamHI and XhoI, and then subjected to gel electrophoresis, and cloned in limited sea organism, and (4) selecting the clone with correct sequencing for amplifying and extracting the plasmid, and storing the extracted plasmid at the temperature of minus 20 ℃ for later use after sterile filtration.

Next, a recombinant expression vector using the verification protein self signal peptide was constructed, designated pNSP: synthesizing polynucleotides with amino acid sequences shown as SEQ ID No. 30-SEQ ID No.38 (the specific sequences are shown in Table 1) by Shanghai bioengineering Co., Ltd, wherein the sequences are SEQ ID No. 48-SEQ ID No.56, and restriction enzyme BamH I restriction sites and Kozak sequences are added at the 5' ends; the 3' end is added with restriction enzyme XhoI cutting site. The synthesized product is subjected to double enzyme digestion by restriction enzymes BamH I and Xho I, then agarose gel electrophoresis is carried out, and the gel is cut to recover the target fragment. Respectively connecting the target fragment with pcNDA3.1(+) (purchased from Invitrogen company) which is also subjected to double enzyme digestion by restriction enzymes BamHI and XhoI, constructing a recombinant eukaryotic expression vector pNSP-IL-17RC/pNSP-CD28/pNSP-PD-1/pNSP-ICOS/pNSP-Decorin/pNSP-Siglec-15/pNSP-IL-7/pNSP-IL-17F/pNSP-CD73, respectively converting Escherichia coli E.coli DH5 alpha, culturing at 37 ℃ for 16h, selecting a single colony for plasmid extraction, carrying out double agarose gel electrophoresis identification on the extracted plasmid after double enzyme digestion by the restriction enzymes BamHI and XhoI, sending the positive clone to Shanghai bioengineering company for sequencing, selecting a correct clone for amplified plasmid sequencing, carrying out sterile filtration and extracting the plasmid after extraction, storing at-20 deg.C for use.

TABLE 1

2. HEK293 cell transfected by recombinant expression vector and target protein expressed

And (3) transfecting HEK293 cells with the constructed recombinant plasmids by using a PEI transfection reagent respectively to express the target protein. HEK293 cells were plated at 1X 10 on the day before transfection⁶passage/mL, culture at 37 ℃. On day of transfection, counts were counted and adjustedCell density of 2X 10⁶mL, survival rate above 95%.

The amounts of the components in the transfection reagent were calculated from the cell density: the corresponding relation between the plasmid dose and the number of cells is 1 × 10⁶cells corresponded to 1. mu.g of plasmid, and the corresponding relationship between PEI dose and plasmid dose was such that the mass of PEI was 3 times the mass of DNA.

Preparing a transfection reagent according to the calculated component usage amount: 50 mu g of recombinant plasmid with the concentration of 200 mu g/mL is added into CD 293TGE culture medium to be 0.5mL (solution A), 150 mu g of PEI transfection reagent with the concentration of 1mg/mL is added into CD 293TGE culture medium to be 0.5mL (solution B), after respectively mixing uniformly, solution B is slowly added into solution A, after mixing uniformly and standing for 10-15min at room temperature, the transfection mixed solution is slowly dripped into 10mL HEK293 cells. 5% CO₂After culturing at 37 ℃ and 135rpm for 96 hours, the supernatant was collected.

3. Separation and purification of target protein

And (3) separating and purifying the target protein by using a His-tag affinity chromatography method, and comparing the effect of the signal peptide of the invention with the effect of the signal peptide of the verification protein. The cell culture supernatant was collected by centrifugation at 1000 Xg for 30min, and the supernatant was filtered and bound to GE HisTrap excel chromatography columns equilibrated in advance with PBS, followed by elution of the target protein with PBS containing 30/100/500mM imidazole in an elution volume of 5CV per concentration, and the protein eluate was collected and subjected to SDS-PAGE analysis.

4. The signal peptide of the invention is used for analyzing the secretion expression effect of the test protein

Under the same conditions, the signal peptide of the invention and the self signal peptide of the verification protein are respectively used for comparing and verifying the secretion expression level of the protein in HEK293 cells, and the SDS-PAGE result is shown in figure 3. The results show that:

1) the signal peptide can improve the secretory expression quantity of the Human IL-17RC protein by more than 10 times;

2) the signal peptide can improve the secretory expression quantity of the Human CD28 protein by more than 10 times;

3) the signal peptide can improve the secretory expression quantity of the Human PD-1 protein by about 4 times;

4) the signal peptide can improve the secretory expression quantity of the Human ICOS protein by more than 5 times;

5) the signal peptide can improve the secretion expression quantity of the Human Decorin protein by more than 5 times;

6) the signal peptide can improve the secretion expression quantity of the Human Siglec-15 protein by about 10 times;

7) the signal peptide can improve the secretory expression quantity of the Human IL-7 protein by about 10 times;

8) the signal peptide can improve the secretory expression quantity of the Human IL-17F protein by about 5 times;

9) the signal peptide can improve the secretory expression quantity of the Human CD73 protein by more than 10 times.

Experimental data show that compared with the signal peptide of the protein, the signal peptide disclosed by the invention has the advantages that the expression quantity of the verification protein in the HEK293 cell is greatly improved, except that the expression quantity of human PD-1 is improved by about 4 times, the expression level of other verification proteins is improved by 5 times or more on average, and the expression level of over 50% verification proteins is improved by 10 times or more.

The results show that the signal peptide is particularly suitable for the secretion expression of the foreign protein of the HEK293 cell, the expression level of the verification protein is improved by 4 times or more, and the signal peptide can be widely used for the high-efficiency secretion expression of the foreign protein in the HEK293 cell.

Example 2

In the embodiment, the Human CD73 protein is selected as a verification protein for the application effect of the signal peptide in CHO cells. Under the same condition, the signal peptide of the invention and the self signal peptide of the verification protein are respectively used for comparing and verifying the secretion expression level of the Human CD73 protein in CHO cells. The specific operation is as follows:

1. construction of recombinant expression vectors

First, a recombinant expression vector using the signal peptide of the present invention (SEQ ID No.1, see Table 1 for a specific sequence) was constructed: the polynucleotide with the sequence of SEQ ID No.29 (the specific sequence is shown in Table 1) is synthesized by Shanghai bioengineering company Limited and has the sequence of SEQ ID No.47, and restriction enzyme Hind II I enzyme cutting site and Kozak sequence are added at the 5' end; the 3' end is added with a restriction enzyme EcoRI cutting site. The synthesized product is subjected to double digestion by restriction enzymes Hind I and EcoR I, then agarose gel electrophoresis is carried out, and the gel is cut to recover the target fragment. Respectively connecting the target fragment with pEE14.4 (purchased from Lonza biologics company) which is also subjected to double enzyme digestion by restriction enzymes HindII and EcoRI (the map of the expression vector pEE14.4 is shown in figure 1B), constructing a recombinant eukaryotic expression vector pCHO-CD73 (the recombinant plasmid is shown in figure 2), transforming Escherichia coli E.coli DH5 alpha, culturing at 37 ℃ for 16h, selecting a single colony for plasmid extraction, carrying out agarose gel electrophoresis identification after double enzyme digestion of the extracted plasmid by the restriction enzymes HindII and EcoRI, sending a positive clone to a marine bioengineering limited company for sequencing, selecting a clone with correct sequencing for amplification extraction of the plasmid, and preserving the extracted plasmid at-20 ℃ for later use after aseptic filtration.

Next, a recombinant expression vector using a signal peptide of the verification protein itself was constructed: the polynucleotide with the sequence of SEQ ID No.38 (the specific sequence is shown in Table 1) is synthesized by Shanghai bioengineering company Limited, the sequence is SEQ ID No.56, and restriction enzyme Hind II I enzyme cutting site and Kozak sequence are added at the 5' end; the 3' end is added with a restriction enzyme EcoRI cutting site. The synthesized product is subjected to double digestion by restriction enzymes Hind I and EcoR I, then agarose gel electrophoresis is carried out, and the gel is cut to recover the target fragment. Respectively connecting the target fragment with pEE14.4 (purchased from Lonza biologics company) which is also subjected to double enzyme digestion by restriction enzymes HindII and EcoRI, constructing a recombinant eukaryotic expression vector pNSP-CHO-CD73, transforming Escherichia coli E.coli DH5 alpha, culturing at 37 ℃ for 16h, selecting a single colony for plasmid extraction, carrying out double enzyme digestion on the extracted plasmid by the restriction enzymes HindII and EcoRI, carrying out agarose gel electrophoresis identification, sending the positive clone to Shanghai bioengineering Co., Ltd for sequencing, selecting the clone with correct sequencing for amplification extraction of the plasmid, and carrying out sterile filtration on the extracted plasmid and storing at-20 ℃ for later use.

2. Recombinant expression vector transfection CHO cell and expression of target protein

Expifeacmine was used^TMCHThe constructed recombinant plasmids pCHO-CD73 and pNSP-CHO-CD73 were transfected into CHO cells by the O Transfection Kit (available from Thermofeisher Co.) to express the target protein. CHO cells were plated at 1X 10 the day before transfection⁶passage/mL, culture at 37 ℃. Cells were counted on the day of transfection and adjusted to a cell density of 2X 10⁶The activity rate is more than 95 percent.

The amounts of the components in the transfection reagent were calculated from the cell density: the corresponding relation between the plasmid dose and the number of cells is 1 × 10⁶cells correspond to 1. mu.g plasmid, Expifeacmine^TMThe corresponding relation between the CHO Transfection Kit Transfection reagent and the plasmid dosage is that the mass of the Transfection reagent is 3 times of the DNA mass.

Preparing a transfection reagent according to the calculated component usage amount: 50. mu.g of the recombinant plasmid at a concentration of 200. mu.g/mL was added to OptiPRO^TMMedium (purchased from Thermofeisher Co.) to 0.5mL (solution A), 150. mu.g Expifactamine at a concentration of 1mg/mL^TMAdding OptiPRO into CHO Transfection Kit Transfection reagent^TMMedium Medium (purchased from Thermofeisher Co.) to 0.5mL (solution B), mixing, slowly adding solution B into solution A, mixing, standing at room temperature for 10min, and slowly dripping the transfection mixture into 10mL CHO cells. 5% CO₂After culturing at 37 ℃ and 135rpm for 96 hours, the supernatant was collected.

3. Separation and purification of target protein

Under the same conditions, the secretion expression level of the protein HumanCD73 in CHO cells is verified by comparing the signal peptide of the invention and the self-signal peptide of the verification protein respectively, and the SDS-PAGE result is shown in FIG. 4. The results show that: the signal peptide can improve the secretion expression quantity of the Human CD73 protein in CHO cells by more than 8 times.

The results show that the signal peptide is not only particularly suitable for the secretion expression of the foreign protein of the HEK293 cell, but also can obviously improve the secretion expression level of the foreign protein in the CHO cell, and the signal peptide can be widely used for the high-efficiency secretion expression of the foreign protein in the mammalian cell.

SEQUENCE LISTING

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Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile

85 90 95

Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly

100 105 110

Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe

115 120 125

Pro Gly Pro Ser Lys Pro

130

<210> 13

<211> 143

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 13

Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala

1 5 10 15

Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe

20 25 30

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

35 40 45

Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln

50 55 60

Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg

65 70 75 80

Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr

85 90 95

Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu

100 105 110

Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro

115 120 125

Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln

130 135 140

<210> 14

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 14

Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile Phe His Asn Gly

1 5 10 15

Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val Gln Gln Phe Lys

20 25 30

Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp Leu Thr Lys Thr

35 40 45

Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu Lys Phe Cys His

50 55 60

Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu Tyr Asn Leu Asp

65 70 75 80

His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser Ile Phe Asp Pro

85 90 95

Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu His Ile Tyr Glu

100 105 110

Ser Gln Leu Cys Cys Gln Leu Lys

115 120

<210> 15

<211> 343

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 15

Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu

1 5 10 15

Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro

20 25 30

Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val

35 40 45

Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu Pro

50 55 60

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

65 70 75 80

Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Ala Leu Ile Leu

85 90 95

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

100 105 110

Val Lys Leu Glu Arg Leu Tyr Leu Ser Lys Asn Gln Leu Lys Glu Leu

115 120 125

Pro Glu Lys Met Pro Lys Thr Leu Gln Glu Leu Arg Ala His Glu Asn

130 135 140

Glu Ile Thr Lys Val Arg Lys Val Thr Phe Asn Gly Leu Asn Gln Met

145 150 155 160

Ile Val Ile Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser Gly Ile Glu

165 170 175

Asn Gly Ala Phe Gln Gly Met Lys Lys Leu Ser Tyr Ile Arg Ile Ala

180 185 190

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

195 200 205

Glu Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala Ser

210 215 220

Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn Ser

225 230 235 240

Ile Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu Arg

245 250 255

Glu Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Pro Gly Gly Leu

260 265 270

Ala Glu His Lys Tyr Ile Gln Val Val Tyr Leu His Asn Asn Asn Ile

275 280 285

Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly His Asn Thr Lys

290 295 300

Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro Val Gln Tyr

305 310 315 320

Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser Ala

325 330 335

Ile Gln Leu Gly Asn Tyr Lys

340

<210> 16

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 16

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

1 5 10 15

Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val Pro Pro Glu

20 25 30

Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys Thr Phe Thr

35 40 45

His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile Trp Arg Ala

50 55 60

Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala Ala Ala Arg

65 70 75 80

Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser His Gly Arg Phe Arg Leu

85 90 95

Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg Val Glu Arg Leu

100 105 110

Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val Glu Phe Ala Gly

115 120 125

Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val Arg Leu His Val

130 135 140

Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu Pro Ser Pro Ala

145 150 155 160

His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu Pro Pro Pro Ala

165 170 175

Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu Ala Ala Val Arg

180 185 190

Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala Glu Leu Pro Ala

195 200 205

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

210 215 220

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

225 230 235 240

Ser Thr

<210> 17

<211> 152

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 17

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

1 5 10 15

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

20 25 30

Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp

35 40 45

Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg

50 55 60

Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu

65 70 75 80

Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val

85 90 95

Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser

100 105 110

Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu

115 120 125

Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys

130 135 140

Ile Leu Met Gly Thr Lys Glu His

145 150

<210> 18

<211> 133

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 18

Arg Lys Ile Pro Lys Val Gly His Thr Phe Phe Gln Lys Pro Glu Ser

1 5 10 15

Cys Pro Pro Val Pro Gly Gly Ser Met Lys Leu Asp Ile Gly Ile Ile

20 25 30

Asn Glu Asn Gln Arg Val Ser Met Ser Arg Asn Ile Glu Ser Arg Ser

35 40 45

Thr Ser Pro Trp Asn Tyr Thr Val Thr Trp Asp Pro Asn Arg Tyr Pro

50 55 60

Ser Glu Val Val Gln Ala Gln Cys Arg Asn Leu Gly Cys Ile Asn Ala

65 70 75 80

Gln Gly Lys Glu Asp Ile Ser Met Asn Ser Val Pro Ile Gln Gln Glu

85 90 95

Thr Leu Val Val Arg Arg Lys His Gln Gly Cys Ser Val Ser Phe Gln

100 105 110

Leu Glu Lys Val Leu Val Thr Val Gly Cys Thr Cys Val Thr Pro Val

115 120 125

Ile His His Val Gln

130

<210> 19

<211> 522

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 19

Trp Glu Leu Thr Ile Leu His Thr Asn Asp Val His Ser Arg Leu Glu

1 5 10 15

Gln Thr Ser Glu Asp Ser Ser Lys Cys Val Asn Ala Ser Arg Cys Met

20 25 30

Gly Gly Val Ala Arg Leu Phe Thr Lys Val Gln Gln Ile Arg Arg Ala

35 40 45

Glu Pro Asn Val Leu Leu Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr

50 55 60

Ile Trp Phe Thr Val Tyr Lys Gly Ala Glu Val Ala His Phe Met Asn

65 70 75 80

Ala Leu Arg Tyr Asp Ala Met Ala Leu Gly Asn His Glu Phe Asp Asn

85 90 95

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

100 105 110

Ile Leu Ser Ala Asn Ile Lys Ala Lys Gly Pro Ala Ser Gln Ile Ser

115 120 125

Gly Leu Tyr Leu Pro Tyr Lys Val Leu Pro Val Gly Asp Glu Val Val

130 135 140

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

145 150 155 160

Gly Thr Asn Leu Val Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro Glu

165 170 175

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

180 185 190

His Ser Gly Phe Glu Met Asp Lys Leu Ile Ala Gln Lys Val Arg Gly

195 200 205

Val Asp Val Val Val Gly Gly His Ser Asn Thr Phe Leu Tyr Thr Gly

210 215 220

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

225 230 235 240

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

245 250 255

Gly Lys Tyr Leu Gly Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly Asn

260 265 270

Val Ile Ser Ser His Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile Pro

275 280 285

Glu Asp Pro Ser Ile Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys Leu

290 295 300

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

305 310 315 320

Gly Ser Ser Gln Ser Cys Arg Phe Arg Glu Cys Asn Met Gly Asn Leu

325 330 335

Ile Cys Asp Ala Met Ile Asn Asn Asn Leu Arg His Thr Asp Glu Met

340 345 350

Phe Trp Asn His Val Ser Met Cys Ile Leu Asn Gly Gly Gly Ile Arg

355 360 365

Ser Pro Ile Asp Glu Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn Leu

370 375 380

Ala Ala Val Leu Pro Phe Gly Gly Thr Phe Asp Leu Val Gln Leu Lys

385 390 395 400

Gly Ser Thr Leu Lys Lys Ala Phe Glu His Ser Val His Arg Tyr Gly

405 410 415

Gln Ser Thr Gly Glu Phe Leu Gln Val Gly Gly Ile His Val Val Tyr

420 425 430

Asp Leu Ser Arg Lys Pro Gly Asp Arg Val Val Lys Leu Asp Val Leu

435 440 445

Cys Thr Lys Cys Arg Val Pro Ser Tyr Asp Pro Leu Lys Met Asp Glu

450 455 460

Val Tyr Lys Val Ile Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp Gly

465 470 475 480

Phe Gln Met Ile Lys Asp Glu Leu Leu Arg His Asp Ser Gly Asp Gln

485 490 495

Asp Ile Asn Val Val Ser Thr Tyr Ile Ser Lys Met Lys Val Ile Tyr

500 505 510

Pro Ala Val Glu Gly Arg Ile Lys Phe Ser

515 520

<210> 20

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 20

Gly Ser His His His His His His

1 5

<210> 21

<211> 470

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 21

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Val Trp

1 5 10 15

Ala Leu Glu Arg Leu Val Gly Pro Gln Asp Ala Thr His Cys Ser Pro

20 25 30

Gly Leu Ser Cys Arg Leu Trp Asp Ser Asp Ile Leu Cys Leu Pro Gly

35 40 45

Asp Ile Val Pro Ala Pro Gly Pro Val Leu Ala Pro Thr His Leu Gln

50 55 60

Thr Glu Leu Val Leu Arg Cys Gln Lys Glu Thr Asp Cys Asp Leu Cys

65 70 75 80

Leu Arg Val Ala Val His Leu Ala Val His Gly His Trp Glu Glu Pro

85 90 95

Glu Asp Glu Glu Lys Phe Gly Gly Ala Ala Asp Ser Gly Val Glu Glu

100 105 110

Pro Arg Asn Ala Ser Leu Gln Ala Gln Val Val Leu Ser Phe Gln Ala

115 120 125

Tyr Pro Thr Ala Arg Cys Val Leu Leu Glu Val Gln Val Pro Ala Ala

130 135 140

Leu Val Gln Phe Gly Gln Ser Val Gly Ser Val Val Tyr Asp Cys Phe

145 150 155 160

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

165 170 175

Arg Tyr Glu Lys Glu Leu Asn His Thr Gln Gln Leu Pro Asp Cys Arg

180 185 190

Gly Leu Glu Val Trp Asn Ser Ile Pro Ser Cys Trp Ala Leu Pro Trp

195 200 205

Leu Asn Val Ser Ala Asp Gly Asp Asn Val His Leu Val Leu Asn Val

210 215 220

Ser Glu Glu Gln His Phe Gly Leu Ser Leu Tyr Trp Asn Gln Val Gln

225 230 235 240

Gly Pro Pro Lys Pro Arg Trp His Lys Asn Leu Thr Gly Pro Gln Ile

245 250 255

Ile Thr Leu Asn His Thr Asp Leu Val Pro Cys Leu Cys Ile Gln Val

260 265 270

Trp Pro Leu Glu Pro Asp Ser Val Arg Thr Asn Ile Cys Pro Phe Arg

275 280 285

Glu Asp Pro Arg Ala His Gln Asn Leu Trp Gln Ala Ala Arg Leu Gln

290 295 300

Leu Leu Thr Leu Gln Ser Trp Leu Leu Asp Ala Pro Cys Ser Leu Pro

305 310 315 320

Ala Glu Ala Ala Leu Cys Trp Arg Ala Pro Gly Gly Asp Pro Cys Gln

325 330 335

Pro Leu Val Pro Pro Leu Ser Trp Glu Asn Val Thr Val Asp Lys Val

340 345 350

Leu Glu Phe Pro Leu Leu Lys Gly His Pro Asn Leu Cys Val Gln Val

355 360 365

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

370 375 380

Leu Gly Pro Leu Lys Asp Asp Val Leu Leu Leu Glu Thr Arg Gly Pro

385 390 395 400

Gln Asp Asn Arg Ser Leu Cys Ala Leu Glu Pro Ser Gly Cys Thr Ser

405 410 415

Leu Pro Ser Lys Ala Ser Thr Arg Ala Ala Arg Leu Gly Glu Tyr Leu

420 425 430

Leu Gln Asp Leu Gln Ser Gly Gln Cys Leu Gln Leu Trp Asp Asp Asp

435 440 445

Leu Gly Ala Leu Trp Ala Cys Pro Met Asp Lys Tyr Ile His Gly Ser

450 455 460

His His His His His His

465 470

<210> 22

<211> 159

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 22

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Val Trp

1 5 10 15

Ala Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp

20 25 30

Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg

35 40 45

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

50 55 60

Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys

65 70 75 80

Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe

85 90 95

Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys

100 105 110

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

115 120 125

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

130 135 140

Phe Pro Gly Pro Ser Lys Pro Gly Ser His His His His His His

145 150 155

<210> 23

<211> 168

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 23

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Val Trp

1 5 10 15

Ala Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro

20 25 30

Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser

35 40 45

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

50 55 60

Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser

65 70 75 80

Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly

85 90 95

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

100 105 110

Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys

115 120 125

Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val

130 135 140

Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln

145 150 155 160

Gly Ser His His His His His His

165

<210> 24

<211> 145

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 24

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Val Trp

1 5 10 15

Ala Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile Phe His Asn

20 25 30

Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val Gln Gln Phe

35 40 45

Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp Leu Thr Lys

50 55 60

Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu Lys Phe Cys

65 70 75 80

His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu Tyr Asn Leu

85 90 95

Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser Ile Phe Asp

100 105 110

Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu His Ile Tyr

115 120 125

Glu Ser Gln Leu Cys Cys Gln Leu Lys Gly Ser His His His His His

130 135 140

His

145

<210> 25

<211> 368

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 25

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Val Trp

1 5 10 15

Ala Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp

20 25 30

Glu Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu

35 40 45

Pro Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg

50 55 60

Val Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu

65 70 75 80

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

85 90 95

Ile Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Ala Leu Ile

100 105 110

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

115 120 125

Leu Val Lys Leu Glu Arg Leu Tyr Leu Ser Lys Asn Gln Leu Lys Glu

130 135 140

Leu Pro Glu Lys Met Pro Lys Thr Leu Gln Glu Leu Arg Ala His Glu

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Ala Asp Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu Pro Pro Ser Leu

210 215 220

Thr Glu Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala

225 230 235 240

Ser Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn

245 250 255

Ser Ile Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu

260 265 270

Arg Glu Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Pro Gly Gly

275 280 285

Leu Ala Glu His Lys Tyr Ile Gln Val Val Tyr Leu His Asn Asn Asn

290 295 300

Ile Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly His Asn Thr

305 310 315 320

Lys Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro Val Gln

325 330 335

Tyr Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser

340 345 350

Ala Ile Gln Leu Gly Asn Tyr Lys Gly Ser His His His His His His

355 360 365

<210> 26

<211> 267

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 26

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Val Trp

1 5 10 15

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

20 25 30

Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val Pro Pro

35 40 45

Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys Thr Phe

50 55 60

Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile Trp Arg

65 70 75 80

Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala Ala Ala

85 90 95

Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser His Gly Arg Phe Arg

100 105 110

Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg Val Glu Arg

115 120 125

Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val Glu Phe Ala

130 135 140

Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val Arg Leu His

145 150 155 160

Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu Pro Ser Pro

165 170 175

Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu Pro Pro Pro

180 185 190

Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu Ala Ala Val

195 200 205

Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala Glu Leu Pro

210 215 220

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

225 230 235 240

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

245 250 255

Ala Ser Thr Gly Ser His His His His His His

260 265

<210> 27

<211> 177

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 27

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Val Trp

1 5 10 15

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

20 25 30

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

35 40 45

Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys

50 55 60

Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu

65 70 75 80

Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu

85 90 95

Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln

100 105 110

Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys

115 120 125

Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp

130 135 140

Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn

145 150 155 160

Lys Ile Leu Met Gly Thr Lys Glu His Gly Ser His His His His His

165 170 175

His

<210> 28

<211> 158

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 28

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Val Trp

1 5 10 15

Ala Arg Lys Ile Pro Lys Val Gly His Thr Phe Phe Gln Lys Pro Glu

20 25 30

Ser Cys Pro Pro Val Pro Gly Gly Ser Met Lys Leu Asp Ile Gly Ile

35 40 45

Ile Asn Glu Asn Gln Arg Val Ser Met Ser Arg Asn Ile Glu Ser Arg

50 55 60

Ser Thr Ser Pro Trp Asn Tyr Thr Val Thr Trp Asp Pro Asn Arg Tyr

65 70 75 80

Pro Ser Glu Val Val Gln Ala Gln Cys Arg Asn Leu Gly Cys Ile Asn

85 90 95

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

100 105 110

Glu Thr Leu Val Val Arg Arg Lys His Gln Gly Cys Ser Val Ser Phe

115 120 125

Gln Leu Glu Lys Val Leu Val Thr Val Gly Cys Thr Cys Val Thr Pro

130 135 140

Val Ile His His Val Gln Gly Ser His His His His His His

145 150 155

<210> 29

<211> 547

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 29

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Val Trp

1 5 10 15

Ala Trp Glu Leu Thr Ile Leu His Thr Asn Asp Val His Ser Arg Leu

20 25 30

Glu Gln Thr Ser Glu Asp Ser Ser Lys Cys Val Asn Ala Ser Arg Cys

35 40 45

Met Gly Gly Val Ala Arg Leu Phe Thr Lys Val Gln Gln Ile Arg Arg

50 55 60

Ala Glu Pro Asn Val Leu Leu Leu Asp Ala Gly Asp Gln Tyr Gln Gly

65 70 75 80

Thr Ile Trp Phe Thr Val Tyr Lys Gly Ala Glu Val Ala His Phe Met

85 90 95

Asn Ala Leu Arg Tyr Asp Ala Met Ala Leu Gly Asn His Glu Phe Asp

100 105 110

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

115 120 125

Pro Ile Leu Ser Ala Asn Ile Lys Ala Lys Gly Pro Ala Ser Gln Ile

130 135 140

Ser Gly Leu Tyr Leu Pro Tyr Lys Val Leu Pro Val Gly Asp Glu Val

145 150 155 160

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

165 170 175

Pro Gly Thr Asn Leu Val Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro

180 185 190

Glu Val Asp Lys Leu Lys Thr Leu Asn Val Asn Lys Ile Ile Ala Leu

195 200 205

Gly His Ser Gly Phe Glu Met Asp Lys Leu Ile Ala Gln Lys Val Arg

210 215 220

Gly Val Asp Val Val Val Gly Gly His Ser Asn Thr Phe Leu Tyr Thr

225 230 235 240

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

245 250 255

Val Thr Ser Asp Asp Gly Arg Lys Val Pro Val Val Gln Ala Tyr Ala

260 265 270

Phe Gly Lys Tyr Leu Gly Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly

275 280 285

Asn Val Ile Ser Ser His Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile

290 295 300

Pro Glu Asp Pro Ser Ile Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys

305 310 315 320

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

325 330 335

Asp Gly Ser Ser Gln Ser Cys Arg Phe Arg Glu Cys Asn Met Gly Asn

340 345 350

Leu Ile Cys Asp Ala Met Ile Asn Asn Asn Leu Arg His Thr Asp Glu

355 360 365

Met Phe Trp Asn His Val Ser Met Cys Ile Leu Asn Gly Gly Gly Ile

370 375 380

Arg Ser Pro Ile Asp Glu Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn

385 390 395 400

Leu Ala Ala Val Leu Pro Phe Gly Gly Thr Phe Asp Leu Val Gln Leu

405 410 415

Lys Gly Ser Thr Leu Lys Lys Ala Phe Glu His Ser Val His Arg Tyr

420 425 430

Gly Gln Ser Thr Gly Glu Phe Leu Gln Val Gly Gly Ile His Val Val

435 440 445

Tyr Asp Leu Ser Arg Lys Pro Gly Asp Arg Val Val Lys Leu Asp Val

450 455 460

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

465 470 475 480

Glu Val Tyr Lys Val Ile Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp

485 490 495

Gly Phe Gln Met Ile Lys Asp Glu Leu Leu Arg His Asp Ser Gly Asp

500 505 510

Gln Asp Ile Asn Val Val Ser Thr Tyr Ile Ser Lys Met Lys Val Ile

515 520 525

Tyr Pro Ala Val Glu Gly Arg Ile Lys Phe Ser Gly Ser His His His

530 535 540

His His His

545

<210> 30

<211> 473

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 30

Met Pro Val Pro Trp Phe Leu Leu Ser Leu Ala Leu Gly Arg Ser Pro

1 5 10 15

Val Val Leu Ser Leu Glu Arg Leu Val Gly Pro Gln Asp Ala Thr His

20 25 30

Cys Ser Pro Gly Leu Ser Cys Arg Leu Trp Asp Ser Asp Ile Leu Cys

35 40 45

Leu Pro Gly Asp Ile Val Pro Ala Pro Gly Pro Val Leu Ala Pro Thr

50 55 60

His Leu Gln Thr Glu Leu Val Leu Arg Cys Gln Lys Glu Thr Asp Cys

65 70 75 80

Asp Leu Cys Leu Arg Val Ala Val His Leu Ala Val His Gly His Trp

85 90 95

Glu Glu Pro Glu Asp Glu Glu Lys Phe Gly Gly Ala Ala Asp Ser Gly

100 105 110

Val Glu Glu Pro Arg Asn Ala Ser Leu Gln Ala Gln Val Val Leu Ser

115 120 125

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

130 135 140

Pro Ala Ala Leu Val Gln Phe Gly Gln Ser Val Gly Ser Val Val Tyr

145 150 155 160

Asp Cys Phe Glu Ala Ala Leu Gly Ser Glu Val Arg Ile Trp Ser Tyr

165 170 175

Thr Gln Pro Arg Tyr Glu Lys Glu Leu Asn His Thr Gln Gln Leu Pro

180 185 190

Asp Cys Arg Gly Leu Glu Val Trp Asn Ser Ile Pro Ser Cys Trp Ala

195 200 205

Leu Pro Trp Leu Asn Val Ser Ala Asp Gly Asp Asn Val His Leu Val

210 215 220

Leu Asn Val Ser Glu Glu Gln His Phe Gly Leu Ser Leu Tyr Trp Asn

225 230 235 240

Gln Val Gln Gly Pro Pro Lys Pro Arg Trp His Lys Asn Leu Thr Gly

245 250 255

Pro Gln Ile Ile Thr Leu Asn His Thr Asp Leu Val Pro Cys Leu Cys

260 265 270

Ile Gln Val Trp Pro Leu Glu Pro Asp Ser Val Arg Thr Asn Ile Cys

275 280 285

Pro Phe Arg Glu Asp Pro Arg Ala His Gln Asn Leu Trp Gln Ala Ala

290 295 300

Arg Leu Gln Leu Leu Thr Leu Gln Ser Trp Leu Leu Asp Ala Pro Cys

305 310 315 320

Ser Leu Pro Ala Glu Ala Ala Leu Cys Trp Arg Ala Pro Gly Gly Asp

325 330 335

Pro Cys Gln Pro Leu Val Pro Pro Leu Ser Trp Glu Asn Val Thr Val

340 345 350

Asp Lys Val Leu Glu Phe Pro Leu Leu Lys Gly His Pro Asn Leu Cys

355 360 365

Val Gln Val Asn Ser Ser Glu Lys Leu Gln Leu Gln Glu Cys Leu Trp

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Glu Tyr Leu Leu Gln Asp Leu Gln Ser Gly Gln Cys Leu Gln Leu Trp

435 440 445

Asp Asp Asp Leu Gly Ala Leu Trp Ala Cys Pro Met Asp Lys Tyr Ile

450 455 460

His Gly Ser His His His His His His

465 470

<210> 31

<211> 160

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 31

Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val

1 5 10 15

Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr

20 25 30

Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser

35 40 45

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

50 55 60

Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser

65 70 75 80

Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr

85 90 95

Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys

100 105 110

Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser

115 120 125

Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro

130 135 140

Leu Phe Pro Gly Pro Ser Lys Pro Gly Ser His His His His His His

145 150 155 160

<210> 32

<211> 175

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 32

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

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

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

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

115 120 125

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

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Gly Ser His His His His His His

165 170 175

<210> 33

<211> 148

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 33

Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys

1 5 10 15

Val Leu Thr Gly Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile

20 25 30

Phe His Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val

35 40 45

Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp

50 55 60

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

65 70 75 80

Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu

85 90 95

Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser

100 105 110

Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu

115 120 125

His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Gly Ser His His

130 135 140

His His His His

145

<210> 34

<211> 367

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 34

Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala

1 5 10 15

Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu

20 25 30

Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro

35 40 45

Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val

50 55 60

Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu Pro

65 70 75 80

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

85 90 95

Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Ala Leu Ile Leu

100 105 110

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

115 120 125

Val Lys Leu Glu Arg Leu Tyr Leu Ser Lys Asn Gln Leu Lys Glu Leu

130 135 140

Pro Glu Lys Met Pro Lys Thr Leu Gln Glu Leu Arg Ala His Glu Asn

145 150 155 160

Glu Ile Thr Lys Val Arg Lys Val Thr Phe Asn Gly Leu Asn Gln Met

165 170 175

Ile Val Ile Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser Gly Ile Glu

180 185 190

Asn Gly Ala Phe Gln Gly Met Lys Lys Leu Ser Tyr Ile Arg Ile Ala

195 200 205

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

210 215 220

Glu Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala Ser

225 230 235 240

Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn Ser

245 250 255

Ile Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu Arg

260 265 270

Glu Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Pro Gly Gly Leu

275 280 285

Ala Glu His Lys Tyr Ile Gln Val Val Tyr Leu His Asn Asn Asn Ile

290 295 300

Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly His Asn Thr Lys

305 310 315 320

Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro Val Gln Tyr

325 330 335

Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser Ala

340 345 350

Ile Gln Leu Gly Asn Tyr Lys Gly Ser His His His His His His

355 360 365

<210> 35

<211> 269

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 35

Met Glu Lys Ser Ile Trp Leu Leu Ala Cys Leu Ala Trp Val Leu Pro

1 5 10 15

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

20 25 30

Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala

85 90 95

Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser His Gly Arg

100 105 110

Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg Val

115 120 125

Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val Glu

130 135 140

Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val Arg

145 150 155 160

Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu Pro

165 170 175

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

180 185 190

Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu Ala

195 200 205

Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala Glu

210 215 220

Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala Asn

225 230 235 240

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

245 250 255

Ser Gly Ala Ser Thr Gly Ser His His His His His His

260 265

<210> 36

<211> 185

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 36

Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile

1 5 10 15

Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys

20 25 30

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

35 40 45

Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe

50 55 60

Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe

65 70 75 80

Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser

85 90 95

Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr

100 105 110

Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala

115 120 125

Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu

130 135 140

Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu

145 150 155 160

Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu

165 170 175

His Gly Ser His His His His His His

180 185

<210> 37

<211> 171

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 37

Met Thr Val Lys Thr Leu His Gly Pro Ala Met Val Lys Tyr Leu Leu

1 5 10 15

Leu Ser Ile Leu Gly Leu Ala Phe Leu Ser Glu Ala Ala Ala Arg Lys

20 25 30

Ile Pro Lys Val Gly His Thr Phe Phe Gln Lys Pro Glu Ser Cys Pro

35 40 45

Pro Val Pro Gly Gly Ser Met Lys Leu Asp Ile Gly Ile Ile Asn Glu

50 55 60

Asn Gln Arg Val Ser Met Ser Arg Asn Ile Glu Ser Arg Ser Thr Ser

65 70 75 80

Pro Trp Asn Tyr Thr Val Thr Trp Asp Pro Asn Arg Tyr Pro Ser Glu

85 90 95

Val Val Gln Ala Gln Cys Arg Asn Leu Gly Cys Ile Asn Ala Gln Gly

100 105 110

Lys Glu Asp Ile Ser Met Asn Ser Val Pro Ile Gln Gln Glu Thr Leu

115 120 125

Val Val Arg Arg Lys His Gln Gly Cys Ser Val Ser Phe Gln Leu Glu

130 135 140

Lys Val Leu Val Thr Val Gly Cys Thr Cys Val Thr Pro Val Ile His

145 150 155 160

His Val Gln Gly Ser His His His His His His

165 170

<210> 38

<211> 556

<212> PRT

<213> Artificial Sequence

<220>

<223> recombinant protein

<400> 38

Met Cys Pro Arg Ala Ala Arg Ala Pro Ala Thr Leu Leu Leu Ala Leu

1 5 10 15

Gly Ala Val Leu Trp Pro Ala Ala Gly Ala Trp Glu Leu Thr Ile Leu

20 25 30

His Thr Asn Asp Val His Ser Arg Leu Glu Gln Thr Ser Glu Asp Ser

35 40 45

Ser Lys Cys Val Asn Ala Ser Arg Cys Met Gly Gly Val Ala Arg Leu

50 55 60

Phe Thr Lys Val Gln Gln Ile Arg Arg Ala Glu Pro Asn Val Leu Leu

65 70 75 80

Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr Ile Trp Phe Thr Val Tyr

85 90 95

Lys Gly Ala Glu Val Ala His Phe Met Asn Ala Leu Arg Tyr Asp Ala

100 105 110

Met Ala Leu Gly Asn His Glu Phe Asp Asn Gly Val Glu Gly Leu Ile

115 120 125

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

130 135 140

Lys Ala Lys Gly Pro Ala Ser Gln Ile Ser Gly Leu Tyr Leu Pro Tyr

145 150 155 160

Lys Val Leu Pro Val Gly Asp Glu Val Val Gly Ile Val Gly Tyr Thr

165 170 175

Ser Lys Glu Thr Pro Phe Leu Ser Asn Pro Gly Thr Asn Leu Val Phe

180 185 190

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

195 200 205

Leu Asn Val Asn Lys Ile Ile Ala Leu Gly His Ser Gly Phe Glu Met

210 215 220

Asp Lys Leu Ile Ala Gln Lys Val Arg Gly Val Asp Val Val Val Gly

225 230 235 240

Gly His Ser Asn Thr Phe Leu Tyr Thr Gly Asn Pro Pro Ser Lys Glu

245 250 255

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

260 265 270

Lys Val Pro Val Val Gln Ala Tyr Ala Phe Gly Lys Tyr Leu Gly Tyr

275 280 285

Leu Lys Ile Glu Phe Asp Glu Arg Gly Asn Val Ile Ser Ser His Gly

290 295 300

Asn Pro Ile Leu Leu Asn Ser Ser Ile Pro Glu Asp Pro Ser Ile Lys

305 310 315 320

Ala Asp Ile Asn Lys Trp Arg Ile Lys Leu Asp Asn Tyr Ser Thr Gln

325 330 335

Glu Leu Gly Lys Thr Ile Val Tyr Leu Asp Gly Ser Ser Gln Ser Cys

340 345 350

Arg Phe Arg Glu Cys Asn Met Gly Asn Leu Ile Cys Asp Ala Met Ile

355 360 365

Asn Asn Asn Leu Arg His Thr Asp Glu Met Phe Trp Asn His Val Ser

370 375 380

Met Cys Ile Leu Asn Gly Gly Gly Ile Arg Ser Pro Ile Asp Glu Arg

385 390 395 400

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

405 410 415

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

420 425 430

Ala Phe Glu His Ser Val His Arg Tyr Gly Gln Ser Thr Gly Glu Phe

435 440 445

Leu Gln Val Gly Gly Ile His Val Val Tyr Asp Leu Ser Arg Lys Pro

450 455 460

Gly Asp Arg Val Val Lys Leu Asp Val Leu Cys Thr Lys Cys Arg Val

465 470 475 480

Pro Ser Tyr Asp Pro Leu Lys Met Asp Glu Val Tyr Lys Val Ile Leu

485 490 495

Pro Asn Phe Leu Ala Asn Gly Gly Asp Gly Phe Gln Met Ile Lys Asp

500 505 510

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

515 520 525

Thr Tyr Ile Ser Lys Met Lys Val Ile Tyr Pro Ala Val Glu Gly Arg

530 535 540

Ile Lys Phe Ser Gly Ser His His His His His His

545 550 555

<210> 39

<211> 1413

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding Signal peptide + Human IL-17RC + poly His tag of the present invention

<400> 39

atgagcgccc tgctgatcct ggccctggtg ggcgccgccg tggtgtgggc cctggagcgc 60

ctggtgggcc cccaggacgc cacccactgc agccccggcc tgagctgccg cctgtgggac 120

agcgacatcc tgtgcctgcc cggcgacatc gtgcccgccc ccggccccgt gctggccccc 180

acccacctgc agaccgagct ggtgctgcgc tgccagaagg agaccgactg cgacctgtgc 240

ctgcgcgtgg ccgtgcacct ggccgtgcac ggccactggg aggagcccga ggacgaggag 300

aagttcggcg gcgccgccga cagcggcgtg gaggagcccc gcaacgccag cctgcaggcc 360

caggtggtgc tgagcttcca ggcctacccc accgcccgct gcgtgctgct ggaggtgcag 420

gtgcccgccg ccctggtgca gttcggccag agcgtgggca gcgtggtgta cgactgcttc 480

gaggccgccc tgggcagcga ggtgcgcatc tggagctaca cccagccccg ctacgagaag 540

gagctgaacc acacccagca gctgcccgac tgccgcggcc tggaggtgtg gaacagcatc 600

cccagctgct gggccctgcc ctggctgaac gtgagcgccg acggcgacaa cgtgcacctg 660

gtgctgaacg tgagcgagga gcagcacttc ggcctgagcc tgtactggaa ccaggtgcag 720

ggccccccca agccccgctg gcacaagaac ctgaccggcc cccagatcat caccctgaac 780

cacaccgacc tggtgccctg cctgtgcatc caggtgtggc ccctggagcc cgacagcgtg 840

cgcaccaaca tctgcccctt ccgcgaggac ccccgcgccc accagaacct gtggcaggcc 900

gcccgcctgc agctgctgac cctgcagagc tggctgctgg acgccccctg cagcctgccc 960

gccgaggccg ccctgtgctg gcgcgccccc ggcggcgacc cctgccagcc cctggtgccc 1020

cccctgagct gggagaacgt gaccgtggac aaggtgctgg agttccccct gctgaagggc 1080

caccccaacc tgtgcgtgca ggtgaacagc agcgagaagc tgcagctgca ggagtgcctg 1140

tgggccgaca gcctgggccc cctgaaggac gacgtgctgc tgctggagac ccgcggcccc 1200

caggacaacc gcagcctgtg cgccctggag cccagcggct gcaccagcct gcccagcaag 1260

gccagcaccc gcgccgcccg cctgggcgag tacctgctgc aggacctgca gagcggccag 1320

tgcctgcagc tgtgggacga cgacctgggc gccctgtggg cctgccccat ggacaagtac 1380

atccacggca gccaccacca ccaccaccac taa 1413

<210> 40

<211> 480

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding Signal peptide + Human CD28+ poly His tag of the present invention

<400> 40

atgagcgccc tgctgatcct ggccctggtg ggcgccgccg tggtgtgggc caacaagatc 60

ctggtgaagc agagccccat gctggtggcc tacgacaacg ccgtgaacct gagctgcaag 120

tacagctaca acctgttcag ccgcgagttc cgcgccagcc tgcacaaggg cctggacagc 180

gccgtggagg tgtgcgtggt gtacggcaac tacagccagc agctgcaggt gtacagcaag 240

accggcttca actgcgacgg caagctgggc aacgagagcg tgaccttcta cctgcagaac 300

ctgtacgtga accagaccga catctacttc tgcaagatcg aggtgatgta cccccccccc 360

tacctggaca acgagaagag caacggcacc atcatccacg tgaagggcaa gcacctgtgc 420

cccagccccc tgttccccgg ccccagcaag cccggcagcc accaccacca ccaccactaa 480

<210> 41

<211> 507

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding signal peptide + Human PD-1+ poly His tag of the present invention

<400> 41

atgagcgccc tgctgatcct ggccctggtg ggcgccgccg tggtgtgggc cctggacagc 60

cccgaccgcc cctggaaccc ccccaccttc agccccgccc tgctggtggt gaccgagggc 120

gacaacgcca ccttcacctg cagcttcagc aacaccagcg agagcttcgt gctgaactgg 180

taccgcatga gccccagcaa ccagaccgac aagctggccg ccttccccga ggaccgcagc 240

cagcccggcc aggactgccg cttccgcgtg acccagctgc ccaacggccg cgacttccac 300

atgagcgtgg tgcgcgcccg ccgcaacgac agcggcacct acctgtgcgg cgccatcagc 360

ctggccccca aggcccagat caaggagagc ctgcgcgccg agctgcgcgt gaccgagcgc 420

cgcgccgagg tgcccaccgc ccaccccagc cccagccccc gccccgccgg ccagttccag 480

ggcagccacc accaccacca ccactaa 507

<210> 42

<211> 438

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding Signal peptide + Human ICOS + poly His tag of the present invention

<400> 42

atgagcgccc tgctgatcct ggccctggtg ggcgccgccg tggtgtgggc cgagatcaac 60

ggcagcgcca actacgagat gttcatcttc cacaacggcg gcgtgcagat cctgtgcaag 120

taccccgaca tcgtgcagca gttcaagatg cagctgctga agggcggcca gatcctgtgc 180

gacctgacca agaccaaggg cagcggcaac accgtgagca tcaagagcct gaagttctgc 240

cacagccagc tgagcaacaa cagcgtgagc ttcttcctgt acaacctgga ccacagccac 300

gccaactact acttctgcaa cctgagcatc ttcgaccccc cccccttcaa ggtgaccctg 360

accggcggct acctgcacat ctacgagagc cagctgtgct gccagctgaa gggcagccac 420

caccaccacc accactaa 438

<210> 43

<211> 1107

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding Signal peptide + Human Decorin + Poly His tag of the present invention

<400> 43

atgagcgccc tgctgatcct ggccctggtg ggcgccgccg tggtgtgggc cggccccttc 60

cagcagcgcg gcctgttcga cttcatgctg gaggacgagg ccagcggcat cggccccgag 120

gtgcccgacg accgcgactt cgagcccagc ctgggccccg tgtgcccctt ccgctgccag 180

tgccacctgc gcgtggtgca gtgcagcgac ctgggcctgg acaaggtgcc caaggacctg 240

ccccccgaca ccaccctgct ggacctgcag aacaacaaga tcaccgagat caaggacggc 300

gacttcaaga acctgaagaa cctgcacgcc ctgatcctgg tgaacaacaa gatcagcaag 360

gtgagccccg gcgccttcac ccccctggtg aagctggagc gcctgtacct gagcaagaac 420

cagctgaagg agctgcccga gaagatgccc aagaccctgc aggagctgcg cgcccacgag 480

aacgagatca ccaaggtgcg caaggtgacc ttcaacggcc tgaaccagat gatcgtgatc 540

gagctgggca ccaaccccct gaagagcagc ggcatcgaga acggcgcctt ccagggcatg 600

aagaagctga gctacatccg catcgccgac accaacatca ccagcatccc ccagggcctg 660

ccccccagcc tgaccgagct gcacctggac ggcaacaaga tcagccgcgt ggacgccgcc 720

agcctgaagg gcctgaacaa cctggccaag ctgggcctga gcttcaacag catcagcgcc 780

gtggacaacg gcagcctggc caacaccccc cacctgcgcg agctgcacct ggacaacaac 840

aagctgaccc gcgtgcccgg cggcctggcc gagcacaagt acatccaggt ggtgtacctg 900

cacaacaaca acatcagcgt ggtgggcagc agcgacttct gcccccccgg ccacaacacc 960

aagaaggcca gctacagcgg cgtgagcctg ttcagcaacc ccgtgcagta ctgggagatc 1020

cagcccagca ccttccgctg cgtgtacgtg cgcagcgcca tccagctggg caactacaag 1080

ggcagccacc accaccacca ccactaa 1107

<210> 44

<211> 804

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding Signal peptide + Human Siglec-15+ poly His tag of the present invention

<400> 44

atgagcgccc tgctgatcct ggccctggtg ggcgccgccg tggtgtgggc cttcgtgcgc 60

accaagatcg acaccaccga gaacctgctg aacaccgagg tgcacagcag ccccgcccag 120

cgctggagca tgcaggtgcc ccccgaggtg agcgccgagg ccggcgacgc cgccgtgctg 180

ccctgcacct tcacccaccc ccaccgccac tacgacggcc ccctgaccgc catctggcgc 240

gccggcgagc cctacgccgg cccccaggtg ttccgctgcg ccgccgcccg cggcagcgag 300

ctgtgccaga ccgccctgag ccacggccgc ttccgcctgc tgggcaaccc ccgccgcaac 360

gacctgagcc tgcgcgtgga gcgcctggcc ctggccgacg accgccgcta cttctgccgc 420

gtggagttcg ccggcgacgt gcacgaccgc tacgagagcc gccacggcgt gcgcctgcac 480

gtgaccgccg ccccccgcat cgtgaacatc agcgtgctgc ccagccccgc ccacgccttc 540

cgcgccctgt gcaccgccga gggcgagccc ccccccgccc tggcctggag cggccccgcc 600

ctgggcaaca gcctggccgc cgtgcgcagc ccccgcgagg gccacggcca cctggtgacc 660

gccgagctgc ccgccctgac ccacgacggc cgctacacct gcaccgccgc caacagcctg 720

cgcagcgagg ccagcgtgta cctgttccgc ttccacggcg ccagcggcgc cagcaccggc 780

agccaccacc accaccacca ctaa 804

<210> 45

<211> 534

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding signal peptide + Human IL-7+ poly His tag of the present invention

<400> 45

atgagcgccc tgctgatcct ggccctggtg ggcgccgccg tggtgtgggc cgactgcgac 60

atcgagggca aggacggcaa gcagtacgag agcgtgctga tggtgagcat cgaccagctg 120

ctggacagca tgaaggagat cggcagcaac tgcctgaaca acgagttcaa cttcttcaag 180

cgccacatct gcgacgccaa caaggagggc atgttcctgt tccgcgccgc ccgcaagctg 240

cgccagttcc tgaagatgaa cagcaccggc gacttcgacc tgcacctgct gaaggtgagc 300

gagggcacca ccatcctgct gaactgcacc ggccaggtga agggccgcaa gcccgccgcc 360

ctgggcgagg cccagcccac caagagcctg gaggagaaca agagcctgaa ggagcagaag 420

aagctgaacg acctgtgctt cctgaagcgc ctgctgcagg agatcaagac ctgctggaac 480

aagatcctga tgggcaccaa ggagcacggc agccaccacc accaccacca ctaa 534

<210> 46

<211> 477

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding Signal peptide + HumanIL-17F + poly His tag of the present invention

<400> 46

atgagcgccc tgctgatcct ggccctggtg ggcgccgccg tggtgtgggc ccgcaagatc 60

cccaaggtgg gccacacctt cttccagaag cccgagagct gcccccccgt gcccggcggc 120

agcatgaagc tggacatcgg catcatcaac gagaaccagc gcgtgagcat gagccgcaac 180

atcgagagcc gcagcaccag cccctggaac tacaccgtga cctgggaccc caaccgctac 240

cccagcgagg tggtgcaggc ccagtgccgc aacctgggct gcatcaacgc ccagggcaag 300

gaggacatca gcatgaacag cgtgcccatc cagcaggaga ccctggtggt gcgccgcaag 360

caccagggct gcagcgtgag cttccagctg gagaaggtgc tggtgaccgt gggctgcacc 420

tgcgtgaccc ccgtgatcca ccacgtgcag ggcagccacc accaccacca ccactaa 477

<210> 47

<211> 1644

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding Signal peptide + Human CD73+ poly His tag of the present invention

<400> 47

atgagcgccc tgctgatcct ggccctggtg ggcgccgccg tggtgtgggc ctgggagctg 60

accatcctgc acaccaacga cgtgcacagc cgcctggagc agaccagcga ggacagcagc 120

aagtgcgtga acgccagccg ctgcatgggc ggcgtggccc gcctgttcac caaggtgcag 180

cagatccgcc gcgccgagcc caacgtgctg ctgctggacg ccggcgacca gtaccagggc 240

accatctggt tcaccgtgta caagggcgcc gaggtggccc acttcatgaa cgccctgcgc 300

tacgacgcca tggccctggg caaccacgag ttcgacaacg gcgtggaggg cctgatcgag 360

cccctgctga aggaggccaa gttccccatc ctgagcgcca acatcaaggc caagggcccc 420

gccagccaga tcagcggcct gtacctgccc tacaaggtgc tgcccgtggg cgacgaggtg 480

gtgggcatcg tgggctacac cagcaaggag acccccttcc tgagcaaccc cggcaccaac 540

ctggtgttcg aggacgagat caccgccctg cagcccgagg tggacaagct gaagaccctg 600

aacgtgaaca agatcatcgc cctgggccac agcggcttcg agatggacaa gctgatcgcc 660

cagaaggtgc gcggcgtgga cgtggtggtg ggcggccaca gcaacacctt cctgtacacc 720

ggcaaccccc ccagcaagga ggtgcccgcc ggcaagtacc ccttcatcgt gaccagcgac 780

gacggccgca aggtgcccgt ggtgcaggcc tacgccttcg gcaagtacct gggctacctg 840

aagatcgagt tcgacgagcg cggcaacgtg atcagcagcc acggcaaccc catcctgctg 900

aacagcagca tccccgagga ccccagcatc aaggccgaca tcaacaagtg gcgcatcaag 960

ctggacaact acagcaccca ggagctgggc aagaccatcg tgtacctgga cggcagcagc 1020

cagagctgcc gcttccgcga gtgcaacatg ggcaacctga tctgcgacgc catgatcaac 1080

aacaacctgc gccacaccga cgagatgttc tggaaccacg tgagcatgtg catcctgaac 1140

ggcggcggca tccgcagccc catcgacgag cgcaacaacg gcaccatcac ctgggagaac 1200

ctggccgccg tgctgccctt cggcggcacc ttcgacctgg tgcagctgaa gggcagcacc 1260

ctgaagaagg ccttcgagca cagcgtgcac cgctacggcc agagcaccgg cgagttcctg 1320

caggtgggcg gcatccacgt ggtgtacgac ctgagccgca agcccggcga ccgcgtggtg 1380

aagctggacg tgctgtgcac caagtgccgc gtgcccagct acgaccccct gaagatggac 1440

gaggtgtaca aggtgatcct gcccaacttc ctggccaacg gcggcgacgg cttccagatg 1500

atcaaggacg agctgctgcg ccacgacagc ggcgaccagg acatcaacgt ggtgagcacc 1560

tacatcagca agatgaaggt gatctacccc gccgtggagg gccgcatcaa gttcagcggc 1620

agccaccacc accaccacca ctaa 1644

<210> 48

<211> 1422

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding self signal peptide + Human IL-17RC + poly His tag

<400> 48

atgcccgtgc cctggttcct gctgagcctg gccctgggcc gcagccccgt ggtgctgagc 60

ctggagcgcc tggtgggccc ccaggacgcc acccactgca gccccggcct gagctgccgc 120

ctgtgggaca gcgacatcct gtgcctgccc ggcgacatcg tgcccgcccc cggccccgtg 180

ctggccccca cccacctgca gaccgagctg gtgctgcgct gccagaagga gaccgactgc 240

gacctgtgcc tgcgcgtggc cgtgcacctg gccgtgcacg gccactggga ggagcccgag 300

gacgaggaga agttcggcgg cgccgccgac agcggcgtgg aggagccccg caacgccagc 360

ctgcaggccc aggtggtgct gagcttccag gcctacccca ccgcccgctg cgtgctgctg 420

gaggtgcagg tgcccgccgc cctggtgcag ttcggccaga gcgtgggcag cgtggtgtac 480

gactgcttcg aggccgccct gggcagcgag gtgcgcatct ggagctacac ccagccccgc 540

tacgagaagg agctgaacca cacccagcag ctgcccgact gccgcggcct ggaggtgtgg 600

aacagcatcc ccagctgctg ggccctgccc tggctgaacg tgagcgccga cggcgacaac 660

gtgcacctgg tgctgaacgt gagcgaggag cagcacttcg gcctgagcct gtactggaac 720

caggtgcagg gcccccccaa gccccgctgg cacaagaacc tgaccggccc ccagatcatc 780

accctgaacc acaccgacct ggtgccctgc ctgtgcatcc aggtgtggcc cctggagccc 840

gacagcgtgc gcaccaacat ctgccccttc cgcgaggacc cccgcgccca ccagaacctg 900

tggcaggccg cccgcctgca gctgctgacc ctgcagagct ggctgctgga cgccccctgc 960

agcctgcccg ccgaggccgc cctgtgctgg cgcgcccccg gcggcgaccc ctgccagccc 1020

ctggtgcccc ccctgagctg ggagaacgtg accgtggaca aggtgctgga gttccccctg 1080

ctgaagggcc accccaacct gtgcgtgcag gtgaacagca gcgagaagct gcagctgcag 1140

gagtgcctgt gggccgacag cctgggcccc ctgaaggacg acgtgctgct gctggagacc 1200

cgcggccccc aggacaaccg cagcctgtgc gccctggagc ccagcggctg caccagcctg 1260

cccagcaagg ccagcacccg cgccgcccgc ctgggcgagt acctgctgca ggacctgcag 1320

agcggccagt gcctgcagct gtgggacgac gacctgggcg ccctgtgggc ctgccccatg 1380

gacaagtaca tccacggcag ccaccaccac caccaccact aa 1422

<210> 49

<211> 483

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding self signal peptide + Human CD28+ poly His tag

<400> 49

atgctgcgcc tgctgctggc cctgaacctg ttccccagca tccaggtgac cggcaacaag 60

atcctggtga agcagagccc catgctggtg gcctacgaca acgccgtgaa cctgagctgc 120

aagtacagct acaacctgtt cagccgcgag ttccgcgcca gcctgcacaa gggcctggac 180

agcgccgtgg aggtgtgcgt ggtgtacggc aactacagcc agcagctgca ggtgtacagc 240

aagaccggct tcaactgcga cggcaagctg ggcaacgaga gcgtgacctt ctacctgcag 300

aacctgtacg tgaaccagac cgacatctac ttctgcaaga tcgaggtgat gtaccccccc 360

ccctacctgg acaacgagaa gagcaacggc accatcatcc acgtgaaggg caagcacctg 420

tgccccagcc ccctgttccc cggccccagc aagcccggca gccaccacca ccaccaccac 480

taa 483

<210> 50

<211> 528

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding self signal peptide + Human PD-1+ poly His tag

<400> 50

atgcagatcc cccaggcccc ctggcccgtg gtgtgggccg tgctgcagct gggctggcgc 60

cccggctggt tcctggacag ccccgaccgc ccctggaacc cccccacctt cagccccgcc 120

ctgctggtgg tgaccgaggg cgacaacgcc accttcacct gcagcttcag caacaccagc 180

gagagcttcg tgctgaactg gtaccgcatg agccccagca accagaccga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgcgt gacccagctg 300

cccaacggcc gcgacttcca catgagcgtg gtgcgcgccc gccgcaacga cagcggcacc 360

tacctgtgcg gcgccatcag cctggccccc aaggcccaga tcaaggagag cctgcgcgcc 420

gagctgcgcg tgaccgagcg ccgcgccgag gtgcccaccg cccaccccag ccccagcccc 480

cgccccgccg gccagttcca gggcagccac caccaccacc accactaa 528

<210> 51

<211> 447

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding self signal peptide + Human ICOS + poly His tag

<400> 51

atgaagagcg gcctgtggta cttcttcctg ttctgcctgc gcatcaaggt gctgaccggc 60

gagatcaacg gcagcgccaa ctacgagatg ttcatcttcc acaacggcgg cgtgcagatc 120

ctgtgcaagt accccgacat cgtgcagcag ttcaagatgc agctgctgaa gggcggccag 180

atcctgtgcg acctgaccaa gaccaagggc agcggcaaca ccgtgagcat caagagcctg 240

aagttctgcc acagccagct gagcaacaac agcgtgagct tcttcctgta caacctggac 300

cacagccacg ccaactacta cttctgcaac ctgagcatct tcgacccccc ccccttcaag 360

gtgaccctga ccggcggcta cctgcacatc tacgagagcc agctgtgctg ccagctgaag 420

ggcagccacc accaccacca ccactaa 447

<210> 52

<211> 1104

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding self signal peptide + Human Decorin + poly His tag

<400> 52

atgaaggcca ccatcatcct gctgctgctg gcccaggtga gctgggccgg ccccttccag 60

cagcgcggcc tgttcgactt catgctggag gacgaggcca gcggcatcgg ccccgaggtg 120

cccgacgacc gcgacttcga gcccagcctg ggccccgtgt gccccttccg ctgccagtgc 180

cacctgcgcg tggtgcagtg cagcgacctg ggcctggaca aggtgcccaa ggacctgccc 240

cccgacacca ccctgctgga cctgcagaac aacaagatca ccgagatcaa ggacggcgac 300

ttcaagaacc tgaagaacct gcacgccctg atcctggtga acaacaagat cagcaaggtg 360

agccccggcg ccttcacccc cctggtgaag ctggagcgcc tgtacctgag caagaaccag 420

ctgaaggagc tgcccgagaa gatgcccaag accctgcagg agctgcgcgc ccacgagaac 480

gagatcacca aggtgcgcaa ggtgaccttc aacggcctga accagatgat cgtgatcgag 540

ctgggcacca accccctgaa gagcagcggc atcgagaacg gcgccttcca gggcatgaag 600

aagctgagct acatccgcat cgccgacacc aacatcacca gcatccccca gggcctgccc 660

cccagcctga ccgagctgca cctggacggc aacaagatca gccgcgtgga cgccgccagc 720

ctgaagggcc tgaacaacct ggccaagctg ggcctgagct tcaacagcat cagcgccgtg 780

gacaacggca gcctggccaa caccccccac ctgcgcgagc tgcacctgga caacaacaag 840

ctgacccgcg tgcccggcgg cctggccgag cacaagtaca tccaggtggt gtacctgcac 900

aacaacaaca tcagcgtggt gggcagcagc gacttctgcc cccccggcca caacaccaag 960

aaggccagct acagcggcgt gagcctgttc agcaaccccg tgcagtactg ggagatccag 1020

cccagcacct tccgctgcgt gtacgtgcgc agcgccatcc agctgggcaa ctacaagggc 1080

agccaccacc accaccacca ctaa 1104

<210> 53

<211> 810

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding self signal peptide + Human Siglec-15+ poly His tag

<400> 53

atggagaaga gcatctggct gctggcctgc ctggcctggg tgctgcccac cggcagcttc 60

gtgcgcacca agatcgacac caccgagaac ctgctgaaca ccgaggtgca cagcagcccc 120

gcccagcgct ggagcatgca ggtgcccccc gaggtgagcg ccgaggccgg cgacgccgcc 180

gtgctgccct gcaccttcac ccacccccac cgccactacg acggccccct gaccgccatc 240

tggcgcgccg gcgagcccta cgccggcccc caggtgttcc gctgcgccgc cgcccgcggc 300

agcgagctgt gccagaccgc cctgagccac ggccgcttcc gcctgctggg caacccccgc 360

cgcaacgacc tgagcctgcg cgtggagcgc ctggccctgg ccgacgaccg ccgctacttc 420

tgccgcgtgg agttcgccgg cgacgtgcac gaccgctacg agagccgcca cggcgtgcgc 480

ctgcacgtga ccgccgcccc ccgcatcgtg aacatcagcg tgctgcccag ccccgcccac 540

gccttccgcg ccctgtgcac cgccgagggc gagccccccc ccgccctggc ctggagcggc 600

cccgccctgg gcaacagcct ggccgccgtg cgcagccccc gcgagggcca cggccacctg 660

gtgaccgccg agctgcccgc cctgacccac gacggccgct acacctgcac cgccgccaac 720

agcctgcgca gcgaggccag cgtgtacctg ttccgcttcc acggcgccag cggcgccagc 780

accggcagcc accaccacca ccaccactaa 810

<210> 54

<211> 558

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding self signal peptide + Human IL-7+ poly His tag

<400> 54

atgttccacg tgagcttccg ctacatcttc ggcctgcccc ccctgatcct ggtgctgctg 60

cccgtggcca gcagcgactg cgacatcgag ggcaaggacg gcaagcagta cgagagcgtg 120

ctgatggtga gcatcgacca gctgctggac agcatgaagg agatcggcag caactgcctg 180

aacaacgagt tcaacttctt caagcgccac atctgcgacg ccaacaagga gggcatgttc 240

ctgttccgcg ccgcccgcaa gctgcgccag ttcctgaaga tgaacagcac cggcgacttc 300

gacctgcacc tgctgaaggt gagcgagggc accaccatcc tgctgaactg caccggccag 360

gtgaagggcc gcaagcccgc cgccctgggc gaggcccagc ccaccaagag cctggaggag 420

aacaagagcc tgaaggagca gaagaagctg aacgacctgt gcttcctgaa gcgcctgctg 480

caggagatca agacctgctg gaacaagatc ctgatgggca ccaaggagca cggcagccac 540

caccaccacc accactaa 558

<210> 55

<211> 516

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding self signal peptide + HumanIL-17F + poly His tag

<400> 55

atgaccgtga agaccctgca cggccccgcc atggtgaagt acctgctgct gagcatcctg 60

ggcctggcct tcctgagcga ggccgccgcc cgcaagatcc ccaaggtggg ccacaccttc 120

ttccagaagc ccgagagctg cccccccgtg cccggcggca gcatgaagct ggacatcggc 180

atcatcaacg agaaccagcg cgtgagcatg agccgcaaca tcgagagccg cagcaccagc 240

ccctggaact acaccgtgac ctgggacccc aaccgctacc ccagcgaggt ggtgcaggcc 300

cagtgccgca acctgggctg catcaacgcc cagggcaagg aggacatcag catgaacagc 360

gtgcccatcc agcaggagac cctggtggtg cgccgcaagc accagggctg cagcgtgagc 420

ttccagctgg agaaggtgct ggtgaccgtg ggctgcacct gcgtgacccc cgtgatccac 480

cacgtgcagg gcagccacca ccaccaccac cactaa 516

<210> 56

<211> 1671

<212> DNA

<213> Artificial Sequence

<220>

<223> sequence encoding self signal peptide + Human CD73+ poly His tag

<400> 56

atgtgccccc gcgccgcccg cgcccccgcc accctgctgc tggccctggg cgccgtgctg 60

tggcccgccg ccggcgcctg ggagctgacc atcctgcaca ccaacgacgt gcacagccgc 120

ctggagcaga ccagcgagga cagcagcaag tgcgtgaacg ccagccgctg catgggcggc 180

gtggcccgcc tgttcaccaa ggtgcagcag atccgccgcg ccgagcccaa cgtgctgctg 240

ctggacgccg gcgaccagta ccagggcacc atctggttca ccgtgtacaa gggcgccgag 300

gtggcccact tcatgaacgc cctgcgctac gacgccatgg ccctgggcaa ccacgagttc 360

gacaacggcg tggagggcct gatcgagccc ctgctgaagg aggccaagtt ccccatcctg 420

agcgccaaca tcaaggccaa gggccccgcc agccagatca gcggcctgta cctgccctac 480

aaggtgctgc ccgtgggcga cgaggtggtg ggcatcgtgg gctacaccag caaggagacc 540

cccttcctga gcaaccccgg caccaacctg gtgttcgagg acgagatcac cgccctgcag 600

cccgaggtgg acaagctgaa gaccctgaac gtgaacaaga tcatcgccct gggccacagc 660

ggcttcgaga tggacaagct gatcgcccag aaggtgcgcg gcgtggacgt ggtggtgggc 720

ggccacagca acaccttcct gtacaccggc aaccccccca gcaaggaggt gcccgccggc 780

aagtacccct tcatcgtgac cagcgacgac ggccgcaagg tgcccgtggt gcaggcctac 840

gccttcggca agtacctggg ctacctgaag atcgagttcg acgagcgcgg caacgtgatc 900

agcagccacg gcaaccccat cctgctgaac agcagcatcc ccgaggaccc cagcatcaag 960

gccgacatca acaagtggcg catcaagctg gacaactaca gcacccagga gctgggcaag 1020

accatcgtgt acctggacgg cagcagccag agctgccgct tccgcgagtg caacatgggc 1080

aacctgatct gcgacgccat gatcaacaac aacctgcgcc acaccgacga gatgttctgg 1140

aaccacgtga gcatgtgcat cctgaacggc ggcggcatcc gcagccccat cgacgagcgc 1200

aacaacggca ccatcacctg ggagaacctg gccgccgtgc tgcccttcgg cggcaccttc 1260

gacctggtgc agctgaaggg cagcaccctg aagaaggcct tcgagcacag cgtgcaccgc 1320

tacggccaga gcaccggcga gttcctgcag gtgggcggca tccacgtggt gtacgacctg 1380

agccgcaagc ccggcgaccg cgtggtgaag ctggacgtgc tgtgcaccaa gtgccgcgtg 1440

cccagctacg accccctgaa gatggacgag gtgtacaagg tgatcctgcc caacttcctg 1500

gccaacggcg gcgacggctt ccagatgatc aaggacgagc tgctgcgcca cgacagcggc 1560

gaccaggaca tcaacgtggt gagcacctac atcagcaaga tgaaggtgat ctaccccgcc 1620

gtggagggcc gcatcaagtt cagcggcagc caccaccacc accaccacta a 1671

Claims

1. A polypeptide of (a) or (b) below:

(a) polypeptide consisting of an amino acid sequence shown in SEQ ID No. 1;

2. The polypeptide of claim 1, wherein the polypeptide consisting of the amino acid sequence shown in SEQ ID No.1 with one or more amino acid substitutions, deletions and/or additions and having the same function as the polypeptide shown in SEQ ID No.1 at least comprises a sequence of at least 9 consecutive amino acids, preferably at least 10 amino acids, more preferably at least 11 amino acids at the C-terminal end of the amino acid sequence shown in SEQ ID No. 1.

3. A polynucleotide encoding the polypeptide of claim 1 or 2.

4. An expression vector comprising the polynucleotide of claim 3.

5. A mammalian cell comprising the polynucleotide of claim 3 or the expression vector of claim 4.

6. Use of the polypeptide of claim 1 or 2 as a signal peptide for secretory expression of a protein of interest in a mammalian cell.

7. A method for secretory expression of a protein of interest in a mammalian cell, the method comprising:

secretory expression of a target protein in a mammalian cell containing the polynucleotide of claim 3 or the expression vector of claim 4.

8. The method of claim 7, wherein the mammalian cells are selected from one or more of Chinese Hamster Ovary (CHO), mouse plasmacytoma (A non-Ig differentiation, non-light-synthesizing subclone of NS-1, NSO), Baby Hamster Kidney (BHK), human embryonic kidney293 (HEK 293) cells.

9. The method according to claim 7, wherein the protein of interest is selected from one or more of Human IL-17RC, Human CD28, Human PD-1, Human ICOS, Human Decorin, Human Siglec-15, Human IL-7, Human IL-17F, and Human CD 73.

10. The method of claim 7, comprising:

ligating a polynucleotide encoding the polypeptide of claim 1 or 2 with a polynucleotide encoding a protein of interest, said polynucleotide encoding the polypeptide of claim 1 or 2 being positioned at the front end of said polynucleotide encoding the protein of interest, and cloning into a mammalian cell expression vector;