NSDP1 micropeptide and its application in repair of demyelination injury

Abstract

The present invention provides an NSDP1 micropeptide and its application in repairing demyelinating damage, belonging to the field of biomedicine technology. The NSDP1 polypeptide of the present invention can significantly promote the remyelination of axonal demyelinating damage, providing a new idea and method for repairing nerve demyelinating damage.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an NSDP1 micropeptide and its application in repairing demyelination damage. Background Art

[0002] Demyelination of the nervous system is a common nerve injury. It refers to the demyelination of the myelin sheath wrapped around the axons of nerve cells due to mechanical damage or immune effects, resulting in nervous system damage. According to the different sites of injury, it can be divided into central nervous system demyelination and peripheral nerve demyelination. Among them, cranial nerve vascular compression syndrome is a common neurosurgical disease caused by vascular compression of the relevant cranial nerves in the cerebellopontine angle (CPA) area near the brainstem. It includes trigeminal neuralgia, hemifacial spasm and spasmodic torticollis. The main manifestations are long-term neuralgic pain, abnormal facial muscle twitching and abnormal head and neck posture, which bring great physical and mental pain to patients. The main pathological feature of cranial nerve vascular compression syndrome is the compression of the trigeminal nerve, facial nerve and accessory nerve at the exit (entry) of the brainstem (REZ area) by blood vessels. Research reports indicate that the REZ is a short transition zone connecting the central nervous system and peripheral nerves, extending from the trigeminal nerve exiting the brainstem (assuming this is 0 mm) to 4.19 mm and the facial nerve exiting the brainstem to 2.86 mm, where central nervous system myelin remains. Furthermore, ultrastructural studies of nerve cells in the REZ of patients with cranial nerve vascular compression syndrome have shown that these patients experience central nervous system demyelination in the REZ, where vascular compression occurs.

[0003] At present, there is no effective means of treating demyelinating diseases clinically. The clinical treatment of cranial nerve vascular compression syndrome mainly uses drugs such as carbamazepine and phenytoin sodium, or surgical treatment with microvascular decompression, but these cannot alleviate the demyelinating damage caused by vascular compression. For example, patent CN201080002517.4 discloses that aminopyridines, such as 4-aminopyridine, can be used to improve conditions with demyelination, such as neurocognitive damage and related neuropsychiatric damage in patients with MS, traumatic brain injury, cerebral palsy, and radiation-induced encephalopathy. Patent CN202011287278.3 discloses the use of dextrorotatory ketamine in the preparation of drugs for the treatment of demyelinating diseases.

[0004] Therefore, it is very necessary to actively seek new and effective prevention and treatment methods to promote the repair of central nervous system demyelinating damage. Summary of the Invention

[0005] In view of the above shortcomings, the present invention provides a NSDP1 micropeptide and its application in repairing demyelinating damage. The NSDP1 polypeptide of the present invention can significantly promote the remyelination of axonal demyelinating damage, providing a new method for the treatment of demyelinating damage in nerve cells.

[0006] It provides new ideas and methods for repairing demyelinating injuries.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides an NSDP1 polypeptide, wherein the amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0009] In another aspect, the present invention provides a series of nucleic acid molecules encoding the polypeptides.

[0010] Specifically, the nucleic acid molecule comprises one or more codon-optimized nucleic acid molecules.

[0011] In another aspect, the present invention provides a series of vectors comprising one or more nucleic acid molecules described in the present application.

[0012] Specifically, the vector includes but is not limited to plasmid, virus, and bacteriophage.

[0013] In another aspect, the present invention provides a series of host cells comprising the above nucleic acid molecules or the above vectors.

[0014] Specifically, the host cells include but are not limited to microorganisms, plants or animal cells, and the vector of the present invention can be introduced into the host cells by methods known to those skilled in the art, such as electroporation, lipofectine transfection, lipofectamin transfection and the like.

[0015] In another aspect, the present invention provides use of the above-mentioned polypeptide, nucleic acid molecule, vector or host cell in the preparation of a drug or reagent for repairing myelin damage.

[0016] In another aspect, the present invention provides a drug or reagent for repairing myelin damage, wherein the drug or reagent comprises the above-mentioned polypeptide, nucleic acid molecule, vector or host cell.

[0017] Specifically, the drug or agent further comprises an optional pharmaceutically acceptable carrier.

[0018] More specifically, the pharmaceutically acceptable carrier includes, but is not limited to, a diluent, an excipient, a filler, a wetting agent, a disintegrant, a flavoring agent, and a binder.

[0019] Specifically, the medicine or agent includes but is not limited to capsules, tablets, lozenges, pills, pellets, suppositories, sprays, creams, patches and the like.

[0020] In certain embodiments, the NSDP1 polypeptide can be obtained by amino acid solid phase synthesis;

[0021] The polypeptide fragment can be obtained by expressing it in a microorganism or host cell and isolating and purifying it. The microorganisms and host cells are well known and commonly used, such as Escherichia coli (E. coli), Actinomycetes (Actinomycetes), Bacillus (Bacillus), Streptomyces (Streptomyces) and other host cells. The polypeptide isolation and purification methods of the present invention are methods and techniques well known to those skilled in the art, such as conventional liquid chromatography for separation and purification.

[0022] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0023] (1) The present invention provides a NSDP1 micropeptide (SEQ ID NO: 1, designated NSDP1NO1), a 13-amino acid peptide derived from MBP. Currently, no relevant functional information has been reported. Bioinformatics analysis results showed that: 1) NSDP1 is derived from amino acids 166-178 of the MBP protein; 2) ProtParam online analysis revealed that PDHPS1 has a fat coefficient of 90 and an average hydrophilicity of 0.292, indicating hydrophobicity and lipophilicity.

[0024] (2) The present invention also provides an NSDP1 micropeptide (SEQ ID NO: 2), which consists of 22 amino acids. Nine arginine residues (R) are added to the N-terminus of the polypeptide represented by SEQ ID NO: 1. Currently, no relevant functional reports have been published. Bioinformatics analysis results showed that PDHPS1 has a fat coefficient of 53.18 and an average hydrophilicity of -1.668, indicating a hydrophobic and lipophilic nature.

[0025] (3) A zebrafish model of demyelinating injury was constructed, and the peptide NSDP1 (SEQ ID NO: 1 or SEQ ID NO: 2) was administered via water. The results showed that the peptide NSDP1 (SEQ ID NO: 2) significantly promoted the remyelination of axonal demyelinating injury. Therefore, the modified NSDP1 can be used to prepare drugs or reagents that promote the repair of demyelinating injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of how the peptide NSDP1 promotes the repair of axonal demyelination damage. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0028] Example 1.

[0029] Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the following sequence shown in SEQ ID NO: 1 by solid phase method: AspThrGlyIleLeuAspSerIleGlyArgGluGluSer.

[0030] The main biological parameters of the peptide sequence obtained by online tools are as follows: isoelectric point (PI) is 4.21, and molecular mass (Mw) is 1427.58.

[0031] The above sequence is a natural sequence derived from the nervous system and can be obtained by amino acid solid-phase synthesis; it can also be obtained by expressing the polypeptide fragment in a microorganism or host cell and isolating and purifying it. The microorganisms and host cells are well known and commonly used, such as Escherichia coli (E. coli), Actinomycetes (Actinomycetes), Bacillus (Bacillus), Streptomyces (Streptomyces) and other host cells. The polypeptide isolation and purification methods described in the present invention are methods and techniques well known to those skilled in the art, for example, separation and purification can be performed by conventional liquid chromatography.

[0032] Example 2.

[0033] Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the following sequence shown in SEQ ID NO: 2 by solid phase method: ArgArgArgArgArgArgArgArgArgAspThrGlyIleLeuAspSerIleGly ArgGluGluSer

[0034] The main biological parameters of the peptide sequence obtained by online tools are as follows: isoelectric point (PI) is 12.37, and molecular mass (Mw) is 2833.26.

[0035] The above sequence represents a modified NSDP1 polypeptide (designated NSDP1NO2), which can be obtained by solid-phase amino acid synthesis; alternatively, the polypeptide fragment can be obtained by expressing it in microorganisms or host cells and then isolating and purifying it. These microorganisms and host cells are well-known and commonly used, such as Escherichia coli, Actinomycetes, Bacillus, and Streptomyces. The polypeptide isolation and purification methods described herein are well-known to those skilled in the art, such as conventional liquid chromatography.

[0036] Example 3.

[0037] Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize the following sequence shown in SEQ ID NO: 3 by solid phase method: ProLysLysLysArgLysValAspThrGlyIleLeuAspSerIleGlyArgGlu GluSer

[0038] The main biological parameters of the peptide sequence obtained by online tools are as follows: isoelectric point (PI) is 10.45, and molecular mass (Mw) is 2292.71.

[0039] The above sequence represents a modified NSDP1 polypeptide (designated NSDP1NO3), which can be obtained by solid-phase amino acid synthesis; alternatively, the polypeptide fragment can be obtained by expressing it in microorganisms or host cells and then isolating and purifying it. These microorganisms and host cells are well-known and commonly used, such as Escherichia coli, Actinomycetes, Bacillus, and Streptomyces. The polypeptide isolation and purification methods described herein are well-known to those skilled in the art, such as conventional liquid chromatography.

[0040] Experimental Example 1.

[0041] 1. Test materials

[0042] Tg(mbp:eEGP) transgenic zebrafish were purchased from the National Zebrafish Resource Center, PTU was purchased from Sigma, lysolecithin was purchased from Sigma, low melting point agarose was purchased from Sigma, and the anesthetic Tricaine was purchased from Sigma.

[0043] 2. Synthesis and dilution of NSDP1 peptide and modified peptide

[0044] The polypeptides in Examples 1 and 2 of this experiment were synthesized by solid-phase synthesis at Nanjing GenScript Co., Ltd. 10 mg of the polypeptide was diluted with sterile ultrapure water to a concentration of 50 mM and stored at -20°C until use.

[0045] 3. In vivo zebrafish experiments to detect the effects of peptide NSDP1 and modified NSDP1 peptides on the repair of axonal demyelination injury

[0046] We used Tg(mbp:eEGP) transgenic zebrafish, which specifically express green fluorescence in the myelin sheaths of spinal cord axons in the zebrafish trunk. This species has been well-established for in vivo studies of demyelinating injury and repair. Zebrafish and the larvae described below were maintained at 28.5°C in a 14-hour light cycle and a 10-hour dark cycle. Tg(mbp:eEGP) male and female fish were placed in a mating box and allowed to mate freely the next day. The fertilized eggs produced by the transgenic zebrafish were collected and cultured in an incubator. After 24 hours, 1 / 20 volume of 0.003% PTU was added to inhibit its pigment growth. When the zebrafish grew to 96 hpf (96 hours after fertilization), the zebrafish were anesthetized with 1% Tricaine and 4nl of 1% lysolecithin was injected into the spinal cord of the zebrafish trunk (above the cloaca). The injected zebrafish were divided into three groups: a control group, a 20μM NSDP1 treatment group, and a 20μM NSDP1 modified polypeptide treatment group. They were continued to be cultured and the demyelination damage and repair near the injection site were observed using laser confocal microscopy (Leica, SP8) 4 hours, 24 hours, and 48 hours after injury.

[0047] The results are as follows Figure 1 shown.

[0048] Test results: Figure 1 As shown in the results, the experiments found that at 4 hours, 24 hours and 48 hours after injury, the NSDP1 polypeptide treatment group could partially promote the repair of early myelin damage, while the modified NSDP1(NO2) polypeptide could significantly promote the repair of demyelination damage, while the modified NSDP1(NO3) had no obvious effect in promoting damage repair, indicating that the modified NSDP1(NO2) polypeptide could significantly promote remyelination after myelin damage.

[0049] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A NSDP1 polypeptide, characterized in that: The amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A nucleic acid molecule encoding the polypeptide of claim 1.

3. A vector comprising the nucleic acid molecule according to claim 2, characterized in that: The vector includes plasmid, virus or phage.

4. A host cell comprising the nucleic acid molecule of claim 2 or the vector of claim 3.

5. The host cell according to claim 4, characterized in that: The host cells include microorganisms or animal cells.

6. Use of the polypeptide according to claim 1, the nucleic acid molecule according to claim 2, the vector according to claim 3 or the host cell according to claim 4 in the preparation of a drug or reagent for repairing myelin damage.

7. A drug or reagent for repairing myelin damage, characterized in that: The drug or reagent comprises the polypeptide according to claim 1, the nucleic acid molecule according to claim 2, the vector according to claim 3 or the host cell according to claim 4.

8. The drug according to claim 7, characterized in that: The drug further comprises optional pharmaceutically acceptable carriers, including but not limited to diluents, excipients, fillers, wetting agents, disintegrants, flavoring agents and binders.

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