Polypeptide for repairing nerve injury as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide for repairing nerve injury and a preparation method and application thereof, and the preparation method of the polypeptide comprises the following steps: S1, swelling resin for synthesis, removing an Fmoc protecting group, and cleaning to obtain resin to be grafted; s2, mixing amino acid to be grafted, a coupling agent and an alkali solution to obtain a mixed reactant, completely immersing resin to be grafted in the mixed reactant, and reacting for a period of time under the protection of inert gas; s3, sequentially replacing the amino acids to be grafted in the step S2 according to a TKDGKQAAW sequence, so that the resin to be grafted is sequentially grafted into the specified amino acids to prepare a crude product; and S4, separating the polypeptide from the resin in the crude product by using a shearing liquid, purifying the polypeptide by using a high performance liquid chromatograph, identifying the target polypeptide by using a mass spectrometer, collecting the target polypeptide, and freeze-drying to obtain the polypeptide.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to a polypeptide for repairing nerve damage, a preparation method 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 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. Because the nervous system is relatively fragile, external damage can easily cause irreversible neurological dysfunction. When nerves are cut by sharp objects, violently pulled or compressed, axons degenerate and disintegrate, resulting in impaired axoplasm transport and swelling and death of neurons, which ultimately cause obstacles to nerve signal transmission and abnormal nerve function. At present, neurotraumatic diseases are an important global public health issue. Its types mainly include: craniocerebral trauma, spinal cord injury and peripheral nerve injury. These diseases are prevalent in people of all ages. The number of patients worldwide is about 300 million, and the number of new patients is increasing year by year. Since neurotrauma can cause limb paralysis, paralysis, and even death in patients, a large amount of drugs are needed for treatment. Therefore, this type of disease not only causes serious physical and psychological harm to the patient himself, but also imposes a huge economic burden on the entire family and society. Since adult nerves have weak self-repair capabilities after being damaged, finding appropriate drug treatments is urgent.

[0003] Studies have shown that after nerve injury, the synthesis and secretion of endogenous neurotrophic factors increase dramatically, and are essential 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 that plays an important role in the growth, development, survival, repair, and functional regulation of the nervous system. In vitro, NGF can promote the growth of dorsal root ganglion (DRG) axons, the formation of neuronal synapses, axon elongation, and the differentiation of Schwann cells (SCs). In vivo, NGF can promote the repair, regeneration, and growth and development of neural tissue, accelerate the formation of blood vessels and the healing of wounds, 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, thereby activating downstream signaling pathways such as PI3K / Akt, MAPK, and PLCγ. NGF has shown its importance in the development and maintenance of the nervous system, pain management, physiological functions of the non-nervous system, protection of the cardiovascular system, immune system regulation, anti-tumor effects, and wound healing. Summary of the invention

[0005] The purpose of the present application is to provide a polypeptide which is easy to artificially synthesize and has a repairing effect on nerve damage.

[0006] In order to achieve the above objectives, the technical solution adopted in the present application is: to provide a polypeptide for repairing nerve damage, including a Loor4 domain, wherein the analyzed amino acid sequence of the Loor4 structure is: TKDGKQAAW.

[0007] The present application also provides a method for preparing a polypeptide for repairing nerve damage, comprising the following steps: S1: swelling the resin used for synthesis, removing the Fmoc protecting group and washing to obtain a resin to be grafted; S2: mixing the amino acid to be grafted, a coupling agent and an alkaline 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: replacing the amino acids to be grafted in the S2 step in sequence according to the TKDGKQAAW sequence, so that the resin to be grafted is grafted with designated amino acids in sequence to obtain a crude product; S4: using a shearing fluid to separate the polypeptide in the crude product from the resin, using a high performance liquid chromatograph to purify the polypeptide, and using a mass spectrometer to identify the target polypeptide, collecting the target polypeptide for freeze-drying to obtain the polypeptide for repairing nerve damage.

[0008] As a preference, the amino acid to be connected, the coupling agent and the base in the alkaline 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-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

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

[0011] As another preference, the shearing fluid 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 fluid.

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

[0014] As another preference, the resin used 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 this application are:

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

[0018] (2) Compared with NGF, the polypeptide NPL prepared in this application 4r It can significantly increase 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 neuronal cells and promote the recovery of motor function. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For different NPL 4r Test results of concentrations that promote neuronal growth;

[0021] Figure 2 This is a microscope image of labeled cell proliferation;

[0022] Figure 3 Crystal violet stained cell microscope images and their corresponding statistical results;

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

[0024] Figure 5 The BBB and inclined board scoring results for each experimental group;

[0025] Figure 6 It is the display result of Nissl staining method and the corresponding statistical results;

[0026] Figure 7 For electrophysiological testing and its corresponding statistical results;

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

[0028] Below, the present application is further described in conjunction with specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0029] The terms "including" and "having" and any variations thereof in the specification and claims of this application are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products or apparatuses.

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

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

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

[0033] The polypeptide NPL for repairing nerve damage of the present application 4r Compared with NGF, it has better stability and biological activity, and from the perspective of artificial synthesis, it is easier to synthesize and has lower preparation costs, making it a potential drug for the treatment of nerve damage diseases.

[0034] The present application provides a solid phase synthesis polypeptide NPL for repairing nerve damage 4r The preparation method comprises the following steps:

[0035] S1: Swelling the resin used for synthesis, removing the Fmoc protecting group and washing to obtain the resin to be grafted;

[0036] S2: Mixing the amino acid to be grafted, the coupling agent and the alkaline 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;

[0037] S3: replacing the amino acids to be grafted in step S2 in sequence according to the TKDGKQAAW sequence, and grafting different Fmoc-protected amino acids into the resin to be grafted in sequence to obtain a crude product;

[0038] S4: Use shearing fluid to separate the peptides in the crude product from the resin, use high performance liquid chromatography to purify the peptides, use mass spectrometry to identify the target peptides, collect the target peptides for freeze-drying, and obtain the peptide NPL 4r .

[0039] In some embodiments, the resin selected for synthesis is Rink-Amide-MBHA resin, which is a modified Rink amide linker connected to the MBHA resin via norleucine. It contains the structure of 4-(2′,4′-dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)-phenoxyacetamido-methylbenzhydrylamine. 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 C-terminal amide-terminated peptides and can be efficiently cleaved from the resin by a single-step acidolysis during the synthesis process.

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

[0041] In some embodiments, the reagents commonly used to remove the Fmoc protecting group include: 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-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and the alkaline solution is diisopropylethylamine (DIPEA).

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

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

[0045] In some embodiments, a high performance liquid chromatography (HPLC) is used to separate and purify the polypeptide, and samples with different peaks in HPLC at different time points are collected. A mass spectrometer is used to identify the molecular weight, and the target polypeptide with a molecular weight of 1667 Da is confirmed.

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

[0047] Example

[0048] Preparation of a peptide NPL for repairing nerve damage by solid phase synthesis 4r , the preparation steps are as follows:

[0049] S1: Weigh the Rink-Amide-MBHA resin and place it in a reaction tube. Soak the resin alternately with dichloromethane (DCM) and N,N-dimethylformamide (DMF). Soak in each solvent for at least 5 minutes. 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 to allow the morpholine to react with the resin for 5-10 minutes. Then, wash the resin alternately with DCM and DMF for 3 times, each time for at least 1 minute, to remove unreacted morpholine and removed Fmoc, and obtain the resin to be grafted.

[0050] S2: Mix the Fmoc-protected amino acid to be connected 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, according to NPL 4r The polypeptide sequence is sequentially connected with different Fmoc protected amino acids until the entire sequence is synthesized to obtain a crude product;

[0052] S4: trifluoroacetic acid, triisopropylsilane and water are mixed in a volume ratio of 38:1:1 to obtain a shearing solution; the crude product is immersed in the shearing solution, and the reaction tube is gently shaken at room temperature for 1 to 1.5 hours to ensure that the polypeptide is completely cut off from the resin; after removing the resin, the obtained polypeptide solution is diluted with 50% acetonitrile solvent to reduce the concentration of trifluoroacetic acid; a high performance liquid chromatography (HPLC) is used to separate and purify the polypeptide, and samples with different peaks in HPLC at different time points are collected; a mass spectrometer is used to identify the molecular weight, and the molecular weight of 1667Da is confirmed as the target polypeptide; the pure peptide liquid with the target peak is collected and placed in a freeze dryer for freeze drying to obtain a solid NPL 4r Peptide.

[0053] Performance Testing

[0054] 1. Extraction and purification of primary neurons

[0055] Soak SD newborn rats at 1 day old in 75% alcohol for 2 minutes, decapitate and remove the brain on a clean bench, peel off the meninges and blood vessels under ice pack precooling, add 2 mg / ml papain and an appropriate amount of DNase and digest in a 37°C incubator for 10 minutes, add a certain volume of DMEM / F12 complete culture medium to terminate digestion, and gently blow the culture medium repeatedly with a 1mL pipette tip to disperse the cells. After standing for 3 minutes, use a 1mL pipette tip to absorb the supernatant cell solution, add the digestion enzyme to the precipitated tissue blocks again, and repeat the above steps 3 times to collect enough cell suspension.

[0056] The cell suspension was centrifuged at 1000r / min for 5min at 4°C, the supernatant was discarded, and DMEM / F12 complete medium was added to seed the cells at a density of 5×109 / L in a poly-lysine-coated plate. After 4h, the original medium was discarded and replaced with Neurobasal complete medium containing cytarabine to inhibit the growth of glial cells and endothelial cells.

[0057] 2. Explore NPL 4r Optimal concentration of peptides to promote neuronal growth

[0058] The extracted primary neurons were seeded in a 96-well plate at a density of 5×109 / L. The next day, the culture medium was aspirated from each well and a gradient concentration of NPL was added. 4r 100 μL of fresh culture medium (0, 50, 100, 250, 500, 1000, 2500, 5000 nM) was added to each well. After culturing for 24 h, 10 μL of CCK-8 solution was added to each well and incubated for 24 h. The absorbance of each well was measured at 450 nm using a microplate reader. The absorbance data of each well is shown in Figure 1 shown.

[0059] analyze Figure 1 The test results show that joining NPL 4r It plays a positive regulatory role in the growth of primary neurons and the effect on cell growth becomes more obvious as the concentration increases. 4r When the concentration increased to 500nM, the promoting effect on neuron growth was the strongest. The promoting effect slightly decreased when the concentration was further increased. Therefore, 500nM was selected as the NPL 4r optimal dosing concentration.

[0060] 3. Explore NPL 4r Promotes the proliferation of neuronal cells

[0061] The extracted primary neurons were seeded in a 24-well plate at a density of 5×109 / L. The next day, the culture medium was aspirated from each well and Neurobasal complete medium containing cytarabine was added. The culture medium contained 500 nM NPL 4r After 24 hours of incubation, 50 μM EdU dye was added to the culture medium and the cells were incubated for 4 hours. The subsequent operations were performed according to the instructions of the EdU kit. At the same time, TRITC-labeled phalloidin (100 μM, red) was used to label the cytoskeleton and imaged using a confocal microscope. The imaging results are shown in Figure 2 As shown, cell proliferation rate (%) = number of cells labeled with EdU / number of cells labeled with DAPI×100%.

[0062] analyze Figure 2 The imaging results showed that the number of positive cells labeled with EdU (green) had no significant difference between the Control group and the NGF group, but it was significantly different in the NPL group. 4r In the NGF group, the number of positive cells labeled with EdU (green) increased significantly, which was significantly different from that in the NGF group (***P﹤0.001). 4r It can significantly increase the proliferation of neurons, while NGF has no such effect.

[0063] 4. Explore NPL 4r Promotes the migration of primary neuronal cells

[0064] Transwell assay was used to evaluate the migration of cells in different groups. Primary neurons at a density of 5×109 / L were implanted in the upper chamber of the Transwell containing Matrigel matrix gel, and the culture medium in the lower chamber contained NGF or NPL4r at a concentration of 500nM. After culturing for 24 hours, the culture medium in the upper chamber was aspirated and the Matrigel and cells in the inner layer of the Transwell chamber were wiped off with a cotton swab, and then stained with crystal violet. After proper air drying, 5 fields of view were selected under a 20x microscope to observe and count the cells. Figure 3 Display results for microscope.

[0065] analyze Figure 3 The results of A show that compared with the Control group, the number of neurons in the NGF group that migrated to the lower chamber increased significantly. 4r group is more obvious. Figure 3 The statistical results of B showed that the number of neuronal migration in the NGF group was significantly higher than that in the Control group. 4r The number of migrations in the group still exceeded that in the NGF group. 4r The effect on the migration of primary neurons is more significant.

[0066] 5. Explore NPL 4r Promoting the maturation of primary neuronal cells

[0067] The maturity of neurons in different groups was evaluated by immunoblotting (WB) experiments. 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 hours of culture, the culture medium in the upper chamber was aspirated and the cells in each group were lysed and the protein concentration was determined. The expression of Synapsin-1 (presynaptic vesicle protein, marking neuronal maturity) and PSD95 (postsynaptic vesicle protein, marking neuronal maturity) in the control group, NGF and NPL groups was detected by WB. 4r The expression of the three groups, the experimental results are as follows Figure 4 shown.

[0068] analyze Figure 4 A test results, compared with the control group, Synapsin-1 and PSD95 in the NGF group and NPL 4r The content of the group increased significantly. Figure 4 B and Figure 4 C. Further statistical analysis of the expression of these two proteins in each group revealed that NGF and NPL 4r It can significantly increase the content of Synapsin-1 and PSD95, and there is no statistical difference between the two. 4r It has the same effect as NGF in promoting the maturation of primary neurons.

[0069] 6. Explore NPL 4r Promoting the recovery of motor function

[0070] To further confirm NPL 4r The SCI model was prepared and the motor function of rats in each group was observed on days 0, 7, 14, 21 and 28 after modeling by BBB scoring and inclined board test. Among them, the SCI model was selected from SD8-week-old female rats. After exposing the spinal cord of the T8 segment, the NYU MASCIS ImpactorⅡ was used to impact this area with a force of 2.00N to form a spinal cord impact injury model. Then, 10μL of NPL4r (2.4μg / mL), NGF (2.4μg / mL) or saline was injected at the impact site and divided into SCI+NPL4r and NGF+NPL4r. 4r group, SCI+NGF group and SCI group. In the sham group, the spinal cord was only exposed without impact. The test results were recorded in Figure 5 middle.

[0071] analyze Figure 5The results showed that after SCI, the BBB scores of the model rats in each group were 0 and the inclined plate scores were very low, indicating that the model rats in each group were completely paralyzed. As time went on, the BBB and inclined plate scores of the SCI group gradually increased, but not as much as those of the SCI+NGF group and SCI+NPL group. 4r The two behavioral scores of the two groups were basically similar and formed a significant statistical difference with the SCI group. Therefore, in terms of the improvement and recovery effect of motor function, NPL 4r The effect is similar to that of NGF.

[0072] 7. Explore NPL 4r Effect on maintaining survival of injured neurons after SCI

[0073] To confirm NPL 4r To determine whether it has an effect on the survival of injured neurons after SCI, the spinal cord tissues of each group were collected after 28 days of repair, and the survival status of neurons in each group was observed by Nissl staining. Since the cytoplasm of neurons is rich in Nissl bodies, Nissl bodies can be effectively stained by Nissl staining. The test results are as follows: Figure 6 As shown, the degree of damage and survival of neurons in the damaged spinal cord tissue can be determined.

[0074] analyze Figure 6 The results of A showed that the neurons in the Sham group were rich in Nissl bodies and had a large number of adult neurons. The neurons in the SCI group were small and sparse in number; while the neurons in SCI+NGF and SCI+NPL 4r The number of colored neurons in the group increased significantly. Figure 6 The statistical data of B show that compared with SCI, SCI+NGF and SCI+NPL 4r The number of surviving neurons in the NPL group increased significantly, and there was no significant difference between the two groups. 4r The peptide has a similar effect as NGF in helping neurons survive.

[0075] 8. Explore NPL 4r Effects on PNI electrical signal conduction

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

[0077] analyze Figure 7 A, The waveform of the PNI group is smaller, while the waveform of the PNI+NPL group is smaller. 4r The waveform and amplitude were larger in the PNI group and PNI+NGF group. Figure 7 B. Statistical analysis of MNCV of each group revealed that the Sham group had the largest MNCV, followed by PNI+NPL 4r group and PNI+NGF group, while the PNI group was the smallest. Figure 7 C Statistics of CAMP, PNI+NPL of each group 4r The CAMP between the PNI group and the PNI+NGF group was similar and significantly greater than that of the PNI group. 4r It can significantly improve the conduction of neural electrical signals after PNI and has a similar therapeutic effect to that of NGF.

[0078] 9. Explore NPL 4r Effects on axon and myelin regeneration after PNI

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

[0080] analyze Figure 8 The staining results of A show that the axons labeled by NF-200 and myelin sheaths labeled by MBP in the sham group are dense and regular, while those labeled by NF-200 in the PNI group are sparse and disordered. 4r The number of labeled axons and myelin sheaths in the PNI+NGF group increased significantly and was densely distributed. Figure 8 B and Figure 8 The statistical results of C showed that compared with the Sham group, the positive area marked by NF-200 and MBP in the PNI group was significantly reduced, while in the PNI+NPL 4r The values ​​of these two indicators in the PNI+NGF group were significantly increased, and there was no significant statistical difference between the two groups. 4r It still has similar biological activity to NGF and is as effective as NGF in promoting axon and myelin regeneration.

[0081] In summary, the present invention provides a NPL for repairing nerve damage. 4r Peptides can significantly increase the proliferation of neurons and have a more significant effect on promoting the migration of primary neurons.

[0082] The above describes 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, and the above embodiments and the specification only describe the principles of the present application. The present application may have various changes and improvements without departing from the spirit and scope of the present application, and these changes and improvements fall within the scope of the present application for which protection is sought. The scope of protection claimed by the present application is defined by the attached claims and their equivalents.

Claims

1. A polypeptide for repairing nerve damage, characterized in that: It includes the Loor4 domain, wherein the resolved amino acid sequence of the Loor4 structure is: TKDGKQAAW.

2. A method for preparing a polypeptide for repairing nerve damage, characterized in that: The following steps are involved: S1: Swelling the resin used for synthesis, removing the Fmoc protecting group and washing to obtain the resin to be grafted; S2: mixing the amino acid to be grafted, the coupling agent and the alkaline 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: replacing the amino acids to be grafted in step S2 in sequence according to the sequence TKDGKQAAW, so that the resin to be grafted is grafted with the designated amino acids in sequence to obtain a crude product; S4: using a shearing fluid to separate the polypeptide in the crude product from the resin, using a high performance liquid chromatography to purify the polypeptide, and using a mass spectrometer to identify the target polypeptide, collecting the target polypeptide for freeze-drying to obtain the polypeptide for repairing nerve damage.

3. The method for preparing a polypeptide for repairing nerve damage according to claim 2, characterized in that: The amino acid to be connected, the coupling agent and the alkali in the alkali solution are mixed in a molar ratio of 1:1:1 to obtain the mixed reactant.

4. The method for preparing a polypeptide for repairing nerve damage according to claim 3, characterized in that: The coupling agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

5. The method for preparing a polypeptide for repairing nerve damage according to claim 3, characterized in that: The alkaline solution is an aqueous solution of diisopropylethylamine.

6. The method for preparing a polypeptide for repairing nerve damage according to claim 2, characterized in that: The shearing fluid is a mixture of trifluoroacetic acid, triisopropylsilane and water.

7. The method for preparing a polypeptide for repairing nerve damage according to claim 6, characterized in that: The trifluoroacetic acid, the triisopropylsilane and the water are mixed in a volume ratio of 38:1:1 to obtain the shearing fluid.

8. The method for preparing a polypeptide for repairing nerve damage according to claim 2, characterized in that: The molecular weight of the polypeptide was 1667 Da as determined by the mass spectrometer.

9. The method for preparing a polypeptide for repairing nerve damage according to claim 2, characterized in that: The resin used 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 to 9.

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