A polypeptide for repairing nerve injury, a preparation method thereof and an application thereof

Abstract

The present application discloses a polypeptide for repairing nerve injury, a preparation method thereof and an application thereof. The preparation method of the polypeptide comprises the following steps: S1: swelling the resin for synthesis, removing the Fmoc protecting group and washing to obtain a resin to be grafted; S2: mixing the amino acid to be incorporated, a coupling agent and an alkali solution to obtain a mixed reactant, completely immersing the resin to be grafted in the mixed reactant, and reacting for a period of time under the protection of an inert gas; S3: sequentially replacing the amino acid to be incorporated in step S2 according to the TKDGKQAAW sequence, so that the resin to be grafted is sequentially incorporated with the specified amino acids to obtain a crude product; S4: separating the polypeptide in the crude product from the resin by using a shearing solution, purifying the polypeptide by using a high performance liquid chromatograph, identifying the target polypeptide by using a mass spectrometer, and collecting the target polypeptide for freeze-drying to obtain the polypeptide.

Description

Technical Field

[0001] This application relates to the field of biological medicine technology, and specifically relates to a polypeptide for repairing nerve injury, a preparation method thereof, and an application thereof. Background Art

[0002] The nervous system is a highly organized system that transmits and processes sensory information and coordinates body functions. It is mainly divided into two major parts: the central nervous system and the peripheral nervous system. The former includes the brain and spinal cord, while the latter includes nerves and sensory organs. Due to the relatively fragile nature of the nervous system, external damage is likely to cause irreversible nerve dysfunction. When a nerve is subjected to sharp cutting, violent traction, or compression, axons undergo degeneration and disintegration, resulting in impaired axoplasmic transport and swelling and death of neurons, ultimately leading to disorders in nerve signal transmission and abnormal nerve function. Currently, traumatic nerve diseases are an important global public health problem, and their types mainly include: craniocerebral trauma, spinal cord injury, and peripheral nerve injury, etc. These diseases are prevalent in all age groups, with the global number of patients being approximately 300 million and the number of newly diagnosed patients increasing year by year. Since nerve trauma can cause patients to have limb paralysis, paralysis, or even death, a large number of drugs are required for treatment. Therefore, this type of disease not only causes serious physical and psychological harm to the patients themselves, but also imposes a huge economic burden on the entire family and society. Since adult nerves have relatively weak self-repair ability after being damaged, it is urgent to find suitable drug treatments.

[0003] Existing studies have shown that after nerve injury, the synthesis and secretion of endogenous neurotrophic factors increase sharply, and are crucial for the survival of neurons and the regeneration of axons. Among them, nerve growth factor (NGF) is an early discovered and well-studied neurotrophic factor, which plays an important role in the growth, development, survival, repair, and function regulation of the nervous system. In vitro, NGF can promote the growth of dorsal root ganglion (DRG) axons, the formation of neuronal synapses, axonal elongation, and the differentiation of Schwann cells (SCs). In vivo, NGF can promote the repair, regeneration, and growth and development of nerve tissue, accelerate blood vessel formation and wound healing, and has broad application prospects in the treatment of neurotraumatic diseases such as cerebral infarction, cerebral hemorrhage, traumatic brain injury, spinal cord injury (SCI), and peripheral nerve injury (PNI).

[0004] NGF exerts these physiological functions mainly by binding to the tyrosine kinase receptor (Trk A) on the cell membrane, and then activating downstream signaling pathways such as PI3K / Akt, MAPK, and PLCγ. NGF has shown its importance in multiple aspects such as the development and maintenance of the nervous system, pain management, non-nervous system physiological functions, cardiovascular system protection, immune system regulation, anti-tumor effects, and wound healing. Summary of the Invention

[0005] The object of the present application is to provide a polypeptide that is easy to synthesize artificially and has a repair effect on nerve injury.

[0006] To achieve the above object, the technical solution adopted in the present application is: to provide a polypeptide for repairing nerve injury, including the Loor4 domain, and the analyzed amino acid sequence of the Loor4 structure is: TKDGKQAAW.

[0007] The present application also provides a preparation method of a polypeptide for repairing nerve injury, including the following steps: S1: Swell the resin for synthesis, remove the Fmoc protecting group and wash it to obtain the resin to be grafted; S2: Mix the amino acid to be incorporated, the coupling agent and the base solution to obtain a mixed reactant, completely immerse the resin to be grafted in the mixed reactant, and react for a period of time under the protection of an inert gas; S3: Replace the amino acid to be incorporated in the step S2 in sequence according to the TKDGKQAAW sequence, so that the resin to be grafted is sequentially incorporated with the specified amino acids to obtain a crude product; S4: Use a shearing solution to separate the polypeptide in the crude product from the resin, purify the polypeptide using a high performance liquid chromatograph, and identify the target polypeptide using a mass spectrometer, and collect the target polypeptide for lyophilization to obtain the polypeptide for repairing nerve injury.

[0008] As a preference, the amino acid to be incorporated, the coupling agent and the base in the base solution are mixed in a molar ratio of 1:1:1 to obtain the mixed reactant.

[0009] As another preference, the coupling agent is 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0010] As another preference, the base solution is an aqueous solution of diisopropylethylamine.

[0011] As another preference, the shearing solution is a mixture of trifluoroacetic acid, triisopropylsilane and water.

[0012] As another preference, the trifluoroacetic acid, the triisopropylsilane and the water are mixed in a volume ratio of 38:1:1 to obtain the shearing solution.

[0013] As another preference, as determined by the mass spectrometer, the molecular weight of the polypeptide is 1667 Da.

[0014] As another preference, the resin for synthesis is Rink-Amide-MBHA resin.

[0015] The present application also provides a pharmaceutical composition, which contains a therapeutically effective amount of the above-mentioned polypeptide or the polypeptide prepared by the above-mentioned preparation method.

[0016] Compared with the prior art, the beneficial effects of the present application are as follows:

[0017] (1) The polypeptide NPL for repairing nerve injury in the present application 4r has better stability and biological activity compared with NGF, and from the perspective of artificial synthesis, it is easier to synthesize and has a lower preparation cost, and can become a potential drug for treating nerve injury diseases;

[0018] (2) Compared with NGF, the polypeptide NPL prepared in the present application 4r can significantly improve the proliferation of neurons, while NGF does not have such an effect;

[0019] (3) The polypeptide NPL4r prepared in the present application has a promoting effect similar to that of NGF, and can promote the maturation of primary neuron cells and the recovery of motor function. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 are the test results of promoting neuron growth at different NPL 4r concentrations;

[0021] Figure 2 are the microscopic imaging diagrams of labeled cell proliferation;

[0022] Figure 3 are the microscopic imaging diagrams of crystal violet-stained cells and their corresponding statistical results;

[0023] Figure 4 are the expression results of Synapsin-1 protein and PSD95 protein and their corresponding statistical results;

[0024] Figure 5 are the BBB and inclined plane score results of each experimental group;

[0025] Figure 6 are the display results of Nissl staining method and their corresponding statistical results;

[0026] Figure 7 are the electrophysiological detections and their corresponding statistical results;

[0027] Figure 8 are the staining results of fluorescent markers and their corresponding statistical results. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] Next, in combination with specific embodiments, the present application will be further described. It should be noted that, on the premise of no conflict, the following-described embodiments or technical features can be arbitrarily combined to form new embodiments.

[0029] The terms "comprising" and "having" in the description and claims of the present application, and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device that comprises a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0030] Nerve growth factor (NGF) has a homodimeric structure. Multiple β-turn loops exposed on each monomer can interact with Trk A. Loop4, which is one of them, is the most important region for NGF to exert its biological activity. The resolved amino acid sequence is: TMDGKQAAW.

[0031] The present application provides a polypeptide NPL for repairing nerve injury 4r , including the Loor4 domain, wherein the resolved amino acid sequence of the Loor4 structure is: TKDGKQAAW. That is, the polypeptide NPL for repairing nerve injury of the present application 4r is obtained by replacing methionine (M) at the second position in the Loop4 domain of NGF with lysine (K).

[0032] The resolved amino acid sequence TKDGKQAAW of the Loor4 structure refers to tyrosine, lysine, aspartic acid, glycine, lysine, glutamine, alanine, alanine and tryptophan in sequence.

[0033] The polypeptide NPL for repairing nerve injury of the present application 4r has better stability and biological activity compared with NGF, and from the perspective of artificial synthesis, it is easier to synthesize and has a lower preparation cost, and can become a potential drug for treating nerve injury diseases.

[0034] The present application provides a method for preparing a solid-phase synthesized polypeptide NPL for repairing nerve injury 4r , comprising the following steps:

[0035] S1: Swell the resin for synthesis, remove the Fmoc protecting group and wash it to obtain the resin to be grafted;

[0036] S2: Mix the amino acid to be incorporated, the coupling agent and the alkali solution to obtain a mixed reactant, completely immerse the resin to be grafted in the mixed reactant, and react for a period of time under the protection of an inert gas;

[0037] S3: Sequentially replace the amino acids to be incorporated in step S2 according to the TKDGKQAAW sequence, and sequentially incorporate different Fmoc-protected amino acids into the resin to be grafted to obtain a crude product.

[0038] S4: Use a cleavage solution to separate the polypeptide from the resin in the crude product, purify the polypeptide using a high-performance liquid chromatograph, and identify the target polypeptide using a mass spectrometer. Collect the target polypeptide and perform lyophilization to obtain polypeptide NPL. 4r 。

[0039] In some embodiments, the resin used for synthesis is selected as Rink-Amide-MBHA resin. Rink-Amide-MBHA resin is connected to the modified Rink amide linker of MBHA resin through norleucine. It contains the structure of 4-(2′,4′-dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)-phenoxyacetamido-methyl-diphenylamine. Rink-Amide-MBHA resin is widely used due to its high efficiency in solid-phase peptide synthesis and its ability to provide high-purity peptide amides. It is suitable for the production of polypeptides with C-terminal amide endings and can be efficiently cleaved from the resin by single-step acidolysis during the synthesis process.

[0040] In some embodiments, Rink-Amide-MBHA resin is alternately soaked in dichloromethane (DCM) and N,N-dimethylformamide (DMF) to ensure that the resin swells sufficiently. Usually, each solvent is soaked for at least 5 minutes and repeated 2 - 3 times.

[0041] In some embodiments, the commonly used reagents for removing the Fmoc protecting group are: piperidine, diethylamine, hydrazine hydrate, 4-methylpiperidine (4MP), piperidine (PP), piperazine (PZ), 50% morpholine solution or DBU (1,8-diazabicyclo[5.4.0]undec-7-ene).

[0042] In some embodiments, the coupling agent is 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and the base solution is selected as diisopropylethylamine (DIPEA).

[0043] In some embodiments, the amino acid, coupling agent, and base solution are mixed according to a molar ratio of 1:1:1 to obtain a mixed reactant.

[0044] In some embodiments, the cleavage solution is a mixture of trifluoroacetic acid, triisopropylsilane, and water. In a more preferred embodiment, trifluoroacetic acid, triisopropylsilane, and water are mixed according to a volume ratio of 38:1:1 to obtain the cleavage solution.

[0045] In some embodiments, a high performance liquid chromatograph (HPLC) is used to separate and purify the polypeptide, and samples with different peak patterns in HPLC at different time points are collected. A mass spectrometer is used for molecular weight identification, and it is confirmed that the target polypeptide has a molecular weight of 1667 Da.

[0046] The present application also provides a pharmaceutical composition containing a therapeutically effective amount of the polypeptide.

[0047] Examples

[0048] Preparation of a polypeptide NPL for repairing nerve injury by solid-phase synthesis method 4r , and the preparation steps are as follows:

[0049] S1: Weigh the Rink-Amide-MBHA resin and place it in a reaction tube. Alternately soak the resin with dichloromethane (DCM) and N,N-dimethylformamide (DMF), soak each solvent for at least 5 minutes, and repeat 2 - 3 times to ensure that the resin is fully swollen; completely immerse the swollen resin in a 50% morpholine solution, gently shake the reaction tube at room temperature, allow morpholine to react with the resin for 5 - 10 minutes, and then alternately wash the resin 3 times with DCM and DMF, each time for at least 1 minute, to remove unreacted morpholine and the removed Fmoc, obtaining the resin to be grafted;

[0050] S2: Mix the Fmoc-protected amino acid to be incorporated with HATU and DIPEA in an equivalent ratio of 1:1:1, add the mixed solution to the resin, ensure that the resin is completely immersed in the solution, and gently shake the reaction tube for 1.5 hours under nitrogen protection to promote the coupling reaction;

[0051] S3: Repeat step S2, and sequentially incorporate different Fmoc-protected amino acids according to the polypeptide sequence of NPL 4r until the entire sequence is synthesized to obtain a crude product;

[0052] S4: Mix trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 38:1:1 to obtain a cleavage solution; immerse the crude product in the cleavage solution, gently shake the reaction tube at room temperature for 1 - 1.5 hours to ensure that the polypeptide is completely cleaved from the resin; after removing the resin, dilute the obtained polypeptide solution with a 50% acetonitrile solvent to reduce the concentration of trifluoroacetic acid; use a high performance liquid chromatograph (HPLC) to separate and purify the polypeptide, collect samples with different peak patterns in HPLC at different time points; use a mass spectrometer for molecular weight identification, and confirm that the target polypeptide has a molecular weight of 1667 Da; collect the pure peptide liquid showing the target peak pattern and place it in a freeze dryer for freeze drying to obtain solid NPL 4r polypeptide.

[0053] Performance detection

[0054] 1. Extraction and purification of primary neurons

[0055] Soak 1-day-old neonatal SD rats in 75% alcohol for 2 min, decapitate them on a laminar flow bench and take out their brains. Under the condition of pre-cooling with an ice pack, peel off the meninges and blood vessels, add 2 mg / ml papain and an appropriate amount of DNase, and digest in an incubator at 37 °C for 10 min. Add a certain volume of DMEM / F12 complete medium to terminate digestion. Gently pipette the culture medium repeatedly with a 1 mL pipette tip to disperse the cells. After standing for 3 min, suck out the supernatant cell suspension with a 1 mL pipette tip, and add the precipitated tissue mass to the digestive enzyme again to repeat the above steps 3 times to collect enough cell suspension.

[0056] Centrifuge the cell suspension at 1000 r / min for 5 min at 4 °C, discard the supernatant, and add DMEM / F12 complete medium to seed in a 96-well plate coated with polylysine at a density of 5×109 / L. After 4 h, discard the original medium and replace it with Neurobasal complete medium containing cytosine arabinoside to inhibit the growth of glial cells and endothelial cells.

[0057] 2. Explore the optimal concentration of NPL 4r Polypeptide promoting the growth of neurons

[0058] Seed the extracted primary neurons in a 96-well plate at a density of 5×109 / L. The next day, aspirate the medium from each well and add 100 μL of fresh medium containing gradient concentrations of NPL 4r (0, 50, 100, 250, 500, 1000, 2500, 5000 nM). After culturing for 24 h, add 10 μL of CCK-8 solution to each well and incubate for 24 h. Measure the absorbance of each well at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The absorbance data of each well are as Figure 1 shown.

[0059] Analysis Figure 1 of the test results shows that adding NPL 4r plays a positive regulatory role in the growth of primary neurons, and with the increase of the concentration, the effect on cell growth is more obvious. When the concentration of NPL 4r increases to 500 nM, the promoting effect on neuron growth is the strongest. Continuing to increase the concentration, the promoting effect slightly decreases. Therefore, 500 nM is selected as the optimal administration concentration of NPL 4r .

[0060] 3. Explore the proliferative effect of NPL 4r on neuronal cells

[0061] The extracted primary neurons were seeded in 24-well plates at a density of 5×109 / L. The next day, the culture medium in each well was aspirated and replaced with Neurobasal complete medium containing cytosine arabinoside, and this medium contained NPL or NGF, both at a concentration of 500 nM. 4r After incubating for 24 h, 50 μM of EdU dye was added to the culture medium and the cells were incubated for 4 h. Then, the subsequent operations were carried out according to the instructions of the EdU kit. Meanwhile, the cytoskeleton was labeled with TRITC-labeled phalloidin (100 μM, red), and imaging was performed using a confocal microscope. The imaging results are as Figure 2 shown. The proliferation rate (%) of the cells = the number of EdU-labeled cells / the number of DAPI-labeled cells × 100%.

[0062] Analysis Figure 2 of the imaging results showed that the number of EdU (green)-labeled positive cells was not significantly different between the Control group and the NGF group, but in the NPL 4r group, the number of EdU (green)-labeled positive cells increased significantly, showing a significant difference compared with the NGF group (***P < 0.001). Therefore, NPL 4r could significantly enhance the proliferation of neurons, while NGF had no such effect.

[0063] 4. Explore the effect of NPL 4r on promoting the migration of primary neuron cells

[0064] The migration of cells in different groups was evaluated using a Transwell assay. Primary neurons at a density of 5×109 / L were seeded in the upper chamber of a Transwell coated with Matrigel matrix gel, and the lower chamber medium contained NGF or NPL4r at a concentration of 500 nM. After culturing for 24 h, the culture medium in the upper chamber was blotted dry, and the Matrigel and cells on the inner layer of the Transwell were wiped off with a cotton swab, and then stained with crystal violet. After appropriate air drying, 5 fields of view were selected under a 20-fold microscope to observe and count the cells, Figure 3 which is the result shown by the microscope.

[0065] Analysis Figure 3 of the results shown in A indicated that compared with the Control group, the number of neurons migrating to the lower chamber in the NGF group increased significantly, and this increasing trend was more obvious in the NPL 4r group. And Figure 3 the statistical results in B showed that the number of neurons migrating in the NGF group was significant compared with the Control group, and the number of migrations in the NPL 4r group still exceeded that in the NGF group. Therefore, compared with NGF, the NPL prepared in this application 4r had a more significant effect on the migration of primary neurons.

[0066] 5. Exploration of NPL 4r Promotion of primary neuron cell maturation

[0067] The maturity of neurons in different groups was evaluated by Western blot (WB) experiment. Primary neurons at a density of 5×109 / L were seeded in 6-well plates. The next day, NGF or NPL at a concentration of 500 nM was added to the culture medium 4r . After 24 h of culture, the culture medium in the upper chamber was aspirated, and the cells in each group were lysed and the protein concentration was measured. The expression levels of two proteins, Synapsin-1 (a presynaptic vesicle protein, marking neuron maturity) and PSD95 (a postsynaptic vesicle protein, marking neuron maturity), were detected by WB in the Control group, NGF, and NPL 4r groups. The test results are shown as Figure 4 follows

[0068] Analysis Figure 4 The test results of A showed that the contents of Synapsin-1 and PSD95 were significantly increased in the NGF group and NPL 4r group compared with the Control group. And Figure 4 B and Figure 4 C further statistically analyzed the expression of these two proteins in each group and found that NGF and NPL 4r could significantly increase the contents of Synapsin-1 and PSD95, and there was no statistical difference between them. Therefore, NPL 4r has the same effect as NGF in promoting the maturation of primary neurons

[0069] 6. Exploration of NPL 4r Promotion of motor function recovery

[0070] To further confirm the repair effect of NPL 4r on the central nervous system, an SCI model was prepared, and the motor function improvement of rats in each group was observed by BBB score and inclined plane test on the 0th, 7th, 14th, 21st, and 28th days after modeling. Among them, for the SCI model, 8-week-old female SD rats were selected. After exposing the spinal cord at the T8 segment, the NYU MASCIS Impactor Ⅱ was used to impact this area with a force of 2.00 N to form a spinal cord impact injury model. Then, 10 μL of NPL4r (2.4 μg / mL), NGF (2.4 μg / mL), or normal saline was injected at the impact site, and the rats were divided into SCI+NPL 4r group, SCI+NGF group, and SCI group. The Sham group only exposed the spinal cord without impact, and the test results were recorded in Figure 5 the following

[0071] Analysis Figure 5The results show that after SCI, the BBB scores of rats in each model group were 0 and the inclined plane scores were very low, indicating that the rats in each model group were completely paralyzed. As time went by, the BBB and inclined plane scores of the SCI group gradually increased, but were lower than those of the SCI+NGF group and the SCI+NPL 4r group. The two behavioral scores of these two groups were basically similar and showed significant statistical differences compared with the SCI group. Therefore, in terms of the improvement and recovery effect of motor function, NPL 4r has a similar effect to NGF.

[0072] 7. Explore the effect of NPL 4r on the maintenance of survival of damaged neurons after SCI

[0073] To confirm whether NPL 4r has the effect of promoting the survival of damaged neurons after SCI, spinal cord tissues repaired for 28 days in each group were collected, and the survival status of neurons in each group was observed by Nissl staining. Since Nissl bodies are rich in the cytoplasm of neurons, Nissl bodies can be effectively stained by Nissl staining method, and the test results are as Figure 6 shown, so as to judge the degree of damage and survival of neurons in the damaged spinal cord tissue.

[0074] Analysis Figure 6 of the results of A shows that neurons in the Sham group were rich in Nissl bodies and there were a large number of adult neurons. The stained neurons in the SCI group were small and sparse in number; while the number of stained neurons in the SCI+NGF and SCI+NPL 4r groups was significantly increased. And by analyzing the statistical data of Figure 6 B, it can be seen that compared with SCI, the number of surviving neurons in the SCI+NGF and SCI+NPL 4r groups was significantly increased, and there was no significant difference between the two. It can be seen that NPL 4r polypeptide has a similar effect to NGF in helping neurons survive.

[0075] 8. Explore the effect of NPL 4r on PNI electrical signal conduction

[0076] To explore the repair effect of NPL 4r on the peripheral nervous system, a PNI model was prepared and the improvement of nerve motor conduction velocity (MNCV) and compound action potential (CAMP) of rats in each group was detected by an electrophysiological instrument on the 28th day after modeling. Among them, for the PNI model, 8-week-old male SD rats were selected. After exposing the right sciatic nerve, an arterial clamp was used to clamp the sciatic nerve with a force of 30 g for 2 min to prepare a sciatic nerve contusion model. Then, 80 ng of NPL was injected at the nerve injury site 4r(100 μL) or an equal dose of NGF or an equal volume of normal saline and administered continuously for 28 days, and they were divided into the PNI + NPL 4r group, PNI + NGF group and PNI group. The Sham group only exposed the sciatic nerve without nerve injury, and the test results are as Figure 7 shown.

[0077] Analysis Figure 7 A. The waveform of the PNI group was smaller, while the waveforms and amplitudes of the PNI + NPL 4r group and PNI + NGF group were larger. Figure 7 B. Statistical analysis of the MNCV of each group found that the Sham group was the largest, followed by the PNI + NPL 4r group and PNI + NGF group, while the PNI group was the smallest. Figure 7 C. Statistical analysis of the CAMP of each group. The CAMP between the PNI + NPL 4r group and PNI + NGF group was similar and significantly greater than that of the PNI group. Thus, it can be seen that NPL 4r can significantly improve the conduction of nerve electrical signals after PNI and has a similar therapeutic effect to NGF.

[0078] 9. Explore the regenerative effect of NPL 4r on axons and myelin sheaths after PNI

[0079] To confirm whether NPL 4r can promote the regeneration of nerves after PNI, on the 28th day of drug administration, the sciatic nerves of rats in each group were collected and frozen sections were made. The regeneration of axons and myelin sheaths at the nerve injury site of each group was observed by double fluorescence staining of NF-200 (nerve filament marker) and MBP (myelin sheath marker), as presented in Figure 8 .

[0080] Analysis Figure 8 A. The staining results showed that the axons labeled by NF-200 and the myelin sheaths labeled by MBP in the Sham group were dense and regular, while the labeled axons and myelin sheaths in the PNI group were sparsely distributed and disorderly arranged, while in the PNI + NPL 4r and PNI + NGF groups, the number of labeled axons and myelin sheaths increased significantly and were densely distributed. Similarly, Figure 8 B and Figure 8 C. The statistical results showed that compared with the Sham group, the positive area of the PNI group labeled by NF-200 and MBP decreased significantly, while in the PNI + NPL 4r and PNI + NGF groups, the values of these two indicators increased significantly, and there was no significant statistical difference between the two. In summary, the NPL of this application 4r still has a biological activity similar to NGF and is equivalent to NGF in promoting axon and myelin sheath regeneration.

[0081] In summary, an NPL 4r polypeptide for repairing nerve injury according to the present application can significantly improve the proliferation of neurons, and has a more significant promoting effect on the migration of primary neurons.

[0082] The foregoing has described the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present application. Without departing from the spirit and scope of the present application, the present application will have various changes and improvements, and these changes and improvements all fall within the scope of the present application claimed. The scope of protection claimed by the present application is defined by the appended claims and their equivalents.

Claims

1. A polypeptide for repairing nerve injury, characterized in that, The amino acid sequence for the analysis of the polypeptide structure is: TKDGKQAAW.

2. The preparation method of the polypeptide for repairing nerve injury according to claim 1, characterized in that, It includes the following steps: S1: Swell the resin for synthesis, remove the Fmoc protecting group and wash it to obtain the resin to be grafted. S2: Mix the amino acid to be incorporated, the coupling agent and the base solution to obtain a mixed reactant. Immerse the resin to be grafted completely in the mixed reactant and react for a period of time under the protection of an inert gas. S3: Replace the amino acid to be incorporated in step S2 successively according to the sequence of TKDGKQAAW, so that the resin to be grafted is successively incorporated with the specified amino acids to obtain a crude product. S4: Use a cleavage solution to separate the polypeptide from the resin in the crude product, purify the polypeptide using a high performance liquid chromatograph, and identify the target polypeptide using a mass spectrometer. Collect the target polypeptide and freeze-dry it to obtain the polypeptide for repairing nerve damage.

3. The preparation method of the polypeptide for repairing nerve injury according to claim 2, wherein, The amino acid to be incorporated, the coupling agent and the base in the base solution are mixed in a molar ratio of 1:1:1 to obtain the mixed reactant.

4. The method for preparing the polypeptide for repairing nerve injury according to claim 3, characterized in that, The coupling agent is 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

5. The preparation method of the polypeptide for repairing nerve injury according to claim 3, wherein The base solution is an aqueous solution of diisopropylethylamine.

6. The preparation method of the polypeptide for repairing nerve injury according to claim 2, characterized in that, The cleavage solution is a mixture of trifluoroacetic acid, triisopropylsilane and water.

7. The preparation method of the polypeptide for repairing nerve injury according to claim 6, wherein, The trifluoroacetic acid, the triisopropylsilane and the water are mixed in a volume ratio of 38:1:1 to obtain the cleavage solution.

8. The preparation method of the polypeptide for repairing nerve injury according to claim 2, characterized in that, Determined by a mass spectrometer analyzer, the molecular weight of 1667 Da is the polypeptide.

9. The preparation method of the polypeptide for repairing nerve injury according to claim 2, wherein, The resin for synthesis is Rink-Amide-MBHA resin.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of the polypeptide of claim 1 or the polypeptide prepared by the preparation method of claims 2-9.

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