Recombinant long-acting human growth hormone fusion protein and preparation method and use thereof

Abstract

The present application relates to a kind of Fc mutant, fusion protein, nucleic acid molecule, expression vector, recombinant cell, pharmaceutical composition and its purposes.Fc mutant has the amino acid sequence as any one of SEQ ID NO:3~7;Fusion protein includes: second peptide segment, second peptide segment is the functional region of bioactive molecule, third peptide segment, third peptide segment includes the amino acid sequence shown in SEQ ID NO:2 or the Fc mutant of preceding, second peptide segment is connected with third peptide segment.The Fc mutant of the present application is monomer form, fusion protein has higher in vitro and in vivo activity and lower ADCC, CDC effect, can effectively treat growth hormone deficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology and medicine, in particular, the present application relates to a recombinant long-acting human growth hormone fusion protein and a preparation method and use thereof, more particularly, the present application relates to an Fc mutant, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition and use thereof. BACKGROUND

[0002] Growth hormone deficiency is a recognized clinical syndrome associated with a number of metabolic abnormalities, including abnormal body composition, decreased physical performance, altered lipid metabolism, decreased bone mass, increased insulin resistance, and reduced quality of life. Most of the metabolic abnormalities associated with growth hormone deficiency can be reversed by recombinant human growth hormone (rhGH) replacement. Traditional growth hormone deficiency treatment includes daily subcutaneous injection of rhGH, but this treatment method is relatively cumbersome and requires daily injection, which is not convenient for many patients, which raises concerns about poor treatment compliance, which can lead to reduced efficacy. Long-acting rhGH formulations or long-acting growth hormone formulations not only reduce the number of injections and improve compliance, but also help improve the efficacy of GH treatment.

[0003] Therefore, there is still an urgent need to develop a new fusion protein for effective treatment of growth hormone deficiency. SUMMARY

[0004] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application provides an Fc mutant, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition and use thereof. The Fc mutant of the present application is prone to form a monomer. The fusion protein of the present application has strong stability and can effectively treat growth hormone deficiency.

[0005] The present application is based on the following findings of the inventors:

[0006] The inventors have found through experiments that mutating specific sites of the Fc fragment of wild-type IgG1, for example, at least one of P228Delete, C229Delete, P230Delete, T366R, L368H, P395K, and K409D / K409T, and L351S, M428Y, and K447A, or P228Delete, C229Delete, T366R, L368H, P395K, and K409D or K409T, can enable the Fc fragment in the form of a natural dimer to form an Fc mutant in the form of a monomer after expression. Furthermore, the inventors have further found through experiments that using the above Fc mutant to link with growth hormone can form a fusion protein in the form of a monomer, which can be purified by Protein A, simplifying the purification process, and the subsequent purification process is simple. Meanwhile, more importantly, the fusion protein has stronger human growth hormone receptor binding activity, cell proliferation promoting activity, and luciferase activity promoting activity of reporter gene cell expression, as well as a longer half-life and better in vivo biological activity.

[0007] To this end, in a first aspect of the present application, the present application provides an Fc mutant. According to an embodiment of the present application, there is provided an Fc mutant, comprising: a first peptide segment, the first peptide segment having mutations at at least one of positions 228, 229, 230, 366, 368, 395, and 409, and positions 351, 428, and 447, or positions 228, 229, 366, 368, 395, and 409, of the Fc fragment of wild-type IgG1. The inventors have found through a large number of experiments that the above mutations are conducive to enabling the Fc fragment in the form of a natural dimer to form an Fc mutant in the form of a monomer after expression, using the Fc mutant in the form of a monomer to fuse with a biologically active molecule, and the subsequent purification can be performed by Protein A, thereby simplifying the purification process; meanwhile, more importantly, the fusion protein prepared using the Fc mutant has higher in vitro and in vivo activity and lower ADCC and CDC effects.

[0008] It should be noted that the Fc fragment of antibody IgG referred to herein refers to the CH2 and CH3 regions of antibody IgG.

[0009] For example, the amino acid sequence (including CH2-CH3) of the Fc fragment of wild-type IgG1 is as follows:

[0010] PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40).

[0011] The amino acid sequence of the hinge region of wild-type IgG1 is as follows:

[0012] DKTHTG (SEQ ID NO: 41).

[0013] The nucleotide sequence of the hinge region of wild-type IgG1 is as follows:

[0014] GACAAGACACACACCGGA (SEQ ID NO: 42).

[0015] In a second aspect of the present application, a fusion protein is provided. According to an embodiment of the present application, the fusion protein comprises: a second peptide segment comprising a functional region of a bioactive molecule; a third peptide segment comprising an Fc mutant having an amino acid sequence as shown in SEQ ID NO: 2 or an Fc mutant as defined in the first aspect, the second peptide segment being connected to the third peptide segment. The fusion protein according to the embodiment of the present application has higher activity in vivo and in vitro and lower ADCC and CDC effects, and can be purified by Protein A subsequently, thus simplifying the purification process.

[0016] In a third aspect of the present application, a fusion protein is provided. According to an embodiment of the present application, the fusion protein has a general structure of X-L-Y or Y-L-X, wherein X is a first bioactive molecule; L is absent or is a connecting peptide; Y is a second bioactive molecule; - is a peptide bond; and the bioactive molecule X or Y is selected from a protein or a protein domain, a polypeptide, an antibody or an antibody fragment.

[0017] It should be noted that "X-L-Y" means that the C terminus of X is connected to the N terminus of L, and the C terminus of L is connected to the N terminus of Y; and "Y-L-X" means that the C terminus of Y is connected to the N terminus of L, and the C terminus of L is connected to the N terminus of X.

[0018] According to embodiments of the present application, the fusion protein of the second aspect or the third aspect can further comprise at least one of the following additional technical features:

[0019] According to embodiments of the present application, the fusion protein is in monomeric form.

[0020] According to embodiments of the present application, the X is human growth hormone. In some embodiments, the amino acid sequence of the human growth hormone comprises a sequence selected from the group consisting of SEQ ID NO: 1 or an amino acid sequence having at least 80%-99% identity thereto or at least a portion of the sequence thereof.

[0021] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 1).

[0022] According to embodiments of the present application, the nucleotide sequence of the human growth hormone comprises a sequence selected from the group consisting of SEQ ID NO: 11 or a nucleotide sequence having at least 80%-99% identity thereto or at least a portion of the sequence thereof or a codon-optimized sequence of the sequence thereof.

[0023] TTTCCCACCATTCCTCTGAGCAGACTGTTCGACAACGCCATGCTGAGAGCCCACAGACTGCACCAGCTGGCCTTTGACACATACCAGGAGTTCGAGGAGGCCTACATCCCCAAGGAGCAGAAGTACAGCTTCCTGCAGAACCCCCAGACCAGCCTGTGCTTCAGCGAGAGCATCCCCACCCCCAGCAATAGAGAGGAGACACAGCAGAAGAGCAACCTGGAGCTGCTGAGAATCAGCCTGCTGCTGATCCAGAGCTGGCTGGAGCCCGTTCAATTTCTGAGAAGCGTGTTCGCCAACAGCCTGGTGTACGGCGCCAGCGATTCTAATGTGTACGACCTGCTGAAGGACCTGGAGGAGGGCATCCAGACCCTGATGGGCAGACTGGAGGACGGCTCTCCTAGAACCGGACAAATTTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAGAACTACGGCCTGCTGTACTGCTTCAGAAAGGACATGGACAAGGTGGAGACATTCCTGAGAATCGTGCAGTGCAGAAGCGTGGAGGGCAGCTGCGGATTC (SEQ ID NO: 11).

[0024] According to embodiments of the present application, the Y is an IgG Fc mutant (referred to as "Fc mutant" hereinafter). In some embodiments, the Y is an IgG1 Fc mutant. In some embodiments, the IgG1 Fc mutant is at least one of 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 having at least 80-99% identity to any one of these sequences, or at least a portion of any one of these sequences.

[0025] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2).

[0026] PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3).

[0027] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA (SEQ ID NO: 4).

[0028] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVYHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5).

[0029] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVYHEALHNHYTQKSLSLSPGA(SEQID NO:6).

[0030] DKTHTGPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:7).

[0031] Compared to the Fc fragment of wild-type IgG1, mutations in the first peptide of SEQ ID NO:2 may include: P228Delete, C229Delete, P230Delete, T366R, L368H, P395K, and K409T.

[0032] Compared to the Fc fragment of wild-type IgG1, mutations in the first peptide of SEQ ID NO:3 and 7 may include: P228Delete, C229Delete, T366R, L368H, P395K, and K409T.

[0033] Compared to the Fc fragment of wild-type IgG1, mutations in the first peptide of SEQ ID NO:4 may include: P228Delete, C229Delete, P230Delete, T366R, L368H, P395K, K409T, and K447A.

[0034] The mutations present in the first peptide segment of SEQ ID NO: 5 compared to the Fc fragment of wild-type IgG1 can include: P228Delete, C229 Delete, P230 Delete, L351S, T366R, L368H, P395K, K409D, and M428Y.

[0035] The mutations present in the first peptide segment of SEQ ID NO: 6 compared to the Fc fragment of wild-type IgG1 can include: P228Delete, C229 Delete, P230 Delete, L351S, T366R, L368H, P395K, K409D, M428Y, and K447A.

[0036] The mutations described above are advantageous for allowing the Fc mutant in the native dimeric form to be expressed in a monomeric form, for allowing the fusion protein to be purified by Protein A, for simplifying the purification process, and for improving the in vivo and in vitro activity.

[0037] According to an embodiment of the present application, the linker peptide includes one or more amino acids selected from the group consisting of glycine, serine, alanine, and threonine.

[0038] According to an embodiment of the present application, the amino acid sequence of the linker peptide includes at least one selected from the group consisting of (GGGGS)n n , or an amino acid sequence having at least 80-99% identity thereto, or at least a portion of any one of them, wherein n is an integer equal to or greater than 1 or n is 1, 2, 3, 4, 5, or 6.

[0039] According to an embodiment of the present application, the amino acid sequence of the linker peptide includes at least one selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or an amino acid sequence having at least 80-99% identity thereto, or at least a portion of any one of them.

[0040] GGGGSGGGGSGGGGS (SEQ ID NO: 8).

[0041] GGGGSGGGGS (SEQ ID NO: 9).

[0042] GGGGS (SEQ ID NO: 10)

[0043] According to embodiments of the present application, the nucleotide sequence of the connecting peptide can comprise at least one selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or a nucleotide sequence having at least 80-99% identity to any one of them, or at least a portion of any one of them, or a codon-optimized sequence of any one of them.

[0044] GGAGGAGGAGGAAGCGGAGGCGGAGGATCTGGAGGAGGAGGAAGC (SEQ ID NO: 21).

[0045] GGCGGCGGAGGATCTGGCGGAGGTGGAAGT (SEQ ID NO: 22).

[0046] GGCGGCGGAGGATCT (SEQ ID NO: 23).

[0047] According to embodiments of the present application, the amino acid sequence of the fusion protein comprises at least one selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or an amino acid sequence having at least 80-99% identity to any one of them, or at least a portion of any one of them.

[0048] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24).

[0049] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25).

[0050] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFDKTHTGPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26).

[0051] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27).

[0052] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28).

[0053] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 29).

[0054] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVYHEALHNHYTQKSLSLSPGK (SEQ ID NO: 30).

[0055] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVYHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 31).

[0056] According to embodiments of the present application, the first biomolecule, the second biomolecule and the linker peptide in the amino acid sequences of SEQ ID NO: 24-31 are as shown in the following table:

[0057]

[0058] According to embodiments of the present application, the nucleotide sequence of the fusion protein comprises at least one of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or a nucleotide sequence having at least 80-99% identity to any one of them, or at least a portion of any one of them, or a codon-optimized sequence of any one of them.

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] In a fourth aspect of the present application, a linker peptide for constructing a fusion protein is provided. According to an embodiment of the present application, the amino acid sequence of the linker peptide comprises at least one selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or an amino acid sequence having at least 80%-99% identity to any one of them, or at least a portion of any one of them.

[0068] In a fifth aspect of the present application, a nucleic acid molecule is provided. According to an embodiment of the present application, the nucleic acid molecule encodes the Fc mutant of the first aspect, the fusion protein of the second or third aspect, or the linker peptide of the fourth aspect. The Fc mutant encoded by the nucleic acid molecule according to an embodiment of the present application is in monomeric form, and the fusion protein obtained by fusing the Fc mutant in monomeric form with a biologically active molecule has higher in vitro and in vivo activity and lower ADCC and CDC effects. The fusion protein encoded by the nucleic acid molecule according to an embodiment of the present application can be subsequently purified by Protein A, simplifying the purification process and having higher in vitro and in vivo activity and lower ADCC and CDC effects.

[0069] According to an embodiment of the present application, the nucleotide sequence comprises at least one selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or a nucleotide sequence having at least 80%-99% identity to any one of them, or at least a portion of any one of them, or a codon-optimized sequence of any one of them.

[0070] The SEQ ID NO: 21 is used to encode SEQ ID NO: 8.

[0071] The SEQ ID NO: 22 is used to encode SEQ ID NO: 9.

[0072] The SEQ ID NO: 23 is used to encode SEQ ID NO: 10.

[0073] According to an embodiment of the present application, the nucleotide sequence comprises at least one selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or a nucleotide sequence having at least 80%-99% identity to any one of them, or at least a portion of any one of them, or a codon-optimized sequence of any one of them.

[0074] The SEQ ID NO: 32 is used to encode SEQ ID NO: 24.

[0075] The SEQ ID NO: 33 is used to encode SEQ ID NO: 25.

[0076] The SEQ ID NO: 34 is used to encode SEQ ID NO: 26.

[0077] The SEQ ID NO: 35 is used to encode SEQ ID NO: 27.

[0078] The SEQ ID NO: 36 is used to encode SEQ ID NO: 28.

[0079] The SEQ ID NO: 37 is used to encode SEQ ID NO: 29.

[0080] The SEQ ID NO: 38 is used to encode SEQ ID NO: 30.

[0081] The SEQ ID NO: 39 is used to encode SEQ ID NO: 31.

[0082] According to an embodiment of the present application, the nucleotide sequence comprises at least one selected from the group consisting of SEQ ID NO: 12, 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, or a nucleotide sequence having at least 80% - 99% identity to any one of them, or at least a portion of any one of them, or a codon-optimized sequence of any one of them.

[0083] The SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16 is used to encode SEQ ID NO: 2.

[0084] The SEQ ID NO: 14 is used to encode SEQ ID NO: 3.

[0085] The SEQ ID NO: 17 is used to encode SEQ ID NO: 4.

[0086] The SEQ ID NO: 18 is used to encode SEQ ID NO: 5.

[0087] The SEQ ID NO: 19 is used to encode SEQ ID NO: 6.

[0088] The SEQ ID NO: 20 is used to encode SEQ ID NO: 7.

[0089] GCCCCTGAACTGCTCGGAGGCCCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGCGGTGTCACGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACCAAGCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCACCCTGACCGTGGACAAGTCCAGATGGCAACAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAA (SEQ ID NO: 12).

[0090] GCTCCTGAACTGCTGGGAGGACCTAGCGTGTTTCTGTTTCCTCCCAAACCTAAAGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTTGATGTGTCTCATGAAGACCCCGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAAACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGCAAGGAATACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAGGCTAAGGGCCAGCCCAGAGAGCCCCAAGTGTATACACTGCCCCCTAGCAGAGATGAACTGACCAAAAACCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATTGCCGTGGAGTGGGAGAGCAATGGCCAACCTGAAAACAATTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCTTTCTTTCTGTATAGCACCCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTATACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAA (SEQ ID NO: 13).

[0091] CCCGCCCCCGAACTGCTGGGAGGACCTTCTGTGTTTCTGTTTCCTCCCAAACCCAAAGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTTGTGGATGTGTCTCATGAGGATCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAGACAAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGAAAGGAATACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACAATTAGCAAGGCCAAGGGCCAGCCCAGAGAACCTCAAGTGTATACCCTGCCTCCCAGCAGAGATGAGCTGACAAAAAATCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTCTACCCCAGCGACATTGCCGTGGAGTGGGAAAGCAATGGCCAACCTGAGAACAACTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCTTTTTTTCTGTACAGCACCCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCATAATCACTACACCCAGAAAAGCCTGAGCCTGAGCCCCGGCAAG (SEQ ID NO: 14).

[0092] GCTCCTGAACTGCTCGGAGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTCGAGGTGCACAACGCCAAGACAAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGCGGTGTCACGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACCAAGCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCACCCTGACCGTGGACAAGTCCAGATGGCAACAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAA (SEQ ID NO: 15).

[0093] GCTCCTGAACTGCTCGGAGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGCGGTGTCACGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACCAAGCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCACCCTGACCGTGGACAAGTCCAGATGGCAACAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAA (SEQ ID NO: 16).

[0094] GCTCCCGAACTGCTGGGAGGACCCAGCGTGTTTCTGTTTCCCCCTAAACCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGATGTGAGCCATGAGGACCCCGAAGTGAAATTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAAGCCAAGGGCCAGCCCAGAGAGCCCCAAGTGTACACATTACCCCCTAGCAGAGATGAACTGACCAAAAACCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGACAACCTGAGAACAACTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCTTTCTTTCTGTACAGCACCCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCATAATCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAGCT (SEQ ID NO: 17).

[0095] GCTCCTGAACTGCTGGGAGGACCTAGCGTGTTTCTGTTTCCTCCTAAACCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGATGTGAGCCATGAAGATCCCGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAAACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGAAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAAGCTAAGGGCCAGCCCAGAGAGCCCCAAGTGTATACAAGCCCTCCCAGCAGGGACGAGCTGACCAAAAATCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATCGCCGTGGAATGGGAAAGCAATGGCCAACCCGAGAACAACTATAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCCTTTTTTCTGTACAGCGACCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGTATCACGAGGCCCTGCACAATCACTACACCCAGAAAAGCCTGAGCCTGAGCCCCGGCAAA (SEQ ID NO: 18).

[0096] GCCCCTGAGCTGCTTGGAGGACCTAGCGTTTTTTTATTTCCCCCCAAACCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTTGTGGATGTGAGCCATGAAGATCCCGAGGTGAAATTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAAACAAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAGGCCAAGGGCCAGCCCAGAGAACCCCAAGTGTATACCAGCCCCCCCAGCAGAGATGAGCTGACAAAAAATCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATCGCCGTGGAGTGGGAATCTAATGGACAACCTGAGAACAACTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGGAGCTTTTTCCTGTATAGCGACCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGTATCACGAGGCCCTGCATAATCACTACACCCAGAAAAGCCTGAGCCTGAGCCCCGGAGCT (SEQ ID NO: 19).

[0097] GACAAGACACACACCGGACCCGCCCCCGAACTGCTGGGAGGACCTTCTGTGTTTCTGTTTCCTCCCAAACCCAAAGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTTGTGGATGTGTCTCATGAGGATCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAGACAAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGAAAGGAATACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACAATTAGCAAGGCCAAGGGCCAGCCCAGAGAACCTCAAGTGTATACCCTGCCTCCCAGCAGAGATGAGCTGACAAAAAATCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTCTACCCCAGCGACATTGCCGTGGAGTGGGAAAGCAATGGCCAACCTGAGAACAACTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCTTTTTTTCTGTACAGCACCCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCATAATCACTACACCCAGAAAAGCCTGAGCCTGAGCCCCGGCAAG (SEQ ID NO: 20).

[0098] In a sixth aspect, the present application provides an expression vector. According to an embodiment of the present application, the expression vector carries the nucleic acid molecule of the fifth aspect. After the expression vector according to an embodiment of the present application is introduced into a suitable recipient cell, the expression of the Fc mutant, fusion protein or linker peptide described above can be effectively realized under the mediation of a regulatory system, so as to obtain the Fc mutant, fusion protein or linker peptide in large quantities.

[0099] In a seventh aspect of the present application, the present application provides a recombinant cell. According to embodiments of the present application, the recombinant cell comprises: the nucleic acid molecule of the fifth aspect; or, expresses the Fc mutant of the first aspect, the fusion protein of the second or third aspect, or the linker peptide of the fourth aspect. The recombinant cell according to embodiments of the present application can be used for in vitro expression and mass acquisition of the Fc mutant, the fusion protein, or the linker peptide as described above.

[0100] In an eighth aspect of the present application, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition comprises: the fusion protein of the second or third aspect. The pharmaceutical composition according to embodiments of the present application can be used for preventing and treating diseases related to abnormality of the biological activity molecule of the fusion protein.

[0101] According to embodiments of the present application, the pharmaceutical composition is for oral, intravenous administration, such as bolus or by continuous infusion for a period of time, subcutaneous, intramuscular, intra-arterial, intraperitoneal, intrapulmonary, intracerebrospinal, intra-articular, intrasynovial, intrathecal, intralesional, or inhalation routes such as intranasal, typically by intravenous or subcutaneous administration of the pharmaceutical composition.

[0102] According to embodiments of the present application, the dosage form of the pharmaceutical composition is a tablet, a capsule, a spray, an injection, a freeze-dried powder injection, or a pre-filled needle injection.

[0103] In a ninth aspect of the present application, the present application provides a method for preparing the fusion protein of the second or third aspect. According to embodiments of the present application, the method comprises: culturing the recombinant cell of the seventh aspect to obtain a culture solution containing the fusion protein; and isolating the fusion protein from the culture solution. Thus, the fusion protein of the second and third aspects can be effectively prepared.

[0104] In a tenth aspect of the present application, the present application provides use of the fusion protein of the second or third aspect, the nucleic acid molecule of the fifth aspect, the expression vector of the sixth aspect, the recombinant cell of the seventh aspect, or the pharmaceutical composition of the eighth aspect in the preparation of a medicament for treating or preventing diseases related to abnormality of growth hormone.

[0105] Advantages

[0106] Compared with the prior art, the present application has at least one of the following advantages:

[0107] (1) The Fc mutant in the fusion protein used in the present application is mutated at a specific site, and the growth hormone fused with the Fc mutant is expressed in a monomer form, which can be purified by Protein A affinity chromatography, and the subsequent purification process is simple.

[0108] (2) The fusion protein provided by the present application has a low EC 50 value and has strong binding activity to human GHR.

[0109] (3) The fusion protein provided by the present application has a low EC 50 value and has a good proliferation effect on Nb2-11 cells.

[0110] (4) The fusion protein provided by the present application has a low EC 50 value and is comparable to or lower than a control sample (Genexine GX-H9), and has excellent activity of promoting luciferase expression in a reporter cell.

[0111] (5) The fusion protein provided by the present application has a longer half-life and better in vivo biological activity than human growth hormone.

[0112] Additional aspects and advantages of the present application will be set forth in part in the description which follows, and in part will become apparent to those having ordinary skill in the art upon examination of the following or can be learned from practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0113] The foregoing and / or additional aspects and advantages of the present application are achieved by providing a fusion protein, a pharmaceutical composition comprising the same, and a method of treating a growth hormone deficiency using the same, as will be described in detail below.

[0114] Figure 1 is a schematic diagram of a monomeric form of the fusion protein in one embodiment of the present application;

[0115] Figure 2 is a result graph of an ADCC effect induced by J2 or J18 in Example 7 of the present application;

[0116] Figure 3 is a result trend graph of weight gain of each group of rats in the determination of in vivo biological activity in Example 9 of the present application. DETAILED DESCRIPTION

[0117] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not to be construed as limiting the present application.

[0118] It should be noted that the terms "first", "second", etc. are used only for descriptive purposes and should not be construed as indicating or implying relative importance or a specific number of the technical features indicated. Thus, the features defined with "first", "second", etc. can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0119] In the present text, the term "room temperature" means ambient temperature, which can be 20-30 °C; in some embodiments, 22-28 °C; in some embodiments, 24-26 °C; in some embodiments, 25 °C.

[0120] In the present text, "antibody fragment" means an antigen-binding fragment of an antibody and antibody analogs, which generally comprise at least a part of the antigen-binding region or variable region (e.g., one or more CDRs) of the parental antibody.

[0121] In the present text, the term "comprising" or "including" is an open expression, i.e., it includes the indicated content of the present invention, but does not exclude other aspects.

[0122] In the present text, the term "optionally", "optional" or "optional" generally means that the event or condition described subsequently can, but need not, occur, and the description includes the case where the event or condition occurs, as well as the case where it does not occur.

[0123] In the present text, the term "variant" or "mutant" generally means any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0124] In the present text, the amino acid numbering of the IgG1 Fc portion is numbered according to the EU numbering system, for example, the 366th position refers to the 366th position numbered according to the EU numbering system; "T366R" means that the threonine at the 366th position numbered according to the EU numbering system is replaced by arginine; "L368H" means that the leucine at the 368th position numbered according to the EU numbering system is replaced by histidine.

[0125] In the present text, the term "(GGGGS) n " means n GGGGS connected, for example, "(GGGGS)3" means GGGGSGGGGSGGGGS.

[0126] In the present context, the term "fusion protein" generally refers to a protein resulting from the fusion of two or more proteins or polypeptides. The genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be linked to each other to form a fusion gene or a fused nucleic acid molecule, which can encode the fusion protein. Translation of the fusion gene results in a single polypeptide having the properties of at least one, and optionally each, of the two or more proteins or polypeptides prior to fusion. Recombinant fusion proteins are artificially created by recombinant DNA technology for biological research or therapy. Recombinant fusion proteins are proteins created by genetic engineering of a fusion gene. The present invention relates to recombinant fusion proteins, and the terms fusion protein and recombinant fusion protein are used herein with the same meaning. The fusion proteins described herein generally comprise at least two domains (A and C), and optionally a third component, a linker, between the two domains. Generation of recombinant fusion proteins is known in the art, and generally involves removing the stop codon from the cDNA sequence encoding the first protein or polypeptide, and then attaching the cDNA sequence of the second protein in frame by ligation or overlap extension PCR. This DNA sequence is then expressed by a cell as a single protein. The protein can be engineered to include the entire sequence of both original proteins or polypeptides, or only a portion thereof.

[0127] The fusion proteins of the present invention are generally prepared by biological synthesis. The nucleic acid encoding the present invention can be readily prepared by those skilled in the art using various known methods according to the nucleotide sequence of the present invention. These methods include, but are not limited to, PCR, DNA artificial synthesis, etc. and the specific methods can be found in J. Sambrook, Molecular Cloning: A Laboratory Manual. As an embodiment of the present invention, the nucleic acid encoding the present invention can be constructed by the method of synthesizing nucleotide sequences in segments followed by overlap extension PCR.

[0128] In the present context, the terms "identity", "homology" or "similarity" are used when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, in terms of the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences determined by conventional means, see, e.g., 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, D.C.). There are a number of algorithms that can be used to align sequences and determine 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 search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLAST family of algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs are available that use these algorithms to compare sequences, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul 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.

[0129] In the present context, the term "nucleotide" generally refers to a ribonucleotide, a deoxynucleotide, or modified forms of either type of nucleotide, and combinations thereof.

[0130] In the present text, the term "at least 90% identity" means at least 90%, which can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence.

[0131] In the present text, the term "at least 80%-99% sequence identity" means at least 80%-99%, at least 81%-99%, at least 82%-99%, at least 83%-99%, at least 84%-99%, at least 85%-99%, at least 86%-99%, at least 87%-99%, at least 88%-99%, at least 89%-99%, at least 90%-99%, at least 91%-99%, at least 92%-99%, at least 93%-99%, at least 94%-99%, at least 95%-99%, at least 96%-99%, at least 97%-99%, at least 98%-99%, or at least 99% sequence identity to each reference sequence.

[0132] In the present text, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into and / or between host cells. The expression vector can include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for the transcription and / or translation of expression of DNA or RNA. The expression vector also includes a vector having a plurality of the above-mentioned functions. The expression vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0133] In the present text, the term "recombinant cell" generally refers to a cell having a unique trait stably inherited by modification or recombination of the genetic material of a host cell using genetic engineering techniques or cell fusion techniques. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell into which a recombinant expression vector can be introduced. The term "transformed" or "transfected" used herein means the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. A suitable host cell can be transformed or transfected with the DNA sequence of the present application, and can be used for the expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (human amniotic fluid-derived cells), and CoS cells.

[0134] In the present context, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any one of the methods well known in the pharmaceutical art. All methods involve the step of bringing the active ingredient into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping or confectioning the resulting mixture.

[0135] In the present context, the term "pharmaceutically acceptable excipients" can include any solvent, solid diluents or other liquid excipients, etc. suitable for the particular intended dosage form. The use of any particular excipient is contemplated to be within the scope of the application except to the extent that any conventional excipient is incompatible with the compound of the application, for example, produces any adverse biological effect or interacts in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition.

[0136] For other pharmaceutically acceptable excipients or processes mentioned herein, reference can be made to the extensive literature on this subject, in particular to the Handbook of Pharmaceutical Excipients, 3rdedition, Arthur H. Kibbe, ed., American Pharmaceutical Association, Washington, USA and Pharmaceutical Press, London; and to the Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete, H.P. Fiedler, ed., 4thedition, Cantor, Aulendorf and earlier editions.

[0137] In the present context, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The fusion proteins of the present application can be administered by any common route, so long as it reaches the intended tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cutaneous, oral, topical, nasal, pulmonary, rectal and smearing, but the present application is not limited to these exemplified modes of administration. Preferably, the fusion proteins of the present application are administered by injection.

[0138] In the present context, the term "growth hormone abnormality related disease" generally refers to a disease caused by abnormality of growth hormone, such as growth hormone deficiency or dysmorphic state, including but not limited to, pediatric growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner's Syndrome, Prader Willi Syndrome, intrauterine growth retardation, idiopathic short stature, renal failure, dysmorphic state during chemotherapy treatment and AIDS treatment. Growth hormone deficiency can include congenital or acquired deficiency. With regard to congenital deficiency, growth hormone deficiency can occur when the pituitary gland does not develop with a disorder in growth hormone secretion. Acquired growth hormone deficiency can occur due to hypoxia resulting from brain tissue damage that is difficult to deliver. Other causes of growth hormone deficiency include pituitary damage caused by radiation used to treat brain tumors or postnatal tuberculous meningitis. Growth hormone deficiency exhibits symptoms such as growth retardation and short stature, and congenital growth hormone deficiency exhibits symptoms of low glucose from the neonatal stage. In addition, children exhibit symptoms such as increased anxiety and decreased energy.

[0139] In the present context, the term "treatment" refers to obtaining a desired pharmacological and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure of a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed or diagnosed with the disease; (b) inhibiting the disease, e.g., arresting its development; or (c) relieving the disease, e.g., causing regression of the symptoms of the disease. "Treatment" as used herein covers any use of a drug or compound to treat, cure, alleviate, improve, reduce the symptoms of, or inhibit a disease in an individual, including but not limited to, administration of a drug containing a compound described herein to an individual in need thereof.

[0140] It is to be understood by those skilled in the art that the monomeric or dimeric form of the Fc mutant determines the monomeric or dimeric form of the fusion protein formed by the Fc mutant.

[0141] The present application provides an Fc mutant, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition, and uses thereof, which will be described in detail below, respectively.

[0142] Fc mutant

[0143] In one aspect of the present application, the present application provides an Fc mutant. According to an embodiment of the present application, there is provided a first peptide segment having mutations at at least one of positions 228, 229, 230, 366, 368, 395 and 409, and positions 351, 428 and 447; or positions 228, 229, 366, 368, 395 and 409, compared to a wild-type IgG1 Fc fragment.

[0144] The inventors have found through a large number of experiments that the above mutations are beneficial to express the Fc fragment in a native dimer form to form an Fc mutant in a monomer form, and the Fc mutant in the monomer form is fused with a bioactive molecule, and the subsequent Protein A purification is simplified, and the purification process is simplified; in addition, the fusion protein prepared by using the Fc mutant has higher in vivo and in vitro activity and lower ADCC and CDC effects.

[0145] According to an embodiment of the present application, the first peptide segment has mutations at at least one of positions 228, 229, 230, 366, 368, 395 and 409, and positions 351, 428 and 447; or positions 228, 229, 230, 351, 366, 368, 395, 409 and 428; or positions 228, 229, 230, 351, 366, 368, 395, 409, 428 and 447, compared to a wild-type IgG1 Fc fragment. Thus, the above mutations can form an Fc mutant in a monomer form.

[0146] According to an embodiment of the present application, the first peptide segment has mutations at at least one of positions T366R, L368H, P395K and K409D or K409T; and L351S, M428Y and K447A, compared to a wild-type IgG1 Fc fragment. Thus, the above mutations can form an Fc mutant in a monomer form.

[0147] According to an embodiment of the present application, the first peptide segment has mutations at the following positions compared to the Fc fragment of wild-type IgGl: 1) P228 Delete, C229 Delete, P230 Delete, T366R, L368H, P395K, K447A, and K409D or K409T; or, 2) P228 Delete, C229 Delete, P230 Delete, L351S, T366R, L368H, P395K, M428Y, and K409D or K409T; or, 3) P228 Delete, C229 Delete, P230 Delete, L351S, T366R, L368H, P395K, M428Y, K447A, and K409D or K409T; or, 4) P228 Delete, C229 Delete, T366R, L368H, P395K, and K409D or K409T. The inventors have found, through extensive experiments, that the Fc mutants in monomeric form described above, when fused to a biologically active molecule, the fusion protein is secreted in monomeric form, and has higher in vitro and in vivo activity and lower ADCC and CDC effects. For example, the fusion protein of the Fc mutant fused to growth hormone has the advantages of strong binding to human growth hormone receptor, good cell proliferation activity, and high in vitro activity.

[0148] Preferably, the first peptide segment has mutations at the following positions compared to the Fc fragment of wild-type IgGl: P228 Delete, C229 Delete, P230 Delete, L351S, T366R, L368H, P395K, M428Y, K447A, and K409D or K409T.

[0149] The inventors have found through a large number of experiments that the Fc mutant is fused with human growth hormone to obtain a fusion protein with higher binding capacity to human growth hormone receptor, higher cell proliferation activity and higher in vitro activity. Exemplarily, the Fc mutant of the fusion protein J16 and J18 according to the embodiments of the present application has different mutation sites, the Fc mutant in J18 contains the above-mentioned site mutations, while J16 does not have L351S and M428Y mutations compared with J18, and the fusion protein J18 has higher binding capacity to human growth hormone receptor, cell proliferation activity and in vitro activity. Moreover, compared with the fusion protein J2 (P228 Delete, C229 Delete, P230 Delete, T366R, L368H, P395K and K409D), the fusion protein J17 has L351S and M428Y mutations added compared with J2, and the mutation sites of the 409th position of J2 and J17 are different (K409T for J17), but the fusion protein J17 has higher cell proliferation activity and in vitro activity. Therefore, the fusion protein prepared by using the Fc mutant with L351S and M428Y mutations has higher binding capacity to human growth hormone receptor, cell proliferation activity and in vitro activity.

[0150] According to the embodiments of the present application, the Fc mutant further comprises a hinge region fragment of wild-type IgG1, and the C-terminal of the hinge region fragment of wild-type IgG1 is connected to the N-terminal of the first peptide segment.

[0151] According to the embodiments of the present application, the hinge region fragment of wild-type IgG1 has an amino acid sequence as shown in SEQ ID NO: 41.

[0152] According to the embodiments of the present application, the Fc mutant has an amino acid sequence as shown in any one of SEQ ID NO: 3-7. The inventors have found that the fusion protein prepared by using the Fc mutant and the bioactive molecule has higher in vivo and in vitro bioactivity.

[0153] According to the embodiments of the present application, the Fc mutant is in a monomer form.

[0154] Fusion protein

[0155] In another aspect of the present application, the present application provides a fusion protein. According to an embodiment of the present application, the fusion protein comprises: a second peptide segment comprising a bioactive molecule functional region; a third peptide segment comprising an Fc mutant having an amino acid sequence as set forth in SEQ ID NO: 2 or an Fc mutant as defined above, the second peptide segment being connected to the third peptide segment. The inventors have found through a large number of experiments that the fusion protein of the present application is in a monomer form, has higher in vivo and in vitro activities, and lower ADCC and CDC effects; and the fusion protein can be subsequently purified by Protein A, thus simplifying the purification process.

[0156] It should be noted that the "monomer" refers to having one Fc mutant, and the specific structure is shown in Figure 1 .

[0157] According to an embodiment of the present application, the second peptide segment comprises a growth hormone, a growth hormone analog, a growth hormone functional region, or a growth hormone analog functional region, and preferably a human growth hormone or a human growth hormone functional region. The inventors have found that the fusion protein of the present application is in a monomer form, has higher in vivo and in vitro activities, and lower ADCC and CDC effects, and has the advantages of strong binding force to human growth hormone receptors, good activity of cell proliferation, and high in vitro activity.

[0158] According to an embodiment of the present application, the human growth hormone has an amino acid sequence as set forth in SEQ ID NO: 1 or has at least 90% identity thereto.

[0159] According to an embodiment of the present application, the N-terminus of the second peptide segment is connected to the C-terminus of the third peptide segment, or the C-terminus of the second peptide segment is connected to the N-terminus of the third peptide segment. In this way, the fusion protein prepared by using the above connection mode has higher in vivo and in vitro activities.

[0160] According to an embodiment of the present application, the N-terminus of the second peptide segment is connected to the C-terminus of the third peptide segment. The inventors have found through a large number of experiments that the fusion protein prepared by using the above connection mode can further improve its binding force to the corresponding receptors, the activity of cell proliferation, and the in vitro activity, for example, the fusion protein prepared by fusing the Fc mutant and the growth hormone has better efficacy in the animal body.

[0161] According to an embodiment of the present application, the fusion protein further comprises a connecting peptide disposed between the second peptide segment and the third peptide segment. In this way, the second peptide segment and the third peptide segment fused by using the above connecting peptide can improve the biological activity of the final obtained fusion protein. For example, the connecting peptides of the fusion proteins J1, J2, J6, and J7 are different, and compared with J1 without the connecting peptide, J2, J6, and J7 have better binding activity to human GHR and better promotion effect on cell proliferation.

[0162] According to an embodiment of the present application, the N-terminus of the connecting peptide is connected to the C-terminus of the third peptide segment, and the C-terminus of the connecting peptide is connected to the N-terminus of the second peptide segment; or the N-terminus of the connecting peptide is connected to the C-terminus of the second peptide segment, and the C-terminus of the connecting peptide is connected to the N-terminus of the third peptide segment.

[0163] According to an embodiment of the present application, the N-terminus of the connecting peptide is connected to the C-terminus of the third peptide segment, and the C-terminus of the connecting peptide is connected to the N-terminus of the second peptide segment. The inventors have found through a large number of experiments that the fusion protein obtained by using the above connection mode can further improve the binding force to the corresponding receptor, the cell proliferation activity and the in-vitro activity, for example, the fusion protein prepared by fusing the Fc mutant and the growth hormone can have better drug efficacy in animals.

[0164] According to an embodiment of the present application, the amino acid sequence of the connecting peptide is (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. n According to an embodiment of the present application, the amino acid sequence of the connecting peptide is (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Thus, the second peptide segment and the third peptide segment fused by using the above connecting peptide can improve the biological activity of the final obtained fusion protein.

[0165] According to an embodiment of the present application, the connecting peptide has an amino acid sequence as shown in any one of SEQ ID NO: 8-10. Thus, the second peptide segment and the third peptide segment fused by using the above connecting peptide can further improve the biological activity of the final obtained fusion protein.

[0166] According to an embodiment of the present application, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NO: 24-31. The inventors have found through experiments that the above fusion protein has strong binding force to human growth hormone receptor, good cell proliferation activity and high in-vitro activity.

[0167] It can be understood by those skilled in the art that the features and advantages described above for the Fc mutant also apply to the fusion protein, which will not be described herein again.

[0168] Nucleic acid molecule

[0169] In another aspect of the present application, a nucleic acid molecule is provided. According to an embodiment of the present application, the nucleic acid molecule encodes the aforementioned Fc mutant or the aforementioned fusion protein. The nucleic acid molecule can be effectively used to express the above Fc mutant or fusion protein, especially in prokaryotic or lower eukaryotic expression systems, and the fusion protein is in a monomer form, has higher in-vivo and in-vitro activity and lower ADCC and CDC effects.

[0170] According to an embodiment of the present application, the nucleic acid molecule is DNA.

[0171] It is understood by those skilled in the art that the features and advantages described above for the Fc mutant and the fusion protein also apply to the nucleic acid molecule, which will not be repeated here.

[0172] Expression vector

[0173] In another aspect of the present application, an expression vector is provided. According to an embodiment of the present application, the expression vector carries the aforementioned nucleic acid molecule. The expression vector can be used to effectively express the aforementioned Fc mutant or fusion protein in a cell, especially in a prokaryotic or lower eukaryotic expression system, and the fusion protein is in a monomeric form, has higher activity in vitro and in vivo, and has lower ADCC and CDC effects.

[0174] According to an embodiment of the present application, the expression vector is a eukaryotic expression vector.

[0175] According to an embodiment of the present application, the expression vector is a lentiviral vector.

[0176] It is understood by those skilled in the art that the features and advantages described above for the Fc mutant, the fusion protein, and the nucleic acid molecule also apply to the expression vector, which will not be repeated here.

[0177] Recombinant cell

[0178] In another aspect of the present application, a recombinant cell is provided. According to an embodiment of the present application, the recombinant cell comprises: carrying the aforementioned nucleic acid molecule; or, expressing the aforementioned Fc mutant or the aforementioned fusion protein. The recombinant cell can be used to effectively express the aforementioned Fc mutant or fusion protein, especially in a prokaryotic or lower eukaryotic expression system, and the fusion protein is in a monomeric form, has higher activity in vitro and in vivo, and has lower ADCC and CDC effects.

[0179] According to an embodiment of the present application, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0180] According to an embodiment of the present application, the recombinant cell is a eukaryotic cell.

[0181] According to an embodiment of the present application, the recombinant cell is a mammalian cell.

[0182] It is understood by those skilled in the art that the features and advantages described above for the Fc mutant, the fusion protein, the nucleic acid molecule, and the expression vector also apply to the recombinant cell, which will not be repeated here.

[0183] Pharmaceutical composition

[0184] In another aspect of the present application, a pharmaceutical composition is provided. According to embodiments of the present application, the pharmaceutical composition comprises the aforementioned fusion protein. The pharmaceutical composition according to embodiments of the present application can be used for preventing and treating growth hormone abnormality related diseases.

[0185] According to embodiments of the present application, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0186] According to embodiments of the present application, the pharmaceutical composition is in the form of an injection.

[0187] According to embodiments of the present application, the administration route of the pharmaceutical composition comprises subcutaneous injection or intravenous injection.

[0188] It is understood by those skilled in the art that the features and advantages described above for the Fc mutant, the fusion protein, the nucleic acid molecule, the expression vector, the recombinant cell, and the pharmaceutical composition are equally applicable to the method for preventing and / or treating growth hormone abnormality related diseases, and thus will not be repeated here.

[0189] Method for preventing and / or treating growth hormone abnormality related diseases

[0190] In another aspect of the present application, a method for preventing and / or treating growth hormone abnormality related diseases is provided. According to embodiments of the present application, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned fusion protein or the aforementioned pharmaceutical composition. According to embodiments of the present application, the method is effective for preventing or treating growth hormone abnormality related diseases.

[0191] According to embodiments of the present application, the administration route of the method comprises subcutaneous injection or intravenous injection.

[0192] According to embodiments of the present application, the growth hormone abnormality related diseases at least include one selected from the group consisting of childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by catabolic state during chemotherapy treatment and AIDS treatment, and intrauterine growth retardation.

[0193] It is understood by those skilled in the art that the features and advantages described above for the Fc mutant, the fusion protein, the nucleic acid molecule, the expression vector, the recombinant cell, and the pharmaceutical composition are equally applicable to the method for preventing and / or treating growth hormone abnormality related diseases, and thus will not be repeated here.

[0194] Use

[0195] In another aspect of the present application, the present application provides use of the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, and the aforementioned pharmaceutical composition in the preparation of a medicament for treating or preventing a growth hormone abnormality related disease.

[0196] According to embodiments of the present application, the growth hormone abnormality related disease at least includes one selected from the group consisting of childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, diseases caused by catabolic states during chemotherapy treatment and AIDS treatment, and intrauterine growth retardation.

[0197] It is understood by those skilled in the art that the features and advantages described above for the Fc mutant, the fusion protein, the nucleic acid molecule, the expression vector, the recombinant cell, and the pharmaceutical composition also apply to the use, which will not be repeated here.

[0198] The solutions of the present application will be explained below in connection with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If no specific techniques or conditions are mentioned in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If no manufacturer of reagents or instruments is mentioned, it is a conventional product that can be obtained commercially.

[0199] Example 1: Preparation of recombinant long-acting human growth hormone fusion protein expression vector

[0200] In the present application, human growth hormone (SEQ ID NO: 1) is connected with Fc mutant (SEQ ID NO: 2 and SEQ ID NO: 7) to construct a fusion protein, obtaining J1 and J3, respectively. In the present application, human growth hormone (SEQ ID NO: 1) is connected with various Fc mutants (SEQ ID NO: 2 and SEQ ID NO: 4-6) through a connecting peptide (SEQ ID NO: 8-10) to construct a fusion protein, obtaining J2, J6, J7, and J16-18, respectively. The specific experimental operations are as follows:

[0201] The nucleotide sequences encoding human growth hormone (SEQ ID NO: 11), the nucleotide sequences encoding the connecting peptide (SEQ ID NO: 21-23), and the nucleotide sequences encoding the Fc mutant of various mutations (SEQ ID NO: 16-20) are combined, and the nucleotide sequences SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39 are cloned into the expression vector pCDNA3.4 (purchased from Thermo Fisher) using gene total synthesis and molecular cloning technology, respectively, to obtain the fusion protein expression vectors of each nucleotide sequence.

[0202] Example 2: Expression of recombinant long-acting human growth hormone fusion protein

[0203] The plasmids obtained in Example 1 are transfected into host cells using Expi CHO-S (Gibco, A29133) as the host cells, and the fusion proteins (i.e., recombinant long-acting human growth hormone fusion proteins) are expressed using the chemical transfection reagent Polyplus-FectoPRO (polyplus, 116-010). The corresponding amino acid sequences are SEQ ID NO: 24-31. The experimental operation is as follows:

[0204] 1. One day before transfection of Expi CHO-S cells, the cells are passaged, and the cell density is adjusted to about 3x10 6 The cell culture flask is placed back on the shaker (37°C, 8% CO2) for continued culture.

[0205] 2. On the day of transfection (1 L per molecule transfection): Expi CHO-S cell solution is taken for cell counting, and the cell density is adjusted to about 6x10 6 cells / mL using culture medium.

[0206] 3. Preparation of transfection complex: 16 sterile cell culture flasks are prepared, 8 labeled "DNA" and 8 labeled "FectoPRO". The transfection reagent Polyplus-FectoPRO is added to the "FectoPRO" flasks. In the 8 "DNA" flasks, 60 mL of Opti-MEM solution is added to each, and 500 μg of the fusion protein expression vector of each nucleotide sequence obtained in Example 1 is added to each "DNA" flask. The fusion protein expression vector diluent is obtained by mixing, and the fusion protein expression vector diluent of each nucleotide sequence is added to the "FectoPRO" flasks and mixed, incubated at room temperature for 10 min, and then added to the cell solution and shaken. The cell culture flasks are placed back on the shaker for continued transfection culture.

[0207] 4. Add an appropriate amount of OPM-CHO ProFeed 18-22 hours after transfection and measure the biochemical parameters of the cell sap. Based on the biochemical parameters, supplement glucose to 6 g / L. Start measuring titers 4 days after transfection, and feed and glucose should be added every other day. When cell viability is <80%, the CHO cell fermentation broth is obtained, and the supernatant can be harvested for purification.

[0208] Example 3: Purification of recombinant long-acting human growth hormone fusion protein

[0209] The CHO cell fermentation broth obtained in Example 2 was subjected to two-stage centrifugation (first stage: 3000×g, 30min; second stage: 12000×g, 20min), and the supernatant was collected and filtered through a 0.2μm filter for later use.

[0210] Protein A affinity chromatography: Equilibrate the column with at least 3 column volumes of an aqueous solution containing 20 mM phosphate and 150 mM sodium chloride at pH 7.2. Load the sample, filter the clear filtrate, and retain the solution on the column for 5 minutes. After loading the sample, equilibrate the column again with at least 1 column volume of an aqueous solution containing 20 mM phosphate and 150 mM sodium chloride at pH 7.2. Elute the target protein sequentially with buffers containing 50 mM acetate (HAc) at pH 4.5, 50 mM HAc at pH 4.0, and 50 mM HAc at pH 3.5. When an absorption peak appears, the peak range is 50 mAU - peak - 50 mAU. A fusion protein with SEC purity meeting the requirements (>98.0%) is obtained, containing the nucleotide sequences SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38. Expi CHO-S cells with ID NO:38 or SEQ ID NO:39 were fermented and then chromatography to obtain J1, J2, J3, J6, J7, J16, J17, and J18 fusion proteins, respectively. The specific structures of the fusion proteins are shown below. Figure 1 As shown, the structures of J1, J2, J3, J6, J7, and J17 are described in [reference needed]. Figure 1 The structure of J16 and J18 is shown in the left figure. Figure 1 The image on the right.

[0211] Example 4: Assay of fusion protein binding to human GHR

[0212] This embodiment describes the in vitro receptor (human growth hormone receptor, or human GHR) binding activity assay of the purified fusion protein obtained in Example 3, with Genexine GX-H9 serving as the control group. The specific experimental procedures are as follows:

[0213] Dilute human GHR to make 0.5 μg / ml coating solution, add 100 μL / well to the enzyme-labeled plate, and coat at 2-8°C for 12 hours or more. Discard the residual solution, add 300 μL of 1% BSA-PBST (1% bovine serum albumin in phosphate buffered saline) per well, and block at 37°C for 1 hour. Wash 3 times with 300 μL of PBST (phosphate buffered saline) per well, dilute recombinant long-acting human growth hormone fusion protein and Genexine GX-H9 to 5 μg / ml, and then dilute 5 times to 8 gradient concentrations, and add 100 μL / well to the enzyme-labeled plate. Incubate at 37°C for 1 hour, wash 3 times with 300 μL of PBST per well, and then add 10000-fold diluted Goat anti human IgG Fc-HRP in 1% BSA-PBST, and add 100 μL / well. Incubate at 37°C for 1 hour, wash 3 times with 300 μL of PBST per well, and then dry by tapping. Add 100 μL of TMB color developing solution per well. After reaction at room temperature for 5 minutes, terminate the reaction with 2M H2SO4 aqueous solution, 100 μL / well. Place the enzyme-labeled plate in which the reaction has been terminated in an enzyme-labeled photometer, and read the absorbance OD at 450 nm 450 Values and calculate the EC50 of each fusion protein for human GHR binding activity, as shown in Table 1, and the results show that, in terms of binding activity, the EC50 value of the fusion protein provided by the present application for human GHR binding is low, and the fusion protein has strong binding activity with human GHR. See the binding activity relative to Genexine GX-H9, which shows that the binding activity with human GHR is similar or better compared to Genexine GX-H9.

[0214] wherein the amino acid sequence of Genexine GX-H9 is:

[0215] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 43).

[0216] Table 1: Results of detection of recombinant long-acting human growth hormone fusion protein for human GHR binding activity test

[0217]

[0218] Example 5: Experiment of recombinant long-acting human growth hormone fusion protein for promoting proliferation of Nb2-11 cells

[0219] The purified fusion protein obtained in Example 3 was subjected to the experiment, and Genexine GX-H9 described above was used as a control group, and the specific experimental operation was as follows:

[0220] The cells were collected and 1 × 10 5Cells were suspended in PRMI 1640 medium (containing 1% FBS and 50 μM β-mercaptoethanol) at a concentration of 100 μL. 50 μL of cell samples were added to each well of a 96-well cell culture plate. The cells were cultured in 50 μL of analytical medium containing various concentrations of recombinant long-acting human growth hormone fusion protein (0.051 ng / mL–3000 ng / mL) and Genexine GX-H9. The cell plates were cultured at 37°C and 5% CO2 for 96 hours, and then 50 μL of ellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571) was added to each well. After 10 minutes, the chemiluminescence signal was detected using a microplate reader. The bioactivity of the recombinant long-acting human growth hormone fusion protein was determined from the obtained dose-response curve, and the results are shown in Table 2. The fusion protein provided by this invention promotes the proliferation of Nb2-11 cells. 50 It has a low value and a good proliferative effect on Nb2-11 cells. Compared with Genexine GX-H9, its activity in promoting Nb2-11 cell proliferation is similar or better.

[0221] Table 2: Experimental results of recombinant long-acting human growth hormone fusion protein promoting Nb2-11 cell proliferation

[0222]

[0223] Example 6: Experiment on the biological activity of recombinant long-acting human growth hormone fusion protein against reporter genes in cells

[0224] This embodiment uses the purified fusion protein obtained in Example 3 for experiments, and uses Genexine GX-H9 as the control group. The specific experimental procedures are as follows:

[0225] Reporter cells in logarithmic growth phase (293-GHR / STAT5 cell line) were harvested, digested with trypsin, and plated (Costar, 3917) at 4×10⁻⁶ m². 4cells / well, 50 μL / well, and the 96-well white plate was incubated in a 37°C, 5% CO2 incubator overnight. The cells were added with gradient-diluted recombinant long-acting human growth hormone fusion protein, with an initial concentration of 100 nM, 5-fold gradient dilution, 10 gradient concentrations, and the dilution was added to the cells with a volume of 50 μL / well. The 96-well white plate was incubated in a 37°C, 5% CO2 incubator for 6 hours. After the incubation, the 96-well white plate and the Nano-Glo Luciferase Assay kit (promega, N112B) were taken out and equilibrated to room temperature. The reaction substrate was added to the sample well with a volume of 50 μL / well, and the sample was placed at room temperature for 10 minutes. The luminescence signal value was recorded by using an enzyme-labeled instrument (Promega, GM2000), with the protein concentration as the X axis and the luminescence signal value as the Y axis. GraphPad Prisim 5 was used for four-parameter fitting, and the EC 50 value was calculated. The results are shown in Table 3. The results show that the fusion protein provided by the present application has a low EC 50 value for promoting the expression of luciferase by the reporter gene cells, and is comparable to or lower than the control group, and has excellent activity in promoting the expression of luciferase by the reporter gene cells.

[0226] Table 3: Experimental detection results of recombinant long-acting human growth hormone fusion protein for promoting the expression of luciferase by reporter gene cells

[0227] Fusion protein number EC 50 (ng / mL) Relative activity (%) relative to Genexine GX-H9 J1 95.47 79.67 J2 70.81 101.71 J3 87.06 82.72 J6 84.14 92.58 J7 89.72 95.29 J16 39.69 215.12 J17 56.46 157.46 J18 33.61 302.59

[0228] Example 7: Test sample induced ADCC effect of effector cell Jurkat-CD16a-luc on target cell CHO-K1-PD-L1 / GHR

[0229] 1. Sample information

[0230] Sample name Concentration (mg / mL) M7824 2.05 2(J2) 0.6 5 (Genexine GX-H9) 1.33 18(J18) 2.66 Acterma 20

[0231] 2. Cell treatment

[0232] Target cell CHO-K1-PD-L1 / GHR (SBTCL015, Shanghai Jingbeier Biomedical Technology Co., Ltd.) treatment: 2 days before the experiment, discard the supernatant, wash the cells with 5 mL of PBS, add 1 mL of 0.25% trypsin-EDTA, digest for 1 min in a 37°C incubator, then add 6 mL of 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 medium 1 (450 mL F12 medium + 50 mL fetal bovine serum (FBS)), and add 120 μL of G418, the G418 concentration is 600 μg / mL, mix well, and continue to culture in the incubator.

[0233] Effector cell Jurkat-CD16a-luc (SBTCL001, Shanghai Jingbeier Biomedical Technology Co., Ltd.) treatment: 2 days before the experiment, mix the cell suspension by blowing, take 5 mL of cell suspension into a new T75 culture bottle, then add 15 mL of medium 2 (450 mL RPMI 1640 medium + 50 mL FBS), and add 320 μL of G418, the G418 concentration is 800 μg / mL, 60 μL of Hygromycin B, the Hygromycin B concentration is 150 μg / mL, mix well, and continue to culture in the incubator.

[0234] 3.3, Experimental procedure

[0235] On the first day, the target cell CHO-K1-PD-L1 / GHR density was adjusted to 1.83 x 10 5 cells / mL, 50 μL / well plated overnight; on the second day, first dilute the test sample (J2 or J18), positive control M7824 and negative control Acterma to 0.1 mg / mL with analysis medium 2 (RPMI 1640 medium) for primary dilution, to 24 μg / mL for secondary dilution, and then 3-fold dilution for 9 gradients, 25 μL / well added to the cell plate, mixed well, and placed in a 37°C incubator for a total of 45 min. After incubation, the cell plate was taken out; the effector cell Jurkat-CD16a-luc density was adjusted to 1.47 x 10 6Cell plate was taken out from incubator and equilibrated at room temperature for 30 min. Then 25 μL / well of diluted sample was added to the cell plate, mixed well and incubated at 37 °C for 5.5 h. After incubation, cell plate was taken out from incubator and equilibrated at room temperature for 30 min. Then 100 μL / well of One-Glo Luciferase Assay System (Promega, E6120) was added to the cell plate, mixed well and incubated at room temperature for 10 min. After incubation, chemiluminescence signal was detected by microplate reader. The results are shown in Figure 8. Figure 2 As shown in Figure 8, after adding positive control PD-L1 antibody M7824 (Shanghai Jiebai Biomedical Technology Co., Ltd.), negative control IL-6R antibody Actemra (Roche) and test samples in the ADCC experiment, the positive control M7824 induced obvious ADCC effect of effector cells Jurkat-CD16a-luc on target cells CHO-K1-PD-L1 / GHR, the negative control Actemra had no ADCC effect, and the test samples had no ADCC effect. Among them, Figure 2 The concentration unit of the abscissa is ng / ml.

[0236] Example 8: Test sample induced CDC effect of human serum complement on target cells CHO-K1-PD-L1 / GHR

[0237] 1. Sample information

[0238] Sample name Concentration (mg / mL) Avelumab 22.65 J2 0.6 Genexine GX-H9 1.33 J18 2.66 Acterma 20

[0239] 2. Cell treatment

[0240] Target cell CHO-K1-PD-L1 / GHR treatment: 2 days before the experiment, the cells were passaged, the supernatant was discarded, the cells were washed with 5 mL of PBS, 1 mL of 0.25% trypsin-EDTA was added, and the cells were digested in a 37 °C incubator for 1 min. Then 6 mL of medium 1 (450 mL F12 medium + 50 mL FBS) was added to terminate the digestion, and the cells were resuspended to a single cell suspension. 5 mL of cell suspension was discarded, 8 mL of medium 1 (450 mL F12 medium + 50 mL FBS) was added, and 120 μL of G418 was added. The concentration of G418 was 600 μg / mL, and the mixture was incubated in an incubator.

[0241] 3. Experimental procedure

[0242] On the first day, the target cells CHO-K1-PD-L1 / GHR were adjusted to a density of 2.0 x 10 5The cells were plated at 50,000 cells / mL, 50 μL / well overnight; the next day, the test sample (i.e., J2 or J18), the positive control Avelumab (PD-L1 antibody, Shanghai Jiebei Biomedical Technology Co., Ltd.), and the negative control Acterma were diluted to 0.1 mg / mL with the differentiation medium (99% F12 medium + 1% FBS) for the first dilution, to 15 μg / mL for the second dilution, and then 3-fold dilution for 7 gradients, 50 μL / well was added to the cell plate, mixed, and placed in a 37°C incubator for a total of 30 min, then the cell plate was removed; 30% complement (containing 70% differentiation medium + 30% complement) was added, 50 μL / well, mixed, and placed in a 37°C incubator for a total of 6 h, then the cell plate was removed and equilibrated at room temperature, One-Glo Luminescent Cell Viability Assay was added, 50 μL / well was added to the cell plate, and the pipette was blown 10 times to mix; after 10 min of lysis at room temperature in the dark, the chemiluminescence signal value was detected by a microplate reader. The results showed that in the CDC experiment, after adding the positive control PD-L1 antibody Avelumab, the negative control IL-6R antibody Acterma, and the SBT123 test sample molecule, the positive control Avelumab induced the human serum complement to have a significant CDC effect on the target cells CHO-K1-PD-L1 / GHR, the negative control Acterma had no CDC effect, and the test sample had no CDC effect.

[0243] Example 9: In vivo biological activity test

[0244] A long-acting in vivo pharmacodynamic study of J2 and J18 fusion proteins was conducted: Four-week-old SPF-grade male SD rats, weighing 60-80g, were selected. Two weeks prior to the experiment, the pituitary gland was surgically removed under clean conditions. A two-week recovery period followed the surgery. Before drug administration, healthy animals whose body weight change was less than ±10% of their pre-operative body weight were selected. The pituitary-removed rats were randomly divided into five groups based on body weight: a model group, a low-dose J2 fusion protein group, a high-dose J2 fusion protein group, a low-dose J18 fusion protein group, and a high-dose J18 fusion protein group; eight rats were in each group. The administration method was subcutaneous injection in the neck. The low-dose J2 fusion protein group received J2 fusion protein at a dose of 9.35 nmol / kg; the low-dose J18 fusion protein group received J18 fusion protein at a dose of 9.35 nmol / kg; the high-dose J2 fusion protein group received J2 fusion protein at a dose of 56.1 nmol / kg; and the high-dose J18 fusion protein group received J18 fusion protein at a dose of 56.1 nmol / kg. The model group received a solvent. Each group received either the above administration or solvent once a week, for a total of two administrations. Each rat was weighed at the same time each day after administration to calculate daily weight changes. The experiment was stopped after 14 days, and the rats were weighed again. Weight gain (g) on ​​a given day after administration was the difference between the animal's weight on that day and its weight before administration. At the end of the experiment, a necropsy was performed to confirm the absence of pituitary remnants in the sella turcica region. The weight gain changes of each group over 14 days were compared, and the results are as follows: Figure 3 As shown, "2#-Low" represents the low-dose J2 fusion protein group, "2#-High" represents the high-dose J2 fusion protein group, "18#-Low" represents the low-dose J18 fusion protein group, and "18#-High" represents the high-dose J18 fusion protein group; 2#-Low vs. model group, p = 0.0083; 2#-High vs. model group, p < 0.0001; 18#-Low vs. model group, p = 0.0030; 18#-High vs. model group, p = 0.0003.

[0245] Depend on Figure 3 The results show that:

[0246] (1) Compared with the model group, both the J2 fusion protein and the J18 fusion protein significantly increased the weight of rats at both low and high doses, indicating that the fusion protein provided by the present invention has good in vivo activity.

[0247] (2) The weight gain in the high-dose group was greater than that in the low-dose group.

[0248] (3) The high-dose group of J18 fusion protein had a more significant effect than the high-dose group of J2.

[0249] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific feature, structure, material or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In the specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any suitable manner in one or more embodiments or examples. In addition, different embodiments or examples described in the specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0250] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application. SEQUENCE LISTING <110> Shenzhen Cospan Biopharma Co., Ltd. <120> Recombinant long-acting human growth hormone fusion protein and preparation method and use thereof <130> PDI220116 <160> 43 <170> PatentIn version 3.3 <210> 1 <211> 191 <212> PRT <213> Artificial <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> 217 <212> PRT <213> Artificial <220> <223> 2 <400> 2 Ala Pro Glu Leu 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 His Glu Asp Pro Glu Val Lys 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 Tyr 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 Ala Leu Pro Ala Pro 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 Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Arg Cys His 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 Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Thr Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 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 Pro Gly Lys 210 215 <210> 3 <211> 218 <212> PRT <213> Artificial <220> <223> 3 <400> 3 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 1 5 10 15 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 20 25 30 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 35 40 45 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 50 55 60 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 65 70 75 80 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 85 90 95 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 100 105 110 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 115 120 125 Leu Thr Lys Asn Gln Val Ser Leu Arg Cys His Val Lys Gly Phe Tyr 130 135 140 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 145 150 155 160 Asn Tyr Lys Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 165 170 175 Leu Tyr Ser Thr Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 180 185 190 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 195 200 205 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 4 <211> 217 <212> PRT <213> Artificial <220> <223> 4 <400> 4 Ala Pro Glu Leu 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 His Glu Asp Pro Glu Val Lys 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 Tyr 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 Ala Leu Pro Ala Pro 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 Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Arg Cys His 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 Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Thr Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 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 Pro Gly Ala 210 215 <210> 5 <211> 217 <212> PRT <213> Artificial <220> <223> 5 <400> 5 Ala Pro Glu Leu 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 His Glu Asp Pro Glu Val Lys 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 Tyr 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 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Ser Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Arg Cys His 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 Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Tyr His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 6 <211> 217 <212> PRT <213> Artificial <220> <223> 6 <400> 6 Ala Pro Glu Leu 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 His Glu Asp Pro Glu Val Lys 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 Tyr 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 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Ser Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gin Val Ser Leu Arg Cys His Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Tyr His Glu Ala Leu His Asn His Tyr Thr Gin 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Ala 210 215 <210> 7 <211> 224 <212> PRT <213> Artificial <220> <223> 7 <400> 7 Asp Lys Thr His Thr Gly Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Arg Cys 130 135 140 His Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Lys Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Thr Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 8 <211> 15 <212> PRT <213> Artificial <220> <223> 8 <400> 8 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 9 <211> 10 <212> PRT <213> Artificial <220> <223> 9 <400> 9 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 10 <211> 5 <212> PRT <213> Artificial <220> <223> 10 <400> 10 Gly Gly Gly Gly Ser 1 5 <210> 11 <211> 573 <212> DNA <213> Artificial <220> <223> 11 <400> 11 tttcccacca ttcctctgag cagactgttc gacaacgcca tgctgagagc ccacagactg 60 caccagctgg cctttgacac ataccaggag ttcgaggagg cctacatccc caaggagcag 120 aagtacagct tcctgcagaa cccccagacc agcctgtgct tcagcgagag catccccacc 180 cccagcaata gagaggagac acagcagaag agcaacctgg agctgctgag aatcagcctg 240 ctgctgatcc agagctggct ggagcccgtt caatttctga gaagcgtgtt cgccaacagc 300 ctggtgtacg gcgccagcga ttctaatgtg tacgacctgc tgaaggacct ggaggagggc 360 atccagaccc tgatgggcag actggaggac ggctctccta gaaccggaca aattttcaag 420 cagacctaca gcaagttcga caccaacagc cacaacgacg acgccctgct gaagaactac 480 ggcctgctgt actgcttcag aaaggacatg gacaaggtgg agacattcct gagaatcgtg 540 cagtgcagaa gcgtggaggg cagctgcgga ttc 573 <210> 12 <211> 651 <212> DNA <213> Artificial <220> <223> 12 <400> 12 gcccctgaac tgctcggagg cccctccgtg tttctgttcc ctccaaagcc taaggacacc 60 ctgatgatca gcagaacccc tgaagtgacc tgcgtggtgg tggatgtgtc ccacgaggat 120 cccgaagtga agttcaattg gtacgtggac ggcgtggaag tgcacaacgc caagaccaag 180 cctagagagg aacagtacaa cagcacctac agagtggtgt ccgtgctgac cgtgctgcac 240 caggattggc tgaacggcaa agagtacaag tgcaaggtgt ccaacaaggc cctgcctgct 300 cctatcgaga aaaccatcag caaggccaag ggccagccta gggaacccca ggtttacaca 360 ctgcctccaa gcagggacga gctgaccaag aatcaggtgt ccctgcggtg tcacgtgaag 420 ggcttctacc cttccgatat cgccgtggaa tgggagagca atggccagcc tgagaacaac 480 tacaagacca ccaagccagt gctggacagc gacggctcat tcttcctgta cagcaccctg 540 accgtggaca agtccagatg gcaacagggc aacgtgttca gctgcagcgt gatgcacgag 600 gccctgcaca accactacac ccagaagtcc ctgagcctgt ctcctggcaa a 651 <210> 13 <211> 651 <212> DNA <213> Artificial <220> <223> 13 <400> 13 gctcctgaac tgctgggagg acctagcgtg tttctgtttc ctcccaaacc taaagacacc 60 ctgatgatca gcagaacccc cgaggtgacc tgcgtggtgg ttgatgtgtc tcatgaagac 120 cccgaagtga agttcaactg gtacgtggac ggcgtggagg tgcacaatgc taaaaccaag 180 cccagagagg agcagtacaa cagcacctac agagtggtga gcgtgctgac cgtgctgcac 240 caagattggc tgaatggcaa ggaatacaag tgcaaggtga gcaacaaggc cctgcccgcc 300 cctattgaaa aaaccattag caaggctaag ggccagccca gagagcccca agtgtataca 360 ctgcccccta gcagagatga actgaccaaa aaccaggtga gcctgagatg ccacgtgaag 420 ggcttttacc ccagcgacat tgccgtggag tgggagagca atggccaacc tgaaaacaat 480 tacaagacca ccaagcccgt gctggacagc gacggatctt tctttctgta tagcaccctg 540 accgtggaca agagcagatg gcagcagggc aatgtgttca gctgcagcgt gatgcacgag 600 gccctgcaca atcactatac ccagaagagc ctgagcctga gccccggcaa a 651 <210> 14 <211> 654 <212> DNA <213> Artificial <220> <223> 14 <400> 14 cccgcccccg aactgctggg aggaccttct gtgtttctgt ttcctcccaa acccaaagac 60 accctgatga tcagcagaac ccccgaggtg acctgcgtgg ttgtggatgt gtctcatgag 120 gatcccgagg tgaagttcaa ctggtacgtg gacggcgtgg aggtgcacaa tgctaagaca 180 aagcccagag aggagcagta caacagcacc tacagagtgg tgagcgtgct gaccgtgctg 240 caccaagatt ggctgaatgg aaaggaatac aagtgcaagg tgagcaacaa ggccctgccc 300 gcccctattg aaaaaacaat tagcaaggcc aagggccagc ccagagaacc tcaagtgtat 360 accctgcctc ccagcagaga tgagctgaca aaaaatcagg tgagcctgag atgccacgtg 420 aagggcttct accccagcga cattgccgtg gagtgggaaa gcaatggcca acctgagaac 480 aactacaaga ccaccaagcc cgtgctggac agcgacggat ctttttttct gtacagcacc 540 ctgaccgtgg acaagagcag atggcagcag ggcaatgtgt tcagctgcag cgtgatgcac 600 gaggccctgc ataatcacta cacccagaaa agcctgagcc tgagccccgg caag 654 <210> 15 <211> 651 <212> DNA <213> Artificial <220> <223> 15 <400> 15 gctcctgaac tgctcggagg cccttccgtg ttcctgtttc ctccaaagcc taaggacacc 60 ctgatgatca gcagaacccc tgaagtgacc tgcgtggtgg tggatgtgtc ccacgaggat 120 cccgaagtga agttcaattg gtacgtggac ggcgtcgagg tgcacaacgc caagacaaag 180 cctagagagg aacagtacaa cagcacctac agagtggtgt ccgtgctgac cgtgctgcac 240 caggattggc tgaacggcaa agagtacaag tgcaaggtgt ccaacaaggc cctgcctgct 300 cctatcgaga aaaccatcag caaggccaag ggccagccta gggaacccca ggtttacaca 360 ctgcctccaa gcagggacga gctgaccaag aatcaggtgt ccctgcggtg tcacgtgaag 420 ggcttctacc cttccgatat cgccgtggaa tgggagagca atggccagcc tgagaacaac 480 tacaagacca ccaagccagt gctggacagc gacggctcat tcttcctgta cagcaccctg 540 accgtggaca agtccagatg gcaacagggc aacgtgttca gctgcagcgt gatgcacgag 600 gccctgcaca accactacac ccagaagtcc ctgagcctgt ctcctggcaa a 651 <210> 16 <211> 651 <212> DNA <213> Artificial <220> <223> 16 <400> 16 gctcctgaac tgctcggagg cccttccgtg ttcctgtttc ctccaaagcc taaggacacc 60 ctgatgatca gcagaacccc tgaagtgacc tgcgtggtgg tggatgtgtc ccacgaggat 120 cccgaagtga agttcaattg gtacgtggac ggcgtggaag tgcacaacgc caagaccaag 180 cctagagagg aacagtacaa cagcacctac agagtggtgt ccgtgctgac cgtgctgcac 240 caggattggc tgaacggcaa agagtacaag tgcaaggtgt ccaacaaggc cctgcctgct 300 cctatcgaga aaaccatcag caaggccaag ggccagccta gggaacccca ggtttacaca 360 ctgcctccaa gcagggacga gctgaccaag aatcaggtgt ccctgcggtg tcacgtgaag 420 ggcttctacc cttccgatat cgccgtggaa tgggagagca atggccagcc tgagaacaac 480 tacaagacca ccaagccagt gctggacagc gacggctcat tcttcctgta cagcaccctg 540 accgtggaca agtccagatg gcaacagggc aacgtgttca gctgcagcgt gatgcacgag 600 gccctgcaca accactacac ccagaagtcc ctgagcctgt ctcctggcaa a 651 <210> 17 <211> 651 <212> DNA <213> Artificial <220> <223> 17 <400> 17 gctcccgaac tgctgggagg acccagcgtg tttctgtttc cccctaaacc taaggacacc 60 ctgatgatca gcagaacccc cgaggtgacc tgcgtggtgg tggatgtgag ccatgaggac 120 cccgaagtga aattcaactg gtacgtggac ggcgtggagg tgcacaatgc taagaccaag 180 cccagagagg agcagtacaa cagcacctac agagtggtga gcgtgctgac cgtgctgcac 240 caagattggc tgaatggcaa agagtacaag tgcaaggtga gcaacaaggc cctgcccgcc 300 cctattgaaa aaaccattag caaagccaag ggccagccca gagagcccca agtgtacaca 360 ttacccccta gcagagatga actgaccaaa aaccaggtga gcctgagatg ccacgtgaag 420 ggcttttacc ccagcgacat cgccgtggag tgggagagca atggacaacc tgagaacaac 480 TACAAGACCA CC AAGCCC GT GCTGGACAGC GACGGATCTT TCTTTCTGTA CAGCACCCTG 540 ACC GTGGACA AGAGCAGATG GCAGCAGGGC AATGTGTTCA GCTGCAGCGT GATGCACGAG 600 GCCCTGCATA ATC ACTACACC C AGAAGAGC CTGAGCCTGA GCCCCGGAGC T 651 <210> 18 <211> 651 <212> DNA <213> Artificial <220> <223> 18 <400> 18 GCTCCTGAAC TGCTGGGAGG ACCTAGCGTG TTTCTGTTTC CTCCTAAACC CAAGGACACC 60 CTGATGATCA GCAGAACCCC CGAGGTGACC TGC GTGGTGGTG GTGAGCCATG AAGAT 120 CCC GAAGTGAA GTTCAACTGG TACGTGGACG GC GTGGAGGTG CACAATGCT AAAACCAAG 180 CCC AGAGAGGA GCAGTACAA CAGCACCTAC AGAGTGGTGA GC GTGCTGACC GTGCTGCAC 240 CAAGATTGGC TGAATGGAAG GAGTACAAGT GCAAGGTGAG CAACAAGGCC CTGCCCGCC 300 CCTATTGAAAA ACCATTAGCA AAGCTAAGGG CCAG CCCAGAGAG CCCCAAGTGT ATACA 360 AGCCCTCCCAGCAGGGACGAGCTGACCAA AAATCAGGTG AGCCTGAGATG CCACGTGAAG 420 ggcttttacc ccagcgacat cgccgtggaa tgggaaagca atggccaacc cgagaacaac 480 tataagacca ccaagcccgt gctggacagc gacggatcct tttttctgta cagcgacctg 540 accgtggaca agagcagatg gcagcagggc aatgtgttca gctgcagcgt gtatcacgag 600 gccctgcaca atcactacac ccagaaaagc ctgagcctga gccccggcaa a 651 <210> 19 <211> 651 <212> DNA <213> Artificial <220> <223> 19 <400> 19 gcccctgagc tgcttggagg acctagcgtt tttttatttc cccccaaacc taaggacacc 60 ctgatgatca gcagaacccc cgaggtgacc tgcgtggttg tggatgtgag ccatgaagat 120 cccgaggtga aattcaactg gtacgtggac ggcgtggagg tgcacaatgc caaaacaaag 180 cccagagagg agcagtacaa cagcacctac agagtggtga gcgtgctgac cgtgctgcac 240 caagattggc tgaacggcaa ggagtacaag tgcaaggtga gcaacaaggc cctgcccgcc 300 cctattgaaa aaaccattag caaggccaag ggccagccca gagaacccca agtgtatacc 360 agccccccca gcagagatga gctgacaaaa aatcaggtga gcctgagatg ccacgtgaag 420 ggcttttacc ccagcgacat cgccgtggag tgggaatcta atggacaacc tgagaacaac 480 tacaagacca ccaagcccgt gctggacagc gacgggagct ttttcctgta tagcgacctg 540 accgtggaca agagcagatg gcagcagggc aatgtgttca gctgcagcgt gtatcacgag 600 gccctgcata atcactacac ccagaaaagc ctgagcctga gccccggagc t 651 <210> 20 <211> 672 <212> DNA <213> Artificial <220> <223> 20 <400> 20 gacaagacac acaccggacc cgcccccgaa ctgctgggag gaccttctgt gtttctgttt 60 cctcccaaac ccaaagacac cctgatgatc agcagaaccc ccgaggtgac ctgcgtggtt 120 gtggatgtgt ctcatgagga tcccgaggtg aagttcaact ggtacgtgga cggcgtggag 180 gtgcacaatg ctaagacaaa gcccagagag gagcagtaca acagcaccta cagagtggtg 240 agcgtgctga ccgtgctgca ccaagattgg ctgaatggaa aggaatacaa gtgcaaggtg 300 agcaacaagg ccctgcccgc ccctattgaa aaaacaatta gcaaggccaa gggccagccc 360 agagaacctc aagtgtatac cctgcctccc agcagagatg agctgacaaa aaatcaggtg 420 agcctgagat gccacgtgaa gggcttctac cccagcgaca ttgccgtgga gtgggaaagc 480 aatggccaac ctgagaacaa ctacaagacc accaagcccg tgctggacag cgacggatct 540 ttttttctgt acagcaccct gaccgtggac aagagcagat ggcagcaggg caatgtgttc 600 agctgcagcg tgatgcacga ggccctgcat aatcactaca cccagaaaag cctgagcctg 660 agccccggca ag 672 <210> 21 <211> 45 <212> DNA <213> Artificial <220> <223> 21 <400> 21 ggaggaggag gaagcggagg cggaggatct ggaggaggag gaagc 45 <210> 22 <211> 30 <212> DNA <213> Artificial <220> <223> 22 <400> 22 ggcggcggag gatctggcgg aggtggaagt 30 <210> 23 <211> 15 <212> DNA <213> Artificial <220> <223> 23 <400> 23 ggcggcggag gatct 15 <210> 24 <211> 408 <212> PRT <213> Artificial <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 lie Gin Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gin lie Phe Lys Gin 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 lie Val Gin Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Ala 180 185 190 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 195 200 205 Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val 210 215 220 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 225 230 235 240 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin 245 250 255 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin 260 265 270 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 275 280 285 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 290 295 300 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 305 310 315 320 Lys Asn Gln Val Ser Leu Arg Cys His Val Lys Gly Phe Tyr Pro Ser 325 330 335 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 340 345 350 Lys Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 355 360 365 Ser Thr Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 370 375 380 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 385 390 395 400 Ser Leu Ser Leu Ser Pro Gly Lys 405 <210> 25 <211> 423 <212> PRT <213> Artificial <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 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro 195 200 205 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 210 215 220 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 225 230 235 240 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 245 250 255 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 260 265 270 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 275 280 285 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 290 295 300 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 305 310 315 320 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 325 330 335 Asn Gln Val Ser Leu Arg Cys His Val Lys Gly Phe Tyr Pro Ser Asp 340 345 350 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 355 360 365 Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 370 375 380 Thr Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 385 390 395 400 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 405 410 415 Leu Ser Leu Ser Pro Gly Lys 420 <210> 26 <211> 415 <212> PRT <213> Artificial <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 Gin Leu Ala Phe Asp Thr Tyr Gin Glu Phe Glu 20 25 30 Glu Ala Tyr He Pro Lys Glu Gin Lys Tyr Ser Phe Leu Gin Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser He Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gin Gin Lys Ser Asn Leu Glu Leu Leu Arg He Ser Leu 65 70 75 80 Leu Leu He Gin Ser Trp Leu Glu Pro Val Gin 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 He Gin Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gin He Phe Lys Gin 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 lie Val Gin Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Asp 180 185 190 Lys Thr His Thr Gly Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 195 200 205 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 210 215 220 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 225 230 235 240 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 245 250 255 Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser 260 265 270 Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys 275 280 285 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 290 295 300 Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro 305 310 315 320 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Arg Cys His 325 330 335 Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn 340 345 350 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Lys Pro Val Leu Asp Ser 355 360 365 Asp Gly Ser Phe Phe Leu Tyr Ser Thr Leu Thr Val Asp Lys Ser Arg 370 375 380 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 385 390 395 400 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 27 <211> 418 <212> PRT <213> Artificial <220> <223> 27 <400> 27 Phe Pro Thr lie 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 lie Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser lie Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg lie Ser Leu 65 70 75 80 Leu Leu lie Gin Ser Trp Leu Glu Pro Val Gin 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 lie Gin Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gin lie Phe Lys Gin 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 lie Val Gin Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly 180 185 190 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Glu Leu Leu Gly Gly 195 200 205 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 210 215 220 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 225 230 235 240 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 245 250 255 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 260 265 270 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 275 280 285 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 290 295 300 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 305 310 315 320 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 325 330 335 Arg Cys His Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 340 345 350 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Lys Pro Val 355 360 365 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Thr Leu Thr Val Asp 370 375 380 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 385 390 395 400 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 405 410 415 Gly Lys <210> 28 <211> 413 <212> PRT <213> Artificial <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 lie Gin Ser Trp Leu Glu Pro Val Gin 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 lie Gin Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gin lie Phe Lys Gin 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 lie Val Gin Cys Arg Ser Val Glu Gly Ser Cys Gly Phe Gly 180 185 190 Gly Gly Gly Ser Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 195 200 205 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu 210 215 220 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 225 230 235 240 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 245 250 255 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 260 265 270 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 275 280 285 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 290 295 300 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 305 310 315 320 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Arg Cys His Val Lys 325 330 335 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 340 345 350 Pro Glu Asn Asn Tyr Lys Thr Thr Lys Pro Val Leu Asp Ser Asp Gly 355 360 365 Ser Phe Phe Leu Tyr Ser Thr Leu Thr Val Asp Lys Ser Arg Trp Gln 370 375 380 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 385 390 395 400 His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 <210> 29 <211> 423 <212> PRT <213> Artificial <220> <223> 29 <400> 29 Ala Pro Glu Leu 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 His Glu Asp Pro Glu Val Lys 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 Tyr 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 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gin Val Ser Leu Arg Cys His Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Thr Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly 210 215 220 Gly Gly Ser Gly Gly Gly Gly Ser Phe Pro Thr He Pro Leu Ser Arg 225 230 235 240 Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gin Leu Ala 245 250 255 Phe Asp Thr Tyr Gin Glu Phe Glu Glu Ala Tyr He Pro Lys Glu Gin 260 265 270 Lys Tyr Ser Phe Leu Gin Asn Pro Gin Thr Ser Leu Cys Phe Ser Glu 275 280 285 Ser lie Pro Thr Pro Ser Asn Arg Glu Glu Thr Gin Gin Lys Ser Asn 290 295 300 Leu Glu Leu Leu Arg lie Ser Leu Leu Leu lie Gin Ser Trp Leu Glu 305 310 315 320 Pro Val Gin Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly 325 330 335 Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly 340 345 350 Ile Gin Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly 355 360 365 Gln lie Phe Lys Gin Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn 370 375 380 Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys 385 390 395 400 Asp Met Asp Lys Val Glu Thr Phe Leu Arg lie Val Gin Cys Arg Ser 405 410 415 Val Glu Gly Ser Cys Gly Phe 420 <210> 30 <211> 423 <212> PRT <213> Artificial <220> <223> 30 <400> 30 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 Ala Pro 195 200 205 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 210 215 220 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 225 230 235 240 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 245 250 255 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 260 265 270 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 275 280 285 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 290 295 300 Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg 305 310 315 320 Glu Pro Gin Val Tyr Thr Ser Pro Pro Ser Arg Asp Glu Leu Thr Lys 325 330 335 Asn Gin Val Ser Leu Arg Cys His Val Lys Gly Phe Tyr Pro Ser Asp 340 345 350 lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 355 360 365 Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 370 375 380 Asp Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 385 390 395 400 Cys Ser Val Tyr His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 405 410 415 Leu Ser Leu Ser Pro Gly Lys 420 <210> 31 <211> 423 <212> PRT <213> Artificial <220> <223> 31 <400> 31 Ala Pro Glu Leu 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 His Glu Asp Pro Glu Val Lys 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 Tyr 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 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Ser Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Arg Cys His 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 Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Tyr His Glu Ala Leu His Asn His Tyr Thr Gin 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly 210 215 220 Gly Gly Ser Gly Gly Gly Gly Ser Phe Pro Thr Ile Pro Leu Ser Arg 225 230 235 240 Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gin Leu Ala 245 250 255 Phe Asp Thr Tyr Gin Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gin 260 265 270 Lys Tyr Ser Phe Leu Gin Asn Pro Gin Thr Ser Leu Cys Phe Ser Glu 275 280 285 Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gin Gin Lys Ser Asn 290 295 300 Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gin Ser Trp Leu Glu 305 310 315 320 Pro Val Gin Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly 325 330 335 Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly 340 345 350 Ile Gin Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly 355 360 365 Gln Ile Phe Lys Gin Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn 370 375 380 Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys 385 390 395 400 Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gin Cys Arg Ser 405 410 415 Val Glu Gly Ser Cys Gly Phe 420 <210> 32 <211> 1224 <212> DNA <213> Artificial <220> <223> 32 <400> 32 ttccccacca ttcctctgag ccggctgttc gacaacgcca tgctgagagc ccacagactg 60 caccagctgg ccttcgacac ctaccaagag ttcgaggaag cctacattcc caaagagcag 120 aagtacagct tcctgcagaa ccctcagacc agcctgtgct tcagcgagag catccccaca 180 cctagcaaca gagaggaaac ccagcagaag tccaacctgg aactgctgcg gatcagcctg 240 ctgctgatcc agtcttggct ggaacccgtg cagttcctga gaagcgtgtt cgccaacagc 300 ctggtgtacg gcgccagcga cagcaacgtt tacgacctgc tgaaggacct ggaagagggc 360 atccagacac tgatgggcag actggaagat ggcagcccta gaaccggcca gatcttcaag 420 cagacctaca gcaagttcga caccaacagc cacaacgacg acgccctgct gaaaaactac 480 ggcctgctgt actgctttcg gaaggacatg gacaaggtgg aaaccttcct gcggatcgtg 540 cagtgcagaa gcgtggaagg cagctgtgga tttgcccctg aactgctcgg aggcccctcc 600 gtgtttctgt tccctccaaa gcctaaggac accctgatga tcagcagaac ccctgaagtg 660 acctgcgtgg tggtggatgt gtcccacgag gatcccgaag tgaagttcaa ttggtacgtg 720 gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caacagcacc 780 tacagagtgg tgtccgtgct gaccgtgctg caccaggatt ggctgaacgg caaagagtac 840 aagtgcaagg tgtccaacaa ggccctgcct gctcctatcg agaaaaccat cagcaaggcc 900 aagggccagc ctagggaacc ccaggtttac acactgcctc caagcaggga cgagctgacc 960 aagaatcagg tgtccctgcg gtgtcacgtg aagggcttct acccttccga tatcgccgtg 1020 gaatgggaga gcaatggcca gcctgagaac aactacaaga ccaccaagcc agtgctggac 1080 agcgacggct cattcttcct gtacagcacc ctgaccgtgg acaagtccag atggcaacag 1140 ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccacta cacccagaag 1200 tccctgagcc tgtctcctgg caaa 1224 <210> 33 <211> 1269 <212> DNA <213> Artificial <220> <223> 33 <400> 33 tttcccacca tccccctgtc tagactgttc gacaatgcca tgctgagagc ccacagactg 60 caccagctgg cctttgacac ataccaggag ttcgaggagg cctacatccc caaggagcag 120 aagtacagct tcctgcagaa cccccagacc agcctgtgtt tcagcgaaag catccccacc 180 CCCAGCAATA GAGAGGAAAC CCAGCAA AAG AGCAACCTGG AGCTGCTGAG AATCAGCCTG 240 CTGCTGATCC AGAGCTGGCT GGAGCCTGTG CAATTTCTGA GAAGCGGTTC GCCAATAGC 300 CTGGTGTACG GCACAGCGAT AGCAACGTGT ATGATCTGCT GAAAGACCTG GAGGAGGGC 360 ATTCA GACAC TGATGGGCAG ACTGGAGGAC GGCAGCCTC TAGAACAGGA CAGATTTCA AG 420 CAGACCTACA GCAAGTTCGA CACCAACAGC CACAACGACG ACGCCCTGCT GAAAACTAC 480 GGCCTGCTGT ACTGCTTCAG AAAGGACATG GACAAGGTGG AGACATTCCT GAGAATCGTG 540 CAGTGCAGAA GC GTGGAGGGC AGCTGTGGAT TTGGAGGAGG AGGAAGCGG AGGC GGAGGA 600 TCTGGAGGAG GAGGAAGCGC TCCTGAAC TGCTGGGAGGACC TAGCGTGTT TC TGT TTC CT 660 CCCAACCTAA AGACACCCCT GATGATCAGC AGAACCCCCG AGGTGACCTG CGTG GTGGTT 720 GATGTGTCTC ATGAAGACCC CGAAGTG AAGTTCAAC TG GTACGTGGAC GGC GTGGAGGTG 780 CACAATGCTA AAACCAAGCCC AGAGAGGAGC AGTACAACAG CCAC TACAG AGTGGTGAGC 840 GTGCTGACCG TGCTGCACCA AGATTGGCTG AATGGCAAGG AATACAAGTG CAAGGTGAGC 900 aacaaggccc tgcccgcccc tattgaaaaa accattagca aggctaaggg ccagcccaga 960 gagccccaag tgtatacact gccccctagc agagatgaac tgaccaaaaa ccaggtgagc 1020 ctgagatgcc acgtgaaggg cttttacccc agcgacattg ccgtggagtg ggagagcaat 1080 ggccaacctg aaaacaatta caagaccacc aagcccgtgc tggacagcga cggatctttc 1140 tttctgtata gcaccctgac cgtggacaag agcagatggc agcagggcaa tgtgttcagc 1200 tgcagcgtga tgcacgaggc cctgcacaat cactataccc agaagagcct gagcctgagc 1260 cccggcaaa 1269 <210> 34 <211> 1245 <212> DNA <213> Artificial <220> <223> 34 <400> 34 ttccctacaa tccccctgag cagactgttc gacaacgcca tgctgagagc ccacagactg 60 caccagttag cctttgatac ctatcaggag ttcgaggagg cctacatccc caaggagcag 120 aagtacagct tcctgcagaa cccccagacc agcctgtgtt tcagcgaaag catccccacc 180 cccagcaaca gagaggagac acagcaaaag agcaacctgg agctgctgag aatcagcctg 240 ctgctgatcc agagctggct ggagcctgtg caattcctga gaagcgtgtt cgccaatagc 300 ctggtgtacg gcgctagcga cagcaacgtg tatgacctgc tgaaggacct ggaggagggc 360 atccaaacac tgatgggcag actggaagat ggcagcccca gaaccggaca aattttcaag 420 cagacctaca gcaagttcga caccaacagc cacaacgacg acgccctgct gaaaaactac 480 ggcctgctgt attgcttcag aaaggacatg gacaaggtgg aaaccttcct gagaatcgtg 540 cagtgcagaa gcgtggaggg cagctgtgga tttgacaaga cacacaccgg acccgccccc 600 gaactgctgg gaggaccttc tgtgtttctg tttcctccca aacccaaaga caccctgatg 660 atcagcagaa cccccgaggt gacctgcgtg gttgtggatg tgtctcatga ggatcccgag 720 gtgaagttca actggtacgt ggacggcgtg gaggtgcaca atgctaagac aaagcccaga 780 gaggagcagt acaacagcac ctacagagtg gtgagcgtgc tgaccgtgct gcaccaagat 840 tggctgaatg gaaaggaata caagtgcaag gtgagcaaca aggccctgcc cgcccctatt 900 gaaaaaacaa ttagcaaggc caagggccag cccagagaac ctcaagtgta taccctgcct 960 cccagcagag atgagctgac aaaaaatcag gtgagcctga gatgccacgt gaagggcttc 1020 taccccagcg acattgccgt ggagtgggaa agcaatggcc aacctgagaa caactacaag 1080 accaccaagc ccgtgctgga cagcgacgga tctttttttc tgtacagcac cctgaccgtg 1140 gacaagagca gatggcagca gggcaatgtg ttcagctgca gcgtgatgca cgaggccctg 1200 cataatcact acacccagaa aagcctgagc ctgagccccg gcaag 1245 <210> 35 <211> 1254 <212> DNA <213> Artificial <220> <223> 35 <400> 35 ttccccacca ttcctctgag ccggctgttc gacaacgcca tgctgagagc ccacagactg 60 caccagctgg ccttcgacac ctaccaagag ttcgaggaag cctacattcc caaagagcag 120 aagtacagct tcctgcagaa ccctcagacc agcctgtgct tcagcgagag catccccaca 180 cctagcaaca gagaggaaac ccagcagaag tccaacctgg aactgctgcg gatcagcctg 240 ctgctgatcc agtcttggct ggaacccgtg cagttcctga gaagcgtgtt cgccaacagc 300 ctggtgtacg gcgccagcga cagcaacgtt tacgacctgc tgaaggacct ggaagagggc 360 atccagacac tgatgggcag actggaagat ggcagcccta gaaccggcca gatcttcaag 420 cagacctaca gcaagttcga caccaacagc cacaacgacg acgccctgct gaaaaactac 480 ggcctgctgt actgctttcg gaaggacatg gacaaggtgg aaaccttcct gcggatcgtg 540 cagtgcagaa gcgtggaagg ctcttgcgga tttggcggcg gaggatctgg cggaggtgga 600 agtgctcctg aactgctcgg aggcccttcc gtgttcctgt ttcctccaaa gcctaaggac 660 accctgatga tcagcagaac ccctgaagtg acctgcgtgg tggtggatgt gtcccacgag 720 gatcccgaag tgaagttcaa ttggtacgtg gacggcgtcg aggtgcacaa cgccaagaca 780 aagcctagag aggaacagta caacagcacc tacagagtgg tgtccgtgct gaccgtgctg 840 caccaggatt ggctgaacgg caaagagtac aagtgcaagg tgtccaacaa ggccctgcct 900 gctcctatcg agaaaaccat cagcaaggcc aagggccagc ctagggaacc ccaggtttac 960 acactgcctc caagcaggga cgagctgacc aagaatcagg tgtccctgcg gtgtcacgtg 1020 aagggcttct acccttccga tatcgccgtg gaatgggaga gcaatggcca gcctgagaac 1080 aactacaaga ccaccaagcc agtgctggac agcgacggct cattcttcct gtacagcacc 1140 ctgaccgtgg acaagtccag atggcaacag ggcaacgtgt tcagctgcag cgtgatgcac 1200 gaggccctgc acaaccacta cacccagaag tcctgagcc tgtctcctgg caaa 1254 <210> 36 <211> 1239 <212> DNA <213> Artificial <220> <223> 36 <400> 36 ttccccacca ttcctctgag ccggctgttc gacaacgcca tgctgagagc ccacagactg 60 caccagctgg ccttcgacac ctaccaagag ttcgaggaag cctacattcc caaagagcag 120 aagtacagct tcctgcagaa ccctcagacc agcctgtgct tcagcgagag catccccaca 180 cctagcaaca gagaggaaac ccagcagaag tccaacctgg aactgctgcg gatcagcctg 240 ctgctgatcc agtcttggct ggaacccgtg cagttcctga gaagcgtgtt cgccaacagc 300 ctggtgtacg gcgccagcga cagcaacgtt tacgacctgc tgaaggacct ggaagagggc 360 GAGAATAGAA ATAGAAATTA GATTAATCAA TATGATGATG ATGATGATTA 60 CAGACCTACA GCAAGTTCGA CACCAACAGC CACAACGACG ACGCCCTGCT GAAAACTAC 480 GGCCTGCTGT ACTGCTTTCG GAAGGACATG GACAAGGTGG AAACCTTCCT GC GGATCGTG 540 CAGTGCAGAA GC GTGGAAGG CTCTTGCGGA TTTGGCGGCG GAGGATCTGC TCCTGAAC TG 600 CTC GGAGGCCCTTCCGTGTT CCTGT TTC CCAAAGCCTA AGGACAC CCT GATGATC AGC 660 AGAACCCCTGA AGTGACCTGC GTGGTGGGTG GATGTGTCCC ACGAGGATCCC GAAGTG AAG 720 TTCAATTGGT AC GTGGACGGCGTGGAAGTG CACAACGCCA AGACCAAGCC TAGAGAGGAA 780 CAGTACAACAGCACCTACA GAGTG GTGTCCGTGCTG ACCGTGCTGCAC CAGGATTGGC TG 840 AACGGCAAAG AGTACAAGTG CAAGGTGTCC AACAAGGCCC TGCCTGCTCC TATCGAGAAA 900 ACCATCAGCA AGGCCAAGGG CCAGCCTAGG GAACCCCAGG TTACACACTG CCTCCAAGC 960 AGGGACGAGC TGACCAAGAA TCAGGTGTCC CTGCGGTGTC ACGTGAAGGG CTTCTAC CCT 1020 TCCGATATCG CC GTGGAATG GGAGAGCAAT GGCCAGCCTG AGAACAAC TA CAAGACCACC 1080 aagccagtgc tggacagcga cggctcattc ttcctgtaca gcaccctgac cgtggacaag 1140 tccagatggc aacagggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 1200 cactacaccc agaagtccct gagcctgtct cctggcaaa 1239 <210> 37 <211> 1269 <212> DNA <213> Artificial <220> <223> 37 <400> 37 gctcccgaac tgctgggagg acccagcgtg tttctgtttc cccctaaacc taaggacacc 60 ctgatgatca gcagaacccc cgaggtgacc tgcgtggtgg tggatgtgag ccatgaggac 120 cccgaagtga aattcaactg gtacgtggac ggcgtggagg tgcacaatgc taagaccaag 180 cccagagagg agcagtacaa cagcacctac agagtggtga gcgtgctgac cgtgctgcac 240 caagattggc tgaatggcaa agagtacaag tgcaaggtga gcaacaaggc cctgcccgcc 300 cctattgaaa aaaccattag caaagccaag ggccagccca gagagcccca agtgtacaca 360 ttacccccta gcagagatga actgaccaaa aaccaggtga gcctgagatg ccacgtgaag 420 ggcttttacc ccagcgacat cgccgtggag tgggagagca atggacaacc tgagaacaac 480 tacaagacca ccaagcccgt gctggacagc gacggatctt tctttctgta cagcaccctg 540 accgtggaca agagcagatg gcagcagggc aatgtgttca gctgcagcgt gatgcacgag 600 gccctgcata atcactacac ccagaagagc ctgagcctga gccccggagc tggaggagga 660 ggaagcggag gaggaggaag cggcggagga ggatcttttc ccacaattcc tctgagcaga 720 ctgttcgaca acgccatgct gagagcccac agactgcacc agctggcctt tgacacatac 780 caggagttcg aggaggccta catccccaag gagcagaagt acagcttcct gcagaacccc 840 cagaccagcc tgtgctttag cgaaagcatt cccaccccca gcaacagaga ggagacacaa 900 caaaagagca acctggagct gctgagaatc agcctgctgc tgatccagag ctggctggag 960 cccgtgcaat ttctgagaag cgtgttcgcc aatagcctgg tgtacggcgc tagcgatagc 1020 aacgtgtatg acctgctgaa ggacctggag gagggcatcc aaaccctgat gggcagactg 1080 gaggacggca gccctagaac aggacagatt tttaagcaga cctacagcaa gttcgacacc 1140 aacagccaca acgacgacgc cctgctgaaa aactacggcc tgctgtactg cttcagaaag 1200 gacatggaca aggtggagac attcctgaga atcgtgcagt gcagaagcgt ggagggcagc 1260 tgtggattt 1269 <210> 38 <211> 1269 <212> DNA <213> Artificial <220> <223> 38 <400> 38 ttccccacaa tccccctgag cagactgttt gacaacgcca tgctgagagc ccacagactg 60 caccagctgg ccttcgatac ataccaggag tttgaggagg cctacatccc caaggagcag 120 aagtacagct tcctgcagaa cccccagacc agcctgtgtt tcagcgagag cattcccaca 180 cccagcaaca gagaggaaac ccagcaaaag agcaacctgg agctgctgag aatcagcctg 240 ctgctgatcc agagctggct ggagcctgtg caattcctga gaagcgtgtt cgccaacagc 300 ctggtgtacg gcgcttctga tagcaatgtg tacgacctgc tgaaggacct ggaggagggc 360 attcagacac tgatgggcag actggaggac ggcagcccta gaacaggaca aattttcaag 420 cagacctaca gcaagttcga caccaacagc cacaacgacg acgccctgct gaaaaactac 480 ggcctgctgt actgcttcag aaaggacatg gacaaggtgg agacattcct gagaatcgtg 540 cagtgcagaa gcgtggaggg cagctgtgga tttggaggcg gaggaagcgg aggaggagga 600 agcggaggag gaggaagcgc tcctgaactg ctgggaggac ctagcgtgtt tctgtttcct 660 cctaaaccca aggacaccct gatgatcagc agaacccccg aggtgacctg cgtggtggtg 720 gatgtgagcc atgaagatcc cgaagtgaag ttcaactggt acgtggacgg cgtggaggtg 780 cacaatgcta aaaccaagcc cagagaggag cagtacaaca gcacctacag agtggtgagc 840 gtgctgaccg tgctgcacca agattggctg aatggaaagg agtacaagtg caaggtgagc 900 aacaaggccc tgcccgcccc tattgaaaaa accattagca aagctaaggg ccagcccaga 960 gagccccaag tgtatacaag ccctcccagc agggacgagc tgaccaaaaa tcaggtgagc 1020 ctgagatgcc acgtgaaggg cttttacccc agcgacatcg ccgtggaatg ggaaagcaat 1080 ggccaacccg agaacaacta taagaccacc aagcccgtgc tggacagcga cggatccttt 1140 tttctgtaca gcgacctgac cgtggacaag agcagatggc agcagggcaa tgtgttcagc 1200 tgcagcgtgt atcacgaggc cctgcacaat cactacaccc agaaaagcct gagcctgagc 1260 cccggcaaa 1269 <210> 39 <211> 1269 <212> DNA <213> Artificial <220> <223> 39 <400> 39 gcccctgagc tgcttggagg acctagcgtt tttttatttc cccccaaacc taaggacacc 60 ctgatgatca gcagaacccc cgaggtgacc tgcgtggttg tggatgtgag ccatgaagat 120 cccgaggtga aattcaactg gtacgtggac ggcgtggagg tgcacaatgc caaaacaaag 180 cccagagagg agcagtacaa cagcacctac agagtggtga gcgtgctgac cgtgctgcac 240 caagattggc tgaacggcaa ggagtacaag tgcaaggtga gcaacaaggc cctgcccgcc 300 cctattgaaa aaaccattag caaggccaag ggccagccca gagaacccca agtgtatacc 360 agccccccca gcagagatga gctgacaaaa aatcaggtga gcctgagatg ccacgtgaag 420 ggcttttacc ccagcgacat cgccgtggag tgggaatcta atggacaacc tgagaacaac 480 tacaagacca ccaagcccgt gctggacagc gacgggagct ttttcctgta tagcgacctg 540 accgtggaca agagcagatg gcagcagggc aatgtgttca gctgcagcgt gtatcacgag 600 gccctgcata atcactacac ccagaaaagc ctgagcctga gccccggagc tggaggagga 660 ggaagcggag gaggaggaag cggaggagga ggatcttttc ctacaattcc tctgagcaga 720 ctgttcgaca acgccatgct gagagcccac agactgcacc agctggcctt tgatacatac 780 caggagttcg aggaggccta catccccaag gagcagaagt acagcttcct gcagaacccc 840 cagaccagcc tgtgttttag cgaaagcatt cccacaccca gcaatagaga agagacacag 900 cagaagagca acctggagct gctgagaatc agcctgctgc tgatccagag ctggctggag 960 cctgtgcaat ttctgagaag cgtgttcgcc aacagcctgg tgtacggcgc tagcgactct 1020 aacgtgtatg atctgctgaa agacctggag gagggcatcc agacactgat gggcagactg 1080 gaggacggca gccccagaac aggacagata tttaagcaaa cctacagcaa gttcgacacc 1140 aacagccaca acgacgacgc cctgctgaaa aactacggcc tgctgtactg cttcagaaag 1200 gacatggaca aggtggagac attcctgaga atcgtgcagt gcagaagcgt ggagggcagc 1260 tgtggattt 1269 <210> 40 <211> 220 <212> PRT <213> Artificial <220> <223> 40 <400> 40 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> 41 <211> 6 <212> PRT <213> Artificial <220> <223> 41 <400> 41 Asp Lys Thr His Thr Gly 1 5 <210> 42 <211> 18 <212> DNA <213> Artificial <220> <223> 42 <400> 42 gacaagacac acaccgga 18 <210> 43 <211> 436 <212> PRT <213> Artificial <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 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

Claims

1. A fusion protein, characterized in that, The amino acid sequence of the fusion protein is shown in any one of SEQ ID NO: 26 or 31.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein, The nucleic acid molecule is DNA.

4. An expression vector, characterized by, carrying the nucleic acid molecule of claim 2 or 3; The expression vector is a eukaryotic expression vector; The expression vector is a lentiviral vector.

5. A recombinant cell, characterized in that, The recombinant cell comprises: carrying the nucleic acid molecule of claim 2 or 3; or, expressing the fusion protein of claim 1.

6. The recombinant cell of claim 5, wherein, The recombinant cell is obtained by introducing the expression vector of claim 4 into a host cell; The recombinant cell is a eukaryotic cell.

7. The recombinant cell of claim 6, wherein, The recombinant cell is a mammalian cell.

8. A pharmaceutical composition, characterized by, comprises: The fusion protein of claim 1.

9. The pharmaceutical composition of claim 8, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 8, wherein, The dosage form of the pharmaceutical composition is an injection; The administration route of the pharmaceutical composition comprises subcutaneous injection or intravenous injection.

11. Use of the fusion protein of claim 1, the nucleic acid molecule of claim 2 or 3, the expression vector of claim 4, the recombinant cell of any one of claims 5-7, or the pharmaceutical composition of any one of claims 8-10 in the preparation of a medicament for treating and / or preventing a growth hormone abnormality related disease. The growth hormone abnormality related disease is: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome.

Images