A growth hormone fusion protein and its application

Abstract

The present invention proposes a growth hormone fusion protein and its application. The fusion protein includes an Fc mutant, which includes: a first peptide chain, wherein the first peptide chain 1) has a mutation at the following sites compared to the wild-type IgG4Fc fragment: at least one of the 234th, 235th, 298th, 299th, and 300th positions; or 231-239th positions; or 2) has a mutation at the following sites compared to the wild-type IgG1Fc fragment: at least one of the 228th-239th positions, and / or at least one of the 268th, 296th, 330th, and 331st positions. The fusion protein has a strong binding activity with human growth hormone receptors, can eliminate ADCC and CDC effects, promote Nb2-11 cell proliferation, and thus effectively treat or prevent diseases related to abnormal human growth hormone.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, specifically, the present invention relates to a growth hormone fusion protein and its application, more specifically, the present invention relates to an Fc mutant, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a composition, a pharmaceutical use and a drug. Background Art

[0002] Growth hormone deficiency is a well-recognized clinical syndrome associated with numerous metabolic abnormalities, including abnormal body composition, decreased physical fitness, altered lipid metabolism, decreased bone mass, increased insulin resistance, and reduced quality of life. Most metabolic abnormalities associated with growth hormone deficiency can be reversed by recombinant human growth hormone (rhGH) replacement. Traditional treatment for growth hormone deficiency involves daily subcutaneous injections of rhGH. However, the injection schedule is too frequent and inconvenient for many patients, raising concerns about poor treatment compliance, which may lead to reduced efficacy. Long-acting rhGH preparations can not only reduce the number of injections, thereby improving compliance, but also help improve the efficacy of GH therapy.

[0003] Studies have been conducted to prepare fusion proteins using immunoglobulin Fc to increase the half-life of fusion proteins (CheolRyong Ku et al., Eur J Endocrinol. 2018 Sep; 179(3): 169-179; Wolfgang Glaesner et al., Diabetes Metab Res Rev. 2010 May; 26(4): 287-96.). Since the biological activity of growth hormone requires a certain spatial effect, improper connection of Fc in the fusion protein will cause a steric hindrance effect. However, studies have found that the use of GGGGSGGGSGGGGS or partial fragments of Genexine GX-H9 to connect with Fc all have problems such as low binding affinity to human growth hormone receptors, low activity in promoting cell proliferation, and low in vitro activity.

[0004] Therefore, there is still an urgent need for a fusion protein that has high binding activity to human growth hormone receptor and can promote T cell proliferation. Summary of the invention

[0005] This application is filed based on the inventor's discovery of the following issues and facts:

[0006] In the first aspect of the present invention, the present invention proposes an Fc mutant. According to an embodiment of the present invention, it comprises: a first peptide chain, wherein the first peptide chain 1) has a mutation at the following sites compared to the wild-type IgG4 Fc fragment: at least one of the 234th, 235th, 298th, 299th, and 300th positions; or 231-239th positions; or 2) has a mutation at the following sites compared to the wild-type IgG1 Fc fragment: at least one of the 228th-239th positions, and / or, at least one of the 268th, 296th, 330th, and 331st positions. The inventors performed the above mutations on the Fc fragment of IgG4 or IgG1 antibodies respectively. Mutations at different sites have different effects. The Fc mutant according to the embodiment of the present invention can effectively reduce the fragment content in the preparation process of the Fc fragment, avoid the formation of half antibodies, and eliminate ADCC and CDC effects. The fusion protein prepared using the Fc mutant described in the present application has a strong binding activity with the human growth hormone receptor, can eliminate ADCC and CDC effects, promote Nb2-11 cell proliferation, and can effectively treat or prevent diseases related to abnormal human growth hormone.

[0007] According to an embodiment of the present invention, the above-mentioned Fc mutant may further include at least one of the following additional technical features:

[0008] According to an embodiment of the present invention, the Fc mutant cannot mutate the amino acid at position 235 to alanine.

[0009] According to an embodiment of the present invention, the sequence of the Fc mutant is not SEQ ID NO:69.

[0010] Amino acid sequence of the Fc mutant exclusion sequence (first peptide chain 6) (having F234V and L235A mutations compared to the Fc fragment of the wild-type IgG4 antibody):

[0011] APEVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:69).

[0012] According to an embodiment of the present invention, the Fc mutant cannot have, and only has, amino acid mutations at positions 234 and 235.

[0013] According to an embodiment of the present invention, the Fc mutant further includes a second peptide chain, the C-terminus of the second peptide chain is connected to the N-terminus of the first peptide chain, and the second peptide chain has the following mutation sites: 1) compared with the hinge region of wild-type IgG4, it has a mutation at the following site: position 228; or 2) has a wild-type IgD fragment 1 or an IgD fragment 1 mutant.

[0014] The hinge region of wild-type IgG4 includes:

[0015] ESKYGPPCPSCP (SEQ ID NO: 72).

[0016] According to an embodiment of the present invention, the wild-type IgD fragment 1 includes amino acids 287-298 of wild-type IgD.

[0017] According to an embodiment of the present invention, the IgD fragment 1 mutant has mutations at the following sites compared to the wild-type IgD fragment 1: at least one of positions 291-296.

[0018] According to an embodiment of the present invention, the Fc mutant further includes a third peptide chain, the C-terminus of the third peptide chain is connected to the N-terminus of the second peptide chain, and the third peptide chain has at least one of the following mutation sites compared to the wild-type IgD fragment 2: position 263, position 264, position 266, position 267, position 268, position 269, position 270, position 271, position 272, position 273, position 274, position 275, position 276, position 277, position 278, position 280, position 281, position 283, position 284, position 285, position 286, position 287, position 288, position 289, position 290, and position 291.

[0019] According to an embodiment of the present invention, the wild-type IgD fragment 2 includes amino acids 262-291 of wild-type IgD.

[0020] According to an embodiment of the present invention, the first peptide chain 1) has mutations at the following sites compared to the wild-type IgG4 Fc fragment: positions 234 and 235; or positions 298, 299, 300; or positions 231-239; or 2) has mutations at the following sites compared to the wild-type IgG1 Fc fragment: positions 228-239, 268, 296, 330 and 331.

[0021] According to an embodiment of the present invention, the second peptide chain 1) has a mutation at the following position compared to the hinge region of wild-type IgG4: position 228; or 2) has a wild-type IgD fragment 1 or an IgD fragment 1 mutant.

[0022] According to an embodiment of the present invention, the IgD fragment 1 mutant has the following mutations compared to the wild-type IgD fragment 1: positions 291-296.

[0023] According to an embodiment of the present invention, the third peptide chain has the following mutation sites compared to the wild-type IgD fragment 2: 1) positions 266, 267, 268, 277, 278, 280, 283, 285, 286 and 287; or 2) positions 266, 277, 278, 280, 283, 285, 286 and 287. and 287th; or 3) 266th, 269th, 270th, 271st, 273rd, 274th, 275th, 277th, 278th, 281st, and 283rd; or 4) 263rd, 264th, 266th, 269th, 270th, 272nd, 274th, 276th, 284th, 287-291st.

[0024] According to an embodiment of the present invention, the first peptide chain 1) has the following mutations compared to the wild-type IgG4 Fc fragment: F234A and L235E; or F234V and L235E; or S298N, T299A and Y300S; or amino acid deletion at positions 231-239; or 2) has the following mutations compared to the wild-type IgG1 Fc fragment: amino acid deletion at positions 228-239, H268Q, Y296F, A330S and P331S. According to a specific embodiment of the present invention, mutations at different sites have different effects, and therefore, the Fc mutants obtained by combining the various mutation sites have different effects, such as, F234V&L235E can eliminate the ADCC effect, and S298N&T299A&Y300S can eliminate the ADCC and CDC effects.

[0025] According to an embodiment of the present invention, the second peptide chain has the following mutation compared to the hinge region of wild-type IgG4: S228P; or 2) has wild-type IgD fragment 1 or IgD fragment 1 mutant. As mentioned above, mutations at different sites have different effects, so the Fc mutants obtained by combining various mutation sites have different effects, among which S228P can prevent the IgG4 constant region from forming a half antibody.

[0026] According to an embodiment of the present invention, the IgD fragment 1 mutant has the following mutations compared to the wild-type IgD fragment 1: P291C, S292H, H293P, T294R, Q295L, P296S.

[0027] According to an embodiment of the present invention, the third peptide chain has mutations such as: 1) R266E, G267R, G268E, K277E, E278K, Q280E, R283E, T285E, K286G and T287E compared to the wild-type IgD fragment 2; or 2) R266K, K277E, E278K, Q280N, R283E, T285E, K286R and T287E; or 3) R266K, E269D, E270D, K271E, K273E, E274D, K275E, K277E, E278K, E281Q and R283G; or 4) N263Q, T264S, R266K, E269D, E270D, K272R, E274D, E276D, E284D and 287-291 deletions.

[0028] Wild-type IgD fragment 2 includes the following amino acid sequence:

[0029] RNTGRGGEEKKKEKEKEEQEERETKTPECP (SEQ ID NO: 44).

[0030] According to an embodiment of the present invention, the first peptide chain has an amino acid sequence shown in any one of SEQ ID NOs: 55, 56, 57, 58, 68, and 69.

[0031] According to an embodiment of the present invention, the second peptide chain has an amino acid sequence shown in any one of SEQ ID NOs: 59, 60, and 61.

[0032] According to an embodiment of the present invention, the third peptide chain has an amino acid sequence shown in any one of SEQ ID NOs: 62, 63, 64, and 65.

[0033] According to an embodiment of the present invention, the wild-type IgG4, wild-type IgG1, and wild-type IgD are human antibodies.

[0034] According to an embodiment of the present invention, the Fc mutant includes: 1) an amino acid sequence shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, and 54; or 2) an amino acid sequence that has at least 90% identity with the amino acid sequence shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, and 54.

[0035] In the second aspect of the present invention, the present invention proposes a fusion protein. According to an embodiment of the present invention, it includes a first peptide segment, the first peptide segment includes a biologically active molecule functional region; a second peptide segment, the second peptide segment includes an Fc mutant, and the Fc mutant is as defined in the first aspect, wherein the N-terminus of the second peptide segment is connected to the C-terminus of the first peptide segment. The fusion protein according to the embodiment of the present invention has a strong binding activity with the human growth hormone receptor, can effectively eliminate ADCC and CDC effects, promote Nb2-11 cell proliferation, and effectively treat or prevent diseases related to abnormal human growth hormone.

[0036] According to an embodiment of the present invention, the above fusion protein may further include at least one of the following additional technical features:

[0037] According to an embodiment of the present invention, the first peptide segment comprises growth hormone or a growth hormone analog, or a growth hormone functional region or a growth hormone analog functional region.

[0038] According to an embodiment of the present invention, the first peptide segment comprises human growth hormone or a functional region of human growth hormone.

[0039] According to an embodiment of the present invention, the human growth hormone has an amino acid sequence as shown in SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0040] According to an embodiment of the present invention, the fusion protein further includes a connecting peptide.

[0041] According to an embodiment of the present invention, the N-terminus of the connecting peptide is connected to the C-terminus of the first peptide segment, and the C-terminus of the connecting peptide is connected to the N-terminus of the second peptide segment.

[0042] According to an embodiment of the present invention, the connecting peptide has the amino acid sequence described in SEQ ID NO:46.

[0043] According to an embodiment of the present invention, compared with the amino acid sequence shown in SEQ ID NO:46, at least one GGGGS of the connecting peptide has at least one of S>T, a G mutation added at the nitrogen terminal, and an L mutation added at the carbon terminal.

[0044] (G4S)n (SEQ ID NO: 46), wherein n is an integer greater than 0, preferably n is 2-6.

[0045] According to an embodiment of the present invention, the connecting peptide has an amino acid sequence shown in SEQ ID NO:11.

[0046] According to an embodiment of the present invention, the fusion protein comprises: 1) an amino acid sequence shown in any one of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28; or 2) an amino acid sequence having at least 90% or at least 95% identity with the amino acid sequence shown in any one of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28. In the present application, the inventor unexpectedly found that the fusion protein obtained by combining the second peptide segment with the connecting peptide has significantly better binding activity to human growth hormone receptor and Nb2-11 cell proliferation promoting effect.

[0047] In the third aspect of the present invention, the present invention proposes a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the optimized Fc mutant described above or the fusion protein described above. The Fc mutant obtained by the nucleic acid molecule according to the embodiment of the present invention can effectively avoid the formation of half antibodies, and eliminate ADCC and CDC effects. The obtained fusion protein has a strong binding activity with the human growth hormone receptor, can effectively eliminate ADCC and CDC effects, promote Nb2-11 cell proliferation, and effectively treat or prevent diseases related to abnormal human growth hormone.

[0048] According to an embodiment of the present invention, the above nucleic acid molecule may further include at least one of the following additional technical features:

[0049] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0050] According to an embodiment of the present invention, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NO:32, 33, 34, 35, 36, 37, 38, 39 or SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20.

[0051] It should be noted that, for the nucleic acids mentioned in the specification and claims of the present invention, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, in the specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequence in the present application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0052] In the fourth aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the aforementioned nucleic acid molecule is carried. When the aforementioned nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control element on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, they are not from the vector itself. Of course, the nucleic acid molecule can be operably connected to the control element. Herein, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can play their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into a suitable receptor cell, the expression of the aforementioned Fc mutant or fusion protein can be effectively achieved under the mediation of the regulatory system, thereby achieving the in vitro acquisition of the Fc mutant or fusion protein in large quantities.

[0053] According to an embodiment of the present invention, the above expression vector may further include at least one of the following additional technical features:

[0054] According to an embodiment of the present invention, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector or a viral vector.

[0055] According to an embodiment of the present invention, the virus comprises a lentivirus.

[0056] In a fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the aforementioned nucleic acid molecule, expression vector, or expresses the aforementioned Fc mutant or fusion protein. The recombinant cell according to an embodiment of the present invention can be used for in vitro expression and mass production of the aforementioned Fc mutant or fusion protein.

[0057] According to an embodiment of the present invention, the above-mentioned recombinant cell may further include at least one of the following additional technical features:

[0058] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0059] According to an embodiment of the present invention, the expression vector is introduced into the host cell by electrotransduction.

[0060] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell.

[0061] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0062] According to some specific embodiments of the present invention, the eukaryotic cells do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0063] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells may be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0064] It should be noted that the "suitable conditions" described in the specification of this application refer to conditions suitable for the expression of the Fc mutant or fusion protein described in this application. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the Fc mutant or fusion protein include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the Fc mutant or fusion protein according to the specific environment of the laboratory.

[0065] In the sixth aspect of the present invention, the present invention proposes a composition. According to an embodiment of the present invention, it includes: the fusion protein, nucleic acid molecule, expression vector or recombinant cell described above. As mentioned above, the fusion protein, and the fusion protein obtained after the expression of the nucleic acid molecule, expression vector or recombinant cell can not only effectively bind to the human growth hormone receptor, but also effectively promote the proliferation of Nb2-11 cells. Therefore, the fusion protein contained in the composition according to the embodiment of the present invention or the expressed fusion protein can bind to the human growth hormone receptor, promote the proliferation of Nb2-11 cells, and effectively treat or prevent diseases related to abnormal growth hormone.

[0066] Those skilled in the art will appreciate that the composition includes food compositions, pharmaceutical compositions, etc. The composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the ingredients contained in the composition can be applied to the subject as a whole, or separately. When the ingredients contained in the composition are applied to the subject separately, each ingredient can be applied to the subject simultaneously or sequentially.

[0067] In the seventh aspect of the present invention, the present invention provides the use of the above-mentioned fusion protein, nucleic acid molecule, expression vector, recombinant cell or composition in the preparation of a drug. According to an embodiment of the present invention, the drug is used to treat or prevent diseases related to abnormal growth hormone.

[0068] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0069] According to an embodiment of the present invention, the abnormal growth hormone-related diseases include at least one selected from the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by the alienation state during chemotherapy and AIDS treatment, and intrauterine growth retardation.

[0070] In the eighth aspect of the present invention, the present invention proposes a drug. According to an embodiment of the present invention, it includes the fusion protein, nucleic acid molecule, expression vector, recombinant cell or composition described above. As mentioned above, the fusion protein, and the fusion protein obtained after the expression of the nucleic acid molecule, expression vector, or recombinant cell can not only effectively bind to the human growth hormone receptor, but also effectively promote the proliferation of Nb2-11 cells. Therefore, the fusion protein contained in the drug according to the embodiment of the present invention or the expressed fusion protein can bind to the human growth hormone receptor, promote the proliferation of Nb2-11 cells, and effectively treat or prevent diseases related to abnormal growth hormone.

[0071] According to an embodiment of the present invention, the above-mentioned medicine may further include at least one of the following additional technical features:

[0072] According to an embodiment of the present invention, the medicine further includes a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, adhesive, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid adhesive, glidant or lubricant, etc., suitable for a specific target dosage form. Except for the scope that any conventional excipient is incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also the scope considered by the present invention.

[0073] For example, the fusion protein of the present invention can be incorporated into a drug suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersants or suspending agents, tablets, pills, powders, liposomes and suppositories. Typical drugs are in the form of injection solutions or infusion solutions. The fusion protein can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0074] In the ninth aspect of the present invention, the present invention proposes a method for preventing or treating diseases related to abnormal growth hormone. According to an embodiment of the present invention, it includes administering to a subject at least one of the following: 1) the fusion protein described above; 2) the nucleic acid molecule described above; 3) the expression vector described above; 4) the recombinant cell described above; 5) the composition described above; and 6) the drug described above. As described above, the fusion protein, and the fusion protein obtained after the expression of the nucleic acid molecule, expression vector, or recombinant cell, can not only effectively bind to the human growth hormone receptor, but also effectively promote the proliferation of Nb2-11 cells. Therefore, the above-mentioned substances and the compositions and drugs containing the above-mentioned substances can effectively treat or prevent diseases related to abnormal growth hormone.

[0075] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0076] According to an embodiment of the present invention, the abnormal growth hormone-related diseases include at least one selected from the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by the alienation state during chemotherapy and AIDS treatment, and intrauterine growth retardation. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 is a schematic diagram of the structure of a fusion protein according to an embodiment of the present invention;

[0078] Figure 2 This is a result diagram of the ADCC effect induced by fusion protein 9# according to an example of the present invention. DETAILED DESCRIPTION

[0079] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0080] In the process of describing the present invention, the relevant terms in this document are explained and illustrated. These explanations and illustrations are only for the convenience of understanding of the scheme and cannot be regarded as limitations on the protection scheme of the present invention.

[0081] The fusion protein of the present invention is usually prepared by a biosynthetic method. According to the nucleotide sequence of the present invention, a person skilled in the art can easily prepare the encoding nucleic acid of the present invention by various known methods. These methods include, but are not limited to, PCR, artificial DNA synthesis, etc. For specific methods, see J. Sambrook, "Molecular Cloning Laboratory Guide". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing the nucleotide sequence in segments and then performing overlap extension PCR.

[0082] In the present application, the "fusion protein" refers to a product obtained by fusing an antibody fragment with other biologically active proteins using genetic engineering technology.

[0083] In this application, the term "mutant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0084] In the present application, the antibody "Fc fragment" includes the CH2 and CH3 regions of the antibody, such as a wild-type IgG4 Fc fragment includes the CH2 and CH3 regions of a wild-type IgG4 antibody, and a wild-type IgG1 Fc fragment includes the CH2 and CH3 regions of a wild-type IgG1 antibody.

[0085] In the present application, "half-antibody" refers to two antibody fragments formed after the disulfide bond of the antibody Fc fragment is broken. For example, the half-antibody formed by the Fc fragment of the wild-type human IgG4 antibody is a fragment containing the CH2 and CH3 regions of the wild-type human IgG4 antibody, and the half-antibody formed by the Fc fragment of the wild-type human IgG1 antibody is a fragment containing the CH2 and CH3 regions of the wild-type human IgG1 antibody.

[0086] In this application, the term "nucleotide" refers to ribonucleotides, deoxynucleotides or modified forms of either type of nucleotide, and combinations thereof.

[0087] The term "host cell" used herein refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The term "transformed" or "transfected" used herein refers to the introduction of nucleic acid (eg, vector) into a cell by various techniques known in the art.

[0088] In this application, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410). et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0089] In the present application, the amino acid numbering of the Fc fragment of IgG4 or IgG1 is based on the EU numbering system. For example, the "S228P" means that the serine at position 228 according to the EU numbering system is replaced by proline.

[0090] In the present application, the amino acid numbering method of the IgD fragment refers to the literature: Takahashi, Nobuhiro, et al. "Complete Amino Acid Sequence of the δ Heavy Chain of Human Immunoglobulin D." Proceedings of the National Academy of Sciences of the United States of America, vol. 79, no. 9, 1982, pp. 2850–54, http: / / www.jstor.org / stable / 11720. Accessed 12 Apr. 2022.

[0091] In some embodiments, the present invention provides a fusion protein, whose general structural formula is represented by XLY, wherein X is a first biologically active molecule; L is absent or a connecting peptide; Y is a second biologically active molecule; - is a peptide bond; and the active molecule X or Y is selected from a protein or protein domain, a polypeptide, an antibody or an antibody fragment.

[0092] According to some specific embodiments of the present invention, the above-mentioned fusion protein may further include at least one of the following additional technical features: According to some specific embodiments of the present invention, the X is human growth hormone.

[0093] According to some specific embodiments of the present invention, the amino acid sequence of human growth hormone includes SEQ ID NO: 1 or an amino acid sequence having at least 80-99% identity thereto or at least a portion of SEQ ID NO: 1.

[0094] Human growth hormone (hGH) amino acid sequence:

[0095] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 1).

[0096] According to some specific embodiments of the present invention, Y is an Fc fragment or a variant thereof.

[0097] According to some specific embodiments of the present invention, the amino acid sequence of the Fc fragment includes SEQ ID NO:29, SEQ ID NO:30, or an amino acid sequence that is at least 80-99% identical to any one of them, or at least a portion of any one of them; or the amino acid sequence of the variant of the Fc fragment includes an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or an amino acid sequence that is at least 80-99% identical to any one of them, or at least a portion of any one of them.

[0098] The amino acid sequence of Fc fragment variant 1 includes:

[0099] ESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:2).

[0100] The amino acid sequence of Fc fragment variant 2 includes:

[0101] ESKYGPPCPPCPAPEVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:3).

[0102] The amino acid sequence of Fc fragment variant 3 includes:

[0103] ESKYGPPCPPCPAPEVEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:4).

[0104] The amino acid sequence of Fc fragment variant 4 includes:

[0105] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:5).

[0106] The amino acid sequence of Fc fragment variant 5 includes:

[0107] TPECCHPRLSLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:6).

[0108] The amino acid sequence of Fc fragment variant 6 includes:

[0109] TPECPSHTQPLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:7).

[0110] The amino acid sequence of the wild-type IgG4 fragment includes:

[0111] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:29).

[0112] The amino acid sequence of the wild-type IgG1 Fc fragment includes:

[0113] PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:30).

[0114] According to some specific embodiments of the present invention, the amino acid sequence of the fusion protein includes an amino acid sequence selected from: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or an amino acid sequence that has at least 80-99% identity with any one of them, or at least a portion of any one of them.

[0115] Amino acid sequence of fusion protein 8# (bioactive molecule functional region-Fc mutant 1):

[0116] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDL LKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRNTGEREEEKKKEKEEKEEEEEEEGEPECPESKYGP PCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 21).

[0117] Amino acid sequence of fusion protein 9# (bioactive molecule functional region-Fc mutant 2):

[0118] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRNTGKGGEEKKKEKEEKENEEEEEREPECPESKYGPPCPPCPAPEVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 22).

[0119] Amino acid sequence of fusion protein 10# (bioactive molecule functional region - Fc mutant 4):

[0120] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRNTGKGGDDEKEDEEEKEQQEGETKTPECPESKYGPPCPPCPAPEVEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:23).

[0121] Amino acid sequence of fusion protein 11# (bioactive molecule functional region-(G4S)3-Fc mutant 5):

[0122] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 24).

[0123] Amino acid sequence of fusion protein 12# (bioactive molecule functional region - GS linker peptide mutant - Fc mutant 3):

[0124] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGGSGGGGTGGGGSLESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 25).

[0125] Amino acid sequence of fusion protein 13# (biologically active molecule functional region-Fc mutant 6):

[0126] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYD LLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRQSGKGGDDKRKDKDKEEQEERDTKESKYGPPCP PCPAPEFLGGGSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO: 26).

[0127] Amino acid sequence of fusion protein 14# (biologically active molecule functional region-(G4S)3-Fc mutant 7):

[0128] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSTPECCHPRLSLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 27).

[0129] Amino acid sequence of fusion protein 15# (bioactive molecule functional region-(G4S)3-Fc mutant 8):

[0130] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSTPECPSHTQPLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28).

[0131] According to some specific embodiments of the present invention, the connecting peptide contains one or more amino acids selected from glycine, serine, alanine and threonine.

[0132] According to some specific embodiments of the present invention, the amino acid sequence of the connecting peptide includes an amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or an amino acid sequence having at least 80-99% identity with any one of them, or at least a portion of any one of them.

[0133] Linker 1 (third peptide chain 1) amino acid sequence:

[0134] RNTGEREEEKKKEKEEKEEEEEEEGEPECP (SEQ ID NO: 8).

[0135] Linker 2 (third peptide chain 2) amino acid sequence:

[0136] RNTGKGGEEKKKEKEEKENEEEEEREPECP (SEQ ID NO: 9).

[0137] Linker 3 (third peptide chain 3) amino acid sequence:

[0138] RNTGKGGDDEKEDEEEKEQQEGETKTPECP (SEQ ID NO: 10).

[0139] Linker 4 amino acid sequence:

[0140] GGGGGSGGGGTGGGGSL (SEQ ID NO: 11).

[0141] Linker 5 (third peptide chain 4) amino acid sequence:

[0142] RQSGKGGDDKRKDKDKEEQEERDTK (SEQ ID NO: 12).

[0143] In some embodiments, the present invention provides a nucleotide sequence encoding the aforementioned connecting peptide.

[0144] In some embodiments, the present invention provides a nucleotide sequence encoding the above-mentioned fusion protein.

[0145] In some embodiments, the present invention provides a vector comprising the above-mentioned nucleotide sequence.

[0146] According to some specific embodiments of the present invention, the above-mentioned carrier may further include at least one of the following additional technical features:

[0147] According to some specific embodiments of the present invention, the vector proposed by the present invention is an expression vector.

[0148] In the present application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0149] In some embodiments, the present invention provides an engineered cell, characterized in that the engineered cell comprises the fusion protein described above, or the engineered cell comprises the nucleotide sequence or expression vector described above.

[0150] In the present application, the term "engineered cell" refers to a cell having unique stable genetic traits obtained by modifying or recombining the genetic material of a host cell using genetic engineering technology or cell fusion technology.

[0151] In some embodiments, the present invention provides a pharmaceutical composition comprising the above-mentioned fusion protein.

[0152] According to some specific embodiments of the present invention, the above-mentioned pharmaceutical composition may further include at least one of the following additional technical features:

[0153] According to some specific embodiments of the present invention, the pharmaceutical composition is a pharmaceutical composition for oral, intravenous administration, such as bolus injection or by continuous infusion over a period of time, by subcutaneous, intramuscular, intraarterial, intraperitoneal, intrapulmonary, intracerebrospinal, intraarticular, intrasynovial, intrathecal, intralesional, or inhalation route such as intranasal, usually by intravenous or subcutaneous administration.

[0154] According to some specific embodiments of the present invention, the dosage form of the pharmaceutical composition is tablet, capsule, spray, injection, lyophilized powder injection or prefilled injection.

[0155] In some embodiments, the present invention provides a method for preparing the aforementioned fusion protein, which comprises: (a) culturing the aforementioned engineered cells to obtain a culture medium containing the fusion protein; and (b) isolating the fusion protein from the culture medium.

[0156] In some embodiments, the present invention provides the use of the aforementioned fusion protein or pharmaceutical composition in the preparation of a drug for treating or preventing a disease caused by lack of human growth hormone.

[0157] In the present application, the term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, especially humans, including: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0158] The names and numbers of the sequences in this application are shown in Table 1:

[0159] Table 1:

[0160]

[0161]

[0162] Compared with the prior art, the present invention has at least one of the following beneficial technical effects:

[0163] (1) Long-acting human growth hormone fusion protein E of the present invention C50 The value is low, and it has strong binding activity with human GHR, especially the binding activity of human growth hormone fusion protein 8#, 9#, 10#, and 15# is significantly better than Genexine GX-H9 and GH+G / Slinker+IgG4-1 protein.

[0164] (2) The long-acting human growth hormone fusion protein of the present invention has a good proliferation effect on Nb2-11 cells; the proliferation activity of human growth hormone fusion proteins 8#, 9#, 10#, and 12# is significantly better than that of Genexine GX-H9 and GH+G / S linker+IgG4-1 protein.

[0165] (3) The long-acting human growth hormone fusion protein of the present invention has a good effect on the activity of luciferase expressed by reporter gene cells. Among them, human growth hormone fusion proteins 8#, 9#, 10#, and 12# are better than Genexine GX-H9 and GH+G / Slinker+IgG4-1 proteins in promoting the activity of luciferase expressed by reporter gene cells.

[0166] (4) The third peptide chain provided by the present invention can enhance the binding activity of the human growth hormone fusion protein comprising the third peptide chain with human GHR, the proliferation activity of Nb2-11 cells and the activity of reporter gene cells expressing luciferase.

[0167] (5) The SEQ ID NO: 54 provided by the present invention is conducive to improving the binding activity between the human growth hormone fusion protein of the second biologically active molecule comprising the amino acid sequence of SEQ ID NO: 54 and human GHR.

[0168] (6) The third peptide chain of SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 provided by the present invention is beneficial to improving the proliferation activity of the human growth hormone fusion protein containing the third peptide chain on Nb2-11 cells.

[0169] (7) The second biologically active molecule shown in SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:50 provided by the present invention is beneficial for improving the proliferation activity of human growth hormone fusion protein containing the second biologically active molecule on Nb2-11 cells.

[0170] (8) The third peptide chain of SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 provided by the present invention is beneficial for improving the activity of luciferase expressed by reporter gene cells of human growth hormone fusion protein containing the third peptide chain.

[0171] (9) The second biologically active molecule shown in SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:50 provided by the present invention is beneficial for increasing the activity of luciferase expressed by reporter gene cells containing the human growth hormone fusion protein containing the second biologically active molecule.

[0172] (10) The fusion protein provided by the present invention has a longer in vivo half-life.

[0173] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0174] Example 1 Preparation of human growth hormone fusion protein expression vector

[0175] In the present application, human growth hormone (SEQ ID NO: 1) is connected with various antibody Fc mutants (SEQ ID NO: 47-54, the bold font in the sequence indicates the second peptide chain) to construct fusion proteins, wherein some fusion proteins have connecting peptides. The specific structure of the fusion protein with connecting peptides is as follows: Figure 1 As shown, the connecting peptide is GGGGSGGGGSGGGGS (SEQ ID NO: 45) or is obtained by mutating it, and the antibody Fc mutant is obtained by mutating the wild-type IgG4 Fc fragment (SEQ ID NO: 71) or the IgG1 Fc fragment (SEQ ID NO: 30), and the mutation method includes point mutation, adding wild-type IgG4 and / or IgD antibody fragments or their corresponding mutants, etc. The specific experimental operation is as follows:

[0176] The nucleotide sequence encoding human growth hormone (SEQ ID NO:31), the nucleotide sequence encoding the connecting peptide and the nucleotide sequences encoding various antibody Fc mutants (SEQ ID NO:32-39) were combined, and the nucleotide sequences SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20 encoding the fusion proteins were cloned into the expression vector pCDNA3.4 (purchased from Thermo Fisher) by total gene synthesis and molecular cloning technology, respectively, to obtain the fusion protein expression vectors of each nucleotide sequence.

[0177] Human growth hormone (hGH) amino acid sequence:

[0178] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 1).

[0179] Amino acid sequence of the first peptide chain 1 (having F234V and L235E mutations compared to the wild-type IgG4 Fc fragment):

[0180] APEVEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:55).

[0181] Amino acid sequence of the first peptide chain 2 (having S298N, T299A and Y300S mutations compared to the wild-type IgG4 Fc fragment):

[0182] APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:56).

[0183] Amino acid sequence of the first peptide chain 3 (compared to the wild-type IgG4 Fc fragment, amino acids 231-239 are missing):

[0184] VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:57).

[0185] Amino acid sequence of the first peptide chain 4 (compared to the wild-type IgG1 Fc fragment, with amino acid deletions at positions 228-239, H268Q, Y296F, A330S and P331S mutations):

[0186] VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:58).

[0187] Amino acid sequence of the first peptide chain 5 (having F234A and L235E mutations compared to the Fc fragment of the wild-type IgG4 antibody):

[0188] APEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:68).

[0189] Amino acid sequence of the second peptide chain 1:

[0190] ESKYGPPCPPCP (SEQ ID NO: 59).

[0191] Amino acid sequence of the second peptide chain 2 (IgD fragment 1 mutant):

[0192] TPECCHPRLSLG (SEQ ID NO: 60).

[0193] Amino acid sequence of the second peptide chain 3 (wild-type IgD fragment 1):

[0194] TPECPSHTQPLG (SEQ ID NO: 61).

[0195] Amino acid sequence of the third peptide chain 1:

[0196] RNTGEREEEKKKEKEEKEEEEEEEGEPECP (SEQ ID NO: 62).

[0197] Amino acid sequence of the third peptide chain 2:

[0198] RNTGKGGEEKKKEKEEKENEEEEEREPECP (SEQ ID NO: 63).

[0199] Amino acid sequence of the third peptide chain 3:

[0200] RNTGKGGDDEKEDEEEKEQQEGETKTPECP (SEQ ID NO: 64).

[0201] Amino acid sequence of the third peptide chain 4:

[0202] RQSGKGGDDKRKDKDKEEQEERDTK (SEQ ID NO: 65).

[0203] Fc mutant 1 (first peptide chain 5-second peptide chain 1-third peptide chain 1) amino acid sequence:

[0204] RNTGEREEEKKKEKEEKEEEEEGEPECPESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:47).

[0205] Amino acid sequence of Fc mutant 2 (first peptide chain 6-second peptide chain 1-third peptide chain 2):

[0206] RNTGKGGEEKKKEKEEKENEEEEEREPECPESKYGPPCPPCPAPEVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:48).

[0207] Amino acid sequence of Fc mutant 3 (first peptide chain 5-second peptide chain 1):

[0208] ESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:49)

[0209] Amino acid sequence of Fc mutant 4 (first peptide chain 1-second peptide chain 1-third peptide chain 3):

[0210] RNTGKGGDDEKEDEEEKEQQEGETKTPECPESKYGPPCPPCPAPEVEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:50).

[0211] Amino acid sequence of Fc mutant 5 (first peptide chain 2-second peptide chain 1):

[0212] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:51).

[0213] Fc mutant 6 (first peptide chain 2-second peptide chain 1-third peptide chain 4) amino acid sequence:

[0214] RQSGKGGDDKRKDKDKEEQEERDTKESKYGPPCPPCPAPEFLGGPSSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNNASRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:52).

[0215] Fc mutant 7 (first peptide chain 3-second peptide chain 2) amino acid sequence:

[0216] TPECCHPRLSLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:53).

[0217] Fc mutant 8 (first peptide chain 4-second peptide chain 3) amino acid sequence:

[0218] TPECPSHTQPLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:54).

[0219] Linker amino acid sequence:

[0220] GGGGGSGGGGTGGGGSL (SEQ ID NO: 11).

[0221] Nucleotide sequence encoding human growth hormone (hGH):

[0222] TTTCCCACCATTCCTCTGAGCAGACTGTTCGACAACGCCATGCTGAGAGCCCACAGACTGCACCAGCTGGCCTTTGACACATACCAGGAGTTCGAGGAGGCCTACATCCCCAAGGAGCAGAAGTACAGCTTCCTGCAGAACCCCCAGACCAGCCTGTGCTTCAGCGAGAGCATCCCCACCCCCAGCAATAGAGAGGAGACACAGCAGAAGAGCAACCTGGAGCTGCTGAGAATCAGCCTGCTGCTGATCCAGAGCTGGCTGGAGCCCGTTCAATTTCTGAGAAGCGTGTTCGCCAACAGCCTGGTGTACGGCGCCAGCGATTCTAATGTGTACGACCTGCTGAAGGACCTGGAGGAGGGCATCCAGACCCTGATGGGCAGACTGGAGGACGGCTCTCCTAGAACCGGACAAATTTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAGAACTACGGCCTGCTGTACTGCTTCAGAAAGGACATGGACAAGGTGGAGACATTCCTGAGAATCGTGCAGTGCAGAAGCGTGGAGGGCAGCTGCGGATTC(SEQ ID NO:31).

[0223] Nucleotide sequence encoding Fc mutant 1:

[0224] AGAAACACCGGCGAGAGAGAGGAAGAGAAGAAGAAAGAGAAAGAGGAAAAAGAAGAGGAAGAGGAAGAAGAGGGCGAGCCTGAGTGCCCCGAGTCTAAGTATGGACCTCCTTGTCCTCCATGTCCAGCTCCAGAAGCTGAAGGCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTATAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:32).

[0225] Nucleotide sequence encoding Fc mutant 2:

[0226] AGAAACACCGGCAAGGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGGAGAAGGAGAACGAGGAGGAGGAGGAGAGAGAGCCCGAGTGCCCTGAAAGCAAATACGGACCCCCTTGCCCTCCCTGCCCTGCTCCTGAAGTTGCTGGAGGACCTAGCGTGTTTCTGTTTCCCCCTAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGAGCCAAGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAAACCAAGCCCAGAGAGGAGCAGTTCAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTATACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGCCAACCCGAAAATAATTACAAGACCACACCTCCCGTGCTGGACAGCGATGGATCATTTTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAGGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:33).

[0227] Nucleotide sequence encoding Fc mutant 3:

[0228] GAATCTAAGTACGGCCCTCCTTGTCCTCCATGTCCAGCTCCAGAAGCTGAAGGCGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTATAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:34).

[0229] Nucleotide sequence encoding Fc mutant 4:

[0230] AGAAACACTGGAAAAGGCGGCGACGACGAGAAAGAGGACGAGGAAGAGAAAGAGCAGCAAGAGGGCGAGACAAAGACCCCTGAGTGCCCCGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCAGCTCCAGAAGTGGAAGGCGGCCCATCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTATAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCCGGTGGCAAGAGGGAAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:35).

[0231] Nucleotide sequence encoding Fc mutant 5:

[0232] GAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCAGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTTAACAACGCCAGCAGAGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:36).

[0233] Nucleotide sequence encoding Fc mutant 6:

[0234] AGACAGTCTGGAAAAGGCGGCGACGACAAGCGCAAGGATAAGGACAAAGAGGAACAAGAAGAACGCGACACCAAAGAGTCTAAGTACGGCCCTCCTTGTCCTCCATGTCCAGCTCCAGAGTTTCTCGGCGGACCCTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTTAACAACGCCAGCAGAGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAA(SEQ ID NO:37).

[0235] Nucleotide sequence encoding Fc mutant 7:

[0236] ACACCTGAGTGCTGTCACCCTAGACTGAGCCTGGGAGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTATAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGTCTCTGGGCAAG(SEQ ID NO:38).

[0237] Nucleotide sequence encoding Fc mutant 8:

[0238] ACACCTGAGTGTCCTAGCCACACACAGCCCCTGGGAGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTATAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCCGGTGGCAGCAGGGCAATGTGTTCAGCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAA(SEQ ID NO:39).

[0239] Nucleotide sequence encoding the third peptide chain 1:

[0240] AGAAACACCGGCGAGAGAGAGGAAGAGAAGAAGAAAGAGAAAGAGGAAAAAGAAGAGGAAGAGGAAGAAGAGGGCGAGCCTGAGTGCCCC(SEQ ID NO:40).

[0241] Nucleotide sequence encoding the third peptide chain 2:

[0242] AGAAACACCGGCAAGGGCGGCGAGGAGAAGAAGAAGGAGAAGGAGGAGAAGGAGAACGAGGAGGAGGAGGAGAGAGCCCGAGTGCCCT (SEQ ID NO: 41).

[0243] Nucleotide sequence encoding the third peptide chain 3:

[0244] AGAAACACTGGAAAAGGCGGCGACGACGAGAAAGAGGACGAGGAAGAGAAAGAGCAGCAAGAGGGCGAGACAAAGACCCCTGAGTGCCCC (SEQ ID NO: 42).

[0245] The nucleotide sequence encoding the third peptide chain 4 is:

[0246] AGACAGTTCTGGAAAAGGCGGCGACGACAAGCGCAAGGATAAGGACAAAGAGGAACAAGAAGAACGCGACACCAAA (SEQ ID NO: 66).

[0247] Nucleotide sequence encoding the linker peptide (Linker4):

[0248] ggcggaggcggaggatctggtggcggaggaacaggcggtggtggaagcctg (SEQ ID NO: 67).

[0249] Nucleotide sequence encoding fusion protein 8#:

[0250]

[0251] Nucleotide sequence encoding fusion protein 9#:

[0252]

[0253] Nucleotide sequence encoding fusion protein 10#:

[0254]

[0255] Nucleotide sequence encoding fusion protein 11#:

[0256]

[0257] Nucleotide sequence encoding fusion protein 12#:

[0258]

[0259] Nucleotide sequence encoding fusion protein 13#:

[0260]

[0261] Nucleotide sequence encoding fusion protein 14#:

[0262]

[0263] Nucleotide sequence encoding fusion protein 15#:

[0264]

[0265] The Fc fragment of wild-type IgG4 includes:

[0266] APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:71).

[0267] Example 2 Expression of human growth hormone fusion protein

[0268] In this example, multiple plasmids obtained in Example 1 were transfected into Expi CHO-S (Gibco, A29133) host cells, and the chemical transfection reagent Polyplus-FectoPRO (polyplus, 116-010) was used to transiently express the long-acting human growth hormone fusion protein, the corresponding amino acid sequence of which is SEQ ID NO: 21-28. The specific experimental operation is as follows:

[0269] (1) Expi CHO-S cells were passaged one day before transient infection and the cell density was adjusted to 3×10 6 Then, the cell culture flask was returned to the shaker (37°C, 8% CO2) for further culture.

[0270] (2) On the day of transient infection (1 L per molecule): Take Expi CHO-S cell fluid for cell counting and adjust the cell density to 6×10 6 cells / mL.

[0271] (3) Preparation of transfection complex: 16 sterile cell culture flasks were taken, 8 of which were marked "DNA" and 8 were marked "FectoPRO". The transfection reagent Polyplus-FectoPRO was added to the "FectoPRO" flask; 60 mL of Opti-MEM solution was added to all 8 "DNA" flasks and 500 μg of the fusion protein expression vectors including each nucleotide sequence obtained in Example 1 was added to each "DNA" flask; the mixture was mixed to obtain a fusion protein expression vector dilution solution, and the fusion protein expression vector dilution solutions including each nucleotide sequence were added to the "FectoPRO" flasks respectively and mixed. After incubation at room temperature for 10 min, the mixture was added to the cell solution and shaken. The cell culture flasks were returned to the shaker for further culture and transfection.

[0272] (4) 18-22 hours after transfection, add appropriate amount of OPM-CHO ProFeed and test the biochemical indexes of the cell fluid. According to the test results of biochemical indexes, supplement glucose to 6g / L. Start testing Titer 4 days after transfection, and feed and sugar every other day. When the cell viability is <80%, CHO cell fermentation broth is obtained, and the supernatant can be harvested for purification.

[0273] Example 3 Purification of human growth hormone fusion protein

[0274] In this example, the CHO cell fermentation broth obtained in Example 2 was subjected to secondary centrifugation (primary centrifugation conditions: 3,000×g, 30 min; secondary centrifugation conditions: 12,000×g, 20 min), and the supernatant was collected and filtered through a 0.2 μm filter for later use.

[0275] Protein A affinity chromatography: Use an aqueous solution containing 20 mM phosphate and 150 mM sodium chloride at pH 7.2 to equilibrate the chromatography column for at least 3 column volumes, load the clarified filtrate after filtration, and retain the feed on the chromatography column for 5 minutes. After loading the sample, use an aqueous solution containing 20 mM phosphate and 150 mM sodium chloride at pH 7.2 to equilibrate the chromatography column for at least 1 column volume, and use a buffer containing 50 mM HAc at pH 4.5, a buffer containing 50 mM HAc at pH 4.0, and a buffer containing 50 mM HAc at pH 3.5 in sequence to elute the target protein. When an absorption peak appears, the peak range is 50 mAU-peak-50 mAU, and a fusion protein with SEC purity that meets the requirements (>98.0) is obtained. ExpiCHO-S cells expressing SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28 were fermented and chromatographed to obtain fusion protein 8#, fusion protein 9#, fusion protein 10#, fusion protein 11#, fusion protein 12#, fusion protein 13#, fusion protein 14# and fusion protein 15#, respectively.

[0276] Example 4 In vitro receptor binding activity assay of human growth hormone fusion protein

[0277] In this example, the purified fusion protein obtained in Example 3 was subjected to in vitro receptor (human growth hormone receptor, Human GHR) binding activity detection, and Genexine GX-H9 (SEQ ID NO: 70) and GH+G / S linker+IgG4-1 (SEQ ID NO: 43) were used as control groups. The specific experimental operation was as follows:

[0278] Human GHR was diluted to prepare 0.5μg / mL coating solution, and 100μL / well was added to the ELISA plate, and the coating was carried out at 2-8℃ for more than 12 hours. The residual solution of the coating plate was discarded, and 1% BSA-PBST was added to each well, 300μL, and blocked at 37℃ for 1 hour. 300μL of PBST was added to each well and washed 3 times. The recombinant long-acting human growth hormone fusion protein, Genexine GX-H9, and GH+G / S linker+IgG4-1 were diluted to 5μg / mL, and then diluted 5 times to 8 gradient concentrations, 100μL / well was added to the ELISA plate, and incubated at 37℃ for 1 hour. After the incubation, 300μL of PBST was added to each well, washed 3 times, and then Goat antihuman IgG Fc-HRP diluted 10000 times with 1% BSA-PBST was added, and 100μL / well was added. After incubation at 37℃ for 1 hour, 300μL of PBST was added to each well, washed 3 times, and patted dry. Add TMB colorimetric solution, 100 μL per well. After 5 minutes of reaction at room temperature, add 2M H2SO4 aqueous solution to terminate the reaction, 100 μL / well. Place the terminated ELISA plate on an ELISA reader and read the absorbance OD at a wavelength of 450 nm. 450 The EC50 of each fusion protein's binding activity to human GHR was calculated.

[0279] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNV YDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGSGGGSGGGGSGGGGSESKYGPPCPPCPA PEFAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:43).

[0280] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNV YDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRNTGRGGEEKKKEKEKEEQEERETKTPEC PSHTQPLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:70).

[0281] The specific experimental results are shown in Table 2. (1) The long-acting human growth hormone fusion protein of the present invention has a low EC50 value and has a strong binding activity with Human GHR. In particular, the human growth hormone fusion proteins 8#, 9#, 10#, and 15# encoded by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 20 have significantly better binding activity to Human GHR than Genexine GX-H9 and GH+G / Slinker+IgG4-1; among them, the human growth hormone fusion protein 15# has the highest binding activity to human GHR, followed by the human growth hormone fusion protein 9#.

[0282] (2) Human growth hormone fusion protein 15# contains the second biologically active molecule encoded by SEQ ID NO:54, indicating that SEQ ID NO:54 is beneficial to improving the binding activity of human growth hormone fusion protein with human GHR.

[0283] Table 2:

[0284]

[0285] Example 5 Effect of human growth hormone fusion protein on Nb2-11 cell proliferation

[0286] This example uses the purified fusion protein obtained in Example 3 for experiment, and uses the above-mentioned Genexine GX-H9 (commercially available product) and GH+G / S linker+IgG4-1 as control groups. The specific experimental operation is as follows:

[0287] The cells were collected and cultured at 1 × 10 5 The concentration of cells was suspended in culture medium (PRMI 1640 culture medium, containing 1% FBS and 50 μM β-mercaptoethanol). Each 50 μL cell sample was added to each well of a 96-well cell culture plate. The above cells were cultured separately with 50 μL of analysis culture medium containing 0.051 ng / mL-3000 ng / mL concentration gradients of long-acting human growth hormone fusion protein, Genexine GX-H9, and GH+G / S linker+IgG4-1. The above cell plate was cultured in a 37°C, 5% CO2 humidified incubator for 96 hours, and then 50 μL ellTiter-Glo Luminescent Cell ViabilityAssay (Promega, G7571) was added to each well. After 10 minutes, the chemiluminescent signal was detected by an enzyme reader. The biological activity of the recombinant long-acting human growth hormone fusion protein was determined by the obtained dose-response curve.

[0288] The specific experimental results are shown in Table 3, among which, (1) the long-acting human growth hormone fusion protein of the present invention has a good proliferation effect on Nb2-11 cells; the proliferation activity of human growth hormone fusion proteins 8#, 9#, 10#, and 12# encoded by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:17 is significantly better than that of Genexine GX-H9 and GH+G / Slinker+IgG4-1; among them, the human growth hormone fusion protein 10# has the highest proliferation activity, followed by human growth hormone fusion protein 8#, and then human growth hormone fusion protein 9#.

[0289] (2) The third peptide chain encoded by SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 is beneficial to improving the proliferation activity of human growth hormone fusion protein on Nb2-11 cells.

[0290] (3) The second biologically active molecule encoded by SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:50 is beneficial to improving the proliferation activity of the human growth hormone fusion protein on Nb2-11 cells.

[0291] Table 3:

[0292]

[0293]

[0294] Example 6 Experiment on biological activity of human growth hormone fusion protein on reporter gene cells

[0295] This example uses the purified human growth hormone fusion protein obtained in Example 3 for experiment, and uses the above-mentioned Genexine GX-H9 (commercially available product) and GH+G / S linker+IgG4-1 as control groups. The specific experimental operation is as follows:

[0296] Reporter gene cells (293-GHR / STAT5 cell line) in logarithmic growth phase were obtained, digested with trypsin and plated (Costar, 3917), 4×10 4 cells / well, 50μL / well, and incubate the 96-well white plate in a 37°C, 5% CO2 incubator overnight. Add gradient dilutions of recombinant long-acting human growth hormone fusion protein to the cells, with a starting concentration of 100nM, 5-fold gradient dilution, 10 gradient concentrations, and add the dilutions to the cells at a volume of 50μL / well. Incubate the 96-well white plate in a 37°C, 5% CO2 incubator for 6 hours. After the incubation, take out the 96-well white plate and Nano-Glo Luciferase Assay kit (promega, N112B), equilibrate to room temperature, add reaction substrate to the sample wells at a volume of 50μL / well, and place at room temperature for 10 minutes. Use an enzyme reader (Promega, GM2000) to record the luminescence signal value, with protein concentration as the X-axis and luminescence signal value as the Y-axis. Use GraphPad Prisim 5 for four-parameter fitting to calculate EC 50 value.

[0297] The specific experimental results are shown in Table 4, where:

[0298] (1) The recombinant long-acting human growth hormone fusion protein of the present invention has a good effect on the activity of luciferase expressed by reporter gene cells. Among them, the human growth hormone fusion proteins 8#, 9#, 10#, and 12# encoded by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:17 have better activities in promoting the expression of luciferase by reporter gene cells than Genexine GX-H9 and GH+G / S linker+IgG4-1; among them, the human growth hormone fusion protein 9# has the highest activity in promoting the expression of luciferase by reporter gene cells, followed by human growth hormone fusion protein 8#, and then human growth hormone fusion protein 10#.

[0299] (2) The third peptide chain encoded by SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 is beneficial to improving the activity of luciferase expressed by the reporter gene cells of the human growth hormone fusion protein.

[0300] (3) The second biologically active molecule encoded by SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:50 is beneficial for increasing the activity of luciferase expressed by reporter gene cells containing human growth hormone fusion protein containing the second biologically active molecule.

[0301] Table 4:

[0302]

[0303]

[0304] Example 7 Detection of ADCC effect of human growth hormone fusion protein-induced effector cells Jurkat-CD16a-luc on target cells CHO-K1-PD-L1 / GHR

[0305] 7.1 Methods

[0306] The sample information is shown in Table 5:

[0307] Table 5:

[0308]

[0309] 3.2 Cell treatment

[0310] Treatment of target cells CHO-K1-PD-L1 / GHR: Perform subculture treatment 2 days before the experiment, discard the supernatant, wash the cells with 5 mL PBS, add 1 mL of 0.25% Trypsin-EDTA, digest for 1 min in a 37°C incubator, add 6 mL of culture medium 1 (450 mL F12 medium + 50 mL FBS) to terminate digestion, resuspend to a single cell suspension, discard 5 mL of cell suspension, add 8 mL of culture medium 1 (450 mL F12 medium + 50 mL FBS), and add 120 μL of G418, the concentration of G418 is 600 μg / mL, mix well, and place in the incubator for continued culture.

[0311] Effector cell Jurkat-CD16a-luc treatment: Perform subculture treatment 2 days before the experiment, mix the cell suspension by pipetting, take 5 mL of cell suspension into a new T75 culture flask, then add 15 mL of culture medium 2 (450 mL RPMI Medium 1640 + 50 mL FBS), 320 μL of G418, the concentration of G418 is 800 μg / mL, 60 μL of Hygromycin B, the concentration of Hygromycin B is 150 μg / mL, mix well, and continue culturing in the incubator.

[0312] 3.3 Experimental process

[0313] On the first day, the target cell CHO-K1-PD-L1 / GHR density was adjusted to 1.83×10 5 cells / mL, 50μL / well plated overnight; the next day, the SBT123 test molecules (molecule 9# and Genexine GX-H9), positive control M7824 and negative control Acterma were diluted to 0.1mg / mL in assay medium 2 (RPMI Medium 1640), diluted to 24μg / mL in the second stage, and then diluted 3-fold in 9 gradients, 25μL / well added to the cell plate, mixed, placed in a 37°C incubator for 45min, and then the cell plate was taken out; the density of the effector cells Jurkat-CD16a-luc was adjusted to 1.47×10 with assay medium 2 (RPMI Medium 1640). 6 cells / mL, 25μL / well was added to the cell plate, mixed, placed in a 37℃ incubator for 5.5h, and then the cell plate was taken out to equilibrate at room temperature, and One-GloLuciferase Assay System was added to the cell plate at 100μL / well, and pipetted 10 times to mix; after lysing at room temperature in the dark for 10min, the chemiluminescent signal value was detected by an enzyme reader. The results are as follows Figure 2 As shown, after adding the positive control PD-L1 antibody M7824, the negative control IL-6R antibody Acterma and the SBT123 test molecule to the ADCC experiment, the positive control M7824 induced effector cells Jurkat-CD16a-luc to have a significant ADCC effect on the target cells CHO-K1-PD-L1 / GHR, the negative control Acterma had no ADCC effect, and the SBT123 test molecule had no ADCC effect.

[0314] Example 8 Human growth hormone fusion protein induces CDC effect of human serum complement on target cells CHO-K1-PD-L1 / GHR

[0315] 8.1 Methods

[0316] The sample information is shown in Table 6:

[0317] Table 6:

[0318]

[0319] (2) Cell treatment

[0320] Treatment of target cells CHO-K1-PD-L1 / GHR: Perform subculture treatment 2 days before the experiment, discard the supernatant, wash the cells with 5 mL PBS, add 1 mL of 0.25% Trypsin-EDTA, digest for 1 min in a 37°C incubator, add 6 mL of culture medium 1 (450 mL F12 medium + 50 mL FBS) to terminate digestion, resuspend to a single cell suspension, discard 5 mL of cell suspension, add 8 mL of culture medium 1 (450 mL F12 medium + 50 mL FBS), and add 120 μL of G418, the concentration of G418 is 600 μg / mL, mix well, and place in the incubator for continued culture.

[0321] (3) Experimental process

[0322] On the first day, the target cell CHO-K1-PD-L1 / GHR density was adjusted to 2.0×10 5cells / mL, 50μL / well plated overnight; on the next day, the SBT123 test molecules (molecule 9# and GenexineGX-H9), positive control Avelumab and negative control Acterma were first diluted to 0.1mg / mL with differentiation medium (99% F12+1% FBS), second diluted to 15μg / mL, and then 3-fold diluted 7 gradients, 50μL / well added to the cell plate, mixed, placed in a 37°C incubator for co-incubation for 30min, and then the cell plate was taken out; 30% complement (containing 70% differentiation medium + 30% complement) was added, 50μL / well, mixed, placed in a 37°C incubator for co-incubation for 6h, the cell plate was taken out and equilibrated at room temperature, and CellTiter-Glo Luminescent Cell Viability Assay was added, 50μL / well added to the cell plate, and the pipette was pipetted 10 times to mix; after lysing at room temperature in the dark for 10min, the chemiluminescent signal value was detected by an enzyme reader. The results showed that after adding the positive control PD-L1 antibody Avelumab, the negative control IL-6R antibody Acterma and the SBT123 test molecule to the CDC experiment, the positive control Avelumab induced human serum complement and had a significant CDC effect on the target cells CHO-K1-PD-L1 / GHR, the negative control Acterma had no CDC effect, and the SBT123 test molecule had no CDC effect.

[0323] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0324] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention. SEQUENCE LISTING <110> Shenzhen Sinovac Pharmaceutical Co., Ltd. <120> A growth hormone fusion protein and its application <130> PDI220115 <160> 72 <170> PatentIn version 3.5 <210> 1 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> 1 <400> 1 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185 190 <210> 2 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> 2 <400> 2 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 3 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> 3 <400> 3 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Val 1 5 10 15 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 4 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> 4 <400> 4 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Val 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 5 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> 5 <400> 5 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Asn 65 70 75 80 Ala Ser Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 6 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> 6 <400> 6 Thr Pro Glu Cys Cys His Pro Arg Leu Ser Leu Gly Val Phe Leu Phe 1 5 10 15 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 35 40 45 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 50 55 60 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 65 70 75 80 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 85 90 95 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 100 105 110 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 115 120 125 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 130 135 140 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 145 150 155 160 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 165 170 175 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 180 185 190 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 195 200 205 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 220 <210> 7 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> 7 <400> 7 Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe 1 5 10 15 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Lys Phe 35 40 45 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 50 55 60 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 65 70 75 80 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 85 90 95 Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 100 105 110 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 115 120 125 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 130 135 140 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 145 150 155 160 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 165 170 175 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 180 185 190 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 195 200 205 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 8 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> 8 <400> 8 Arg Asn Thr Gly Glu Arg Glu Glu Glu Lys Lys Lys Glu Lys Glu Glu 1 5 10 15 Lys Glu Glu Glu Glu Glu Glu Glu Gly Glu Pro Glu Cys Pro 20 25 30 <210> 9 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> 9 <400> 9 Arg Asn Thr Gly Lys Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Glu 1 5 10 15 Lys Glu Asn Glu Glu Glu Glu Glu Arg Glu Pro Glu Cys Pro 20 25 30 <210> 10 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> 10 <400> 10 Arg Asn Thr Gly Lys Gly Gly Asp Asp Glu Lys Glu Asp Glu Glu Glu 1 5 10 15 Lys Glu Gln Gln Glu Gly Glu Thr Lys Thr Pro Glu Cys Pro 20 25 30 <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 11 <400> 11 Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser 1 5 10 15 Leu <210> 12 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> 12 <400> 12 Arg Gln Ser Gly Lys Gly Gly Asp Asp Lys Arg Lys Asp Lys Asp Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Asp Thr Lys 20 25 <210> 13 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> 13 <400> 13 tttccgacca ttccgctgag ccgcctgttt gataacgcga tgctgcgcgc gcatcgcctg 60 catcagctgg cgtttgatac ctatcaggaa tttgaagaag cgtatattcc gaaagaacag 120 aaatatagct ttctgcagaa cccgcagacc agcctgtgct ttagcgaaag cattccgacc 180 ccgagcaacc gcgaagaaac ccagcagaaa agcaacctgg aactgctgcg cattagcctg 240 ctgctgattc agagctggct ggaaccggtg cagtttctgc gcagcgtgtt tgcgaacagc 300 ctggtgtatg gcgcgagcga tagcaacgtg tatgatctgc tgaaagatct ggaagaaggc 360 attcagaccc tgatgggccg cctggaagat ggcagcccgc gcaccggcca gatttttaaa 420 cagacctata gcaaatttga taccaacagc cataacgatg atgcgctgct gaaaaactat 480 ggcctgctgt attgctttcg caaagattg gataaagtgg aaacctttct gcgcattgtg 540 cagtgccgca gcgtggaagg cagctgcgg tttcgcaaca ccggcgaacc cgaagaagaa 600 aaaaaaaaaag aaaaagaga aaaagagaa gaagagaag aagaaggcga accggaatgc 660 720 ccgagcgtgt ttctgtttcc gccgaaaccg aaagataccc tgatgattag ccgcaccccg 780 gaagtgacct gcgtggtggt ggatgtgagc caggaagatc cggaagtgca gtttaactgg 840 tatgtggatg gcgtggaagt gcataacgcg aaaaccaaac cgcgcgaaga acagtttaac 900 agcacctatc gcgtggtgag cgtgctgacc gtgctgcatc aggattggct gaacggcaaa 960 gaatataaat gcaaagtgag caacaaaggc ctgccgagca gcattgaaaa aaccattagc 1020 aaagcgaaag gccagccgcg cgaaccgcag gtgtataccc tgccgccgag ccaggaagaa 1080 atgaccaaaa accaggtgag cctgacctgc ctggtgaaag gcttttatcc gagcgatatt 1140 gcggtggaat gggaaagcaa cggccagccg gaaaacaact ataaaaccac cccgccggtg 1200 ctggatagcg atggcagctt ttttctgtat agccgcctga ccgtggataa aagccgctgg 1260 caggaaggca acgtgtttag ctgcagcgtg atgcatgaag cgctgcataa ccattatacc 1320 cagaaaagcc tgagcctgag cctgggcaaa 1350 <210> 14 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> 14 <400> 14 tttccgacca ttccgctgag ccgcctgttt gataacgcga tgctgcgcgc gcatcgcctg 60 catcagctgg cgtttgatac ctatcaggaa tttgaagaag cgtatattcc gaagaacag 120 aaatatagct ttctgcagaa cccgcagacc agcctgtgct ttagcgaaag cattccgacc 180 ccgagcaacc gcgaagaaac ccagcagaaa agcaacctgg aactgctgcg cattagcctg 240 ctgctgattc agagctggct ggaaccggtg cagtttctgc gcagcgtgtt tgcgaacagc 300 ctggtgtatg gcgcgagcga tagcaacgtg tatgatctgc tgaaagatct ggaagaaggc 360 attcagaccc tgatgggccg cctggaagat ggcagcccgc gcaccggcca gatttttaaa 420 cagacctata gcaaatttga taccaacagc cataacgatg atgcgctgct gaaaaactat 480 ggcctgctgt attgctttcg caaagattg gataaagtgg aaacctttct gcgcattgtg 540 cagtgccgca gcgtggaagg cagctgcgg tttcgcaaca ccggcaaagg cggcgaagaa 600 aaaaaaaaaag aaaaagaaga aaaagaaaac gaagagaag aagaacgcga accggaatgc 660 ccggaaagca aatatggccc gccgtgcccg ccgtgcccgg cgccggaagt ggcgggcggc 720 ccgagcgtgt ttctgtttcc gccgaaaccg aaagataccc tgatgattag ccgcaccccg 780 gaagtgacct gcgtggtggt ggatgtgagc caggaagaatc cggaagtgca gtttaactgg 840 tatgtggatg gcgtggaagt gcataacgcg aaaaccaaac cgcgcgaaga acagtttaac 900 agcacctatc gcgtggtgag cgtgctgacc gtgctgcatc aggattggct gaacggcaaa 960 gaatataaat gcaaagtgag caacaaaggc ctgccgagca gcattgaaaa aaccattagc 1020 aaagcgaaag gccagccgcg cgaaccgcag gtgtataccc tgccgccgag ccaggaagaa 1080 atgaccaaaa accaggtgag cctgacctgc ctggtgaaag gcttttatcc gagcgatatt 1140 gcggtggaat gggaaagcaa cggccagccg gaaaacaact ataaaaccac cccgccggtg 1200 ctggatagcg atggcagctt ttttctgtat agccgcctga ccgtggataa aagccgctgg 1260 caggaaggca acgtgtttag ctgcagcgtg atgcatgaag cgctgcataa ccattatacc 1320 cagaaaagcc tgagcctgag cctgggcaaa 1350 <210> 15 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> 15 <400> 15 tttccgacca ttccgctgag ccgcctgttt gataacgcga tgctgcgcgc gcatcgcctg 60 catcagctgg cgtttgatac ctatcaggaa tttgaagaag cgtatattcc gaaagaacag 120 aaatatagct ttctgcagaa cccgcagacc agcctgtgct ttagcgaaag cattccgacc 180 ccgagcaacc gcgaagaaac ccagcagaaa agcaacctgg aactgctgcg cattagcctg 240 ctgctgattc agagctggct ggaaccggtg cagtttctgc gcagcgtgtt tgcgaacagc 300 ctggtgtatg gcgcgagcga tagcaacgtg tatgatctgc tgaaagatct ggaagaaggc 360 attcagaccc tgatgggccg cctggaagat ggcagcccgc gcaccggcca gatttttaaa 420 cagacctata gcaaatttga taccaacagc cataacgatg atgcgctgct gaaaaactat 480 ggcctgctgt attgctttcg caaagatatg gataaagtgg aaacctttct gcgcattgtg 540 cagtgccgca gcgtggaagg cagctgcggc tttcgcaaca ccggcaaagg cggcgatgat 600 gaaaaagaag atgaagaaga aaaagaacag caggaaggcg aaaccaaaac cccggaatgc 660 ccggaaagca aatatggccc gccgtgcccg ccgtgcccgg cgccggaagt ggaaggcggc 720 ccgagcgtgt ttctgtttcc gccgaaaccg aaagataccc tgatgattag ccgcaccccg 780 gaagtgacct gcgtggtggt ggatgtgagc caggaagaatc cggaagtgca gtttaactgg 840 tatgtggatg gcgtggaagt gcataacgcg aaaaccaaac cgcgcgaaga acagtttaac 900 agcacctatc gcgtggtgag cgtgctgacc gtgctgcatc aggattggct gaacggcaaa 960 gaatataaat gcaaagtgag caacaaaggc ctgccgagca gcattgaaaa aaccattagc 1020 aaagcgaaag gccagccgcg cgaaccgcag gtgtataccc tgccgccgag ccaggaagaa 1080 atgaccaaaa accaggtgag cctgacctgc ctggtgaaag gcttttatcc gagcgatatt 1140 gcggtggaat gggaaagcaa cggccagccg gaaaacaact ataaaaccac cccgccggtg 1200 ctggatagcg atggcagctt ttttctgtat agccgcctga ccgtggataa aagccgctgg 1260 caggaaggca acgtgtttag ctgcagcgtg atgcatgaag cgctgcataa ccattatacc 1320 cagaaaagcc tgagcctgag cctgggcaaa 1350 <210> 16 <211> 1305 <212> DNA <213> Artificial Sequence <220> <223> 16 <400> 16 tttccgacca ttccgctgag ccgcctgttt gataacgcga tgctgcgcgc gcatcgcctg 60 catcagctgg cgtttgatac ctatcaggaa tttgaagaag cgtatattcc gaaagaacag 120 aaatatagct ttctgcagaa cccgcagacc agcctgtgct ttagcgaaag cattccgacc 180 ccgagcaacc gcgaagaaac ccagcagaaa agcaacctgg aactgctgcg cattagcctg 240 ctgctgattc agagctggct ggaaccggtg cagtttctgc gcagcgtgtt tgcgaacagc 300 ctggtgtatg gcgcgagcga tagcaacgtg tatgatctgc tgaaagatct ggaagaaggc 360 attcagaccc tgatgggccg cctggaagat ggcagcccgc gcaccggcca gatttttaaa 420 cagacctata gcaaatttga taccaacagc cataacgatg atgcgctgct gaaaaactat 480 ggcctgctgt attgctttcg caaagatatg gataaagtgg aaacctttct gcgcattgtg 540 cagtgccgca gcgtggaagg cagctgcggc tttggcggcg gcggcagcgg cggcggcggc 600 agcggcggcg gcggcagcga aagcaaatat ggcccgccgt gcccgccgtg cccggcgccg 660 gaatttctgg gcggcccgag cgtgtttctg tttccgccga aaccgaaaga taccctgatg 720 attagccgca ccccggaagt gacctgcgtg gtggtggatg tgagccagga agatccggaa 780 gtgcagttta actggtatgt ggatggcgtg gaagtgcata acgcgaaaac caaccgcgc 840 gaaacagt ttaacaacgc gagccgcgtg gtgagcgtgc tgaccgtgct gcatcaggat 900 tggctgaacg gcaaagaata taaatgcaaa gtgagcaaca aaggcctgcc gagcagcatt 960 gaaaaaacca ttagcaaagc gaaggccag ccgcgcgaac cgcaggtgta taccctgccg 1020 ccgagccagg aagaaatgac caaaaaccag gtgagcctga cctgcctggt gaaaggcttt 1080 1140 accaccccgc cggtgctgga tagcgatggc agcttttttc tgtatagccg cctgaccgtg 1200 gataaaagcc gctggcagga aggcaacgtg tttagctgca gcgtgatgca tgaagcgctg 1260 cataaccat atacccagaa aagcctgagc ctgagcctgg gcaaa 1305 <210> 17 <211> 1368 <212> DNA <213> Artificial Sequence <220> <223> 17 <400> 17 atggagttcg gcctgagctg gctgttcctg gtggctattc tgaaaggcgt gcaatgcttc 60 cccaccattc ctctgagccg gctgttcgac aacgccatgc tgagagccca cagactgcac 120 cagctggcct tcgacaccta ccaagagttc gaggaagcct acattcccaa agagcagaag 180 tacagcttcc tgcagaaccc tcagaccagc ctgtgcttca gcgagagcat ccccacacct 240 agcaacagag aggaaaccca gcagaagtcc aacctggaac tgctgcggat cagcctgctg 300 ctgatccagt cttggctgga acccgtgcag ttcctgagaa gcgtgttcgc caacagcctg 360 gtgtacggcg ccagcgacag caacgtttac gacctgctga aggacctgga agagggcatc 420 cagacactga tgggcagact ggaagatggc agccctagaa ccggccagat cttcaagcag 480 acctacagca agttcgacac caacagccac aacgacgacg ccctgctgaa aaactacggc 540 ctgctgtact gctttcggaa ggacatggac aaggtggaaa ccttcctgcg gatcgtgcag 600 tgcagaagcg tggaaggctc ttgtggcttt ggcggaggcg gaggatctgg tggcggagga 660 acaggcggtg gtggaagcct ggaatctaag tacggccctc cttgtcctcc atgtccagct 720 ccagaagctg aaggcggccc ttccgtgttc ctgtttcctc caaagcctaa ggacaccctg 780 atgatcagca gaacccctga agtgacctgc gtggtggtgg acgtgtccca agaggatcct 840 gaggtgcagt tcaattggta cgtggacggc gtggaagtgc aaacgccaa gaccaagcct 900 960 gattggctga acggcaaaga gtacaagtgc aaggtgtcca acaagggcct gcctagcagc 1020 atcgagaaaa ccatcagcaa ggccaagggc cagccaaggg aaccccaggt ttacacactg 1080 cctccaagcc aaggaaat gaccaagaac caggtgtccc tgacctgcct ggtcaagggc 1140 ttctaccctt ccgatatcgc cgtggaatgg gagagcaatg gccagcctga gaacactac 1200 aagaccacac ctcctgtgct ggacagcgac ggctcattct tcctgtacag cagactgacc 1260 gtggcaaga gcagatggca agagggcaac gtgttcagct gcagcgtgat gcacgaggcc 1320 ctgcacaacc actacaccca gaagtctctg agcctgagcc tgggcaaa 1368 <210> 18 <211> 1335 <212> DNA <213> Artificial Sequence <220> <223> 18 <400> 18 tttccgacca ttccgctgag ccgcctgttt gataacgcga tgctgcgcgc gcatcgcctg 60 catcagctgg cgtttgatac ctatcaggaa tttgaagaag cgtatattcc gaaagaacag 120 aaatatagct ttctgcagaa cccgcagacc agcctgtgct ttagcgaaag cattccgacc 180 ccgagcaacc gcgaagaaac ccagcagaaa agcaacctgg aactgctgcg cattagcctg 240 ctgctgattc agagctggct ggaaccggtg cagtttctgc gcagcgtgtt tgcgaacagc 300 ctggtgtatg gcgcgagcga tagcaacgtg tatgatctgc tgaaagatct ggaagaaggc 360 attcagaccc tgatgggccg cctggaagat ggcagcccgc gcaccggcca gatttttaaa 420 cagacctata gcaaatttga taccaacagc cataacgatg atgcgctgct gaaaaactat 480 ggcctgctgt attgctttcg caaagatatg gataaagtgg aaacctttct gcgcattgtg 540 cagtgccgca gcgtggaagg cagctgcggc tttcgccaga gcggcaaagg cggcgatgat 600 aaacgcaaag ataaagataa agagaacag gaaacgcg ataccaaaga aagcaaatat 660 ggcccgccgt gcccgccgtg cccggcgccg gaatttctgg gcggccccgag cgtgtttctg 720 tttccgccga aaccgaaaga taccctgatg attagccgca ccccggaagt gacctgcgtg 780 gtggtggatg tgagccagga agatccggaa gtgcagttta actggtatgt ggatggcgtg 840 gaagtgcata acgcgaaaac caaaccgcgc gaaacagt ttaacaacgc gagccgcgtg 900 gtgagcgtgc tgaccgtgct gcatcaggat tggctgaacg gcaaagaata taaatgcaaa 960 gtgagcaaca aaggcctgcc gagcagcatt gaaaaaacca ttagcaaagc gaaggccag 1020 ccgcgcgaac cgcaggtgta taccctgccg ccgagccagg aagaaatgac caaaaaccag 1080 gtgagcctga cctgcctggt gaaaggcttt tatccgagcg atattgcggt ggaatgggaa 1140 agcaacggcc agccggaaaa caactataaa accaccccgc cggtgctgga tagcgatggc 1200 agcttttttc tgtatagccg cctgaccgtg gataaaagcc gctggcagga aggcaacgtg 1260 tttagctgca gcgtgatgca tgaagcgctg cataaccatt atacccagaa aagcctgagc 1320 ctgagcctgg gcaaa 1335 <210> 19 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> 19 <400> 19 tttccgacca ttccgctgag ccgcctgttt gataacgcga tgctgcgcgc gcatcgcctg 60 catcagctgg cgtttgatac ctatcaggaa tttgaagaag cgtatattcc gaaagaacag 120 aaatatagct ttctgcagaa cccgcagacc agcctgtgct ttagcgaaag cattccgacc 180 ccgagcaacc gcgaagaaac ccagcagaaa agcaacctgg aactgctgcg cattagcctg 240 ctgctgattc agagctggct ggaaccggtg cagtttctgc gcagcgtgtt tgcgaacagc 300 ctggtgtatg gcgcgagcga tagcaacgtg tatgatctgc tgaaagatct ggaagaaggc 360 attcagaccc tgatgggccg cctggaagat ggcagcccgc gcaccggcca gatttttaaa 420 cagacctata gcaaatttga taccaacagc cataacgatg atgcgctgct gaaaaactat 480 ggcctgctgt attgctttcg caaagatatg gataaagtgg aaacctttct gcgcattgtg 540 cagtgccgca gcgtggaagg cagctgcggc tttggcggcg gcggcagcgg cggcggcggc 600 agcggcggcg gcggcagcac cccggaatgc tgccatccgc gcctgagcct gggcgtgttt 660 ctgtttccgc cgaaaccgaa agataccctg atgattagcc gcaccccgga agtgacctgc 720 gtggtggtgg atgtgagcca ggaagatccg gaagtgcagt ttaactggta tgtggatggc 780 gtggaagtgc ataacgcgaa aaccaaaccg cgcgaagaac agtttaacag cacctatcgc 840 gtggtgagcg tgctgaccgt gctgcatcag gattggctga acggcaaaga atataaatgc 900 aaagtgagca acaaaggcct gccgagcagc attgaaaaaa ccattagcaa agcgaaaggc 960 cagccgcgcg aaccgcaggt gtataccctg ccgccgagcc aggaagaaat gaccaaaaac 1020 caggtgagcc tgacctgcct ggtgaaaggc ttttatccga gcgatattgc ggtggaatgg 1080 gaaagcaacg gccagccgga aaacaactat aaaaccaccc cgccggtgct ggatagcgat 1140 ggcagctttt ttctgtatag ccgcctgacc gtggataaaa gccgctggca ggaaggcaac 1200 gtgtttagct gcagcgtgat gcatgaagcg ctgcataacc attataccca gaaaagcctg 1260 agcctgagcc tgggcaaa 1278 <210> 20 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> 20 <400> 20 tttccgacca ttccgctgag ccgcctgttt gataacgcga tgctgcgcgc gcatcgcctg 60 catcagctgg cgtttgatac ctatcaggaa tttgaagaag cgtatattcc gaaagaacag 120 aaatatagct ttctgcagaa cccgcagacc agcctgtgct ttagcgaaag cattccgacc 180 ccgagcaacc gcgaagaaac ccagcagaaa agcaacctgg aactgctgcg cattagcctg 240 ctgctgattc agagctggct ggaaccggtg cagtttctgc gcagcgtgtt tgcgaacagc 300 ctggtgtatg gcgcgagcga tagcaacgtg tatgatctgc tgaaagatct ggaagaaggc 360 attcagaccc tgatgggccg cctggaagat ggcagcccgc gcaccggcca gatttttaaa 420 cagacctata gcaaatttga taccaacagc cataacgatg atgcgctgct gaaaaactat 480 ggcctgctgt attgctttcg caaagatatg gataaagtgg aaacctttct gcgcattgtg 540 cagtgccgca gcgtggaagg cagctgcggc tttggcggcg gcggcagcgg cggcggcggc 600 agcggcggcg gcggcagcac cccggaatgc ccgagccata cccagccgct gggcgtgttt 660 ctgtttccgc cgaaaccgaa agataccctg atgattagcc gcaccccgga agtgacctgc 720 gtggtggtgg atgtgagcca ggaagatccg gaagtgaaat ttaactggta tgtggatggc 780 gtggaagtgc ataacgcgaa aaccaaaccg cgcgaagaac agtttaacag cacctatcgc 840 gtggtgagcg tgctgaccgt gctgcatcag gattggctga acggcaaaga atataaatgc 900 aaagtgagca acaaagcgct gccgagcagc attgaaaaaa ccattagcaa agcgaaaggc 960 cagccgcgcg aaccgcaggt gtataccctg ccgccgagcc gcgatgaact gaccaaaaac 1020 caggtgagcc tgacctgcct ggtgaaaggc ttttatccga gcgatattgc ggtggaatgg 1080 gaaagcaacg gccagccgga aaacaactat aaaaccaccc cgccggtgct ggatagcgat 1140 ggcagctttt ttctgtatag caaactgacc gtggataaaa gccgctggca gcagggcaac 1200 gtgtttagct gcagcgtgat gcatgaagcg ctgcataacc attataccca gaaaagcctg 1260 agcctgagcc cgggcaaa 1278 <210> 21 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> 21 <400> 21 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Arg 180 185 190 Asn Thr Gly Glu Arg Glu Glu Glu Lys Lys Lys Glu Lys Glu Glu Lys 195 200 205 Glu Glu Glu Glu Glu Glu Glu Gly Glu Pro Glu Cys Pro Glu Ser Lys 210 215 220 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445 Gly Lys 450 <210> 22 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> 22 <400> 22 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Arg 180 185 190 Asn Thr Gly Lys Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Glu Lys 195 200 205 Glu Asn Glu Glu Glu Glu Glu Arg Glu Pro Glu Cys Pro Glu Ser Lys 210 215 220 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Val Ala Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445 Gly Lys 450 <210> 23 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> 23 <400> 23 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Arg 180 185 190 Asn Thr Gly Lys Gly Gly Asp Asp Glu Lys Glu Asp Glu Glu Glu Lys 195 200 205 Glu Gln Gln Glu Gly Glu Thr Lys Thr Pro Glu Cys Pro Glu Ser Lys 210 215 220 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Val Glu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445 Gly Lys 450 <210> 24 <211> 435 <212> PRT <213> Artificial Sequence <220> <223> 24 <400> 24 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly 180 185 190 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser 195 200 205 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly 210 215 220 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 225 230 235 240 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 245 250 255 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 260 265 270 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Asn Ala Ser 275 280 285 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 290 295 300 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 305 310 315 320 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 325 330 335 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 340 345 350 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 355 360 365 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 370 375 380 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 385 390 395 400 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 405 410 415 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 420 425 430 Leo Gly Lys 435 <210> 25 <211> 437 <212> PRT <213> Artificial Sequence <220> <223> 25 <400> 25 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly 180 185 190 Gly Gly Gly Gly Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser Leu 195 200 205 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala 210 215 220 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 225 230 235 240 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 245 250 255 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 260 265 270 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 275 280 285 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 290 295 300 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 305 310 315 320 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 325 330 335 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 340 345 350 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 355 360 365 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 370 375 380 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 385 390 395 400 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 405 410 415 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 420 425 430 Leu Ser Leu Gly Lys 435 <210> 26 <211> 445 <212> PRT <213> Artificial Sequence <220> <223> 26 <400> 26 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Arg 180 185 190 Gln Ser Gly Lys Gly Gly Asp Asp Lys Arg Lys Asp Lys Asp Lys Glu 195 200 205 Glu Gln Glu Glu Arg Asp Thr Lys Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Asn Ala Ser Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 27 <211> 426 <212> PRT <213> Artificial Sequence <220> <223> 27 <400> 27 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly 180 185 190 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Pro 195 200 205 Glu Cys Cys His Pro Arg Leu Ser Leu Gly Val Phe Leu Phe Pro Pro 210 215 220 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 225 230 235 240 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 245 250 255 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 260 265 270 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 275 280 285 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 290 295 300 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 305 310 315 320 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 325 330 335 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 340 345 350 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 355 360 365 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 370 375 380 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 385 390 395 400 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 405 410 415 Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 420 425 <210> 28 <211> 426 <212> PRT <213> Artificial Sequence <220> <223> 28 <400> 28 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly 180 185 190 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Pro 195 200 205 Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro 210 215 220 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 225 230 235 240 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Lys Phe Asn Trp 245 250 255 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 260 265 270 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 275 280 285 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 290 295 300 Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 305 310 315 320 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 325 330 335 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 340 345 350 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 355 360 365 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 370 375 380 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 385 390 395 400 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 405 410 415 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 420 425 <210> 29 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> 29 <400> 29 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 30 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> 30 <400> 30 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 1 5 10 15 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 35 40 45 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 50 55 60 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 65 70 75 80 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 85 90 95 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 100 105 110 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 115 120 125 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 130 135 140 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 145 150 155 160 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 165 170 175 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 180 185 190 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 195 200 205 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 31 <211> 573 <212> DNA <213> Artificial Sequence <220> <223> 31 <400> 31 tttcccacca ttcctctgag cagactgttc gatacgcca tgctgagagc ccacagactg 60 caccagctgg ccttgacac ataccaggag ttcgaggagg cctacatccc caggagcag 120 aagtacagct tcctgcagaa cccccagacc agcctgtgct tcagcgagag catccccacc 180 cccagcaata gagaggac acagcagaag agcaacctgg agctgctgag atcagcctg 240 ctgctgatcc agagctggct ggagccgtt caatttctga gaagcgtgtt cgccacagc 300 ctggtgtacg gcgccagcga ttctaatgtg tacgacctgc tgaaggacct ggaggaggc 360 atccagaccc tgatgggcag actggaggac ggctctccta gaaccggaca aattttcaag 420 cagacctaca gcaagttcga caccacagc cacaacgacg acgccctgct gagaactac 480 ggcctgctgt actgcttcag aaggacatg ugaraggtgg agacattcct gagaatcgtg 540 cagtgcagaa gcgtggagggg cagctgcgga ttc 573 <210> 32 <211> 777 <212> DNA <213> Artificial Sequence <220> <223> 32 <400> 32 agaaacaccg gcgagagaga gggaagaaaagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa roager 60 gaggaagaag agggcgagcc tgagtgcccc gagtctaagt atggacctcc ttgtcctcca 120 tgtccagctc cagaagctga aggcggccct tccgtctcc tgtttcctcc aaagcctaag 180 gandaccctga tgatcagcag aacccctgaa gtgacctgcg tggtggtgga cgtgtcccaa 240 gaggatcctg aggtgcagtt caatggtac gtggacggcg tggagtgca caacgccaag 300 accaagccta gagaggaaca gttcaacagc acctatagag tggtgtccgt gctgaccgtg 360 ctgcaccagg attggctgaa cggcaagg tacaagtgca aggtgtccaa caagggcctg 420 cctagcagca tcgagaaaac catcagcaag gccaagggcc agccaaggga acccaggtt 480 tacacactgc ctccaagcca agaggaatg accagaacc aggtgtccct gacctgcctg 540 gtcaagggct tctacccttc cgatatcgcc gtggaatggg agagcaatgg ccagcctgag 600 aacaactaca agaccacacc tcctgtgctg gatagcgacg gctcattctt cctgtacagc 660 agactgaccg tggacaagag cagatggcaa gagggcaacg tgttcagctg cagcgtgatg 777. cacgaggccc tgcacaacca ctacacccag aagtctctga gcctgagcct gggcaaa <210> 33 <211> 777 <212> DNA <213> Artificial Sequence <220> <223> 33 <400> 33 agaaacaccg gcaagggcgg cgaggagag aagaaggaga aggaggagagagacgag gaggaggagg agagagagcc cgagtgccct gaaagcaaat acggacccc ttgccctccc tgccctgctc ctgaagttgc tggaggacct agcgtgtttc tgtttccccc tagcctaag 180 240. gacaccctga tgatcagcag aacccccga gtgacctgcg tggtggtgga tgtgagccaa gaggaccccg aggtgcagtt caattggtac gtggacggcg tggaggtgca caacgccaaa accaagccca gagaggagca gttcaacagc acctacagag tggtgagcgt gctgaccgtg ctgcaccagg attggctgaa cggcaaggag tacaagtgca aggtgagcaa caagggcctg cccagcagca tcgagagac catcagcag gccaagggcc agcccagaga gccccaggtg 480 tataccctgc cccctagcca agaggagatg accagaacc aggtgagcct gacctgccctg 540 gtgaagggct tctaccccag cgacatcgcc gtggagtggg agagcaatgg ccaacccgaa 600 aaattaca agaccacacc tcccgtgctg ggagcgatg gatcattttt cctgtacagc 660 agactgaccg tggacaagg cagatggcag gaggcaacg tgttcagctg cagcgtgatg 720 cacgaggccc tgcacaatca ctacaccag aagagcctga gcctgagcct gggcaaa 777 <210> 34 <211> 687 <212> DNA <213> Artificial Sequence <220> <223> 34 <400> 34 gatctaagt acggccctcc ttgtcctcca tgtccagctc cagaagctga aggcggccct 60 tccgtgttcc tgtttcctcc aaagcctaag gataccctga tgatcagcag aacccctgaa 120 gtgacctgcg tggtggtgga cgtgtcccaa gaggatcctg aggtgcagtt caatggtac 180 gtggacggcg tggagtgca caacgccaag accaagccta gagaggaca gttcacacc 240 acctatagag tggtgtccgt gctgaccgtg ctgcaccagg attggctgaa cggcaagg 300 tacaagtgca aggtgtccaa caagggcctg cctagcagca tcgagaaaac catcagcaag 360 gccaagggcc agccaaggga accccaggtt tacacactgc ctccaagcca agaggaatg 420 accagaacc aggtgtccct gacctgcctg gtcaagggct tctaccttc cgatatcgcc 480 gtggaatggg agagcaatgg ccagcctgag aacaactaca agaccacacc tcctgtgctg 540 gagagcgacg gctcattctt cctgtacagc agactgaccg tggacaagg cagatggcaa 600 gagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660 aagtctctga gcctgagcct gggcaaa 687 <210> 35 <211> 777 <212> DNA <213> Artificial Sequence <220> <223> 35 <400> 35 agaaacactg gaaaaggcgg cgacgacgag aagaggacg agggaaaaaaaggcg 60 gagggcgaga caaagacccc tgagtgcccc gagtctaagt acggccctcc ttgtcctcca 120 tgtccagctc cagaagtgga aggcggcca tccgttttc tgttccctcc aaagcctaag 180 gandaccctga tgatcagcag aacccctgaa gtgacctgcg tggtggtgga cgtgtcccaa 240 gaggatcctg aggtgcagtt caatggtac gtggacggcg tggagtgca caacgccaag 300 accaagccta gagaggaaca gttcaacagc acctatagag tggtgtccgt gctgaccgtg 360 ctgcaccagg attggctgaa cggcaagg tacaagtgca aggtgtccaa caagggcctg 420 cctagcagca tcgagaaaac catcagcaag gccaagggcc agccaaggga acccaggtt 480 tacacactgc ctccaagcca agaggaatg accagaacc aggtgtccct gacctgcctg 540 gtcaagggct tctacccttc cgatatcgcc gtggaatggg agagcaatgg ccagcctgag 600 aacaactaca agaccacacc tcctgtgctg gatagcgacg gctcattctt cctgtacagc 660 agactgaccg tggacagag ccggtggcaa gagggaacg tgttcagctg cagcgtgatg 720 cacgaggcccc tgcacaacca ctacaccag aagtctctga gcctgagcct gggcaaa 777 <210> 36 <211> 687 <212> DNA <213> Artificial Sequence <220> <223> 36 <400> 36 gagtctaagt acggccctcc tgtcctcca tgtccagctc cagagttttct cggcggaccc 60 tccgtgttcc tgtttcctcc aaagcctaag gataccctga tgatcagcag aacccctgaa 120 gtgacctgcg tggtggtgga cgtgtcccaa gaggatcctg aggtgcagtt caatggtac 180 gtggacggcg tggagtgca caacgccaag accaagccta gagaggaca gtttaacaac 240 gccagcagag tggtgtccgt gctgacagtg ctgcaccagg attggctgaa cggcaagg 300 tacaagtgca aggtgtccaa caagggcctg cctagcagca tcgagaaaac catcagcaag 360 gccaagggcc agccaaggga accccaggtt tacacactgc ctccaagcca agaggaatg 420 accagaacc aggtgtccct gacctgcctg gtcaagggct tctaccttc cgatatcgcc 480 gtggaatggg agagcaatgg ccagcctgag aacaactaca agaccacacc tcctgtgctg 540 gagagcgacg gctcattctt cctgtacagc agactgaccg tggacaagg cagatggcaa 600 gagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660 aagtctctga gcctgagcct gggcaaa 687 <210> 37 <211> 762 <212> DNA <213> Artificial Sequence <220> <223> 37 <400> 37 agacagtctg gaaaggcgg cgacgacaag cgcaaggata agcaaaga ggacaagaa 60 gaacgcgaca ccaaagagtc tagtacggc cctccttgtc ctccatgtcc agctccagag 120 tttctcggcg gaccctccgt gttcctgttt cctccaaagc ctaagggac cctgatgatc 180 agcagaaccc ctgaagtgac ctgcgtggtg gtggacgtgt cccaagagga tcctgaggtg 240 cagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcctagagag 300 gaacagttta aaacgccag cagagtggtg tccgtgctga cagtgctgca ccaggattgg 360 ctgaacggca aagagtacaa gtgcaaggtg tccaacaagg gcctgcctag cagcatcgag 420 aaaaccatca gcaaggccaa gggccagcca agggaacccc aggtttacac actgcctcca 480 agccaagagg aaatgaccaa gaaccaggtg tccctgacct gcctggtcaa gggcttctac 540 ccttccgata tcgccgtgga atgggagagc aatggccagc ctgagaacaa ctacaagacc 600 acacctcctg tgctggacag cgacggctca ttcttcctgt acagcagact gaccgtggac 660 aagagcagat ggcaagaggg caacgtgttc agctgcagcg tgatgcacga ggccctgcac 720 aaccactaca cccagaagtc tctgagcctg agcctgggca aa 762 <210> 38 <211> 660 <212> DNA <213> Artificial Sequence <220> <223> 38 <400> 38 acacctgagt gctgtcaccc tagactgagc ctgggagtgt tcctgtttcc tccaaagcct 60 areacaccc tgatgatcag cagaacccct gaagtgacct gcgtggtggt ggacgtgtcc 120 caagagatc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc 180 aagaccaagc ctagagag agagttcaac agcacctata gagtggtgtc cgtgctgacc 240 gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc 300 ctgcctagca gcatcgagaa aaccatcagc aaggccaagg gccagccaag ggaacccag 360 gtttacacac tgcctccaag ccaagagaa atgaccaaga accaggtgtc cctgacctgc 420 ctggtcaagg gcttctaccc ttccgatatc gccgtggaat gggagagcaa tggccagcct 480 gagaacact aaagaccac acctcctgtg ctggacagcg acggctcatt cttcctgtac 540 agcagactga ccgtggacaa gagcagatgg caagagggca acgtgttcag ctgcagcgtg 600 atgcacgagg ccctgcacaa ccactacacc cagaagtctc tgagcctgtc tctgggcaag 660 <210> 39 <211> 660 <212> DNA <213> Artificial Sequence <220> <223> 39 <400> 39 acacctgagt gtcctagcca cacacagccc ctgggagtgt tcctgtttcc tccaaagcct 60 areacaccc tgatgatcag cagaacccct gaagtgacct gcgtggtggt ggacgtgtcc 120 caaggatc ccgaagtgaa gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc 180 aagaccaagc ctagagag agagttcaac agcacctata gagtggtgtc cgtgctgacc 240 gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaaggcc 300 ctgcctagca gcatcgagaa aaccatcagc aaggccaagg gccagcctag ggaaccccag 360 gtttacacac tgcctccaag cagggacgag ctgaccaaga atcaggtgtc cctgacctgc 420 ctggtcagg gctttaccc ttccgatatc gccgtggaat gggagagcaa tggccagcct 480 gagacaacact acagaccac acctcctgtg ctggacagcg acggctcatt cttcctgtac 540 agcaagctga cagtggacaa gagccggtgg cagcagggca atgtgttcag ctgtagcgtg 600 atgcacgagg ccctgcacaa ccactacacc cagaagtccc tgagcctgtc tcctggcaaa 660 <210> 40 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> 40 <400> 40 agaaacaccg gcgagagaga gggaagaaaagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa roager 60 gaggaagagggcgagcc tgagtgcccc 90 <210> 41 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> 41 <400> 41 agaaacaccg gcaagggcgg cgaggagaag agaaggaga agggagagagagaagggag 60 gaggagg aggagaggcc cgagtgccct 90 <210> 42 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> 42 <400> 42 agaaacactg gaaaaggcgg cgacgacgag aaagaggacg aggaagagaa agagcagcaa 60 gagggcgaga caaagacccc tgagtgcccc 90 <210> 43 <211> 436 <212> PRT <213> Artificial Sequence <220> <223> 43 <400> 43 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly 180 185 190 Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 195 200 205 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Ala 210 215 220 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 225 230 235 240 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 245 250 255 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 260 265 270 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 275 280 285 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 290 295 300 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 305 310 315 320 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 325 330 335 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 340 345 350 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 355 360 365 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 370 375 380 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 385 390 395 400 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 405 410 415 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 420 425 430 Ser Leu Gly Lys 435 <210> 44 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> 44 <400> 44 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro 20 25 30 <210> 45 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> 45 <400> 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 46 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 46 <220> <221> MISC_FEATURE <222> (1)..(5) <223> n GGGGS sequences are linked together, where n is an integer greater than 0 <400> 46 Gly Gly Gly Gly Ser 1 5 <210> 47 <211> 259 <212> PRT <213> Artificial Sequence <220> <223> 47 <400> 47 Arg Asn Thr Gly Glu Arg Glu Glu Glu Lys Lys Lys Glu Lys Glu Glu 1 5 10 15 Lys Glu Glu Glu Glu Glu Glu Glu Gly Glu Pro Glu Cys Pro Glu Ser 20 25 30 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly 35 40 45 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 50 55 60 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 65 70 75 80 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 85 90 95 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 100 105 110 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 115 120 125 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 130 135 140 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 145 150 155 160 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 165 170 175 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 180 185 190 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 195 200 205 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 210 215 220 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 225 230 235 240 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 245 250 255 Leu Gly Lys <210> 48 <211> 259 <212> PRT <213> Artificial Sequence <220> <223> 48 <400> 48 Arg Asn Thr Gly Lys Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Glu 1 5 10 15 Lys Glu Asn Glu Glu Glu Glu Glu Arg Glu Pro Glu Cys Pro Glu Ser 20 25 30 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Val Ala Gly 35 40 45 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 50 55 60 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 65 70 75 80 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 85 90 95 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 100 105 110 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 115 120 125 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 130 135 140 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 145 150 155 160 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 165 170 175 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 180 185 190 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 195 200 205 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 210 215 220 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 225 230 235 240 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 245 250 255 Leu Gly Lys <210> 49 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> 49 <400> 49 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 50 <211> 259 <212> PRT <213> Artificial Sequence <220> <223> 50 <400> 50 Arg Asn Thr Gly Lys Gly Gly Asp Asp Glu Lys Glu Asp Glu Glu Glu 1 5 10 15 Lys Glu Gln Gln Glu Gly Glu Thr Lys Thr Pro Glu Cys Pro Glu Ser 20 25 30 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Val Glu Gly 35 40 45 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 50 55 60 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 65 70 75 80 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 85 90 95 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 100 105 110 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 115 120 125 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 130 135 140 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 145 150 155 160 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 165 170 175 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 180 185 190 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 195 200 205 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 210 215 220 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 225 230 235 240 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 245 250 255 Leu Gly Lys <210> 51 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> 51 <400> 51 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Asn 65 70 75 80 Ala Ser Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 52 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> 52 <400> 52 Arg Gln Ser Gly Lys Gly Gly Asp Asp Lys Arg Lys Asp Lys Asp Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Asp Thr Lys Glu Ser Lys Tyr Gly Pro Pro 20 25 30 Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 35 40 45 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 50 55 60 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 65 70 75 80 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 85 90 95 Lys Pro Arg Glu Glu Gln Phe Asn Asn Ala Ser Arg Val Val Ser Val 100 105 110 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 115 120 125 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 130 135 140 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 145 150 155 160 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 165 170 175 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 180 185 190 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 195 200 205 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 210 215 220 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 225 230 235 240 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 245 250 <210> 53 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> 53 <400> 53 Thr Pro Glu Cys Cys His Pro Arg Leu Ser Leu Gly Val Phe Leu Phe 1 5 10 15 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 35 40 45 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 50 55 60 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 65 70 75 80 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 85 90 95 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 100 105 110 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 115 120 125 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 130 135 140 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 145 150 155 160 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 165 170 175 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 180 185 190 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 195 200 205 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 220 <210> 54 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> 54 <400> 54 Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe 1 5 10 15 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 20 25 30 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Lys Phe 35 40 45 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 50 55 60 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 65 70 75 80 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 85 90 95 Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 100 105 110 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 115 120 125 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 130 135 140 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 145 150 155 160 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 165 170 175 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 180 185 190 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 195 200 205 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 55 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> 55 <400> 55 Ala Pro Glu Val Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 56 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> 56 <400> 56 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Asn Ala Ser Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 57 <211> 208 <212> PRT <213> Artificial Sequence <220> <223> 57 <400> 57 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 1 5 10 15 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 20 25 30 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 35 40 45 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 50 55 60 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 65 70 75 80 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 85 90 95 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 100 105 110 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 115 120 125 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 130 135 140 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 145 150 155 160 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 165 170 175 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 180 185 190 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 195 200 205 <210> 58 <211> 208 <212> PRT <213> Artificial Sequence <220> <223> 58 <400> 58 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 1 5 10 15 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 20 25 30 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 35 40 45 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 50 55 60 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 65 70 75 80 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr 85 90 95 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 100 105 110 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 115 120 125 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 130 135 140 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 145 150 155 160 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 165 170 175 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 180 185 190 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 195 200 205 <210> 59 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 59 <400> 59 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 60 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 60 <400> 60 Thr Pro Glu Cys Cys His Pro Arg Leu Ser Leu Gly 1 5 10 <210> 61 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 61 <400> 61 Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly 1 5 10 <210> 62 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> 62 <400> 62 Arg Asn Thr Gly Glu Arg Glu Glu Glu Lys Lys Lys Glu Lys Glu Glu 1 5 10 15 Lys Glu Glu Glu Glu Glu Glu Glu Gly Glu Pro Glu Cys Pro 20 25 30 <210> 63 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> 63 <400> 63 Arg Asn Thr Gly Lys Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Glu 1 5 10 15 Lys Glu Asn Glu Glu Glu Glu Glu Arg Glu Pro Glu Cys Pro 20 25 30 <210> 64 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> 64 <400> 64 Arg Asn Thr Gly Lys Gly Gly Asp Asp Glu Lys Glu Asp Glu Glu Glu 1 5 10 15 Lys Glu Gln Gln Glu Gly Glu Thr Lys Thr Pro Glu Cys Pro 20 25 30 <210> 65 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> 65 <400> 65 Arg Gln Ser Gly Lys Gly Gly Asp Asp Lys Arg Lys Asp Lys Asp Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Asp Thr Lys 20 25 <210> 66 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> 66 <400> 66 agacagtctg gaaaaggcgg cgacgacaag cgcaaggata aggacaaaga ggaacaagaa 60 gaacgcgaca ccaaa 75 <210> 67 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> 67 <400> 67 ggcggaggcg gaggatctgg tggcggagga acaggcggtg gtggaagcct g 51 <210> 68 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> 68 <400> 68 Ala Pro Glu Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 69 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> 69 <400> 69 Ala Pro Glu Val Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 70 <211> 436 <212> PRT <213> Artificial Sequence <220> <223> 70 <400> 70 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Arg 180 185 190 Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu 195 200 205 Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr 210 215 220 Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 225 230 235 240 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 245 250 255 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 260 265 270 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 275 280 285 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 290 295 300 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 305 310 315 320 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 325 330 335 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 340 345 350 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 355 360 365 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 370 375 380 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 385 390 395 400 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 405 410 415 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 420 425 430 Ser Leu Gly Lys 435 <210> 71 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> 71 <400> 71 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 72 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 72 <400> 72 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 1 5 10

Claims

1. An Fc mutant, characterized in that: The amino acid sequence of the Fc mutant is shown in SEQ ID NO:

47.

2. Use of the Fc mutant according to claim 1 in preparing fusion proteins.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Fc mutant of claim 1.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleic acid molecule is DNA.

5. The nucleic acid molecule according to claim 3, characterized in that The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

32.

6. An expression vector, characterized in that: Carrying the nucleic acid molecule according to any one of claims 3 to 5.

7. The expression vector according to claim 6, characterized in that The expression vector is a eukaryotic expression vector, a prokaryotic expression vector or a viral vector.

8. The expression vector according to claim 7, characterized in that The virus includes a lentivirus.

9. A recombinant cell, characterized in that Carrying the nucleic acid molecule according to any one of claims 3 to 5, or the expression vector according to any one of claims 6 to 8, or Expressing the Fc mutant of claim 1.

10. The recombinant cell according to claim 9, characterized in that The recombinant cell is obtained by introducing the expression vector according to any one of claims 6 to 8 into a host cell.

11. The recombinant cell according to claim 10, characterized in that The expression vector is introduced into the host cell by electrotransduction.

12. The recombinant cell according to claim 9, characterized in that The recombinant cell is a eukaryotic cell.

13. The recombinant cell according to claim 9, characterized in that The recombinant cell is a mammalian cell.

14. A fusion protein, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO:

21.

15. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the fusion protein of claim 14.

16. The nucleic acid molecule according to claim 15, characterized in that The nucleic acid molecule is DNA.

17. The nucleic acid molecule according to claim 15, characterized in that The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

13.

18. An expression vector, characterized in that: Carrying the nucleic acid molecule according to any one of claims 15 to 17.

19. The expression vector according to claim 18, characterized in that The expression vector is a eukaryotic expression vector, a prokaryotic expression vector or a viral vector.

20. The expression vector according to claim 19, characterized in that The virus includes a lentivirus.

21. A recombinant cell, characterized in that Carrying the nucleic acid molecule according to any one of claims 15 to 17, the expression vector according to any one of claims 18 to 20, or Express the fusion protein of claim 14.

22. The recombinant cell according to claim 21, characterized in that The recombinant cell is obtained by introducing the expression vector according to any one of claims 18 to 20 into a host cell.

23. The recombinant cell according to claim 22, characterized in that The expression vector is introduced into the host cell by electrotransduction.

24. The recombinant cell according to claim 22, characterized in that The recombinant cell is a eukaryotic cell.

25. The recombinant cell according to claim 22, characterized in that The recombinant cell is a mammalian cell.

26. A composition, characterized in that include: The fusion protein of claim 14, the nucleic acid molecule of any one of claims 15 to 17, the expression vector of any one of claims 18 to 20, or the recombinant cell of any one of claims 21 to 25.

27. Use of the fusion protein of claim 14, the nucleic acid molecule of any one of claims 15 to 17, the expression vector of any one of claims 18 to 20, the recombinant cell of any one of claims 21 to 25, or the composition of claim 26 in the preparation of a drug for treating or preventing a disease related to abnormal growth hormone; The disease associated with abnormal growth hormone is selected from at least one of the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, and Prader-Willi syndrome.

28. A drug, characterized in that include: The fusion protein of claim 14, the nucleic acid molecule of any one of claims 15 to 17, the expression vector of any one of claims 18 to 20, the recombinant cell of any one of claims 21 to 25, or the composition of claim 26.

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