CN107286233A - Low pain nerve growth factor mutant - Google Patents
Low pain nerve growth factor mutant Download PDFInfo
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- CN107286233A CN107286233A CN201710225746.6A CN201710225746A CN107286233A CN 107286233 A CN107286233 A CN 107286233A CN 201710225746 A CN201710225746 A CN 201710225746A CN 107286233 A CN107286233 A CN 107286233A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/48—Nerve growth factor [NGF]
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Abstract
The present invention relates to low pain nerve growth factor mutant, belong to field of biological pharmacy.Low pain nerve growth factor mutant, is sequence table SEQ ID No：3 to SEQ ID No：The amino acid sequence of any one in 49.The advantage of the invention is that：Nerve growth factor mutant has low painful, can mitigate pain side effect.
Description
Technical field
The present invention relates to low pain nerve growth factor mutant, belong to field of biological pharmacy.
Background technology
Pain can be divided into sensitivity pain and the class of neurogenic pain two according to its neuro-physiology mechanism, and the former is direct
Caused by noxious stimulation, also known as inflammatory pain relevant with tissue damage or inflammatory reaction；The latter is by somatesthesia nerveous system
The chronic ache that the damage of system or disease are directly contributed.
Nerve growth factor (Nerve Growth Factor, NGF) be Italian scientist Levi-Montlcini in
First neurotrophic factor that nineteen fifty-three finds in mice sarcoma cell, NGF is a kind of with neurotrophic and promotion
A kind of nerve growth regulatory factor of enation double biological function, its development to maincenter and peripheral neurons,
Differentiation, growth, regeneration and the expression of functional characteristic are respectively provided with important regulating and controlling effect.NGF includes tri- subunits of α, β, γ, β
Subunit is active region, is formed by two single-stranded combined by non-covalent bond.Levi-Montalcini because being found that NGF and
Obtain the Nobel Prize.At present, multiple NGF launch are had both at home and abroad, are clinically mainly used in treating nervous system development
It is bad, including the disease such as amblyopia, neuroma, various neurotrosises and nervous system lesion.
NGF is present in a variety of species, in male mice salivary gland, ox refining, snake venom, cavy prostate and Human plactnta group
Knit middle rich content.Wherein mouse NGF reaches 90% with people's NGF amino acid sequence homologies.Human body is applied in view of mouse NGF
Species difference and the potential virulence factor risk that has as raw material of mouse and the limit of human placenta raw material
System, develops technique for gene engineering Prepare restructuring people NGF (rhNGF, recombinant human NGF) to replace the mouse of extraction
NGF and people NGF have good application prospect.
The NGF of cylinder mature exists in the form of homodimer, and every peptide chain includes 120 amino acid.People's ngf gene
On No. 1 the short arm of a chromosome, complete NGF extrons are made up of 241 amino acid, commonly referred to as prepro NGF precursors,
The signal peptide of prepro NGF precursors is cut in endoplasmic reticulum, forms pro NGF precursors (223 amino acid), pro
NGF precursors exist in endoplasmic reticulum in homodimer form, are then transferred in golgiosome, precursor portions are through Furin enzymes
Cut, form ripe NGF dimers (monomer contains 120 amino acid), be transported to it is extracellular, while also having part without cutting
The pro NGF precursors cut are secreted to extracellular.
Although recombination human source NGF avoids some potentially pathogenic risks, but still can exist in actual use
Very big problem：1) NGF bioactivity is kept, as other protein, NGF bioactivity is depended on secondly level and three-level knot
Structure, therefore keep its bioactivity especially important during during preparing, purify, store and being administered；2) meeting during NGF uses
Cause more serious pain, some patients can not be resistant to, therefore cause and be limited using upper part.NGF take part in the disease of pain
Physiology course is managed, by influenceing the release of inflammatory mediator, the opening of ion channel and promoting the growth of nerve fibre to cause pain,
And developed by modulation of ion channels and molecular signal so as to participate in pain.There is scholar to speculate that NGF is also possible to by promoting
Entering the expression of algogenic substance causes pain, and can change after body injury the budding and regeneration of neuron.Current research hair
It is existing：In the mankind, it is 0.03 μ g/kg (Pettyet al., 1994--29) that will not cause hyperalgesic maximum dose.But,
So low dosage limits NGF application, while also limit the expansion of its indication, is such as used for central nervous system.
Therefore, to avoid above mentioned problem, need to seek it is a kind of can mitigate pain side effect even painless restructuring hNGF, from
And dosage and tested crowd can be increased, to expand indication and providing possibility applied to central nervous system.
The content of the invention
There is low painful it is an object of the invention to provide one group or even painless nerve growth factor mutant, that is, recombinate
hNGF。
To achieve the above object, the present invention uses following technical scheme：
Low pain nerve growth factor mutant, is sequence table SEQ ID No：3 to SEQ ID No：The institute of any one in 49
The amino acid sequence shown.
The nucleotide sequence of the described low pain nerve growth factor mutant of coding.
Described nucleotide sequence, including genomic DNA, cDNA, the DNA and RNA of synthesis.It is preferred that DNA, is more preferably compiled
The cDNA of code sequence.Present invention also contemplates that the nucleotides sequence of enzyme or albumen of the coding with special properties as herein defined
Row.Term " nucleotide sequence " used herein refers to oligonucleotide sequence or polynucleotide sequence and its variant, homologue, piece
Section and derivative (such as its part).The nucleotide sequence can be derived from genome or synthetic or restructuring thing, and it can be
Double-strand or single-stranded (no matter it represents positive-sense strand or antisense strand) nucleic acid molecules.It can be passed through by ripe standard method
Synthesis encodes the nucleotide sequence of the low pain nerve growth factor mutant to prepare, and utilizes and is synthesized on automatic dna synthesizer
Oligonucleotides, is then expanded, digestion is connected in appropriate carrier.
Low pain restructuring hNGF mutant of the present invention is in wild type hNGF sequences (SEQ ID No：2) carried out on the basis of
What single-point or multipoint mutation were obtained, preferably following NGF mutant, its amino acid sequence is as follows：
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (wild type hNGF amino acid sequences:SEQ ID No：2)
ATGTCCATGTTGTTCTACACTCTGATCACAGCTTTTCTGATCGGCATACAGGCGGAACCACACTCAGAGAGCAATGT
CCCTGCAGGACACACCATCCCCCAAGCCCACTGGACTAAACTTCAGCATTCCCTTGACACTGCCCTTCGCAGAGCCC
GCAGCGCCCCGGCAGCGGCGATAGCTGCACGCGTGGCGGGGCAGACCCGCAACATTACTGTGGACCCCAGGCTGTTT
AAAAAGCGGCGACTCCGTTCACCCCGTGTGCTGTTTAGCACCCAGCCTCCCCGTGAAGCTGCAGACACTCAGGATCT
GGACTTCGAGGTCGGTGGTGCTGCCCCCTTCAACAGGACTCACAGGAGCAAGCGGTCATCATCCCATCCCATCTTCC
ACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGC
AAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCG
GGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACA
CCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGT
GTGCTCAGCAGGAAGGCTGTGAGAAGAGCCTGA (wild type hNGF DNA sequence dna SEQ ID No：1)
K115A：Amino acid sequence is SEQ ID No：3；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRAAVRRA (amino acid sequences:SEQ ID No：3)
R114A：Amino acid sequence is SEQ ID No：4；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSAKAVRRA (amino acid sequences:SEQ ID No：4)
R119A：Amino acid sequence is SEQ ID No：5；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRAA (amino acid sequences:SEQ ID No：5)
L112A：Amino acid sequence is SEQ ID No：6；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVASRKAVRRA (amino acid sequences:SEQ ID No：6)
S113M：Amino acid sequence is SEQ ID No：7；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLMRKAVRRA (amino acid sequences:SEQ ID No：7)
D105N：Amino acid sequence is SEQ ID No：8；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRINTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：8)
D105A：Amino acid sequence is SEQ ID No：9；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIATACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：9)
R103K：Amino acid sequence is SEQ ID No：10；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIKIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：10)
R103A：Amino acid sequence is SEQ ID No：11；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIAIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：11)
A97E：Amino acid sequence is SEQ ID No：12；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQEAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：12)
M92E：Amino acid sequence is SEQ ID No：13；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTEDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：13)
K88M：Amino acid sequence is SEQ ID No：14；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVMALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：14)
K88A：Amino acid sequence is SEQ ID No：15；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVAALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：15)
H84A：Amino acid sequence is SEQ ID No：16；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTATFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：16)
Y79A：Amino acid sequence is SEQ ID No：17；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SACTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：17)
T81A：Amino acid sequence is SEQ ID No：18；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCATTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：18)
N77A：Amino acid sequence is SEQ ID No：19；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWA
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：19)
H75A：Amino acid sequence is SEQ ID No：20；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKAWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：20)
K74A：Amino acid sequence is SEQ ID No：21；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSAHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：21)
D72A：Amino acid sequence is SEQ ID No：22；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIASKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：22)
R69A：Amino acid sequence is SEQ ID No：23；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCAGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：23)
R59A：Amino acid sequence is SEQ ID No：24；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCADPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：24)
R59K：Amino acid sequence is SEQ ID No：25；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCKDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：25)
K57A：Amino acid sequence is SEQ ID No：26；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETACRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：26)
E55A：Amino acid sequence is SEQ ID No：27；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFATKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：27)
Y52F：Amino acid sequence is SEQ ID No：28；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQFFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：28)
K50A：Amino acid sequence is SEQ ID No：29；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFAQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：29)
S47A：Amino acid sequence is SEQ ID No：30；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNAVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：30)
N46K：Amino acid sequence is SEQ ID No：31；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINKSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：31)
E41A：Amino acid sequence is SEQ ID No：32；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGAVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：32)
K32A：Amino acid sequence is SEQ ID No：33；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIAGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：33)
K32R：Amino acid sequence is SEQ ID No：34；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIRGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：34)
K34A：Amino acid sequence is SEQ ID No：35；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGAEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：35)
E35A：Amino acid sequence is SEQ ID No：36；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKAVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：36)
I31N：Amino acid sequence is SEQ ID No：37；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATDNKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：37)
D30N：Amino acid sequence is SEQ ID No：38；
SSSHPIFHRGEFSVCDSVSVWVGDKTTATNIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：38)
T27E：Amino acid sequence is SEQ ID No：39；
SSSHPIFHRGEFSVCDSVSVWVGDKTEATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：39)
T26K：Amino acid sequence is SEQ ID No：40；
SSSHPIFHRGEFSVCDSVSVWVGDKKTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：40)
K25Q：Amino acid sequence is SEQ ID No：41；
SSSHPIFHRGEFSVCDSVSVWVGDQTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：41)
D24A：Amino acid sequence is SEQ ID No：42；
SSSHPIFHRGEFSVCDSVSVWVGAKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：42)
D16A：Amino acid sequence is SEQ ID No：43；
SSSHPIFHRGEFSVCASVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：43)
S13M：Amino acid sequence is SEQ ID No：44；
SSSHPIFHRGEFMVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：44)
F12A：Amino acid sequence is SEQ ID No：45；
SSSHPIFHRGEASVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：45)
E11A：Amino acid sequence is SEQ ID No：46；
SSSHPIFHRGAFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：46)
H8A：Amino acid sequence is SEQ ID No：47；
SSSHPIFARGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：47)
P5A：Amino acid sequence is SEQ ID No：48；
SSSHAIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：48)
H4D：Amino acid sequence is SEQ ID No：49.
SSSDPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWN
SYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequences:SEQ ID No：49)
It is obtaining or wherein that the NGF mutant can be that one in above-mentioned amino acid mutation site is mutated
The NGF mutant that two sites, three even multiple site simultaneous mutations are obtained.Such as：In wild type hNGF amino acid sequence
Row basis, can obtain D30N and tri- site simultaneous mutations of NGF mutant, K34A, E35A and I31N of T27E simultaneous mutations
NGF mutant etc..
A kind of expression vector, (nucleotides sequence of low pain nerve growth factor is encoded containing described nucleotide sequence
Row).
The expression vector is in the group being made up of DNA vector and viral vector.
The DNA vector is selected from and couples body and combination by DNA plasmid carrier, the liposome with reference to it, with reference to its molecule
In the group that its polymer is constituted；Preferably, the DNA plasmid carrier is eukaryotic expression vector, procaryotic cell expression
Carrier or filamentous fungi expression vector；The viral vector is selected from by gland relevant viral vector, slow virus carrier, adenovirus vector
And in the group that is constituted of baculovirus vector.It is highly preferred that the DNA plasmid carrier is eukaryotic expression vector；The disease
Poisonous carrier is in the group being made up of gland relevant viral vector, slow virus carrier and adenovirus vector.
A kind of method for expressing the expression vector, described expression vector is converted to host cell, culture gained weight
Group cell expression obtains low pain nerve growth factor mutant.
Host cell containing the expression vector.
The host cell is mammalian cell, yeast cells, insect cell, Escherichia coli, filamentous fungi or plant
Cell.Preferably, described host cell is mammalian cell.
The mammalian cell is Chinese hamster ovary cell, human embryo kidney 293 cells, COS cells or Hela cells.Institute
Insect cell is stated for SF9 cells, SF21 cells, SF900 cells.
A kind of pharmaceutical composition, described pharmaceutical composition contains pharmaceutically acceptable excipient, and above-mentioned refers to
One or more in low pain nerve growth factor mutant, the above-mentioned expression vector referred to and the above-mentioned host cell referred to.
The diversified forms such as injection, capsule, tablet or pulvis, the medicine of above-mentioned various formulations can be made in the medicine of the present invention
Thing can be prepared according to the conventional method of pharmaceutical field.
Described pharmaceutical composition is preferably parenteral solution, and the parenteral solution includes pharmaceutically acceptable excipient and above-mentioned
The low pain nerve growth factor mutant referred to.
When needs, one or more pharmaceutically acceptable carriers can also be added in aforementioned pharmaceutical compositions,
The carrier includes conventional diluent, stabilizer, surfactant and preservative of pharmaceutical field etc..
Described low pain nerve growth factor mutant is used to prepare the purposes in the medicine for the treatment of the nervous system disease.
Described low pain nerve growth factor mutant is used to prepare the purposes in the medicine effectively lost weight.
Described low pain nerve growth factor mutant is used to prepare the purposes in the medicine for the treatment of ophthalmology relevant disease.
Described low pain nerve growth factor mutant is used to prepare the use in the medicine for the treatment of reproductive system relevant disease
On the way.
Described low pain nerve growth factor mutant is used to prepare the use in the medicine for the treatment of autoimmune-associated diseases
On the way.
Described low pain nerve growth factor mutant is used to prepare the purposes in the medicine for the treatment of bone related disease.
Sequence table SEQ ID No：3 to SEQ ID No：Amino acid sequence in 49 shown in any one is used to prepare low pain
The purposes of nerve growth factor preparation.
Sequence table SEQ ID No：3 to SEQ ID No：Amino acid sequence in 49 shown in any one is painless for preparing
The purposes of nerve growth factor preparation.
The advantage of the invention is that：For wild type nerve growth factor of the prior art, the disclosure it is low
It is even painless that pain nerve growth factor can mitigate pain side effect, can increase patient to the compliance of nerve growth factor and resistance to
By property, dosage and tested crowd can be increased, to expand indication and providing possibility applied to central nervous system.
Below in conjunction with the drawings and specific embodiments, the present invention will be further described, not limitation of the invention, all
This area equivalent substitution carried out according to the disclosure of invention, belongs to the scope of the present invention.
Brief description of the drawings
Fig. 1 is the SDS PAGE electrophoresis results for the wild type hNGF that embodiment 4Superdex75 posts are purified
Fig. 2 is the short-term administration mouse Determination of Pain Threshold result of embodiment 6
Fig. 3 (A) and Fig. 3 (B) is the long term administration mouse Determination of Pain Threshold result of embodiment 6
Fig. 4 is the mice behavior experimental result of embodiment 7, and mutant, wild type hNGF, observation mouse lift leg are injected respectively
Hold time
Embodiment
The wild type h NGF of embodiment 1. and its mutant plasmid construction
1st, wild type hNGF DNA sequence dna expression plasmid is built
Synthesize wild type hNGF DNA sequence dna (sequence table SEQ ID NO：1), using primer (F：
GGAATTCATGTCCATGTTG (sequence table SEQ ID NO：50), R：CAAGCTTTCAGGCTCTTCT (sequence table SEQ ID
NO：51) enter performing PCR amplification to aim sequence, carried out using EcorI (NEB#R0101S) after digestion, digestion products are used
HindIII (NEB#R0104S) carries out secondary digestion.PcDNA3.1 (-) expression vector is subjected to digestion using same method.
Digestion carrier and PCR amplifications purpose fragment are entered into row agarose gel electrophoresis, purpose fragment is cut, uses DNA glue reclaim reagents
Box (TIANGEN, #DP209-03) is separately recovered after the carrier after digestion and target DNA fragment, uses DNA ligase kit
(Takara/6022) 16 DEG C of connection 1h, complete wild type hNGF plasmid construction.
2nd, the DNA sequence dna expression plasmid of hNGF mutant is built
The same above method, we have been respectively synthesized polynucleotide SEQ ID NO：3 to SEQ ID No：Shown in 49
The gene of low pain nerve growth factor mutant, and the plasmid containing above-mentioned encoding gene is constructed respectively.
The plasmid conversion and extraction of embodiment 2, hNGF and its mutant
1st, convert
HNGF and its mutant plasmid progress that above-described embodiment 1 is built are heat-shock transformed, by Top10 competent cells
(Tiangeng/CB104-02) is immediately placed on after being taken out from -70 DEG C of refrigerators thaws on ice, takes 50ul to be used to convert.2ul is added thereto
Plasmid, flicks mixing, ice bath 30min.In 42 DEG C of dry bath 90s, it is immediately placed on ice after during which should not vibrating centrifuge tube, taking-up
2min.Add 500ul, 150rpm/min, 37 DEG C of shaking table culture 45min of nonreactive LB/SOC culture mediums.Liquid in pipe will be centrifuged complete
Portion is poured on LB flat boards, uniform spreadable.After flat board dries, incubator culture 16h is inverted in.
2nd, plasmid is carried greatly
The single bacterium colony that the above-mentioned step of converting of picking is obtained is inoculated in 500ul LB fluid nutrient mediums, and 37 DEG C are cultivated 7h, simultaneously
Bacterium solution censorship is sequenced.Correct bacterium solution will be sequenced to carry out largely shaking bacterium, 500ul bacterium solutions are inoculated in 500mlLB culture mediums, 37 DEG C
Cultivate 16h.4 DEG C incubated overnight is collected by centrifugation bacterium solution, 6000xg centrifuge 10 minutes, abundant supernatant discarding proposes greatly examination using plasmid
The big upgrading grain of agent box (being purchased from QIAGEN, article No. 12163), measurement concentration is standby.
The wild type hNGF of embodiment 3. and its mutant expression
Wild type hNGF that above-described embodiment 2 is raised greatly and and its mutant plasmid transfection 293F cells in, transfection
Expression supernatant is collected within 4th afterwards and quantitative.
Experimental procedure：
1st, the day before transfection, is inoculated with 293F cells, 0.5 × 106/ ml, common 900ml, 300ml/ bottles.
2nd, the transfection same day counts, cell density about 1.0 × 106/ ml, motility rate more than 99%.
3rd, transfect：36ml cell culture mediums are taken to 125ml blake bottles；Plus 360ug plasmids, mix；Again plus 1080ug PEI,
Mix.It is stored at room temperature 15 minutes；Mixed with cell, about 12.3ml/ bottles；37 DEG C, 8%CO2, 120RPM cultures.
4th, cell conditioned medium is collected within the 4th day after transfecting, 10000g is centrifuged 20 minutes.
5th, supernatant is collected, 0.45um filterings obtain the albumen supernatant of wild type hNGF and its mutant.
6th, SDS-PAGE is detected, cma staining is quantitative.
The wild type hNGF of embodiment 4. and its mutant purifying
The NGF and its albumen supernatant of mutant obtained in purifying above-described embodiment 3.
1st, cation-exchange chromatography：The albumen supernatant of wild type hNGF and its mutant is first adjusted using acetic acid and water to pH
For 4.0.CM Sepharose FF chromatographic columns loading, loading after 0.05mol/L acetate buffers (pH4.0) fully balance
After the completion of, rinsed with equilibrium liquid to baseline, miscellaneous peak is eluted to baseline with 0.05mol/L Tris-HC1 (pH9.0) buffer solution, then
With 0.05mol/LTris-HC1 and 0.05mol/L Tris-HC1-0.4mol/L NaCl (pH9.0) gradient elution, according to ultraviolet
Absorbing state collects target peak, is shown on Ultraviolet Detector and starts to collect when numeral is begun to ramp up, and stops receiving when being reduced to baseline
Ji Mubiaodanbaifeng.
2nd, hydrophobic chromatography：Butyl Sepharose 4FF chromatographic columns are through 0.02mol/L phosphate (pH6.8) -1.5mol/L
Sodium chloride buffer is fully balanced.Solid sodium chloride is added in the target peak feed liquid that step 1 is collected makes sodium chloride in feed liquid
Final concentration of 1.5mol/L, loading after sodium to be chlorinated fully dissolves, speed is to be rinsed with equilibrium liquid after 120cm/h, completion of the sample
To baseline, then target peak is collected with 0.02mol/L phosphate (pH6.8) elution.
3rd, gel exclusion chromatography：Superdex 75prep grade chromatographic columns with pH6.8,0.05mol/L phosphate-
The target peak collected after 0.15mol/L sodium chloride buffers are fully balanced in loading step/2, shows number on Ultraviolet Detector
Word when baseline is begun to ramp up collect, stop collecting target protein peak when being reduced to baseline.
The wild type hNGF of Superdex75 posts purifying SDS PAGE are as shown in figure 1, the NGF purity that explanation is prepared
It is higher.The wild type hNGF of collection and its mutant target protein peak sample are concentrated into 0.4mg/ml, 4 DEG C of guarantors using super filter tube
Deposit for subsequent experimental.
The chick embryo method of embodiment 5. determines wild type hNGF and its mutant activity
1st, chick embryonic dorsal root ganglion method measurement wild type hNGF and its mutant activity
The wild type hNGF and its mutant sample that are obtained in above-described embodiment 4 are diluted：A liquid：The open country that 6ng is extracted
Raw type hNGF and its mutant sample add 1ml serum-free DMEM nutrient solutions to dissolve；B liquid：The μ l of A liquid 50 plus serum-free DMEM are taken to cultivate
Liquid 4.95ml；C liquid：The μ l of B liquid 60 plus serum-free DMEM nutrient solution 2.94ml (total amount 3ml) is taken to make final concentration of (3AU/ml).A、B
Liquid is diluted in centrifuge tube, and C liquid is using C liquid as headpin, then 3 times of gradient dilutions in cell bottle：No. 2, No. 3, No. 4,5
Number, No. 6 prepare liquids.Each prepare liquid adds 1 blake bottle, 2ml/ bottles.Simultaneously using serum-free DMEM nutrient solutions as blank control,
Using the standard items purchased from National Institute for Food and Drugs Control as positive control (reference material).Add the chicken embryo Dorsal root god of 8 ages in days
5%CO is placed in after warp knuckle2, in 37 DEG C of saturated humidity incubators, result is observed after 24 hours.
NGF content is used as 1 active unit (AU) in every milliliter of testing sample during growing preferably.It is negative right from occurring
The conduct for starting to take growth best in back the 3rd and the 4th two dilution factor of number according to the dilution factor of result judges endpoint calculation potency.
Reference material is the standard items purchased from Zhong Jian institutes, and every loading amount is 1000AU.
NGF is than calculation formula living：
Active (AU/ml) × [the sample pre-dilution multiple × correspondence reference material of ratio work (AU/mg)=reference material of testing sample
Activity (AU/ml)/reference material actual measurement at dilution point is active (AU/ml)]
Survey slip-knot fruit and show that low pain hNGF mutant remains wild type hNGF activity.
Whether induced pain (pain threshold) is detected for embodiment 6.NGF and its mutant
Experimental principle：Normergic to the pain sensation qualified mouse of screening, inject doses NGF samples (wild type or
Its mutant), the bent pawl of mouse is determined by mechanical stimulus and reacts pain threshold, statistical analysis is carried out, it is final whether to determine sample
Cause mouse hyperalgia.
6-1, the observation of short-term induced pain situation
First, experiment material
Dynamic plantar tactile instrument, Italy/Ugo Basile, model 37450.
2nd, experiment content
1st, qualified mouse screening
SPF rank CD-1 mouse are ordered, mouse specification is male, body weight 30-35g.
Using the sufficient average threshold of dynamic plantar tactile instrument [Italy/Ugo Basile, model 37450] screening two in 7.5-
Between 10 and same mouse or so pin threshold P values, P values>0.05 experimental animal is qualified mouse.
Random packet, is divided into experimental group and blank control group, experimental group is divided into small according to different samples and dosage again
Group, every group 10.
2nd, NGF samples administration design
2.1 wild-type samples induced pain dose screenings
Medicine is prepared：Positive control NGF wild-type samples, NGF S47A mutant, NGF I31N mutant samples is equal
It is diluted using stock sample solution (50mM PB, 150mM NaCl, PH6.8).
Blank control：For NGF stock sample solutions.
Administering mode and dosage：20ul is subcutaneously administered in mouse or so pin vola respectively；
Short-term administration lowest dose level is 1.25 μ g/.
3rd, Determination of Pain Threshold
Mechanical threshold measure is carried out, and records numerical value in 1 hour, 2 hours respectively upon administration, is given in short term
The observation of medicine (in 2 hours).
4th, result statistical analysis
Graph making and result statistical analysis, relatively more each dosage group and control group are carried out using GraphPad Prism softwares
Between mechanical threshold it is whether variant, analysis wild-type samples and mutant sample induced pain.
As shown in Figure 2, when administration lowest dose level is 1.25 μ g/, the performance of control group mice without pain, and the sun of wild type
Property control group, pain threshold 1h be significantly lower than less than 5, pain is obvious；And each mutant experimental group pain threshold is 6 to 7
Left and right, compared with positive controls, pain substantially mitigates.
The long-term induced pain situation observations of 6-2
Except set this experiment 3 dosages as：0.2 μ g/, 0.5 μ g/, 1.25 μ g/, daily administration one
Secondary, continuous 3 weeks, during which outside the measure of continual progress pain threshold, remaining was according in 6-1 " short-term induced pain situation is observed "
Method carry out.
As a result as shown in Fig. 3 (A) and 3 (B), show：Wild type NGF three dosage within 17 days show that the threshold of pain gradually drops
It is low, engender pain.The experimental result of low high three dosage groups was shown in 14 days in S47A mutant, I31N mutant
In addition to except the S47A mutant high dose groups threshold of pain, substantially reduction causes pain, remaining dosage group threshold of pain all maintains more than 6 substantially, with
Wild type, which is compared, to be played the role of substantially to mitigate pain.
Whether the behaviouristics of induced pain is detected for the wild type hNGF of embodiment 7. and its mutant
Mouse joint is administered for wild type hNGF and its mutant sample, by animal behavioral study mouse lift the leg time and
Number of times come investigate sample whether induced pain.
Experiment content
1st, mouse is ordered
SPF rank CD-1 mouse are ordered, mouse specification is male, body weight 30-35g.Random packet, is divided into experimental group, sky
White control group (abbreviation control group) and positive controls, each group are divided into three groups according to dosage again, and each group selects 6 at random
Only.
2nd, dosage and time
2.1 dosage
Positive control：It is 1.25 using stock sample solution (50mM PB, 150mM NaCl, PH6.8) dilution wild type hNGF
μg/10ul；
Experimental group：The same positive control of mutant method for preparation of drug, dilution NGF I31N mutant is 1.25 μ g/10ul；
Blank control：Physiological saline.
2.2 administering mode
In mouse back leg intra-articular injection medicine, 10ul is administered in each articular cavity.
2.3 administration time：
Single is carried out per dosage group to be administered within continuous 3-4 days.I.e. the 10 points one morning, and afterwards the 2nd, 3,4 days it is identical
Time point is administered.
3rd, animal behavioral study
2 after being administered on the day of each experimental group and control group, observed within 4 hours, and be administered the 2nd, 3,4 days it is identical when
Between point observed.
Observation index：The mouse time (second) that spontaneously number of times of lift leg and each lift leg are maintained, calculates lift in 2 minutes
Leg maintains the cumulative time.
4th, result statistical analysis
Held time using GraphPad Prism softwares two-way analysis of variance lift leg, whether comparative analysis difference sample
Induced pain.
Experimental result is as shown in Figure 4：Each detection time point of the wild type hNGF groups after injection medicine is responsible for substantially
Pain, I31N mutant experimental group successive administration pain phenomenons are obvious, and lift leg maintains cumulative time card side below 1 second
Analysis, which is learnt in each detection time point positive controls and experimental group, notable difference, illustrates that I31N mutant can substantially mitigate
The pain reaction of drug injection.
Sequence table
<110>SHUTAISHEN（Beijing）Bio-pharmaceuticals limited company
<120>Low pain nerve growth factor mutant
<130>
<160> 51
<170> PatentIn version 3.5
<210> 1
<211> 726
<212> DNA
<213>Wild type hNGF DNA sequence dna
<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 acttcgaggt cggtggtgct 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>Wild type hNGF amino acid sequence
<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
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> 3
<211> 120
<212> PRT
<213>Genus Homo, ethnic group：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 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 Ala Ala Val Arg Arg Ala
115 120
<210> 4
<211> 120
<212> PRT
<213>Genus Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：NGF mutant R103K
<400> 10
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 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 Homo, ethnic group：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 Homo, ethnic group：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 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
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 Homo, ethnic group：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 Homo, ethnic group：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
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 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 Homo, ethnic group：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 Homo, ethnic group：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 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> 17
<211> 120
<212> PRT
<213>Genus Homo, ethnic group：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 Homo, ethnic group：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
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210> 19
<211> 120
<212> PRT
<213>Genus Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 40 45
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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 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> 31
<211> 120
<212> PRT
<213>Genus Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：NGF mutant E35A
<400> 36
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 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 Homo, ethnic group：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 Homo, ethnic group：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 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> 39
<211> 120
<212> PRT
<213>Genus Homo, ethnic group：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 Homo, ethnic group：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 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> 41
<211> 120
<212> PRT
<213>Genus Homo, ethnic group：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 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> 42
<211> 120
<212> PRT
<213>Genus Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Ala Val Arg Arg Ala
115 120
<210> 46
<211> 120
<212> PRT
<213>Genus Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：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 Homo, ethnic group：NGF mutant H4D
<400> 49
Ser Ser Ser Asp 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 Arg Ala
115 120
<210> 50
<211> 19
<212> DNA
<213>It is artificial synthesized, primers F
<400> 50
ggaattcatg tccatgttg 19
<210> 51
<211> 19
<212> DNA
<213>It is artificial synthesized, primer R
<400> 51
caagctttca ggctcttct 19
Claims (10)
1. low pain nerve growth factor mutant, it is characterised in that：For sequence table SEQ ID No：3 to SEQ ID No：Appoint in 49
One amino acid sequence of meaning.
2. encode the nucleotide sequence of the nerve growth factor mutant described in claim 1；Preferably, the nucleotide sequence
For genomic DNA, cDNA, the DNA or RNA synthesized；It is preferred that DNA, more preferably coded sequence cDNA.
3. a kind of expression vector, it is characterised in that：The expression vector contains the nucleotide sequence described in Claims 2 or 3；It is excellent
Selection of land, the expression vector is in the group being made up of DNA vector and viral vector；Preferably, the DNA vector be selected from by
In DNA plasmid carrier, the liposome with reference to it, the group for coupling with reference to its molecule body and being constituted with reference to its polymer；It is excellent
Selection of land, the DNA plasmid carrier is eukaryotic expression vector；The viral vector is selected from by gland relevant viral vector, slow disease
In the group that poisonous carrier and adenovirus vector are constituted.
4. a kind of method for expressing expression vector described in claim 3, it is characterised in that：Expression described in claim 4 is carried
Body is converted to host cell, and culture gained recombinant cell expression obtains low pain nerve growth factor mutant.
5. the host cell containing expression vector described in claim 3；Preferably, the host cell is mammalian cell；
Preferably, the mammalian cell is Chinese hamster ovary cell, human embryo kidney 293 cells, COS cells or Hela cells.
6. a kind of pharmaceutical composition, it is characterised in that described pharmaceutical composition contains pharmaceutically acceptable excipient, Yi Jixuan
From the low pain nerve growth factor mutant described in claim 1, the expression vector described in claim 3 and claim 5 institute
One or more in the host cell stated；Preferably, described pharmaceutical composition is parenteral solution, and the parenteral solution is included pharmaceutically
Low pain nerve growth factor mutant described in acceptable excipient and claim 1.
7. the low pain nerve growth factor mutant described in claim 1 is used to prepare in the medicine for treating the nervous system disease
Purposes.
8. the low pain nerve growth factor mutant described in claim 1 is used to prepare the use in the medicine effectively lost weight
On the way.
9. sequence table SEQ ID No：3 to SEQ ID No：Amino acid sequence shown in any one in 49 is used to prepare low pain god
Purposes through growth-factor preparation.
10. sequence table SEQ ID No：3 to SEQ ID No：Amino acid sequence shown in any one in 49 is painless for preparing
The purposes of nerve growth factor preparation.
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| WO2022105847A1 (en) | 2020-11-19 | 2022-05-27 | Staidson (Beijing) Biopharmaceuticals Co. , Ltd. | Long-acting nerve growth factor polypeptides and uses 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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| CN112409471A (en) | 2021-02-26 |
| CN107286233B (en) | 2020-11-06 |
| CN112409471B (en) | 2022-07-29 |
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