Rabies virus derived peptide and application thereof

Abstract

The invention discloses a rabies virus derived peptide and application thereof. The rabies virus derived peptide provided by the invention has an amino acid sequence as shown in SEQ ID NO: 1 or a sequence with 75%, 80%, 85%, 90%, 95% or more than 100% homology with the amino acid sequence as shown in SEQ ID NO: 1, and can deliver bioactive macromolecules and other active micromolecules which are difficult to pass through a blood brain barrier to nerve cells in a targeted manner, so that the treatment effect on brain diseases is improved; the polypeptide has good development and application prospects in the fields of new drug research and development and clinical treatment of cerebral diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a polypeptide and its application, in particular to a rabies virus-derived peptide and its application in pharmaceutical manufacturing. Background Art

[0002] Brain diseases such as Alzheimer's disease, brain tumors, and mental illnesses are on the rise, posing a serious threat to human health. One of the bottlenecks in treating brain diseases lies in the blood-brain barrier. Over 95% of potentially therapeutic bioactive macromolecules, such as proteins and nucleic acids, are unable to cross the blood-brain barrier and enter the brain, rendering them ineffective. Furthermore, a crucial challenge in treating brain diseases is how to achieve targeted drug delivery to the brain to minimize the significant side effects of systemic drug delivery.

[0003] There are three main types of brain-targeted drug delivery methods: the first is invasive and damaging direct drug delivery, including hyperosmotic shock, carotid artery injection of vasoactive substances, and intrathecal or intraventricular injection. However, this method is prone to brain infection and surgical damage, and is significantly invasive to the brain. The second is to increase the ability of drugs to penetrate the blood-brain barrier, but this method has high requirements for the physical and chemical properties of the drug itself and has significant limitations. The third is drug transport mediated by brain-targeted drug delivery vehicles. This method can increase the targeted uptake of drugs in the brain and greatly reduce invasive damage to the brain. Therefore, finding a vehicle that can carry bioactive substances through the blood-brain barrier to achieve brain-targeted drug delivery is the focus and difficulty in the current research of drugs for treating brain diseases. Summary of the Invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a novel rabies virus-derived peptide and its use in the preparation of a brain-targeted drug delivery vehicle.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present 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 100% or greater homology to the amino acid sequence as 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 the present invention, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds having amino acid residues covalently linked by peptide bonds.

[0010] In the description of the present invention, the term "amino acid" refers to the basic unit that constitutes protein, which gives the protein a specific molecular structure and shape, and makes its molecule have biochemical activity. For example, the "amino acid" used in the present 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 polypeptides described in the present invention also include polypeptides formed by replacing, deleting or adding one or more (2, 3, 4, 5, 6) amino acid residues of the polypeptide shown in SEQ ID NO: 1, and having the same function.

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

[0013] In the present invention, polypeptide sequences obtained by modification also fall within the scope of protection of the present invention. 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 the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in an amino acid sequence. "Insertion" or "addition" refers to a change in an amino acid sequence that results in the increase of one or more amino acids compared to a 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 the present invention, the terms "polynucleotide," "nucleic acid molecule," and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules.

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

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

[0019] Furthermore, the vector includes plasmid and viral vector.

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

[0021] In the present invention, the term "vector" refers to a non-chromosomal nucleic acid containing an intact replicon so that when placed in a permissive cell, the vector can be replicated, for example, by a transformation process. The vector can replicate in one cell type (e.g., bacteria), but has a limited ability to replicate in another cell type (e.g., mammalian cells). The vector can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; and DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers (e.g., heterogeneous polylysine, fixed-length oligopeptides, and polyethyleneimine), in some cases also contained in liposomes.

[0022] In some embodiments, the vector of the present invention comprises a plasmid (expression plasmid, cloning vector, minicircle, microvector, double minichromosome), a lentiviral vector, an adenoviral vector or a retroviral vector.

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

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

[0025] Preferably, the vector further comprises one or more regulatory elements.

[0026] Preferably, the regulatory elements include a promoter, an enhancer, a ribosome binding site for translation initiation, a terminator, a polyadenylation 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 comprises the nucleic acid molecule described in the second aspect of the present invention or the vector molecule described in the third aspect of the present invention.

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

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

[0032] Furthermore, the brain targeting and / or nerve 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 nerve cell delivery system further comprises a biologically active substance.

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

[0035] The rabies virus-derived peptide provided by the present invention can provide an efficient, safe, and non-invasive method for the brain-targeted transport of bioactive macromolecules and other active small molecules that have difficulty crossing 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 the half-life, such as cyclization modification, acetylation modification, PAS modification, PEG modification, fatty acid modification, albumin modification, albumin affinity peptide modification, NOTA modification, radionuclide modification, tumor homing peptide coupling, membrane-penetrating peptide coupling, nanocarrier coupling and other foreseeable chemical modification methods.

[0037] As used herein, the term "biologically active substance" refers to, for the purposes of the present invention, a biologically active modulator of physiological functions and a substance that can be used to treat all diseases caused by an imbalance or dysfunction of physiological functions. Biologically active substances may be nucleic acids, peptides, polypeptides, antibodies, or chemical compounds (chemotherapeutics), but the present invention is not limited thereto.

[0038] A 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 comprises the following steps: culturing the host cell described in the fourth aspect of the present invention to obtain a culture product, and isolating 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 use of the rabies virus-derived peptide described in the first aspect of the present invention in the preparation of a product 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 use of the rabies virus-derived peptide described in the first aspect of the present invention in preparing a carrier for delivering biologically active substances to nerve cells.

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

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

[0047] In another aspect, the present invention provides a pharmaceutical composition for treating brain diseases, comprising any one or more materials selected from the group consisting of: a peptide of SEQ ID NO: 1 or a modified peptide thereof, a peptide or a dimer thereof, and a biocompatible polymer, a liposome, a vesicle, and a nanomaterial, a therapeutically effective amount of a complex comprising a biologically active substance; and (b) a pharmaceutically acceptable carrier.

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

[0049] The terms "preferably," "more preferably," and the like, used herein refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are unsuitable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0050] In the description of the present invention, the term "and / or" includes all and any combinations of one or more related listed items.

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

[0052] Beneficial effects of the present invention:

[0053] The present invention provides a rabies virus-derived peptide and its application, which can target and deliver bioactive macromolecules and other active small molecules that have difficulty crossing the blood-brain barrier to nerve cells, helping to improve the therapeutic effect of brain diseases and has good development and application prospects in the field of new drug research and development and clinical treatment of brain diseases. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0055] Figure 2This figure shows the results of using a luciferase detection kit to detect the expression of Luc in Neuro-2a cells after treatment with rabies virus-derived peptides. DETAILED DESCRIPTION

[0056] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without inventive work are all within the scope of protection of the present invention.

[0057] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional 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 example is KSVWTWNEKPPSKGCLCVGGMCHPPVNGGGRRRRRRRRR (SEQ ID NO: 1). A 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 neuronal properties, such as the ability to differentiate, expression of neuronal markers, and responsiveness to neurotransmitters and drugs. They are often used to study neurological diseases, neurodevelopmental processes, and neurotoxicity.

[0061] 1. Experimental Methods

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

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

[0064] 3. Add the plasmid pmax-GFP-peptide complex to the SH-SY5Y cells that have been plated, set up a negative control, and continue culturing in a 37°C carbon dioxide cell culture incubator.

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

[0066] 2. Experimental Results

[0067] The results are as follows Figure 1 As shown, there was no GFP green fluorescence 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 plasmid 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 example is KSVWTWNEKPPSKGCLCVGGMCHPPVNGGGRRRRRRRRR (SEQ ID NO: 1). A polypeptide was synthesized based on this amino acid sequence and named 2D3.

[0070] 1. Experimental Methods

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

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

[0073] 3. Add the plasmid pLuc-peptide complex to the plated Neuro-2a cells and set up a negative control. Continue culturing in a 37°C CO2 cell culture incubator.

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

[0075] 2. Experimental Results

[0076] The results are as follows Figure 2 As shown, the Luc expression level in the control group was extremely low, while the Luc expression level in Neuro-2a cells treated with rabies virus-derived peptide 2D3 was very high, indicating that the rabies virus-derived peptide 2D3 has strong neurotropism and can mediate the entry of Luc plasmid into Neuro2a cells.

[0077] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A rabies virus-derived peptide, characterized in that: 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 100% or greater homology to the amino acid sequence as shown in SEQ ID NO:

1.

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

3. A carrier molecule, characterized in that The vector molecule comprises the nucleic acid molecule of claim 2; Preferably, the vector comprises a plasmid or a viral vector; Preferably, the viral vector includes a lentivirus, an adenovirus, or an adeno-associated virus vector.

4. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 2 or the vector molecule of claim 3; Preferably, the host cells include prokaryotic cells and eukaryotic cells.

5. A brain-targeted and / or nerve cell-targeted delivery system, characterized in that: The brain targeting and / or neural cell delivery system comprises the rabies virus-derived peptide of claim 1; Preferably, the brain targeting and / or neural cell delivery system further comprises a biologically active substance; Preferably, the bioactive substance is any one selected from the group consisting of nucleic acids, peptides, antibodies, and chemotherapeutic agents.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the rabies virus-derived peptide according to claim 1.

7. A method for preparing the rabies virus-derived peptide according to claim 1, characterized in that: The method comprises the following steps: culturing the host cell according to claim 4 to obtain a culture product, and separating and purifying the rabies virus-derived peptide according to claim 1 from the culture product.

8. Use of the rabies virus-derived peptide according to claim 1 in the preparation of a product for promoting cytotoxicity in tumor cells and / or inhibiting tumor cell proliferation; Preferably, the tumor cells are neuroblastoma cells.

9. Use of the rabies virus-derived peptide according to claim 1 in the preparation of a carrier for delivering a biologically active substance to nerve cells; Preferably, the bioactive substance is any one selected from the group consisting of nucleic acids, peptides, antibodies, and chemotherapeutic agents.

10. Use of the rabies virus-derived peptide according to claim 1 in the preparation of a brain-targeted drug delivery vehicle.

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