Linear peptide with nerve growth factor biological activity and application

Abstract

The invention relates to the field of biological medicine, in particular to a linear peptide with nerve growth factor biological activity and application. The invention provides NPL2m with a sequence as shown in SEQ ID NO: 1, experimental results show that the NPL2m has a remarkable promoting effect on proliferation and migration of a PC12 nerve cell line, extension of primary dorsal root ganglion axons and growth of Schwann cells can be remarkably promoted, in addition, the NPL2m can remarkably improve the motor function and nerve electrophysiological signal transduction of animals, the effect is similar to that of NGF, and the NPL2m can be used for preparing a medicine for preventing and treating the diseases of the animals. Therefore, the NPL2m disclosed by the invention can greatly reduce the cost and has higher stability while retaining the biological activity of the NGF.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a linear peptide NPL having the biological activity of nerve growth factor 2m and applications. Background Art

[0002] After the nervous system is damaged by different factors (including traffic accidents, sharp cutting, ischemia and hypoxia, poisoning, etc.), there will be different degrees of motor and sensory disorders and neuronal death, leading to nerve damage. According to the different nerve structures, it can be divided into central nervous system and peripheral nerve damage. The former mainly includes brain injury and spinal cord injury, while the latter is mainly peripheral nerve injury. According to statistics, the number of new cases of brain injury in the world is about 27 million to 69 million each year. At the same time, about 15.4 million patients basically lose sensation and / or motor function below the injury plane. In addition, about 4.5 million people suffer from peripheral nerve injury, which brings huge economic burden to patients' families and society. Because adult neurons have weak regenerative ability, they have limited repair ability after being damaged by the outside world, and need external conditions to effectively enhance their own structure and functional recovery.

[0003] Nerve growth factor (NGF) is a trace active protein that protects nerves and promotes nerve growth. In vitro, NGF can promote the growth of dorsal root ganglion (DRG) axons and the differentiation of Schwann cells (SCs); in vivo, injection of NGF can significantly inhibit neuronal apoptosis and induce axon regeneration and oligodendrocyte remyelination, which can significantly improve motor and sensory dysfunction caused by nerve injury. Therefore, NGF is effective in treating neurotraumatic diseases such as brain injury, spinal cord injury and peripheral nerve injury (PNI). However, the molecular weight of NGF is about 26 kDa, which is a macromolecular drug with the disadvantages of complex preparation process, high price and poor stability. Therefore, it is imperative to develop drugs with NGF biological activity and overcome NGF defects.

[0004] Peptide drugs are a class of small molecule compounds formed by connecting different amino acids through peptide bonds. They have high safety and target affinity, good thermal stability, low synthesis cost, high yield and purity, etc. At present, there are more than 80 peptide drugs on the market worldwide, which are widely used in the prevention, diagnosis and treatment of tumors, cardiovascular and cerebrovascular diseases, metabolic disorders, infections, and blood system diseases. Summary of the invention

[0005] In view of this, the present invention provides a linear peptide NPL having the biological activity of nerve growth factor 2m And application, the small molecule polypeptide NPL disclosed in the present invention 2mThe amino acid sequence is: VNIKNSVFR (SEQ ID NO: 1), which is modified from the hairpin structure LOOP II on NGF (amino acid sequence: VNINNSVF, SEQ ID NO: 2), that is, the fourth amino acid asparagine (N) on LOOP II is replaced by lysine (K), and an arginine (R) is added at the end.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a polypeptide having:

[0008] (1) the amino acid sequence shown in SEQ ID NO: 1; or

[0009] (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0010] (3) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (1) or (2);

[0011] The plurality is 2 to 5.

[0012] The present invention also provides the use of the above polypeptide in any of the following items:

[0013] (A1), promote the proliferation of nerve cells;

[0014] (A2), promote the migration of nerve cells;

[0015] (A3) Preparation of drugs or preparations for promoting the proliferation of nerve cells;

[0016] (A4) Preparation of drugs or preparations for promoting nerve cell migration.

[0017] The present invention also provides the use of the above polypeptide in any of the following items:

[0018] (B1), promote the growth of dorsal root ganglion axons;

[0019] (B2), promote Schwann cell migration;

[0020] (B3) Preparation of drugs or preparations for promoting the growth of dorsal root ganglion axons;

[0021] (B4) Prepare drugs or preparations that promote Schwann cell migration.

[0022] In some specific embodiments of the present invention, the above-mentioned application of promoting the growth of dorsal root ganglion axons comprises:

[0023] (a) increase the number of dorsal root ganglion axons; and / or

[0024] (b) Promote the extension of dorsal root ganglion axons.

[0025] In some specific embodiments of the present invention, the promoting Schwann cell migration in the above application includes increasing the migration distance of Schwann cells.

[0026] The present invention also provides the use of the polypeptide in preparing a medicine for restoring the body's motor function.

[0027] In some specific embodiments of the present invention, the above-mentioned drug achieves the recovery of the body's motor function by repairing nerve damage.

[0028] The present invention also provides the use of the polypeptide in repairing nerve damage.

[0029] In some specific embodiments of the present invention, the repairing of nerve damage in the above application includes improving the conduction of nerve electrical signals.

[0030] In some specific embodiments of the present invention, the above-mentioned application of improving the conduction of neural electrical signals comprises:

[0031] (C1), increase the amplitude of compound muscle action potential;

[0032] (C2) Increase nerve conduction velocity.

[0033] The present invention also provides a medicine, comprising the above polypeptide and acceptable adjuvants or auxiliary agents.

[0034] In some specific embodiments of the present invention, the polypeptide of the above-mentioned drug is administered at a concentration of 200 nM, 400 nM, 600 nM, 800 nM, 1000 nM, 1200 nM, 1400 nM or 1600 nM.

[0035] The present invention also provides a method for restoring the body's motor function, comprising: administering the above-mentioned polypeptide or the above-mentioned drug to achieve the restoration of the body's motor function. In some specific embodiments of the present invention, the administration concentration is 200 nM, 400 nM, 600 nM, 800 nM, 1000 nM, 1200 nM, 1400 nM or 1600 nM.

[0036] The present invention also provides a method for repairing nerve damage, comprising: administering the above-mentioned polypeptide or the above-mentioned drug to restore the body's motor function. In some specific embodiments of the present invention, the administration concentration is 200 nM, 400 nM, 600 nM, 800 nM, 1000 nM, 1200 nM, 1400 nM or 1600 nM.

[0037] NPL of the present invention 2m Its application has the following effects:

[0038] In vitro results showed that NPL 2m The optimal administration concentration is 400 nM, which has a significant promoting effect on the proliferation and migration of PC12 neural cell lines. At the same time, it can significantly promote the extension of primary dorsal root ganglion axons and the growth of Schwann cells. The results of in vivo animal models also showed that NPL 2m It can significantly improve the motor function and neurophysiological signal conduction of animals and its effect is similar to that of NGF. 2m While retaining the biological activity of NGF, the price is greatly reduced and it has stronger stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0040] Figure 1 NPL 2m Screening results of the optimal concentration for PC12 cell growth;

[0041] Figure 2 Cell images showing PC12 cell proliferation (A) and cell proliferation rate (B);

[0042] Figure 3 The results of PC12 cell migration experiment are shown, where A is the cell image of the cell scratch experiment, B is the cell image of the Transwell experiment, C is the statistical graph of cell migration rate, and D is the statistical graph of cell number;

[0043] Figure 4 Shows the growth status of DRG, where A is the DRG cell image, B is the DRG axon length statistical diagram, and C is the SCs migration distance statistical diagram;

[0044] Figure 5 The results of the PNI rat footstep imprint experiment are shown in Figure 1, where A is the footstep imprint image, B shows the SFI determination method, and C is the SFI value statistical chart;

[0045] Figure 6 The results of nerve electrophysiological conduction evaluation are shown in Figure 1, where A is the electromechanical graph, B is the compound muscle action potential statistical graph, and C is the nerve conduction velocity statistical graph. DETAILED DESCRIPTION

[0046] The invention discloses a linear peptide NPL having biological activity of nerve growth factor 2mThe present invention relates to the present invention and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0047] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0048] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0049] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0050] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0051] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0052] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0053] The present invention will be further described below in conjunction with the embodiments.

[0054] Example 1: NPL 2m Synthesis and purification of

[0055] Using the solid phase synthesis method, Rink-Amide-MBHA resin was swollen in dichloromethane (DCM) and N,N-dimethylformamide (DMF), and the Fmoc protecting group was removed using 50% morpholine; then, after washing with DCM and DMF alternately for 3 times, the amino acid to be connected was mixed with 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and diisopropylethylamine (DIPEA) in an equivalent ratio of 1:1:1 and reacted under nitrogen blowing for 1.5 h. According to the above steps, according to NPL 2m After the linear peptide resin is synthesized, shearing fluid (V 三氟乙酸 :V 三异丙基硅烷 :V 纯水 =95:2.5:2.5) and shake for lysis for 1-1.5 h; after removing the resin, add 50% acetonitrile solvent, separate and purify the peptide by high performance liquid chromatography (HPLC), collect samples with different peaks at different time points, identify the molecular weight by mass spectrometer, obtain the target peptide with a molecular weight of 1448 Da, collect the pure peptide liquid with the peak and put it into freeze dryer for freeze drying, so as to obtain solid NPL 2m .

[0056] Example 2: Synthesis and purification of LOOP II

[0057] Using the solid phase synthesis method, Rink-Amide-MBHA resin was swollen in dichloromethane (DCM) and N,N-dimethylformamide (DMF), and the Fmoc protecting group was removed using 50% morpholine; then, after washing with DCM and DMF alternately for 3 times, the amino acid to be connected was mixed with 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and diisopropylethylamine (DIPEA) in an equivalent ratio of 1:1:1 and reacted under nitrogen blowing for 1.5 h. According to the above steps, different Fmoc protected amino acids were connected in sequence according to the peptide sequence of LOOP II. After the linear peptide resin was synthesized, a shearing solution (V 三氟乙酸 :V 三异丙基硅烷 :V 纯水 =95:2.5:2.5) and shake for lysis for 1-1.5 h; after removing the resin, add 50% acetonitrile solvent, separate and purify the peptide by high performance liquid chromatography (HPLC), collect samples with different peaks on HPLC at different time points, identify the molecular weight by mass spectrometer, obtain the target peptide with a molecular weight of 1320 Da, collect the pure peptide liquid with the peak and put it into a freeze dryer for freeze drying, so as to obtain solid LOOP II.

[0058] Example 3: NPL 2m Screening of the optimal concentration for promoting cell growth

[0059] PC12 cells cultured for more than 3 generations were seeded in a 96-well plate at a density of 5000 / well. The next day, the culture medium was aspirated from each well and a gradient concentration of NPL was added. 2m 100 μL of fresh culture medium with 500 nM (0 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, 1600 nM) was added to each well. After 24 h of culture, 10 μL of CCK-8 solution was added to each well. After the cells were incubated for 24 h, the absorbance of each well was measured at 450 nm using a microplate reader.

[0060] Depend on Figure 1 The results show that joining NPL 2m It has a positive regulatory effect on the growth of PC12 cells and the effect on cell growth becomes more obvious with increasing concentration. 2m After the concentration of NPL increased to 400 nM, its promoting effect on cell growth remained stable. Therefore, 400 nM was selected as the NPL 2m optimal dosing concentration.

[0061] Example 4: NPL 2m Promote cell proliferation

[0062] PC12 cells cultured for more than three generations were cultured at 5×10 4 The density of 100 μg / mL was planted in a 24-well plate with sterile slides. The next day, the culture medium was aspirated from each well and NPL 2m 300 μL of fresh medium containing LOOP II or NGF was added to make NPL 2m The final concentrations of LOOP II and NGF were all 400 nM. After 24 h of cell culture, 50 μM EdU dye was added to the culture medium and the cells were incubated for 4 h. Then, the subsequent operations were performed according to the experimental steps of the EdU kit instructions. At the same time, TRITC-labeled phalloidin (100 μM, red) was used to label the cytoskeleton. Confocal microscopy was used for imaging. The cell proliferation rate (%) = the number of cells labeled with EdU / the number of cells labeled with DAPI × 100%.

[0063] Depend on Figure 2 The results showed that PC12 cells in each group showed a spindle-shaped morphology (red mark). Compared with the normal (Control) group, the number of EdU-labeled nuclei in the LOOP II group increased, but not as much as that in the NPL group. 2m group and NGF group (green, Figure 2The statistical results also showed that the cell proliferation rate of the LOOP II group was significantly higher than that of the normal (Control) group ( * P < 0.05), but less than NPL 2m group and NGF group, there was no statistical difference in cell proliferation rate between the latter two groups ( Figure 2 B in ). Therefore, compared with LOOPII, the modified NPL 2m It has a significant promoting effect on cell proliferation and its effect is comparable to that of NGF.

[0064] Example 5: NPL 2m Promote cell migration

[0065] PC12 cells cultured for more than three generations were cultured at 5×10 4 When the cells grew to about 80% of the wells, a straight line was drawn along the center of each well with a 1 mL pipette tip. Then, each well was washed twice with PBS, and fresh culture medium containing 400 nM NPL was added. 2m , LOOP II or NGF, and images were taken using an optical microscope at 0 h, 24 h, and 48 h after scratching.

[0066] In addition, the Transwell assay was used to evaluate the migration of cells in different groups. PC12 cells of the same density were seeded in the upper chamber of the Transwell containing Matrigel matrix gel, and the culture medium in the lower chamber contained 400 nM NPL 2m , LOOPII or NGF. After 24 h of culture, the culture medium in the upper chamber was aspirated and the Matrigel and cells in the inner layer of the Transwell chamber were wiped clean with a cotton swab, and then stained with crystal violet. After proper air drying, 5 fields of view were selected under a 10x microscope to observe and count the cells.

[0067] The results show that LOOP II, NPL 2m Both NGF and NF-κB can promote the growth of PC12 cells along the scratch boundary toward the inside, but the latter two drugs have a more significant effect in accelerating the disappearance of the scratch area ( Figure 3 The statistical results showed that at 24 h and 48 h after administration, NPL 2m and NGF promoted cell migration rates significantly better than LOOP II ( Figure 3 C). The results of Transwell experiments also found that compared with LOOP II, the addition of NPL 2m Or NGF can significantly increase the number of PC12 cells that penetrate from the upper chamber into the lower chamber ( Figure 3 B), but there was no significant difference in the number of cells counted between the two ( Figure 3D in ). Therefore, NPL 2m Both LOOP II and NGF can significantly promote cell migration, and the effect is better than LOOP II.

[0068] Example 6: NPL 2m Promotes the growth of DRG axons and SCs

[0069] To further explore NPL 2m The biological activity of DRG was extracted from newborn mice and implanted on a 24-well plate slide. 2m The cells were then cultured for 3 days with 300 μL of Neurobasal medium containing LOOP II or NGF (final concentration of 400 nM). The growth of DRG in each group was observed by double staining with NF-200 and S-100 fluorescence.

[0070] Depend on Figure 4 The results showed that under normal culture conditions, the number of axons and SCs emitted from the neuronal cell bodies of DRG was small and the extension distance was short; when LOOP II and NPL were added to the culture medium, 2m After treatment with NGF or NGF, the distance of axons from DRG cell bodies extended outward increased, accompanied by an increase in the number and migration of SCs with different effects. However, the latter two drugs were more effective in promoting axon growth and SC migration ( Figure 4 A in Figure 1). The results of the statistical analysis of the axon length and SCs migration distance of each group are as follows: NPL 2m Group > NGF group > LOOP II group > Control group ( Figure 4 B and C in Figure 1). Therefore, compared with the entity LOOPII, the NPL with changed amino acid sequence 2m In terms of promoting the growth of axons and SCs, the effect was greatly enhanced, and this enhanced effect was even better than that of NGF.

[0071] Example 7: NPL 2m Promote recovery of motor function after PNI

[0072] Using the PNI model to characterize NPL in vivo 2m The role of NPL in repairing damaged nerves was further explored. After anesthetizing 8-week-old SD male rats, surgical scissors and glass needles were used to expose and separate the sciatic nerve. The sciatic nerve was clamped with a 30 g venous clamp for 2 minutes to create a sciatic nerve contusion model. The muscles and skin were then sutured and 80 ng of NPL was injected in situ at the injury site. 2m(100 μL), LOOP II (100 μL), or NGF (100 μL) were administered for 28 consecutive days. Then, the improvement of motor function of rats was evaluated using the footstep imprinting test. In this experiment, the footprints left by each rat on white paper were measured for PL (the distance between the third toe and the heel), TS (the distance between the first and fifth toes), and IT (the distance between the second and fourth toes) (e.g. Figure 5 The measured results were substituted into the following formula to calculate the sciatic nerve function index (SFI), SFI = -38.3 × (EPL-NPL) / NPL + 109.5 × (ETS-NTS) / NTS + 13.3 × (EIT-NIT) / NIT-8.8, where E is the injured side and N is the contralateral side. The larger the SFI value, the better the animal's motor function recovery.

[0073] Depend on Figure 5 The results showed that the sham group rats had clear footsteps and the toes were clearly separated, while the footprints left by the hind limbs on the operated side of the PNI group could not be stretched, and the toes were curled together. 2m After treatment with NGF or LOOP II, the footprints of PNI were clearer and the toes were more spread out, and this effect was better than that of LOOP II ( Figure 5 A in the figure). The statistical analysis of the SFI values ​​of the groups showed that the SFI of the Sham group was close to 0, the average SFI of the PNI group was about -34.7, the average SFI of the LOOP II group was about -31.4, and the average SFI of the NPL group was about -34.7. 2m The mean SFI values ​​of WT and NGF were -25.4 and -23.6, respectively, and there was no statistical difference between them ( Figure 5 C in ). Therefore, NPL 2m It can significantly improve the recovery of motor function in PNI rats and the effect is similar to that of NGF.

[0074] Example 8: NPL 2m Promote the conduction of nerve electrophysiology after PNI

[0075] The changes in the bioelectrical activity of the nerves and muscles of the rats in each group after the sciatic nerves were stimulated by electrical signals were detected by electrophysiological instrument. On the 28th day after surgery, the rats in each group were anesthetized with pentobarbital, and the sciatic nerves on the surgical side were re-exposed. The stimulating electrode of the instrument was placed at the site of sciatic nerve contusion, and the recording electric shock was inserted into the gastrocnemius muscle. The parameters of the instrument were set as follows: stimulation intensity = 50 mV; frequency = 1 Hz; duration = 5 ms. The electromechanical diagrams of the rats in each group were recorded, and the nerve conduction velocity (MNCV) and compound muscle action potential (CMAP) were calculated.

[0076] Depend on Figure 6 The results show that the waveform of PNI is smaller, while that of LOOP II and NPL2m The waveform and amplitude of the NGF group increased to varying degrees ( Figure 6 A in the figure). The CMAP of each group was statistically analyzed and found that the Control group had the largest CMAP, followed by the NPL group. 2m and NGF-treated groups, followed by the LOOP II group, while the PNI group was the smallest ( Figure 6 Similarly, NPL 2m The MNCV between the NGF2 group and the LOOP II group was similar and significantly higher than that of the PNI group ( Figure 6 C in ). Therefore, NPL 2m It can significantly improve the conduction of neural electrical signals after PNI and has a similar therapeutic effect to that of NGF.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide obtained by modifying the amino acid sequence of LOOP II, wherein LOOP II is a part of the amino acid sequence of nerve growth factor, characterized in that: The sequence of the polypeptide is SEQ ID NO:

1.

2. Use of the polypeptide according to claim 1 in any of the following: (A1), promote the proliferation of nerve cells; (A2), promote neuronal cell migration; (A3) Preparation of drugs or preparations for promoting the proliferation of nerve cells; (A4) Preparation of drugs or preparations for promoting nerve cell migration.

3. Use of the polypeptide according to claim 1 in any of the following: (B1), promote the growth of dorsal root ganglion axons; (B2), promote Schwann cell migration; (B3) preparing drugs or preparations for promoting the growth of dorsal root ganglion axons; (B4) Prepare drugs or preparations that promote Schwann cell migration.

4. The use according to claim 3, characterized in that The method for promoting the growth of dorsal root ganglion axons comprises: (a) increase the number of dorsal root ganglion axons; and / or (b) Promote the extension of dorsal root ganglion axons.

5. The use according to claim 3 or 4, characterized in that: The promoting Schwann cell migration includes increasing the migration distance of Schwann cells.

6. Use of the polypeptide according to claim 1 in preparing a drug for repairing nerve damage.

7. Use of the polypeptide according to claim 1 in the preparation of a drug for restoring the body's motor function.

8. The use according to claim 7, characterized in that The medicine restores the body's motor function by repairing nerve damage.

9. A drug, characterized in that The invention comprises the polypeptide according to claim 1, and acceptable adjuvants or auxiliary agents.

10. The drug according to claim 9, characterized in that The administration concentration of the polypeptide is above 200 nM.

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