CN107286233B - Low-pain nerve growth factor mutant - Google Patents
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Abstract
The invention relates to a low-pain nerve growth factor mutant, belonging to the field of biological pharmacy. The low-pain nerve growth factor mutant is shown in a sequence table SEQ ID No: 3 to SEQ ID No: 49 with a sequence of any one of seq id no. The invention has the advantages that: the nerve growth factor mutant has low pain and can relieve pain side effect.
Description
Technical Field
The invention relates to a low-pain nerve growth factor mutant, belonging to the field of biological pharmacy.
Background
Pain can be classified into nociceptive pain and neuropathic pain according to the neurophysiological mechanism, the former is directly caused by nociceptive stimulation and is related to tissue injury or inflammatory reaction, also called inflammatory pain; the latter is chronic pain directly caused by injury or disease of the somatosensory nervous system.
Nerve Growth Factor (NGF) is the first neurotrophic Factor discovered by Italian scientist Levi-Montlcini in 1953 in mouse sarcoma cells, and NGF is a Nerve cell Growth regulator with the biological functions of both neuron nutrition and promotion of neurite outgrowth, and has important regulation and control effects on development, differentiation, Growth, regeneration and expression of functional characteristics of central and peripheral neurons. NGF comprises three subunits, alpha, beta and gamma, the beta subunit is the active region and is formed by combining two single chains through non-covalent bonds. Levi-Montalcini received the Nobel prize for the discovery of NGF. At present, a plurality of NGF products are sold on the market at home and abroad, and are mainly used for treating the dysplasia of the nervous system clinically, including amblyopia, neuroma, various nerve injuries, pathological changes of the nervous system and other diseases.
NGF is present in a variety of species, abundant in male mouse submaxillary gland, bovine seminal plasma, snake venom, guinea pig prostate and human placental tissue. Wherein the amino acid sequence homology of mouse NGF and human NGF reaches 90%. Considering the species difference of the mouse NGF applied to human bodies, the potential risk of pathogenic factors of mice used as raw materials and the limitation of human placenta tissue raw materials, the development of the genetic engineering technology for preparing the recombinant human NGF (rhNGF) to replace the extracted mouse NGF and human NGF has good application prospect.
NGF matured in vivo exists as homodimers, each peptide chain comprising 120 amino acids. The human NGF gene is located on the short arm of chromosome 1, the complete NGF exon is composed of 241 amino acids, and is generally called prepro NGF precursor, a signal peptide of prepro NGF precursor is cleaved in endoplasmic reticulum to form a pro NGF precursor (223 amino acids), the pro NGF precursor exists in homodimer form in endoplasmic reticulum and then is transferred to golgi, the precursor is partially digested by Furin to form mature NGF dimer (monomer contains 120 amino acids), and the mature NGF dimer is transferred to the outside of cells, and meanwhile, part of uncleaved pro NGF precursor is secreted to the outside of cells.
Although recombinant humanized NGF avoids some potential pathogenic risks, the practical use process still has great problems: 1) the maintenance of NGF biological activity, which, like other proteins, is dependent on its secondary and tertiary structure, is particularly important during preparation, purification, storage and administration; 2) NGF causes severe pain during use and is partially intolerant to some patients, thus causing partial limitation in use. NGF is involved in the pathophysiological processes of pain, causes pain by affecting the release of inflammatory mediators, the opening of ion channels and the promotion of nerve fiber growth, and participates in the development of pain by regulating ion channels and molecular signals. It is speculated by the scholars that NGF may also cause pain by promoting the expression of pain-causing substances and may alter the sprouting and regeneration of neurons after bodily injury. The current research finds that: in humans, the maximum dose that does not cause hyperalgesia is 0.03 μ g/kg (Pettyet al., 1994-29). However, such low doses limit the use of NGF and also limit the expansion of its indications, such as for the central nervous system.
Therefore, in order to avoid the above problems, it is desired to find a recombinant hNGF that can alleviate the side effects of pain or even be painless, thereby increasing the dosage and the population of subjects, and providing the possibility of expanding the indications and applying to the central nervous system.
Disclosure of Invention
The invention aims to provide a group of nerve growth factor mutants with low pain or even no pain, namely recombinant hNGF.
In order to achieve the purpose, the invention adopts the following technical scheme:
the low-pain nerve growth factor mutant is shown in a sequence table SEQ ID No: 3 to SEQ ID No: 49 according to any one of the preceding claims.
The nucleotide sequence of the low-pain nerve growth factor mutant is coded.
The nucleotide sequence comprises genomic DNA, cDNA, synthetic DNA and RNA. Preferably DNA, more preferably cDNA of the coding sequence. The invention also encompasses nucleotide sequences encoding enzymes or proteins having the specific properties as defined herein. The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or a polynucleotide sequence and variants, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be a double-stranded or single-stranded (whether representing a sense or antisense strand) nucleic acid molecule. The nucleotide sequence encoding the mutant low-pain nerve growth factor can be prepared by synthesis by well-established standard methods, and oligonucleotides are synthesized on an automatic DNA synthesizer, amplified, and enzymatically ligated into an appropriate vector.
The low-pain recombinant hNGF mutant is obtained by carrying out single-point or multi-point mutation on the basis of a wild type hNGF sequence (SEQ ID No: 2), and preferably the following NGF mutants are selected, and the amino acid sequences of the NGF mutants are as follows:
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (wild type hNGF amino acid sequence: SEQID No: 2)
ATGTCCATGTTGTTCTACACTCTGATCACAGCTTTTCTGATCGGCATACAGGCGGAACCACACTCAGAGAGCAATGTCCCTGCAGGACACACCATCCCCCAAGCCCACTGGACTAAACTTCAGCATTCCCTTGACACTGCCCTTCGCAGAGCCCGCAGCGCCCCGGCAGCGGCGATAGCTGCACGCGTGGCGGGGCAGACCCGCAACATTACTGTGGACCCCAGGCTGTTTAAAAAGCGGCGACTCCGTTCACCCCGTGTGCTGTTTAGCACCCAGCCTCCCCGTGAAGCTGCAGACACTCAGGATCTGGACTTCGAGGTCGGTGGTGCTGCCCCCTTCAACAGGACTCACAGGAGCAAGCGGTCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGCAAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCGGGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACACCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGTGTGCTCAGCAGGAAGGCTGTGAGAAGAGCCTGA (DNA sequence of wild type hNGF SEQ ID NO: 1)
K115A: the amino acid sequence is SEQ ID No: 3;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRAAVRRA (amino acid sequence: SEQ ID No: 3)
R114A: the amino acid sequence is SEQ ID No: 4;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSAKAVRRA (amino acid sequence: SEQ ID No: 4)
R119A: the amino acid sequence is SEQ ID No: 5;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRAA (amino acid sequence: SEQ ID No: 5)
L112A: the amino acid sequence is SEQ ID No: 6;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVASRKAVRRA (amino acid sequence: SEQ ID No: 6)
S113M: the amino acid sequence is SEQ ID No: 7;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLMRKAVRRA (amino acid sequence: SEQ ID No: 7)
D105N: the amino acid sequence is SEQ ID No: 8;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRINTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 8)
D105A: the amino acid sequence is SEQ ID No: 9;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIATACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 9)
R103K: the amino acid sequence is SEQ ID No: 10;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIKIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 10)
R103A: the amino acid sequence is SEQ ID No: 11;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIAIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 11)
A97E: the amino acid sequence is SEQ ID No: 12;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQEAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 12)
M92E: the amino acid sequence is SEQ ID No: 13;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTEDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 13)
K88M: the amino acid sequence is SEQ ID No: 14;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVMALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 14)
K88A: the amino acid sequence is SEQ ID No: 15;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVAALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 15)
H84A: the amino acid sequence is SEQ ID No: 16;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTATFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 16)
Y79A: the amino acid sequence is SEQ ID No: 17;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSACTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 17)
T81A: the amino acid sequence is SEQ ID No: 18;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCATTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 18)
N77A: the amino acid sequence is SEQ ID No: 19;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWASYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 19)
H75A: the amino acid sequence is SEQ ID No: 20;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKAWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 20)
K74A: the amino acid sequence is SEQ ID No: 21;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSAHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 21)
D72A: the amino acid sequence is SEQ ID No: 22;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIASKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 22)
R69A: the amino acid sequence is SEQ ID No: 23;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCAGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 23)
R59A: the amino acid sequence is SEQ ID No: 24;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCADPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 24)
R59K: the amino acid sequence is SEQ ID No: 25;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCKDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 25)
K57A: the amino acid sequence is SEQ ID No: 26;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETACRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 26)
E55A: the amino acid sequence is SEQ ID No: 27;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFATKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 27)
Y52F: the amino acid sequence is SEQ ID No: 28;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQFFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 28)
K50A: the amino acid sequence is SEQ ID No: 29;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFAQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 29)
S47A: the amino acid sequence is SEQ ID No: 30, of a nitrogen-containing gas;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNAVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 30)
N46K: the amino acid sequence is SEQ ID No: 31;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINKSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 31)
E41A: the amino acid sequence is SEQ ID No: 32, a first step of removing the first layer;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGAVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 32)
K32A: the amino acid sequence is SEQ ID No: 33;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIAGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 33)
K32R: the amino acid sequence is SEQ ID No: 34;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIRGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 34)
K34A: the amino acid sequence is SEQ ID No: 35;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGAEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 35)
E35A: the amino acid sequence is SEQ ID No: 36;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKAVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 36)
I31N: the amino acid sequence is SEQ ID No: 37;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDNKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 37)
D30N: the amino acid sequence is SEQ ID No: 38;
SSSHPIFHRGEFSVCDSVSVWVGDKTTATNIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 38)
T27E: the amino acid sequence is SEQ ID No: 39;
SSSHPIFHRGEFSVCDSVSVWVGDKTEATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 39)
T26K: the amino acid sequence is SEQ ID No: 40;
SSSHPIFHRGEFSVCDSVSVWVGDKKTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 40)
K25Q: the amino acid sequence is SEQ ID No: 41;
SSSHPIFHRGEFSVCDSVSVWVGDQTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 41)
D24A: the amino acid sequence is SEQ ID No: 42;
SSSHPIFHRGEFSVCDSVSVWVGAKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 42)
D16A: the amino acid sequence is SEQ ID NO: 43;
SSSHPIFHRGEFSVCASVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 43)
S13M: the amino acid sequence is SEQ ID No: 44;
SSSHPIFHRGEFMVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 44)
F12A: the amino acid sequence is SEQ ID No: 45, a first step of;
SSSHPIFHRGEASVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 45)
E11A: the amino acid sequence is SEQ ID No: 46;
SSSHPIFHRGAFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 46)
H8A: the amino acid sequence is SEQ ID No: 47;
SSSHPIFARGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 47)
P5A: the amino acid sequence is SEQ ID No: 48;
SSSHAIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 48)
H4D: the amino acid sequence is SEQ ID No: 49.
SSSDPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 49)
The NGF mutant can be obtained by mutating one of the amino acid mutation sites, or can be obtained by mutating two, three or even more sites simultaneously. Such as: based on the amino acid sequence of wild hNGF, NGF mutants with simultaneous mutation of D30N and T27E, NGF mutants with simultaneous mutation of three sites of K34A, E35A and I31N, and the like can be obtained.
An expression vector containing the nucleotide sequence (namely the nucleotide sequence for coding the low-pain nerve growth factor).
The expression vector is selected from the group consisting of a DNA vector and a viral vector.
The DNA vector is selected from the group consisting of a DNA plasmid vector, a liposome bound thereto, a molecular conjugate bound thereto, and a polymer bound thereto; preferably, the DNA plasmid vector is a eukaryotic cell expression vector, a prokaryotic cell expression vector or a filamentous fungus expression vector; the viral vector is selected from the group consisting of adeno-associated viral vectors, lentiviral vectors, adenoviral vectors, and baculovirus vectors. More preferably, the DNA plasmid vector is a eukaryotic cell expression vector; the viral vector is selected from the group consisting of adeno-associated viral vectors, lentiviral vectors, and adenoviral vectors.
The expression vector is transformed into a host cell, and the recombinant cell obtained by culturing is expressed to obtain the low-pain nerve growth factor mutant.
A host cell containing the expression vector.
The host cell is a mammalian cell, a yeast cell, an insect cell, an escherichia coli, a filamentous fungus, or a plant cell. Preferably, the host cell is a mammalian cell.
The mammalian cell is Chinese hamster ovary cell, human embryonic kidney 293 cell, COS cell or Hela cell. The insect cells are SF9 cells, SF21 cells and SF900 cells.
A pharmaceutical composition comprising a pharmaceutically acceptable excipient, and one or more of the aforementioned low-pain nerve growth factor mutant, the aforementioned expression vector and the aforementioned host cell.
The medicine of the invention can be prepared into various forms such as injection, capsule, tablet or powder, and the medicines of the various forms can be prepared according to the conventional method in the pharmaceutical field.
The pharmaceutical composition is preferably an injection solution comprising a pharmaceutically acceptable excipient and the aforementioned low-pain nerve growth factor mutant.
If necessary, one or more pharmaceutically acceptable carriers may be further added to the above pharmaceutical composition, and the carriers include diluents, stabilizers, surfactants, preservatives and the like which are conventional in the pharmaceutical field.
The low-pain nerve growth factor mutant is used for preparing a medicament for treating nervous system diseases.
The use of the low-pain nerve growth factor mutant for preparing a medicament for effectively reducing weight.
The application of the low-pain nerve growth factor mutant in preparing a medicament for treating ophthalmic related diseases.
The low-pain nerve growth factor mutant is used for preparing a medicine for treating diseases related to the reproductive system.
The low-pain nerve growth factor mutant is used for preparing a medicament for treating autoimmune related diseases.
The low-pain nerve growth factor mutant is used for preparing a medicament for treating bone related diseases.
Sequence listing SEQ ID No: 3 to SEQ ID No: 49 for preparing a low-pain nerve growth factor preparation.
Sequence listing SEQ ID No: 3 to SEQ ID No: 49 for preparing a painless nerve growth factor preparation.
The invention has the advantages that: compared with the wild nerve growth factor in the prior art, the low-pain nerve growth factor disclosed by the invention can relieve pain side effects even has no pain, can increase the compliance and tolerance of a patient to the nerve growth factor, can increase the use dosage and the tested population, and provides possibility for expanding indications and applying to a central nervous system.
The invention is further described with reference to the following drawings and detailed description, but the invention is not limited thereto, and equivalents in the art made in accordance with the disclosure are intended to fall within the scope of the invention.
Drawings
FIG. 1 is the SDS PAGE result of wild-type hNGF purified by Superdex75 column of example 4
FIG. 2 shows the results of the pain threshold measurement of the mice administered with the short-term drug of example 6
FIGS. 3(A) and 3(B) are the results of measuring the pain threshold of mice administered for a long period of time in example 6
FIG. 4 shows the experimental results of mouse behavioural experiments in example 7, in which mutant and wild type hNGF were injected, respectively, to observe the leg-lifting maintenance time of the mouse
Detailed Description
Example 1 plasmid construction of wild-type h NGF and its mutants
1. Construction of DNA sequence expression plasmid of wild type hNGF
A DNA sequence of wild-type hNGF (SEQ ID NO: 1 of the sequence Listing) was synthesized, a target sequence was PCR-amplified using primers (F: GGAATTCATGTCCATGTTG (SEQ ID NO: 50 of the sequence Listing), R: CAAGCTTTCAGGCTCTTCT (SEQ ID NO: 51 of the sequence Listing), digested with Ecori (NEB # R0101S), and the digested product was digested with HindIII (NEB # R0104S) twice, and pcDNA3.1(-) expression vector was digested with the same method, the digested vector and the PCR-amplified target fragment were subjected to agarose gel electrophoresis, the target fragment was excised, and the digested vector and target DNA fragment were recovered with a DNA gel recovery kit (TIANGEN, # DP209-03), and then ligated at 16 ℃ for 1 hour using a DNA ligase kit (Takara/6022) to complete plasmid construction of wild-type hNGF.
2. Construction of DNA sequence expression plasmid of hNGF mutant
Also in the above process, we have synthesized the coding sequences of SEQ ID NOs: 3 to SEQ ID No: 49, and plasmids containing the coding genes are respectively constructed.
Example 2 plasmid transformation and extraction of hNGF and mutants thereof
1. Transformation of
hNGF and its mutant plasmids constructed in example 1 above were transformed by heat shock, Top10 competent cells (Tiangen/CB 104-02) were removed from a refrigerator at-70 deg.C and immediately thawed on ice, and 50ul was used for transformation. Add 2ul plasmid into it, mix gently, ice-bath for 30 min. The tube was dry-bathed at 42 ℃ for 90s without shaking, and immediately removed and placed on ice for 2 min. Adding non-resistant LB/SOC culture medium 500ul, 150rpm/min, and shake culturing at 37 deg.C for 45 min. The liquid in the centrifuge tube was poured onto an LB plate and spread out evenly. After the plate is dried, the plate is placed in an incubator upside down for culturing for 16 h.
2. Plasmid major grape
And (3) selecting a single colony obtained in the conversion step, inoculating the single colony in 500ul of LB liquid culture medium, culturing at 37 ℃ for 7h, and simultaneously detecting and sequencing the bacterial liquid. And (3) carrying out mass bacteria shaking on the bacteria liquid with the correct sequencing, inoculating 500ul of the bacteria liquid into 500ml of LB culture medium, and culturing for 16h at 37 ℃. The overnight cultured broth was collected by centrifugation at 4 ℃ and centrifuged at 6000Xg for 10 minutes, and the supernatant was sufficiently discarded to obtain a plasmid by mass extraction using a plasmid mass extraction kit (purchased from QIAGEN, cat. No. 12163), and the concentration was measured for use.
Example 3 expression of wild-type hNGF and mutants thereof
The wild type hNGF and its mutant plasmid obtained in example 2 above were transfected into 293F cells, and the expression supernatant was collected and quantified on the fourth day after transfection.
The experimental steps are as follows:
1. the day before transfection, 293F cells, 0.5X 10 cells were seeded6900 ml/ml, 300 ml/bottle.
2. Cell density on day of transfectionAbout 1.0X 106The activity rate is more than 99 percent.
3. Transfection: taking 36ml of cell culture medium to a 125ml culture bottle; adding 360ug of plasmid, and mixing; then 1080ug PEI is added and mixed evenly. Standing for 15 minutes at room temperature; mixing with cells, about 12.3 ml/bottle; 37 ℃ and 8% CO2Cultivation was carried out at 120 RPM.
4. Cell supernatants were collected the fourth day after transfection and centrifuged at 10000g for 20 min.
5. And collecting the supernatant, and filtering by 0.45um to obtain the protein supernatant of the wild type hNGF and the mutant thereof.
6. And (4) detecting by SDS-PAGE, and quantifying silver nitrate staining.
Example 4 purification of wild-type hNGF and mutants thereof
The protein supernatant of NGF and its mutants obtained in example 3 above was purified.
1. Cation exchange chromatography: the protein supernatant of wild-type hNGF and mutants thereof was first adjusted to pH4.0 using acetic acid and water. The CM Sepharose FF chromatographic column is fully equilibrated with 0.05mol/L acetate buffer solution (pH4.0) and then loaded, after loading is completed, the column is washed to a base line by an equilibration solution, a mixed peak is eluted to the base line by 0.05mol/L Tris-HC1(pH9.0) buffer solution, then 0.05mol/L Tris-HC1 and 0.05mol/L Tris-HC1-0.4mol/L NaCl (pH9.0) are used for gradient elution, a target peak is collected according to the ultraviolet absorption condition, the collection is started when the number displayed on an ultraviolet detector starts to rise, and the collection of the target protein peak is stopped when the number is reduced to the base line.
2. Hydrophobic chromatography: butyl Sepharose 4FF column was equilibrated well with 0.02mol/L phosphate (pH6.8) -1.5mol/L NaCl buffer. And (2) adding sodium chloride solid into the target peak feed liquid collected in the step (1) to ensure that the final concentration of sodium chloride in the feed liquid is 1.5mol/L, loading the sample at the speed of 120cm/h after the sodium chloride is fully dissolved, flushing the sample to a baseline by using a balance liquid after the loading is finished, and eluting by using 0.02mol/L phosphate (pH6.8) to collect a target peak.
3. Gel exclusion chromatography: and (3) fully balancing the Superdex75 prep grade chromatographic column by using 0.05mol/L phosphate-0.15 mol/L sodium chloride buffer solution with pH6.8, loading the target peak collected in the step/2, starting to collect when the number displayed on an ultraviolet detector starts to rise from a baseline, and stopping collecting the target protein peak when the number decreases to the baseline.
SDS PAGE of wild type hNGF purified by Superdex75 column is shown in FIG. 1, which indicates that the purity of the prepared NGF is high. The collected wild type hNGF and its mutant target protein peak samples were concentrated to 0.4mg/ml using an ultrafiltration tube and stored at 4 ℃ for subsequent experiments.
Example 5 chick embryo assay for Activity of wild-type hNGF and mutants thereof
1. Method for measuring activity of wild type hNGF and mutant thereof by chick embryo dorsal root ganglion method
The wild-type hNGF obtained in example 4 above and mutant samples thereof were diluted: solution A: 6ng of the extracted wild type hNGF and mutant samples thereof are dissolved by adding 1ml of serum-free DMEM culture solution; and B, liquid B: taking 50 mu l of the A liquid, and adding 4.95ml of serum-free DMEM culture solution; and C, liquid C: 60. mu.l of the B solution was added to 2.94ml (total amount: 3ml) of serum-free DMEM medium to give a final concentration (3 AU/ml). A. And B liquid is diluted in a centrifuge tube, C liquid is in a cell bottle, C liquid is taken as a No. 1 bottle, and then the solution is diluted by 3 times in a gradient manner: no. 2, No. 3, No. 4, No. 5 and No. 6 solutions to be tested. Each solution to be tested was added to 1 flask, 2 ml/flask. Serum-free DMEM culture solution is used as a blank control, and a standard substance purchased from China food and drug testing research institute is used as a positive control (reference substance). Adding 8-day-old chick embryo dorsal root ganglion, and placing in 5% CO2And in a saturated humidity incubator at 37 ℃ for 24 hours, the results were observed.
The NGF content per ml of the sample to be tested at the best growth was taken as 1 Activity Unit (AU). The titer was calculated as the endpoint of the determination, starting from the dilution at which the negative control result appeared and counting back to the 3 rd and 4 th dilutions from which the best growth was obtained. The reference product is a standard product purchased from a hospital center, and the content per unit is 1000 AU.
The calculation formula of the NGF specific activity is as follows:
specific activity (AU/mg) of the sample to be tested is reference activity (AU/ml) x [ pre-dilution multiple of sample x activity (AU/ml) at corresponding dilution point of reference)/actual activity (AU/ml) of reference substance ]
The activity test results show that the low-pain hNGF mutant retains the activity of wild type hNGF.
Example 6 determination of whether NGF and its mutants cause pain (pain threshold)
The experimental principle is as follows: screening qualified mice with normal hyperalgesia response, injecting a certain dose of NGF sample (wild type or mutant thereof), measuring the mouse curved paw response pain threshold value through mechanical stimulation, and performing statistical analysis to finally determine whether the sample causes the mouse hyperalgesia.
6-1, short-term pain-causing Observation
First, experimental material
Dynamic plantar tactile device, Italy/Ugo Basile, model 37450.
Second, the experimental contents
1. Screening of qualified mice
SPF grade CD-1 mice were ordered, male-sized, and 30-35g in weight.
The experimental animals with mean threshold value of both feet between 7.5 and 10 and threshold value P of the left and right feet of the same mouse, P value >0.05, were screened as qualified mice using dynamic plantar tactile instrument [ Italy/Ugo Basile, model 37450 ].
The test groups are divided into 10 groups according to different samples and administration doses.
2. NGF sample dosing design
2.1 wild-type sample analgesic dose screening
Preparing the medicine: positive control NGF wild-type samples, NGF S47A mutant, and NGF I31N mutant samples were diluted with sample stock (50mM PB,150mM NaCl, pH 6.8).
Blank control: is an NGF sample stock.
Administration mode and dose: the subcutaneous administration of 20ul of the left and right feet of the mouse is respectively carried out;
the minimum dose for short-term administration was 1.25. mu.g/mouse.
3. Threshold of pain determination
Mechanical threshold measurements were performed at 1 hour and 2 hours after dosing, respectively, and values were recorded for short-term dosing (over 2 hours) observations.
4. Statistical analysis of results
Graph drawing and result statistical analysis are carried out by adopting GraphPad Prism software, whether mechanical threshold values of various dose groups are different from those of a control group is compared, and pain-causing performance of a wild type sample and a mutant sample is analyzed.
As can be seen from FIG. 2, when the lowest dose of 1.25 μ g/mouse is administered, the control mice have no pain, while the wild-type positive control group has a pain threshold value of significantly less than 5 within 1 hour, and the pain is significant; and the pain threshold of each mutant experimental group is about 6 to 7, and compared with the positive control group, the pain is obviously reduced.
6-2 Long-term pain observation
Except for setting 3 doses in this experiment: the pain threshold was measured once a day for 3 weeks at 0.2. mu.g/patient, 0.5. mu.g/patient, 1.25. mu.g/patient, and the rest was performed as in 6-1 "short-term pain condition observation".
The results are shown in FIGS. 3(A) and 3(B), indicating that: three doses of wild-type NGF over 17 days showed a progressive decrease in pain threshold and a progressive appearance of pain sensation. The experimental results of the low-dose and high-dose groups of the S47A mutant and the I31N mutant show that the pain threshold of the groups of the other doses is basically maintained above 6 except that the pain threshold of the group of the high-dose mutant of the S47A mutant is obviously reduced to cause pain within 14 days, and the effect of obviously reducing the pain is realized compared with the wild type.
Example 7 behavioural test of whether wild-type hNGF and its mutants cause pain
Wild type hNGF and mutant samples thereof are administered to mouse joints, and whether the samples cause pain or not is examined by observing leg lifting time and times of the mice by behaviours.
Content of the experiment
1. Mouse ordering
SPF grade CD-1 mice were ordered, male-sized, and 30-35g in weight. The groups are divided into an experimental group, a blank control group (referred to as a control group for short) and a positive control group, each group is divided into three groups according to the dosage, and each group randomly selects 6 animals.
2. Dosage and time of administration
2.1 dosage administration
Positive control: wild-type hNGF was diluted to 1.25. mu.g/10 ul using sample stock (50mM PB,150mM NaCl, pH 6.8);
experimental groups: the preparation method of the mutant medicament is the same as that of a positive control, and the NGF I31N mutant is diluted to be 1.25 mu g/10 ul;
blank control: physiological saline.
2.2 modes of administration
The mice were injected with drugs in the hind leg joints, 10ul of each joint cavity was administered.
2.3 administration time:
each dose group was given a single continuous 3-4 days. I.e., 10 am the first day, and the same time points on days 2, 3, and 4 thereafter.
3. Behavioral observations
The observation was performed 2 and 4 hours after the same day of administration for each experimental group and control group, and the observation was performed at the same time point on the 2 nd, 3 rd and 4 th days of administration.
Observation indexes are as follows: the cumulative time for raising the legs was calculated based on the number of times the mouse spontaneously raised the legs within 2 minutes and the time (seconds) for each leg raising.
4. Statistical analysis of results
Leg lifting maintenance time is analyzed by adopting GraphPad Prism software two-factor variance analysis, and whether different samples cause pain or not is analyzed in a comparative way.
The results of the experiment are shown in FIG. 4: the wild type hNGF group can cause obvious pain at each detection time point after the injection of the drug, the I31N mutant experimental group has no obvious continuous administration pain phenomenon, and chi-square analysis that the cumulative time of leg lifting maintenance is below 1 second shows that the positive control group and the experimental group have obvious difference at each detection time point, which shows that the I31N mutant can obviously relieve the pain reaction of the drug injection.
Sequence listing
<110> Shutaishen (Beijing) biopharmaceutical corporation
<120> Low-pain nerve growth factor mutant
<130>
<160>51
<170>PatentIn version 3.5
<210>1
<211>726
<212>DNA
<213> DNA sequence of wild-type hNGF
<400>1
atgtccatgt tgttctacac tctgatcaca gcttttctga tcggcataca ggcggaacca 60
cactcagaga gcaatgtccc tgcaggacac accatccccc aagcccactg gactaaactt 120
cagcattccc ttgacactgc ccttcgcaga gcccgcagcg ccccggcagc ggcgatagct 180
gcacgcgtgg cggggcagac ccgcaacatt actgtggacc ccaggctgtt taaaaagcgg 240
cgactccgtt caccccgtgt gctgtttagc acccagcctc cccgtgaagc tgcagacact 300
caggatctgg acttcgaggtcggtggtgct gcccccttca acaggactca caggagcaag 360
cggtcatcat cccatcccat cttccacagg ggcgaattct cggtgtgtga cagtgtcagc 420
gtgtgggttg gggataagac caccgccaca gacatcaagg gcaaggaggt gatggtgttg 480
ggagaggtga acattaacaa cagtgtattc aaacagtact tttttgagac caagtgccgg 540
gacccaaatc ccgttgacag cgggtgccgg ggcattgact caaagcactg gaactcatat 600
tgtaccacga ctcacacctt tgtcaaggcg ctgaccatgg atggcaagca ggctgcctgg 660
cggtttatcc ggatagatac ggcctgtgtg tgtgtgctca gcaggaaggc tgtgagaaga 720
gcctga 726
<210>2
<211>120
<212>PRT
<213> amino acid sequence of wild-type hNGF
<400>2
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
6570 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>3
<211>120
<212>PRT
<213> genus, race: NGF mutant K115A
<400>3
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly LysGln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Ala Ala Val Arg Arg Ala
115 120
<210>4
<211>120
<212>PRT
<213> genus, race: NGF mutant R114A
<400>4
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Ala Lys Ala Val Arg Arg Ala
115 120
<210>5
<211>120
<212>PRT
<213> genus, race: NGF mutant R119A
<400>5
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Ala Ala
115 120
<210>6
<211>120
<212>PRT
<213> genus, race: NGF mutant L112A
<400>6
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Ala
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>7
<211>120
<212>PRT
<213> genus, race: NGF mutant S113M
<400>7
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Met Arg Lys Ala Val Arg Arg Ala
115 120
<210>8
<211>120
<212>PRT
<213> genus, race: NGF mutant D105N
<400>8
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asn Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>9
<211>120
<212>PRT
<213> genus, race: NGF mutant D105A
<400>9
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Ala Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>10
<211>120
<212>PRT
<213> genus, race: NGF mutant R103K
<400>10
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
15 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Lys Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>11
<211>120
<212>PRT
<213> genus, race: NGF mutant R103A
<400>11
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Ala Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>12
<211>120
<212>PRT
<213> genus, race: NGF mutant A97E
<400>12
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu ValMet Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Glu Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>13
<211>120
<212>PRT
<213> genus, race: NGF mutant M92E
<400>13
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Glu Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>14
<211>120
<212>PRT
<213> genus, race: NGF mutant K88M
<400>14
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
5055 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Met Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>15
<211>120
<212>PRT
<213> genus, race: NGF mutant K88A
<400>15
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Ala Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>16
<211>120
<212>PRT
<213> genus, race: NGF mutant H84A
<400>16
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr Ala Thr Phe Val LysAla Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>17
<211>120
<212>PRT
<213> genus, race: NGF mutant Y79A
<400>17
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Ala Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>18
<211>120
<212>PRT
<213> genus, race: NGF mutant T81A
<400>18
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Ala Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>19
<211>120
<212>PRT
<213> genus, race: NGF mutant N77A
<400>19
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Ala Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>20
<211>120
<212>PRT
<213> genus, race: NGF mutant H75A
<400>20
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys Ala Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>21
<211>120
<212>PRT
<213> genus, race: NGF mutant K74A
<400>21
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Ala His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>22
<211>120
<212>PRT
<213> genus, race: NGF mutant D72A
<400>22
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Ala Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>23
<211>120
<212>PRT
<213> genus, race: NGF mutant R69A
<400>23
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Ala Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>24
<211>120
<212>PRT
<213> genus, race: NGF mutant R59A
<400>24
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Ala Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>25
<211>120
<212>PRT
<213> genus, race: NGF mutant R59K
<400>25
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Lys Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>26
<211>120
<212>PRT
<213> genus, race: NGF mutant K57A
<400>26
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 4045
Phe Lys Gln Tyr Phe Phe Glu Thr Ala Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>27
<211>120
<212>PRT
<213> genus, race: NGF mutant E55A
<400>27
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Ala Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>28
<211>120
<212>PRT
<213> genus, race: NGF mutant Y52F
<400>28
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Phe Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>29
<211>120
<212>PRT
<213> genus, race: NGF mutant K50A
<400>29
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Ala Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>30
<211>120
<212>PRT
<213> genus, race: NGF mutant S47A
<400>30
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ala Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 9095
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>31
<211>120
<212>PRT
<213> genus, race: NGF mutant N46K
<400>31
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Lys Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>32
<211>120
<212>PRT
<213> genus, race: NGF mutant E41A
<400>32
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Ala Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>33
<211>120
<212>PRT
<213> genus, race: NGF mutant K32A
<400>33
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Ala
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>34
<211>120
<212>PRT
<213> genus, race: NGF mutant K32R
<400>34
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Arg
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>35
<211>120
<212>PRT
<213> genus, race: NGF mutant K34A
<400>35
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Ala Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>36
<211>120
<212>PRT
<213> genus, race: NGF mutant E35A
<400>36
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu PheSer Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Ala Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>37
<211>120
<212>PRT
<213> genus, race: NGF mutant I31N
<400>37
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Asn Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>38
<211>120
<212>PRT
<213> genus, race: NGF mutant D30N
<400>38
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asn Ile Lys
20 2530
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>39
<211>120
<212>PRT
<213> genus, race: NGF mutant T27E
<400>39
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Glu Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>40
<211>120
<212>PRT
<213> genus, race: NGF mutant T26K
<400>40
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Lys Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn ProVal
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>41
<211>120
<212>PRT
<213> genus, race: NGF mutant K25Q
<400>41
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Gln Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys ArgGly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>42
<211>120
<212>PRT
<213> genus, race: NGF mutant D24A
<400>42
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Ala Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>43
<211>120
<212>PRT
<213> genus, race: NGF mutant D16A
<400>43
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Ala
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>44
<211>120
<212>PRT
<213> genus, race: NGF mutant S13M
<400>44
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Met Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>45
<211>120
<212>PRT
<213> genus, race: NGF mutant F12A
<400>45
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Ala Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys AlaVal Arg Arg Ala
115 120
<210>46
<211>120
<212>PRT
<213> genus, race: NGF mutant E11A
<400>46
Ser Ser Ser His Pro Ile Phe His Arg Gly Ala Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>47
<211>120
<212>PRT
<213> genus, race: NGF mutant H8A
<400>47
Ser Ser Ser His Pro Ile Phe Ala Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>48
<211>120
<212>PRT
<213> genus, race: NGF mutant P5A
<400>48
Ser Ser Ser His Ala Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>49
<211>120
<212>PRT
<213> genus, race: NGF mutant H4D
<400>49
Ser Ser Ser AspPro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210>50
<211>19
<212>DNA
<213> Artificial Synthesis, primer F
<400>50
ggaattcatg tccatgttg 19
<210>51
<211>19
<212>DNA
<213> Artificial Synthesis, primer R
<400>51
caagctttca ggctcttct 19
Claims (19)
1. A low-pain nerve growth factor mutant, characterized by: is shown in a sequence table SEQ ID No: 37.
2. A nucleotide sequence encoding the nerve growth factor mutant of claim 1.
3. The nucleotide sequence of claim 2, characterized in that: the nucleotide sequence is genomic DNA, cDNA, synthetic DNA or RNA.
4. The nucleotide sequence of claim 2, characterized in that: the nucleotide sequence is DNA.
5. The nucleotide sequence of claim 4, characterized in that: the DNA is cDNA of a coding sequence.
6. An expression vector, characterized in that: the expression vector contains the nucleotide sequence of any one of claims 2 to 5.
7. The expression vector of claim 6, wherein: the expression vector is selected from the group consisting of a DNA vector and a viral vector.
8. The expression vector of claim 7, wherein: the DNA vector is selected from the group consisting of a DNA plasmid vector, a liposome bound thereto, a molecular conjugate bound thereto, and a polymer bound thereto.
9. The expression vector of claim 8, wherein: the DNA plasmid vector is a eukaryotic cell expression vector.
10. The expression vector of claim 7, wherein: the viral vector is selected from the group consisting of an adeno-associated viral vector, a lentiviral vector, and an adenoviral vector.
11. A method of expressing the expression vector of any one of claims 6 to 10, characterized in that: transforming the expression vector of any one of claims 6 to 10 into a host cell, and culturing the obtained recombinant cell to express the recombinant cell to obtain the low-pain nerve growth factor mutant.
12. A host cell comprising the expression vector of any one of claims 6 to 10.
13. The host cell of claim 12, wherein the host cell is a mammalian cell.
14. The host cell of claim 13, wherein the mammalian cell is a chinese hamster ovary cell, a human embryonic kidney 293 cell, a COS cell, or a Hela cell.
15. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more selected from the group consisting of the low-pain nerve growth factor mutant of claim 1, the expression vector of any one of claims 6 to 10, and the host cell of any one of claims 12 to 14.
16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is an injection solution comprising a pharmaceutically acceptable excipient and the low pain nerve growth factor mutant of claim 1.
17. Use of the low pain nerve growth factor mutant of claim 1 for the manufacture of a medicament for the treatment of a neurological disease.
18. Use of the low pain nerve growth factor mutant of claim 1 for the manufacture of a medicament effective for weight loss.
19. Sequence listing SEQ ID No: 37 for preparing a low-pain nerve growth factor preparation.
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| IT201800009384A1 (en) * | 2018-10-11 | 2020-04-11 | Cosmo Srl | Peptide for cosmetic application |
| CN112439053B (en) * | 2019-08-29 | 2024-08-02 | 江苏中新医药有限公司 | Use of long-acting protein preparation for improving sexual dysfunction |
| CN114829384B (en) | 2020-11-19 | 2023-12-12 | 舒泰神（北京）生物制药股份有限公司 | Long-acting nerve growth factor polypeptide and application thereof |
| WO2024140625A1 (en) * | 2022-12-28 | 2024-07-04 | 舒泰神（北京）生物制药股份有限公司 | Method for screening and/or identifying mutable site in ngf, and method for screening and/or identifying ngf mutant |
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| CA2710141A1 (en) * | 2007-12-20 | 2009-07-02 | Cytos Biotechnology Ag | Ngf conjugates and uses thereof |
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