Amyloid fusion protein and its preparation method and application

Abstract

The present invention relates to the technical field of biochemistry, molecular biology and cell biology, and provides an amyloid fusion protein and a preparation method and application thereof. The amyloid fusion protein has a connected amyloid accumulation core fragment and a target protein specific binding fragment, and can specifically recruit the target protein in the cell through the inclusion body formed by the accumulation of the fusion protein, and specifically reduce the availability of the target protein in the cell to interfere with the normal biological function thereof. The design concept of the amyloid fusion protein of the present invention can be applied to the research and development of targeted transcription factor drugs and the design of amyloid fusion protein-related drugs using mRNA transfection or protein delivery methods.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, molecular biology and cell biology, and in particular to an amyloid fusion protein and a preparation method and application thereof. Background Art

[0002] The accumulation, interaction and recruitment of proteins are often closely related to the occurrence and development of diseases. Taking polyglutamine (PolyQ) protein as an example, the repeated extension of glutamine in PolyQ protein can cause the protein to misfold and accumulate. The accumulated protein can recruit active proteins in the cell, such as transcription factors, molecular chaperones, ubiquitin system components, etc., through its own domains and interactions mediated by RNA and ubiquitin, resulting in a decrease in the soluble proportion of the protein factor in the cell, thereby affecting its normal biological function.

[0003] Currently, research on the accumulation and recruitment of proteins mainly focuses on their relationship with the occurrence and development of diseases, and there are no reports on their use in research such as cell function regulation and drug design. Summary of the invention

[0004] Based on this, the purpose of the embodiments of the present application includes providing an amyloid fusion protein and a preparation method and application thereof.

[0005] In the first aspect of the present application, an amyloid fusion protein is provided, which has a connected amyloid accumulation core fragment and a target protein-specific binding fragment, and can accumulate the target protein bound by the target protein-specific binding fragment by utilizing the accumulation and recruitment effect of the amyloid accumulation core fragment.

[0006] In some embodiments of the present application, the amyloid accumulation core fragment includes one or more of the NAC region fragment of α-Synuclein, the GAV motif of α-Synuclein, Aβ42, Aβ44, Aβ46, the TDP-25 fragment of TDP-43, and the 311-360 region fragment of TDP-43;

[0007] Alternatively, the amyloid accumulating core fragment comprises a polyglutamine fragment or has conservative substitutions relative to the polyglutamine fragment and the amyloid accumulating properties are not altered.

[0008] In some embodiments of the present application, the polyglutamine fragment has 30-200 glutamines, optionally 50-150 glutamines, and further optionally 80-110 glutamines.

[0009] In some embodiments of the present application, the amyloid accumulation core fragment includes AR (androgen receptor), TBP (TATA binding protein), Ataxin-1, Ataxin-2, Ataxin-3, Ataxin-7, Huntingtin, CACNA1A, Atrophin-1, Htt 100Q -N552 and Atx7 93Q -N172, Atx2 96Q -One or more of N317.

[0010] In some embodiments of the present application, the target protein-specific binding fragment is linked to the C-terminus of the amyloid accumulation core fragment.

[0011] In some embodiments of the present application, the number of the target protein-specific binding fragments is one or more, and when the number of the target protein-specific binding fragments is multiple, the multiple target protein-specific binding fragments are continuously distributed in the amyloid fusion protein.

[0012] In some embodiments of the present application, the number of the target protein-specific binding fragments is 1-5, and further optionally 1-3.

[0013] In some embodiments of the present application, an increase in the protein level of the target protein bound by the target protein-specific binding fragment may lead to the occurrence and / or development of a disease.

[0014] In some embodiments of the present application, the target protein bound by the target protein-specific binding fragment includes an enzyme and / or a transcription factor.

[0015] In some embodiments of the present application, the enzyme includes HDM2 and / or USP7.

[0016] In some embodiments of the present application, the target protein-specific binding fragment includes PMI and / or IRF.

[0017] In some embodiments of the present application, the amyloid fusion protein further comprises one or more of an identification tag and a nuclear localization fragment.

[0018] In some embodiments of the present application, the recognition tag comprises an antibody recognition tag. Further optionally, the recognition tag is selected from one or more of FLAG and HA.

[0019] In some embodiments of the present application, the nuclear localization fragment includes an NLS fragment.

[0020] In some embodiments of the present application, the nuclear localization fragment is linked to the N-terminus of the amyloid accumulation core fragment.

[0021] In some embodiments of the present application, the amino acid sequence is shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8.

[0022] In the second aspect of the present application, a nucleic acid molecule is provided, which encodes the amyloid fusion protein described in the first aspect.

[0023] In some embodiments of the present application, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7.

[0024] In the third aspect of the present application, a recombinant vector is provided, which comprises the nucleic acid molecule described in the second aspect.

[0025] In some embodiments of the present application, the recombinant vector is a eukaryotic expression vector.

[0026] In some embodiments of the present application, the eukaryotic expression vector comprises a pcDNA3.1 plasmid.

[0027] In the fourth aspect of the present application, a recombinant cell is provided, which comprises the nucleic acid molecule described in the second aspect of the present application and the recombinant vector described in the third aspect.

[0028] In some embodiments of the present application, the recombinant cell is a eukaryotic cell.

[0029] In some embodiments of the present application, the eukaryotic cells include HEK 293T cells or A549 cells.

[0030] In the fifth aspect of the present application, a method for producing the amyloid fusion protein described in the first aspect is provided, comprising the following steps:

[0031] The recombinant cell described in the fourth aspect of the present application is cultured, and the amyloid fusion protein is isolated from the obtained cell lysate.

[0032] In the sixth aspect of the present application, the amyloid fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, and the recombinant cell described in the fourth aspect are provided for use in the preparation of disease treatment drugs, P53 stabilizers, or P53 downstream regulatory gene expression promoters.

[0033] In some embodiments of the present application, the P53 downstream regulatory genes include P21 and / or PUMA.

[0034] In the seventh aspect of the present application, a disease treatment drug is provided, which comprises the amyloid fusion protein described in the first aspect, and pharmaceutically acceptable excipients.

[0035] In an eighth aspect of the present application, a method for intervening in biological functions of target cells is provided, comprising the following steps:

[0036] The amyloid fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, and the recombinant vector described in the third aspect are introduced into the target cells, thereby achieving intervention in the biological function of the target cells.

[0037] In the ninth aspect of the present application, a method for treating a disease in a subject is provided, comprising the following steps: administering to the subject a therapeutically effective amount of the disease treating drug described in the eighth aspect.

[0038] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0040] Figure 1 This is the technical route of amyloid fusion protein based on accumulation and recruitment in the embodiment of this application;

[0041] Figure 2 The DNA fragments and polypeptide fragments of PQE-IRF and PQE-3IRF in the examples of this application;

[0042] Figure 3 The DNA fragments and polypeptide fragments of PQE-PMI and PQE-3PMI in the examples of this application;

[0043] Figure 4 This is a schematic diagram of the eukaryotic expression vector of the fusion protein PQE-Pept in the examples of this application;

[0044] Figure 5 In the examples of this application, the PolyQ-IRF and PolyQ-3IRF fusion proteins can interact with exogenous USP7 through the IRF peptide segment;

[0045] Figure 6In the examples of this application, the PQE-IRF and PQE-3IRF fusion proteins recruit USP7 in cells into inclusion bodies;

[0046] Figure 7 In the examples of this application, fluorescence microscopy was used to observe the recruitment effect of PQE-IRF and PQE-3IRF fusion proteins on USP7 in cells;

[0047] Figure 8 In the examples of this application, the PQE-IRF and PQE-2IRF fusion proteins can improve the stability of the P53 protein;

[0048] Fig. 9 In the examples of this application, the PolyQ-PMI and PolyQ-3PMI fusion proteins can interact with exogenous HDM2;

[0049] Fig.10 In the examples of this application, the PQE-PMI and PQE-3PMI fusion proteins do not affect the expression of HDM2 protein in cells;

[0050] Fig.11 In the examples of this application, PQE-PMI and PQE-3PMI can recruit HDM2 in cells into inclusion bodies;

[0051] Fig.12 In the examples of this application, fluorescence microscopy was used to observe the recruitment effect of PQE-PMI and PQE-3PMI on HDM2 in cells;

[0052] Fig.13 In the examples of this application, PQE-PMI and PQE-3PMI can improve the stability of P53 protein;

[0053] Fig.14 In the examples of the present application, PQE-PMI / 3PMI promotes the mRNA levels of its downstream genes PUMA and P21 by increasing the stability of P53. DETAILED DESCRIPTION

[0054] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

[0056] the term

[0057] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0058] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0059] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0060] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0061] In the present application, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0062] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0063] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0064] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0065] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0066] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0067] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0068] In the present invention, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0069] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0070] The first aspect of the present application

[0071] The present application provides an amyloid fusion protein having a connected amyloid accumulation core fragment and a target protein-specific binding fragment, which can accumulate the target protein bound by the target protein-specific binding fragment by utilizing the accumulation and recruitment effect of the amyloid accumulation core fragment.

[0072] The present application relates to a fusion protein technology based on accumulation and recruitment. This technology utilizes the characteristics of amyloid accumulation core fragments (such as: glutamine-extended PolyQ protein, Aβ42 peptide, accumulation core (311-360) region in TDP-43 protein, etc.) that can form inclusion bodies, and fuses peptides that can specifically bind to target proteins (such as PMI, IRF, etc.), and specifically recruits target proteins in cells through the inclusion bodies formed by the accumulation of the fusion protein, and specifically reduces the availability of target proteins in cells to interfere with the performance of their normal biological functions. This technology can be applied to the research and development of targeted transcription factor drugs and the design of amyloid fusion protein-related drugs using mRNA transfection or protein delivery methods.

[0073] The present application does not specifically limit the type and quantity of amyloid accumulation core fragments. In some examples, the amyloid accumulation-like core fragment includes one or more of the NAC region fragment of α-Synuclein, the GAV motif of α-Synuclein, Aβ42, Aβ44, Aβ46, the TDP-25 fragment of TDP-43, and the 311-360 region fragment of TDP-43. In some examples, the amyloid accumulation core fragment contains a polyglutamine fragment or has a conservative substitution relative to the polyglutamine fragment and the amyloid accumulation property is not changed. In the art, conservative substitutions with PolyQ proteins with similar properties or similar amino acid sequences generally do not change the function of the protein or polypeptide. Optionally, the polyglutamine fragment has 30-200 (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200) glutamines, further optionally 50-150 glutamines, and further optionally 80-110 glutamines. Optionally, the starch accumulation-like core fragment includes AR, TBP, Ataxin-1, Ataxin-2, Ataxin-3, Ataxin-7, Huntingtin, CACNA1A, Atrophin-1, Htt 100Q -N552 and Atx7 93Q -N172, Atx2 96Q - one or more of N317. The motifs or polypeptide fragments in the present application include but are not limited to: polypeptide fragments or motifs that have been reported in the literature to interact with specific proteins, and polypeptides and motifs that interact between proteins found through in vitro or in vivo experiments.

[0074] In the amyloid fusion protein of the present application, the target protein-specific binding fragment can be connected to the N-terminus of the amyloid accumulation core fragment, or it can be connected to the C-terminus of the amyloid accumulation core fragment. In some examples, the target protein-specific binding fragment is connected to the C-terminus of the amyloid accumulation core fragment.

[0075] The present application does not particularly limit the number of the target protein specific binding fragments in the amyloid fusion protein, which can be 1 or multiple. When it is multiple, the multiple target protein specific binding fragments can be distributed in the amyloid fusion protein at intervals, or they can be continuously distributed. The continuous distribution described in the present application refers to the N-terminus of a target protein specific binding fragment directly connected to the C-terminus of another target protein specific binding fragment, and there are no other connecting amino acids or polypeptides between each other. Optionally, multiple target protein specific binding fragments are continuously distributed in the amyloid fusion protein. Optionally, the number of the target protein specific binding fragments is 1-5 (for example, 1, 2, 3, 4, 5), and further optionally 1-3.

[0076] The present application does not specifically limit the target protein specific binding fragment. Based on the design concept of the fusion protein of the present application, the corresponding target protein specific binding fragment can be designed according to the needs to capture the target protein. In some examples, the increase in the protein level of the target protein bound by the target protein specific binding fragment can lead to the occurrence and / or development of the disease. Optionally, the target protein bound by the target protein specific binding fragment includes an enzyme and / or a transcription factor.

[0077] The application does not specifically limit the type of the enzyme, which may be HDM2 (E3 ubiquitin ligase) or USP7 (deubiquitinase). Further, the target protein specific binding fragment includes PMI (amino acid sequence is TSFAEYWNLLSP) or / and IRF (amino acid sequence is SPGEGPAGTG).

[0078] In one example, the amyloid fusion protein of the present application includes an amyloid accumulation core and a polypeptide PMI that can specifically bind to HDM2, and the amyloid fusion protein does not affect the expression of HDM2 in cells, but only affects the biological function by reducing the availability of HDM2 in cells. Specifically, the amyloid fusion protein has a polypeptide PMI that can specifically bind to positions 25-109 of HDM2, can accumulate in cells, and specifically recruit HDM2 to inclusion bodies, and interfere with the function of HDM2 and upregulate the stability of P53 by reducing the soluble ratio of HDM2 in cells, thereby achieving the effect of tumor suppression.

[0079] Although small molecule inhibitors of HDM2 (such as RG7388, KRT-232, APG-115, etc.) can effectively inhibit the activity of HDM2, they all have great limitations. First, these inhibitors require a large dose to be effective; second, a large amount of wild-type P53 is required in the body; at the same time, the functions of these compounds may be too specific to fully activate the function of P53. Compared with HDM2 inhibitors, the embodiments of the present application have many unique advantages as follows: (1) PQE-PMI fusion protein recruits HDM2 in cells through dense inclusion bodies. Compared with toxic oligomers and small molecule compounds, inclusion bodies are less toxic to cells. (2) The polypeptide sequence PMI in the PQE-PMI fusion protein only binds to HDM2. Compared with the broad spectrum of HDM2 inhibitors, the PQE-PMI fusion protein is more specific in targeting HDM2.

[0080] In one example, the amyloid fusion protein of the present application includes an amyloid accumulation core and a polypeptide IRF (sequence: SPGEGPAGTG) that can specifically bind to USP7, and the fusion protein does not affect the expression of USP7 in cells, but only affects the biological function of USP7 by reducing the availability of USP7 in cells. The fusion protein can accumulate in cells and specifically recruit USP7 into inclusion bodies, thereby affecting the biological function of USP7 by reducing the soluble proportion of USP7 in cells.

[0081] In some examples, the amyloid fusion protein further comprises one or more of an identification tag and a nuclear localization fragment. The identification tag and the nuclear localization fragment can be independently connected to the N-terminus or the C-terminus of the amyloid accumulation core fragment. Optionally, the nuclear localization fragment is connected to the N-terminus of the amyloid accumulation core fragment. The present application does not specifically limit the type of the identification tag, including but not limited to antibody recognition tags, such as one or more of FLAG and HA. The present application does not specifically limit the type of the nuclear localization fragment, such as an NLS fragment.

[0082] In some examples, the amino acid sequence of the amyloid fusion protein is shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8.

[0083] The amyloid fusion protein with accumulation and recruitment functions of the present application, such as the "PQE-Pept fusion protein", "PQE-IRF fusion protein" and "PQE-PMI fusion protein" involved in the embodiments, all refer to active proteins or polypeptides that can be expressed in eukaryotic vectors. Of course, those skilled in the art can also easily obtain the fusion protein through chemical synthesis and genetic recombination methods.

[0084] As used herein, the terms "amyloid fusion protein technology", "PolyQ fusion protein technology", and "PolyQ-Pept fusion protein" refer to a PolyQ protein or a fragment containing a polyglutamine sequence that contains the amyloid accumulation core, and is fused with a peptide segment that interacts with the target protein, specifically recruiting the target protein in the cell, changing the state of the target protein in the cell, and thus affecting its biological function. "PolyQ-PMI fusion protein" refers to a PolyQ protein or a fragment containing a polyglutamine sequence that is fused with a PMI peptide segment that specifically binds to HDM2. P53 stability is improved by interfering with the function of HDM2.

[0085] The second aspect of the present application

[0086] The present application provides a nucleic acid molecule encoding the amyloid fusion protein described in the first aspect.

[0087] In some examples, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7.

[0088] The full-length nucleotide or partial fragments of the fusion protein with accumulation and recruitment function of the present application can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0089] The third aspect of the present application

[0090] The present application provides a recombinant vector, which comprises the nucleic acid molecule described in the second aspect.

[0091] In some examples, the recombinant vector is a eukaryotic expression vector. The present application does not specifically limit the type of the eukaryotic expression vector, which may be, but is not limited to, a pcDNA3.1 plasmid.

[0092] The fourth aspect of the present application

[0093] The present application provides a recombinant cell, which comprises the nucleic acid molecule described in the second aspect of the present application and the recombinant vector described in the third aspect.

[0094] In some examples, the recombinant cell is a eukaryotic cell. The present application does not specifically limit the type of the eukaryotic cell, which may be but is not limited to HEK293T cells and A549 cells.

[0095] The fifth aspect of the present application

[0096] The present application provides a method for producing the amyloid fusion protein described in the first aspect, which comprises the following steps:

[0097] The recombinant cell described in the fourth aspect of the present application is cultured, and the amyloid fusion protein is isolated from the obtained cell lysate.

[0098] The sixth aspect of the present application

[0099] The present application provides the use of the amyloid fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, and the recombinant cell described in the fourth aspect in the preparation of disease treatment drugs, P53 stabilizers, or P53 downstream regulatory gene expression promoters.

[0100] In some embodiments of the present application, the P53 downstream regulatory genes include P21 and / or PUMA.

[0101] The seventh aspect of the present application

[0102] The present application provides a disease treatment drug, which comprises the amyloid fusion protein described in the first aspect, and pharmaceutically acceptable excipients.

[0103] The present application does not specifically limit the types of excipients, and appropriate pharmaceutical excipients can be selected and the dosage of the excipients can be adjusted according to the dosage form required clinically.

[0104] As used herein, "pharmaceutically acceptable" refers to those ligands, materials, compositions, and / or dosage forms that are suitable for administration to a patient within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0105] In this application, "pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. As used herein, the phrase "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with drug administration. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Suitable examples include, but are not limited to: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch, potato starch and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc. Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffered saline; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0106] In the present application, "excipients" include but are not limited to mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium ethylenediaminetetraacetic acid (disodium EDTA), calcium sodium EDTA, carbonates, acetates, phosphates or aqueous solutions of monovalent alkali metals, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, oligofructose, dextran, glycine, starch, sucrose, dextrin (such as maltodextrin), lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, and magnesium stearate.

[0107] The design concept of the fusion protein of the present application can be applied to the design and development of drugs related to PolyQ fusion proteins using mRNA transfection or protein delivery methods. In addition, the amyloid fusion protein of the present application can be expressed in eukaryotic cells, and the localization of the fusion protein in the cell can be changed by modifying the sequence, thereby better recruiting protein factors in the cell. Compared with small molecule compounds, the drugs designed based on the concept of the present application have the characteristics of less toxicity and stronger specificity, providing a potential possibility for disease treatment, and also providing a new idea for the development and application of drugs targeting intracellular protein factors.

[0108] The eighth aspect of the present application

[0109] The present application provides a method for intervening in the biological function of a target cell, which comprises the following steps:

[0110] The amyloid fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, and the recombinant vector described in the third aspect are introduced into the target cells, thereby achieving intervention in the biological function of the target cells.

[0111] In the present application, the target cell can be a eukaryotic cell. The source of the eukaryotic cell can be an animal, optionally a mammal. "Mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, humans, etc., preferably primates, more preferably humans, especially, derived from patients with diseases, disorders and / or symptoms.

[0112] In the present application, the method of introduction can be through mRNA transfection technology, injecting mRNA encoding the fusion protein into the target cells for expression; or through protein delivery technology, transferring the amyloid fusion protein chemically synthesized or genetically expressed in vitro into the cells, thereby achieving the effect of modulating cell function.

[0113] In one example, a PolyQ extended protein fragment was used as the amyloid accumulation core and fused with a polypeptide that can bind to the target protein. The product, the PolyQ extended protein, has the characteristics of forming dense accumulation inclusions in the cell and can specifically bind to the target protein in the cell. Then, by virtue of the accumulation effect of the amyloid core and the specific binding effect of the peptide fragment, the target protein in the cell is recruited, the cell function is modulated, and the expression of downstream genes or signal transduction is affected.

[0114] Ninth aspect of the present application

[0115] The present application provides a method for treating a disease in a subject, comprising the following steps: administering to the subject a therapeutically effective amount of the disease treating drug described in the eighth aspect.

[0116] In the present application, "subject" is an animal, optionally a mammal, more preferably a human, and the subject includes but is not limited to a patient with a disease, condition and / or symptom. "Mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, humans, etc., preferably primates, more preferably humans.

[0117] In this application, "effective amount" will vary depending on the specific state, disease, disorder or condition being treated and its severity, as well as the age, weight, physical condition and responsiveness of the individual being treated. Therefore, one or more of these parameters can be used to select and adjust the therapeutically effective amount of the therapeutic drug.

[0118] The diseases in this application are mainly diseases related to the accumulation and recruitment of proteins, such as diseases related to PolyQ proteins. At present, there are nine known PolyQ proteins related to diseases, namely Androgen Receptor (AR), TATA-Binding Protein (TBP), Ataxin-1, Ataxin-2, Ataxin-3, Ataxin-7, Huntingtin, CACNA1A and Atrophin-1. Studies have shown that: PolyQ-extended Ataxin-7 can recruit USP22 into inclusion bodies through its own zinc finger domain; PolyQ-extended Ataxin-3 can recruit VCP / P97 proteins in cells into inclusion bodies through its own VBM motif, thereby affecting the biological function of its downstream NEDD8; PolyQ-extended TBP can affect the transcription level of its downstream gene INPP5A by recruiting Sp1 of cells. A large number of studies have shown that the recruitment effect of PolyQ-extended proteins on specific proteins in cells often leads to impaired function of the target protein. As a tumor suppressor protein, P53 has important biological functions in inhibiting tumor occurrence and maintaining cell homeostasis.

[0119] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions of this area (such as the conditions described in "Molecular cloning: A laboratory manual, 3rd ed., Sambrook et al., Cold Spring Harbor Laboratory, 2001"), can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0120] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0121] The embodiment of the present application is based on the amyloid fusion protein technology route of accumulation and recruitment (see Figure 1 ):

[0122] Combination Figure 1 Figure A in the middle shows that a new fusion protein is constructed by introducing a polypeptide fragment that specifically binds to the target protein into a protein containing an amyloid accumulation core. By leveraging the recruitment effect of the amyloid accumulation core and the binding effect between the fusion peptide fragment and the target protein, it is able to specifically recruit proteins in cells, thereby reducing the availability of the target protein.

[0123] Combination Figure 1 Figure B, taking the amyloid fusion protein PQE-Pept as an example: using glutamine-extended PolyQ protein fragments (length of 30-200 glutamine residues, such as Atx7 93Q -N172) as the amyloid accumulation core, the peptides that specifically bind to the target protein are selected as the peptide IRF that can specifically interact with USP7 (the number of repeats of this peptide is usually 1-5) and the peptide PMI that can specifically interact with HDM2 (the number of repeats of this peptide is usually 1-5). Antibody recognition tags (such as FLAG, HA) and nuclear localization signals (NLS) can be selected as appropriate.

[0124] The inventors of the present application have found through long-term and in-depth research that by extending the amyloid accumulation core-PolyQ-Atx7 93Q-N172 and PMI polypeptide sequence fusion can obtain a new fusion protein. Studies have shown that the fusion protein (PQE-PMI) can specifically recruit HDM2 in cells into inclusion bodies, thereby significantly reducing the soluble proportion of HDM2 in cells, affecting the biological function of HDM2. At the same time, PolyQ extension protein can also fuse polypeptide motifs or domains that interact with other target proteins to achieve the purpose of targeted interference with protein expression. This technology can be used to design and develop drugs related to fusion proteins based on accumulation and recruitment using mRNA transfection or protein delivery methods. Compared with traditional siRNA or small molecule compounds, fusion protein technology based on accumulation and recruitment can specifically recruit components in cells through dense inclusion bodies in cells to interfere with the performance of their biological functions. Fusion protein technology based on accumulation and recruitment is more specific than traditional small molecule compounds, and may have less toxicity and side effects.

[0125] Example 1

[0126] 1. DNA and amino acid sequences of PQE-IRF / 3IRF

[0127] The known PQE gene fragments were analyzed, and the PQE-IRF fusion plasmid and PQE-3IRF fusion plasmid were designed and constructed by PCR method.

[0128] like Figure 2 As shown, select PolyQ-extended Atx7 93Q The -N172 fragment serves as the amyloid accumulation core, and a nuclear localization signal is introduced at the N-terminus (see the dotted box parts in Figures A and B), and one IRF fragment and three IRF repeat fragments are introduced at the C-terminus (see the solid box parts in Figures A and B), among which the number of glutamine residues in the extended part of glutamine (see the underlined parts in Figures A and B) is 93.

[0129] The DNA sequence of PQE-IRF is shown in SEQ ID NO.1:

[0130] ATGCCGAAAAAAAAACGTAAAGTAGAATTCATGTCGGAGCGGGCCGCGGATGACGTCAGGGGGGAGCCGCGCCGCGCGGCGGCGGCGGCGGGCGGAGCAGCGGCCGCGGCCGCCCGGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCCGCCGCCTCCGCAGCCCCAGCGGCAGCAGCACCCGCCACCGCCGCCACGGCGCACACGGCCGGAGGACGGCGGGCCCGGCGCCGCCTCCACCTCGGCCGCCGCAATGGCGACGGTCGGGGAGCGCAGGCCTCTGCCCAGTCCTGAAGTGATGCTGGGACAGTCGTGGAATCTGTGGGTTGAGGCTTCCAAACTTCCTGGGAAGGACGGGACAGAATTGGACGAAAGTTTCAAGGAGTTTGGGAAAAACCGCGAAGTCATGGGGCTCTGTCGGGAAGACATGCCAATATTTGGTTTCTGTCCAGCCCATGATGATTTCTACTTGGTGGTGTGTAACGACTGTAATCAGGTTGTCAAACCGCAGGCATTTCAATCACATTATGAAAGAAGACATAGCTCACTCGAGTCCCCTGGCGAGGGCCCTTCCGGCACTGGCTAA。

[0131] The amino acid sequence of PQE-IRF is shown in SEQ ID NO.2:

[0132] MPKKKRKVEFMSERAADDVRGEPRRAAAAAGGAAAAAARQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQPPPPQPQRQQHPPPPPRRTRPEDGGPGAASTSAAAMATVGERRPLPSPEVMLGQSWNLWVEASKLPGKDGTELDESFKEFGKNREVMGLCREDMPIFGFCPAHDDFYLVVCNDCNQVVKPQAFQSHYERRHSSLESPGEGPSGTG。

[0133] The sequence of the DNA of PQE-3IRF is shown in SEQ ID NO.3:

[0134] ATGCCGAAAAAAAAACGTAAAGTAGAATTCATGTCGGAGCGGGCCGCGGATGACGTCAGGGGGGAGCCGCGCCGCGCGGCGGCGGCGGCGGGCGGAGCAGCGGCCGCGGCCGCCCGGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCCGCCGCCTCCGCAGCCCCAGCGGCAGCAGCACCCGCCACCGCCGCCACGGCGCACACGGCCGGAGGACGGCGGGCCCGGCGCCGCCTCCACCTCGGCCGCCGCAATGGCGACGGTCGGGGAGCGCAGGCCTCTGCCCAGTCCTGAAGTGATGCTGGGACAGTCGTGGAATCTGTGGGTTGAGGCTTCCAAACTTCCTGGGAAGGACGGGACAGAATTGGACGAAAGTTTCAAGGAGTTTGGGAAAAACCGCGAAGTCATGGGGCTCTGTCGGGAAGACATGCCAATATTTGGTTTCTGTCCAGCCCATGATGATTTCTACTTGGTGGTGTGTAACGACTGTAATCAGGTTGTCAAACCGCAGGCATTTCAATCACATTATGAAAGAAGACATAGCTCACTCGAGTCCCCTGGCGAGGGCCCTTCCGGCACTGGCTCCCCTGGCGAGGGCCCTTCCGGCACTGGCTCCCCTGGCGAGGGCCCTTCCGGCACTGGCTAA。

[0135] The amino acid sequence of PQE-3IRF is shown in SEQ ID NO.4:

[0136] MPKKKRKVEFMSERAADDVRGEPRRAAAAAGGAAAAAARQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ QQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQPPPPQPQRQQHPPPPPR RTRPEDGGPGAASTSAAAMATVGERRPLPSPEVMLGQSWNLWVEASKLPGKDGTELDESFKEFGKNREVMGLCREDMPIFGFCPAHDDFYLVVCNDCNQVVKPQAFQSHYERRHSSLESPGEGPSGTGSPGEGPSGTGSPGPGPSGTG.

[0137] 2. Construction of eukaryotic expression vector of fusion protein PQE-Pept

[0138] Select two restriction sites, BamH1 and Xba1, respectively, and construct the DNA fragment of PQE-Pept on the pcDNA3.1-N-FLAG plasmid vector to obtain the eukaryotic expression plasmid of PQE-Pept fusion protein. Figure 4 .

[0139] The DNA fragments of PQE-Pept mainly include the DNA fragments of PQE-IRF or PQE-3IRF, and a PQE (NLS-Atx7 93Q -N172), PQE-IRF and PQE-3IRF fusion protein eukaryotic expression plasmids.

[0140] 3. PolyQ-IRF fusion protein and PolyQ-3IRF fusion protein can interact with exogenous USP7 through IRF peptide

[0141] To test the interaction between the fusion fragment and the target protein, we used Atx7 10Q -N172 was used to identify the interaction between fusion proteins of IRF or 3IRF. 10Q -N172, NLS-Atx7 10Q -N172-IRF and NLS-Atx7 10Q -N172-3IRF fusion plasmid was transfected into HEK 293T cells using PolyJet reagent (purchased from SigaGEN).

[0142] The cells were cultured in a constant temperature incubator at 37°C and 5% CO2. The medium was changed 6 hours after transfection. After 48 hours, the cells were washed with PBS buffer and collected. After lysis, the supernatant was taken and incubated with magnetic beads coupled with FLAG (purchased from Abmart) for four times overnight. The supernatant was discarded the next day, and the magnetic beads were washed with PBS three times for 5 minutes each time. An appropriate amount of protein loading buffer was added and boiled at 100°C for 5 minutes, and western blotting was performed. FLAG antibody (purchased from Abmart) was used to detect NLS-Atx7, 10Q -N172, NLS-Atx7 10Q -N172-IRF and NLS-Atx7 10Q -The expression of N172-3IRF fusion protein was detected, and the interaction between exogenous USP7 and it was detected using HA antibody (purchased from Abmart).

[0143] See the results Figure 5 The results showed that after fusion expression with the peptide IRF, HA-USP7 could bind to NLS-Atx7 through the peptide 10Q -N172-IRF and NLS-Atx7 10Q -N172-3IRF fusion protein interaction.

[0144] 4. PQE-IRF and PQE-3IRF fusion proteins can recruit USP7 in cells to inclusion bodies

[0145] The supernatant / precipitate separation method was used to detect the distribution of USP7 in cells.

[0146] Empty plasmid, PQE, PQE-IRF and PQE-3IRF fusion proteins were overexpressed in A549 cells, respectively. After 48 hours, the cells were collected and lysed on ice for 30 minutes at 13,000 rpm using RIPA lysis buffer containing protease inhibitors. The supernatant was collected by centrifugation at 4°C for 15 minutes, and the precipitate was washed three times with RIPA lysis buffer for 10 minutes each time. Then, protein electrophoresis loading buffer containing 8 M urea was added to the precipitate to dissolve the precipitate, and the precipitate was boiled at 100°C for 5 minutes. An appropriate amount of protein electrophoresis loading buffer was added to the collected supernatant, and the supernatant was boiled at 100°C for 5 minutes to completely denature the protein before electrophoresis.

[0147] Western blotting technique was used to detect the USP7 content in the supernatant and precipitate of overexpressing cells with USP7 antibody (purchased from Novus), and FLAG antibody was used to detect the expression of PQE, PQE-IRF and PQE-3IRF fusion proteins.

[0148] See the results Figure 6 . Figure 6 middle:

[0149] Figure B shows that after transfection of PQE-IRF or PQE-3IRF fusion plasmid, the protein content of USP7 in the cells was significantly reduced in the supernatant and significantly increased in the precipitate, and the difference was statistically significant *, p<0.05; **, p<0.01; ***, p<0.001.

[0150] At the same time, we also lysed the cells with RIPA lysis buffer containing 8M urea and detected the changes in the total USP7 protein in the cells (Figure A). The results showed that after expressing the PQE-IRF / 3IRF fusion protein, the total amount of USP7 in the cells did not change, and there was no significant difference in NS.

[0151] 5. Observation of the recruitment of USP7 by PQE-IRF and PQE-3IRF fusion proteins in cells using fluorescence microscopy

[0152] Immunofluorescence technology was used to observe the co-localization of USP7 and the inclusion bodies formed by glutamine-extended PolyQ protein.

[0153] PQE(NLS-Atx7 93Q -N), PQE-IRF and PQE-3IRF fusion plasmids were transfected into A549 cells using PolyJet reagent, respectively. The medium was changed 6 hours after transfection. After 36 hours, the cells were fixed with 4% paraformaldehyde solution at room temperature for 15 minutes, and then the cells were washed three times with PBS buffer, each time for 15 minutes. The antibody was diluted with cell perforation solution (5% BSA 0.1% Triton-100 PBS buffer Ph8.0), and the primary antibody USP7 (1:200) was added, and incubated at 4°C overnight.

[0154] The next day, the primary antibody was recovered and the cells were washed three times with PBS buffer for 10 minutes each time. Then, the corresponding fluorescent secondary antibody (1:200) and the nuclear dye Hoechst (1:200) were added and incubated at room temperature for 1 hour. The fluorescent secondary antibody was discarded, and the cells were washed three times with PBS buffer for 10 minutes each time. Then, the co-localization of USP7 and inclusion bodies was observed using a Leica SP8 full-spectrum microscope.

[0155] See the results Figure 7 , Figure 7 middle:

[0156] After overexpressing PQE in A549 cells, the inclusion bodies formed by the PQE protein did not co-localize with the USP7 protein in the cells, that is, PQE and USP7 did not co-localize, as shown in the first row of figures;

[0157] After overexpressing PQE-IRF fusion protein in A549 cells, the inclusion bodies formed by PQE-IRF fusion protein co-localized with USP7 in the cells, that is, PQE-IRF and USP7 co-localized, indicating that PQE-IRF fusion protein can recruit USP7 in cells, as shown in the second row of figures;

[0158] After overexpressing PQE-3IRF fusion protein in A549 cells, the inclusion bodies formed by PQE-3IRF fusion protein co-localized with USP7 protein in the cells, that is, PQE-3IRF and USP7 co-localized, indicating that PQE-3PMI fusion protein can recruit USP7 in cells, see the third row of figures.

[0159] 6. PQE-IRF / 2IRF fusion protein can improve the stability of P53 protein

[0160] The empty plasmid and PQE, PQE-IRF, PQE-2IRF and PQE-3IRF fusion plasmids were transfected into A549 cells using PolyJet reagent, respectively. The medium was changed 6 hours after transfection, and the cells were cultured in a constant temperature incubator at 37°C and 5% CO2 for 48 hours. The cells were washed and collected with PBS buffer, and the cells were lysed on ice for 30 minutes with RIPA lysis buffer containing 8M urea and protease inhibitors. After that, an appropriate amount of protein electrophoresis loading buffer was added, and the cells were boiled at 100°C for 5 minutes to completely denature the protein for electrophoresis.

[0161] Western blotting technique was used to detect the expression of endogenous P53 in cells using P53 antibody (purchased from Santa Cruz), and FLAG antibody was used to detect the expression of fusion protein.

[0162] See the results Figure 8 . Figure 8 middle:

[0163] Figure A shows the expression of empty plasmid, PQE (NLS-Atx7 93Q -N172), PQE-IRF, PQE-2IRF and 3IRF fusion proteins, and their expression was detected by FLAG antibody; Figure A shows that after transfection of PQE-IRF or PQE-2IRF fusion plasmid, the content of P53 in cells increased significantly;

[0164] Figure B shows the statistical content of P53 protein in cells; Figure B shows that it is statistically significant*, p<0.05; NS, no significant difference.

[0165] Example 2

[0166] 1. DNA and amino acid sequences of PQE-PMI and PQE-3PMI

[0167] The known PQE gene sequence was analyzed, and the PQE-PMI and PQE-3PMI fusion plasmids were designed and constructed by PCR.

[0168] like Figure 3 As described, select PolyQ-extended Atx7 93Q The -N172 fragment serves as the amyloid accumulation core, and a nuclear localization signal is introduced at the N-terminus (see the parts marked with dotted boxes in Figures A and B), and one PMI sequence and three PMI repeat fragments are introduced at the C-terminus (see the parts marked with solid boxes in Figures A and B), among which the number of glutamine residues in the extended part of glutamine (marked in green) is 93.

[0169] The sequence of the DNA of PQE-PMI is shown in SEQ ID NO.5: ATGCCGAAAAAAAAACGTAAAGTAGAATTCATGTCGGAGCGGGCCGCGGATGACGTCAGGGGGGAGCCGCGCCGCGCGGCGGCGGCGGCGGGCGGAGCAGCGGCCGCGGCCGCCCGGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCCGCCGCCTCCGCAGCCCCAGCGGCAGCAGCACCCGCCACCGCCGCCACGGCGCACACGGCCGGAGGACGGCGGGCCCGGCGCCGCCTCCACCTCGGCCGCCGCAATGGCGACGGTCGGGGAGCGCAGGCCTCTGCCCAGTCCTGAAGTGATGCTGGGACAGTCGTGGAATCTGTGGGTTGAGGCTTCCAAACTTCCTGGGAAGGACGGGACAGAATTGGACGAAAGTTTCAAGGAGTTTGGGAAAAACCGCGAAGTCATGGGGCTCTGTCGGGAAGACATGCCAATATTTGGTTTCTGTCCAGCCCATGATGATTTCTACTTGGTGGTGTGTAACGACTGTAATCAGGTTGTCAAACCGCAGGCATTTCAATCACATTATGAAAGAAGACATAGCTCACTCGAGACGAGCTTCGCAGAATACTGGAACCTACTGAGCCCGTGA。

[0170] The amino acid sequence of PQE-PMI is shown in SEQ ID NO.6:

[0171] MPKKKRKVEFMSERAADDVRGEPRRAAAAAGGAAAAAARQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQPPPPQPQRQQHPPPPPRRTRPEDGGPGAASTSAAAMATVGERRPLPSPEVMLGQSWNLWVEASKLPGKDGTELDESFKEFGKNREVMGLCREDMPIFGFCPAHDDFYLVVCNDCNQVVKPQAFQSHYERRHSSLETSFAEYWNLLSP。

[0172] The sequence of the DNA of PQE-3PMI is shown in SEQ ID NO.7:

[0173] ATGCCGAAAAAAAAACGTAAAGTAGAATTCATGTCGGAGCGGGCCGCGGATGACGTCAGGGGGGAGCCGCGCCGCGCGGCGGCGGCGGCGGGCGGAGCAGCGGCCGCGGCCGCCCGGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCCGCCGCCTCCGCAGCCCCAGCGGCAGCAGCACCCGCCACCGCCGCCACGGCGCACACGGCCGGAGGACGGCGGGCCCGGCGCCGCCTCCACCTCGGCCGCCGCAATGGCGACGGTCGGGGAGCGCAGGCCTCTGCCCAGTCCTGAAGTGATGCTGGGACAGTCGTGGAATCTGTGGGTTGAGGCTTCCAAACTTCCTGGGAAGGACGGGACAGAATTGGACGAAAGTTTCAAGGAGTTTGGGAAAAACCGCGAAGTCATGGGGCTCTGTCGGGAAGACATGCCAATATTTGGTTTCTGTCCAGCCCATGATGATTTCTACTTGGTGGTGTGTAACGACTGTAATCAGGTTGTCAAACCGCAGGCATTTCAATCACATTATGAAAGAAGACATAGCTCACTCGAGACGAGCTTCGCAGAATACTGGAACCTACTGAGCCCGACGAGCTTCGCAGAATACTGGAACCTACTGAGCCCGACGAGCTTCGCAGAATACTGGAACCTACTGAGCCCGTGA。

[0174] The amino acid sequence of PQE-3PMI is shown in SEQ ID NO.8:

[0175] MPKKKRKVEFMSERAADDVRGEPRRAAAAAGGAAAAAARQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ QQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQPPPPQPQRQQHPPPPPRRTR PEDGGPGAASTSAAAMATVGERRPLPSPEVMLGQSWNLWVEASKLPGKDGTELDESFKEFGKNREVMGLCREDMPIFGFCPAHDDFYLVVCNDCNQVVKPQAFQSHYERRHSSLETSFAEYWNLLSPTSFAEYWNLLSPTSFAEYWNLLSP.

[0176] 2. Construction of eukaryotic expression vector of fusion protein PQE-Pept

[0177] Select two restriction sites, BamH1 and Xba1, respectively, and construct the DNA fragment of PQE-Pept on the pcDNA3.1-N-FLAG plasmid vector to obtain the eukaryotic expression plasmid of PQE-Pept fusion protein. Figure 4 .

[0178] The DNA fragments of PQE-Pept mainly include the DNA fragments of PQE-PMI and PQE-3PMI, which together construct a PQE (NLS-Atx7 93Q -N172), PQE-PMI, and PQE-3PMI fusion protein eukaryotic expression plasmids.

[0179] 3. PolyQ-PMI and PolyQ-3PMI fusion proteins can interact with exogenous HDM2

[0180] To test the interaction between the fusion fragment and the target protein, we used Atx7 10Q The interaction between HA-HDM2 and the fusion protein of PMI or 3PMI was identified. 10Q -N172, NLS-Atx7 10Q -N172-PMI and NLS-Atx7 10Q-N172-3PMI fusion plasmid was transfected into HEK293T cells by PolyJet reagent. The cells were cultured in a constant temperature incubator at 37°C and 5% CO2. The medium was changed 6 hours after transfection. After 48 hours, the cells were washed and collected with PBS buffer. After lysis, the supernatant was taken and incubated with magnetic beads coupled with FLAG for four times overnight. The supernatant was discarded the next day, and the magnetic beads were washed with PBS three times for 5 minutes each time. The appropriate amount of protein loading buffer was added and boiled at 100°C for 5 minutes, and Western blotting was performed. NLS-Atx7 was detected with FLAG antibody respectively. 10Q -N172, NLS-Atx7 10Q -N172-PMI and NLS-Atx7 10Q -The expression of N172-3PMI fusion protein was examined, and the interaction between exogenous HDM2 and it was examined using HA antibody.

[0181] See the results Fig. 9 The results showed that after fusion expression with the peptide PMI, HA-HDM2 could bind to NLS-Atx7 through the peptide 10Q -N172-PMI and NLS-Atx7 10Q -N172-3PMI fusion protein interaction.

[0182] 4. PQE-PMI and PQE-3PMI do not affect the expression of HDM2 in cells

[0183] The empty plasmid and PQE (NLS-Atx7 93Q -N172), PQE-PMI and PQE-3PMI fusion plasmids were transfected into HEK293T cells by PolyJet reagent. The cells were cultured in a constant temperature incubator at 37°C and 5% CO2. The medium was changed 6 hours after transfection. After 48 hours, the cells were washed and collected with PBS buffer. The cells were lysed on ice for 15 minutes with RIPA lysis buffer containing 8M urea and protease inhibitors. Then, an appropriate amount of protein electrophoresis loading buffer was added, and the proteins were completely denatured by boiling at 100°C for 5 minutes before electrophoresis.

[0184] Western blotting technique was used to detect the expression of endogenous HDM2 in cells using HDM2 antibody (purchased from Abcam), and FLAG antibody (purchased from Abmart) was used to detect the expression of PQE, PQE-PMI or PQE-3PMI fusion protein.

[0185] See the results Fig.10 , Fig.10 middle:

[0186] Figure A shows the overexpression of empty plasmid, PQE (NLS-Atx7 93Q -N172), PQE-PMI and PQE-3PMI fusion proteins; Figure A shows that after transfection of PQE (NLS-Atx7 93Q -N172), PQE-PMI or PQE-3PMI fusion plasmids, the intracellular HDM2 content did not change;

[0187] Figure B shows the statistical content of intracellular HDM2 protein levels; Figure B shows that there is no statistical significance, NS, no significant difference.

[0188] 5. PQE-PMI and PQE-3PMI can recruit HDM2 in cells to inclusion bodies

[0189] The supernatant / precipitate separation method was used to detect the distribution of HDM2 in cells.

[0190] Empty plasmid and PQE, PQE-PMI or PQE-3PMI fusion protein were overexpressed in HEK293T cells, and the cells were collected after 48 hours. The cells were lysed on ice for 30 minutes at 13,000 rpm using RIPA lysis buffer containing protease inhibitors; the supernatant was collected by centrifugation at 4°C for 15 minutes, and the precipitate was washed three times with RIPA lysis buffer for 10 minutes each time. Then, protein electrophoresis loading buffer containing 8 M urea was added to the precipitate to dissolve the precipitate, and the precipitate was boiled at 100°C for 5 minutes; an appropriate amount of protein electrophoresis loading buffer was added to the collected supernatant, and the supernatant was boiled at 100°C for 5 minutes to completely denature the protein before electrophoresis.

[0191] Western blotting technique was used to detect the HDM2 content in the supernatant and precipitate of cells with HDM2 antibody, and the expression of PQE, PQE-PMI or PQE-3PMI fusion protein was detected with FLAG antibody.

[0192] See the results Fig.11 , Fig.11 middle:

[0193] Figure A shows that after transfection of PQE-PMI or PQE-3PMI fusion plasmid, the protein content of HDM2 in the cell was significantly reduced in the supernatant and significantly increased in the precipitate. Figure B shows that it is statistically significant *, p<0.05; **, p<0.01.

[0194] 6. Observation of the recruitment of HDM2 by PQE-PMI / 3PMI in cells using fluorescence microscopy

[0195] Immunofluorescence technology was used to observe the co-localization of HDM2 and the inclusion bodies formed by glutamine-extended PolyQ protein.

[0196] PQE(NLS-Atx7 93Q -N), PQE-PMI and PQE-3PMI fusion plasmids were transfected into HEK293T cells by PolyJet reagent, and the medium was changed 6 hours after transfection. 36 hours later, the cells were fixed with 4% paraformaldehyde solution at room temperature for 15 minutes, and then the cells were washed 3 times with PBS buffer, each time for 15 minutes, and the antibody was diluted with cell perforation solution (5% BSA 0.1% Triton-100 PBS buffer Ph8.0), and the primary antibody HDM2 (1:200) was added, and incubated at 4°C overnight.

[0197] The next day, the primary antibody was recovered and the cells were washed three times with PBS buffer for 10 minutes each time. Then, the corresponding fluorescent secondary antibody (1:200) and the nuclear dye Hoechst (1:200) were added and incubated at room temperature for 1 hour. The fluorescent secondary antibody was discarded, and the cells were washed three times with PBS buffer for 10 minutes each time. Then, the co-localization of HDM2 and inclusion bodies was observed using a Leica SP8 full-spectrum microscope.

[0198] Results Fig.12 , Fig.12 middle:

[0199] After overexpressing PQE in HEK293T cells, the inclusion bodies formed by the PQE protein did not co-localize with the HDM2 protein in the cells, that is, there was no co-localization between PQE and HDM2, as shown in the first row of figures;

[0200] After overexpressing the PQE-PMI fusion protein in HEK293T cells, the inclusion bodies formed by the PQE-PMI fusion protein co-localized with HDM2 in the cells, that is, PQE-PMI co-localized with HDM2, indicating that the PQE-PMI fusion protein can recruit HDM2 in the cells, as shown in the second row of figures;

[0201] After overexpressing PQE-3PMI fusion protein in HEK293T cells, the inclusion bodies formed by PQE-3PMI fusion protein co-localized with HDM2 protein in the cells, indicating that PQE-3PMI fusion protein can recruit HDM2 in cells, see the third row of figures.

[0202] 7. PQE-PMI and PQE-3PMI can improve the stability of P53 protein

[0203] HEK293T cells were transfected with empty plasmid and PQE, PQE-PMI or PQE-3PMI fusion plasmid using PolyJet reagent. The medium was changed 6 hours after transfection and the cells were cultured in a constant temperature incubator at 37°C and 5% CO2 for 48 hours. The cells were washed and collected with PBS buffer and lysed on ice for 30 minutes with RIPA lysis buffer containing 8M urea and protease inhibitors. After that, an appropriate amount of protein electrophoresis loading buffer was added and the cells were boiled at 100°C for 5 minutes to completely denature the protein for electrophoresis.

[0204] Western blotting technique was used to detect the expression of endogenous P53 in cells using P53 antibody (purchased from Santa Cruz), and FLAG antibody was used to detect the expression of PQE, PQE-PMI or PQE-3PMI fusion protein.

[0205] See the results Fig.13 , Fig.13 middle:

[0206] Figure A shows the overexpression of empty plasmid, PQE (NLS-Atx7 93Q -N172), PQE-PMI or PQE-3PMI fusion protein. Their expression was detected by FLAG antibody, and the level of P53 in cells was checked by P53 antibody; Figure A shows that after transfection of PQE-PMI or PQE-3PMI fusion plasmid, the content of P53 in cells increased significantly;

[0207] Figure B shows the statistical content of intracellular P53 protein levels; Figure B shows that it is statistically significant, *, p<0.05; **, p<0.01.

[0208] 8. PQE-PMI and PQE-3PMI promote the mRNA expression of downstream genes PUMA and P21 by improving the stability of P53

[0209] RT-PCR was used to detect the mRNA levels of P21 and PUMA, the downstream genes of P53, after overexpression of PQE, PQE-PMI or PQE-3PMI fusion protein in HEK293T cells.

[0210] HEK293T cells were transfected with empty plasmid and PQE, PQE-PMI or PQE-3PMI fusion plasmid by PolyJet reagent. The medium was changed 6 hours after transfection, and the cells were then cultured in a constant temperature incubator at 37°C and 5% CO2 for 48 hours. The cells were washed with PBS buffer and collected. 1 ml of Trizon was added to lyse the cells on ice for 10 minutes. 200 μl of chloroform was added and vortexed to mix. After standing on ice for 5 minutes, the cells were centrifuged at 13000 rpm and 4°C for 15 minutes. The supernatant was collected and an equal volume of isopropanol was added to precipitate RNA. After centrifugation at 13000 rpm and 4°C for 15 minutes, the precipitate was collected and the RNA was washed twice with pre-cooled 75% ethanol for 5 minutes each time. After drying the residual ethanol, 50 μl of DEPC water was added to dissolve the RNA. After the concentration was measured by NanoDrop2000 (Thermo Fisher), reverse transcription was performed. 2 μg was reversed each time. After the reverse transcription was completed, the sample was diluted tenfold and stored at -80°C.

[0211] Take 2 μl of the diluted reverse transcription product and perform the experiment according to the following system:

[0212] Table 1

[0213]

[0214]

[0215] The mRNA levels of P21 and PUMA genes in the samples were detected by Roche LC480 real-time fluorescence quantitative PCR instrument.

[0216] Table 2

[0217]

[0218] Fig.14 PQE-PMI / 3PMI promotes the mRNA levels of its downstream genes PUMA and P21 by improving the stability of P53; in the figure:

[0219] Figure A, empty plasmid, PQE (NLS-Atx7 93Q -N172), PQE-PMI and PQE-3PMI fusion proteins, and RT-PCR was used to detect the mRNA level of P21. The results in Figure A show that the mRNA level expression of P21 in HEK293T cells increased significantly with the expression of PQE-PMI or PQE-3PMI fusion protein;

[0220] Figure B shows the overexpression of empty plasmid, PQE (NLS-Atx7 93Q-N172), PQE-PMI and PQE-3PMI fusion proteins, and RT-PCR was used to detect the mRNA level of PUMA. Figure B shows that the mRNA level of PUMA in HEK293T cells increased significantly with the expression of PQE-PMI or PQE-3PMI fusion protein*, p<0.05; **, p<0.01.

[0221] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0222] The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solutions of the present invention in detail, but cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. An amyloid fusion protein having an amyloid-accumulating core fragment and a target protein-specific binding fragment linked thereto.

2. The amyloid fusion protein according to claim 1, wherein The amyloid accumulation core fragment includes one or more of the NAC region fragment of α-Synuclein, the GAV motif of α-Synuclein, Aβ42, Aβ44, Aβ46, the TDP-25 fragment of TDP-43, and the 311-360 region fragment of TDP-43; Alternatively, the amyloid accumulating core fragment comprises a polyglutamine fragment or has conservative substitutions relative to the polyglutamine fragment and the amyloid accumulating properties are not altered.

3. The amyloid fusion protein according to claim 2, wherein The polyglutamine fragment has 30-200 glutamines, optionally 50-150 glutamines, further optionally 80-110 glutamines.

4. The amyloid fusion protein according to claim 3, wherein The amyloid accumulation core fragments include AR, TBP, Ataxin-1, Ataxin-2, Ataxin-3, Ataxin-7, Huntingtin, CACNA1A, Atrophin-1, Htt 100Q -N552 and Atx7 93Q -N172, Atx2 96Q -One or more of N317.

5. The amyloid fusion protein according to claim 1, wherein The target protein-specific binding fragment is linked to the C-terminus of the amyloid accumulation core fragment.

6. The amyloid fusion protein according to any one of claims 1 to 5, wherein The number of the target protein specific binding fragments is one or more, and when the number of the target protein specific binding fragments is more than one, the multiple target protein specific binding fragments are continuously distributed in the amyloid fusion protein; Optionally, the number of the target protein-specific binding fragments is 1-5, further optionally 1-3.

7. The amyloid fusion protein according to any one of claims 1 to 5, wherein An increase in the protein level of the target protein bound by the target protein-specific binding fragment may lead to the occurrence and / or development of a disease; Optionally, the target protein bound by the target protein-specific binding fragment includes an enzyme and / or a transcription factor.

8. The amyloid fusion protein according to claim 7, wherein The amyloid fusion protein further comprises one or more of a recognition tag and a nuclear localization fragment; Optionally, the nuclear localization fragment is linked to the N-terminus of the amyloid accumulation core fragment. 9 . The amyloid fusion protein according to claim 1 , wherein the amino acid sequence is shown as SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO.

8.

10. A nucleic acid molecule encoding the amyloid fusion protein according to any one of claims 1 to 9; Optionally, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.

7.

11. A recombinant vector comprising the nucleic acid molecule according to claim 10; Optionally, the recombinant vector is a eukaryotic expression vector.

12. A recombinant cell comprising the nucleic acid molecule of claim 10 or the recombinant vector of claim 11; Optionally, the recombinant cell is a eukaryotic cell.

13. The method for producing the amyloid fusion protein according to any one of claims 1 to 9, comprising the following steps: Cultivating the recombinant cell according to claim 12, and isolating the amyloid fusion protein from the obtained cell lysate.

14. Use of the amyloid fusion protein according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the recombinant vector according to claim 11 or the recombinant cell according to claim 12 in the preparation of a drug for treating a disease. 15 . A drug for treating a disease, comprising the amyloid fusion protein according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, and pharmaceutically acceptable excipients.

16. A method for intervening in biological functions of target cells, comprising the following steps: The amyloid fusion protein according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10 or the recombinant vector according to claim 11 is introduced into a target cell, thereby intervening in the biological function of the target cell.

17. A method for treating a disease in a subject, comprising the step of administering a therapeutically effective amount of the disease treating drug according to claim 15 to the subject.

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