Rabies virus-derived peptide and use thereof

Abstract

The application discloses a rabies virus-derived peptide and application thereof. The rabies virus-derived peptide provided by the application has an amino acid sequence as shown in SEQ ID NO:1 or a sequence with 75%, 80%, 85%, 90%, 95% or 100% homology with the amino acid sequence shown in SEQ ID NO:1, can target delivery of bioactive macromolecules and other active small molecules which are difficult to cross the blood-brain barrier to nerve cells, helps to improve the treatment effect of brain diseases, and has a good development and application prospect in the field of new drug research and development and clinical treatment of brain diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a polypeptide and its application, particularly to a rabies virus-derived peptide and its application in pharmaceuticals. Background Technology

[0002] The incidence of brain diseases such as Alzheimer's disease, brain tumors, and mental illnesses is increasing daily, seriously threatening human health. One of the bottlenecks in treating brain diseases lies in the blood-brain barrier; more than 95% of bioactive macromolecules with therapeutic potential, such as proteins and nucleic acids, cannot cross the blood-brain barrier to enter the brain and thus cannot exert their therapeutic effects. Furthermore, a crucial issue that needs to be addressed in treating brain diseases is how to achieve targeted drug delivery to the brain to reduce the significant side effects associated with systemic administration.

[0003] There are three main types of brain-targeted drug delivery methods: First, invasive direct drug delivery, including hyperosmolar shock, carotid artery injection of vasoactive substances, and intrathecal or intraventricular injection. However, this method easily causes brain infection and surgical damage, posing a significant threat to the brain. Second, methods that increase the drug's ability to cross the blood-brain barrier, but this method requires high-quality physicochemical properties of the drug itself and has considerable limitations. Third, drug transport mediated by brain-targeted drug delivery carriers. This method can increase targeted drug uptake in the brain and greatly reduce invasive damage to the brain. Therefore, finding carriers capable of carrying bioactive substances across the blood-brain barrier to achieve brain-targeted drug delivery is currently a key focus and challenge in drug research for treating brain diseases. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the purpose of this invention is to provide a novel rabies virus-derived peptide and its application in the preparation of a brain-targeted drug delivery carrier.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of this invention provides a rabies virus-derived peptide.

[0007] Furthermore, the rabies virus-derived peptide has an amino acid sequence as shown in SEQ ID NO:1 or a sequence having 75%, 80%, 85%, 90%, 95% or more of homology with the amino acid sequence shown in SEQ ID NO:1.

[0008] Furthermore, the amino acids in the amino acid sequence of the rabies virus-derived peptide are L-type or D-type amino acids.

[0009] In this invention, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds having amino acid residues covalently linked by a peptide bond.

[0010] In the description of this invention, the term "amino acid" refers to the basic unit that constitutes a protein, giving the protein a specific molecular structure and morphology, and endowing its molecules with biochemical activity. For example, the "amino acid" used in this invention includes the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y).

[0011] In some embodiments, the polypeptide of the present invention also includes a polypeptide having the same function formed by substituting, deleting or adding one or more (2, 3, 4, 5 or 6) amino acid residues of the polypeptide shown in SEQ ID NO:1.

[0012] In this invention, the terms "homology" and "identity" are used interchangeably. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0013] In this invention, the modified polypeptide sequences also fall within the scope of protection. The term "modification" refers to any chemical modification of an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids with different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence resulting in an increase of one or more amino acids compared to the naturally occurring molecule.

[0014] A second aspect of the present invention provides a nucleic acid molecule.

[0015] Furthermore, the nucleic acid molecule encodes the rabies virus-derived peptide described in the first aspect of the present invention.

[0016] In this invention, the terms "polynucleotide," "nucleic acid molecule," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, namely deoxyribonucleotides or ribonucleotides or their analogues. Polynucleotides may be further modified after polymerization, for example by conjugation with labeled components. The term also refers to double-stranded and single-stranded molecules.

[0017] A third aspect of the present invention provides a carrier molecule.

[0018] Furthermore, the carrier molecule comprises the nucleic acid molecule described in the second aspect of the present invention.

[0019] Furthermore, the vector includes plasmids and viral vectors.

[0020] Furthermore, the viral vector includes lentivirus, adenovirus, and adeno-associated virus vector.

[0021] In this invention, the term "vector" refers to a nonchromosomal nucleic acid containing a complete replicon, such that the vector can be replicated when placed within a permitted cell, for example, through a transformation process. A vector can replicate in one cell type (e.g., bacteria) but has limited ability to replicate in another cell type (e.g., mammalian cells). Vectors can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; and DNA, alone or in combination with a cationic polymer, in a cationic lipid complex; anionic and cationic liposomes; DNA-protein complexes; and particles containing DNA condensed with cationic polymers (such as heteropolymers of polylysine, fixed-length oligopeptides, and polyethyleneimine), and in some cases, also contained in liposomes.

[0022] In some embodiments, the vectors described in this invention include plasmids (expression plasmids, cloning vectors, small loops, microvectors, double microchromosomes), lentiviral vectors, adenoviral vectors, or retroviral vectors.

[0023] Preferably, the lentiviral vector includes a recombinant lentiviral vector from primates, namely a recombinant human immunodeficiency virus (HIV) vector or a recombinant simian immunodeficiency virus (SIV) vector.

[0024] Preferably, the lentiviral vector includes a non-primate recombinant lentiviral vector, namely recombinant equine infectious anemia virus (EIAV), recombinant feline immunodeficiency virus (FIV), or recombinant caprine arthritis-encephalitis virus (CAEV).

[0025] Preferably, the carrier further includes one or more control elements.

[0026] Preferably, the regulatory element comprises a promoter, an enhancer, a ribosome binding site for translation initiation, a terminator, a polyadenylate sequence, and a selection marker gene.

[0027] Preferably, the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

[0028] A fourth aspect of the present invention provides a host cell.

[0029] Furthermore, the host cell contains the nucleic acid molecule described in the second aspect of the present invention or the carrier molecule described in the third aspect of the present invention.

[0030] Furthermore, the host cells include prokaryotic cells and eukaryotic cells.

[0031] The fifth aspect of the present invention provides a brain-targeting and / or nerve cell delivery system.

[0032] Furthermore, the brain-targeting and / or neural cell delivery system comprises the rabies virus-derived peptide described in the first aspect of the present invention.

[0033] Furthermore, the brain-targeting and / or neural cell delivery system also contains bioactive substances.

[0034] Furthermore, the bioactive substance is selected from any of the following: nucleic acids, peptides, antibodies, and chemotherapeutic agents.

[0035] The rabies virus-derived peptides provided by this invention offer an efficient, safe, and non-invasive method for the brain-targeted transport of bioactive macromolecules and other small bioactive molecules that are difficult to cross the blood-brain barrier, thereby improving the therapeutic effect of brain diseases.

[0036] In some embodiments of the present invention, while retaining the activity of the rabies virus-derived peptide, the rabies virus-derived peptide can be chemically modified to prolong its half-life, such as cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, albumin affinity peptide modification, NOA modification, radionuclide modification, tumor homing peptide coupling, membrane-penetrating peptide coupling, nanocarrier coupling, and a series of other predictable chemical modification methods.

[0037] As used herein, the term "bioactive substance" refers to a physiologically active regulator of physiological function and a substance that can be used to treat all diseases caused by imbalances or dysfunctions of physiological function, in relation to the purposes of this invention. Bioactive substances may be nucleic acids, peptides, polypeptides, antibodies, or chemical compounds (chemotherapeutic agents), but this invention is not limited thereto.

[0038] The sixth aspect of the present invention provides a pharmaceutical composition.

[0039] Furthermore, the pharmaceutical composition comprises the rabies virus-derived peptide described in the first aspect of the present invention.

[0040] The seventh aspect of the present invention provides a method for preparing the rabies virus-derived peptide described in the first aspect of the present invention.

[0041] Furthermore, the method includes the following steps: culturing the host cells described in the fourth aspect of the present invention to obtain a culture product, and separating and purifying the rabies virus-derived peptide described in the first aspect of the present invention from the culture product.

[0042] The eighth aspect of the present invention provides the use of the rabies virus-derived peptide described in the first aspect of the present invention in the preparation of products for promoting cytotoxicity in tumor cells and / or inhibiting tumor cell proliferation.

[0043] Preferably, the tumor cells are neuroblastoma cells.

[0044] The ninth aspect of the present invention provides the use of the rabies virus-derived peptide described in the first aspect of the present invention in the preparation of a carrier for delivering bioactive substances to nerve cells.

[0045] Furthermore, the bioactive substance is selected from any of the following: nucleic acids, peptides, antibodies, and chemotherapeutic agents.

[0046] The ninth aspect of this invention provides the application of the rabies virus-derived peptide described in the first aspect of this invention in the preparation of a brain-targeted drug delivery carrier.

[0047] In another aspect of the invention, the invention provides a pharmaceutical composition for treating brain diseases comprising (b) a material selected from any one or more of the following: a peptide of SEQ ID NO:1 or a modified peptide thereof, a peptide or a dimer thereof, and a biocompatible polymer, liposomes, vesicles and nanomaterials, a therapeutically effective amount of a complex containing a bioactive substance; and (b) a pharmaceutically acceptable carrier.

[0048] Furthermore, the brain diseases mentioned include, but are not limited to, brain tumors, central degenerative diseases (Parkinson's disease (PD), Alzheimer's disease (AD), etc.), cerebrovascular lesions, and bacterial and viral infections of the brain.

[0049] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0050] In the description of this invention, the term "and / or" includes all and any combination of one or more of the associated listed items.

[0051] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0052] The beneficial effects of this invention are:

[0053] This invention provides a rabies virus-derived peptide and its application, which can target and deliver bioactive macromolecules and other bioactive small molecules that are difficult to cross the blood-brain barrier to nerve cells, thus helping to improve the treatment effect of brain diseases and showing good development and application prospects in the field of new drug development and clinical treatment of brain diseases. Attached Figure Description

[0054] Figure 1 Figure showing the results of observing GFP expression in SH-SY5Y cells after treatment with rabies virus-derived peptides using a fluorescence inverted microscope;

[0055] Figure 2The figure shows the results of detecting the expression of Luc in Neuro-2a cells after treatment with rabies virus-derived peptides using a luciferase assay kit. Detailed Implementation

[0056] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0057] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0058] Example 1: Transport of rabies virus-derived peptides in human neuroblastoma SH-SY5Y cells

[0059] The amino acid sequence of the rabies virus-derived peptide used in this embodiment is KSVWTWNEKPPSKGCLCVGGMCHPPVNGGGRRRRRRRRR (SEQ ID NO:1). The polypeptide was synthesized based on this amino acid sequence and named 2D3.

[0060] The human neuroblastoma SH-SY5Y cell line is a commonly used neural cell line derived from the neuroblastoma cell line SK-N-SH. SH-SY5Y cells possess certain characteristics of neurons, such as the ability to differentiate, express neuron-specific markers, and respond to neurotransmitters and drugs. They are frequently used to study nervous system diseases, neural development processes, and neurotoxicity.

[0061] I. Experimental Methods

[0062] 1. Seed SH-SY5Y cells in 24-well plates (2×10⁻⁶ cells / well). 5 Cells / well), incubated overnight in a 37°C CO2 cell culture incubator.

[0063] 2. Add plasmid pmax-GFP and peptide 2D3 to 50 μL of Opti-MEM medium at a mass ratio of 2 μg: 2 μg, mix well, and incubate at room temperature for 30 min using a microplate constant temperature shaker.

[0064] 3. Add the pmax-GFP-peptide complex plasmid to the pre-coated SH-SY5Y cells, and set up a negative control. Continue culturing in a 37°C carbon dioxide cell incubator.

[0065] 4. After 24 hours, the expression of GFP was observed using a fluorescence inverted microscope.

[0066] II. Experimental Results

[0067] The results are as follows Figure 1 As shown, no GFP green fluorescence was observed in the control group, while GFP green fluorescence was observed in SH-SY5Y cells treated with rabies virus-derived peptide 2D3, indicating that rabies virus-derived peptide 2D3 has strong neurotropism and can mediate the entry of GFP plasmids into SH-SY5Y cells.

[0068] Example 2: Transport of rabies virus-derived peptides in mouse neuroblastoma Neuro-2a cells

[0069] The amino acid sequence of the rabies virus-derived peptide used in this embodiment is KSVWTWNEKPPSKGCLCVGGMCHPPVNGGGRRRRRRRRR (SEQ ID NO:1). The polypeptide was synthesized based on this amino acid sequence and named 2D3.

[0070] I. Experimental Methods

[0071] 1. Seed Neuro-2a cells in 24-well plates (2 × 10⁻⁶ cells / well). 5 Cells / well), incubated overnight in a 37°C CO2 cell culture incubator.

[0072] 2. Add plasmid pLuc and peptide 2D3 to 50 μL of Opti-MEM medium at a mass ratio of 2 μg: 8 μg, mix well, and incubate at room temperature for 30 min using a microplate constant temperature shaker.

[0073] 3. Add the pLuc-peptide plasmid complex to the prepared Neuro-2a cells, and set up a negative control. Continue culturing in a 37°C carbon dioxide cell incubator.

[0074] 4. After 24 hours, the expression of Luc was detected using a luciferase assay kit.

[0075] II. Experimental Results

[0076] The results are as follows Figure 2 As shown, the expression level of Luc in the control group was extremely low, while the expression level of Luc in Neuro-2a cells treated with rabies virus-derived peptide 2D3 was very high, indicating that rabies virus-derived peptide 2D3 has strong neurotropism and can mediate the entry of Luc plasmid into Neuro-2a cells.

[0077] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A rabies virus-derived peptide, characterized in that, The amino acid sequence of the rabies virus-derived peptide is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the rabies virus-derived peptide of claim 1.

3. A carrier molecule, characterized in that, The carrier molecule comprises the nucleic acid molecule of claim 2.

4. The carrier molecule according to claim 3, characterized in that, The vector molecules include plasmids and viral vectors.

5. The carrier molecule according to claim 4, characterized in that, The viral vectors include lentiviruses, adenoviruses, and adeno-associated virus vectors.

6. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 2 or the carrier molecule of any one of claims 3-5.

7. The host cell according to claim 6, characterized in that, The host cells include prokaryotic cells and eukaryotic cells.

8. A brain-targeting and / or nerve cell-targeting delivery system, characterized in that, The brain-targeting and / or neural cell delivery system comprises the rabies virus-derived peptide of claim 1.

9. The brain-targeting and / or nerve cell-targeting delivery system according to claim 8, characterized in that, The brain-targeting and / or neural cell delivery system also contains a bioactive substance, which is a nucleic acid.

10. A method for preparing the rabies virus-derived peptide of claim 1, characterized in that, The method includes the following steps: culturing the host cells as described in claim 6 or 7 to obtain a culture product, and separating and purifying the rabies virus-derived peptide as described in claim 1 from the culture product.

11. The use of the rabies virus-derived peptide of claim 1 in the preparation of a carrier for delivering a bioactive substance to nerve cells; wherein the bioactive substance is a nucleic acid.

12. The application of the rabies virus-derived peptide according to claim 1 in the preparation of a brain nerve cell targeted drug delivery carrier, characterized in that, The drug in question is a nucleic acid.

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