Transmembrane protein and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a cell-penetrating protein and application thereof. The invention provides a cell-penetrating peptide. Different from a ZF5.3 structure which only comprises one strand of alpha helix and cannot clearly determine whether the spatial distribution of the combination of the positive charges and the alpha helix has other diversity or not, the protein designed by the invention has three strands of alpha helixes, so that the positive charge distribution is not limited to the same strand of alpha helix for the first time; meanwhile, two or three alpha helixes are distributed, and the membrane penetrating function can also be achieved. Experiments of flow cytometry, cell fluorescence, cell proliferation, cell apoptosis and the like prove that the cell-penetrating peptide can effectively carry FITC / GFP / KLA apoptotic peptide to enter cells to play a role, and has no obvious biotoxicity. The cell-penetrating peptide provided by the invention can be widely applied to the field of drug delivery.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a transmembrane protein and an application thereof. Background Art

[0002] Protein drugs account for about 20% of the market share of all types of drugs and are unique and important in the field of tumor treatment. Among them, the most widely used protein drugs are a series of monoclonal antibodies, such as trastuzumab (HER2 monoclonal antibody), slulizumab (PD-1 monoclonal antibody), infliximab (TNF-α monoclonal antibody), bevacizumab (VEGF monoclonal antibody), etc., all of which are antibody drugs that can specifically bind to tumor cell surface targets. In addition to cell surface targets, there are also a large number of protein-protein interactions closely related to tumor occurrence and development inside tumor cells, including p53-MDM2, Bcl2-Bid, c-Myc / Max, etc. Combined with proteolysis-targeting chimeras (PROTACs), antibody-drug conjugates (ADC) and other technologies, new protein drugs that regulate intracellular PPI are a new field of tumor treatment with great potential and yet to be developed.

[0003] Due to the huge molecular weight of antibodies, it is difficult to be efficiently transported into cells, which is not conducive to effectively targeting intracellular targets. On the one hand, in order to solve this problem, new skeleton proteins with small molecular weight that can accommodate complex interaction interfaces and new protein drugs have emerged. They include de novo designed ankyrin repeat proteins (DARPins), affibodies, etc. On the other hand, drug delivery systems also provide new solutions for the development of large molecular weight protein drugs. Among them, the simplest delivery tool is cell penetrating peptides (CPPs), also known as cell penetrating peptides, a short peptide consisting of no more than 30 amino acids that can penetrate the cell membrane autonomously. In nature, there are many natural proteins that have the ability to penetrate the cell membrane. In the 1970s, studies found that HIV trans-transcription factor TAT can enter cells autonomously, and only 13 amino acids are needed to mediate this function. Since then, cell penetrating peptides have gradually been valued by the academic community, and hundreds of cell penetrating peptide sequences have been discovered one after another. Compared with viral vectors, membrane-penetrating peptides have the advantages of high efficiency, low toxicity, and easy production. They can effectively deliver small molecules, proteins, nucleic acids, nanoparticles, etc., and are considered to be a delivery tool with great development prospects.

[0004] The biological mechanism of cell entry of membrane-penetrating peptides is not single, but often mixed with multiple modes, which brings challenges to the mechanism research and design of CPP. It is currently believed that CPP entry mainly depends on two ways: endocytosis and direct transmembrane. The former can be divided into clathrin-mediated endocytosis, macropinocytosis, and lipid valve-mediated endocytosis, and the latter can be divided into transient perforation model, carpet model, and flip particle model. For the study of endocytosis mechanism, researchers mainly use different endocytosis inhibitors to treat, such as chlorpromazine that inhibits clathrin-mediated endocytosis pathway, EIPA that inhibits macropinocytosis, and MβCD that can remove cholesterol components of cell membranes. In contrast, the study of direct transmembrane is more difficult. In the past, it was believed that there was a direct transmembrane mode after excluding endocytosis. For example, in the study of the cell entry mechanism of TAT, previous studies believed that TAT was not inhibited by classic endocytosis inhibitors and energy metabolism inhibitors, and it was believed that TAT entered the cell by directly transmembrane. In 2003, Fittipaldi.A. et al. showed through kinetic studies that TAT does enter cells through lipid valve-mediated endocytosis rather than directly crossing the membrane, indicating that there is still no direct evidence of direct transmembrane transport.

[0005] In contrast, the physical and chemical properties that affect transmembrane performance are constantly being revealed, among which the most generalizable characteristics are the number of positive charges and the degree of helicity. A study on the transmembrane ability of natural proteins showed that when the average positive charge level reaches +0.75 / kD, the protein has the ability to penetrate the membrane; when it is lower than this level, it does not have the ability to penetrate the membrane. More importantly, many of the discovered CPPs basically carry positively charged amino acids, which shows that the positive charge characteristic is an important determinant of transmembrane performance. The research group of Alanna Schepartz at the University of California proposed that the simultaneous distribution of arginine on one side of the helix can enable proteins with a helical structure to have transmembrane ability, and used this to develop the first new transmembrane protein ZF5.3 with a clear secondary structure, and systematically verified it in terms of cellular mechanisms and target delivery.

[0006] With the rise of computer-aided protein design technology, many new de novo designed proteins with different structural forms have emerged in recent years. Among them, α-helix is ​​a common secondary structure element that can form a key part of the large, usually uncharacterized interface region of many therapeutically relevant protein-protein interactions, which provides a better choice for the design of new binding proteins involving complex interaction interfaces. At present, scholars have successfully designed a series of new proteins with drug binding ability, such as influenza virus HA binding protein, SARS-Cov2 Spike binding protein, etc., and have good stability and great clinical transformation potential. However, there are some significant limitations in the design and application of current membrane-penetrating peptides. First, the existing design rules are extremely lacking, and they are mainly focused on the design of amino acid sequences, lacking membrane-penetrating design specifications for secondary structures or even tertiary structures. This makes it extremely difficult to rationally develop new membrane-penetrating peptides, limiting the application of membrane-penetrating peptides in new protein drugs. Secondly, most membrane-penetrating peptides are linear short peptides, which can usually only be fused and expressed at the N-terminus or C-terminus of the target protein. This fusion method may change the original function of the target protein, thereby affecting its biological activity and therapeutic effect. In addition, linear short peptides cannot effectively combine protein design technology to develop new membrane-penetrating multifunctional proteins. As the first transmembrane peptide discovered to have a stable secondary structure, ZF5.3 explains that the distribution of positive charges on one side of the α-helix is ​​the key to its cell penetration ability. However, ZF5.3 contains only one α-helix, and whether this transmembrane feature can be applied to the transmembrane modification of multi-stranded α-helical proteins has not yet been explained and verified. Therefore, a new method is urgently needed to obtain transmembrane skeleton proteins with tertiary structures, so as to overcome the shortcomings of existing transmembrane peptides in design and function, and promote their widespread application in biomedicine and related fields.

[0007] References:

[0008] Chevalier A,Silva DA,Rocklin GJ,et al.Massively parallel de novoprotein design for targeted therapeutics.Nature.2017;550(7674):74-79.

[0009] Fleishman SJ,Leaver-Fay A,Corn JE,et al.RosettaScripts: a scriptinglanguage interface to the Rosetta macromolecular modeling suite.PLoSOne.2011;6(6):e20161. Summary of the invention

[0010] The first aspect of the present invention aims to provide a protein or a derivative thereof.

[0011] The second aspect of the present invention aims to provide biological materials related to the protein or its derivatives shown in the first aspect.

[0012] The purpose of the third aspect of the present invention is to provide a method for preparing the protein or its derivatives described in the first aspect of the present invention.

[0013] The fourth aspect of the present invention aims to provide a fusion protein.

[0014] The fifth aspect of the present invention aims to provide an application.

[0015] The sixth aspect of the present invention aims to provide a medicine.

[0016] The seventh aspect of the present invention aims to provide a delivery system.

[0017] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0018] The first aspect of the present invention provides a protein or a derivative thereof, wherein the amino acid sequence of the protein is as shown in any one of a1) to a3):

[0019] a1) any one of SEQ ID NO.1-5;

[0020] a2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 1 to 5;

[0021] a3) an amino acid sequence having at least 95% homology with any one of SEQ ID NOs. 1 to 5.

[0022] In the first aspect of the present invention, the common substitutions of proteins with the same function in a2) are considered conservative substitutions, such as substitutions among aliphatic amino acids Ala, Val, Leu and Ile, interchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr.

[0023] In the first aspect of the present invention, a3) means that the given amino acid sequence has at least 95% identity with the reference sequence, for example, 95%, 96%, 97%, 98%, 99%. Alternatively, it means that the given amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid differences with the reference sequence. For proteins, such differences are preferably amino acid substitutions or deletions. The difference in identity here can come from conservative substitutions of amino acids, or from the introduction of tag proteins, or the introduction of nuclear import sequences, or the introduction and modification of signal peptide sequences. For example, adding His, GFP, HA or other tags to the C-terminus of SEQ ID NO.1 results in a change in identity, or adding a signal peptide to the N-terminus of SEQ ID NO.1, or adding a conventional nuclear import sequence to the N-terminus, thereby resulting in a change in identity.

[0024] Preferably, in a2), the amino acid sequence of the protein with the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 to 5 is as shown in SEQ ID NO.6.

[0025] Preferably, the derivative comprises at least one of a pharmaceutically acceptable salt or a pharmaceutically acceptable modification.

[0026] Preferably, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0027] Preferably, the metal salt comprises an alkali metal salt or an alkaline earth metal salt.

[0028] Preferably, the alkali metal salt includes at least one of a sodium salt and a potassium salt.

[0029] Preferably, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt and aluminum salt.

[0030] Preferably, the salt formed with the organic base includes a salt formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N,N'-dibenzylethylenediamine.

[0031] Preferably, the salt formed with an inorganic acid includes a salt formed with the following inorganic acids: at least one of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0032] Preferably, the salt formed with the organic acid includes a salt formed with the following organic acids: at least one of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0033] Preferably, the salt formed with a basic amino acid includes a salt formed with the following basic amino acids: at least one of arginine, lysine and ornithine.

[0034] Preferably, the salt formed with the acidic amino acid includes a salt formed with the following acidic amino acids: at least one of aspartic acid and glutamic acid.

[0035] Preferably, the pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, halogenation, polyethylene glycol modification, polysialic acid modification, and fatty acid modification. For example, PEG modification can be used to shield and hinder the surface of the protein, so that the enzymatic hydrolysis rate of the modified protein is significantly reduced, the stability is improved, and the half-life in vivo is prolonged; or fatty acid modification is used to react with the protein to undergo acylation modification, and the affinity of the protein to serum albumin can be increased by exposing carboxyl residues on the surface of the protein, thereby increasing its circulation time in the blood, thereby prolonging the half-life of the protein drug. Those skilled in the art can select the corresponding modification method according to the actual use of the drug.

[0036] The second aspect of the present invention provides biological materials related to the protein or its derivatives described in the first aspect of the present invention, including any one of b1 to b12);

[0037] b1) a nucleic acid molecule encoding the protein described in any one of a1) to a3);

[0038] b2) an expression cassette comprising the nucleic acid molecule described in b1);

[0039] b3) a vector comprising the nucleic acid molecule described in b1);

[0040] b4) a vector comprising the expression cassette described in b2);

[0041] b5) a transgenic cell line comprising the nucleic acid molecule described in b1);

[0042] b6) a transgenic cell line comprising the expression cassette described in b2);

[0043] b7) a transgenic cell line comprising the vector described in b3);

[0044] b8) a transgenic cell line comprising the vector described in b4);

[0045] b9) a microorganism comprising the nucleic acid molecule described in b1);

[0046] b10) a microorganism comprising the expression cassette described in b2);

[0047] b11) a microorganism comprising the carrier described in b3);

[0048] b12) A microorganism comprising the vector described in b4).

[0049] Preferably, the transgenic cell line comprises no reproductive material.

[0050] Preferably, the nucleic acid molecule b1) is as shown in any one of SEQ ID NOs. 8 to 13;

[0051] Preferably, the vector comprises a fusion expression tag.

[0052] Preferably, the fusion expression tag includes but is not limited to common fusion expression tags such as GFP, His, and GST.

[0053] Preferably, the microorganism is a transformant, which is obtained by introducing the above-mentioned recombinant expression vector into a host.

[0054] Preferably, the host is Escherichia coli; further preferably, the Escherichia coli is E. coli BL21 (DE3) cells.

[0055] The third aspect of the present invention provides a method for preparing the protein or its derivatives according to the first aspect of the present invention:

[0056] Prepared by culturing the biomaterial described in the second aspect of the present invention;

[0057] Alternatively, it can be obtained by a peptide chemical synthesis method, purified by high performance liquid chromatography (HPLC) to ensure a purity higher than 95%, and finally identified by mass spectrometry (MS).

[0058] Preferably, the culture method is a conventional method in the art. For example, for the molecular cloning part, reference may be made to "Molecular Cloning Laboratory Manual" (sequential coding system: (US) MR Green (Michael R. Green), (US) J. Sambrook (Joseph. Sambrook), He Fuchu. Molecular Cloning Laboratory Manual (Original Book Fourth Edition) (Volumes 1, 2, and 3) [M]. Beijing: Science Press, 2017); for the cell culture part, reference may be made to "Cell Biology Laboratory Handbook" (sequential coding system: Julio E. Celis [ed.]. Cell Biology Laboratory Handbook. Volume 1. Volume 1: Introduction Edition | Cell biology alaboratory handbook. Volume 1 Photocopy Edition [M]. Beijing: Science Press, 2008)

[0059] The fourth aspect of the present invention provides a fusion protein, comprising the protein or its derivatives described in the first aspect of the present invention;

[0060] and a coupling portion, wherein the coupling portion comprises at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

[0061] Preferably, the detectable marker is selected from a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a label protein, a colored substance, or any combination thereof.

[0062] Preferably, the tag protein includes but is not limited to at least one of His, Flag, Myc, GST, GFP, and HA.

[0063] Preferably, the conjugate is selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activated enzymes, chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0064] Preferably, the drug comprises an apoptotic peptide.

[0065] Preferably, the apoptotic peptide comprises KLA apoptotic peptide, and the amino acid sequence of the KLA apoptotic peptide is shown in SEQ ID NO.7: KLAKLAKKLAKLAK.

[0066] Preferably, the fusion protein has the following structural formula:

[0067] X1-X-X2(Ⅰ)

[0068] In the formula,

[0069] X is a core fragment, wherein the sequence of the core fragment is selected from the protein or its derivatives described in the first aspect of the present invention;

[0070] X1 and X2 are independently none or a coupling moiety.

[0071] Among them, X1 and X, and X and X2 are connected by a peptide bond, a peptide linker (such as a flexible linker composed of 1-15 amino acids) or other linkers.

[0072] The fifth aspect of the present invention provides the use of the protein or its derivative described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, and the fusion protein described in the fourth aspect of the present invention in at least one of c1) to c2):

[0073] c1) preparing a product for drug delivery;

[0074] c2) Preparation of products for treating tumors.

[0075] Preferably, the product comprises at least one of a drug and a drug delivery system.

[0076] Preferably, the tumor comprises at least one of a solid tumor and a hematological tumor; and further comprises a solid tumor.

[0077] Preferably, the solid tumor comprises liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, adrenocortical carcinoma, pararenal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, heart tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, embryonic tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, renal cancer, Langerhans cell tissue cell At least one of: leukocyte proliferation, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, sinonasal cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumors, islet cell tumors, papillomatosis, paraganglioma, sinonasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer and single myeloma.

[0078] Preferably, the blood tumor is selected from at least one of B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia and preleukemia.

[0079] In some embodiments of the present invention, the tumor is at least one of cervical cancer (HeLa) and nasopharyngeal carcinoma (CNE2, HK1).

[0080] The sixth aspect of the present invention provides a drug comprising at least one of the protein or its derivative described in the first aspect of the present invention and the fusion protein described in the fourth aspect of the present invention.

[0081] Preferably, the medicament comprises pharmaceutically acceptable excipients.

[0082] Preferably, the pharmaceutically acceptable excipients include at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, a carrier, a lubricant, a sweetener and a flavoring agent.

[0083] Preferably, the carrier comprises a solid carrier or a liquid carrier.

[0084] Preferably, the liquid carrier comprises: saline, buffer, glucose solution, aqueous solution, glycerol, ethanol.

[0085] Preferably, the solid carrier comprises at least one of low molecular weight dextran, polyethylene glycol 400, polyethylene glycol 6000, cyclodextrin, mannitol, lactose, sucrose, sodium chloride and sorbitol.

[0086] Preferably, the drug is administered by one or more methods selected from the following group: oral, injection, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration.

[0087] Preferably, the dosage form of the drug comprises at least one of capsules, tablets, microcapsule preparations, lyophilized powder injection preparations, injections, suppositories, sprays, powders, soft capsules, pellets, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets.

[0088] The seventh aspect of the present invention provides a delivery system, comprising at least one of the protein or its derivative according to the first aspect of the present invention, the fusion protein according to the fourth aspect of the present invention, and the drug according to the sixth aspect of the present invention;

[0089] and delivery vehicles.

[0090] Preferably, the delivery vehicle comprises at least one of a liposome and an extracellular vesicle.

[0091] Preferably, the liposome comprises at least one of polyethylene glycol, phospholipids and cholesterol.

[0092] Preferably, the polyethylene glycol comprises at least one of phosphatidylcholine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, and distearoylphosphatidylcholine-polyethylene glycol.

[0093] Preferably, the phospholipids include at least one of egg yolk lecithin, egg phosphatidylglycerol, egg phosphatidylinositol, egg phosphatidylserine, hydrogenated egg phosphatidylcholine, hydrogenated egg phosphatidylglycerol, hydrogenated egg phosphatidylinositol, hydrogenated egg phosphatidylserine, hydrogenated phosphatidylethanolamine, phosphatidic acid, hydrogenated soybean lecithin, hydrogenated soybean phosphatidylglycerol, hydrogenated soybean phosphatidylserine, hydrogenated soybean phosphatidylinositol, hydrogenated soybean phosphatidylethanolamine, and hydrogenated soybean phosphatidic acid.

[0094] Preferably, the phospholipid comprises at least one of hydrogenated soybean lecithin and egg yolk lecithin.

[0095] Preferably, the sterol includes at least one of cholesterol, lanosterol, sitosterol, stigmasterol and ergosterol.

[0096] The beneficial effects of the present invention are:

[0097] The present invention provides a membrane-penetrating peptide. Different from the structure of ZF5.3 which only contains one strand of α-helix, it is impossible to clarify whether there is other diversity in the spatial distribution of the combination of positive charge and α-helix. The protein designed by the present invention has three strands of α-helix, which is the first time to illustrate that the distribution of positive charge is not limited to the same strand of α-helix, and that the membrane-penetrating function can also be obtained by distributing it in two or three strands of α-helix. Through experiments such as flow cytometry, cell fluorescence, cell proliferation, and cell apoptosis, it is proved that the membrane-penetrating peptide involved in the present invention can effectively carry FITC / GFP / KLA apoptotic peptides into cells to function, and has no obvious biological toxicity. The membrane-penetrating peptide provided by the present invention can be widely used in the field of drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 Schematic diagram of the high-throughput design and screening process of transmembrane skeleton proteins.

[0099] Figure 2 Figure 3 is the density distribution result of the designed skeleton protein library in the secondary structure classification, where A is the kernel density estimation map of different amino acid sequence lengths in the skeleton protein library, B is the kernel density estimation map of the amino acid position where the helical segment is located in the skeleton protein library, C is the kernel density estimation map of the amino acid position where the β-folded segment is located in the skeleton protein library, and D is the kernel density estimation map of the amino acid position where the loop segment is located in the skeleton protein library.

[0100] Figure 3 This is a correlation analysis between the results of two replicates of the second-generation sequencing.

[0101] Figure 4 These are the enriched candidate transmembrane proteins obtained through next-generation sequencing enrichment analysis.

[0102] Figure 5The soluble expression results of CPP-GFP fusion protein, wherein A is the size exclusion chromatography result of CPP-GFP fusion protein, and B is the SDS-PAGE electrophoresis result of CPP-GFP fusion protein.

[0103] Figure 6 The results of cell flow cytometry detection of CPP-GFP fusion protein.

[0104] Figure 7 Fluorescence microscopy results of CPP-GFP fusion protein.

[0105] Figure 8 The tertiary structure and charge distribution results of the new transmembrane protein.

[0106] Fig. 9 Schematic diagram of the optimization and transformation of CPP11D36R, where A is the tertiary structure of CPP11, B is the tertiary structure of CPP11D36R, C is the tertiary structure of ZF5.3, D is the spiral wheel diagram of CPP11, E is the spiral wheel diagram of CPP11D36R, and F is the spiral wheel diagram of ZF5.3.

[0107] Fig.10 This is the secondary structure detection result after CPP11D36R modification.

[0108] Fig.11 These are the results of CPP11D36R carrying FITC into cells, where A is the flow cytometry result of CPP11D36R carrying FITC into HK1 cells, B is the flow cytometry result of CPP11D36R carrying FITC into CNE2 cells, C is the fluorescence microscopy result of CPP11D36R carrying FITC into HK1 cells, and D is the fluorescence microscopy result of CPP11D36R carrying FITC into CNE2 cells.

[0109] Fig.12 This is the result of CPP11D36R carrying GFP into cells.

[0110] Fig.13 This is the cytotoxicity test result of CPP11D36R.

[0111] Fig.14 These are the results of CPP11D36R carrying KLA apoptotic peptide into cells, where A is the flow cytometry result of CPP11D36R carrying KLA apoptotic peptide into HeLa cells, CNE2 cells and HK1 cells, B is the cytotoxicity detection result of CPP11D36R carrying KLA apoptotic peptide into HeLa cells, CNE2 cells and HK1 cells, and C is the cloning result of CPP11D36R carrying KLA apoptotic peptide into CNE2 cells.

[0112] Fig.15 These are the results of CPP11D36R carrying KLA apoptotic peptide into cells to exert apoptotic effects, where A is the Western blot detection result of CPP11D36R carrying KLA apoptotic peptide into CNE2 cells, and B is the flow cytometry apoptosis detection result of CPP11D36R carrying KLA apoptotic peptide into CNE2 cells. DETAILED DESCRIPTION

[0113] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0114] Significance description: The data of the following examples of the present invention are expressed as mean ± standard deviation (n = 3), and * and different letters represent significant differences between treatments (P < 0.05). When only two groups were compared, unpaired two-tailed T-test was used for statistical analysis; when more than two groups were compared, one-way ANOVA was used.

[0115] The experimental data were collated using Excel 2019, and statistical analysis and graphing were performed using GraphPad Prism 9.0.

[0116] Example 1 Design and construction of α-helical protein library

[0117] In this example, Rosetta Commons was first used to generate a protein library with a three-stranded α-helix as the skeleton. In brief, a three-stranded α-helix scaffold without disulfide bonds was generated by Rosetta Remodel, and then the side chain sequence was designed using the Fast Design method, optimized by conformational adjustment, and the designed proteins with scores in the top 1% were screened according to the overall energy score. (References: Chevalier A, Silva DA, Rocklin GJ, et al. Massively parallel de novo protein design for targeted therapeutics. Nature. 2017; 550(7674): 74-79. Fleishman SJ, Leaver-Fay A, Corn JE, et al. Rosetta Scripts: a scripting language interface to the Rosetta macromolecular modeling suite. PLoS One. 2011; 6(6): e20161.).

[0118] Example 2 Screening of transmembrane proteins by phage display technology

[0119] 1. Establishment of phage display library

[0120] The codon optimization tool (https: / / www.genscript.com.cn / gensmart-free-gene-codon-optimization.html) was used to select Escherichia coli as the expression host, and the SacI / SpeI restriction sites were avoided. The amino acid sequence of the α-helical protein library was converted into a nucleic acid sequence suitable for synthesis by GenScript Biotech. The linear nucleic acid library was used as a template for PCR amplification, and SacI / SpeI restriction sites were introduced at both ends. The specific PCR conditions are as follows: a high-fidelity Q5 DNA polymerase (NEB, M0491L); 25-30 PCR cycles to avoid bias caused by preferential amplification of some sequences during amplification; electrophoresis verification and gel recovery purification of amplified PCR products; SacI / SpeI double digestion of purified PCR library (NEB, R3156L; NEB, R3133M) to generate sticky ends; SacI / SpeI double digestion of pComb3XSS plasmid to generate sticky ends; Cycle Pure PCR purification kit (Omega, D6492-02) to purify the PCR library and pComb3XSS vector after restriction digestion; the purified PCR fragment and pComb3XSS vector were ligated with T4 DNA ligase to obtain a DNA plasmid library. The specific conditions are as follows: the molar ratio of insert to vector is (3-5):1; the ligation system is gently mixed to avoid centrifugation; it is treated at room temperature for 5 minutes; it is ligated with Cycle Pure The PCR purification kit was used to purify the plasmid product after ligation to avoid contamination by salt, ethanol, protein and buffer, which would reduce the efficiency of electrotransformation. The DNA plasmid library was transformed into Escherichia coli TG1 by electrotransformation. After electrotransformation, 1 mL of 2×YT medium without resistance was immediately added to the TG1 bacterial solution, and the solution was placed in a 37°C shaker for recovery culture for 1 hour. The bacterial solution was then evenly spread on a 2×YT solid plate containing resistance, and the plate was placed upside down in a 37°C incubator for overnight culture for 13-17 hours to obtain a bacterial library. 20 single clones were selected for Sanger sequencing to preliminarily evaluate whether the target DNA sequence was successfully transformed into TG1 bacteria. The TG1 bacteria that successfully grew on the solid plate were scraped, and a portion was added to 100 mL of 2×YT medium containing 50 μg / mL kanamycin and 4% glucose for expansion culture. The remaining portion was used for phagemid extraction to verify the composition and sequence abundance of the initial phage library by second-generation sequencing. When the OD600 value of the bacterial solution reached 0.35-0.At 45, helper phage M13KO7 was added according to the ratio of helper phage number / bacterial number = (10-20), and the cells were cultured in a 37°C bacterial shaker for 30 minutes to assemble the helper phage with the pIII protein displaying the target protein into a complete phage to obtain a phage library; the bacterial solution was collected and centrifuged at 3000 rcf for 10 minutes at room temperature; the bacterial blocks were collected and resuspended in 2×YT medium containing no glucose, 50 μg / mL kanamycin and 100 μg / mL ampicillin, and cultured in a 30°C bacterial shaker overnight; a solution containing 10% PEG8000 and 2.5M NaCl was prepared and sterilized by high pressure; the M13 phage was purified by the PEG8000 precipitation method; finally, the purified phage was aliquoted and frozen at -80°C for later use. .

[0121] 2. Library secondary structure identification

[0122] The original protein library sequence obtained by second-generation sequencing was classified into secondary structure using the DSSP (Defne Secondary Structure of Proteins) algorithm (reference: Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec; 22 (12): 2577-637).

[0123] like Figure 2 As shown in Figure 2, the median length of the amino acid sequence in the generated protein library is 49 ( Figure 2 A), and the amino acids located in the helical region have the highest frequency ( Figure 2 In B), the amino acid frequency in the loop region is second ( Figure 2 D), while the amino acids located in the β-fold region have the lowest frequency ( Figure 2 Middle C).

[0124] 3. Preliminary evaluation of library enrichment by phage titer assay

[0125] In order to screen for proteins with cell penetration function, in this example, HeLa cells were cultured and incubated with the phage library at 37°C for 1 hour, and then the incubation system was acid-washed with 100mM glycine (pH 2.5), and then the cells were lysed with a cell lysis solution (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% NP40, 5mM EDTA) to collect the enriched phages. The above phage bioenrichment process was repeated three times, and the phage titer was measured to preliminarily evaluate whether the library was enriched after screening. The following are the steps for phage titer determination: inoculate TG1 bacteria into 1 mL of 2×YT medium and culture overnight in a 37°C bacterial shaker; add 10 μL of the phage to be tested into 990 μL PBS as the first concentration gradient; dilute with PBS at a ratio of 1:10 to obtain the 2nd to 10th concentration gradients with a total volume of 1 mL of phage; inoculate 1 mL of TG1 bacterial solution into 100 mL 2×YT culture medium, placed in a 37°C bacterial shaker and cultured until the OD600 value is 0.35-0.45; take 450 μL of the above TG1 bacterial solution, add 50 μL of phage; place in a 37°C metal bath for 30 minutes, 600 rpm; take 100 μL of TG1 bacteria containing phage, and evenly spread it on a 2×YT agar plate containing resistance; place in a 37°C incubator and culture for about 12-16 hours; count the plate clones; if the number of clones grown in the Nth concentration gradient coating is M, the phage titer is: M / (10 N-5 ) PFU / mL. The degree of enrichment is reflected by the enrichment factor, which is calculated as follows: enrichment factor = output ratio in the Nth round / output ratio in the first round.

[0126] As shown in Table 1, after three rounds of cell-penetrating screening, the enrichment factor was 2.02, indicating that the screening system can effectively enrich the phage library.

[0127] Table 1. Preliminary evaluation of the enrichment of the phage library by phage titer determination

[0128]

[0129] * Output ratio (%) = output phage amount / input phage amount x 100%.

[0130] ** Enrichment factor = output ratio in the Nth round / output ratio in the first round.

[0131] Example 3 Next Generation Sequencing

[0132] 1. Data quality control

[0133] In order to avoid the interference caused by the large error rate at the end of the second-generation sequencing read length, this embodiment adopts the method of bidirectional sequencing and sequence splicing to determine the complete sequence. In order to verify the reliability of the sequencing data, this embodiment uses FastQC software to evaluate the sequencing data from the perspectives of per base sequencing quality, per tiles sequence quality, per sequence quality scores, per base sequence content at each position, per sequence GC content, per base N content, sequence length distribution, and sequence duplication levels. Then, Cutadapt software is used to eliminate the read length sequences with the following situations: the average quality score is too low; the sequence is too short; it contains a large number of uncertain bases; the GC content is seriously biased. At the same time, the starting segment and end of the sequence and the sequencing ligand sequence region are shielded. In order to eliminate the problems that may introduce mutations during library construction and sequencing, this embodiment repeats all the above-mentioned library construction and second-generation sequencing processes for a second time, and performs correlation analysis on the sequencing results of different batches.

[0134] like Figure 3 As shown, the correlation analysis results of the two repeated sequencing results of libraries G0, G1, G2, G3 and G4 showed that the correlation coefficients were all 1.00, indicating that there was no batch difference in the library construction and sequencing process, and the repeatability was excellent.

[0135] 2. Enrichment analysis

[0136] In this example, transmembrane proteins before and after phage display were enriched according to the following criteria: 1) Transmembrane proteins with significant enrichment were defined as those with an adjusted p value less than 0.01 (adjusted p value<0.01) and an absolute value of the logarithmic enrichment frequency fold change greater than 1 (log2 fold change>1 or log2 fold change<-1); 3) The frequency of occurrence of CPP candidate proteins in each round was higher than that in the previous round (sequence frequency of round 3>sequence frequency of round 2>sequence frequency of round 1>sequence frequency of round 0).

[0137] like Figure 4 As shown in the volcano plot, there are 84 red dots showing the enriched sequences in the R3 library relative to the R0 library. R0 represents the initial library; R3 represents the library after three rounds of phage screening.

[0138] Example 4 Soluble purification of prokaryotic expressed protein

[0139] In order to evaluate the solubility of the backbone protein CPP and facilitate the detection of the cell penetration ability of the backbone protein CPP, this example adds a GFP tag to the C-terminus of CPP and performs soluble purification instead of denaturation purification. In order to facilitate protein purification, this example introduces a 6×His tag to the C-terminus of CPP-GFP. Among them, the nucleotide sequences encoding each CPP protein are shown in the following table.

[0140]

[0141]

[0142] The following are the steps for soluble purification of prokaryotic expressed proteins: transform the prokaryotic expression plasmid pET28a(+) expressing the target protein into the Escherichia coli Rosetta (DE3) strain; evenly spread the transformed Escherichia coli on a bacterial plate containing 32 mg / L chloramphenicol and 30 mg / L kanamycin, and culture it in a 37°C incubator for 12-16 hours; pick a single clone from the bacterial plate, place it in 8 mL of TB culture medium containing 32 mg / L chloramphenicol and 30 mg / L kanamycin, and culture it at 37°C, 150 rpm for 12 hours; transfer 8 mL of the bacterial solution to 800 mL of TB culture medium containing 32 mg / L chloramphenicol and 30 mg / L kanamycin for expansion culture, and culture it at 37°C, 120 rpm until the OD value of the bacterial solution is between 0.6 and 0.8; adjust the temperature of the bacterial oscillator to 18°C, pre-cool and culture for 1 hour; add 1 mM IPTG, 18°C, 120rpm shaking culture for 12-16 hours to induce protein expression; collect bacterial solution and centrifuge, 4°C, 4000rpm, 10 minutes; discard the supernatant, collect bacterial blocks, and fully resuspend with washing buffer to make the resuspended volume greater than 20mL; use a low-temperature pressure crusher to crush the resuspended bacterial solution, the pressure value is 600-700, and the crushing is 30s; the crushed bacterial solution is centrifuged, 4°C, 18,000rcf, 1 hour; take the supernatant after centrifugation; add the supernatant to the nickel ion affinity chromatography column and collect the column fluid; use the column fluid as the supernatant, repeat 3-5 times; add 10 times the column volume of washing buffer containing 30mM imidazole to the column to remove impurity proteins, repeat 3-5 times; add elution buffer containing 500mM imidazole to collect the target protein; pass through a concentrated ultrafiltration tube (Merck The eluted protein was further concentrated to 1 mL by Millipore (UFC9010); the protein sample was subjected to high-speed centrifugation at 4°C, 20,000 rcf, for 10 minutes to further remove potential precipitation; a Superdex 75increase 10 / 300GL column (Cytiva) was selected through a liquid chromatography system to further separate and purify the protein; the protein at the peak position was collected for SDS-PAGE analysis; the gel was stained with Coomassie Brilliant Blue and decolorized; the protein with the expected molecular weight consistent with the target protein was collected; the protein was concentrated and ultrafiltered again, and centrifuged to the ideal volume under the conditions of 4°C, 3800 rcf; the protein concentration was determined using a BCA kit (ThermoFisher, 23225); the sample was quickly frozen in liquid nitrogen and aliquoted and stored at -80°C for later use.

[0143] like Figure 5As shown in the figure, CPP7, CPP11, CPP55, CPP109 and CPP112 can be expressed and purified in a soluble manner after being fused with GFP, and the molecular sieve shows that the peak position is slightly earlier than that of GFP, and the SDS-PAGE band is also slightly larger than that of GFP, which is consistent with the fact that the CPP candidate protein consists of only 49 amino acids, indicating that the above CPP-GFP fusion protein can be fully expressed. Considering that good solubility and protein stability are the basic prerequisites for transmembrane skeleton proteins, other candidate proteins were eliminated due to low yield of soluble expression or inability to express soluble expression.

[0144] in,

[0145] The amino acid sequence of CPP7 is shown in SEQ ID NO. 1: DQDKVLIKIIAKYLKVDEEQVKEYLSRQNLSPLELVRRILLNQRVAVDP

[0146] The amino acid sequence of CPP11 is shown in SEQ ID NO. 2: LKEVLKIVQHLIALGVTPSQIKKILLSLGIDPREVDTAVKLVGRRRGDP;

[0147] The amino acid sequence of CPP55 is shown in SEQ ID NO. 3: DEEIEIIIEKLLAAGYNEEQITKILRKNGVSDDQLKKVQKIVRRRRGGS;

[0148] The amino acid sequence of CPP109 is shown in SEQ ID NO. 4: SEEAKKILTQIQQIDPRVVEEIIILRQMGISDDQIVTLLVKAGVVAVDP;

[0149] The amino acid sequence of CPP112 is shown in SEQ ID NO.5:

[0150] DEEIEKIIVDLLLAGYNEEQITKILRKNGVSDDQLKKVQKIVRRRRGGS.

[0151] Example 5 Identification of cell membrane penetration ability

[0152] 1. Detection of GFP cell uptake rate by flow cytometry

[0153] Cells were seeded into 96-well plates and cultured overnight. Purified CPP-GFP protein was diluted in culture medium. Cells were incubated with CPP-GFP protein at 37°C for approximately 4 hours. After incubation, cells were washed twice with culture medium and then digested with 0.25% trypsin-EDTA at 37°C for 10 minutes. The cellular uptake level of CPP-GFP protein was calculated by median fluorescence intensity and normalized to GFP or blank control.

[0154] like Figure 6 As shown, compared with ZF5.3, the cellular uptake efficiency of GFP by CPP11 was increased by four times, that of CPP55 and CPP112 was increased by three times, and that of CPP7 and CPP109 was increased by 2.5 times, and the differences were statistically significant.

[0155] 2. Observation of GFP distribution in living cells using an inverted fluorescence microscope

[0156] Cells were seeded into 24-well plates and cultured overnight. Purified CPP-GFP protein or FITC-labeled peptide was diluted in culture medium. Cells were then incubated with CPP-GFP protein for 4 hours at 37°C. To reduce the fluorescence of CPP-GFP or GFP that may be non-specifically bound to the cell surface, cells were rinsed with 5 mg / ml trypan blue before washing the culture medium twice. Fluorescence signals within cells were observed using a fluorescence microscope to evaluate the cellular uptake of CPP-GFP.

[0157] like Figure 7 As shown in the figure, there are obvious green fluorescence signals inside the cells of the CPP11, CPP55 and CPP112 treatment groups, while there are weaker green fluorescence signals in the CPP7-GFP and CPP109-GFP treatment groups, which is consistent with the results of flow cytometry. In addition, no green fluorescence signals were observed on the cell surface and in the intercellular space, indicating that the washing process after incubation is sufficient to remove nonspecific adhesion proteins on the surface.

[0158] Example 6 Identification of Enriched Protein Structure

[0159] 1. AlphaFold2 structure prediction method

[0160] In order to accurately evaluate the three-dimensional structure of artificially designed proteins, this example selected AlphaFold2, the protein structure model currently recognized to have the highest accuracy, for structure prediction. The AlphaFold2 prediction tool used is provided by Colab, and the specific website is as follows: https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynbThe predictions were made using the default model parameters. The confidence of the predicted structure was evaluated by the pLDDT index with a value range of 0 to 100, with pLDDT>90 as the cutoff point for high accuracy and a lower critical value of pLDDT>70 corresponding to the cutoff point for generally correct skeleton prediction.

[0161] like Figure 8 As shown, the AlphaFold2 predicted structure suggests that the discovered transmembrane protein ZF5.3 only contains a single α-helix, and the positive charge is concentrated on one side of the α-helix; while CPP11, CPP7, CPP55, CPP109, and CPP112 are all composed of three α-helices, and each helix surface has different degrees of positively charged amino acids distributed.

[0162] 2. Optimization and transformation of CPP11

[0163] Based on the two major factors affecting transmembrane penetration, α-helix and positive charge distribution, this example further optimizes the screened transmembrane protein CPP11 to improve the transmembrane effect of CPP11. First, the structure of CPP11 was predicted using AlphaFold2, and the amino acids of the three-stranded α-helical structural segments of CPP11 were determined to be 1-14, 18-28, and 33-46, respectively. Then, the Helical Wheel diagram of the predicted three-stranded α-helical structural segments was drawn using the HeliQuset tool, further clarifying the positive charge distribution of the three-stranded helix of CPP11 on the hydrophilic surface. In order to improve the positive charge level of the single-stranded α-helix of CPP11, Helix3, which exhibits the highest number of positive charges, was selected for modification, and the aspartic acid (D) at the 36th amino acid was mutated to arginine (R). It was named CPP11D36R, and its amino acid sequence is shown in SEQ ID NO.6: LKEVLKIVQHLIALGVTPSQIKKILLSLGIDPREVRTAVKLVGRRRGDP.

[0164] like Fig. 9As shown, CPP11 exhibits a classic amphipathic structure in Helix1, Helix2 and Helix3, which contains both a hydrophilic surface composed of polar amino acids and a hydrophobic surface composed of hydrophobic amino acids. Among them, the positive charge numbers displayed on the hydrophilic surfaces of Helix1, Helix2 and Helix3 are 2, 2, and 3 respectively; the net charge numbers displayed on the hydrophilic surfaces of Helix1, Helix2 and Helix3 are 1, 2, and 1 respectively. The total number of positive charges displayed by CPP11 in all helical segments is 7, the total net charge number is 4, and the average number of positive charges displayed per amino acid is 0.143. In contrast, the helical segment of ZF5.3 displays a positive charge number of 4, a net charge number of 4, and an average number of positive charges displayed per amino acid is 0.148. This indicates that not only is the overall positive charge level of CPP11 lower than that of ZF5.3, but the positive charge level of the single α-helix is ​​also lower than that of ZF5.3, which may explain why CPP11 has a lower membrane penetration effect than ZF5.3. Compared with CPP11, the number of positive charges displayed on the hydrophilic surface of Helix3 of the mutated CPP11D36R increased from 3 to 4, and the net charge displayed on the hydrophilic surface of Helix3 increased from 1 to 3, and the backbone structure of the three-stranded α-helix was retained before and after the mutation.

[0165] 3. Determination of protein secondary structure composition by circular dichroism

[0166] In order to verify whether CPP11 can maintain its original α-helical structure after modification, ChiRAScan (Applied Photophysics, Ltd.) was used to collect circular dichroism spectra of CPP11 and CPP11D36R. First, the buffer was scanned alone, and then the protein dissolved in the buffer was scanned; 3 repeated scans were collected; after the sample scan, the cuvette was washed with water and buffer, inverted and dried, and the next sample to be tested was added; a cuvette with a path length of 0.5 mm was used, and the range of 190-250 nm was recorded, with a sensitivity of 100 mdeg, a resolution of 0.2 nm, a response time of 8 s, a bandwidth of 1.0 nm, and a scanning speed of 10 nm / min; in the cuvette, the protein concentration remained unchanged in all measurements, and the total volume in the cuvette was 200 μL. The sample signal value was subtracted from the buffer control group and the average was calculated, and the spectral signal was converted into molar ellipticity [θ] by CDtoolX software. MR .

[0167] like Fig.10As shown, the circular dichroism (CD) spectrum confirmed the presence of helical structures in CPP11 and CPP11D36R, which was manifested by a clear negative peak at a wavelength of about 222nm and a positive minimum optical activity signal at a wavelength of about 190nm. Compared with CPP11, the helical ratio of CPP11D36R did not decrease. As a control, ZF5.3, because it is composed of a segment of α helix and a segment of β fold, its CD spectrum is different from that of CPP11 / CPP1136R, which has α helix as the main structural unit.

[0168] Example 7 CPP11D36R can efficiently carry GFP and FITC into cells

[0169] The specific operation process is the same as that of Example 5 (identification of cell transmembrane ability), except that the cell treatment protein is replaced with CPP11D36R-GFP or FITC-CPP11D36R.

[0170] As shown in the figure, through the cell membrane penetration experiment, the cells carrying FITC ( Fig.11 ) and carrying GFP( Fig.12 ) and verified that its membrane-penetrating ability was significantly better than that of CPP11. More interestingly, CPP11D36R was also significantly better than the reported ZF5.3 protein. This shows that a highly efficient new membrane-penetrating protein was successfully obtained from a backbone protein based on a triple α-helix structure.

[0171] Example 8 KLA carrying intracellular function experiment

[0172] 1. Cell Viability Assay

[0173] In order to exclude the possibility that the transmembrane protein CPP11D36R itself has cytotoxicity, this example treats cells with a series of different concentrations of CPP11 or CPP11D36R at 37°C for 48 hours, followed by cell activity detection. On the basis of clarifying that CPP11D36R itself is not cytotoxic, in order to further clarify whether the transmembrane protein CPP11D36R can play the role of intracellular protein delivery and maintain the function of the delivered protein, this example constructs the 11D36R-KLA-GFP fusion protein to explore whether it can induce cytotoxicity. Cell activity detection is completed by CCK8 reagent (Selleck, B34304), and the specific steps are as follows: cells are seeded in 96-well plates and cultured overnight to facilitate cell adhesion. After cell attachment, the cells are treated with different proteins and cultured for another 24 hours. After treatment, 10 μL of CCK8 reagent is added to each well and further cultured for about 1 hour. Optical density (OD) at 450 nm was measured using a microplate reader, and cell viability was calculated based on OD450 minus OD630. The amino acid sequence of KLA apoptotic peptide is shown in SEQ ID NO. 7: KLAKLAKKLAKLAK.

[0174] First, by Fig.13 It can be seen that the CPP11D36R protein treatment group had no obvious cytotoxic effect at a concentration of 0-40μM, and there was no significant difference from the unmodified and CPP11 / 55 treatment groups. The uptake efficiency of 1D36R-KLA-GFP fusion protein in different cells was detected by flow cytometry. Compared with the GFP control group, 1D36R-KLA-GFP fusion protein could be significantly taken up by HeLa cells, CNE2 cells and HK1 cells, and the difference was statistically significant ( Fig.14 Next, the cell activity was measured to reflect the cell killing function of 11D36R-KLA-GFP fusion protein in different cells. The results showed that in HeLa cells, CNE2 cells and HK1 cells, 11D36R-KLA-GFP fusion protein could inhibit cell activity by about 50% ( Fig.14 (middle B).

[0175] 2. Cell clone formation experiment

[0176] Cells were seeded at a density of 1,000 cells per well in 6-well plates and cultured overnight. Then, cells were treated with KLA or CPP11D36R-KLA-GFP and cultured for 7-14 days until cell colonies formed. Subsequently, methanol was added to fix the cells for 10 minutes, followed by staining with 0.5% crystal violet solution for 30 minutes. Finally, the cells were washed with PBS and the stained cell colonies were photographed.

[0177] like Fig.14As shown in C, the results showed that CPP11D36R-KLA-GFP effectively inhibited cell growth and proliferation in a dose-dependent manner.

[0178] 3. Western Blot Analysis

[0179] Cells were seeded in 24-well plates and cultured overnight. Then, cells were treated with KLA, GFP, 11D36R-KLA-GFP, or culture medium for 24 h. Cells were collected and lysed using 1× sample buffer containing β-mercaptoethanol to extract total protein. Proteins were denatured at 95°C for 5 min. Samples were loaded on SDS-PAGE gels and electrophoresed. Subsequently, proteins were transferred to PVDF membranes, and the membranes were blocked with 5% skim milk powder for 30 min at room temperature. The membranes were then incubated with primary antibodies overnight, followed by incubation with secondary antibodies for another 30 min. Enhanced chemiluminescent substrates were used for detection, and signals were captured using the ChemiDoc MP system (BioRad).

[0180] like Fig.15 As shown in Figure 5A, mechanistically, caspase-3 cleavage products were detected in the CPP11D36R-KLA-GFP group, suggesting that the apoptosis pathway was activated.

[0181] 4. Cell apoptosis detection

[0182] Cells were seeded in 24-well plates and cultured overnight. Then, cells were treated with KLA, GFP, 11D36R-KLA-GFP or culture medium for 12 hours. After treatment, cells were collected, washed with PBS, and then resuspended in 1× binding buffer, and cells were stained with an apoptosis detection kit (Elabscience, E-CK-A213) according to the instructions, then incubated in the dark for 15 minutes, and samples were analyzed by flow cytometry. The total ratio of early apoptotic cells and late apoptotic cells was used as an indicator reflecting the degree of apoptosis.

[0183] like Fig.15 As shown in B, the results of the apoptosis experiment showed that the apoptosis rate induced by CPP11D36R-KLA-GFP was as high as 38.70%, while the apoptosis rates of the KLA and GFP groups were 12.37% and 5.31%, respectively. This further illustrates that the KLA apoptotic peptide successfully enters the cytoplasm under the mediation of the transmembrane protein CPP11D36R, thereby exerting the apoptotic function of the KLA apoptotic peptide.

Claims

1. A protein or a derivative thereof, characterized in that: The amino acid sequence of the protein is shown in any one of a1) to a3): a1) any one of SEQ ID NO.1-5; a2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 1 to 5; a3) an amino acid sequence having at least 95% homology with any one of SEQ ID NOs. 1 to 5.

2. The protein according to claim 1, characterized in that: The amino acid sequence of a2) is shown in SEQ ID NO.

6.

3. The protein according to claim 1, characterized in that: The derivative includes at least one of a pharmaceutically acceptable salt or a pharmaceutically acceptable modification.

4. A biological material related to the protein or its derivative according to any one of claims 1 to 3, including any one of b1 to b12); b1) a nucleic acid molecule encoding the protein described in any one of a1) to a3); b2) an expression cassette comprising the nucleic acid molecule described in b1); b3) a vector comprising the nucleic acid molecule described in b1); b4) a vector comprising the expression cassette described in b2); b5) a transgenic cell line comprising the nucleic acid molecule described in b1); b6) a transgenic cell line comprising the expression cassette described in b2); b7) a transgenic cell line comprising the vector described in b3); b8) a transgenic cell line comprising the vector described in b4); b9) a microorganism comprising the nucleic acid molecule described in b1); b10) a microorganism comprising the expression cassette described in b2); b11) a microorganism comprising the carrier described in b3); b12) a microorganism comprising the carrier described in b4); Preferably, the nucleic acid molecule b1) is as shown in any one of SEQ ID NOs. 8 to 13; Preferably, the vector comprises a fusion expression tag; Preferably, the microorganism comprises Escherichia coli.

5. A method for preparing the protein or its derivative according to any one of claims 1 to 3, comprising: Prepared using the biomaterial according to claim 4; Or, obtained by chemical synthesis of polypeptides.

6. A fusion protein, characterized in that: A protein or a derivative thereof comprising any one of claims 1 to 3; and a coupling moiety, the coupling moiety comprising at least one of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme; Preferably, the drug comprises an apoptotic peptide; Preferably, the amino acid sequence of the apoptotic peptide is as shown in SEQ ID NO.

7.

7. Use of the protein or derivative thereof according to any one of claims 1 to 3, the biomaterial according to claim 4, or the fusion protein according to claim 6 in at least one of c1) to c2): c1) preparing a product for drug delivery; c2) Preparation of products for treating tumors.

8. The use according to claim 7, characterized in that: The product includes at least one of a drug and a drug delivery system.

9. A drug comprising the protein or its derivative according to any one of claims 1 to 3, or the fusion protein according to claim 6; The drug includes pharmaceutically acceptable excipients.

10. A delivery system, characterized in that: The delivery system comprises at least one of the protein or derivative thereof according to any one of claims 1 to 3, the fusion protein according to claim 6, and the drug according to claim 9; and delivery vehicles; Preferably, the delivery vehicle comprises at least one of a liposome and an extracellular vesicle.

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