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CN107286233B - low pain nerve growth factor mutant - Google Patents

low pain nerve growth factor mutantDownload PDF

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CN107286233B
CN107286233BCN201710225746.6ACN201710225746ACN107286233BCN 107286233 BCN107286233 BCN 107286233BCN 201710225746 ACN201710225746 ACN 201710225746ACN 107286233 BCN107286233 BCN 107286233B
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陈志�
王超
马磊
杜天飞
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Staidson Beijing Biopharmaceutical Co Ltd
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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

Translated fromChinese
低痛神经生长因子突变体low pain nerve growth factor mutant

技术领域technical field

本发明涉及低痛神经生长因子突变体，属于生物制药领域。The invention relates to a low-pain nerve growth factor mutant and belongs to the field of biopharmaceuticals.

背景技术Background technique

疼痛根据其神经生理学机制可分为感受性疼痛和神经病理性疼痛两类，前者直接由伤害性刺激引起，与组织损伤或炎症反应有关，又称炎症性疼痛；后者由躯体感觉神经系统的损伤或疾病直接造成的慢性疼痛。According to its neurophysiological mechanism, pain can be divided into two types: receptive pain and neuropathic pain. The former is directly caused by noxious stimuli and is related to tissue damage or inflammatory response, also known as inflammatory pain; the latter is caused by damage to the somatosensory nervous system or Chronic pain as a direct result of disease.

神经生长因子(Nerve Growth Factor，NGF)是意大利科学家Levi-Montlcini于1953年在小鼠肉瘤细胞内发现的第一个神经营养因子，NGF是一种具有神经元营养和促进突起生长双重生物学功能的一种神经细胞生长调节因子，它对中枢及周围神经元的发育、分化、生长、再生和功能特性的表达均具有重要的调控作用。NGF包含α、β、γ三个亚单位，β亚单位是活性区，由两条单链通过非共价键结合而成。Levi-Montalcini因为发现了NGF而获得了诺贝尔奖。目前，国内外已有多个NGF产品上市，临床上主要用于治疗神经系统发育不良，包括弱视、神经瘤、各种神经损伤及神经系统病变等疾病。Nerve Growth Factor (NGF) is the first neurotrophic factor discovered in mouse sarcoma cells by Italian scientist Levi-Montlcini in 1953. NGF is a dual biological function of neuron nutrition and neurite outgrowth. It plays an important role in regulating the development, differentiation, growth, regeneration and expression of functional properties of central and peripheral neurons. NGF contains three subunits, α, β, and γ. The β subunit is the active region, which is formed by two single chains bound together by non-covalent bonds. Levi-Montalcini won the Nobel Prize for the discovery of NGF. At present, there are a number of NGF products on the market at home and abroad, which are mainly used in clinical treatment of dysplasia of the nervous system, including amblyopia, neuroma, various nerve injuries and diseases of the nervous system.

NGF存在于多种物种中，在雄性小鼠颌下腺、牛精浆、蛇毒、豚鼠前列腺和人胎盘组织中含量丰富。其中小鼠NGF与人NGF氨基酸序列同源性达到90％。考虑到鼠NGF应用于人体的种属差异性及小鼠作为原材料所具有的潜在的致病因子风险及人胎盘组织原材料的限制，发展基因工程技术制备重组人NGF(rhNGF，recombinant human NGF)来取代提取的鼠NGF和人NGF具有很好的应用前景。NGF is present in a variety of species and is abundant in male mouse submandibular glands, bovine seminal plasma, snake venom, guinea pig prostate and human placental tissue. The amino acid sequence homology between mouse NGF and human NGF reaches 90%. Considering the species differences of murine NGF applied to humans, the potential risk of pathogenic factors in mice as raw materials and the limitation of human placental tissue raw materials, the development of genetic engineering technology to prepare recombinant human NGF (rhNGF, recombinant human NGF) to It has a good application prospect to replace the extracted murine NGF and human NGF.

体内成熟的NGF以同源二聚体的形式存在，每条肽链包含120个氨基酸。人NGF基因位于1号染色体短臂上，完整的NGF外显子由241个氨基酸组成，通常称为prepro NGF前体，在内质网中prepro NGF前体的信号肽被切割下来，形成pro NGF前体(223个氨基酸)，proNGF前体在内质网中以同源二聚体形式存在，然后转移至高尔基体中，前体部分经Furin酶切，形成成熟的NGF二聚体(单体含有120个氨基酸)，被转运至细胞外，同时也有部分未经切割的pro NGF前体分泌至细胞外。Mature NGF in vivo exists in the form of homodimers, and each peptide chain contains 120 amino acids. The human NGF gene is located on the short arm ofchromosome 1. The complete NGF exon consists of 241 amino acids, which is usually called prepro NGF precursor. The signal peptide of prepro NGF precursor in the endoplasmic reticulum is cleaved to form pro NGF. The precursor (223 amino acids), the proNGF precursor exists as a homodimer in the endoplasmic reticulum, and is then transferred to the Golgi apparatus. The precursor part is cleaved by Furin to form a mature NGF dimer (monomer) (contains 120 amino acids), is transported to the outside of the cell, and some uncleaved pro NGF precursors are also secreted to the outside of the cell.

重组人源NGF虽然避免了一些潜在的致病风险，但在实际使用过程中仍然会存在很大问题：1)保持NGF生物活性，像其他蛋白质一样，NGF的生物活性依赖于其二级和三级结构，故在制备、纯化、存储及给药期间过程中保持其生物活性特别重要；2)NGF使用过程中会引起较严重的疼痛，部分病人无法耐受，故造成了使用上的部分受限。NGF参与了疼痛的病理生理过程，通过影响炎症介质的释放、离子通道的开放及促进神经纤维的生长导致疼痛，并通过调控离子通道及分子信号从而参与疼痛的发生发展。有学者推测NGF还可能通过促进致痛物质的表达导致疼痛，并在机体损伤后可改变神经元的出芽和再生。目前的研究发现：在人类中，不会引起痛觉过敏的最大剂量为0.03μg/kg(Pettyet al.,1994--29)。但是，如此低的剂量限制了NGF的应用，同时也限制了其适应症的扩大，如用于中枢神经系统。Although recombinant human NGF avoids some potential pathogenic risks, there are still great problems in the actual use process: 1) To maintain the biological activity of NGF, like other proteins, the biological activity of NGF depends on its secondary and tertiary Therefore, it is particularly important to maintain its biological activity during preparation, purification, storage and administration; 2) NGF can cause severe pain during use, which some patients cannot tolerate, resulting in some suffering from use. limit. NGF participates in the pathophysiological process of pain. It induces pain by affecting the release of inflammatory mediators, the opening of ion channels, and the growth of nerve fibers. It also participates in the occurrence and development of pain by regulating ion channels and molecular signals. Some scholars speculate that NGF may also cause pain by promoting the expression of pain-causing substances, and may alter neuron sprouting and regeneration after injury. The current study found that: in humans, the maximum dose that does not cause hyperalgesia is 0.03 μg/kg (Petty et al., 1994--29). However, such a low dose limits the application of NGF, as well as the expansion of its indications, such as for the central nervous system.

因此，为避免上述问题，需寻求一种可以减轻疼痛副作用甚至无痛的重组hNGF，从而可加大使用剂量及受试人群，为扩大适应症以及应用于中枢神经系统提供了可能。Therefore, in order to avoid the above problems, it is necessary to seek a recombinant hNGF that can reduce painful side effects or even be painless, so that the dosage and the test population can be increased, and it is possible to expand the indications and apply to the central nervous system.

发明内容SUMMARY OF THE INVENTION

本发明的目的是提供一组具有低疼痛性甚至无痛的神经生长因子突变体，即重组hNGF。The purpose of the present invention is to provide a group of nerve growth factor mutants with low pain or even no pain, namely recombinant hNGF.

为实现上述目的，本发明采用以下技术方案：To achieve the above object, the present invention adopts the following technical solutions:

低痛神经生长因子突变体，为序列表SEQ ID No：3至SEQ ID No：49中任意一个所示的氨基酸序列。The low-pain nerve growth factor mutant has the amino acid sequence shown in any one of SEQ ID No: 3 to SEQ ID No: 49 in the sequence listing.

编码所述的低痛神经生长因子突变体的核苷酸序列。The nucleotide sequence encoding the low-pain NGF mutant.

所述的核苷酸序列，包括基因组DNA、cDNA、合成的DNA和RNA。优选DNA，更优选为编码序列的cDNA。本发明还涵盖编码具有如本文所定义的特定性质的酶或蛋白的核苷酸序列。本文所用的术语"核苷酸序列"是指寡核苷酸序列或多核苷酸序列和其变体、同源物、片段和衍生物(如其部分)。所述核苷酸序列可以是源自基因组或合成物或重组物，其可以是双链的或单链的(无论其代表正义链还是反义链)核酸分子。可以通过成熟的标准方法通过合成来制备编码所述低痛神经生长因子突变体的核苷酸序列，利用自动DNA合成仪上合成寡核苷酸，然后将其扩增、酶切连接至适当的载体中。The nucleotide sequence includes genomic DNA, cDNA, synthetic DNA and RNA. DNA is preferred, more preferably cDNA encoding the sequence. The present invention also encompasses nucleotide sequences encoding enzymes or proteins having specific properties as defined herein. The term "nucleotide sequence" as used herein refers to oligonucleotide sequences or polynucleotide sequences and variants, homologues, fragments and derivatives thereof (eg portions thereof). The nucleotide sequence may be derived from a genome or a synthetic or recombinant nucleic acid molecule, which may be double-stranded or single-stranded (whether it represents the sense or antisense strand). Nucleotide sequences encoding the low-pain NGF mutants can be prepared synthetically by well-established standard methods, oligonucleotides are synthesized on an automated DNA synthesizer, then amplified, digested and ligated into appropriate oligonucleotides. in the carrier.

本发明所述低痛重组hNGF突变体是在野生型hNGF序列(SEQ ID No：2)基础上进行单点或多点突变得到的，优选以下NGF突变体，其氨基酸序列如下：The low-pain recombinant hNGF mutant of the present invention is obtained by single-point or multi-point mutation on the basis of the wild-type hNGF sequence (SEQ ID No: 2), preferably the following NGF mutants, whose amino acid sequences are as follows:

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(野生型hNGF氨基酸序列:SEQID No：2)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (wild-type hNGF amino acid sequence: SEQ ID No: 2)

ATGTCCATGTTGTTCTACACTCTGATCACAGCTTTTCTGATCGGCATACAGGCGGAACCACACTCAGAGAGCAATGTCCCTGCAGGACACACCATCCCCCAAGCCCACTGGACTAAACTTCAGCATTCCCTTGACACTGCCCTTCGCAGAGCCCGCAGCGCCCCGGCAGCGGCGATAGCTGCACGCGTGGCGGGGCAGACCCGCAACATTACTGTGGACCCCAGGCTGTTTAAAAAGCGGCGACTCCGTTCACCCCGTGTGCTGTTTAGCACCCAGCCTCCCCGTGAAGCTGCAGACACTCAGGATCTGGACTTCGAGGTCGGTGGTGCTGCCCCCTTCAACAGGACTCACAGGAGCAAGCGGTCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGCAAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCGGGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACACCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGTGTGCTCAGCAGGAAGGCTGTGAGAAGAGCCTGA(野生型hNGF的DNA序列SEQ IDNo：1)ATGTCCATGTTGTTCTACACTCTGATCACAGCTTTTCTGATCGGCATACAGGCGGAACCACACTCAGAGAGCAATGTCCCTGCAGGACACACCATCCCCCAAGCCCACTGGACTAAACTTCAGCATTCCCTTGACACTGCCCTTCGCAGAGCCCGCAGCGCCCCGGCAGCGGCGATAGCTGCACGCGTGGCGGGGCAGACCCGCAACATTACTGTGGACCCCAGGCTGTTTAAAAAGCGGCGACTCCGTTCACCCCGTGTGCTGTTTAGCACCCAGCCTCCCCGTGAAGCTGCAGACACTCAGGATCTGGACTTCGAGGTCGGTGGTGCTGCCCCCTTCAACAGGACTCACAGGAGCAAGCGGTCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGCAAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCGGGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACACCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGTGTGCTCAGCAGGAAGGCTGTGAGAAGAGCCTGA(野生型hNGF的DNA序列SEQ IDNo：1)

K115A：氨基酸序列为SEQ ID No：3；K115A: the amino acid sequence is SEQ ID No: 3;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRAAVRRA(氨基酸序列:SEQ ID No：3)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRAAVRRA (amino acid sequence: SEQ ID No: 3)

R114A：氨基酸序列为SEQ ID No：4；R114A: the amino acid sequence is SEQ ID No: 4;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSAKAVRRA(氨基酸序列:SEQ ID No：4)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSAKAVRRA (amino acid sequence: SEQ ID No: 4)

R119A：氨基酸序列为SEQ ID No：5；R119A: the amino acid sequence is SEQ ID No: 5;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRAA(氨基酸序列:SEQ ID No：5)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRAA (amino acid sequence: SEQ ID No: 5)

L112A：氨基酸序列为SEQ ID No：6；L112A: the amino acid sequence is SEQ ID No: 6;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVASRKAVRRA(氨基酸序列:SEQ ID No：6)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVASRKAVRRA (amino acid sequence: SEQ ID No: 6)

S113M：氨基酸序列为SEQ ID No：7；S113M: the amino acid sequence is SEQ ID No: 7;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLMRKAVRRA(氨基酸序列:SEQ ID No：7)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLMRKAVRRA (amino acid sequence: SEQ ID No: 7)

D105N：氨基酸序列为SEQ ID No：8；D105N: the amino acid sequence is SEQ ID No: 8;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRINTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：8)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRINTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 8)

D105A：氨基酸序列为SEQ ID No：9；D105A: the amino acid sequence is SEQ ID No: 9;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIATACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：9)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIATACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 9)

R103K：氨基酸序列为SEQ ID No：10；R103K: the amino acid sequence is SEQ ID No: 10;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIKIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：10)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIKIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 10)

R103A：氨基酸序列为SEQ ID No：11；R103A: the amino acid sequence is SEQ ID No: 11;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIAIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：11)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIAIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 11)

A97E：氨基酸序列为SEQ ID No：12；A97E: the amino acid sequence is SEQ ID No: 12;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQEAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：12)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQEAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 12)

M92E：氨基酸序列为SEQ ID No：13；M92E: the amino acid sequence is SEQ ID No: 13;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTEDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：13)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTEDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 13)

K88M：氨基酸序列为SEQ ID No：14；K88M: the amino acid sequence is SEQ ID No: 14;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVMALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：14)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVMALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 14)

K88A：氨基酸序列为SEQ ID No：15；K88A: the amino acid sequence is SEQ ID No: 15;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVAALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：15)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVAALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 15)

H84A：氨基酸序列为SEQ ID No：16；H84A: the amino acid sequence is SEQ ID No: 16;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTATFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：16)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTATFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 16)

Y79A：氨基酸序列为SEQ ID No：17；Y79A: the amino acid sequence is SEQ ID No: 17;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSACTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：17)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSACTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 17)

T81A：氨基酸序列为SEQ ID No：18；T81A: the amino acid sequence is SEQ ID No: 18;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCATTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：18)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCATTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 18)

N77A：氨基酸序列为SEQ ID No：19；N77A: the amino acid sequence is SEQ ID No: 19;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWASYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：19)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWASYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 19)

H75A：氨基酸序列为SEQ ID No：20；H75A: the amino acid sequence is SEQ ID No: 20;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKAWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：20)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKAWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 20)

K74A：氨基酸序列为SEQ ID No：21；K74A: the amino acid sequence is SEQ ID No: 21;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSAHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：21)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSAHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 21)

D72A：氨基酸序列为SEQ ID No：22；D72A: the amino acid sequence is SEQ ID No: 22;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIASKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：22)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIASKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 22)

R69A：氨基酸序列为SEQ ID No：23；R69A: the amino acid sequence is SEQ ID No: 23;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCAGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：23)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCAGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 23)

R59A：氨基酸序列为SEQ ID No：24；R59A: the amino acid sequence is SEQ ID No: 24;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCADPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：24)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCADPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 24)

R59K：氨基酸序列为SEQ ID No：25；R59K: the amino acid sequence is SEQ ID No: 25;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCKDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：25)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCKDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 25)

K57A：氨基酸序列为SEQ ID No：26；K57A: the amino acid sequence is SEQ ID No: 26;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETACRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：26)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETACRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 26)

E55A：氨基酸序列为SEQ ID No：27；E55A: the amino acid sequence is SEQ ID No: 27;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFATKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：27)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFATKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 27)

Y52F：氨基酸序列为SEQ ID No：28；Y52F: the amino acid sequence is SEQ ID No: 28;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQFFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：28)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQFFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 28)

K50A：氨基酸序列为SEQ ID No：29；K50A: the amino acid sequence is SEQ ID No: 29;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFAQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序:SEQ ID No：29)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFAQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 29)

S47A：氨基酸序列为SEQ ID No：30；S47A: the amino acid sequence is SEQ ID No: 30;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNAVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：30)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNAVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 30)

N46K：氨基酸序列为SEQ ID No：31；N46K: the amino acid sequence is SEQ ID No: 31;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINKSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：31)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINKSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 31)

E41A：氨基酸序列为SEQ ID No：32；E41A: the amino acid sequence is SEQ ID No: 32;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGAVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：32)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGAVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 32)

K32A：氨基酸序列为SEQ ID No：33；K32A: the amino acid sequence is SEQ ID No: 33;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIAGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：33)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIAGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 33)

K32R：氨基酸序列为SEQ ID No：34；K32R: the amino acid sequence is SEQ ID No: 34;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIRGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：34)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIRGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 34)

K34A：氨基酸序列为SEQ ID No：35；K34A: the amino acid sequence is SEQ ID No: 35;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGAEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：35)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGAEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 35)

E35A：氨基酸序列为SEQ ID No：36；E35A: the amino acid sequence is SEQ ID No: 36;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKAVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：36)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKAVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 36)

I31N：氨基酸序列为SEQ ID No：37；I31N: the amino acid sequence is SEQ ID No: 37;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATDNKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：37)SSSHPIFHRGEFSVCDSVSVWVGDKTTATDNKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 37)

D30N：氨基酸序列为SEQ ID No：38；D30N: the amino acid sequence is SEQ ID No: 38;

SSSHPIFHRGEFSVCDSVSVWVGDKTTATNIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：38)SSSHPIFHRGEFSVCDSVSVWVGDKTTATNIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 38)

T27E：氨基酸序列为SEQ ID No：39；T27E: the amino acid sequence is SEQ ID No: 39;

SSSHPIFHRGEFSVCDSVSVWVGDKTEATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：39)SSSHPIFHRGEFSVCDSVSVWVGDKTEATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 39)

T26K：氨基酸序列为SEQ ID No：40；T26K: the amino acid sequence is SEQ ID No: 40;

SSSHPIFHRGEFSVCDSVSVWVGDKKTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：40)SSSHPIFHRGEFSVCDSVSVWVGDKKTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 40)

K25Q：氨基酸序列为SEQ ID No：41；K25Q: the amino acid sequence is SEQ ID No: 41;

SSSHPIFHRGEFSVCDSVSVWVGDQTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：41)SSSHPIFHRGEFSVCDSVSVWVGDQTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 41)

D24A：氨基酸序列为SEQ ID No：42；D24A: the amino acid sequence is SEQ ID No: 42;

SSSHPIFHRGEFSVCDSVSVWVGAKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：42)SSSHPIFHRGEFSVCDSVSVWVGAKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 42)

D16A：氨基酸序列为SEQ ID No：43；D16A: the amino acid sequence is SEQ ID No: 43;

SSSHPIFHRGEFSVCASVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：43)SSSHPIFHRGEFSVCASVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 43)

S13M：氨基酸序列为SEQ ID No：44；S13M: the amino acid sequence is SEQ ID No: 44;

SSSHPIFHRGEFMVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：44)SSSHPIFHRGEFMVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 44)

F12A：氨基酸序列为SEQ ID No：45；F12A: the amino acid sequence is SEQ ID No: 45;

SSSHPIFHRGEASVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：45)SSSHPIFHRGEASVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 45)

E11A：氨基酸序列为SEQ ID No：46；E11A: the amino acid sequence is SEQ ID No: 46;

SSSHPIFHRGAFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：46)SSSHPIFHRGAFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 46)

H8A：氨基酸序列为SEQ ID No：47；H8A: the amino acid sequence is SEQ ID No: 47;

SSSHPIFARGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：47)SSSHPIFARGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 47)

P5A：氨基酸序列为SEQ ID No：48；P5A: the amino acid sequence is SEQ ID No: 48;

SSSHAIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：48)SSSHAIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 48)

H4D：氨基酸序列为SEQ ID No：49。H4D: The amino acid sequence is SEQ ID No:49.

SSSDPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA(氨基酸序列:SEQ ID No：49)SSSDPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA (amino acid sequence: SEQ ID No: 49)

所述NGF突变体可以是上述氨基酸突变位点中的一个突变得到的，也可以是其中两个位点、三个甚至是多个位点同时突变得到的NGF突变体。如：在野生型hNGF的氨基酸序列基础，可得到D30N与T27E同时突变的NGF突变体、K34A、E35A及I31N三个位点同时突变的NGF突变体等。The NGF mutant may be obtained by mutation of one of the above amino acid mutation sites, or may be an NGF mutant obtained by simultaneous mutation of two sites, three or even multiple sites. For example, based on the amino acid sequence of wild-type hNGF, NGF mutants in which D30N and T27E are mutated at the same time, and NGF mutants in which three sites K34A, E35A and I31N are mutated at the same time can be obtained.

一种表达载体，含有所述的核苷酸序列(即编码低痛神经生长因子的核苷酸序列)。An expression vector containing the nucleotide sequence (ie the nucleotide sequence encoding low pain nerve growth factor).

所述表达载体选自由DNA载体和病毒载体所组成的组中。The expression vector is selected from the group consisting of DNA vectors and viral vectors.

所述DNA载体选自由DNA质粒载体、结合其的脂质体、结合其的分子耦联体和结合其的多聚物所组成的组中；优选地，所述DNA质粒载体为真核细胞表达载体、原核细胞表达载体或丝状真菌表达载体；所述病毒载体选自由腺相关病毒载体、慢病毒载体、腺病毒载体及杆状病毒载体所组成的组中。更优选地，所述DNA质粒载体为真核细胞表达载体；所述病毒载体选自由腺相关病毒载体、慢病毒载体和腺病毒载体所组成的组中。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 expressed in eukaryotic cells A vector, a prokaryotic cell expression vector or a filamentous fungus expression vector; the viral vector is selected from the group consisting of adeno-associated virus vector, lentivirus vector, adenovirus vector and baculovirus vector. More preferably, the DNA plasmid vector is a eukaryotic cell expression vector; and the viral vector is selected from the group consisting of adeno-associated virus vector, lentivirus vector and adenovirus vector.

一种表达所述表达载体的方法，将所述的表达载体转化至宿主细胞，培养所得重组细胞表达得到低痛神经生长因子突变体。A method for expressing the expression vector, which comprises transforming the expression vector into a host cell, and culturing the obtained recombinant cell to express a low-pain nerve growth factor mutant.

含有所述表达载体的宿主细胞。A host cell containing the expression vector.

所述宿主细胞为哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌、丝状真菌或植物细胞。优选地，所述的宿主细胞为哺乳动物细胞。The host cells are mammalian cells, yeast cells, insect cells, E. coli, filamentous fungi or plant cells. Preferably, the host cells are mammalian cells.

所述哺乳动物细胞为中国仓鼠卵巢细胞、人胚肾293细胞、COS细胞或Hela细胞。所述昆虫细胞为SF9细胞、SF21细胞、SF900细胞。The mammalian cells are Chinese hamster ovary cells, human embryonic kidney 293 cells, COS cells or Hela cells. The insect cells are SF9 cells, SF21 cells and SF900 cells.

一种药物组合物，所述药物组合物含有药学上可接受的赋形剂，以及上述提及的低痛神经生长因子突变体、上述提及的表达载体和上述提及的宿主细胞中的一种或几种。A pharmaceutical composition comprising a pharmaceutically acceptable excipient, and one of the above-mentioned low-pain nerve growth factor mutant, the above-mentioned expression vector and the above-mentioned host cell. species or several.

本发明的药物可以制成注射剂、胶囊、片剂或粉剂等多种形式，上述各种剂型的药物均可以按照药学领域的常规方法制备。The medicament of the present invention can be made into various forms such as injection, capsule, tablet or powder, and the medicaments of the above-mentioned various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.

所述药物组合物优选为注射液，所述注射液包括药学上可接受的赋形剂以及上述提及的低痛神经生长因子突变体。The pharmaceutical composition is preferably an injection solution, which includes a pharmaceutically acceptable excipient and the aforementioned low-pain NGF mutant.

需要的时候，在上述药物组合物中还可以加入一种或多种药学上可接受的载体，所述载体包括药学领域常规的稀释剂、稳定剂、表面活性剂和防腐剂等。When necessary, one or more pharmaceutically acceptable carriers can also be added to the above-mentioned pharmaceutical compositions, and the carriers include conventional diluents, stabilizers, surfactants, preservatives and the like in the pharmaceutical field.

所述的低痛神经生长因子突变体用于制备治疗神经系统疾病的药物中的用途。Use of the low-pain nerve growth factor mutant for preparing a medicament for treating nervous system diseases.

所述的低痛神经生长因子突变体用于制备有效减轻体重的药物中的用途。Use of the low-pain nerve growth factor mutant for preparing a medicine for effectively reducing body weight.

所述的低痛神经生长因子突变体用于制备治疗眼科相关疾病的药物中的用途。Use of the low-pain nerve growth factor mutant for preparing a medicament for treating ophthalmology-related diseases.

所述的低痛神经生长因子突变体用于制备治疗生殖系统相关疾病的药物中的用途。The use of the low-pain NGF mutant in the preparation of a medicament for treating reproductive system-related diseases.

所述的低痛神经生长因子突变体用于制备治疗自身免疫相关疾病的药物中的用途。Use of the low-pain nerve growth factor mutant for preparing a medicament for treating autoimmune-related diseases.

所述的低痛神经生长因子突变体用于制备治疗骨相关疾病的药物中的用途。Use of the low-pain NGF mutant for preparing a medicament for treating bone-related diseases.

序列表SEQ ID No：3至SEQ ID No：49中任意一条所示的氨基酸序列用于制备低痛神经生长因子制剂的用途。Use of the amino acid sequence shown in any one of SEQ ID No: 3 to SEQ ID No: 49 in the sequence listing for preparing a low-pain nerve growth factor preparation.

序列表SEQ ID No：3至SEQ ID No：49中任意一条所示的氨基酸序列用于制备无痛神经生长因子制剂的用途。Use of the amino acid sequence shown in any one of SEQ ID No: 3 to SEQ ID No: 49 in the sequence listing for the preparation of painless nerve growth factor preparations.

本发明的优点在于：相对于现有技术中的野生型神经生长因子而言，本公开的低痛神经生长因子可以减轻疼痛副作用甚至无痛，可增加患者对神经生长因子的依从性和耐受性，可加大使用剂量及受试人群，为扩大适应症以及应用于中枢神经系统提供了可能。The advantages of the present invention are: compared with the wild-type nerve growth factor in the prior art, the low-pain nerve growth factor disclosed in the present disclosure can reduce pain side effects or even be painless, and can increase the compliance and tolerance of the patient to the nerve growth factor It can increase the dosage and test population, and provide the possibility for expanding the indications and applying it to the central nervous system.

以下结合附图和具体实施方式对本发明做进一步说明，并非对本发明的限定，凡依照本发明公开内容所进行的本领域等同替换，均属于本发明保护范围。The present invention will be further described below with reference to the accompanying drawings and specific embodiments, which are not intended to limit the present invention, and all equivalent replacements in the field according to the disclosure content of the present invention belong to the protection scope of the present invention.

附图说明Description of drawings

图1为实施例4Superdex75柱纯化的野生型hNGF的SDS PAGE电泳结果Fig. 1 is the SDS PAGE electrophoresis result of wild-type hNGF purified by Superdex75 column in Example 4

图2为实施例6短期给药小鼠痛阈测定结果Fig. 2 isembodiment 6 short-term administration mouse pain threshold measurement result

图3(A)和图3(B)为实施例6长期给药小鼠痛阈测定结果Fig. 3(A) and Fig. 3(B) are the results of pain threshold measurement in mice with long-term administration of Example 6

图4为实施例7小鼠行为学实验结果，分别注射突变体、野生型hNGF，观测小鼠抬腿维持时间Figure 4 shows the results of the behavioral experiment of mice in Example 7. The mutant and wild-type hNGF were injected respectively, and the maintenance time of the mice raising their legs was observed.

具体实施方式Detailed ways

实施例1.野生型h NGF及其突变体的质粒构建Example 1. Plasmid construction of wild-type hNGF and its mutants

1、构建野生型hNGF的DNA序列表达质粒1. Construction of the DNA sequence expression plasmid of wild-type hNGF

合成野生型hNGF的DNA序列(序列表SEQ ID NO：1)，使用引物(F：GGAATTCATGTCCATGTTG(序列表SEQ ID NO：50)，R：CAAGCTTTCAGGCTCTTCT(序列表SEQ IDNO：51)对目的序列进行PCR扩增，使用EcorI(NEB#R0101S)进行酶切后，将酶切产物使用HindIII(NEB#R0104S)进行二次酶切。将pcDNA3.1(-)表达载体使用同样的方法进行酶切。将酶切载体和PCR扩增目的片段进行琼脂糖凝胶电泳，切下目的片段，使用DNA胶回收试剂盒(TIANGEN，#DP209-03)分别回收酶切后的载体和目的DNA片段后，使用DNA连接酶试剂盒(Takara/6022)16℃连接1h，完成野生型hNGF的质粒构建。The DNA sequence of wild-type hNGF (SEQ ID NO: 1 in the sequence listing) was synthesized, and the target sequence was amplified by PCR using primers (F: GGAATTCATGTCCATGTTG (SEQ ID NO: 50 in the sequence listing), R: CAAGCTTTCAGGCTCTTCT (SEQ ID NO: 51 in the sequence listing). In addition, after using EcorI (NEB#R0101S) for digestion, the digestion product was digested with HindIII (NEB#R0104S). The pcDNA3.1(-) expression vector was digested using the same method. The enzyme Divide the vector and PCR-amplified target fragments for agarose gel electrophoresis, cut out the target fragments, and use a DNA gel recovery kit (TIANGEN, #DP209-03) to recover the digested vector and target DNA fragments, respectively, and then use DNA ligation. The enzyme kit (Takara/6022) was ligated at 16°C for 1 h to complete the plasmid construction of wild-type hNGF.

2、构建hNGF突变体的DNA序列表达质粒2. Construction of hNGF mutant DNA sequence expression plasmid

同样上述方法，我们分别合成了编码序列表SEQ ID NO：3至SEQ ID No：49所示的低痛神经生长因子突变体的基因，并分别构建了含有上述编码基因的质粒。In the same way as above, we synthesized the genes encoding the low-pain nerve growth factor mutants shown in SEQ ID NO: 3 to SEQ ID No: 49 in the sequence listing, and constructed plasmids containing the above-mentioned encoding genes.

实施例2、hNGF及其突变体的质粒转化及提取Example 2. Plasmid transformation and extraction of hNGF and its mutants

1、转化1. Conversion

将上述实施例1构建好的hNGF及其突变体质粒进行热激转化，将Top10感受态细胞(天根/CB104-02)从-70℃冰箱取出后立即置于冰上解冻，取50ul用于转化。向其中加入2ul质粒，轻弹混匀，冰浴30min。于42℃干浴90s，期间不要振荡离心管，取出后立即置于冰上2min。加入无抗LB/SOC培养基500ul，150rpm/min，37℃摇床培养45min。将离心管内液体全部倒入LB平板上，均匀涂开。待平板晾干后，倒置于培养箱培养16h。The hNGF and its mutant plasmids constructed in the above Example 1 were subjected to heat shock transformation, and the Top10 competent cells (Tiangen/CB104-02) were taken out from the -70°C refrigerator and thawed on ice immediately, and 50ul was used for thawing. transform. 2ul plasmid was added to it, mixed by flicking, and ice-bathed for 30min. Dry bath at 42°C for 90s, do not shake the centrifuge tube during this period, and immediately place it on ice for 2min. Add 500ul of anti-anti-LB/SOC medium, 150rpm/min, and incubate at 37°C for 45min. Pour all the liquid in the centrifuge tube onto the LB plate and spread it evenly. After the plates were air-dried, they were placed upside down in an incubator for 16 h.

2、质粒大提2. Plasmid lift

挑取上述转化步骤得到的单菌落接种于500ul LB液体培养基，37℃培养7h，同时菌液送检测序。将测序正确的菌液进行大量摇菌，将500ul菌液接种于500mlLB培养基，37℃培养16h。4℃离心收集过夜培养的菌液，6000xg离心10分钟，充分弃去上清使用质粒大提试剂盒(购自QIAGEN，货号12163)大提质粒，测量浓度备用。The single colony obtained in the above transformation step was picked and inoculated into 500ul LB liquid medium, cultivated at 37°C for 7h, and the bacterial liquid was sent to the detection sequence at the same time. Shake the bacteria with the correct sequencing in a large amount, inoculate 500ul of the bacteria into 500ml of LB medium, and cultivate at 37°C for 16h. Collect overnight cultured bacterial liquid by centrifugation at 4°C, centrifuge at 6000×g for 10 minutes, fully discard the supernatant, and use a plasmid extraction kit (purchased from QIAGEN, Cat. No. 12163) to maximize the plasmid, and measure the concentration for later use.

实施例3.野生型hNGF及其突变体的表达Example 3. Expression of wild-type hNGF and its mutants

将上述实施例2大提得到的野生型hNGF和及其突变体质粒转染293F细胞中，转染后第四日收集表达上清并定量。The wild-type hNGF and its mutant plasmids obtained in Example 2 above were transfected into 293F cells, and the expression supernatant was collected and quantified on the fourth day after transfection.

实验步骤：Experimental steps:

1、转染前一天，接种293F细胞，0.5×106/ml，共900ml，300ml/瓶。1. One day before transfection, inoculate 293F cells, 0.5×106 /ml, a total of 900ml, 300ml/vial.

2、转染当天计数，细胞密度约1.0×106/ml，活率99％以上。2. Count on the day of transfection, the cell density is about 1.0×106 /ml, and the viability rate is over 99%.

3、转染：取36ml细胞培养基到125ml培养瓶；加360ug质粒，混匀；再加1080ug PEI，混匀。室温静置15分钟；与细胞混匀，约12.3ml/瓶；37℃，8％CO2，120RPM培养。3. Transfection: Take 36ml of cell culture medium into a 125ml culture flask; add 360ug of plasmid, mix well; add 1080ug of PEI, mix well. Let stand for 15 minutes at room temperature; mix with cells, about 12.3ml/flask; cultivate at 37°C, 8% CO2 , 120RPM.

4、转染后第四日收集细胞上清，10000g离心20分钟。4. The cell supernatant was collected on the fourth day after transfection, and centrifuged at 10,000g for 20 minutes.

5、收集上清，0.45um过滤，得到野生型hNGF及其突变体的蛋白上清液。5. Collect the supernatant and filter with 0.45um to obtain the protein supernatant of wild-type hNGF and its mutants.

6、SDS-PAGE检测，硝酸银染色定量。6. Detected by SDS-PAGE and quantified by silver nitrate staining.

实施例4.野生型hNGF及其突变体的纯化Example 4. Purification of wild-type hNGF and its mutants

纯化上述实施例3中得到的NGF及其突变体的蛋白上清液。The protein supernatant of NGF and its mutants obtained in Example 3 above was purified.

1、阳离子交换层析：先使用乙酸和水调野生型hNGF及其突变体的蛋白上清液至pH为4.0。CM Sepharose FF层析柱用0.05mol/L乙酸盐缓冲液(pH4.0)充分平衡后上样，上样完成后，用平衡液冲洗至基线，用0.05mol/L Tris-HC1(pH9.0)缓冲液洗脱杂峰至基线，再用0.05mol/LTris-HC1和0.05mol/L Tris-HC1-0.4mol/L NaCl(pH9.0)梯度洗脱，根据紫外吸收情况收集目标峰，在紫外检测仪上显示数字开始上升时开始收集，降为基线时停止收集目标蛋白峰。1. Cation-exchange chromatography: First, adjust the protein supernatant of wild-type hNGF and its mutants to pH 4.0 with acetic acid and water. The CM Sepharose FF chromatography column was fully equilibrated with 0.05mol/L acetate buffer (pH4.0) and loaded. 0) Elute the impurity peak to the baseline with buffer, then use 0.05mol/L Tris-HCl and 0.05mol/L Tris-HCl-0.4mol/L NaCl (pH9.0) gradient elution, collect the target peak according to the UV absorption, Start collecting when the number displayed on the UV detector starts to rise, and stop collecting the target protein peak when it falls to the baseline.

2、疏水层析：Butyl Sepharose 4FF层析柱经0.02mol/L磷酸盐(pH6.8)-1.5mol/L氯化钠缓冲液充分平衡。在步骤1收集的目标峰料液中加入氯化钠固体使料液中氯化钠的终浓度为1.5mol/L，待氯化钠充分溶解后上样，速度为120cm/h，上样完成后用平衡液冲洗至基线，然后用0.02mol/L磷酸盐(pH6.8)洗脱收集目标峰。2. Hydrophobic chromatography: Butyl Sepharose 4FF chromatography column is fully equilibrated with 0.02mol/L phosphate (pH6.8)-1.5mol/L sodium chloride buffer. Add sodium chloride solid to the target peak feed liquid collected instep 1 so that the final concentration of sodium chloride in the feed liquid is 1.5mol/L. After the sodium chloride is fully dissolved, the sample is loaded at a speed of 120cm/h, and the sample loading is completed. Afterwards, it was washed to the baseline with equilibration solution, and then eluted with 0.02mol/L phosphate (pH6.8) to collect the target peak.

3、凝胶排阻层析：Superdex 75prep grade层析柱用pH6.8、0.05mol/L磷酸盐-0.15mol/L氯化钠缓冲液充分平衡后上样步骤/2中收集的目标峰，在紫外检测仪上显示数字从基线开始上升时开始收集，降为基线时停止收集目标蛋白峰。3. Gel size exclusion chromatography: The target peak collected in the sample loading step/2 after the Superdex 75prep grade chromatography column was fully equilibrated with pH6.8, 0.05mol/L phosphate-0.15mol/L sodium chloride buffer, When the number displayed on the UV detector starts to rise from the baseline, the collection starts, and when it falls to the baseline, the collection of the target protein peak is stopped.

Superdex75柱纯化的野生型hNGF的SDS PAGE如图1所示，说明制备得到的NGF纯度较高。将收集的野生型hNGF及其突变体目标蛋白峰样品使用超滤管浓缩至0.4mg/ml，4℃保存用于后续实验。The SDS PAGE of wild-type hNGF purified by Superdex75 column is shown in Figure 1, indicating that the prepared NGF is of high purity. The collected wild-type hNGF and its mutant target protein peak samples were concentrated to 0.4 mg/ml using an ultrafiltration tube, and stored at 4°C for subsequent experiments.

实施例5.鸡胚法测定野生型hNGF及其突变体活性Example 5. Determination of wild-type hNGF and its mutant activity by chicken embryo method

1、鸡胚背根神经节法测量野生型hNGF及其突变体活性1. Chick embryo dorsal root ganglion method to measure the activity of wild-type hNGF and its mutants

将上述实施例4中得到的野生型hNGF及其突变体样品进行稀释：A液：6ng提取的野生型hNGF及其突变体样品加1ml无血清DMEM培养液溶解；B液：取A液50μl加无血清DMEM培养液4.95ml；C液：取B液60μl加无血清DMEM培养液2.94ml(总量3ml)使终浓度为(3AU/ml)。A、B液稀释在离心管中，C液在细胞瓶内，将C液作为1号瓶，再3倍梯度稀释为：2号、3号、4号、5号、6号待测液。每个待测液加入1个培养瓶，2ml/瓶。同时以无血清DMEM培养液为空白对照，以购自中国食品药品检定研究院的标准品为阳性对照(参考品)。加入8日龄的鸡胚背根神经节后置于5％CO2、37℃饱和湿度培养箱中，24小时后观察结果。Dilute the wild-type hNGF and its mutant samples obtained in the above Example 4: liquid A: 6 ng of the extracted wild-type hNGF and its mutant samples are dissolved in 1 ml of serum-free DMEM medium; liquid B: take 50 μl of liquid A and add Serum-free DMEM medium 4.95ml; C liquid: take 60μl of B liquid and add serum-free DMEM medium 2.94ml (total 3ml) to make the final concentration (3AU/ml). The solution A and B are diluted in centrifuge tubes, and the solution C is in the cell flask. The solution C is used as the No. 1 bottle, and then 3-fold gradient dilution is: No. 2, No. 3, No. 4, No. 5, No. 6 to be tested. Add 1 culture bottle to each solution to be tested, 2ml/bottle. At the same time, the serum-free DMEM medium was used as blank control, and the standard substance purchased from China National Institute for Food and Drug Control was used as positive control (reference substance). 8-day-old chick embryo dorsal root ganglion was added and placed in a 5% CO2 , 37° C. saturated humidity incubator, and the results were observed after 24 hours.

以生长最好时每毫升待测样品中NGF的含量作为1个活性单位(AU)。从出现阴性对照结果的稀释度开始往回数第3和第4两个稀释度中取生长最好的作为判定终点计算效价。参考品为购自中检院的标准品，每支装量为1000AU。The content of NGF in each milliliter of the sample to be tested when the growth was the best was taken as 1 activity unit (AU). The titer with the best growth is taken as the end point to determine the titer among the third and fourth dilutions from the dilution at which the negative control result appears. The reference product is the standard product purchased from the China National Inspection and Quarantine Institute, and the volume of each bottle is 1000AU.

NGF比活的计算公式为：The formula for calculating the specific activity of NGF is:

待测样品的比活(AU/mg)＝参考品活性(AU/ml)×[样品预稀释倍数×对应参考品稀释点处的活性(AU/ml)/参考品实测活性(AU/ml)]The specific activity of the sample to be tested (AU/mg) = the activity of the reference product (AU/ml) × [the pre-dilution multiple of the sample × the activity at the dilution point of the corresponding reference product (AU/ml) / the measured activity of the reference product (AU/ml) ]

测活结果表明，低痛hNGF突变体，均保留了野生型hNGF的活性。The test results showed that the low-pain hNGF mutants retained the activity of wild-type hNGF.

实施例6.NGF及其突变体是否致痛(疼痛阈值)检测Example 6. Detection of whether NGF and its mutants cause pain (pain threshold)

实验原理：筛选对痛觉反应正常的合格小鼠，注射一定剂量的NGF样品(野生型或其突变体)，通过机械刺激测定小鼠曲爪反应疼痛阈值，进行统计分析，最终确定样品是否引起小鼠痛觉过敏。Experimental principle: Screen qualified mice with normal pain response, inject a certain dose of NGF sample (wild type or its mutant), measure the pain threshold of the mouse's paw-curving response through mechanical stimulation, and conduct statistical analysis to determine whether the sample induces pain. Rat hyperalgesia.

6-1、短期致痛情况观测6-1. Observation of short-term pain

一、实验材料1. Experimental materials

动态足底触觉仪，意大利/Ugo Basile，型号37450。Dynamic plantar haptics, Italy/Ugo Basile, model 37450.

二、实验内容2. Experimental content

1、合格小鼠筛选1. Screening of qualified mice

订购SPF级别CD-1小鼠，小鼠规格为雄性，体重30-35g。Order SPF grade CD-1 mice, the mouse specification is male, weighing 30-35g.

使用动态足底触觉仪[意大利/Ugo Basile，型号37450]筛选两足平均阈值在7.5-10之间且同一只小鼠左右脚阈值P值，P值>0.05的实验动物为合格小鼠。Using a dynamic plantar tactile instrument [Italy/Ugo Basile, model 37450], the average threshold of both feet was between 7.5-10 and the P value of the left and right foot thresholds of the same mouse, and the experimental animals with P value>0.05 were qualified mice.

随机分组，分为实验组和空白对照组，实验组又按照不同样品及给药剂量分为小组，每组10只。Randomly divided into experimental group and blank control group, the experimental group was divided into groups according to different samples and doses, 10 in each group.

2、NGF样品给药设计2. NGF sample administration design

2.1野生型样品致痛剂量筛选2.1 Screening of pain-inducing dose of wild-type samples

药物配制：将阳性对照NGF野生型样品、NGF S47A突变体、NGF I31N突变体样品均使用样品储备液(50mM PB,150mM NaCl，PH6.8)进行稀释。Drug formulation: Positive control NGF wild-type samples, NGF S47A mutant, NGF I31N mutant samples were all diluted with sample stock solution (50 mM PB, 150 mM NaCl, pH 6.8).

空白对照：为NGF样品储备液。Blank control: NGF sample stock solution.

给药方式及剂量：小鼠左右脚足底皮下分别给药20ul；Mode of administration and dosage: 20ul subcutaneously on the left and right feet of mice;

短期给药最低剂量为1.25μg/只。The lowest dose for short-term administration is 1.25 μg/only.

3、痛阈测定3. Pain threshold determination

在给药后分别在1个小时、2个小时进行机械阈值测定，并且记录数值，进行短期给药(2小时内)的观测。Mechanical threshold measurements were performed at 1 hour and 2 hours after administration, and the values were recorded for observation of short-term administration (within 2 hours).

4、结果统计分析4. Statistical analysis of results

采用GraphPad Prism软件进行图表绘制及结果统计分析，比较各剂量组与对照组之间机械阈值是否有差异，分析野生型样品以及突变体样品的致痛性。GraphPad Prism software was used for graph drawing and statistical analysis of the results, to compare whether there was a difference in mechanical threshold between each dose group and the control group, and to analyze the pain-inducing properties of wild-type samples and mutant samples.

由图2可知，给药最低剂量为1.25μg/只时，对照组小鼠无疼痛表现，而野生型的阳性对照组，疼痛阈值在1h明显低于5以下，疼痛明显；而各突变体实验组疼痛阈值均在6至7左右，与阳性对照组相比，疼痛明显减轻。It can be seen from Figure 2 that when the lowest dose of 1.25 μg/mice was administered, the mice in the control group showed no pain, while in the wild-type positive control group, the pain threshold was significantly lower than 5 for 1 h, and the pain was obvious; The pain thresholds of the groups were all around 6 to 7, and the pain was significantly reduced compared with the positive control group.

6-2长期致痛情况观测6-2 Observation of long-term pain

除设定本次实验的3个给药剂量为：0.2μg/只、0.5μg/只、1.25μg/只，每天给药一次，连续3周，期间不间断的进行疼痛阈值的测定外，其余均按照6-1“短期致痛情况观测”中的方法进行。Except that the 3 doses in this experiment were set as: 0.2 μg/a, 0.5 μg/a, 1.25 μg/a, administered once a day for 3 consecutive weeks, and the pain threshold was measured continuously during the period. All were carried out according to the method in 6-1 "Short-term Pain Observation".

结果如图3(A)和3(B)所示，表明：野生型NGF在17天以内三个剂量显示痛阈逐渐降低，逐渐出现疼痛感。S47A突变体、I31N突变体中低高三个剂量组的实验结果显示在14天内除S47A突变体高剂量组痛阈明显降低引起疼痛外，其余剂量组痛阈基本都维持在6以上，与野生型相比有明显减轻疼痛的作用。The results are shown in Figures 3(A) and 3(B), indicating that the three doses of wild-type NGF within 17 days gradually decreased the pain threshold and gradually appeared pain. The experimental results of the three dose groups of S47A mutant and I31N mutant showed that in 14 days, except for the pain threshold of S47A mutant high-dose group was significantly reduced, the pain thresholds of other dose groups were basically maintained above 6, which was similar to the wild type. significantly relieves pain.

实施例7.野生型hNGF及其突变体是否致痛的行为学检测Example 7. Behavioral detection of whether wild-type hNGF and its mutants cause pain

野生型hNGF及其突变体样品对小鼠关节给药，通过行为学观察小鼠抬腿时间以及次数来考察样品是否致痛。Wild-type hNGF and its mutant samples were administered to the joints of mice, and whether the samples caused pain was observed by behavioral observation of the time and times of leg raising.

实验内容Experimental content

1、小鼠订购1. Mice order

订购SPF级别CD-1小鼠，小鼠规格为雄性，体重30-35g。随机分组，分为实验组、空白对照组(简称对照组)及阳性对照组，各组根据剂量又分为三个小组，每个小组随机挑选6只。Order SPF grade CD-1 mice, the mouse specification is male, weighing 30-35g. Randomly divided into experimental group, blank control group (referred to as control group) and positive control group, each group was divided into three groups according to the dose, and 6 mice were randomly selected from each group.

2、给药剂量与时间2. Dosage and time of administration

2.1给药剂量2.1 Dosage

阳性对照：采用样品储备液(50mM PB,150mM NaCl，PH6.8)稀释野生型hNGF为1.25μg/10ul；Positive control: use sample stock solution (50mM PB, 150mM NaCl, pH6.8) to dilute wild-type hNGF to 1.25μg/10ul;

实验组：突变体药物配制方法同阳性对照，稀释NGF I31N突变体为1.25μg/10ul；Experimental group: The preparation method of mutant drug is the same as that of positive control, and the dilution of NGF I31N mutant is 1.25μg/10ul;

空白对照：生理盐水。Blank control: saline.

2.2给药方式2.2 Mode of administration

在小鼠后腿关节内注射药品，每个关节腔给药10ul。The drug was injected intra-articularly in the hind legs of mice, and 10ul was administered to each joint cavity.

2.3给药时间：2.3 Administration time:

每剂量组进行单次连续3-4天给药。即第一天上午10点，以及之后第2、3、4天相同时间点给药。Each dose group was administered as a single dose for 3-4 consecutive days. That is, 10:00 am on the first day, and the same time point on the 2nd, 3rd, and 4th days after that.

3、行为学观察3. Behavioral observation

各实验组以及对照组当天给药后2、4小时进行观察，以及给药的第2、3、4天相同时间点进行观察。Each experimental group and control group were observed 2 and 4 hours after administration on the same day, and at the same time points on the 2nd, 3rd, and 4th days of administration.

观察指标：2分钟内小鼠自发性抬腿的次数以及每次抬腿维持的时间(秒)，计算抬腿维持累计时间。Observation indicators: The number of spontaneous leg lifts within 2 minutes and the maintenance time (seconds) of each leg lift, and the cumulative leg lift maintenance time was calculated.

4、结果统计分析4. Statistical analysis of results

采用GraphPad Prism软件双因素方差分析抬腿维持时间，比较分析不同样品是否致痛。Two-way ANOVA of GraphPad Prism software was used to analyze the maintenance time of leg raising, and to compare and analyze whether different samples caused pain.

实验结果如图4所示：野生型hNGF组在注射药物后的各检测时间点均能引起明显的疼痛，I31N突变体实验组连续给药疼痛现象不明显，抬腿维持累计时间均在1秒以下卡方分析得知在各检测时间点阳性对照组与实验组均有明显差异，说明I31N突变体可明显减轻药物注射的疼痛反应。The experimental results are shown in Figure 4: the wild-type hNGF group can cause obvious pain at each detection time point after the injection of the drug, while the I31N mutant experimental group has no obvious pain after continuous administration, and the cumulative time of leg raising is within 1 second. The following chi-square analysis shows that there are significant differences between the positive control group and the experimental group at each detection time point, indicating that the I31N mutant can significantly reduce the pain response to drug injection.

序列表sequence listing

<110> 舒泰神（北京）生物制药股份有限公司<110> Shutaishen (Beijing) Biopharmaceutical Co., Ltd.

<120> 低痛神经生长因子突变体<120> low pain nerve growth factor mutant

<130><130>

<160> 51<160> 51

<170> PatentIn version 3.5<170> PatentIn version 3.5

<210> 1<210> 1

<211> 726<211> 726

<212> DNA<212> DNA

<213> 野生型hNGF的DNA序列<213> DNA sequence of wild-type hNGF

<400> 1<400> 1

atgtccatgt tgttctacac tctgatcaca gcttttctga tcggcataca ggcggaacca 60atgtccatgt tgttctacac tctgatcaca gcttttctga tcggcataca ggcggaacca 60

cactcagaga gcaatgtccc tgcaggacac accatccccc aagcccactg gactaaactt 120cactcagaga gcaatgtccc tgcaggacac accatcccccc aagcccactg gactaaactt 120

cagcattccc ttgacactgc ccttcgcaga gcccgcagcg ccccggcagc ggcgatagct 180cagcattccc ttgacactgc ccttcgcaga gcccgcagcg ccccggcagc ggcgatagct 180

gcacgcgtgg cggggcagac ccgcaacatt actgtggacc ccaggctgtt taaaaagcgg 240gcacgcgtgg cggggcagac ccgcaacatt actgtggacc ccaggctgtt taaaaagcgg 240

cgactccgtt caccccgtgt gctgtttagc acccagcctc cccgtgaagc tgcagacact 300cgactccgtt caccccgtgt gctgtttagc acccagcctc cccgtgaagc tgcagacact 300

caggatctgg acttcgaggt cggtggtgct gcccccttca acaggactca caggagcaag 360caggatctgg acttcgaggt cggtggtgct gcccccttca acaggactca caggagcaag 360

cggtcatcat cccatcccat cttccacagg ggcgaattct cggtgtgtga cagtgtcagc 420cggtcatcat cccatcccat cttccacagg ggcgaattct cggtgtgtga cagtgtcagc 420

gtgtgggttg gggataagac caccgccaca gacatcaagg gcaaggaggt gatggtgttg 480gtgtgggttg gggataagac caccgccaca gacatcaagg gcaaggaggt gatggtgttg 480

ggagaggtga acattaacaa cagtgtattc aaacagtact tttttgagac caagtgccgg 540ggagaggtga acattaacaa cagtgtattc aaacagtact ttttttgagac caagtgccgg 540

gacccaaatc ccgttgacag cgggtgccgg ggcattgact caaagcactg gaactcatat 600gacccaaatc ccgttgacag cgggtgccgg ggcattgact caaagcactg gaactcatat 600

tgtaccacga ctcacacctt tgtcaaggcg ctgaccatgg atggcaagca ggctgcctgg 660tgtaccacga ctcacacctt tgtcaaggcg ctgaccatgg atggcaagca ggctgcctgg 660

cggtttatcc ggatagatac ggcctgtgtg tgtgtgctca gcaggaaggc tgtgagaaga 720cggtttatcc ggatagatac ggcctgtgtg tgtgtgctca gcaggaaggc tgtgagaaga 720

gcctga 726gcctga 726

<210> 2<210> 2

<211> 120<211> 120

<212> PRT<212> PRT

<213> 野生型hNGF的氨基酸序列<213> Amino acid sequence of wild-type hNGF

<400> 2<400> 2

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 3<210> 3

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体K115A<213> Homo, species: NGF mutant K115A

<400> 3<400> 3

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Ala Ala Val Arg Arg AlaSer Arg Ala Ala Val Arg Arg Ala

115 120 115 120

<210> 4<210> 4

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体R114A<213> Homo, species: NGF mutant R114A

<400> 4<400> 4

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Ala Lys Ala Val Arg Arg AlaSer Ala Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 5<210> 5

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体R119A<213> Homo, species: NGF mutant R119A

<400> 5<400> 5

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Ala AlaSer Arg Lys Ala Val Arg Ala Ala

115 120 115 120

<210> 6<210> 6

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体L112A<213> Homo, species: NGF mutant L112A

<400> 6<400> 6

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val AlaAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Ala

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 7<210> 7

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体S113M<213> Homo, species: NGF mutant S113M

<400> 7<400> 7

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Met Arg Lys Ala Val Arg Arg AlaMet Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 8<210> 8

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体D105N<213> Homo, species: NGF mutant D105N

<400> 8<400> 8

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asn Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asn Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 9<210> 9

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体D105A<213> Homo, species: NGF mutant D105A

<400> 9<400> 9

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Ala Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Ala Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 10<210> 10

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体R103K<213> Homo, species: NGF mutant R103K

<400> 10<400> 10

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Lys Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Lys Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 11<210> 11

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体R103A<213> Homo, species: NGF mutant R103A

<400> 11<400> 11

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Ala Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Ala Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 12<210> 12

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体A97E<213> Homo, species: NGF mutant A97E

<400> 12<400> 12

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Glu Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuGlu Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 13<210> 13

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体M92E<213> Homo, species: NGF mutant M92E

<400> 13<400> 13

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Glu Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Glu Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 14<210> 14

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体K88M<213> Homo, species: NGF mutant K88M

<400> 14<400> 14

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Met Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Met Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 15<210> 15

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体K88A<213> Homo, species: NGF mutant K88A

<400> 15<400> 15

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Ala Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Ala Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 16<210> 16

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体H84A<213> Homo, species: NGF mutant H84A

<400> 16<400> 16

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr Ala Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr Ala Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 17<210> 17

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体Y79A<213> Homo, species: NGF mutant Y79A

<400> 17<400> 17

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Ala CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Ala Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 18<210> 18

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体T81A<213> Homo, species: NGF mutant T81A

<400> 18<400> 18

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Ala Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnAla Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 19<210> 19

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体N77A<213> Homo, species: NGF mutant N77A

<400> 19<400> 19

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Ala Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Ala Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 20<210> 20

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体H75A<213> Homo, species: NGF mutant H75A

<400> 20<400> 20

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys Ala Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys Ala Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 21<210> 21

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体K74A<213> Homo, species: NGF mutant K74A

<400> 21<400> 21

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Ala His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Ala His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 22<210> 22

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体D72A<213> Homo, species: NGF mutant D72A

<400> 22<400> 22

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Ala Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Ala Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 23<210> 23

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体R69A<213> Homo, species: NGF mutant R69A

<400> 23<400> 23

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Ala Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Ala Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 24<210> 24

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体R59A<213> Homo, species: NGF mutant R59A

<400> 24<400> 24

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Ala Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Ala Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 25<210> 25

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体R59K<213> Homo, species: NGF mutant R59K

<400> 25<400> 25

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Lys Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Lys Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 26<210> 26

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体K57A<213> Homo, species: NGF mutant K57A

<400> 26<400> 26

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Ala Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Ala Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 27<210> 27

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体E55A<213> Homo, species: NGF mutant E55A

<400> 27<400> 27

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Ala Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Ala Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 28<210> 28

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体Y52F<213> Homo, species: NGF mutant Y52F

<400> 28<400> 28

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Phe Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Phe Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 29<210> 29

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体K50A<213> Homo, species: NGF mutant K50A

<400> 29<400> 29

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Ala Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Ala Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 30<210> 30

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体S47A<213> Homo, species: NGF mutant S47A

<400> 30<400> 30

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ala ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ala Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 31<210> 31

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体N46K<213> Homo, species: NGF mutant N46K

<400> 31<400> 31

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Lys Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Lys Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 32<210> 32

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体E41A<213> Homo, species: NGF mutant E41A

<400> 32<400> 32

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Ala Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Ala Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 33<210> 33

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体K32A<213> Homo, species: NGF mutant K32A

<400> 33<400> 33

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile AlaSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Ala

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 34<210> 34

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体K32R<213> Homo, species: NGF mutant K32R

<400> 34<400> 34

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile ArgSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Arg

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 35<210> 35

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体K34A<213> Homo, species: NGF mutant K34A

<400> 35<400> 35

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Ala Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Ala Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 36<210> 36

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体E35A<213> Homo, species: NGF mutant E35A

<400> 36<400> 36

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Ala Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Ala Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 37<210> 37

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体I31N<213> Homo, species: NGF mutant I31N

<400> 37<400> 37

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Asn LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Asn Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 38<210> 38

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体D30N<213> Homo, species: NGF mutant D30N

<400> 38<400> 38

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asn Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asn Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 39<210> 39

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体T27E<213> Homo, species: NGF mutant T27E

<400> 39<400> 39

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Glu Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Glu Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 40<210> 40

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体T26K<213> Homo, species: NGF mutant T26K

<400> 40<400> 40

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Lys Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Lys Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 41<210> 41

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体K25Q<213> Homo, species: NGF mutant K25Q

<400> 41<400> 41

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Gln Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Gln Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 42<210> 42

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体D24A<213> Homo, species: NGF mutant D24A

<400> 42<400> 42

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Ala Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Ala Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 43<210> 43

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体D16A<213> Homo, species: NGF mutant D16A

<400> 43<400> 43

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AlaSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Ala

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 44<210> 44

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体S13M<213> Homo, species: NGF mutant S13M

<400> 44<400> 44

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Met Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Met Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 45<210> 45

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体F12A<213> Homo, species: NGF mutant F12A

<400> 45<400> 45

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Ala Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Glu Ala Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 46<210> 46

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体E11A<213> Homo, species: NGF mutant E11A

<400> 46<400> 46

Ser Ser Ser His Pro Ile Phe His Arg Gly Ala Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe His Arg Gly Ala Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 47<210> 47

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体H8A<213> Homo, species: NGF mutant H8A

<400> 47<400> 47

Ser Ser Ser His Pro Ile Phe Ala Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Pro Ile Phe Ala Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 48<210> 48

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体P5A<213> Homo, species: NGF mutant P5A

<400> 48<400> 48

Ser Ser Ser His Ala Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser His Ala Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 49<210> 49

<211> 120<211> 120

<212> PRT<212> PRT

<213> 人属，人种：NGF突变体H4D<213> Homo, species: NGF mutant H4D

<400> 49<400> 49

Ser Ser Ser Asp Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys AspSer Ser Ser Asp Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 151 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile LysSer Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30 20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser ValGly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45 35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro ValPhe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60 50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr CysAsp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 8065 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys GlnThr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95 85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val LeuAla Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110 100 105 110

Ser Arg Lys Ala Val Arg Arg AlaSer Arg Lys Ala Val Arg Arg Ala

115 120 115 120

<210> 50<210> 50

<211> 19<211> 19

<212> DNA<212> DNA

<213> 人工合成，引物F<213> Synthetic, primer F

<400> 50<400> 50

ggaattcatg tccatgttg 19ggaattcatg tccatgttg 19

<210> 51<210> 51

<211> 19<211> 19

<212> DNA<212> DNA

<213> 人工合成，引物R<213> Synthetic, primer R

<400> 51<400> 51

caagctttca ggctcttct 19caagctttca ggctcttct 19

Claims (19)

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