CN118344440B - Targeted NGF-binding polypeptide and its application - Google Patents

Abstract

The present invention relates to the field of biomedicine, and in particular to a targeted binding NGF polypeptide and its application. The polypeptide has an amino acid sequence shown in Formula I. The polypeptide provided by the present invention has a high affinity with NGF and can inhibit the binding of NGF to its receptor, thereby preventing and/or treating diseases mediated by it.

Description

Polypeptide capable of targeting NGF binding and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a polypeptide for targeting NGF binding and application thereof.

Background

Nerve Growth Factor (NGF) is the first member to be found in the neurotrophic factor family. Nerve growth factor is a nerve growth regulator, has double biological functions of providing nutrition for neurons and promoting neurite growth, and plays an important role in regulating the development, differentiation, growth, regeneration and functional expression of central and peripheral neurons. Nerve growth factor comprises three subunits, α, β and γ. The beta subunit is an active region and is formed by combining two single chains through non-covalent bonds.

For the nervous system, nerve growth factors have functions of promoting neuronal development, axon growth, transmitter synthesis, inhibiting neuronal apoptosis, etc., and have been developed clinically for the treatment of certain nervous system diseases such as glaucoma and alzheimer's disease. In addition, nerve growth factors are mainly involved in regulating immune system functions, inhibiting mitosis of part of tumor cells, promoting wound healing, etc. for other systems such as cardiovascular, immune, and reproductive systems.

NGF exerts biological effects by binding to two different receptors, the TrkA receptor and the p75 receptor. p75 belongs to the tumor necrosis factor receptor superfamily, binds all neurotrophins with the same affinity, and is a non-selective low affinity receptor. TrkA is a 140kDa transmembrane glycoprotein encoded by the NTRK1 gene, consisting of an extracellular domain, a transmembrane region and an intracellular domain containing a tyrosine kinase domain, belonging to the family of tyrosine kinase receptors (including TrkA, trkB and TrkC), is a specific high affinity receptor for NGF, mediating most biological functions and being a functional receptor therefor. NGF binding to TrkA receptor can lead to receptor dimerization, activating intrinsic kinase activity, leading to activation of a variety of signaling pathways, including Ras/MAPK, PI3K/Akt and plcγ signaling pathway (Norman B H,McDermott J S.Targeting the nerve growth factor(NGF)pathway in drug discovery[J].Journal ofmedicinal chemistry,2017,60(1):66-88),, associated with the pathophysiological processes of pain and tumor development, proliferation, angiogenesis and metastasis.

Studies have shown that peripheral nociceptors are activated to produce and release NGF under thermal, chemical or mechanical stimulation. After NGF binds to and activates TrkA receptor, on the one hand, nociceptive pain is promoted by the influx of Ca ⁺ by modulating the expression and activity of transient receptor potential cation channel (TRPV 1). On the other hand, the expression of Calcitonin Gene Related Peptide (CGRP) and Substance P (SP) is promoted, and these neuropeptides are transported to the central end of the dorsal horn of the spinal cord and released, and then bind to the central neuronal NMDA receptor to produce long-term depolarization, leading to central sensitization. In addition, the NGF/TrkA complex formed is endocytosed and retrograde transported to the neuronal cell body, modulating bradykinin receptor (BK), acid Sensitive Ion Channel (ASIC), voltage-gated sodium channel (VGSC) and transient receptor potential cation channel (TRPV 1) that are involved in nociceptive cell surface receptor expression, thereby eliciting peripheral sensitization and pain hypersensitivity. In addition, NGF stimulates mast cells to express histamine, 5-hydroxytryptamine, H ⁺, and NGF, forming a positive feedback loop, resulting in local amplification of pain signals (Shang X,Wang Z,Tao H.Mechanism and therapeutic effectiveness ofnerve growth factor in osteoarthritis pain[J].Therapeutics and clinical riskmanagement,2017,13:951).

To date, several preclinical and clinical studies have shown that targeting NGF-TrkA signaling pathways is effective in treating pain. Preclinical study data of an anti-TrkA monoclonal antibody CRB0089 is disclosed in US20130336964 A1. In the test, complete Freund's adjuvant was injected into the right hind paw pad of male Wistar rats to induce them to produce mechanical hyperalgesia, CRB0089 (5 or 20. Mu.g) was subcutaneously injected after 72 hours, PWT was measured by using a Randall Selitto type analgesic apparatus after 18 hours, and the in vivo analgesic activity of the drug was evaluated. The results show that CRB0089 has significant analgesic effects in a complete freund adjuvant induced inflammatory pain (CFA) model, and can dose-dependently reduce mechanical hyperalgesia in rats following CFA injury. US20190263932A1 discloses in vivo and in vitro activity data of an anti-TrkA monoclonal antibody, and the results also show that the anti-TrkA antibody can effectively relieve pain, and 1mg/kg anti-TrkA antibody can significantly improve four pain indicators of rats after CFA induction.

In addition, there have been studies showing that TrkA participates in the development of tumors in two ways, one is that the fusion of TrkA encoding gene NTRK1 with other genes produces chimeric oncogenes, resulting in sustained activation of TrkA kinase no longer regulated and controlled by NGF, and this structural rearrangement of TrkA is a direct cause of tumorigenesis. The other is that TrkA is over-expressed in various tumors such as breast cancer, cervical squamous carcinoma, lung squamous cell carcinoma, epithelial ovarian cancer, head and neck squamous cell carcinoma and the like, and through overactivation in a mode of depending on NGF, angiogenesis, peri-nerve infiltration (PNI) and epithelial-mesenchymal transition (EMT) of the tumors are promoted. Thus, NGF or TrkA inhibitors are able to reverse NGF-induced tumor EMT by blocking the overactivation of TrkA kinase by NGF, thereby inhibiting tumor invasion and metastasis.

Therefore, the treatment of diseases with NGF-TrkA signaling pathway as a target is the research foundation for developing analgesic or antitumor drugs.

The polypeptide is a bioactive substance composing various cell functions in a organism, has the characteristics of small relative molecular mass, high specificity, easy absorption, easy synthesis, easy transformation, capability of improving the immunity of the organism, high safety and the like, and has higher application value in clinical treatment.

In view of this, it is of great importance to develop a pharmaceutical polypeptide capable of targeting NGF binding.

Disclosure of Invention

The invention aims to provide a drug polypeptide capable of targeting NGF, and the drug polypeptide has high affinity with NGF.

To achieve the above object, a first aspect of the present invention provides a polypeptide that targets binding to NGF, or a pharmaceutically acceptable salt thereof, the polypeptide having an amino acid sequence shown in formula I:

X₁-X₂-C₁-X₃-R-X₄-X₅-Q-X₆-X₇-X₈-W-X₉-C₂-X₁₀-X₁₁-X₁₂ The compound of the formula I,

Wherein X ₁ is selected from W, M or F;

x ₂ is selected from Y, K, N, T or H;

x ₃ is selected from W, F, Y, H or M;

X ₄ is selected from M, E, Y, A, T, Q, V, K, L, or D;

X ₅ is selected from E, Q or H;

X ₆ is selected from F or W;

x ₇ is selected from H or Q;

X ₈ is selected from N, D or E;

x ₉ is selected from T, V, E, Q, S, I or A;

X ₁₀ is selected from D, E, T, I, V, K or H;

x ₁₁ is selected from Y, A, W, Q, G or H;

x ₁₂ is selected from L, G, D, N, M, T, L, V, S, W, F, Y or H.

In a second aspect the invention provides a gene whose nucleotide sequence is a nucleotide sequence capable of encoding an amino acid sequence of a polypeptide which targets NGF binding as described in the first aspect.

In a third aspect, the present invention provides a vector comprising the gene described in the second aspect.

In a fourth aspect the invention provides a host cell comprising a vector as described in the third aspect.

In a fifth aspect the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide or a pharmaceutically acceptable salt thereof which targets NGF in the first aspect.

A sixth aspect of the invention provides the use of at least one of a polypeptide targeted to bind NGF as described in the first aspect hereinbefore or a pharmaceutically acceptable salt thereof, a gene as described in the second aspect hereinbefore, a vector as described in the third aspect hereinbefore, a host cell as described in the fourth aspect hereinbefore, a pharmaceutical composition as described in the fifth aspect hereinbefore, in the manufacture of a medicament for the prophylaxis and/or treatment of a disease mediated by NGF.

The polypeptide provided by the invention is obtained through phage screening, has higher affinity with NGF, and can effectively inhibit the binding of NGF and a receptor thereof, thereby being capable of preventing and/or treating the mediated diseases.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Detailed Description

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

It is noted that unless otherwise defined herein, scientific and technical terms used in the present invention should have meanings commonly understood by those skilled in the art.

In the present invention, the term "sequence identity" refers to the degree to which two sequences (e.g., amino acids) have identical residues at identical positions after alignment. For example, "an amino acid sequence is identical to SEQ ID NO: YX%" means that the amino acid sequence is identical to SEQ ID NO: Y by X%, and is described as having X% of the residues in the amino acid sequence identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using a computer program. The computer program for comparing and aligning pairs of sequences may be, for example, ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman,1988; pearson, 1990), gapped BLAST (Altschul et al, 1997), BLASTP, BLASTN or GCG (Devereux et al, 1984). In addition, in determining the degree of sequence identity between two amino acid sequences, one skilled in the art can consider "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue having a similar chemical structure, which have little or no effect on the function, activity, or other biological properties of the polypeptide. Such "conservative" amino acids may be amino acids known in the art.

As previously described, a first aspect of the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, which is targeted to bind NGF, the polypeptide having an amino acid sequence shown in formula I:

X₁-X₂-C₁-X₃-R-X₄-X₅-Q-X₆-X₇-X₈-W-X₉-C₂-X₁₀-X₁₁-X₁₂ The compound of the formula I,

Wherein X ₁ is selected from W, M or F;

x ₂ is selected from Y, K, N, T or H;

x ₃ is selected from W, F, Y, H or M;

X ₄ is selected from M, E, Y, A, T, Q, V, K, L, or D;

X ₅ is selected from E, Q or H;

X ₆ is selected from F or W;

x ₇ is selected from H or Q;

X ₈ is selected from N, D or E;

x ₉ is selected from T, V, E, Q, S, I or A;

X ₁₀ is selected from D, E, T, I, V, K or H;

x ₁₁ is selected from Y, A, W, Q, G or H;

x ₁₂ is selected from L, G, D, N, M, T, L, V, S, W, F, Y or H.

The inventors of the present invention have found that when the position and/or sequence of one or more amino acid residues in C ₁、R、Q、W、C₂ in formula I described above is changed, the resulting polypeptide has significantly reduced affinity for NGF. In view of this, the present invention provides the above-described polypeptide having the amino acid sequence shown in formula I.

Preferably, the polypeptide has a first amino acid sequence as set forth in any one of SEQ ID NOs 1 to 18, or an amino acid sequence having at least 90% sequence identity to the first amino acid sequence.

The first amino acid sequence shown in any one of SEQ ID NO 1-18 is obtained by adopting phage display technology. Specifically, the method comprises the following steps:

Constructing a single-chain DNA mutation random library, transferring the mutation random library into an escherichia coli strain SS320 cell infected by using M13K07 auxiliary phage to obtain a phage display library, taking NGF as a target protein, taking the phage display library as a mobile phase for co-incubation, and sequentially washing, eluting and amplifying to obtain the target polypeptide.

The method for constructing the vector and phage is not particularly limited, and those skilled in the art can select the vector and phage in combination with known technical means in the art. The present invention is provided by way of example with a method that should not be construed as limiting the invention to those skilled in the art. Specifically, reference may be made to the method in Liu,B.,et al.Improving the mutagenesis efficiency ofthe Kunkel methodby codon optimization and annealing temperature adjustment.New Biotechnology.56(2019).

The method for constructing the single-stranded DNA mutant random library is not particularly limited, and may be selected by those skilled in the art in combination with known technical means. The present invention is provided by way of example as a method of construction and those skilled in the art should not be construed as limiting the invention. Specifically, reference may be made to the method in Tonikian,R.,et al.Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries.Nature Protocol.2.6(2007):1368-1386.

The method for obtaining the target polypeptide is not particularly limited, and may be selected by those skilled in the art in combination with known technical means. The present invention is exemplified by a screening method, and those skilled in the art should not be construed as limiting the invention. Specifically, reference may be made to the method in Padmanaban,G.,et al.Identification of peptides that selectively bind to myoglobin by biopanning of phage displayed-peptide library.Journal ofBiotechnology.2.6(2007):1368-1386.

Preferably, disulfide bonds are formed between C ₁ and C ₂.

Preferably, the N-terminal and/or C-terminal of the amino acid sequence of the polypeptide is modified with at least one connecting peptide of combination A consisting of the following connecting peptides:

SAAG、GSGS、GSGC、SAAGG、AGGGGSG、GAGGGGSG、GGGGS。

Further preferably, the combination a consists of the following connecting peptides:

SAAG、GSGS。

particularly preferably, the C-terminal of the amino acid sequence of the polypeptide is modified with a linker peptide SAAG, and the N-terminal of the amino acid sequence of the polypeptide is modified with a linker peptide GSGS. The inventors of the present invention found that the polypeptides in this preferred case have a higher affinity.

According to a preferred embodiment, the polypeptide has any one of the following structures:

Preferably, the pharmaceutically acceptable salt is selected from at least one of trifluoroacetate, acetate, hydrochloride and phosphate.

In the present invention, the amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 18 are shown in Table 1 below:

TABLE 1

	Amino acid sequence
		SEQ ID NO:1	WYCWRMEQFHNWTCDYM
SEQ ID NO:2	WKCWREQQFHNWTCEAV
		SEQ ID NO:3	MYCWRYEQWHDWVCEWG
SEQ ID NO:4	WNCFRAQQFHDWTCTQS
		SEQ ID NO:5	WTCFRTQQFHEWTCIAV
SEQ ID NO:6	WNCWRQQQFHDWTCEYV
		SEQ ID NO:7	WHCYRVHQFHDWECEYV
SEQ ID NO:8	FTCYRKEQFHDWTCIWG
		SEQ ID NO:9	WTCWRLHQFQDWQCEWT
SEQ ID NO:10	WTCHRDQQFHEWQCVWL
		SEQ ID NO:11	WKCWRKEQWHNWSCEWT
SEQ ID NO:12	MYCWRVQQFHDWQCDWM
		SEQ ID NO:13	WHCYRVEQWHDWICKGG
SEQ ID NO:14	WYCWRDQQWHEWSCVHN
		SEQ ID NO:15	WKCWRDEQFHDWSCHWG
SEQ ID NO:16	WTCWRLEQFHDWVCVGD
		SEQ ID NO:17	WHCFREQQWHDWVCKGG
SEQ ID NO:18	WYCFRVEQFHEWACKQL

。

As previously described, the second aspect of the present invention provides a gene whose nucleotide sequence is a nucleotide sequence capable of encoding the amino acid sequence of a polypeptide which targets binding to NGF described in the first aspect.

As described above, the third aspect of the present invention provides a vector comprising the gene described in the second aspect.

As previously mentioned, a fourth aspect of the invention provides a host cell comprising a vector as described in the third aspect.

As previously mentioned, a fifth aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide or a pharmaceutically acceptable salt thereof which targets binding to NGF as described in the first aspect.

Preferably, the composition further comprises a pharmaceutically acceptable carrier.

The type of the vector is not particularly limited, and those skilled in the art can select the vector according to the technical means known in the art, so long as the polypeptide provided in the present invention can target NGF.

As previously mentioned, a sixth aspect of the invention provides the use of at least one of a polypeptide targeted to bind NGF as described in the first aspect hereinbefore or a pharmaceutically acceptable salt thereof, a gene as described in the second aspect hereinbefore, a vector as described in the third aspect hereinbefore, a host cell as described in the fourth aspect hereinbefore, a pharmaceutical composition as described in the fifth aspect hereinbefore, in the manufacture of a medicament for the prevention and/or treatment of an NGF mediated disease.

Preferably, the NGF-mediated disease is selected from at least one of pain, cancer.

More preferably, the pain is chronic pain and the cancer is at least one selected from colorectal cancer, breast cancer, cervical squamous cell carcinoma, lung squamous cell carcinoma, epithelial ovarian cancer, head and neck squamous cell carcinoma.

The invention will be described in detail below by way of examples.

In the examples below, all the raw materials used are commercially available, unless otherwise specified.

In the examples below, the room temperature is 25.+ -. 5 ℃ unless otherwise indicated.

Example 1 screening of target Polypeptides

After the beads (streptavidin T1 beads, manufacturer: invitrogen, cat# 65602) were resuspended and washed as described in the instructions, 60. Mu.g of NGF (target protein) was immobilized on 105. Mu.L of the beads, and 60. Mu.L of PBS was added, and a total volume of 180. Mu.L was finally obtained. Placing on a rotary mixer, and uniformly mixing for 20+/-4 hours at the temperature of 4 ℃. After the incubation, the magnetic beads bound to the target protein were precipitated with a magnetic rack, the supernatant was removed, and the magnetic beads were washed 4 times with 1mL of 0.1wt% tween in PBS (PBST). Subsequently, the beads were blocked, and the beads were blocked with 0.5wt% BSA-PBS at room temperature for 30min. In round 1, 5. Mu.L of 1X10 ¹¹ pfu of M13K07 helper phage library (PBS dissolved) was added to the blocked beads and incubated for 1h with gentle mixing at 4 ℃. After the incubation was completed, unbound phage were repeatedly washed with PBST, and in the next round of screening, the tween concentration was gradually increased from 0.1wt% to 0.4wt% according to the 0.1 wt%/round of amplification. Elution was performed with 0.1M citrate (ph=3.1) for 2min, yielding phage binding to the target protein, and immediate neutralization with 1M Tris-HCl (ph=9.1). 20. Mu.L was used for phage titer determination, the remaining phages were amplified using E.coli strain SS320, the phages were purified and used for the next round of screening, each round of phage input was 1X10 ¹¹ pfu.

EXAMPLE 2 ELISA identification of target polypeptide

1. Coating the ELISA plate with 50 mu L of 50mM sodium bicarbonate (pH=8.5) after diluting SA to 5 mu g/mL at 37 ℃ for 2h;

2. blocking, namely blocking the ELISA plate at 4 ℃ overnight by adopting TBST+20mg/mL BSA blocking solution;

3. Washing the ELISA plate with 200. Mu.L of washing solution (25 mM Tris-HCl (pH=7.2) +150mM NaCl+0.1wt% BSA+0.05wt% Tween 20) 3 times for 3min each;

4. binding target protein 200ng NGF was incubated with the ELISA plates at 37℃for 1h;

5. Washing with 200. Mu.L of washing solution (25 mM Tris-HCl (pH=7.2) +150mM NaCl+0.1wt% BSA+0.05wt% Tween 20) for 3min each;

6. Phage binding 1X 10 ⁹/1×10¹⁰ pfu of phage supernatant was incubated with NGF coated ELISA plates for 1h at 37 ℃;

7. Washing with 200. Mu.L of washing solution (25 mM Tris-HCl (pH=7.2) +150mM NaCl+0.1wt% BSA+0.05wt% Tween 20) for 3min each time;

8. binding antibody 100. Mu.L of M13K07 helper phage antibody at a concentration of 0.1. Mu.g/mL was incubated with phage in the ELISA plates above at 37℃for 1h;

9. washing with 200. Mu.L of washing solution (25 mM Tris-HCl (pH=7.2) +150mM NaCl+0.1wt% BSA+0.05wt% Tween 20) for 3min each time;

10. color development, namely adding 100 mu L of A, B equal-volume TMB color development liquid, and developing for 5min at room temperature;

11. Termination by adding 100. Mu.L of H ₂SO₄ at a concentration of 2M;

12. Scanning, namely, reading OD450 by adopting an enzyme-labeled instrument to obtain the target polypeptide with high affinity with NGF.

EXAMPLE 3 Synthesis of polypeptide Compounds

The polypeptide compound and the derivative thereof provided by the invention are synthesized into a straight-chain precursor by adopting a solid-phase synthesis method, and the target polypeptide crude product is obtained after cutting. Wherein the solid phase carrier is 2-Chlotrityl Resin resin. In the synthesis process, firstly, fully swelling 2-Chlotrityl Resin resin in N, N-Dimethylformamide (DMF), then repeatedly condensing the swelled solid phase carrier with activated amino acid derivatives, washing, deprotecting Fmoc, washing, condensing the next round of amino acid to reach the required length of the synthesized polypeptide chain, finally, reacting the mixed solution of trifluoroacetic acid, water, triisopropylsilane and phenylsulfide (v: v: v=90:2.5:2.5:5) with the solid phase resin to crack the polypeptide from the solid phase carrier, and settling the polypeptide by freezing methyl tertiary butyl ether to obtain the crude product of the target polypeptide. Purifying and separating the target polypeptide crude product in an acetonitrile/water system of 0.1% trifluoroacetic acid by a C18 reversed phase preparation chromatographic column to obtain a pure product of the polypeptide and the derivative thereof. The following specific methods for synthesizing polypeptide compound 1, wherein table 2 is the reagents used in the synthesis.

TABLE 2

Synthesis of polypeptide compound 1:

Step 1, coupling Fmoc-Ser (tBu) -OH as the first amino acid

0.1Mmol of the 2-Chlorotrityl chloride resin was fully swollen in DCM for 1h. 0.08mmol of Fmoc-Ser (tBu) -OH and 0.32mmol of DIEA were weighed into 5mL of DCM and the swollen resin was added and reacted at room temperature for 2h. After completion of the reaction 10mL of blocking solution (DCM: methanol: DIEA (v: v: v=85:10:5) was added and blocked for 10min at room temperature.

Step 2 Synthesis of straight-chain precursor peptide chain

Linear precursor peptide chain of polypeptide compound 1:

S-A-A-G-W-Y-C-W-R-M-E-Q-F-H-N-W-T-C-D-Y-M-G-S-G-S。

the resin obtained in step 1 was fully swollen in DMF for 1h, after which it was synthesized in the order of the linear precursor sequence from the second carboxy-terminal to the amino-terminal. Each coupling cycle proceeds as follows:

(1) Fmoc-deprotection of the swollen resin was performed twice for 8min each with 10mL of a mixture of piperidine and DMF (v: v=20:80);

(2) Washing the resin with DMF for 6-8 times until the pH of the liquid is neutral;

(3) 0.5mmol Fmoc-AA, 0.5mmol HCTU and 1mmol NMM were dissolved in DMF and the washed resin was added and reacted at room temperature for 1h;

(4) Washing the resin with DMF 4-6 times before the next amino acid coupling;

after synthesis of the linear precursor peptide chain, the resin was washed 5 times with DMF and then with DCM and the washed resin was dried in vacuo for use.

Step3 cleavage of the Linear precursor peptide chain

10ML of a freshly prepared mixture of trifluoroacetic acid and water and triisopropylsilane and phenylsulfide (v: v: v: v=90:2.5:2.5:5) was added to the resin obtained in step 2, and the reaction was carried out at room temperature with shaking for 2 hours. After the reaction, the reaction solution was filtered, the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of frozen MTBE to give crude polypeptide. The crude polypeptide is washed 3 times by MTBE and then is pumped out in vacuum for standby.

Step 4 formation of intramolecular disulfide bonds

The crude product obtained in step 3 was added with 20% (v: v) DMSO for complete dissolution, then 2mM GSH was added to 50mM ammonium bicarbonate buffer (pH=8.0, containing 30vol% acetonitrile), and the dissolved polypeptide solution was slowly added dropwise to the above buffer at a final concentration of 1mg/mL and shaken at room temperature for 16h. LC-MS monitors the reaction result, and the purification preparation is directly carried out after the reaction is finished.

Step 5 purification of crude polypeptide

After the crude polypeptide was dissolved in 20vol% acetonitrile aqueous solution, the solution was filtered through a 0.45 μm membrane and separated by a reversed phase high performance liquid chromatography system, wherein the buffer solution was phase A (0.1 wt% trifluoroacetic acid aqueous solution) and phase B (0.1 wt% trifluoroacetic acid acetonitrile solution). Wherein the chromatographic column is BR-C18 (Seikovia technology) reversed phase chromatographic column, the detection wavelength of the chromatograph is set to 230nm, the flow rate is 15mL/min, and the gradient is that the phase B is changed from 20% to 50% within 0-40 min. And collecting the relevant fractions of the product, combining the fractions with the purity of more than 95% after HPLC identification, and freeze-drying to obtain the polypeptide pure product.

Step 6, detection and characterization method

And (3) determining the purity of the polypeptide pure product obtained in the step (5) through the combination of analytical high performance liquid chromatography and liquid chromatography/mass spectrometry, and forming intramolecular disulfide bonds by the compound.

Other polypeptide compounds of the present invention may be synthesized by reference to the synthesis method of polypeptide compound 1.

Example 4 determination of affinity of target polypeptide to NGF by ELISA

NGF protein (concentration: 0.2. Mu.g/mL) was immobilized on 384-well plates at 25. Mu.L/well, each of the above-mentioned target polypeptide solutions was transferred to the 384-well plates at 12.5. Mu.L/well by a workstation, a premix solution (TrkA protein (concentration: 1.2. Mu.g/mL) and peroxidase-conjugated affinity pure goat anti-human IgG-Fcγ fragment (concentration: 0.2. Mu.g/mL) were added to the 384-well plates at 12.5. Mu.L/well, mixed at room temperature for 1h at a volume ratio of 1:1), and the resultant solution was subjected to instantaneous centrifugation to remove air bubbles and then placed in a 37℃incubator for incubation for 2h. After the liquid in the wells was discarded, the plates were washed 4 times with 80 μl of 1×tbst wash at ph=7.4 for 4min each. The 384-well plates were drained, 25. Mu.L of TMB color development solution was added to each well, and incubation was continued in an incubator at 37℃for 30min. Finally, 25. Mu.L of stop solution (1M HCl) was added to each well to terminate the reaction. Absorbance at 450nm was read for each well using an microplate reader. After the inhibition rate of the concentration of each target polypeptide is calculated according to the formula (1), the IC50 value of each polypeptide is calculated by drawing by using graphpaprism software, and the specific result is shown in the table 3. The calculation formula of the inhibition rate is as follows:

Inhibition% = (1- (absorbance of sample-absorbance of background)/(absorbance of positive-absorbance of background)) ×100%;

Wherein,

Adding polypeptide into the reaction system;

positive indicates that no polypeptide is added into the reaction system;

the background is that no polypeptide and NGF protein are added into the reaction system.

TABLE 3 IC ₅₀ values for target polypeptides

From the results in Table 3, it can be seen that the polypeptide provided by the invention has higher affinity with NGF, and can effectively inhibit NGF-TrkA binding.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims (10)

1. A polypeptide that targets and binds to NGF or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 3 or 14. 2 . The polypeptide or a pharmaceutically acceptable salt thereof according to claim 1 , wherein a disulfide bond is formed between the first cysteine and the second cysteine in the amino acid sequence of the polypeptide. 3. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the N-terminus and/or C-terminus of the amino acid sequence of the polypeptide is modified with at least one connecting peptide in combination A; The combination A consists of the following connecting peptides: SAAG, GSGS, GSGC, SAAGG, AGGGGSG, GAGGGGSG, GGGGS. 4. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the N-terminus and/or C-terminus of the amino acid sequence of the polypeptide is modified with at least one connecting peptide in combination A, wherein combination A consists of the following connecting peptides: SAAG, GSGS. 5. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the C-terminus of the amino acid sequence of the polypeptide is modified with a connecting peptide SAAG; and the N-terminus of the amino acid sequence of the polypeptide is modified with a connecting peptide GSGS. 6. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the polypeptide has any one of the following structures: 7. A gene, characterized in that the nucleotide sequence of the gene is a nucleotide sequence that can encode the amino acid sequence of the polypeptide targeting and binding to NGF as described in any one of claims 1-6. 8. A vector, characterized in that it contains the gene according to claim 7. 9. A host cell, characterized in that the host cell contains the vector according to claim 8. 10. A pharmaceutical composition, characterized in that it contains a therapeutically effective amount of the polypeptide targeting and binding to NGF as described in any one of claims 1-6 or a pharmaceutically acceptable salt thereof.