Use of IL-27 protein in the manufacture of a product for the treatment and / or delay of Alzheimer's disease

Abstract

The present invention provides the use of IL-27 protein in the treatment of Alzheimer's disease and / or the manufacture of a delaying product, and belongs to the field of biomedical technology. The IL-27 protein is a recombinant IL-27 protein, which is directed against the therapeutic target IL-27 receptor, and includes mouse-derived IL-27, human-derived IL-27, and mammalian IL-27 other than the mouse-derived IL-27 and the human-derived IL-27. The recombinant IL-27 protein provided by the present invention can effectively alleviate Alzheimer's disease caused by Aβ deposition, can selectively and specifically bind to its receptor, and guarantees the accuracy of the detection results. The specificity of this protein receptor is highly expressed in the hippocampal dentate gyrus region, which maximally guarantees the drug targeting property. The recombinant IL-27 protein has good application prospects, can rapidly and effectively improve behaviors such as memory impairment in Alzheimer's disease mice, and has clinical transformation value.

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority of a Chinese application filed with the China National Intellectual Property Administration on June 30, 2023, with application number 202310798677.3 and title of invention "Use of IL - 27 protein in the manufacture of therapeutic products for Alzheimer's disease", the entire content of which is incorporated herein by reference.

Technical Field

[0002] The present invention relates to the field of biopharmaceutical technology, and in particular to the use of IL - 27 protein in the treatment and / or manufacture of delaying products for Alzheimer's disease.

Background Art

[0003] Alzheimer's disease (AD) has a profound impact on human health. With the increase in the average human lifespan, the aging society is advancing, and the prevalence of AD is also rising. AD patients usually show behavioral manifestations such as memory decline and weakened learning ability, which greatly affect the development of individuals, families, and ultimately society. As a neurodegenerative disease with a high disability rate and high mortality rate, AD brings a heavy social and family burden.

[0004] The main pathological feature of AD is neuronal degenerative death in brain regions such as the hippocampus, and the behavior is manifested as learning and cognitive dysfunction. Synapses are important structures for neurotransmission and neural plasticity, and are the basis for learning, memory, and cognitive functions. The learning and memory process dominated by hippocampal neurons conducts neural signal transmission and neural plasticity regulation through synaptic structure and functional plasticity, and participates in various behavioral scenes such as learning spatial memory and working memory. Currently, the pathological molecular mechanism of AD is unknown, and there are many theories that classical amyloid (Aβ) deposition, neurofibrillary tangles (NFTs), or phosphorylation of tau protein affect neuronal function and survival, etc. However, most of the clinical experimental effects of recent molecular target drug development for Aβ and the like have ended in failure, suggesting the possible multiple pathogenesis mechanisms of AD and the need for new drug treatment targets.

[0005] Interleukin 27 (IL-27) is composed of two subunits, EBI-3 and p28, and is mainly secreted from dendritic cells (DCs) and macrophages. The IL-27 receptor contains two subunits, gp130 and IL-27R (also called TCCR or WSX1), and is mainly expressed on immune cells such as T cells, B cells, NK cells, and myeloid cells.

[0006] IL-27 has a bidirectional regulatory effect on the immune response. On the one hand, IL-27 can promote the differentiation of naive CD4+ T cells into Th1 cells. During viral infection, IL-27 can induce the overactivation of CD4+ T cells into Th1 cells and IFN-γ production, which can further lead to the death of mice. On the other hand, IL-27 can promote the expression of inhibitory molecules such as SOCSs from T cells and directly inhibit the differentiation and function of Th2 and Th17 cells. In addition, IL-27 can limit the production of IL-2 and macrophage colony-stimulating factor (GM-CSF), induce IL-10, promote the function of the Treg cell subgroup, and indirectly inhibit the inflammatory response mediated by Th17 and Th1 cells.

[0007] In summary, currently IL-27 has attracted wide attention in the differentiation and function of immune cells, but the function of IL-27 in Alzheimer's disease is still blank.

Summary of the Invention

Means for Solving the Problems

[0008] To solve the above problems, the present invention provides the use of IL-27 protein in the manufacture of a therapeutic and / or delaying product for Alzheimer's disease, wherein the IL-27 protein is a recombinant IL-27 protein, which is for a therapeutic target IL-27 receptor, and includes mouse-derived IL-27, human-derived IL-27, and mammalian IL-27 other than the mouse-derived IL-27 and the human-derived IL-27.

[0009] Preferably, the recombinant IL-27 protein is A. a protein having the amino acid sequence shown in SEQ ID No. 1, B. any one of the proteins derived from SEQ ID No. 1 or its mammalian homologous protein sequence, in which one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1 are substituted, deleted or added, and which has the same activity.

[0010] Preferably, the gene encoding the IL-27 protein is the nucleotide sequence shown in SEQ ID No. 2, or a nucleotide sequence in which one or more nucleotides in the nucleotide sequence shown in SEQ ID No. 2 are substituted, deleted, and / or added, and which encodes a protein having the same activity, or a nucleotide sequence containing the nucleotide sequence encoding the amino acid sequence of SEQ ID No. 1.

[0011] Preferably, the recombinant expression plasmid, viral vector or mRNA fragment of the IL-27 protein has A. a feature containing the recombinant IL-27 protein therein, B. a feature containing the nucleotide sequence of the recombinant IL-27 protein therein, C. any one of the features containing a fragment of the nucleotide sequence of the recombinant IL-27 protein therein.

[0012] Preferably, the liposomal formulation of the recombinant expression plasmid, viral vector or mRNA fragment of the IL-27 protein is obtained by liposome-modifying the nucleic acid product of the recombinant IL-27 protein.

[0013] Preferably, the recombinant expression plasmid or viral vector is used for expressing the IL-27 recombinant protein in Escherichia coli, insect cells, mammalian cells, and in vivo.

[0014] Preferably, the host cell based on the IL-27 protein is further included, and the recombinant expression plasmid is included in the host cell.

[0015] Preferably, a cloning plasmid based on the IL-27 protein is further included, and the cloning plasmid A. has a feature of containing the recombinant IL-27 protein as described above therein; B. has a feature of containing the nucleotide sequence of the recombinant IL-27 protein therein; C. has any one of the features of containing a fragment of the nucleotide sequence of the recombinant IL-27 protein therein.

[0016] Preferably, the product is any one of a drug, a detection reagent, a kit, and a diagnostic reagent.

[0017] Preferably, the drug contains the IL-27 protein and a pharmaceutically acceptable vector therein, the pharmaceutically acceptable vector contains at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, and a lubricant, and the dosage form of the drug includes a paste, a powder, a tablet, a capsule, and granules.

[0018] The present invention provides the use of an IL-27 protein in the treatment of Alzheimer's disease and / or the manufacture of a delaying product, and belongs to the field of biomedical technology. Among them, the recombinant IL-27 protein is a protein with the amino acid sequence shown in SEQ ID No. 1, or a protein derived from SEQ ID No. 1 in which one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1 are substituted, deleted, or added and have the same activity. The recombinant IL-27 protein provided by the present invention is directed against a new therapeutic target, the RmIL-27 receptor, and can effectively relieve Alzheimer's disease caused by Aβ deposition, providing a new method for the treatment and alleviation of Alzheimer's disease.

Effects of the Invention

[0019] In summary, the recombinant IL-27 protein provided by the present invention can effectively delay and treat the damage of receptor proteins in memory impairment caused by Alzheimer's disease, and has the following beneficial effects.

[0020] (1) IL-27 can specifically and selectively bind to its receptor, ensuring the accuracy of detection results.

[0021] (2) The specificity of this protein receptor is highly expressed in the hippocampal dentate gyrus region, maximizing drug targeting.

[0022] (3) The recombinant IL-27 protein has good application prospects, can quickly and effectively improve and relieve behaviors such as memory impairment in Alzheimer's disease model mice, and has extremely important clinical transformation value.

Brief Description of the Drawings

[0023]

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[0024] The realization, functional features and advantages of the object of the present invention will be further described in conjunction with the embodiments with reference to the drawings.

Modes for Carrying Out the Invention

[0025] Hereinafter, the technical solutions of the present invention will be clearly and completely described in relation to the embodiments. Obviously, the described embodiments are some of the embodiments of the present invention, not all of them. All other embodiments obtained by those skilled in the art without creative labor based on the embodiments in the present invention belong to the protection scope of the present invention.

[0026] Unless otherwise specifically defined below, the meanings of all technical terms and scientific terms used in the specific embodiments of the present invention are the same as those generally understood by those skilled in the art. The following terms are considered to be well understood by those skilled in the art, but the following definitions are described to better explain the present invention.

[0027] As used in the present invention, the terms "comprising," "including," "having," "containing," or "relating to" are inclusive or open-ended and do not exclude other unenumerated elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "including." In the following, when defined as a group including at least a certain number of embodiments, it should also be understood that this preferably discloses a group consisting of only these embodiments.

[0028] When referring to a singular noun, the indefinite or definite article used, e.g., "a" or "an," "the," includes the plural form of this noun.

[0029] The following are provided merely to assist in understanding the present invention. These definitions should not be understood to be less than what those skilled in the art would understand.

[0030] Hereinafter, the technical solutions of the present invention will be described in more detail in connection with specific embodiments, but this does not constitute any limitation to the present invention. Any modification made as long as it is within the scope of the claims of the present invention is within the scope of the claims of the present invention.

[0031] The present invention provides the use of an IL-27 protein in the treatment of Alzheimer's disease and / or the manufacture of a delaying product, wherein the IL-27 protein is a recombinant IL-27 protein, is directed against a therapeutic target IL-27 receptor, and includes mouse-derived IL-27, human-derived IL-27, and mammalian IL-27 other than the mouse-derived IL-27 and the human-derived IL-27.

[0032] As described above, the use in the present invention includes the treatment of Alzheimer's disease and also includes alleviating the symptoms of patients.

[0033] Furthermore, the recombinant IL-27 protein is any one of the following proteins. A. A protein having the amino acid sequence shown in SEQ ID No.1, B. A protein derived from SEQ ID No.1 or its mammalian homologous protein sequence, in which one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1 are substituted, deleted or added, and which has the same activity.

[0034] As described above, the recombinant IL-27 protein can contain either A or B.

[0035] In the present invention, the amino acid sequence shown in SEQ ID No.1 is specifically MGQVTGDLGWRLSLLLLPLLLVQAGSWGFPTDPLSLQELRREFTVSLYLARKLLSEVQGYVHSFAESRLPGVNLDLLPLGYHLPNVSLTFQAWHHLSDSERLCFLATTLRPFPAMLGGLGTQGTWTSSEREQLWAMRLDLRDLHRHLRFQVLAAGFKCSKEEEDKEEEEEEEEEEKKLPLGALGGPNQVSSQVSWPQLLYTYQLLHSLELVLSRAVRDLLLLSLPRRPGSAWDS.

[0036] The IL-27 protein consists of two subunits, EBI-3EBI3 (NCBI Reference Sequence: NM_015766) and P28 (NCBI Reference Sequence: NM_145636), and SEQ ID No.1 is the amino acid sequence of the P28 subunit.

[0037] The IL-27 protein provided by the present invention, namely the recombinant IL-27 protein, is directed against a new therapeutic target, the IL-27 receptor, and can include mouse-derived IL-27, human-derived IL-27, and other mammalian IL-27. The recombinant IL-27 protein can effectively alleviate Alzheimer's disease caused by Aβ deposition. IL-27 can selectively and specifically bind to its receptor, ensuring the accuracy of test results. The specificity of this protein receptor is highly expressed in the hippocampal dentate gyrus region, maximizing drug targeting. The recombinant IL-27 protein has good application prospects, can rapidly and effectively improve and alleviate behaviors such as memory impairment in Alzheimer's disease mice, and has extremely important clinical translational value. In short, the recombinant IL-27 protein provided by the present invention provides a new method for the treatment and alleviation of Alzheimer's disease.

[0038] Furthermore, the gene encoding the recombinant IL-27 protein includes (1) the nucleotide sequence shown in SEQ ID No.2, (2) or a nucleotide sequence in which one or more nucleotides of the nucleotide sequence shown in SEQ ID No.2 are substituted, deleted, and / or added, and which encodes a protein with the same activity, (3) or any nucleotide sequence that includes one of the nucleotide sequences encoding the amino acid sequence of SEQ ID No.1.

[0039] ctcagcagtcaagaacacagtttgtcttacaaaagacccaagtttgagtcccagcatccacatagtgactcacaaccatcctcaaatccagttccaggggacctgatgtctcgtctcacccccttaagcactgcacacatgtggtacacacccatacacattaaataaataaataaataaataataggaatgttaaagggaaaagaaataaaatgcaagatttaaataaagacatgactagattagataaagactttgaccctgtaagtctgagggagaggctaccagagcaaccagggcctgtggttactgatgtcactggaggtgatgggcagcagaaggaatggagactctctctggttcttgatgtggccctgtgtgtctttcctcataggtgctggctgcaggattcaaatgttcaaaggaggaggaggacaaggaggaagaggaagaggaggaagaagaagaaaagaagctgcccctaggggctctgggtggccccaatcaggtgtcatcccaagtgtcctggccccagctgctctatacctaccagctccttcactccctggagcttgtcctgtctcgggctgttcgggacctgctgctgctgtccctgcccaggcgcccaggctcagcctgggattcctaacacctagcttcaagccctatggagtgaccttccagctccctccctcgcccgttaagactctaaggctggagtctggccaatcacaggacaggctctagctcgtttgccttagaccaggcagggcttcactagctcccagccctgacccaataatttaaaagccctccagtccttaccagatatttatttcttggatatttatttatttttaagaaatggtttatttattgtttcactcttgagttaggccaccatgctgggtgcctaataaagccatccagcccgg is as follows. In the present invention, the nucleotide sequence shown in SEQ ID No. 2 is a genomic sequence encoding the P28 subunit.

[0040] In addition, in the use of the present invention, a recombinant expression plasmid, a viral vector or an mRNA fragment based on the IL-27 protein is provided, A. It has a feature including the recombinant IL-27 protein as described above, B. It has a feature including the nucleotide sequence of the recombinant IL-27 protein, C. It has any one of the features including a fragment of the nucleotide sequence of the recombinant IL-27 protein.

[0041] Furthermore, the liposomal preparation of the recombinant expression plasmid, viral vector or mRNA fragment of the IL-27 protein is obtained by liposome-modifying the nucleic acid product of the recombinant IL-27 protein.

[0042] Furthermore, the recombinant expression plasmid or viral vector is used to express the IL-27 recombinant protein in Escherichia coli, insect cells, mammalian cells, and in vivo.

[0043] In addition, in the use of the present invention, a production method for constructing a recombinant protein expressed in HEK293 cells based on the recombinant expression plasmid or viral vector is provided, including the following. S1. A gene fragment with a nucleotide sequence of SEQ ID No. 2 is directly cloned into the pTriEx-1.1 Hygro expression vector to obtain a recombinant expression plasmid. S2. The recombinant expression plasmid is transiently transfected into HEK293 cells to express the recombinant protein. S3. After separating and purifying the expressed protein, the recombinant protein is obtained.

[0044] As described above, pTriEx-1.1 Hygro is a two-hybrid plasmid for protein expression. It is an expression vector that can efficiently express recombinant proteins in E. coli. This vector contains multiple cloning sites and a selection marker, including a T7 RNA polymerase promoter, a lac operon, a His6 label, a TEV sequence, etc. In addition, pTriEx-1.1 Hygro has an antibiotic resistance gene and can be screened and amplified under a variety of different culture conditions.

[0045] As described above, HEK293 cells are a human embryonic kidney cell line and have high quality and stability. They are easy to culture, amplify, and transfect in vitro and can express a variety of proteins, including complex recombinant proteins and antibodies.

[0046] As described above, transient transfection is a method of introducing exogenous DNA or RNA into cells and is usually an unstable transfection technology that can rapidly and efficiently express exogenous proteins or RNA in target cells. Transient transfection is different from stable transfection, where the latter requires the integration of exogenous DNA or RNA into the genome to achieve long-term expression.

[0047] Furthermore, the S2 transiently transfects the recombinant expression plasmid into HEK293 cells to express the recombinant protein. S21: Mix the recombinant expression plasmid and a liposome transfection reagent. S22: Transfect the mammalian cells HEK293 when the cell density reaches 50% - 80% and culture them in an incubator at 37°C and 5% CO2 for 48 h, 72 h, and 96 h to transiently express the recombinant protein with a 6×His label fused to the C-terminus.

[0048] As described above, culturing the cells after transfection for different periods of time, namely 48 h, 72 h, and 96 h, is for optimizing the expression efficiency and yield of the recombinant protein. Different proteins require different expression times to reach their maximum expression levels.

[0049] In the case of transient transfection, since the exogenous DNA / RNA is not integrated into the host genome, the expression time is short.

[0050] Therefore, by collecting and analyzing the expressing cells at different time points, the optimal expression time point can be determined, and recombinant proteins with higher expression levels and purity can be obtained. In this method, the recombinant proteins have a 6×His tag fused to their C-terminus, so they can be separated and purified using affinity chromatography (such as nickel ion affinity chromatography) techniques.

[0051] The manufacturing method provided in this example aims to produce the desired recombinant IL-27 protein. This method adopts transient transfection technology, which has a fast expression rate, a high yield, and can obtain the required recombinant protein in a relatively short time. It is constructed using HEK293 cells, has good stability and scalability, and can still maintain a high expression level during long-term culture. Since the recombinant protein has a 6×His tag fused to its C-terminus, simple separation and purification methods such as affinity chromatography can be used to increase the purity and yield of the product.

[0052] In addition, the manufacturing method provided in the present invention does not require large-scale culturing of cells or the use of expensive equipment and media compared with other manufacturing methods, thus reducing production costs.

[0053] In summary, this manufacturing method has advantages such as efficiency, stability, ease of purification, and economy, and can be widely used in the manufacture and production of recombinant proteins.

[0054] In addition, for the use of the present invention, host cells based on the IL-27 protein are included, and the recombinant expression plasmid is included in the host cells.

[0055] As described above, a host cell refers to a vector used for gene expression in a recombinant DNA or RNA expression system, and it may be a known cell line or strain. In the present invention, an exogenous gene (the recombinant expression plasmid in the present invention) is introduced into the host cell, and the necessary protein or RNA product is generated by its metabolism and mechanism.

[0056] Furthermore, in the use of the present invention, a clone plasmid based on the IL-27 protein is provided, and the clone plasmid A. has the characteristic of containing the recombinant IL-27 protein as described above; B. has the characteristic of containing the nucleotide sequence of the recombinant IL-27 protein; C. has any one of the characteristics of containing a fragment of the nucleotide sequence of the recombinant IL-27 protein.

[0057] Furthermore, the product in the use of the present invention is any one of a drug, a detection reagent, a kit, and a diagnostic reagent.

[0058] Specifically, the drug in the use of the present invention contains the IL-27 protein and a pharmaceutically acceptable vector. The pharmaceutically acceptable vector contains at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, and a lubricant. The dosage form of the drug includes paste, powder, tablet, capsule, and granule.

[0059] As described above, it is a detection reagent, a kit, and a diagnostic reagent for detecting an IL-27Ra agonist drug by a receptor. It may also be detection equipment, a detection device, a detection system, etc.

[0060] Furthermore, the present invention provides the use of IL-27 protein in the treatment and / or delay of Alzheimer's disease. The IL-27 protein is a recombinant IL-27 protein and is directed against the therapeutic target IL-27 receptor. The IL-27 protein includes IL-27 proteins derived from mammals.

[0061] In the present invention, the IL-27 protein preferably includes IL-27 proteins derived from mice and / or humans.

[0062] In the present invention, the administration method of the IL-27 protein preferably includes injection. The injection preferably includes intracranial injection. Based on mice, the intracranial administration amount of the IL-27 protein is preferably 100 ng / side.

[0063] Furthermore, the present invention provides a method for treating Alzheimer's disease, which includes the step of administering IL-27 protein to a patient with Alzheimer's disease. The IL-27 protein is a recombinant IL-27 protein and is directed against the therapeutic target IL-27 receptor. The IL-27 protein includes IL-27 proteins derived from mammals.

[0064] In the present invention, the IL-27 protein includes IL-27 proteins derived from mice and / or humans.

[0065] Hereinafter, the present invention will be further described by specific examples, but it should be understood that these examples are only for more detailed explanation and are not intended to limit the present invention in any form.

[0066] Example 1: In this example, the specific overexpression of IL-27 receptor in the hippocampal region of mice was verified by experiments.

[0067] 1. Experimental method: (1) C57BL / 6J wild-type male mice (8 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were raised in the Experimental Animal Center of Nanjing University. The animals were raised in a breeding room with a 12h light-dark cycle at a constant temperature and humidity. Feed and water were available for free intake. All animal procedures were conducted in accordance with the program approved by the Animal Welfare and Ethics Committee. (2) The hippocampal tissues of the mice were collected for in situ hybridization examination of the IL-27 receptor gene.

[0068] 2. Experimental Results: As shown in Figures 1 - 5, Figures 1 - 5 show the expression status of IL-27Ra in the hippocampal tissue. Among them, as shown in Figures 1 - 5, after in situ hybridization detection of the IL-27 receptor gene in C57BL / 6J experimental mice, they are immunohistochemical staining diagrams of DAPI, IL27Ra, VGlut, Vgat, and Merge respectively.

[0069] As mentioned above, DAPI in Figure 1 stains the DNA in the cell nucleus and is a DNA staining agent that exhibits blue fluorescence. In Figure 2, IL27RA is the abbreviation of interleukin 27 receptor alpha subunit, that is, the detected molecule. In the same figure, IL27RA is immunohistochemically stained using a specific antibody and exhibits a green fluorescence signal. In Figure 3, VGlut is a glutamate transporter protein, which is mainly present in glutamatergic neurons. In the same figure, VGlut is immunohistochemically stained using a specific antibody and exhibits a red fluorescence signal. In Figure 4, Vgat is a GABA transporter protein, which is mainly present in gamma-aminobutyric acid (GABA)-ergic neurons. Among them, Vgat is immunohistochemically stained using a specific antibody and exhibits a yellow fluorescence signal. In Figure 5, Merge (merged overlay figure) refers to a color image formed by merging all signals to display the relationship and overlay status between different tags.

[0070] It was discovered that the IL-27 receptor is specifically highly expressed mainly in the hippocampal dentate gyrus region of mice by in situ hybridization examination technology. Furthermore, when analyzing the subtypes of neurons, it was found that the IL-27 receptor is mainly expressed in glutamatergic neurons.

[0071] Example 2: In this example, it was experimentally proven that IL-27 gene-deficient mice and IL-27 receptor gene-deficient mice exhibit significant learning and memory ability impairments.

[0072] 1. Experimental method: During the experiment, there are at least 6 mice in each group of IL-27KO and IL-27RaKO female mice (gene-deficient mice, purchased from Jaxlab, USA). For 6- to 8-week-old mice, behavioral detections were performed to learn memory behaviors such as object recognition, location recognition, and Barnes maze.

[0073] 2. Experimental results: As shown in Figures 6 to 9, Figures 6 to 9 are all behavioral detection tests. Here, Figure 6 is the discrimination index in the object discrimination experiment (NOR). Figure 7 is the discrimination index in the location discrimination experiment (NOL) of gene-deficient mice. Figure 8 is the delay time diagram of gene-deficient mice reaching the target hole in the Barnes maze experiment. Figure 9 is the quantity diagram of gene-deficient mice entering the wrong holes in the Barnes maze experiment.

[0074] From the results of Figures 6 to 9, compared with the WT group, the IL-27KO mice and IL-27RaKO mice had similar exploration times for new and old objects and new and old locations, and clearly had memory impairments. Also, in the Barnes maze experiment, the IL-27KO mice and IL-27RaKO mice had an extended delay time to reach the target box compared to the WT group.

[0075] From the above experimental results, it was clearly shown that the IL-27 signaling pathway is important for maintaining learning and memory ability.

[0076] Example 3: In this example, a test was conducted on the improvement of FAD5X AD experimental mouse Alzheimer's disease by IL-27.

[0077] 1. Experimental method: (1) FAD5X AD female mice (36 weeks old, Nanjing Jicui Yakang Vivarium Technology Co., Ltd.) were A. Control group (corresponding to FAD5X + ACSF in the figure), B. RM IL-27 group (corresponding to FAD5X + IL27 in the figure), and were randomly divided. Among them, there were at least 7 mice in each group.

[0078] They were positioned and administered in the hippocampal region. Administration frequency: once. Among them, A. Control group: An equal amount of artificial cerebrospinal fluid (ACSF) was administered to the mice daily. B. Rm IL-27 group: Recombinant IL-27 protein (dissolved in sterile water) was administered into the mouse skull according to a dose of 100 ng / side (each side of the left and right brains).

[0079] (2) At two time points, 1 h and 24 h after administration, behavioral detections of object recognition, location recognition, and Barnes maze were performed on the mice respectively.

[0080] 2. Experimental results: The results are shown in Figures 10 to 14. Figures 10 to 14 are all behavioral detection tests. Among them, Figure 10 shows the discrimination index in the object recognition experiment (NOR) at the 2-hour time point for FAD5X AD (FAD) experimental mice (RM IL-27 group and control group). Figure 11 shows the recognition index in the location recognition experiment (NOL) at the 2-hour time point for FAD5X AD experimental mice (RM IL-27 group and control group). Figure 12 shows the discrimination index in the object recognition experiment (NOR) at the 24-hour time point for FAD5X AD experimental mice (RM IL-27 group and control group). Figure 13 shows the recognition index in the location recognition experiment (NOL) at the 24-hour time point for FAD5X AD experimental mice (RM IL-27 group and control group). Figure 14 shows the delay time graph of FAD5X AD experimental mice (RM IL-27 group and control group) reaching the target hole in the Barnes maze experiment.

[0081] Overall, Figures 10 to 14 can show the influence of IL-27 on experimental Alzheimer's disease. From the results of Figures 10 to 14, compared with the control group, the IL-27 group had a significantly increased sniffing time for new objects and new locations. In the Barnes maze, the delay time for mice to reach the target box was significantly shortened. It was found that IL-27 delayed and treated the progression of experimental mouse Alzheimer's disease.

[0082] Example 4: In this example, an improvement degree test of FAD5X AD experimental mouse Alzheimer's disease by the IL-27-mRNA group was conducted.

[0083] 1. Experimental method: (1) FAD5X AD female mice (36 weeks old, Nanjing Jicui Yakangwei Experimental Animal Technology Co., Ltd.) were A. Divided randomly into a control group (corresponding to +ACSF in the figure, that is, FAD5X + ACSF), B. An IL-27-mRNA group (corresponding to +IL27mRNA in the figure, that is, FAD5X + IL27mRNA). Among them, there were at least 7 mice in each group.

[0084] They were positioned and administered in the hippocampal region. Administration frequency: 1 time. Among them, A. Control group: An equal amount of artificial cerebrospinal fluid was injected into the mice daily. B. IL-27-mRNA group: IL-27-mRNA liposome nanoparticles (LNP) (dissolved in sterile water) were injected into the intracranial cavity of the mice according to a dose of 100 ng / side.

[0085] (2) For the mice, behavioral detections of object recognition, location recognition, and Barnes maze were performed at two time points: 36 h after administration and 72 h after administration.

[0086] 2. Experimental results: The results are shown in Figures 15 to 17. Figures 15 to 17 are all behavioral detection tests. Among them, Figure 15 is a comparison diagram of recognition indices in the object recognition test (NOR) at 36 h after administration and 72 h after administration for FAD5X AD (FAD) test mice (IL-27-mRNA group and control group). Figure 16 is a comparison diagram of recognition indices in the location recognition test (NOL) at 36 h after administration and 72 h after administration for FAD5X AD (FAD) test mice (IL-27-mRNA group and control group). Figure 17 is a diagram of the delay time for FAD5X AD (FAD) experimental mice (IL-27-mRNA group and control group) to reach the target hole in the Barnes maze experiment.

[0087] Figures 15 to 17 can show the influence of IL-27-mRNA on experimental Alzheimer's disease. From the results of Figures 15 to 17, compared with the control group, the mRNA-IL-27 group had a significantly increased sniffing time for new objects and new locations, and in the Barnes maze, the delay time for the mice to reach the target box was significantly shortened.

[0088] The experimental results showed that the IL-27-mRNA delivery system could effectively delay and treat the progression of experimental murine Alzheimer's disease.

[0089] The above are the preferred embodiments of the present invention and the corresponding examples. For those skilled in the art, on the premise of not departing from the creative concept of the present invention, several modifications and improvements can be made, including but not limited to the adjustment of proportion, flow, usage amount, and reaction vessel, such as using a continuous flow reactor falling within the protection scope of the present invention. It should be pointed out that all of these belong to the protection scope of the present invention. The above are the preferred embodiments of the present invention and the corresponding examples. For those skilled in the art, on the premise of not departing from the creative concept of the present invention, several modifications and improvements can be made, including but not limited to the adjustment of proportion, flow, usage amount, and reaction vessel, such as using a continuous flow reactor falling within the protection scope of the present invention. It should be pointed out that all of these belong to the protection scope of the present invention.

Claims

1. Use of an IL-27 protein in the manufacture of a product for treating and / or delaying Alzheimer's disease, wherein the IL-27 protein is a recombinant IL-27 protein and is directed against a therapeutic target IL-27 receptor, and the IL-27 protein comprises an IL-27 protein derived from a mammal, characterized by use of the IL-27 protein in the manufacture of a product for treating and / or delaying Alzheimer's disease.

2. The use according to claim 1, characterized in that the IL-27 protein comprises a mouse-derived IL-27 protein and / or a human-derived IL-27 protein.

3. The recombinant IL-27 protein is A. a protein having the amino acid sequence shown in SEQ ID No. 1, B. one of the proteins derived from SEQ ID No. 1 or its mammalian homologous protein sequence, wherein one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1 are substituted, deleted or added and have the same activity,

4. The gene encoding the recombinant IL-27 protein is the nucleotide sequence shown in SEQ ID No. 2, or a nucleotide sequence in which one or more nucleotides in the nucleotide sequence shown in SEQ ID No. 2 are substituted, deleted, and / or added and encode a protein having the same activity, or a nucleotide sequence encoding the amino acid sequence of SEQ ID No. 1, characterized by the use according to claim 3.

5. The recombinant expression plasmid, viral vector or mRNA fragment expressing the recombinant IL-27 protein has B. a feature of containing the nucleotide sequence encoding the recombinant IL-27 protein, C. any one of the features of containing a fragment of the nucleotide sequence encoding the recombinant IL-27 protein, characterized by the use according to claim 1.

6. The liposome-based formulation expressing the recombinant expression plasmid, viral vector or mRNA fragment of the IL-27 protein is obtained by liposome-modifying a nucleic acid product encoding the recombinant IL-27 protein, characterized by the use according to claim 5.

7. The recombinant expression plasmid or viral vector expressing the recombinant IL-27 protein is used for expressing the IL-27 recombinant protein in Escherichia coli, insect cells, mammalian cells, and in vivo, as characterized in the use according to claim 5.

8. The host cell expressing the recombinant IL-27 protein contains the recombinant expression plasmid, as characterized in the use according to claim 5.

9. The cloning plasmid encoding the recombinant IL-27 protein B. Characterized by containing the nucleotide sequence encoding the recombinant IL-27 protein, C. Having any one of the characteristics of containing a fragment of the nucleotide sequence encoding the recombinant IL-27 protein, as characterized in the use according to claim 1.

10. The product is any one of a drug, a detection reagent, a kit, a diagnostic reagent, a detection facility, a detection device, or a detection system, as characterized in the use according to claim 1.

11. The drug contains the IL-27 protein and a pharmaceutically acceptable vector, The pharmaceutically acceptable vector is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, and a lubricant, The dosage form of the drug includes paste, powder, tablets, capsules, and granules, as characterized in the use according to claim 10.

12. Use of the IL-27 protein in the treatment and / or delay of Alzheimer's disease, The IL-27 protein is a recombinant IL-27 protein and is directed against the therapeutic target IL-27 receptor, The IL-27 protein includes the IL-27 protein derived from mammals, Characterized by the use of the IL-27 protein in the treatment and / or delay of Alzheimer's disease.

13. The IL-27 protein includes the IL-27 protein derived from mice and / or the IL-27 protein derived from humans, as characterized in the use according to claim 12.

14. The administration method of the IL-27 protein includes injection, as characterized in the use according to claim 12.

15. The administration method of the IL-27 protein includes injection, and the injection includes intracranial injection, as characterized in the use according to claim 14.

16. The intracranial administration amount of the IL-27 protein is 100 ng / side based on mice, and the use according to claim 15 is characterized by this.

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