CN115873127A - Recombinant long-acting human growth hormone fusion protein and preparation method and application thereof - Google Patents

Recombinant long-acting human growth hormone fusion protein and preparation method and application thereof Download PDF

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CN115873127A
CN115873127A CN202210395423.2A CN202210395423A CN115873127A CN 115873127 A CN115873127 A CN 115873127A CN 202210395423 A CN202210395423 A CN 202210395423A CN 115873127 A CN115873127 A CN 115873127A
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秦锁富
李娟�
周冬梅
游琼英
张伟
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Shenzhen Kexing Pharmaceutical Co ltd
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Abstract

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

Description

Recombinant long-acting human growth hormone fusion protein and preparation method and application thereof
Technical Field
The invention relates to the fields of molecular biology and medicines, in particular to a recombinant long-acting human growth hormone fusion protein and a preparation method and application thereof, and more particularly relates to an Fc mutant, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition and application thereof.
Background
Growth hormone deficiency is a recognized clinical syndrome associated with a number of metabolic abnormalities, including abnormalities in body composition, decreased physical performance, altered lipid metabolism, decreased bone mass, increased insulin resistance, and decreased quality of life. Most of the metabolic abnormalities associated with growth hormone deficiency can be reversed by recombinant human growth hormone (rhGH) substitutes. Conventional treatment of growth hormone deficiency includes daily subcutaneous injection of rhGH, however, the treatment is cumbersome, requires daily injections, is inconvenient for many patients, raises concerns about poor compliance with the treatment, and may result in reduced efficacy. The long-acting rhGH preparation or the long-acting growth hormone preparation not only can reduce the injection times and improve the compliance, but also is helpful to improve the curative effect of GH treatment.
Therefore, there is still a need to develop a new fusion protein effective in treating growth hormone deficiency.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. To this end, the invention provides an Fc mutant, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition, and uses thereof. The Fc mutants of the present invention are susceptible to formation of monomeric forms. The fusion protein has strong stability and can effectively treat growth hormone deficiency.
The present invention has been completed based on the following findings of the inventors:
the inventor finds out through experiments that mutation of specific sites of the Fc fragment of wild-type IgG1, such as at least one of P228Delete, C229Delete, P230 Delete, T366R, L368H, P395K and K409D/K409T, and L351S, M428Y and K447A, or P228Delete, C229Delete, T366R, L368H, P395K and K409D or K409T, can make the Fc fragment in a natural dimer form expressed to form an Fc mutant in a monomer form. Moreover, experiments further show that the Fc mutant is connected with growth hormone to form the fusion protein in a monomer form, so that the fusion protein can be purified by ProteinA, the purification process is simplified, and the subsequent purification process is simple. Meanwhile, more importantly, the fusion protein obtains stronger human growth hormone receptor binding activity, promotes cell proliferation activity and promotes reporter gene cell to express luciferase activity, and has longer half-life and better in vivo biological activity.
To this end, in a first aspect of the invention, the invention proposes an Fc mutant. According to an embodiment of the invention, comprising: a first peptidyl fragment having mutations compared to the Fc fragment of wild-type IgG1 at the following positions: at least one of 228 th bit, 229 th bit, 230 th bit, 366 th bit, 368 th bit, 395 th bit and 409 th bit, and 351 th bit, 428 th bit and 447 th bit; or 228 th bit, 229 th bit, 366 th bit, 368 th bit, 395 th bit and 409 th bit. The inventor finds through a large number of experiments that the mutation is beneficial to enabling the Fc fragment in the form of natural dimer to be expressed and then form the Fc mutant in the form of monomer, the Fc mutant in the form of monomer is adopted to be fused with bioactive molecules, and the subsequent purification can be realized through ProteinA, so that the purification process is simplified; more importantly, the fusion protein prepared by the Fc mutant has higher in vivo and in vitro activity and lower ADCC and CDC effects.
It should be noted that the Fc fragment of the antibody IgG referred to herein refers to the CH2 region and the CH3 region of the antibody IgG.
For example, the amino acid sequence (including CH2-CH 3) of the Fc fragment of wild-type IgG1 is as follows:
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:40)。
the amino acid sequence of the Hinge region (Hinge) of wild-type IgG1 is shown below:
DKTHTG(SEQ ID NO:41)。
the nucleotide sequence of the Hinge region (Hinge) of wild-type IgG1 is shown below:
GACAAGACACACACCGGA(SEQ ID NO:42)。
in a second aspect of the invention, a fusion protein is provided. According to an embodiment of the invention, the fusion protein comprises: a second peptide segment comprising a functional region of a biologically active molecule; a third peptide stretch comprising an Fc mutant having the amino acid sequence shown in SEQ ID NO 2 or as defined in the first aspect, the second peptide stretch being linked to the third peptide stretch. The fusion protein provided by the embodiment of the invention has higher in vitro and in vivo activity and lower ADCC and CDC effects, and can be purified by ProteinA subsequently, so that the purification process is simplified.
In a third aspect of the invention, a fusion protein is provided. According to an embodiment of the invention, the general structural formula of the fusion protein is represented as X-L-Y or Y-L-X, wherein X is a first bioactive molecule; l is absent or is a linker peptide; y is a second biologically active molecule; -is a peptide bond; and the active molecule X or Y is selected from a protein or protein domain, a polypeptide, an antibody or an antibody fragment.
It should be noted that "X-L-Y" means that the C-terminal of X is connected to the N-terminal of L, and the C-terminal of L is connected to the N-terminal of Y; the term "Y-L-X" refers to the condition that the C end of Y is connected with the N end of L, and the C end of L is connected with the N end of X.
According to an embodiment of the present invention, the fusion protein according to the second or third aspect may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the fusion protein is in monomeric form.
According to an embodiment of the invention, said X is human growth hormone. In some embodiments, the amino acid sequence of human growth hormone comprises at least a portion of an amino acid sequence selected from SEQ ID No. 1 or an amino acid sequence having at least 80% to 99% identity thereto, or a sequence thereof.
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO:1)。
According to an embodiment of the invention, the nucleotide sequence of human growth hormone comprises a codon optimized sequence selected from SEQ ID NO 11 or a nucleotide sequence having at least 80% -99% identity thereto or at least a part of the sequence thereof or the sequence thereof.
TTTCCCACCATTCCTCTGAGCAGACTGTTCGACAACGCCATGCTGAGAGCCCACAGACTGCACCAGCTGGCCTTTGACACATACCAGGAGTTCGAGGAGGCCTACATCCCCAAGGAGCAGAAGTACAGCTTCCTGCAGAACCCCCAGACCAGCCTGTGCTTCAGCGAGAGCATCCCCACCCCCAGCAATAGAGAGGAGACACAGCAGAAGAGCAACCTGGAGCTGCTGAGAATCAGCCTGCTGCTGATCCAGAGCTGGCTGGAGCCCGTTCAATTTCTGAGAAGCGTGTTCGCCAACAGCCTGGTGTACGGCGCCAGCGATTCTAATGTGTACGACCTGCTGAAGGACCTGGAGGAGGGCATCCAGACCCTGATGGGCAGACTGGAGGACGGCTCTCCTAGAACCGGACAAATTTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAGAACTACGGCCTGCTGTACTGCTTCAGAAAGGACATGGACAAGGTGGAGACATTCCTGAGAATCGTGCAGTGCAGAAGCGTGGAGGGCAGCTGCGGATTC(SEQ ID NO:11)。
According to an embodiment of the invention, said Y is an IgG Fc mutant (abbreviated as "Fc mutant"). In some embodiments, the Y is an Ig G1 Fc mutant. In some embodiments, the Ig G1 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 thereof, or at least a portion of any one thereof.
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:2)。
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3)。
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA(SEQ ID NO:4)。
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVYHEALHNHYTQKSLSLSPGK(SEQ ID NO:5)。
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVYHEALHNHYTQKSLSLSPGA(SEQ ID NO:6)。
DKTHTGPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:7)。
The presence of a mutation in the first peptidyl fragment of SEQ ID NO:2 compared to the Fc fragment of wild type IgG1 may comprise: p228Delete, C229Delete, P230 Delete, T366R, L368H, P395K, and K409T.
The presence of mutations in the first peptidyl fragments of SEQ ID NOs 3 and 7 compared to the Fc fragment of wild-type IgG1 may include: p228Delete, C229Delete, T366R, L368H, P395K, and K409T.
The presence of a mutation in the first peptidyl fragment of SEQ ID NO:4 compared to the Fc fragment of wild type IgG1 may comprise: p228Delete, C229Delete, P230 Delete, T366R, L368H, P395K, K409T, and K447A.
The presence of a mutation in the first peptidyl fragment of SEQ ID NO:5 compared to the Fc fragment of wild type IgG1 may comprise: p228Delete, C229Delete, P230 Delete, L351S, T366R, L368H, P395K, K409D and M428Y.
The presence of a mutation in the first peptidyl fragment of SEQ ID NO:6 compared to the Fc fragment of wild type IgG1 may comprise: p228Delete, C229Delete, P230 Delete, L351S, T366R, L368H, P395K, K409D, M428Y and K447A.
The mutation is beneficial to the formation of a monomer form after the Fc mutant in a natural dimer form is expressed, and is beneficial to the purification of the fusion protein through ProteinA, the purification process is simplified, and the in vivo and in vitro activity is improved.
According to an embodiment of the invention, the linker peptide comprises 1 or more amino acids selected from the group consisting of glycine, serine, alanine and threonine.
According to an embodiment of the invention, the amino acid sequence of the linker peptide comprises a sequence selected from (GGGGS) n Or an amino acid sequence having at least 80% to 99% identity to any one of the sequences or at least a portion of any one of the sequences, wherein n is an integer of 1 or more or n is 1, 2, 3, 4, 5 or 6.
According to an embodiment of the invention, 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% to 99% identity to any one of them or at least a part of any one of them.
GGGGSGGGGSGGGGS(SEQ ID NO:8)。
GGGGSGGGGS(SEQ ID NO:9)。
GGGGS(SEQ ID NO:10)
According to an embodiment of the present invention, the nucleotide sequence of the linker peptide may 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% to 99% identity to any one of them or a codon optimized sequence of at least a part of any one of them or any one of them.
GGAGGAGGAGGAAGCGGAGGCGGAGGATCTGGAGGAGGAGGAAGC(SEQ ID NO:21)。
GGCGGCGGAGGATCTGGCGGAGGTGGAAGT(SEQ ID NO:22)。
GGCGGCGGAGGATCT(SEQ ID NO:23)。
According to an embodiment of the invention, 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 the sequences or at least a part of any one of the sequences.
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQ QKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:24)。
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:25)。
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFDKTHTGPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:26)。
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:27)。
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:28)。
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO:29)。
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVYHEALHNHYTQKSLSLSPGK(SEQ ID NO:30)。
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVYHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO:31)。
According to an embodiment of the present invention, the first biomolecule, the second biomolecule and the linker peptide in the amino acid sequences shown in SEQ ID NO:24-31 are shown in the following table:
Figure BDA0003597162480000051
according to an embodiment of the invention, the nucleotide sequence of the fusion protein comprises at least one selected from the group consisting of SEQ ID NO 32, 33, 34, 35, 36, 37, 38, 39 or a nucleotide sequence having at least 80% -99% identity to any one of them or a codon optimized sequence of at least a part of any one of them or any one of them.
TTCCCCACCATTCCTCTGAGCCGGCTGTTCGACAACGCCATGCTGAGAGCCCACAGACTGCACCAGCTGGCCTTCGACACCTACCAAGAGTTCGAGGAAGCCTACATTCCCAAAGAGCAGAAGTACAGCTTCCTGCAGAACCCTCAGACCAGCCTGTGCTTCAGCGAGAGCATCCCCACACCTAGCAACAGAGAGGAAACCCAGCAGAAGTCCAACCTGGAACTGCTGCGGATCAGCCTGCTGCTGATCCAGTCTTGGCTGGAACCCGTGCAGTTCCTGAGAAGCGTGTTCGCCAACAGCCTGGTGTACGGCGCCAGCGACAGCAACGTTTACGACCTGCTGAAGGACCTGGAAGAGGGCATCCAGACACTGATGGGCAGACTGGAAGATGGCAGCCCTAGAACCGGCCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAAAACTACGGCCTGCTGTACTGCTTTCGGAAGGACATGGACAAGGTGGAAACCTTCCTGCGGATCGTGCAGTGCAGAAGCGTGGAAGGCAGCTGTGGATTTGCCCCTGAACTGCTCGGAGGCCCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGCGGTGTCACGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACCAAGCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCACCCTGACCGTGGACAAGTCCAGATGGCAACAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAA(SEQ ID NO:32)。
TTTCCCACCATCCCCCTGTCTAGACTGTTCGACAATGCCATGCTGAGAGCCCACAGACTGCACCAGCTGGCCTTTGACACATACCAGGAGTTCGAGGAGGCCTACATCCCCAAGGAGCAGAAGTACAGCTTCCTGCAGAACCCCCAGACCAGCCTGTGTTTCAGCGAAAGCATCCCCACCCCCAGCAATAGAGAGGAAACCCAGCAAAAGAGCAACCTGGAGCTGCTGAGAATCAGCCTGCTGCTGATCCAGAGCTGGCTGGAGCCTGTGCAATTTCTGAGAAGCGTGTTCGCCAATAGCCTGGTGTACGGCGCCAGCGATAGCAACGTGTATGATCTGCTGAAAGACCTGGAGGAGGGCATTCAGACACTGATGGGCAGACTGGAGGACGGCAGCCCTAGAACAGGACAGATTTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAAAACTACGGCCTGCTGTACTGCTTCAGAAAGGACATGGACAAGGTGGAGACATTCCTGAGAATCGTGCAGTGCAGAAGCGTGGAGGGCAGCTGTGGATTTGGAGGAGGAGGAAGCGGAGGCGGAGGATCTGGAGGAGGAGGAAGCGCTCCTGAACTGCTGGGAGGACCTAGCGTGTTTCTGTTTCCTCCCAAACCTAAAGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTTGATGTGTCTCATGAAGACCCCGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAAACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGCAAGGAATACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAGGCTAAGGGCCAGCCCAGAGAGCCCCAAGTGTATACACTGCCCCCTAGCAGAGATGAACTGACCAAAAACCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATTGCCGTGGAGTGGGAGAGCAATGGCCAACCTGAAAACAATTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCTTTCTTTCTGTATAGCACCCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTATACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAA(SEQ ID NO:33)。
TTCCCTACAATCCCCCTGAGCAGACTGTTCGACAACGCCATGCTGAGAGCCCACAGACTGCACCAGTTAGCCTTTGATACCTATCAGGAGTTCGAGGAGGCCTACATCCCCAAGGAGCAGAAGTACAGCTTCCTGCAGAACCCCCAGACCAGCCTGTGTTTCAGCGAAAGCATCCCCACCCCCAGCAACAGAGAGGAGACACAGCAAAAGAGCAACCTGGAGCTGCTGAGAATCAGCCTGCTGCTGATCCAGAGCTGGCTGGAGCCTGTGCAATTCCTGAGAAGCGTGTTCGCCAATAGCCTGGTGTACGGCGCTAGCGACAGCAACGTGTATGACCTGCTGAAGGACCTGGAGGAGGGCATCCAAACACTGATGGGCAGACTGGAAGATGGCAGCCCCAGAACCGGACAAATTTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAAAACTACGGCCTGCTGTATTGCTTCAGAAAGGACATGGACAAGGTGGAAACCTTCCTGAGAATCGTGCAGTGCAGAAGCGTGGAGGGCAGCTGTGGATTTGACAAGACACACACCGGACCCGCCCCCGAACTGCTGGGAGGACCTTCTGTGTTTCTGTTTCCTCCCAAACCCAAAGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTTGTGGATGTGTCTCATGAGGATCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAGACAAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGAAAGGAATACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACAATTAGCAAGGCCAAGGGCCAGCCCAGAGAACCTCAAGTGTATACCCTGCCTCCCAGCAGAGATGAGCTGACAAAAAATCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTCTACCCCAGCGACATTGCCGTGGAGTGGGAAAGCAATGGCCAACCTGAGAACAACTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCTTTTTTTCTGTACAGCACCCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCATAATCACTACACCCAGAAAAGCCTGAGCCTGAGCCCCGGCAAG(SEQ ID NO:34)。
TTCCCCACCATTCCTCTGAGCCGGCTGTTCGACAACGCCATGCTGAGAGCCCACAGACTGCACCAGCTGGCCTTCGACACCTACCAAGAGTTCGAGGAAGCCTACATTCCCAAAGAGCAGAAGTACAGCTTCCTGCAGAACCCTCAGACCAGCCTGTGCTTCAGCGAGAGCATCCCCACACCTAGCAACAGAGAGGAAACCCAGCAGAAGTCCAACCTGGAACTGCTGCGGATCAGCCTGCTGCTGATCCAGTCTTGGCTGGAACCCGTGCAGTTCCTGAGAAGCGTGTTCGCCAACAGCCTGGTGTACGGCGCCAGCGACAGCAACGTTTACGACCTGCTGAAGGACCTGGAAGAGGGCATCCAGACACTGATGGGCAGACTGGAAGATGGCAGCCCTAGAACCGGCCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAAAACTACGGCCTGCTGTACTGCTTTCGGAAGGACATGGACAAGGTGGAAACCTTCCTGCGGATCGTGCAGTGCAGAAGCGTGGAAGGCTCTTGCGGATTTGGCGGCGGAGGATCTGGCGGAGGTGGAAGTGCTCCTGAACTGCTCGGAGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTCGAGGTGCACAACGCCAAGACAAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGCGGTGTCACGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACCAAGCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCACCCTGACCGTGGACAAGTCCAGATGGCAACAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAA(SEQ ID NO:35)。
TTCCCCACCATTCCTCTGAGCCGGCTGTTCGACAACGCCATGCTGAGAGCCCACAGACTGCACCAGCTGGCCTTCGACACCTACCAAGAGTTCGAGGAAGCCTACATTCCCAAAGAGCAGAAGTACAGCTTCCTGCAGAACCCTCAGACCAGCCTGTGCTTCAGCGAGAGCATCCCCACACCTAGCAACAGAGAGGAAACCCAGCAGAAGTCCAACCTGGAACTGCTGCGGATCAGCCTGCTGCTGATCCAGTCTTGGCTGGAACCCGTGCAGTTCCTGAGAAGCGTGTTCGCCAACAGCCTGGTGTACGGCGCCAGCGACAGCAACGTTTACGACCTGCTGAAGGACCTGGAAGAGGGCATCCAGACACTGATGGGCAGACTGGAAGATGGCAGCCCTAGAACCGGCCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAAAACTACGGCCTGCTGTACTGCTTTCGGAAGGACATGGACAAGGTGGAAACCTTCCTGCGGATCGTGCAGTGCAGAAGCGTGGAAGGCTCTTGCGGATTTGGCGGCGGAGGATCTGCTCCTGAACTGCTCGGAGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGCGGTGTCACGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACCAAGCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCACCCTGACCGTGGACAAGTCCAGATGGCAACAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAA(SEQ ID NO:36)。
GCTCCCGAACTGCTGGGAGGACCCAGCGTGTTTCTGTTTCCCCCTAAACCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGATGTGAGCCATGAGGACCCCGAAGTGAAATTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAAGCCAAGGGCCAGCCCAGAGAGCCCCAAGTGTACACATTACCCCCTAGCAGAGATGAACTGACCAAAAACCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGACAACCTGAGAACAACTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCTTTCTTTCTGTACAGCACCCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCATAATCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAGCTGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGCGGAGGAGGATCTTTTCCCACAATTCCTCTGAGCAGACTGTTCGACAACGCCATGCTGAGAGCCCACAGACTGCACCAGCTGGCCTTTGACACATACCAGGAGTTCGAGGAGGCCTACATCCCCAAGGAGCAGAAGTACAGCTTCCTGCAGAACCCCCAGACCAGCCTGTGCTTTAGCGAAAGCATTCCCACCCCCAGCAACAGAGAGGAGACACAACAAAAGAGCAACCTGGAGCTGCTGAGAATCAGCCTGCTGCTGATCCAGAGCTGGCTGGAGCCCGTGCAATTTCTGAGAAGCGTGTTCGCCAATAGCCTGGTGTACGGCGCTAGCGATAGCAACGTGTATGACCTGCTGAAGGACCTGGAGGAGGGCATCCAAACCCTGATGGGCAGACTGGAGGACGGCAGCCCTAGAACAGGACAGATTTTTAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAAAACTACGGCCTGCTGTACTGCTTCAGAAAGGACATGGACAAGGTGGAGACATTCCTGAGAATCGTGCAGTGCAGAAGCGTGGAGGGCAGCTGTGGATTT(SEQ ID NO:37)。
TTCCCCACAATCCCCCTGAGCAGACTGTTTGACAACGCCATGCTGAGAGCCCACAGACTGCACCAGCTGGCCTTCGATACATACCAGGAGTTTGAGGAGGCCTACATCCCCAAGGAGCAGAAGTACAGCTTCCTGCAGAACCCCCAGACCAGCCTGTGTTTCAGCGAGAGCATTCCCACACCCAGCAACAGAGAGGAAACCCAGCAAAAGAGCAACCTGGAGCTGCTGAGAATCAGCCTGCTGCTGATCCAGAGCTGGCTGGAGCCTGTGCAATTCCTGAGAAGCGTGTTCGCCAACAGCCTGGTGTACGGCGCTTCTGATAGCAATGTGTACGACCTGCTGAAGGACCTGGAGGAGGGCATTCAGACACTGATGGGCAGACTGGAGGACGGCAGCCCTAGAACAGGACAAATTTTCAAGCAGACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAAAACTACGGCCTGCTGTACTGCTTCAGAAAGGACATGGACAAGGTGGAGACATTCCTGAGAATCGTGCAGTGCAGAAGCGTGGAGGGCAGCTGTGGATTTGGAGGCGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGCTCCTGAACTGCTGGGAGGACCTAGCGTGTTTCTGTTTCCTCCTAAACCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGATGTGAGCCATGAAGATCCCGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAAACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGAAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAAGCTAAGGGCCAGCCCAGAGAGCCCCAAGTGTATACAAGCCCTCCCAGCAGGGACGAGCTGACCAAAAATCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATCGCCGTGGAATGGGAAAGCAATGGCCAACCCGAGAACAACTATAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCCTTTTTTCTGTACAGCGACCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGTATCACGAGGCCCTGCACAATCACTACACCCAGAAAAGCCTGAGCCTGAGCCCCGGCAAA(SEQ ID NO:38)。
GCCCCTGAGCTGCTTGGAGGACCTAGCGTTTTTTTATTTCCCCCCAAACCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTTGTGGATGTGAGCCATGAAGATCCCGAGGTGAAATTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAAACAAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAGGCCAAGGGCCAGCCCAGAGAACCCCAAGTGTATACCAGCCCCCCCAGCAGAGATGAGCTGACAAAAAATCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATCGCCGTGGAGTGGGAATCTAATGGACAACCTGAGAACAACTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGGAGCTTTTTCCTGTATAGCGACCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGTATCACGAGGCCCTGCATAATCACTACACCCAGAAAAGCCTGAGCCTGAGCCCCGGAGCTGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGATCTTTTCCTACAATTCCTCTGAGCAGACTGTTCGACAACGCCATGCTGAGAGCCCACAGACTGCACCAGCTGGCCTTTGATACATACCAGGAGTTCGAGGAGGCCTACATCCCCAAGGAGCAGAAGTACAGCTTCCTGCAGAACCCCCAGACCAGCCTGTGTTTTAGCGAAAGCATTCCCACACCCAGCAATAGAGAAGAGACACAGCAGAAGAGCAACCTGGAGCTGCTGAGAATCAGCCTGCTGCTGATCCAGAGCTGGCTGGAGCCTGTGCAATTTCTGAGAAGCGTGTTCGCCAACAGCCTGGTGTACGGCGCTAGCGACTCTAACGTGTATGATCTGCTGAAAGACCTGGAGGAGGGCATCCAGACACTGATGGGCAGACTGGAGGACGGCAGCCCCAGAACAGGACAGATATTTAAGCAAACCTACAGCAAGTTCGACACCAACAGCCACAACGACGACGCCCTGCTGAAAAACTACGGCCTGCTGTACTGCTTCAGAAAGGACATGGACAAGGTGGAGACATTCCTGAGAATCGTGCAGTGCAGAAGCGTGGAGGGCAGCTGTGGATTT(SEQ ID NO:39)。
In a fourth aspect of the invention, the invention provides a linker peptide for use in the construction of fusion proteins. According to an embodiment of the invention, 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% to 99% identity to any one of them or at least a part of any one of them.
In a fifth aspect of the invention, a nucleic acid molecule is presented. According to an embodiment of the invention, the nucleic acid molecule encodes the Fc mutant of the first aspect, the fusion protein of the second aspect or the third aspect or the linker peptide of the fourth aspect. The Fc mutant encoded by the nucleic acid molecule of the embodiment of the invention is in a monomer form, and the fusion protein obtained by fusing the Fc mutant in the monomer form and a bioactive molecule has higher in vitro and in vivo activity and lower ADCC and CDC effects. The fusion protein encoded by the nucleic acid molecule can be purified by ProteinA subsequently, so that the purification process is simplified, and the fusion protein has higher in vitro and in vivo activity and lower ADCC and CDC effects.
According to an embodiment of the invention, the nucleotide sequence comprises a codon optimized sequence selected from at least one of SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23 or a nucleotide sequence having at least 80% to 99% identity to any of them or at least a part of any of them or any of them.
The SEQ ID NO. 21 is used for coding SEQ ID NO. 8.
The SEQ ID NO. 22 is used for coding SEQ ID NO. 9.
The SEQ ID NO. 23 is used for coding SEQ ID NO. 10.
According to an embodiment of the invention, the nucleotide sequence comprises a codon optimized sequence selected from at least one of SEQ ID NO 32, 33, 34, 35, 36, 37, 38, 39 or a nucleotide sequence having at least 80% -99% identity to any of them or at least a part of any of them or any of them.
The SEQ ID NO. 32 is used for coding SEQ ID NO. 24.
The SEQ ID NO. 33 is used for coding SEQ ID NO. 25.
The SEQ ID NO. 34 is used for coding SEQ ID NO. 26.
The SEQ ID NO. 35 is used for coding the SEQ ID NO. 27.
The SEQ ID NO. 36 is used for coding SEQ ID NO. 28.
The SEQ ID NO. 37 is used for coding SEQ ID NO. 29.
The SEQ ID NO 38 is used for coding the SEQ ID NO 30.
The SEQ ID NO 39 is used for coding SEQ ID NO 31.
According to an embodiment of the invention, the nucleotide sequence comprises at least one selected from the group consisting of SEQ ID NO 12, 13, 14, 15, 16, 17, 18, 19 and 20 or a nucleotide sequence having at least 80% -99% identity to any one of them or at least a part of any one of them or a codon optimized sequence of any one of them.
The SEQ ID NO 12, 13, 15 and 16 are used for coding SEQ ID NO 2.
The SEQ ID NO. 14 is used for coding SEQ ID NO. 3.
The SEQ ID NO. 17 is used for coding SEQ ID NO. 4.
The SEQ ID NO. 18 is used for coding SEQ ID NO. 5.
The SEQ ID NO. 19 is used for coding SEQ ID NO. 6.
The SEQ ID NO. 20 is used for coding SEQ ID NO. 7.
GCCCCTGAACTGCTCGGAGGCCCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGCGGTGTCACGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACCAAGCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCACCCTGACCGTGGACAAGTCCAGATGGCAACAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAA(SEQ ID NO:12)。
GCTCCTGAACTGCTGGGAGGACCTAGCGTGTTTCTGTTTCCTCCCAAACCTAAAGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTTGATGTGTCTCATGAAGACCCCGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAAACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGCAAGGAATACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAGGCTAAGGGCCAGCCCAGAGAGCCCCAAGTGTATACACTGCCCCCTAGCAGAGATGAACTGACCAAAAACCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATTGCCGTGGAGTGGGAGAGCAATGGCCAACCTGAAAACAATTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCTTTCTTTCTGTATAGCACCCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTATACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAA（SEQ ID NO:13）。
CCCGCCCCCGAACTGCTGGGAGGACCTTCTGTGTTTCTGTTTCCTCCCAAACCCAAAGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTTGTGGATGTGTCTCATGAGGATCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAGACAAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGAAAGGAATACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACAATTAGCAAGGCCAAGGGCCAGCCCAGAGAACCTCAAGTGTATACCCTGCCTCCCAGCAGAGATGAGCTGACAAAAAATCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTCTACCCCAGCGACATTGCCGTGGAGTGGGAAAGCAATGGCCAACCTGAGAACAACTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCTTTTTTTCTGTACAGCACCCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCATAATCACTACACCCAGAAAAGCCTGAGCCTGAGCCCCGGCAAG（SEQ ID NO:14）。
GCTCCTGAACTGCTCGGAGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTCGAGGTGCACAACGCCAAGACAAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGCGGTGTCACGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACCAAGCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCACCCTGACCGTGGACAAGTCCAGATGGCAACAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAA（SEQ ID NO:15）。
GCTCCTGAACTGCTCGGAGGCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGCGGTGTCACGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACCAAGCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCACCCTGACCGTGGACAAGTCCAGATGGCAACAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAA（SEQ ID NO:16）。
GCTCCCGAACTGCTGGGAGGACCCAGCGTGTTTCTGTTTCCCCCTAAACCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGATGTGAGCCATGAGGACCCCGAAGTGAAATTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAAGCCAAGGGCCAGCCCAGAGAGCCCCAAGTGTACACATTACCCCCTAGCAGAGATGAACTGACCAAAAACCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGACAACCTGAGAACAACTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCTTTCTTTCTGTACAGCACCCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCATAATCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAGCT(SEQ ID NO:17)。
GCTCCTGAACTGCTGGGAGGACCTAGCGTGTTTCTGTTTCCTCCTAAACCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGATGTGAGCCATGAAGATCCCGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAAACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGAAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAAGCTAAGGGCCAGCCCAGAGAGCCCCAAGTGTATACAAGCCCTCCCAGCAGGGACGAGCTGACCAAAAATCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATCGCCGTGGAATGGGAAAGCAATGGCCAACCCGAGAACAACTATAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCCTTTTTTCTGTACAGCGACCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGTATCACGAGGCCCTGCACAATCACTACACCCAGAAAAGCCTGAGCCTGAGCCCCGGCAAA(SEQ ID NO:18)。
GCCCCTGAGCTGCTTGGAGGACCTAGCGTTTTTTTATTTCCCCCCAAACCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTTGTGGATGTGAGCCATGAAGATCCCGAGGTGAAATTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAAACAAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACCATTAGCAAGGCCAAGGGCCAGCCCAGAGAACCCCAAGTGTATACCAGCCCCCCCAGCAGAGATGAGCTGACAAAAAATCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTTTACCCCAGCGACATCGCCGTGGAGTGGGAATCTAATGGACAACCTGAGAACAACTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGGAGCTTTTTCCTGTATAGCGACCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGTATCACGAGGCCCTGCATAATCACTACACCCAGAAAAGCCTGAGCCTGAGCCCCGGAGCT(SEQ ID NO:19)。
GACAAGACACACACCGGACCCGCCCCCGAACTGCTGGGAGGACCTTCTGTGTTTCTGTTTCCTCCCAAACCCAAAGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTTGTGGATGTGTCTCATGAGGATCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCTAAGACAAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGATTGGCTGAATGGAAAGGAATACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCTATTGAAAAAACAATTAGCAAGGCCAAGGGCCAGCCCAGAGAACCTCAAGTGTATACCCTGCCTCCCAGCAGAGATGAGCTGACAAAAAATCAGGTGAGCCTGAGATGCCACGTGAAGGGCTTCTACCCCAGCGACATTGCCGTGGAGTGGGAAAGCAATGGCCAACCTGAGAACAACTACAAGACCACCAAGCCCGTGCTGGACAGCGACGGATCTTTTTTTCTGTACAGCACCCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCATAATCACTACACCCAGAAAAGCCTGAGCCTGAGCCCCGGCAAG(SEQ ID NO:20)。
In a sixth aspect of the invention, an expression vector is provided. According to an embodiment of the invention, the expression vector carries a nucleic acid molecule according to the fifth aspect. After the expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, the expression of the Fc mutant, the fusion protein or the connecting peptide described above can be effectively achieved under the mediation of a regulatory system, so that the Fc mutant, the fusion protein or the connecting peptide can be obtained in a large amount.
In a seventh aspect of the invention, the invention features a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises: carrying a nucleic acid molecule according to the fifth aspect; or, expressing the Fc mutant of the first aspect, the fusion protein of the second aspect or the third aspect, or the connecting peptide of the fourth aspect. The recombinant cells according to embodiments of the present invention can be used for in vitro expression and mass production of the aforementioned Fc mutants, fusion proteins or connecting peptides.
In an eighth aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: the fusion protein of the second or third aspect. The pharmaceutical composition according to the embodiment of the present invention can be used for preventing and treating diseases associated with abnormality of biologically active molecules of the fusion protein.
According to an embodiment of the invention, the pharmaceutical composition is for oral, intravenous administration, such as bolus injection or by continuous infusion for a period of time, subcutaneous, intramuscular, intraarterial, intraperitoneal, intrapulmonary, intracerebrospinal, intraarticular, intrasynovial, intrathecal, intralesional, or inhalation route, such as intranasal, typically intravenous or subcutaneous administration.
According to the embodiment of the invention, the dosage form of the pharmaceutical composition is tablets, capsules, spray, injection, freeze-dried powder injection or pre-filled injection.
In a ninth aspect of the invention, the invention provides a method for preparing the fusion protein of the second or third aspect. According to an embodiment of the present invention, the preparation method comprises: culturing the recombinant cell of the seventh aspect to obtain a culture solution containing the fusion protein; separating the fusion protein from the culture medium. Thereby, the fusion proteins according to the second and third aspects can be efficiently produced.
In a tenth aspect, the present invention provides a 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 a disease associated with growth hormone abnormality.
Advantageous effects
Compared with the prior art, the invention at least has one of the following beneficial technical effects:
(1) The Fc mutant in the fusion protein used by the invention is mutated at a specific site, the growth hormone fused with the Fc mutant is secreted and expressed in a monomer form, and can be purified by ProteinA affinity chromatography, and the subsequent purification process is simple.
(2) The fusion protein provided by the invention binds to human GHR 50 Low value and strong binding activity with human GHR.
(3) EC of fusion protein provided by the invention for promoting Nb2-11 cell proliferation 50 Low value, and good proliferation effect on Nb2-11 cells.
(4) The fusion protein provided by the invention promotes the reporter gene cell to express the EC of luciferase 50 The value is low, and is equivalent to or lower than that of a control sample (Genexine GX-H9), and the luciferase activity expressed by reporter gene cells is excellent.
(5) Compared with human growth hormone, the fusion protein provided by the invention has longer half-life and better in-vivo biological activity.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a schematic representation of the monomeric form of a fusion protein in one embodiment of the invention;
FIG. 2 is a graph showing the results of J2 or J18 induced ADCC effect in example 7 of the present invention;
FIG. 3 is a graph showing the trend of the results of the weight gain of rats in each group measured for in vivo biological activity in example 9 of the present invention.
Detailed Description
The following describes in detail embodiments of the present invention. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
Herein, the term "room temperature" means ambient temperature, which may be from 20 ℃ to 30 ℃; in some embodiments, from 22 ℃ to 28 ℃; in some embodiments, from 24 ℃ to 26 ℃; and in some embodiments, 25 ℃.
Herein, "antibody fragment" means antigen-binding fragments and antibody analogs of antibodies, which typically comprise at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parent antibody (parent antibody).
In this document, the terms "comprise" or "comprise" are open-ended expressions that include the elements indicated in the present invention, but do not exclude other elements.
As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs, and instances where it does not.
As used herein, the term "variant" or "mutant" refers generally to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.
Herein, the amino acid numbering of the IgG1 Fc moiety is according to EU numbering system, e.g., position 366 refers to position 366 according to EU numbering system numbering; the expression "T366R" means that the threonine at position 366 according to the EU numbering system is replaced by arginine; "L368H" means a substitution of leucine at position 368 by histidine according to the EU numbering system.
In this context, the term "(GGGGS) n "indicates that n GGGGS are connected, e.g.," (GGGGS) 3 "indicates GGGGSGGGGSGGGGS.
As used herein, the term "fusion protein" generally refers to a protein resulting from the fusion of two or more proteins or polypeptides. Genes or nucleic acid molecules encoding the two or more proteins or polypeptides may be linked to each other to form a fusion gene or a fused nucleic acid molecule, which may encode the fusion protein. Translation of the fusion gene results in a single polypeptide having the properties of at least one, and even each, of the two or more proteins or polypeptides prior to fusion. Recombinant fusion proteins are created artificially by recombinant DNA techniques for biological research or therapy. Recombinant fusion proteins are proteins created by genetic engineering of fusion genes. 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 typically comprise at least two domains (a and C), and optionally a third component, a linker between the two domains. The generation of recombinant fusion proteins is known in the art and typically involves removing a stop codon from a cDNA sequence encoding a first protein or polypeptide and then attaching the cDNA sequence of a second protein in frame by ligation or overlap extension PCR. The DNA sequence will then be expressed by the cell as a single protein. The protein may be engineered to include the entire sequence of both original proteins or polypeptides, or only a portion thereof.
The fusion proteins of the invention are typically prepared by biosynthetic methods. The encoding nucleic acids of the present invention can be readily prepared by a variety of known methods by those skilled in the art based on the nucleotide sequences of the present invention. Such methods are for example but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in sambrook, molecular cloning guidelines. As an embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing nucleotide sequences by segmentation and then performing overlap extension PCR.
As used herein, the terms "identity", "homology" or "similarity" are used to describe amino acid sequences or nucleic acid sequences relative to a reference sequence, and the determination of the percentage of identical amino acids or nucleotides between two amino acid or nucleic acid sequences is performed by conventional methods, e.g., see Ausubel et al, eds (1995), current Protocols in Molecular Biology, chapter 19 (Greene Publishing and Wiley-Interscience, new York); and the ALIGN program (Dayhoff (1978), atlas of Protein Sequence and Structure 5 (National biological Research Foundation, washington, D.C.). There are many algorithms for aligning sequences and determining Sequence identity, including the homology alignment algorithm of Needleman et al (1970) J.mol.biol.48: 443; the local homology algorithm of Smith et al (1981) adv.Appl.Math.2: 482; methods for similarity searching by Pearson et al (1988) Proc. Natl.Acad.Sci.85:2444, the Smith-Waterman algorithm (meth. Mol.biol.70:173-187 (1997)), and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J.mol.biol.215: 403-410) computer programs utilizing these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al, meth.Enzym., 266-480 (1996)), or GAP, BESTFIT, BLAST Altschul et al, supra, TA, and TFASTA, provided in Genetics computer Computing Group (GCG) package, 8 th edition, madisin, USA, and FAS, the programs available in U.A., USA, and U.S.A..
As used herein, the term "nucleotide" generally refers to a modified form that can be a ribonucleotide, a deoxynucleotide, or any type of nucleotide, as well as combinations thereof.
Herein, the term "at least 90% identity" refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence.
As used herein, the term "at least 80% -99% sequence identity" refers to 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.
In this context, the term "expression vector" generally refers to a nucleic acid molecule capable of inserting into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells. The expression vectors may include vectors primarily for the insertion of DNA or RNA into a cell, vectors primarily for the replication of DNA or RNA, and vectors primarily for the expression of transcription and/or translation of DNA or RNA. The expression vector also includes vectors having a variety of the above-described functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce the desired expression product by culturing a suitable host cell containing the expression vector.
In this context, the term "recombinant cell" generally refers to a host cell whose genetic material has been modified or recombined by genetic engineering techniques or cell fusion techniques to obtain a cell with a unique trait of stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The term "transformed" or "transfected" as used herein refers to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the invention and can be used for expression and/or secretion of the target protein. Examples of suitable host cells that can be used in the present invention 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.
The term "pharmaceutical composition" as used herein generally refers to a unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. Generally, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely divided solid carrier, or both.
As used herein, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, and the like, suitable for the particular intended dosage form. Except insofar as any conventional adjuvant is incompatible with the compounds of the invention, e.g., any adverse biological effect produced or interaction in a deleterious manner with any other component of a pharmaceutically acceptable composition, their use is contemplated by the present invention.
Reference may be made to a number of other pharmaceutically acceptable adjuvants or processes mentioned herein for this subject matter, see in particular Handbook of Pharmaceutical Excipients, 3 rd edition, edited by Arthur h. Kibbe, american Pharmaceutical Association, washington, USA and Pharmaceutical Press, london; and Lexikon der Hilfsstuffe fur Pharmazie, kosmetik and angrenzene Gebiete, edited by H.P. Fiedler, 4 th edition, cantor, aulentorf and earlier versions.
As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by some suitable means. The fusion protein of the present invention can be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, rectal, and topical, but the invention is not limited to these exemplified modes of administration. Preferably, the fusion protein of the invention is administered by injection.
Herein, the term "growth hormone abnormality-related disease" generally refers to a disease caused by growth hormone abnormality, such as a related disease caused by growth hormone deficiency or a dissimilatory state, including, but not limited to, childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, turner's Syndrome (TurnersSyndrome), prader Willi Syndrome, intrauterine growth retardation, idiopathic short stature, renal failure, chemotherapy treatment, and dissimilatory state during AIDS treatment. Growth hormone deficiency may include congenital or acquired deficiency. Regarding the congenital defect, a growth hormone defect may occur when the pituitary does not develop growth hormone secretion disorders. Acquired growth hormone deficiency may occur due to brain tissue damage caused by hypoxia resulting from difficult delivery. Other causes of growth hormone deficiency include pituitary damage caused by radiation used to treat brain tumors or postnatal tubercular meningitis. Growth hormone deficiency exhibits symptoms such as growth retardation and short stature, and innate growth hormone deficiency exhibits low glucose symptoms, beginning with newborns. In addition, children exhibit symptoms such as increased anxiety and decreased vitality.
As used herein, the term "treating" is intended to mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce, or inhibit a disease in the individual, including, but not limited to, administering a drug containing a compound described herein to an individual in need thereof.
It is to be understood by those skilled in the art that the monomeric or dimeric form of an Fc mutant determines the monomeric or dimeric form of the fusion protein it forms.
The present invention 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.
Fc mutants
In one aspect of the invention, the invention provides an Fc mutant. According to an embodiment of the invention, comprising: a first peptidyl fragment having mutations compared to the Fc fragment of wild-type IgG1 at the following positions: at least one of 228 th bit, 229 th bit, 230 th bit, 366 th bit, 368 th bit, 395 th bit and 409 th bit, and 351 th bit, 428 th bit and 447 th bit; or 228 th bit, 229 th bit, 366 th bit, 368 th bit, 395 th bit and 409 th bit.
The inventor finds through a large number of experiments that the mutation is beneficial to enabling the Fc fragment in the form of natural dimer to be expressed and then form the Fc mutant in the form of monomer, the Fc mutant in the form of monomer is adopted to be fused with bioactive molecules, and the subsequent purification can be realized through ProteinA, so that the purification process is simplified; in addition, the fusion protein prepared by the Fc mutant has higher in vivo and in vitro activity and lower ADCC and CDC effects.
According to an embodiment of the invention, the first peptide fragment has mutations compared to the Fc fragment of wild-type IgG1 at the following positions: 1) 228, 229, 230, 366, 368, 395, 409, and 447 bits; or, 2) 228 th bit, 229 th bit, 230 th bit, 351 th bit, 366 th bit, 368 th bit, 395 th bit, 409 th bit and 428 th bit; or, 3) 228 th bit, 229 th bit, 230 th bit, 351 th bit, 366 th bit, 368 th bit, 395 th bit, 409 th bit, 428 th bit and 447 th bit. Thus, the above mutations may form Fc mutants in monomeric form.
According to an embodiment of the invention, the first peptide fragment has mutations compared to the Fc fragment of wild-type IgG1 at the following positions: T366R, L368H, P395K and K409D or K409T; and at least one of L351S, M428Y and K447A. Thus, the above mutations may form Fc mutants in monomeric form.
According to an embodiment of the invention, the first peptide fragment has mutations compared to the Fc fragment of wild-type IgG1 at the following positions: 1) P228Delete, C229Delete, P230 Delete, T366R, L368H, P395K, K447A and K409D or K409T; or, 2) P228Delete, C229Delete, P230 Delete, L351S, T366R, L368H, P395K, M428Y and K409D or K409T; or, 3) P228Delete, C229Delete, P230 Delete, L351S, T366R, L368H, P395K, M428Y, K447A and K409D or K409T; or, 4) P228Delete, C229Delete, T366R, L368H, P395K and K409D or K409T. The inventor carries out a large number of experiments, and obtains the monomer-form Fc mutant, the fusion protein of the Fc mutant and bioactive molecules after fusion is secreted and expressed in a monomer form, and the fusion protein has higher in vivo and in vitro activity and lower ADCC and CDC effects.
Preferably, the first peptide fragment has mutations compared to the Fc fragment of wild-type IgG1 at the following positions: p228Delete, C229Delete, P230 Delete, L351S, T366R, L368H, P395K, M428Y, K447A and K409D or K409T.
The inventor finds out through a large number of experiments that the Fc mutant is fused with human growth hormone, and the obtained fusion protein has higher binding force to a human growth hormone receptor, higher cell proliferation activity and higher in vitro activity. Illustratively, the Fc mutants of fusion proteins J16 and J18 according to the embodiments of the present invention have different mutation sites, and the Fc mutant of J18 contains the above site mutations, whereas J16 has no L351S and M428Y mutations compared to J18, and fusion protein J18 has stronger binding force to human growth hormone receptor, cell proliferation activity and in vitro activity. Furthermore, fusion protein J17 increased L351S and M428Y mutations compared to fusion protein J2 (P228 Delete, C229Delete, P230 Delete, T366R, L368H, P395K and K409D) and J2 and J17 had different mutation sites at position 409 (J17 is K409T), but the J17 fusion protein was more active on cell proliferation and in vitro activity. Therefore, the fusion protein prepared by adopting the Fc mutant with L351S and M428Y mutation has higher human growth hormone receptor binding force, cell proliferation activity and in vitro activity.
According to an embodiment of the present invention, the Fc mutant further comprises: a hinge region fragment of wild type IgG1, wherein the C end of the hinge region fragment of wild type IgG1 is connected with the N end of the first peptide segment.
According to an embodiment of the present invention, the hinge region fragment of wild type IgG1 has the amino acid sequence shown in SEQ ID NO: 41.
According to an embodiment of the present invention, the Fc mutant has an amino acid sequence as shown in any one of SEQ ID NOs 3 to 7. The inventor finds that the fusion protein obtained by fusing the Fc mutant and bioactive molecules has high in vivo and in vitro bioactivity.
According to an embodiment of the invention, the Fc mutant is in monomeric form.
Fusion proteins
In yet another aspect of the invention, a fusion protein is provided. According to an embodiment of the invention, the fusion protein comprises: a second peptide segment comprising a biologically active molecular domain; a third peptide stretch comprising an Fc mutant having the amino acid sequence shown in SEQ ID NO 2 or an Fc mutant as defined previously, the second peptide stretch being linked to the third peptide stretch. The inventor finds through a large number of experiments that the fusion protein is in a monomer form, and has higher in vivo and in vitro activity and lower ADCC and CDC effects; moreover, the fusion protein can be purified through ProteinA subsequently, and the purification process is simplified.
It should be noted that "monomer" refers to 1 Fc mutant, and the specific structure is shown in fig. 1.
According to an embodiment of the invention, said second peptide segment comprises growth hormone, a growth hormone analogue, a growth hormone functional region or a growth hormone analogue functional region, preferably human growth hormone or a human growth hormone functional region. The inventor finds that the fusion protein is in a monomer form, has higher in vivo and in vitro activity and lower ADCC and CDC effects, and has the advantages of strong binding force to human growth hormone receptors, good cell proliferation activity, high in vitro activity and the like.
According to an embodiment of the invention, the human growth hormone has the amino acid sequence as SEQ ID No. 1 or an amino acid sequence having at least 90% identity thereto.
According to an embodiment of the invention, the N-terminus of the second peptide stretch is linked to the C-terminus of the third peptide stretch; or the C end of the second peptide segment is connected with the N end of the third peptide segment. Therefore, the fusion protein prepared by adopting the connection mode has higher in vivo and in vitro activity.
According to an embodiment of the invention, the N-terminus of the second peptidyl fragment is linked to the C-terminus of the third peptidyl fragment. The inventors have found through a large number of experiments that the fusion protein obtained by the above connection method can further improve the binding force to the corresponding receptor, the cell proliferation activity and the in vitro activity, such as: the fusion protein prepared by fusing the Fc mutant and the growth hormone has better drug effect in animals.
According to an embodiment of the present invention, the fusion protein further comprises a linker peptide disposed between the second peptide segment and the third peptide segment. Therefore, the second peptide segment and the third peptide segment which can be fused by the connecting peptide are adopted, and the biological activity of the finally obtained fusion protein is improved. Illustratively, the fusion proteins J1, J2, J6 and J7 differ in their connecting peptides, such that J2, J6 and J7 have better binding activity for human GHR and better promotion of cell proliferation than J1 without the connecting peptide.
According to an embodiment of the present invention, the N-terminus of the linker peptide is linked to the C-terminus of the third peptide fragment, and the C-terminus of the linker peptide is linked to the N-terminus of the second peptide fragment; or the N end of the connecting peptide is connected with the C end of the second peptide segment, and the C end of the connecting peptide is connected with the N end of the third peptide segment.
According to an embodiment of the invention, the N-terminus of the linker peptide is linked to the C-terminus of the third peptide stretch, and the C-terminus of the linker peptide is linked to the N-terminus of the second peptide stretch. The inventors have found through a large number of experiments that the fusion protein obtained by the above connection method can further improve the binding force to the corresponding receptor, the cell proliferation activity and the in vitro activity, such as: the fusion protein prepared by fusing the Fc mutant and the growth hormone has better drug effect in animals.
According to an embodiment of the present invention, the amino acid sequence of the linker 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 use of the second peptide fragment and the third peptide fragment, which are fused by the linker peptide, can improve the biological activity of the finally obtained fusion protein.
According to an embodiment of the invention, the linker peptide has an amino acid sequence as shown in any one of SEQ ID NOs 8 to 10. Thus, the use of the second peptide fragment and the third peptide fragment, which are fused by the linker peptide, can further improve the biological activity of the finally obtained fusion protein.
According to an embodiment of the invention, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs 24 to 31. The inventor finds that the fusion protein has strong binding force to human growth hormone receptors, good cell proliferation activity and high in vitro activity through experiments.
It will be appreciated by those skilled in the art that the features and advantages described above for the Fc mutants apply equally to the fusion proteins and are not described in detail here.
Nucleic acid molecules
In another aspect of the invention, the invention features a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the aforementioned Fc mutant or the aforementioned fusion protein. The nucleic acid molecule can be effectively used for expressing the Fc mutant or the fusion protein, particularly can effectively express the fusion protein in a prokaryotic organism or lower eukaryotic organism expression system, and the fusion protein is in a monomer form and has higher in vitro and in vivo activity and lower ADCC and CDC effects.
According to an embodiment of the invention, the nucleic acid molecule is DNA.
It will be appreciated by those skilled in the art that the features and advantages described above for the Fc mutants and fusion proteins apply equally to the nucleic acid molecules and will not be described in further detail herein.
Expression vector
In another aspect of the invention, the invention features an expression vector. According to an embodiment of the invention, the expression vector carries the aforementioned nucleic acid molecule. The Fc mutant or the fusion protein can be effectively expressed in cells by using the expression vector, particularly the fusion protein can be effectively expressed in a prokaryotic organism or lower eukaryotic organism expression system, and the fusion protein is in a monomer form and has higher in-vivo and in-vitro activity and lower ADCC and CDC effects.
According to an embodiment of the invention, the expression vector is a eukaryotic expression vector.
According to an embodiment of the invention, the expression vector is a lentiviral vector.
It will be appreciated by those skilled in the art that the features and advantages described above for the Fc mutants, fusion proteins and nucleic acid molecules apply equally to the expression vector and are not described in detail here.
Recombinant cell
In another aspect of the invention, the invention features a recombinant cell. According to an embodiment of the invention, 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 for effectively expressing the Fc mutant or the fusion protein, particularly the fusion protein can be effectively expressed in a prokaryotic organism or lower eukaryotic organism expression system, and the fusion protein is in a monomer form and has higher in vitro and in vivo activity and lower ADCC and CDC effects.
According to an embodiment of the present invention, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.
According to an embodiment of the invention, the recombinant cell is a eukaryotic cell.
According to an embodiment of the invention, the recombinant cell is a mammalian cell.
It will be appreciated by those skilled in the art that the features and advantages described above for the Fc mutants, fusion proteins, nucleic acid molecules and expression vectors apply equally to the recombinant cells and will not be described in detail here.
Pharmaceutical composition
In another aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: the aforementioned fusion protein. The pharmaceutical composition according to the embodiment of the invention can be used for preventing and treating diseases related to growth hormone abnormality.
According to an embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
According to an embodiment of the present invention, the pharmaceutical composition is in the form of an injection.
According to an embodiment of the invention, the route of administration of the pharmaceutical composition comprises subcutaneous injection or intravenous injection.
It will be appreciated by those skilled in the art that the features and advantages described above for the Fc mutants, fusion proteins, nucleic acid molecules, expression vectors and recombinant cells apply equally to the pharmaceutical composition and will not be described in further detail herein.
Method for preventing and/or treating diseases related to growth hormone abnormality
In another aspect of the present invention, the present invention provides a method for preventing and/or treating diseases associated with growth hormone abnormality. According to an embodiment of the invention, the method comprises: administering to the subject a pharmaceutically acceptable amount of the aforementioned fusion protein or the aforementioned pharmaceutical composition. According to embodiments of the present invention, the method is effective for preventing or treating diseases associated with growth hormone abnormality.
According to an embodiment of the invention, the route of administration of the method comprises subcutaneous injection or intravenous injection.
According to an embodiment of the present invention, the disease associated with growth hormone abnormality includes at least one selected from the group consisting of: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, turner's syndrome, prader-willi syndrome, renal failure, diseases caused by dissimilatory states during chemotherapy treatment and AIDS treatment, intrauterine growth retardation.
It will be appreciated by those skilled in the art that the features and advantages described above for the Fc mutants, fusion proteins, nucleic acid molecules, expression vectors, recombinant cells and pharmaceutical compositions are equally applicable to the method for preventing and/or treating diseases associated with growth hormone abnormalities and will not be described in detail herein.
Use of
In another aspect of the present invention, the present invention provides a 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 disease associated with growth hormone abnormality.
According to an embodiment of the present invention, the disease associated with growth hormone abnormality includes at least one selected from the group consisting of: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, turner's syndrome, prader-willi syndrome, renal failure, diseases caused by dissimilatory states during chemotherapy treatment and AIDS treatment, intrauterine growth retardation.
It will be appreciated by those skilled in the art that the features and advantages described above for the Fc mutants, fusion proteins, nucleic acid molecules, expression vectors, recombinant cells and pharmaceutical compositions are equally applicable to this use and will not be described in further detail herein.
The scheme of the invention will be explained with reference to the following examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: preparation of recombinant long-acting human growth hormone fusion protein expression vector
In the present application, human growth hormone (SEQ ID NO: 1) was linked to Fc mutants (SEQ ID NO:2 and SEQ ID NO: 7) to construct fusion proteins, to obtain J1 and J3, respectively. In the 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 connecting peptides (SEQ ID NO: 8-10) to construct fusion proteins, and J2, J6, J7 and J16-18 are obtained respectively. The specific experimental procedures were as follows:
nucleotide sequences (SEQ ID NO: 11) for coding human growth hormone, nucleotide sequences (SEQ ID NO: 21-23) for coding connecting peptide and nucleotide sequences (SEQ ID NO: 16-20) for coding various mutant Fc mutants 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 respectively cloned into an expression vector pCDNA3.4 (purchased from Thermo Fisher) by adopting gene total synthesis and molecular cloning technologies, so as to respectively obtain fusion protein expression vectors of the nucleotide sequences.
Example 2: recombinant long-acting human growth hormone fusion protein expression
The host cells were transfected with the plasmids obtained in example 1 using Expi CHO-S (Gibco, A29133) as host cells, and the fusion proteins (i.e., recombinant long-acting human growth hormone fusion proteins) were transiently expressed using a chemical transfection reagent Polyplus-FectoPRO (Polyplus, 116-010), and the corresponding amino acid sequences were SEQ ID NO:24-31. The experimental procedure was as follows:
1. the cells were passaged one day before transient infection with Expi CHO-S cells, and the cell density was adjusted to 3X 10 with the medium 6 cells/mL or so. The cell culture flask was returned to the shaker (37 ℃, 8% CO) 2 ) And (5) continuing culturing.
2. Day of transient infection (1L per molecule transfection): taking Expi CHO-S cell fluid to count cells, adjusting the cell density to 6 x 10 by using a culture medium 6 cells/mL or so.
3. Preparing a transfection complex: taking 16 sterile cell culture bottles, marking 8 marked DNA and 8 marked FectoPRO, and adding a transfection reagent Polyplus-FectoPRO into the FectoPRO bottle; adding 60mL of Opti-MEM solution to 8 DNA bottles and 500. Mu.g of each of the fusion protein expression vectors of the nucleotide sequences obtained in example 1 to each DNA bottle; mixing to obtain fusion protein expression vector diluent, adding the fusion protein expression vector diluent obtained from each nucleotide sequence into a FectoPRO bottle respectively, mixing, incubating at room temperature for 10min, adding into cell liquid, shaking, and placing the cell culture bottle back to the shaker for continuous culture and transfection.
4. After transfection for 18-22h, adding a proper amount of OPM-CHO Profeed, and measuring the biochemical indexes of cell fluid. According to the biochemical index, glucose is supplemented to 6g/L. Titer assay was started 4d after transfection and supplemented with sugar every other day. The CHO cell fermentation liquor is obtained when the cell survival rate is less than 80 percent, and supernatant can be harvested for purification.
Example 3: recombinant long-acting human growth hormone fusion protein purification
The CHO cell fermentation liquid obtained in example 2 was subjected to secondary centrifugation (primary: 3000 Xg, 30min; secondary: 12000 Xg, 20 min), and the supernatant was collected and filtered through a 0.2 μm filter for use.
Protein a affinity chromatography: the chromatography column is equilibrated for at least 3 column volumes using an aqueous solution of pH7.2 containing 20mM phosphate and 150mM sodium chloride, the filtered clear filtrate is loaded, the feed solution is retained on the chromatography column for 5 minutes, after the sample is loaded, the chromatography column is equilibrated for at least 1 column volume using an aqueous solution of pH7.2 containing 20mM phosphate and 150mM sodium chloride, the target protein is eluted using a buffer solution of pH 4.5 containing 50mM acetic acid (HAc), a buffer solution of pH4.0 containing 50mM HAc, and a buffer solution of pH 3.5 containing 50mM HAc, respectively, and when an absorption peak occurs, the peak absorption range is 50mAU-peak-50mAU, a fusion protein having a purity SEC (> 98.0) is obtained, and the fusion protein containing the nucleotide sequence 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, or SEQ ID: 39, SEQ ID NO:39, CHO, SEQ ID NO:16, CHO, 18J, 17J, 18J, 18, 17, 18J, 18, respectively, and the fusion protein is fermented. The specific structure of the fusion protein is shown in FIG. 1, wherein the structures of J1, J2, J3, J6, J7 and J17 are shown in the left picture of FIG. 1, and the structures of J16 and J18 are shown in the right picture of FIG. 1.
Example 4: fusion protein binding assay to human GHR
In this example, the purified fusion protein obtained in example 3 is subjected to in vitro receptor (human growth hormone receptor, abbreviated as human GHR) binding activity detection, and Genexine GX-H9 is used as a control group, and the specific experimental operations are as follows:
diluting human GHR to obtain 0.5 μ g/ml coating solution, adding 100 μ L/well of the coating solution into an ELISA plate, and coating at 2-8 deg.C for more than 12 hr. The plate-coated residual solution was discarded, and 1% BSA-PBST (1% bovine serum albumin in phosphate Tween buffer) was added thereto, at 300. Mu.L per well, and blocked at 37 ℃ for 1 hour. Adding 300 mu L of PBST (phosphate Tween buffer) into each well, washing for 3 times, diluting the recombinant long-acting human growth hormone fusion protein and the Genexine GX-H9 to 5 mu g/ml, diluting the diluted recombinant long-acting human growth hormone fusion protein and the Genexine GX-H9 to 8 gradient concentrations by 5 times, and adding 100 mu L of the fusion protein into an ELISA plate. Incubation at 37 ℃ for 1 hour300 μ L of PBST was added to each well, washed 3 times, and then 1% of Goat anti human IgG Fc-HRP diluted 10000 times with BSA-PBST was added, and 100 μ L/well was added. After incubation for 1 hour at 37 deg.C, 300. Mu.L of PBST was added to each well, washed 3 times, and patted dry. TMB developing solution was added thereto in an amount of 100. Mu.L per well. After 5 minutes reaction at room temperature, 2M H was added 2 SO 4 The reaction was stopped with 100. Mu.L/well of aqueous solution. Placing the enzyme label plate on an enzyme label instrument, and reading the absorbance OD at the wavelength of 450nm 450 The EC50 value of the human GHR binding activity of each fusion protein is calculated, and the result is shown in Table 1, and the result shows that the fusion protein provided by the invention has low EC50 value of the human GHR binding activity and strong binding activity with the human GHR. See the binding activity relative to Genexine GX-H9, which shows that the binding activity to human GHR is similar or better than that of Genexine GX-H9.
Wherein the amino acid sequence of the Genexine GX-H9 is as follows:
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:43)。
table 1: detection result of recombinant long-acting human growth hormone fusion protein in human GHR (growth hormone receptor) binding activity test
Figure BDA0003597162480000191
Example 5: experiment for promoting Nb2-11 cell proliferation by recombinant long-acting human growth hormone fusion protein
The purified fusion proteins obtained in example 3 were tested, and the above Genexine GX-H9 was used as a control, and the following specific test procedures were performed:
cells were collected and cultured at 1X 10 per ml 5 Individual cell concentrations were suspended in culture medium (PRMI)1640 medium, 1% FBS and 50. Mu.M. Beta. -mercaptoethanol). Each 50 μ L cell sample was added to each well of a 96-well cell culture plate. The cells were cultured in 50. Mu.L of assay medium containing various concentrations of recombinant long-acting human growth hormone fusion protein from 0.051ng/mL to 3000ng/mL, and Genexine GX-H9. At 37 ℃ C, 5% CO 2 The Cell plates were cultured in a wet incubator for 96 hours, and then 50. Mu.L of an ellTiter-Glo luminescennt Cell Viability Assay (Promega, G7571) was added to each well. After 10 minutes, the microplate reader detects the chemiluminescent signal. The biological activity of the recombinant long-acting human growth hormone fusion protein can be measured by the obtained dose response curve, and the result is shown in Table 2, and the EC of the fusion protein provided by the invention on promoting the proliferation of Nb2-11 cells 50 Low value, and good proliferation effect on Nb2-11 cells. Compared with Genexine GX-H9, the relative activity of promoting the cell proliferation of Nb2-11 is similar or better.
Table 2: experimental detection result of recombinant long-acting human growth hormone fusion protein for promoting Nb2-11 cell proliferation
Figure BDA0003597162480000201
Example 6: cell biological activity experiment of recombinant long-acting human growth hormone fusion protein on reporter gene
In this example, experiments were performed on the purified fusion protein obtained in example 3, and the above Genexine GX-H9 was used as a control group, and the specific experimental operations were as follows:
taking reporter gene cells (293-GHR/STAT 5 cell line) in logarithmic growth phase, plating after trypsinization (costar, 3917), 4 × 10 4 cells/well, 50. Mu.L/well, 96-well white plate at 37 ℃ C. And 5% CO 2 Incubate overnight in the incubator. Adding gradient diluted recombinant long-acting human growth hormone fusion protein to the cells, starting at 100nM, 5-fold gradient dilution, 10 gradient concentrations, adding the dilution to the cells, adding the volume of 50 μ L/well, placing 96-well white plate at 37 deg.C, and 5% CO 2 Incubate in incubator for 6 hours. After the incubation was completed, the 96-well white plate and the Nano-Glo Luciferase Assay kit (promega, N112B) were removed and the samples were averagedThe reaction substrate was added to the wells at room temperature, the wells were left at 50. Mu.L/well for 10 minutes at room temperature, the luminescence signal values were recorded using a microplate reader (Promega, GM 2000) with the protein concentration as the X-axis and the luminescence signal values as the Y-axis, and the EC was calculated using GraphPad Prisim 5 as four-parameter fit 50 The results are shown in Table 3. The results show that the fusion protein provided by the invention promotes the reporter gene cell to express the EC of luciferase 50 The value is low, and is equivalent to or lower than that of a control group, and the luciferase activity expressed by reporter gene cells is excellent.
Table 3: experimental detection result of recombinant long-acting human growth hormone fusion protein for promoting reporter gene cell to express luciferase
Fusion protein numbering EC 50 (ng/mL) Relative Activity with respect 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
Example 7: the test sample induces ADCC Effect of Effector cells Jurkat-CD16a-luc on target cells CHO-K1-PD-L1/GHR
1. Sample information
Sample name Concentration (mg/mL)
M7824 2.05
2(J2) 0.6
5(Genexine GX-H9) 1.33
18(J18) 2.66
Acterma 20
2. Cell processing
Target cells CHO-K1-PD-L1/GHR (Shanghai Jiabei biological medicine technology Co., ltd., SBTCL 015) treatment: subculture was carried out 2 days before the experiment, the supernatant was discarded, the cells were washed with 5mL of PBS, 1mL of 0.25% Trypsin-EDTA was added, after digestion in an incubator at 37 ℃ for 1min, 6mL of Medium 1 (450mL F12 Medium +50mL FBS) was added to stop the digestion, and the suspension was resuspended to a single cell suspension, 5mL of the cell suspension was discarded, 8mL of Medium 1 (450mL F12 Medium +50mL Fetal Bovine Serum (FBS)) was added, 120. Mu.L of G418 was added at a concentration of 600. Mu.g/mL, mixed well, and placed in the incubator to continue the culture.
Effector cell Jurkat-CD16a-luc (Shanghai Jiabei Biopharmaceutical technology, inc., SBTCL 001) treatment: subculturing 2 days before experiment, blowing and beating the mixed cell suspension, taking 5mL of cell suspension to a new T75 culture flask, adding 15mL of culture medium 2 (450mL of RPMI 1640 culture medium +50mL of FBS), adding 320 μ L of G418, uniformly mixing the mixture with 60 μ L of Hygromycin B (Hygromycin B) and 150 μ G/mL of Hygromycin B, and putting the mixture into an incubator for continuous culture.
3.3 Experimental procedure
The first day, target cells CHO-K1-PD-L1/GHR density was adjusted to 1.83X 10 with assay Medium 1 (40mL F12 Medium +0.4mL FBS) 5 cells/mL, 50 μ L/well plated overnight; the next day, the test sample (i.e. J2 or J18), the positive control M7824 and the negative control Acterma were first diluted to 0.1mg/mL with the analytical medium 2 (RPMI 1640 medium), second diluted to 24. Mu.g/mL, then diluted 3 times to 9 gradients, 25. Mu.L/well was added to the cell plate, mixed well, incubated in a 37 ℃ incubator for 45min, and the fine particles were removedTaking out the cell plate; effector cell Jurkat-CD16a-luc Density adjusted to 1.47X 10 with analytical Medium 2 (RPMI 1640 Medium) 6 Adding 25 mu L/hole of cells/mL into a cell plate, uniformly mixing, placing the cell plate in an incubator at 37 ℃ for co-incubation for 5.5h, taking the cell plate out, balancing the cell plate at room temperature, adding One-Glo Luciferase Assay System (Promega, E6120), adding 100 mu L/hole of the cell plate, blowing by a pipette for 10 times, and uniformly mixing; and (4) after the mixture is cracked for 10min in a dark place at room temperature, detecting a chemiluminescence signal value by using an enzyme-labeling instrument. As shown in FIG. 2, after adding the positive control PD-L1 antibody M7824 (Shanghai Behcet Biomedicine technology Co., ltd.), the negative control IL-6R antibody Acterma (Roche) and the test sample to the ADCC experiment, the positive control M7824 induces significant ADCC effect of the effector cell Jurkat-CD16a-luc on the target cell CHO-K1-PD-L1/GHR, no ADCC effect of the negative control Acterma and no ADCC effect of the test sample, wherein the concentration unit of the abscissa of FIG. 2 is ng/ml.
Example 8: test samples induce CDC Effect of human serum complement on target cells CHO-K1-PD-L1/GHR
1. Sample information
Sample name Concentration (mg/mL)
Avelumab 22.65
J2 0.6
Genexine GX-H9 1.33
J18 2.66
Acterma 20
2. Cell processing
CHO-K1-PD-L1/GHR treatment of target cells: subculture was carried out 2 days before the experiment, the supernatant was discarded, the cells were washed with 5mL of PBS, 1mL of 0.25% Trypsin-EDTA was added, after digestion in an incubator at 37 ℃ for 1min, 6mL of Medium 1 (450mL F12 Medium +50mL FBS) was added to stop the digestion, and the suspension was resuspended to a single cell suspension, 5mL of the cell suspension was discarded, 8mL of Medium 1 (450mL F12 Medium +50mL FBS) was added, 120. Mu.L of G418 was added, and the concentration of G418 was 600. Mu.g/mL, mixed well, and placed in the incubator to continue the culture.
3. Experimental procedure
The first day, the target cells CHO-K1-PD-L1/GHR density was adjusted to 2.0X 10 with differentiation medium (99% F12 medium +1% FBS) 5 cells/mL, 50 μ L/well plated overnight; the next day, the test sample (i.e., J2 or J18), the positive control Avelumab (PD-L1 antibody, shanghai bei bio-pharmaceutical technology limited), and the negative control Acterma were first diluted to 0.1mg/mL with differentiation medium (99% f12 medium +1 fbs), second diluted to 15 μ g/mL, further diluted 3-fold for 7 gradients, 50 μ L/well added to the cell plate, mixed well, placed in a 37 ℃ incubator for 30min, and then the cell plate was removed; adding 30% complement (containing 70% differentiation medium and 30% complement), 50 uL/hole, mixing, placing in an incubator at 37 ℃ for incubation for 6h, taking out the Cell plate, balancing the Cell plate at room temperature, adding One-Glo Luminescent Cell vitality Assay, adding 50 uL/hole into the Cell plate, blowing by a pipette for 10 times, and mixing; after cracking for 10min at room temperature in dark, the chemiluminescence signal value is detected by an enzyme-linked immunosorbent assay. The result shows that after the positive control PD-L1 antibody Avelumab, the negative control IL-6R antibody Acterma and the SBT123 test sample molecules are added in a CDC experiment, the positive control Avelumab induces that human serum complement has obvious CDC effect on the target cell CHO-K1-PD-L1/GHR, the negative control Acterma has no CDC effect, and the test sample has no CDC effect.
Example 9: in vivo biological Activity assay
Pharmacodynamic studies of long-acting in vivo growth promotion of J2 fusion proteins and J18 fusion proteins: selecting SPF male SD rats with the body weight of 60g-80g and the age of 4 weeks. The hypophysis is removed under the clean condition of 2 weeks before the experiment, 2 weeks after the hypophysis removal operation is a recovery period, qualified healthy animals with the weight change of rats less than +/-10% of the weight before the operation are selected before administration, and the hypophysis removal rats are uniformly and randomly divided into 5 groups according to the weight, namely a model group, a J2 fusion protein low-dose group, a J2 fusion protein high-dose group, a J18 fusion protein low-dose group and a J18 fusion protein high-dose group; each group had 8. The administration mode is neck subcutaneous injection, and the J2 fusion protein is administered to the J2 fusion protein low-dose group according to the administration amount of 9.35 nmol/kg; the J18 fusion protein low-dose group is used for administering the J18 fusion protein according to the administration amount of 9.35 nmol/kg; the high-dose group of the J2 fusion protein administers the J2 fusion protein according to the administration amount of 56.1 nmol/kg; the high-dose group of the J18 fusion protein administers the J18 fusion protein according to the administration amount of 56.1 nmol/kg; vehicle administration to the model group; each group was dosed or administered vehicle once a week, co-dosed or administered vehicle 2 times as described above. After dosing, each rat was weighed at the same time each day, daily rat weight changes were calculated, the test was stopped after 14 days, and the rats were weighed. The weight gain (g) on a day after administration was the difference between the daily weight and the weight before administration for each animal. At the end of the experiment, dissection examination was performed to confirm that no pituitary residue remained in the sphenoid saddle region. The results of comparing the change in body weight gain of each group over 14 days are shown in fig. 3, where "2# -low" is the J2 fusion protein low dose group, "2# -high" is the J2 fusion protein high dose group, "18# -low" is the J18 fusion protein low dose group, and "18# -high" is the J18 fusion protein high dose group; 2# -low vs model group, p =0.0083;2# -high vs model group, p < 0.0001;18# -low vs model set, p =0.0030;18# -high vs model group, p =0.0003.
From the results of fig. 3, it can be seen that:
(1) Compared with a model group, the weight of a rat can be obviously improved by the J2 fusion protein and the J18 fusion protein under the conditions of low dose and high dose, and the fusion protein provided by the invention has good in vivo activity.
(2) The high dose group showed a greater magnitude of body weight gain than the low dose group.
(3) The J18 fusion protein high dose group had a more significant effect than the J2 high dose group.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen Konjing pharmaceutical Limited
<120> recombinant long-acting human growth hormone fusion protein, preparation method and application thereof
<130> PDI220116
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<170> PatentIn version 3.3
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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 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 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 Gln 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 ccaagcccgt gctggacagc gacggatctt tctttctgta cagcaccctg 540
accgtggaca agagcagatg gcagcagggc aatgtgttca gctgcagcgt gatgcacgag 600
gccctgcata atcactacac ccagaagagc 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 tgcgtggtgg tggatgtgag ccatgaagat 120
cccgaagtga agttcaactg gtacgtggac ggcgtggagg tgcacaatgc taaaaccaag 180
cccagagagg agcagtacaa cagcacctac agagtggtga gcgtgctgac cgtgctgcac 240
caagattggc tgaatggaaa ggagtacaag tgcaaggtga gcaacaaggc cctgcccgcc 300
cctattgaaa aaaccattag caaagctaag ggccagccca gagagcccca agtgtataca 360
agccctccca gcagggacga gctgaccaaa aatcaggtga gcctgagatg 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 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 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 Ile 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 Gln
245 250 255
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
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 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 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 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
260 265 270
Val Leu Thr Val Leu His Gln 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 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
305 310 315 320
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Arg Cys His
325 330 335
Val Lys Gly Phe Tyr Pro Ser Asp Ile 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 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 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 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 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 Ile 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 Gln 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 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 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 Gln Leu Ala
245 250 255
Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln
260 265 270
Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu
275 280 285
Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn
290 295 300
Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu
305 310 315 320
Pro Val Gln 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 Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly
355 360 365
Gln Ile Phe Lys Gln 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 Gln 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 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
305 310 315 320
Glu Pro Gln Val Tyr Thr Ser 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
Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
385 390 395 400
Cys Ser Val Tyr His Glu Ala Leu His Asn His Tyr Thr Gln 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 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 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 Gln Leu Ala
245 250 255
Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln
260 265 270
Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu
275 280 285
Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn
290 295 300
Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu
305 310 315 320
Pro Val Gln 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 Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly
355 360 365
Gln Ile Phe Lys Gln 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 Gln 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 ccagcaaaag agcaacctgg agctgctgag aatcagcctg 240
ctgctgatcc agagctggct ggagcctgtg caatttctga gaagcgtgtt cgccaatagc 300
ctggtgtacg gcgccagcga tagcaacgtg tatgatctgc tgaaagacct ggaggagggc 360
attcagacac tgatgggcag actggaggac ggcagcccta gaacaggaca gattttcaag 420
cagacctaca gcaagttcga caccaacagc cacaacgacg acgccctgct gaaaaactac 480
ggcctgctgt actgcttcag aaaggacatg gacaaggtgg agacattcct gagaatcgtg 540
cagtgcagaa gcgtggaggg cagctgtgga tttggaggag gaggaagcgg aggcggagga 600
tctggaggag gaggaagcgc tcctgaactg ctgggaggac ctagcgtgtt tctgtttcct 660
cccaaaccta aagacaccct gatgatcagc agaacccccg aggtgacctg cgtggtggtt 720
gatgtgtctc atgaagaccc cgaagtgaag ttcaactggt acgtggacgg cgtggaggtg 780
cacaatgcta aaaccaagcc cagagaggag cagtacaaca gcacctacag 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 tccctgagcc 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
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 gaggatctgc tcctgaactg 600
ctcggaggcc cttccgtgtt cctgtttcct ccaaagccta aggacaccct gatgatcagc 660
agaacccctg aagtgacctg cgtggtggtg gatgtgtccc acgaggatcc cgaagtgaag 720
ttcaattggt acgtggacgg cgtggaagtg cacaacgcca agaccaagcc tagagaggaa 780
cagtacaaca gcacctacag agtggtgtcc gtgctgaccg tgctgcacca ggattggctg 840
aacggcaaag agtacaagtg caaggtgtcc aacaaggccc tgcctgctcc tatcgagaaa 900
accatcagca aggccaaggg ccagcctagg gaaccccagg tttacacact gcctccaagc 960
agggacgagc tgaccaagaa tcaggtgtcc ctgcggtgtc acgtgaaggg cttctaccct 1020
tccgatatcg ccgtggaatg ggagagcaat ggccagcctg agaacaacta 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 (10)

1. An Fc mutant, comprising:
a first peptide stretch having mutations compared to the Fc fragment of wild-type IgG1 at the following positions:
at least one of 228 th bit, 229 th bit, 230 th bit, 366 th bit, 368 th bit, 395 th bit and 409 th bit, and 351 th bit, 428 th bit and 447 th bit; or
228 th bit, 229 th bit, 366 th bit, 368 th bit, 395 th bit and 409 th bit;
optionally, the first peptidyl fragment has mutations compared to the Fc fragment of wild-type IgG1 at the following positions:
1) 228 th, 229 th, 230 th, 366 th, 368 th, 395 th, 409 th and 447 th bits; or the like, or, alternatively,
2) 228 th, 229 th, 230 th, 351 th, 366 th, 368 th, 395 th, 409 th and 428 th bits; or the like, or, alternatively,
3) 228, 229, 230, 351, 366, 368, 395, 409, 428, and 447 bits;
optionally, the first peptide fragment has the following mutations compared to the Fc fragment of wild-type IgG 1:
1) P228Delete, C229Delete, P230 Delete, T366R, L368H, P395K, K447A and K409D or K409T; or the like, or, alternatively,
2) P228Delete, C229Delete, P230 Delete, L351S, T366R, L368H, P395K, M428Y and K409D or K409T; or the like, or, alternatively,
3) P228Delete, C229Delete, P230 Delete, L351S, T366R, L368H, P395K, M428Y, K447A and K409D or K409T; or
4) P228Delete, C229Delete, T366R, L368H, P395K and K409D or K409T;
optionally, further comprising:
a hinge region fragment of wild type IgG1, wherein the C end of the hinge region fragment of wild type IgG1 is connected with the N end of the first peptide segment;
optionally, the hinge region fragment of the wild type IgG1 has an amino acid sequence shown as SEQ ID NO. 41;
optionally, the Fc mutant has an amino acid sequence shown as any one of SEQ ID NO 3-7;
optionally, the Fc mutant is in monomeric form.
2. A fusion protein, comprising:
a second peptide segment comprising a functional region of a biologically active molecule;
a third peptide stretch comprising an Fc mutant having an amino acid sequence as set forth in SEQ ID NO 2 or as defined in claim 1, the second peptide stretch being linked to the third peptide stretch.
3. The fusion protein of claim 2, wherein the second peptide segment comprises a growth hormone, a growth hormone analog, a growth hormone functional region, or a growth hormone analog functional region, preferably a human growth hormone or a human growth hormone functional region;
optionally, the human growth hormone has an amino acid sequence as set forth in SEQ ID NO 1 or at least 90% identity thereto;
optionally, the N-terminus of the second peptide fragment is linked to the C-terminus of the third peptide fragment; alternatively, the first and second electrodes may be,
and the C end of the second peptide segment is connected with the N end of the third peptide segment.
4. The fusion protein of claim 2 or 3, further comprising a linker peptide disposed between the second and third peptide segments;
optionally, the N-terminus of the linker peptide is linked to the C-terminus of the third peptide fragment, and the C-terminus of the linker peptide is linked to the N-terminus of the second peptide fragment; alternatively, the first and second electrodes may be,
the N end of the connecting peptide is connected with the C end of the second peptide segment, and the C end of the connecting peptide is connected with the N end of the third peptide segment;
optionally, the amino acid sequence of the linker 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;
optionally, the connecting peptide has an amino acid sequence shown in any one of SEQ ID NO 8-10.
5. The fusion protein of claim 2, wherein the fusion protein has an amino acid sequence represented by any one of SEQ ID NOs 24 to 31.
6. A nucleic acid molecule encoding the Fc mutant of claim 1 or the fusion protein of any one of claims 2 to 5;
optionally, the nucleic acid molecule is DNA.
7. An expression vector carrying the nucleic acid molecule of claim 6;
optionally, the expression vector is a eukaryotic expression vector;
preferably, the expression vector is a lentiviral vector.
8. A recombinant cell, wherein the recombinant cell comprises:
carrying the nucleic acid molecule of claim 6; or the like, or, alternatively,
expressing the Fc mutant according to claim 1 or the fusion protein according to any one of claims 2 to 5;
optionally, the recombinant cell is obtained by introducing the expression vector of claim 7 into a host cell;
optionally, the recombinant cell is a eukaryotic cell;
preferably, the recombinant cell is a mammalian cell.
9. A pharmaceutical composition, comprising:
the fusion protein of any one of claims 2 to 5;
optionally, the pharmaceutical composition further comprises pharmaceutically acceptable excipients;
optionally, the pharmaceutical composition is in the form of an injection;
optionally, the route of administration of the pharmaceutical composition comprises subcutaneous injection or intravenous injection.
10. Use of the fusion protein according to any one of claims 2 to 5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7, the recombinant cell according to claim 8, the pharmaceutical composition according to claim 9 for the preparation of a medicament for the treatment and/or prevention of a disease associated with growth hormone abnormalities;
optionally, the disease associated with abnormal growth hormone comprises at least one selected from the group consisting of:
childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, turner's syndrome, prader-willi syndrome, renal failure, diseases caused by dissimilatory states during chemotherapy treatment and AIDS treatment, intrauterine growth retardation.
CN202210395423.2A 2021-11-26 2022-04-14 Recombinant long-acting human growth hormone fusion protein and preparation method and application thereof Pending CN115873127A (en)

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CN109517059B (en) * 2011-06-30 2023-03-28 中外制药株式会社 Heterodimerised polypeptides
ES2876009T3 (en) * 2012-12-27 2021-11-11 Chugai Pharmaceutical Co Ltd Heterodimerized polypeptide
EP3842455A1 (en) * 2014-01-15 2021-06-30 F. Hoffmann-La Roche AG Fc-region variants with improved protein a-binding
EP3583126A1 (en) * 2017-02-17 2019-12-25 Sanofi Multispecific binding molecules having specificity to dystroglycan and laminin-2
SG11202006420TA (en) * 2018-01-10 2020-08-28 Denali Therapeutics Inc Transferrin receptor-binding polypeptides and uses thereof
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