P53-targeting polypeptide and its application in preparing medicine for treating cancer

Abstract

The present invention discloses a polypeptide targeting P53 and its use in the preparation of a drug for treating cancer. The polypeptide specifically binds to a P53 mutant and is selected from any one or more of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, etc. The present invention uses a peptide chip to screen for promising peptide fragments on a large scale, and then uses in vitro and in vivo screening and verification to identify novel peptide compounds with anti-cancer activity. These peptides have the advantages of target specificity, low toxicity, and few side effects as drugs, and therefore have potential application value in developing anti-cancer drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polypeptide targeting P53 and an application thereof in preparing a drug for treating cancer. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women. According to the latest 2020 global cancer burden data released by the World Health Organization's International Agency for Research on Cancer (IARC), 2.26 million new cases of breast cancer were diagnosed worldwide in 2020, surpassing lung cancer (2.21 million cases) for the first time to become the world's leading cancer. China is a major breast cancer country, with approximately 420,000 new cases and nearly 120,000 deaths in 2020. Breast cancer typically develops in the glandular epithelium of the breast and is most common in women. Male breast cancer accounts for 0.5% to 1% of all breast cancer cases. Treatment options for breast cancer include surgical resection, radiotherapy, chemotherapy, and hormone therapy. In recent years, molecular targeted therapy, as a new treatment for breast cancer, has demonstrated promising results and is gaining increasing attention in the academic community. Molecular targeted therapy targets specific gene fragments in tumor cells, modulating or blocking their function to achieve therapeutic effects. Molecular targeted therapy for breast cancer targets signaling pathways involved in the development and progression of breast cancer and the expression products of oncogenes involved. Molecularly targeted drugs block signal transduction in tumor cells or related cells, thereby controlling changes in cellular gene expression and inhibiting or killing tumor cells. Targeted therapy is highly specific, effective, and largely harmless to normal tissues, making it the most promising approach for cancer treatment.

[0003] p53 is a collective name for a family of homologous proteins known as tumor suppressor proteins (also called p53 proteins or p53 tumor proteins). p53 is a tumor suppressor protein and transcription factor that activates multiple transcriptional targets in response to cellular stress or DNA damage. p53 coordinates multiple responses, including cell cycle arrest, DNA repair, metabolic alterations, antioxidant effects, anti-angiogenesis, autophagy, senescence, and apoptosis. p53 maintains genomic stability and prevents or reduces mutations, earning it the nickname "guardian of the genome." p53 plays a crucial role in mechanisms that prevent cancer, such as apoptosis, cell senescence, genomic stability, and angiogenesis inhibition. TP53 (the gene encoding p53) mutations are the most common genetic mutations in cancer, with partial or complete loss of function in over 50% of tumors. p53 mutations provide tumor cells with a selective advantage, enabling them to circumvent cell cycle checkpoints, avoid apoptosis and senescence, and proliferate under conditions where normal cells cannot. Mutated p53 proteins may lose their ability to effectively bind to DNA, preventing the formation of p21 proteins and the signal to stop cell division. Consequently, damaged cells continue to divide uncontrollably, ultimately forming tumors. In recent years, increasing research has focused on developing drugs targeting mutant p53 as an anti-cancer target in Traditional Chinese Medicine (TCM). However, reports of drugs that restore the function of mutant p53 proteins to inhibit cancer cell growth remain lacking.

[0004] Peptides are compounds consisting of three or more amino acid molecules linked by peptide bonds. As one of the most important biological substances, peptides are widely present in living organisms, regulating the functional activities of various systems, organs, and cells. In recent years, peptide drugs have attracted considerable attention in the development of anti-tumor drugs due to their advantages such as high targeting, low immunogenicity, high tissue penetration, and safety. In 2019, the global market for anti-tumor peptide therapeutics was valued at US$8.6 billion and is expected to maintain steady growth over the next decade. Peptides, as small molecule compounds, can effectively bind to mutant p53 proteins, thereby restoring their normal function and inhibiting the development of cancer. Therefore, the development of effective peptide drugs that can restore the activity of mutant p53 proteins not only promotes research on tumor growth but also has significant clinical value.

[0005] Therefore, developing peptide drug molecules with potential medicinal value using P53 as an anti-cancer target is a hot issue in the development of anti-tumor drugs. Summary of the Invention

[0006] The present invention aims to provide a polypeptide targeting P53 and its use in preparing a drug for treating cancer, so as to provide more polypeptide drug molecules with potential medicinal value for the development of anti-cancer drugs.

[0007] To achieve the above object, according to one aspect of the present invention, a polypeptide targeting a P53 mutant is provided. The polypeptide specifically binds to a P53 mutant, and the polypeptide is selected from any one or more of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

[0008] Furthermore, the P53 mutant is a mutant having at least one mutation selected from the following sites: Y220C, R175H or R273H; preferably, the polypeptide is a modified peptide segment; preferably, the modification is a chemical group modification or an amino acid modification; preferably, the chemical group modification is any one or more of PEG modification, acetylation modification or amidation modification; preferably, the PEG modification is a linear PEG modification, a PEG modification with a single functional group or a PEG modification with a bifunctional group; preferably, the PEG modification site is selected from any one or more of the N-terminus, C-terminus, Lys side chain and Cys side chain of the polypeptide; preferably, the PEG modification is a PEG modification with a molecular weight of 500 to 40,000; preferably, the N-terminus and C-terminus of the polypeptide have acetylation modification and amidation modification, respectively; preferably, the polypeptide The N-terminus has a fatty acid modification, more preferably a Myr modification; preferably, the amino acid modification is a hydrophilic amino acid modification or a cysteine ​​modification; preferably, the hydrophilic amino acid modification is the addition of 1-4 hydrophilic amino acids to the N-terminus, C-terminus or NC-terminal ends of the polypeptide, more preferably, the hydrophilic amino acid is Glu, Lys, Ser or Gly; further preferably, the 1-4 hydrophilic amino acids are selected from any one of the following: Glu-Glu, Lys-Lys or Ser-Gly-Ser; preferably, the cysteine ​​modification is the addition of cysteine ​​at any of the following positions of the polypeptide: N-terminus, C-terminus, NC-terminal ends or in the middle of the peptide chain; more preferably, adding cysteine ​​in the middle of the peptide chain includes inserting one or more cysteines in the middle of the peptide chain, or one or more cysteines are connected to the middle of the peptide chain in the form of a branch.

[0009] According to another aspect of the present invention, a polypeptide drug is provided, which comprises any one or more of the above polypeptides and pharmaceutically optional excipients.

[0010] Furthermore, the polypeptide drug is an anticancer drug, preferably an anti-breast cancer drug; preferably, the concentration of the polypeptide in the polypeptide drug is 0.1 μM to 100 μM.

[0011] According to another aspect of the present invention, there is provided a use of the above polypeptide in the preparation of a drug for treating individual tumors or cancers. Optionally, the drug is a polypeptide drug or a combination drug.

[0012] Furthermore, the cancer includes any one or more of the following: breast cancer, lung cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, liver cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, uterine cancer, ovarian cancer, fat cancer, brain tumor, squamous cell carcinoma, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymus cancer and central nervous system cancer; preferably, the central nervous system cancer is brain cancer; preferably, the application includes the function of inhibiting the proliferation of human breast cancer cells through the polypeptide.

[0013] According to another aspect of the present invention, a kit for detecting P53 mutants is provided, which comprises any one or more polypeptides described above.

[0014] Furthermore, the polypeptide exists in the form of a polypeptide-protein conjugate; preferably, the protein in the polypeptide-protein conjugate is selected from any one of the following: bovine serum albumin, ovalbumin, keyhole limpet hemocyanin or casein; more preferably, the polypeptide is coupled to the protein via a linker sequence to form a polypeptide-protein conjugate, and further preferably, the linker sequence is CGSG.

[0015] Furthermore, the polypeptide is coated on a solid phase carrier; preferably, the solid phase carrier includes an enzyme labeling plate, a membrane carrier or a microsphere; preferably, the membrane carrier includes a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane; preferably, the membrane carrier is also coated with a positive control substance, and the polypeptide and the positive control substance are sequentially arranged on the membrane carrier in the detection order; preferably, the kit further includes at least one of the following: (1) an enzyme-labeled secondary antibody, more preferably the enzyme-labeled secondary antibody is an HRP-labeled secondary antibody; (2) a colloidal gold binding pad, the colloidal gold binding pad is coated with a colloidal gold-labeled antigen and a specific binding substance of the positive control substance; (3) a labeling pad, the labeling pad is coated with fluorescently labeled microspheres, and the microspheres are loaded with a specific binding substance of the positive control substance; preferably, the positive control substance is selected from mouse immunoglobulin, human immunoglobulin, sheep immunoglobulin or rabbit immunoglobulin, and accordingly, the specific binding substance of the positive control substance is selected from anti-mouse immunoglobulin, anti-human immunoglobulin, anti-sheep immunoglobulin or anti-rabbit immunoglobulin.

[0016] Furthermore, the kit includes a chip on which a polypeptide array composed of polypeptides is pre-installed.

[0017] The present invention uses the P53 mutant protein as the target protein for anticancer drugs, screens potential polypeptide fragments on a large scale through polypeptide chips, and then uses in vivo and in vitro screening and verification methods to address the technical problems existing in existing treatment methods and find new polypeptide compounds with anticancer activity; these polypeptides have the advantages of target specificity, low toxicity and few side effects as drugs, and therefore have potential application value in being developed into anticancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0019] Figure 1a-1g Graph showing the results of HPLC detection of polypeptide synthesis in an embodiment of the present invention;

[0020] Figure 2a-2cThe MTT results of the anticancer polypeptides on MDA-MB-231 cell proliferation in the embodiments of the present invention are shown; and

[0021] Figure 3a-3c The graph shows the effect of the polypeptide in the embodiment of the present invention on the inhibition of MDA-MB-231 cell proliferation. DETAILED DESCRIPTION

[0022] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0023] Explanation of terms:

[0024] Polypeptide: In this application, it refers to any peptide segment predicted or screened to be able to specifically bind to the target P53 mutant.

[0025] Polypeptide-carrier protein conjugate: In this application, it refers to a conjugate formed by coupling a polypeptide to a carrier protein, wherein a carrier protein can be coupled to one or more polypeptides. When multiple polypeptides are coupled, the multiple polypeptides have the same amino acid sequence. Depending on the differences in the physicochemical properties of the specific coupled polypeptide sequence, the different types of specific carrier proteins, and the different coupling methods, the number of polypeptides coupled to each carrier protein varies. In this application, 2 to 50 polypeptides are preferred, and 3 to 45, 5 to 40, 5 to 35, 5 to 30, 8 to 30, 10 to 30, 12 to 30, and 15 to 30 polypeptides are more preferred; or, more preferably, any one of 6 to 36, 8 to 32, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, and 10 to 15 polypeptides are more preferred.

[0026] Peptide chip technology is a detection technology based on peptide chips. It utilizes the contact between a wide variety of peptides on a peptide chip and a sample. Image acquisition technology is then used to capture the characteristic signals on the peptide chip (specifically, fluorescent images carrying each characteristic signal). The signal intensity of each characteristic on the chip is then output as the peptide chip detection result data. Based on the sample detection signal output from the peptide chip detection result data, analysis of analytes in the sample that have bound to the peptides on the peptide chip, as well as sample analysis, can be performed.

[0027] Motif: In biology, a motif is a mathematical statistical model based on data, typically a sequence, but can also be a structure, a sequence prediction for a specific group. For example, a DNA sequence can be defined as a transcription factor binding site, meaning that the sequence tends to be bound by transcription factors. For proteins, a sequence motif can be defined as a protein sequence belonging to a given protein family. A simple motif can be, for example, a pattern shared by all members of a group.

[0028] As mentioned in the background art, there is an urgent need to develop anticancer polypeptide drugs targeting different targets in the prior art. The present invention selects P53 mutant protein as the target protein of anticancer drugs, screens potential polypeptide fragments on a large scale through a polypeptide chip, and then quickly screens out polypeptide drugs with anticancer activity through in vitro and in vivo screening and verification. iCXOncP11a, iCXOncP12a, iCXOncP13a, iCXOncP15a, iCXOncP17a, iCXOncP18a, iCXOncP19a, iCXOncP22a, iCXOncP31a, iCXOncP32a, iCXOncP33a, iCXOncP34a, iCXOncP35a, iCXOncP36a, iCXOncP16a, iCXOncP20a, iCXOncP Twenty-two peptides, including iCXOncP21a, iCXOncP23a, iCXOncP24a, iCXOncP25a, iCXOncP27a, and iCXOncP29a (sequence information is shown in Table 1), showed significant inhibition of breast cancer cell growth. Among them, iCXOncP12a, iCXOncP22a, iCXOncP16a, iCXOncP20a, iCXOncP21a, and iCXOncP29a showed the most significant effects, providing ideas for the discovery of new active compounds in subsequent drug development. Furthermore, the novel anticancer peptide sequences we provide can serve as lead compounds for subsequent drug development.

[0029] Based on the above research results, the applicant proposed a series of technical solutions of this application. In a typical embodiment, a polypeptide targeting P53 is provided, which polypeptide specifically binds to a P53 mutant, and the polypeptide is selected from any one or more of the following: RRGPARVSQVPKHL (SEQ ID NO: 1), RRWLPPFGVFS (SEQ ID NO: 2), RRNDYVLRLNKHS (SEQ ID NO: 3), RRYSRAPWSG (SEQ ID NO: 4), RRVEHENAFG (SEQ ID NO: 5), RRPNFPLAQSSD (SEQ ID NO: 6), RRFESKKRSG (SEQ ID NO: 7), RRYWYKNHLYHG (SEQ ID NO: 8), RRLLDAGPSEG (SEQ ID NO: 9), RREPAKSYWSQVE (SEQ ID NO: 10), RRPALGNRLWDAQVE (SEQ ID NO: 11), RRPNYLDQFADG (SEQ ID NO: 12), RRNHHQPADG (SEQ ID NO: 13), RRNAYGQVFD (SEQ ID NO: 14), RRFNFYRQVG (SEQ ID NO: 15), ID NO: 15), RRWRYYFWKED (SEQ ID NO: 16), RRGHEWPLDG (SEQ ID NO: 17), RNDEFAQKVS (SEQ ID NO: 18), RRFYQWKELED (SEQ ID NO: 19), RRHNVKWED (SEQ ID NO: 20), RRSRGGDG (SEQ ID NO: 21), or RRSHPNAHED (SEQ ID NO: 22).

[0030] As described above, the present invention uses the p53 mutant protein as the target protein for anticancer drugs. Through large-scale screening of promising polypeptide fragments using peptide microarrays, and then through in vitro and in vivo screening and verification, novel polypeptide compounds with anticancer activity have been identified, addressing the technical issues existing in existing treatments. These polypeptides have the advantages of target specificity, low toxicity, and minimal side effects, and thus have potential application value as anticancer drugs. Among the aforementioned polypeptides, compounds represented by SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 22 are preferred, as they exhibit the highest anticancer activity, for example, higher activity against breast cancer than other polypeptides.

[0031] The above-mentioned P53 mutant is any mutant that can cause the loss of its tumor suppressor function, and in the present application, includes but is not limited to mutants with at least one of the following site mutations: Y220C, R175H or R273H.

[0032] Polypeptides have a significant structural advantage. Without affecting the original functional polypeptide fragment, new functional chemical modifications can be introduced at one or both ends of the polypeptide through solid-phase synthesis or biosynthesis to obtain multifunctionality. This multifunctional chemical modification has the mechanism of pH-sensitive liposomes with receptor / ligand binding ability to enter cells, which will induce tumor tissue cells to actively internalize liposomes and then release drugs. Common polypeptide modifications can be divided into four categories according to the modification site: C-terminal modification (amidation, sulfation, etc.), N-terminal modification (acetylation, fatty acidization, etc.), intermediate residue modification (glycosylation modification bound to Ser-, Tyr-, Asn-, Thr-; phosphorylation modification bound to Ser-, Tyr-, Thr-, etc.) and cyclization modification.

[0033] Therefore, according to the needs of drug development, the above polypeptides can also be modified in various ways as needed during development. In a preferred embodiment, the polypeptide is a modified peptide segment; preferably, the modification is a chemical group modification or an amino acid modification.

[0034] For example, in order to further improve the affinity of certain peptide segments, in a preferred embodiment, the above-mentioned chemical group modification is PEG modification; preferably, the PEG modification is a straight-chain PEG modification, a PEG modification with a single functional group, or a PEG modification with a bifunctional group; preferably, the PEG modification site is selected from any one or more of the N-terminus, C-terminus, Lys side chain, and Cys side chain of the polypeptide; preferably, the PEG modification is a PEG modification with a molecular weight of 500 to 40,000; preferably, the amino acid modification is a hydrophilic amino acid modification or a cysteine ​​modification; preferably, the hydrophilic amino acid modification is the addition of 1-4 hydrophilic amino acids to the N-terminus, C-terminus, or both ends of the NC of the polypeptide, more preferably, the hydrophilic amino acid is Glu, Lys, Ser, or Gly; further preferably, the 1-4 hydrophilic amino acids are selected from any one of the following: Glu-Glu, Lys-Lys, or Ser-Gly-Ser.

[0035] Since chemically synthesized polypeptides often carry free amino and carboxyl groups, and the sequence of a polypeptide often represents the sequence of the parent protein in which it is derived, in order to make the synthesized protein more similar to the parent protein in terms of sequence and activity, the ends of the polypeptide are usually blocked, generally by acetylation of the N-terminus and amidation of the C-terminus. Such modifications reduce the overall charge of the polypeptide, lowering its solubility, and further allowing the polypeptide to mimic the original state of the α-amino and carboxyl groups in the parent protein. Therefore, in other embodiments, the above-mentioned chemical group modification is a double-terminal modification of the polypeptide, more preferably, the N-terminus and C-terminus of the polypeptide are acetylated and amidated, respectively.

[0036] In certain embodiments, in order to better achieve directional coupling of peptide segments, it is preferred to modify the polypeptide with cysteine. Specifically, it includes but is not limited to adding cysteine ​​at the N-terminus, C-terminus, or both ends of the NC of the peptide segment, or adding cysteine ​​in the middle of the peptide chain of the polypeptide. When adding cysteine ​​in the middle of the peptide chain of the peptide segment, one or more cysteines can be inserted in the middle of the peptide chain (i.e., inserted between two amino acid residues), or one or more cysteines can be connected to the middle of the peptide chain in the form of a branched chain (i.e., as a side chain of an amino acid in the middle of the peptide chain).

[0037] It should be noted that the modification methods of the above-mentioned polypeptides can adopt the most mature and widely used chemical modification methods, including liquid phase method and solid phase method.

[0038] In a second exemplary embodiment of the present application, a polypeptide drug is provided, comprising any of the above-mentioned polypeptides and pharmaceutically optional excipients. The drug comprising the above-mentioned polypeptide molecule has the advantages of high targeting ability to bind to P53 mutants, high anticancer activity, low toxicity, and few side effects.

[0039] The pharmaceutically optional excipients mentioned above may vary depending on the dosage form and / or administration method and route of administration of the drug to be prepared, and can be reasonably selected from existing pharmaceutical excipients. They include, but are not limited to, pharmaceutically acceptable carriers, excipients, or adjuvants. Moreover, the polypeptide drug can be prepared into different dosage forms to accommodate a variety of administration routes, such as oral administration, injection, or transdermal administration. The dosage forms of the drug include capsules, tablets, oral solutions, injections, or transdermal absorbable formulations.

[0040] Specifically, the medicine of the present application can be made into tablet capsules, tablets, oral liquids, injections or transdermal absorbents according to methods known in the pharmaceutical industry. Preferably, the injection is an intravenous injection. When preparing capsules, tablets, and oral liquids suitable for oral administration, sucrose, lactose, galactose, corn starch, gelatin, microcrystalline cellulose, carboxymethyl cellulose, etc. can be used as carriers or excipients. In addition, the medicine of the present application can also be made into solutions and suspensions suitable for oral administration using methods and auxiliary ingredients known in the pharmaceutical industry. If solutions and suspensions suitable for parenteral administration are prepared, distilled water, water for injection, isotonic sodium chloride or glucose solution, or low concentration (e.g., 1-100 mm) phosphate buffered saline (PBS) can be used as carriers or diluents. One or more other auxiliary ingredients or additives can be added to these parenteral preparations, for example, ascorbic acid can be used as an antioxidant, and sodium benzoate, etc. can be used as a preservative. Other solubilizers, disintegrants, colorants, dispersants, or surfactants can also be contained in these preparations.

[0041] The above-mentioned polypeptide drug can be used as an anticancer drug, preferably an anti-breast cancer drug, because it has anti-cancer activity. In the above-mentioned polypeptide drug, the concentration of the polypeptide varies depending on the dosage form or administration method. In a preferred embodiment, the concentration of the polypeptide in the polypeptide drug is 0.1μM to 100μM, and it has anti-cancer activity within this concentration range. More preferably, the concentration is 1μM to 100μM, and further preferably 10μM to 100μM. Specifically, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100μM.

[0042] In a third exemplary embodiment of the present application, the use of any of the above-mentioned polypeptides in the preparation of a drug for treating cancer is provided. Since P53 is a tumor suppressor protein, it plays a crucial role in inhibiting the pathogenesis of cancer. Therefore, the polypeptides screened in this application that specifically bind to P53 mutants have potential for development as pharmaceuticals for treating cancer, particularly breast cancer. These drugs are used to inhibit the proliferation of human breast cancer cells, thereby achieving the goal of treating breast cancer.

[0043] The aforementioned cancers include, but are not limited to, any one or more of the following: breast cancer, lung cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, uterine cancer, ovarian cancer, fatty liver cancer, brain tumor, squamous cell carcinoma, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymus cancer, and central nervous system cancer; preferably, the central nervous system cancer is brain cancer. Preferably, the aforementioned cancer is breast cancer.

[0044] It should be noted that the above-mentioned treatment includes different degrees of inhibition of cancer cell proliferation and other effects. The specific inhibition degrees include more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or even 100% of cancer cell proliferation.

[0045] In a fourth typical embodiment of the present application, a kit for detecting P53 mutants is provided, the kit comprising any of the above-mentioned polypeptides. Since the above-mentioned polypeptides have been verified by peptide chips combined with AI-assisted screening and experiments to have the ability to specifically target and bind to P53 mutants, and thus have anti-cancer activity by binding to P53 mutants, in particular, the anti-breast cancer cell activity of the polypeptides represented by any one of SEQ ID NO: 1 to SEQ ID NO: 22 or their modified sequences. Therefore, the use of a kit comprising the above-mentioned polypeptides can specifically and accurately detect the presence or absence of P53 mutants and the level of expression.

[0046] Since the polypeptide molecules are relatively small, in order to improve the detection capability, the above-mentioned polypeptides can be prepared into the form of polypeptide-carrier protein conjugates for detection. According to the requirements of the preparation of the polypeptide-carrier protein conjugate, a specific suitable carrier protein can be selected to form the polypeptide-carrier protein conjugate. In some preferred embodiments, the polypeptide exists in the form of a polypeptide-protein conjugate. The carrier proteins in this application include but are not limited to BSA (bovine serum albumin), OVA (ovalbumin), KLH (keyhole limpet hemocyanin) or CS (casein). According to the amino acid sequence composition of different polypeptides, in order to facilitate coupling with the carrier protein, it is necessary to couple with the carrier protein through a connecting sequence (also called a linker or linker). In this application, the connecting sequence is preferably CGSG.

[0047] It should be noted that the aforementioned polypeptide-carrier protein conjugates are recombinantly synthesized and expressed proteins. In addition to detecting P53 mutant proteins, based on protein homology, these recombinantly expressed polypeptide-carrier protein conjugates can also be used to detect proteins that are homologous to P53 or have different mutation sites.

[0048] The number of polypeptides that can be coupled to each carrier protein varies depending on the physicochemical properties of the polypeptide amino acids, the different carrier proteins used, and the different coupling methods. Taking into account the coupling efficiency and the antibody recognition and binding ability, it is preferred that each carrier protein be coupled to 2 to 50 polypeptides, more preferably 3 to 45, 5 to 40, 5 to 35, 5 to 30, 8 to 30, 10 to 30, 12 to 30, or 15 to 30; or more preferably any one of 6 to 36, 8 to 32, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, and 10 to 15.

[0049] The above-mentioned kit can be prepared into a variety of different types of detection kits according to specific needs. However, from the perspective of convenient detection and easy judgment of the test results, the polypeptide in the kit can be set to be pre-coated on a solid phase carrier. The specific pre-coated solid phase carrier is reasonably designed according to needs. More preferably, the solid phase carrier includes an ELISA plate (mostly made of polystyrene materials), a membrane carrier or a microsphere; further preferably, the membrane carrier includes a nitrocellulose membrane (the most widely used), a glass cellulose membrane or a nylon membrane, and further preferably, the membrane carrier is also coated with a positive control substance, and the polypeptide-carrier protein conjugate and the positive control substance are sequentially arranged on the nitrocellulose membrane in the order of detection.

[0050] Depending on the specific detection method of the kit, the specific supporting reagents in the kit will also vary accordingly, but all supporting reagents can be combined according to the preparation method of known kits. Preferably, the above kit also includes at least one of the following: (1) an enzyme-labeled secondary antibody, more preferably an HRP-labeled secondary antibody (corresponding to an ELISA detection kit); (2) a colloidal gold conjugate pad coated with a colloidal gold-labeled polypeptide-carrier protein conjugate and a specific binding substance of the positive control substance (corresponding to an immunocolloidal gold detection kit); (3) a labeling pad coated with fluorescently labeled microspheres, and the microspheres are loaded with a specific binding substance of the positive control substance (corresponding to an immunofluorescence detection kit).

[0051] The above-mentioned immunocolloidal gold detection kit and immunofluorescence detection kit are relatively more convenient for detection, and only the C line of the positive control and the T line of the test sample need to be established. The positive control substance pre-coated at the C line of the positive control can be a specific binding substance with a detection label that can be carried over during the serum chromatography process of the test sample, and there is no special limitation on the specific antigen or antibody of the specific positive control substance. Preferably, the above-mentioned positive control substance is selected from mouse immunoglobulin, human immunoglobulin, sheep immunoglobulin or rabbit immunoglobulin, and accordingly, the specific binding substance of the positive control substance is selected from anti-mouse immunoglobulin, anti-human immunoglobulin, anti-sheep immunoglobulin or anti-rabbit immunoglobulin.

[0052] The anti-mouse immunoglobulin can be a goat anti-mouse immunoglobulin or a rabbit anti-mouse immunoglobulin, or an anti-mouse immunoglobulin from another immunizable animal, depending on the target of the immunization. Similarly, the anti-human immunoglobulin, anti-goat immunoglobulin, or anti-rabbit immunoglobulin can also be anti-immunoglobulins from different species, depending on the animal being immunized. The immunoglobulin can be any one of IgM, IgG, IgA, IgD, or IgE. These anti-immunoglobulin antibodies can be monoclonal antibodies or polyclonal antibodies.

[0053] In the above-mentioned kits, the specifications of the ELISA plates used vary depending on the number of samples to be tested, and can be appropriately selected from 12- to 384-well ELISA plates. In pre-coated ELISA plates, the amount of peptide-carrier protein conjugate coated per well also varies depending on the target of detection in the peptide-carrier protein conjugate. Similarly, the amount of peptide-carrier protein conjugate coated on a membrane support (e.g., nitrocellulose membrane) also varies. For example, it can be 0.8-8 μg / cm, more preferably 0.8-7 μg / cm, 0.8-6 μg / cm, 0.8-5 μg / cm, 0.8-4 μg / cm, 0.8-3 μg / cm, 0.8-2 μg / cm, 0.8-1.8 μg / cm, 0.8-1.7 μg, 0.8-1.6 μg / cm, 0.8-1.5 μg / cm, 0.8-1.4 μg / cm or 0.8-1.2 μg / cm.

[0054] In some preferred embodiments, the kit includes a chip pre-loaded with polypeptides, wherein the polypeptides comprise a polypeptide array comprising the aforementioned polypeptides. Detection of the aforementioned polypeptides on the polypeptide chip not only enhances detection specificity by leveraging the polypeptides' targeted binding ability to P53 mutants, but also increases detection throughput by utilizing the array format, making it suitable for large-scale cancer screening.

[0055] The beneficial effects of the present application will be further illustrated below with reference to specific embodiments. It should be noted that the embodiments of the present application mainly include the following parts:

[0056] The present invention is to investigate the inhibitory activity IC of candidate anticancer peptides on MDA-MB-231 cells (human breast cancer cells) in vitro at different concentrations (0.1 μM, 1 μM, 10 μM, 100 μM). 50 The anti-cancer activity of 22 polypeptides was determined by the determination of the values, namely the anti-breast cancer iCXOncP11a, iCXOncP12a, iCXOncP13a, iCXOncP15a, iCXOncP17a, iCXOncP18a, iCXOncP19a, iCXOncP22a, iCXOncP31a, iCXOncP32a, iCXOncP33a, iCXOncP34a, iCXOncP35a, iCXOncP36a, iCXOncP16a, iCXOncP20a, iCXOncP21a, iCXOncP23a, iCXOncP24a, iCXOncP25a, iCXOncP27a and iCXOncP29a polypeptides. And through verification and comparison with the currently available peptide library data (NCBI peptide group), we found that all the peptides in this application are completely new sequences.

[0057] Specific implementation plan:

[0058] (1) Incubate the peptide chip to screen the peptide library.

[0059] (2) Computer docking.

[0060] (3) Synthesis of candidate polypeptide sequences.

[0061] (4) Determination of the biological activity of each candidate peptide, based on the determination of anti-cancer cells, the working concentration of the peptide was detected, and the in vitro inhibitory activity IC was estimated 50 value.

[0062] (5) The cytotoxicity of each candidate peptide was verified using 293T cells.

[0063] Example 1

[0064] Peptide chip screening of peptide library

[0065] 1) Screening of binding peptide collections

[0066] A, candidate Y220C, R175H, and R273H mutant P53 proteins were selected and fluorescently labeled (Kit: Alexa 555 Protein Labeling Kit, A20174), wherein the sources of the above-mentioned P53 mutant proteins are shown in Table 1.

[0067] Table 1 Sources of three P53 mutant proteins:

[0068] protein company Item No. Specification P53 mutant Y220C Cusabio CSB-BP024077HU(M1) 0.5mg P53mutant R175H Cusabio CSB-BP024077HU(M2) 0.5mg P53mutant R273H Cusabio CSB-BP024077HU(M3) 0.5mg

[0069] B, High-throughput short peptide array chip detection of fluorescently labeled P53 mutant protein.

[0070] a. The fluorescently labeled samples were diluted according to seven concentration gradients of 1:500, 1:1000, 1:5000, 1:10000, 1:50000, 1:500000 and 1:5000000, with the median of the final dilution concentration being 1:10000.

[0071] b. Place the chip in a chip hydration apparatus and add ultrapure water to cover the chip. Hydrate on an orbital shaker at 55 ± 5 rpm for 20 minutes. Then, spray the chip surface with isopropyl alcohol and centrifuge to dry. Assemble the dried chip into an assay cassette according to the experimental design.

[0072] c. Add the diluted sample to the assembled chip at 90 μL / well and incubate on a constant temperature shaker for 1 hour.

[0073] d. Place the assay cassette in a plate washer for washing.

[0074] e. The chips in the assay cassette are disassembled, cleaned, and dried before being assembled into the imaging cassette. Scanning and imaging are performed using a Molecular Devices ImageXpress micro 4 imager. Each sample is scanned and imaged using a TIFF image file, representing the raw data.

[0075] C. Sort the polypeptide sequences in the polypeptide set according to the binding signal strength; select a predetermined number of top-ranked polypeptide sequences as candidate polypeptides for the target protein.

[0076] 2) Antibody incubation to screen effective peptides

[0077] A, The IST chip was supersaturated with the above-mentioned P53 mutant protein to allow the mutant protein to bind to the peptides in the chip.

[0078] B. The antibody PAB1620 (Merck catalog number: MABE339) binds to the normal structure of P53. Detecting the binding of PAB1620 can indicate the recovery of the P53 protein structure. Therefore, PAB1620 was used for high-throughput short peptide array chip detection to screen for peptides that can restore the structural activity of P53.

[0079] C. By analyzing the signal data detected by immune indication technology, specific binding peptides with stable specific signals are found.

[0080] D, matched with the peptide sequence of the initial screening, and finally obtained a peptide that can stably bind to mutant P53 and restore its structural activity.

[0081] Example 2

[0082] Computer docking (simulating the combination of candidate peptides from Example 1 for further screening and verification).

[0083] 1) Extracting receptor protein coordinates: The 4MZR PDB file contains protein, ligand, and water molecules; first extract the protein coordinates.

[0084] 2) Hydrogenation: Crystal structures often lack coordinates for hydrogen atoms (because hydrogen atoms have few electrons and their protons have a weak attraction to electrons, making them difficult to locate). However, during docking, hydrogen atoms, especially polar hydrogen atoms, are essential for calculating electrostatic interactions. Therefore, it is necessary to add hydrogen atoms to the protein.

[0085] 3) Define the 3D search space for ligand binding: If the binding site is unknown, theoretically one can define a rectangular box that contains the entire protein or any specific region.

[0086] 4) Prepare reference ligand

[0087] The atomic positions of the reference ligand were extracted from the 4MZR PDB structure.

[0088] Similar to protein structures, ligand structures also lack hydrogen atoms, so it is necessary to add hydrogen atoms and define which bonds are rotatable for flexible docking.

[0089] 5) Prepare the docking configuration file, which contains the input receptor (protein), ligand (compound) and default search parameters.

[0090] 6) Docking D and using PyMol to visualize the docking results.

[0091] 7) Based on the strength of the binding affinity and combined with professional knowledge, potential functional peptides were selected for synthesis and further functional verification of the peptide activity. The relevant results are shown in Table 2.

[0092] Table 2

[0093]

[0094]

[0095] Example 3

[0096] Peptide synthesis

[0097] The candidate peptides were commissioned to be synthesized by Gill Biotechnology: the terminal peptides were modified with Myr, and the detailed information is shown in Table 3.

[0098] Table 3

[0099]

[0100]

[0101] The HPLC chromatograms of the synthesis of the polypeptides represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 22 are as follows: Figures 1a to 1g shown.

[0102] Example 4

[0103] Peptide biological function verification test

[0104] 1. Chemicals and Reagents: Dulbecco's Modified Eagle Medium (DMEM) was purchased from Invitrogen (CA, USA). Fetal bovine serum (FBS) was from HyClone Laboratories, Inc. (USA). Dimethyl sulfoxide (DMSO) was purchased from Sigma, and the MTT kit was purchased from Sangon Biotechnology (E606334-0500).

[0105] 2. MTT assay steps:

[0106] 1. All tubes in the kit need to be centrifuged before opening.

[0107] 2. Preparation of MTT Solution: Dissolve MTT Reagent (Component A) in MTT Solvent (Component B) to prepare a 5 mg / ml MTT solution. Use immediately or store directly at -20°C in the dark. Alternatively, aliquot as needed and store at -20°C in the dark.

[0108] 3. Add 10 μL of MTT solution to each well to make the final concentration of MTT in each well 0.5 mg / ml.

[0109] 4. After gentle mixing, incubate in a 5% CO2, 37°C incubator for 4 hours.

[0110] 5. Carefully aspirate the medium from each well to prevent the cell monolayer from breaking.

[0111] 6. Add 100 μL of Formazan Solubilization Solution (Component C) to each well.

[0112] 7. Place the 96-well plate on a shaker and gently shake for 10 minutes until the formazan is completely dissolved as observed under a normal optical microscope.

[0113] 8. Measure the absorbance at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0114] 3. Cell culture:

[0115] 1) MDA-MB-231 (source: ATCC) human breast cancer cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and cultured at 37°C and 5% carbon dioxide (CO2) content.

[0116] 2) Detection of the biological activity of the peptide in vitro.

[0117] MTT (USE) assay was used to detect cell proliferation. MDA-MB-231 cells were first seeded in a 96-well plate, with 5×10 3 Cells were plated in 100 μL of DMEM containing 10% FBS per well, leaving 8 wells empty as blank controls. Incubate overnight (37°C, 5% CO2) to allow cells to adhere. Different concentrations (0.1 μM, 1 μM, 10 μM, 100 μM) of the designed peptide dissolved in DMSO were added to each well. DMEM containing 2% FBS was added to each well. Incubate (37°C, 5% CO2) for 48 hours to allow the peptide to take effect.

[0118] Add 0.05% MTT solution to each well. Incubate (37°C, 5% CO2) for 3 hours to allow MTT to metabolize. Discard the culture medium and resuspend formazan (MTT metabolite) in 100 μL DMSO. Mix the formazan into the solvent. Read the optical density at 570 nm and record the result. Draw a growth curve with concentration as the horizontal axis and cell viability as the vertical axis. The results are shown in Figure 2. Figure 2a 、 Figure 2b and Figure 2c shown.

[0119] Depend on Figure 2a The results showed that all seven peptides exhibited anticancer activity to varying degrees, with iCXOncP12a showing the most significant anticancer activity. It could effectively inhibit cancer cell growth at a concentration of 10-100 μM. The IC value of iCXOncP11a was calculated based on the above data. 50 =37.64μM, IC of iCXOncP12a 50=12.98μM, IC of iCXOncP13a 50 =41.23 μM, IC of iCXOncP15a 50 =40.95μM, IC of iCXOncP17a 50 =44.36μM, IC of iCXOncP18a 50 =48.52μM, IC of iCXOncP19a 50 =31.3 μM, IC of the positive control peptide ReACp53 50 =52.96μM (Positive control reference: Soragni A, Janzen DM, Johnson LM, Lindgren AG, Thai-Quynh Nguyen A, Tiourin E, Soriaga AB, Lu J, Jiang L, Faull KF, Pellegrini M, Memarzadeh S, Eisenberg DS. ADesigned Inhibitor of p53 Aggregation Rescues p53 Tumor Suppression inOvarian Carcinomas. Cancer Cell. 2016Jan 11;29(1):90-103.).

[0120] Depend on Figure 2b The results showed that the other seven peptides all exhibited anticancer activity to varying degrees, with iCXOncP22a showing the most significant anticancer activity. It could effectively inhibit cancer cell growth at a concentration of 10-100 μM. The IC value of iCXOncP22a was calculated based on the above results. 50 =6.739μM, IC of iCXOncP31a 50 =20.82μM, IC of iCXOncP32a 50 =14.27 μM, IC of iCXOncP33a 50 =47.64μM, IC of iCXOncP34a 50 =42.47 μM, IC of iCXOncP35a 50 =72.62μM, IC of iCXOncP36a 50 =33.57 μM, IC of the positive control peptide ReACp53 50 =52.96μM.

[0121] Depend on Figure 2cThe results showed that all eight peptides exhibited anticancer activity to varying degrees, with iCXOncP20a showing the most significant anticancer activity. It could effectively inhibit cancer cell growth at a concentration of 10-100 μM. The IC value of iCXOncP16a was calculated based on the above data. 50 =26.04μM, IC of iCXOncP20a 50 =8.652μM, IC of iCXOncP21a 50 =29.65μM, IC of iCXOncP23a 50 =32.92μM, IC of iCXOncP24a 50 =28.39μM, IC of iCXOncP25a 50 =30.45 μM, IC of iCXOncP27a 50 =24.33 μM, IC of iCXOncP29a 50 =29.34 μM, IC of the positive control peptide ReACp53 50 =52.96μM.

[0122] From this, we can conclude that all 22 peptides in this application have certain anti-cancer effects, and most of these effects are better than those of the positive control. Subsequent research on these peptides will further confirm their drugability.

[0123] The above IC 50 IC value calculation method: Based on the inhibition rate of peptide on tumor cells at different concentrations, a regression curve is drawn with the peptide concentration on the horizontal axis and the inhibition rate of tumor cells on the vertical axis. The regression equation and R value are obtained, and the y value of 50% inhibition rate is used to calculate IC value. 50 The value of .

[0124] In another experiment, breast cancer cells were treated with 22 peptides at different concentrations (1 μM, 10 μM, 100 μM). Figure 3a 、 Figure 3b and Figure 3c As shown, cancer cells grew slowly after polypeptide treatment, and no cancer cells survived under treatment with 100 μM.

[0125] Example 5

[0126] Peptide cytotoxicity assay

[0127] Under the action of different concentrations of peptides, MTT was used to detect the cell viability of renal epithelial cells 293T (source: ATCC), and the LD values ​​of 22 peptides for 293T cells were calculated based on the results. 50 The results are shown in Table 4. 50 and IC 50The results, IC 50 Both are much smaller than LD 50 , compared with other peptides, it has higher medicinal development value.

[0128] Table 4

[0129]

[0130]

[0131] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present invention selects P53 mutants as the target protein for anticancer drugs, screens potential polypeptide fragments on a large scale through polypeptide chips, and then quickly screens out polypeptide drugs with anticancer activity through in vitro and in vivo screening and verification. The twenty-two polypeptides of the present invention, iCXOncP11a, iCXOncP12a, iCXOncP13a, iCXOncP15a, iCXOncP17a, iCXOncP18a, iCXOncP19a, iCXOncP22a, iCXOncP31a, iCXOncP32a, iCXOncP33a, iCXOncP34a, iCXOncP35a, iCXOncP36a, iCXOncP16a, iCXOncP20a, iCXOncP21a, iCXOncP23a, iCXOncP24a, iCXOncP25a, iCXOncP27a and iCXOncP29a (sequence information is shown in Table 3), showed significant inhibitory effects on the growth of breast cancer cells. Taking into account the IC 50 Value size, IC 50 With LD 50 The size comparison and the survival of breast cancer cells after peptide treatment taken under a microscope (as listed in Table 5) show that iCXOncP12a, iCXOncP22a, iCXOncP16a, iCXOncP20a, iCXOncP21a, and iCXOncP29a have the most significant effects.

[0132] Table 5

[0133]

[0134]

[0135] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention. Sequence Listing <110> Zhuhai Carbon Cloud Intelligent Technology Co., Ltd. <120> P53-targeting polypeptide and its application in preparing medicine for treating cancer <130> PN167666SZTY <160> twenty two <170> SIPOSequenceListing 1.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(14) <223> Peptides targeting P53 <400> 1 Arg Arg Gly Pro Ala Arg Val Ser Gln Val Pro Lys His Leu 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Peptides targeting P53 <400> 2 Arg Arg Trp Leu Pro Pro Phe Gly Val Phe Ser 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(13) <223> Peptides targeting P53 <400> 3 Arg Arg Asn Asp Tyr Val Leu Arg Leu Asn Lys His Ser 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Peptides targeting P53 <400> 4 Arg Arg Tyr Ser Arg Ala Pro Trp Ser Gly 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Peptides targeting P53 <400> 5 Arg Arg Val Glu His Glu Asn Ala Phe Gly 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(12) <223> Peptides targeting P53 <400> 6 Arg Arg Pro Asn Phe Pro Leu Ala Gln Ser Ser Asp 1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Peptides targeting P53 <400> 7 Arg Arg Phe Glu Ser Lys Lys Arg Ser Gly 1 5 10 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(12) <223> Peptides targeting P53 <400> 8 Arg Arg Tyr Trp Tyr Lys Asn His Leu Tyr His Gly 1 5 10 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Peptides targeting P53 <400> 9 Arg Arg Leu Leu Asp Ala Gly Pro Ser Glu Gly 1 5 10 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(13) <223> Peptides targeting P53 <400> 10 Arg Arg Glu Pro Ala Lys Ser Tyr Trp Ser Gln Val Glu 1 5 10 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(15) <223> Peptides targeting P53 <400> 11 Arg Arg Pro Ala Leu Gly Asn Arg Leu Trp Asp Ala Gln Val Glu 1 5 10 15 <210> 12 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(12) <223> Peptides targeting P53 <400> 12 Arg Arg Pro Asn Tyr Leu Asp Gln Phe Ala Asp Gly 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Peptides targeting P53 <400> 13 Arg Arg Asn His His Gln Pro Ala Asp Gly 1 5 10 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Peptides targeting P53 <400> 14 Arg Arg Asn Ala Tyr Gly Gln Val Phe Asp 1 5 10 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Peptides targeting P53 <400> 15 Arg Arg Phe Asn Phe Tyr Arg Gln Val Gly 1 5 10 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Peptides targeting P53 <400> 16 Arg Arg Trp Arg Tyr Tyr Phe Trp Lys Glu Asp 1 5 10 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Peptides targeting P53 <400> 17 Arg Arg Gly His Glu Trp Pro Leu Asp Gly 1 5 10 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Peptides targeting P53 <400> 18 Arg Asn Asp Glu Phe Ala Gln Lys Val Ser 1 5 10 <210> 19 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Peptides targeting P53 <400> 19 Arg Arg Phe Tyr Gln Trp Lys Glu Leu Glu Asp 1 5 10 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Peptides targeting P53 <400> 20 Arg Arg His Asn Val Lys Trp Glu Asp 1 5 <210> twenty one <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> Peptides targeting P53 <400> twenty one Arg Arg Ser Arg Gly Gly Asp Gly 1 5 <210> twenty two <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Peptides targeting P53 <400> twenty two Arg Arg Ser His Pro Asn Ala His Glu Asp 1 5 10

Claims

1. A polypeptide targeting a P53 mutant, characterized in that: The polypeptide specifically binds to a P53 mutant, and the polypeptide is: SEQ ID NO:

8.

2. A polypeptide drug, characterized in that: The polypeptide drug comprises the polypeptide according to claim 1 and pharmaceutically optional excipients.

3. The drug according to claim 2, characterized in that The concentration of the polypeptide in the polypeptide drug is 0.1 μM to 100 μM.

4. Use of the polypeptide according to claim 1 in the preparation of a medicament for treating cancer, wherein the cancer is breast cancer.

5. The use according to claim 4, characterized in that The drug is a polypeptide drug or a combined drug.

6. The use according to claim 4, characterized in that The application includes inhibiting the proliferation of human breast cancer cells through the polypeptide.

7. A kit for detecting P53 mutants, characterized in that: The kit comprises the polypeptide of claim 1.

8. The kit according to claim 7, characterized in that The polypeptide is coated on a solid support.

9. The kit according to claim 8, characterized in that The solid phase carrier includes an ELISA plate, a membrane carrier or a microsphere.

10. The kit according to claim 9, characterized in that The membrane carrier includes nitrocellulose membrane, glass cellulose membrane or nylon membrane.

11. The kit according to claim 10, characterized in that The membrane carrier is also coated with a positive control substance, and the polypeptide and the positive control substance are sequentially arranged on the membrane carrier according to the detection order.

12. The kit according to claim 11, characterized in that The kit further comprises at least one of the following: (1) Enzyme-labeled secondary antibody; (2) a colloidal gold binding pad coated with a specific binding substance of the colloidal gold-labeled antigen and the positive control; (3) A labeling pad, wherein the labeling pad is coated with fluorescently labeled microspheres, and the microspheres are loaded with a specific binding substance of the positive control substance.

13. The kit according to claim 12, characterized in that The enzyme-labeled secondary antibody is an HRP-labeled secondary antibody.

14. The kit according to claim 11, characterized in that The positive control substance is selected from mouse immunoglobulin, human immunoglobulin, sheep immunoglobulin or rabbit immunoglobulin. Correspondingly, the specific binding substance of the positive control substance is selected from anti-mouse immunoglobulin, anti-human immunoglobulin, anti-sheep immunoglobulin or anti-rabbit immunoglobulin.

15. The kit according to any one of claims 7 to 14, characterized in that The kit comprises a chip, on which a polypeptide array composed of the polypeptides is pre-installed.

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