A cell-penetrating peptide and use thereof

Abstract

The application discloses a cell penetrating peptide and application thereof; the cell penetrating peptide is selected from SEQ ID NO. 1, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26 or SEQ ID NO. 28; the cell penetrating peptide of the application has more excellent cell penetration rate and cell permeability compared with the cell penetrating peptide H16 which is recognized in the art; and the cell penetrating peptide has no cytotoxicity; and further has no immunogenicity, and can be used for human body imaging or treatment, and expands the application range of the existing cell penetrating peptide. The fusion protein with tumor cell penetration and tumor environment specific activation is designed and constructed for GSDME, and a new idea is provided for development of an anticancer targeted drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a cell-penetrating peptide and its applications. Background Technology

[0002] The cell membrane is an effective barrier preventing the uptake of most molecules by cells through inactive transport, thus hindering the delivery of therapeutic substances. Except for molecules with specific molecular weights, polarities, and / or electrostatic charges that can (passively) diffuse across the cell membrane, other molecules must be actively transported via receptor-mediated transport or by ATP-bound transport molecules. Furthermore, methods such as electroporation, cationic lipids / liposomes, microinjection, viral delivery, or polymer encapsulation can artificially force molecules across the cell membrane. However, these methods are limited to in vitro use and cannot be used as tools for delivering drugs directly to cells to prevent or treat medical diseases.

[0003] Cell-penetratinig peptides (CPPs), also known as membrane ectopic sequences, are used to overcome the impermeability of the plasma membrane, cross the cell membrane, and enter the cell interior. CPPs are highly useful mediators for transporting "cargo" (such as proteins, oligonucleotides, peptides, nucleic acids, and other pharmacologically active compounds with very limited activity periods) into cells to achieve desired purposes. Furthermore, CPPs have the advantage of being able to transfect substances into cells in recombinant or mixed forms. Therefore, CPPs provide a viable method for molecules that cannot pass through the cell membrane.

[0004] Many CPPs have serious side effects on the cells to which they are applied because, in fact, most of the proteins derived from most CPPs can act as antimicrobial agents or toxins. For example, CPPs can cause cytoplasmic leakage due to membrane rupture and can also interfere with the normal function of membrane proteins. CPPs can also exhibit cytotoxic effects; many CPPs function only under certain very specific conditions that cannot be met in the in vivo environment. Another drawback is that CPPs can degrade rapidly in cells. Finally, because many known CPPs are derived from non-human proteins, toxic and / or immunogenic effects are often observed, which can interfere with the utilization of these peptides, for example, in therapeutic applications in humans.

[0005] Therefore, it is crucial to ensure that CPP possesses superior cell penetration rate and permeability compared to recognized cell-penetrating peptides in the art, its suitability as a fusion protein, and its proprietary technological advantage of lacking immunogenic effects. This invention screens four peptides with cell-penetrating functions from the gasdermin family of proteins that possess cell-penetrating capabilities in the human body. Among these, the cell-penetrating functional segment of gasdermin C is optimized and truncated to obtain a short peptide GCP (gasderminC cell-penetratinig peptide, GCP) of only 30 amino acids. It exhibits superior cell penetration rate and permeability compared to the recognized cell-penetrating peptide H16; it can be fused with green fluorescent protein (EGFP) for expression and internalize EGFP, which cannot independently penetrate the cell membrane; it is also non-cytotoxic; most importantly, this CPP is derived from the human body's own protein, lacks immunogenicity, and can be used for human imaging or therapy. Summary of the Invention

[0006] The first aspect of the present invention is to provide a cell-penetrating peptide.

[0007] A second aspect of the present invention is to provide a composite.

[0008] The third aspect of this invention relates to biomaterials related to the aforementioned penetrating peptides or complexes.

[0009] The fourth aspect of this invention aims to provide a method for preparing the above-mentioned complex.

[0010] The fifth aspect of this invention aims to provide the application of the above-mentioned penetrating peptide or complex.

[0011] The sixth aspect of this invention aims to provide a product.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the present invention provides a cell-penetrating peptide GCP, the amino acid sequence of which is:

[0014] a) As shown in any one of SEQ ID NO.1, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, or SEQ ID NO.28; or

[0015] b) The amino acid sequence shown in a) is modified by substitution, deletion or addition of one or more amino acids, and has the same or similar function.

[0016] The cell-penetrating peptide is a peptide that mediates the delivery of bioactive molecules into cells.

[0017] Preferably, the amino acid sequence of the cell-penetrating peptide GCP is shown in SEQ ID NO.1.

[0018] A second aspect of the invention provides a complex comprising: the cell-penetrating peptide described in the first aspect of the invention; and a cargo molecule.

[0019] Preferably, the cargo molecule is fused to the N-terminus or C-terminus of the cell-penetrating peptide.

[0020] Preferably, the cargo molecule is selected from at least one of molecules with pharmaceutical activity, molecules with labeling function, and molecules with targeting function.

[0021] Preferably, the drug-active molecule includes at least one of a polypeptide, protein, nucleic acid, or compound.

[0022] Preferably, the labeling molecule includes at least one of a tag protein, a radionuclide, or a radioactive reagent.

[0023] Preferably, the targeting molecules are, for example, peptides or antibodies that specifically bind to CD34, CD56, CD3, epidermal growth factor receptor HER, and vascular endothelial growth factor receptor VEGFR.

[0024] Preferably, the tagged protein includes SUMO, His6, MBP, Flag, HA, fluorescent reagent, etc.

[0025] Preferably, the fluorescent reagent includes EGFP, ECFP, EYFP, mCherry, fluorescent dyes, CY3, CY5, etc.

[0026] Preferably, the fluorescent protein is green fluorescent protein (EGFP), and its amino acid sequence is as follows:

[0027] a)MSRVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK(SEQ ID NO.2); or

[0028] b) An amino acid sequence with the same or similar function after one or more amino acids have been substituted, deleted or added to the amino acid sequence shown in SEQ ID NO.2.

[0029] Preferably, the recombinant drug delivery system is GCP-EGFP, and its amino acid sequence is as follows:

[0030] a) Any one of SEQ ID NO.3, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, or SEQ ID NO.27;

[0031] b) The amino acid sequence shown in a) is modified by substitution, deletion or addition of one or more amino acids, and has the same or similar function.

[0032] Preferably, the drug-active molecule includes GSDME, and the recombinant drug delivery system 2 is TRAP2, whose amino acid sequence is:

[0033] a) (SEQ ID NO.4) (Where the single underline represents the SUMO tag sequence, with emphasis on the amino acid sequence of the penetrant peptide GCP; the double underline represents the amino acid sequence of GSDME); or

[0034] b) The cysteine ​​sequence shown in SEQ ID NO.4 mutated to alanine or other amino acids, with the same or similar function; or

[0035] c) The dmpdaah amino acid sequence recognized by caspase 3 (as shown in SEQ ID NO. 4) is mutated to an amino acid sequence recognized by matrix metalloproteinases (MMPs); or

[0036] d) The solubilizing protein SUMO shown in SEQ ID NO.4 is mutated into other non-human homologous proteins and / or other hydrophilic proteins; or

[0037] e) Adjustment of the positional order of the protein elements in the fusion protein shown in SEQ ID NO.4.

[0038] A third aspect of the present invention provides a protein-related biological material, said related biological material being any one of the following (B1) to (B8):

[0039] (B1) A nucleic acid molecule encoding the penetrating peptide described in the first aspect of the present invention or the complex described in the second aspect of the present invention;

[0040] (B2) An expression cassette containing the nucleic acid molecule described in (B1);

[0041] (B3) A recombinant vector containing the nucleic acid molecule described in (B1);

[0042] (B4) A recombinant vector containing the expression cassette described in (B2);

[0043] (B5) Recombinant microorganisms containing the nucleic acid molecules described in (B1);

[0044] (B6) Recombinant microorganisms containing the expression cassette described in (B2);

[0045] (B7) Recombinant microorganisms containing the recombinant vector described in (B3);

[0046] (B8) Recombinant microorganisms containing the recombinant vector described in (B4).

[0047] Preferably, the microorganisms include prokaryotic cells and eukaryotic cells.

[0048] A fourth aspect of the present invention provides a method for preparing the penetrating peptide or the complex described in the first aspect of the present invention, wherein the host cells described in the third aspect of the present invention are cultured to obtain the penetrating peptide or the complex.

[0049] A fifth aspect of the present invention provides the use of the penetrating peptide described in the first aspect of the present invention, or the complex described in the second aspect of the present invention, or the related biomaterial described in the third aspect of the present invention, in the preparation of products.

[0050] Preferably, the product has at least one of the functions of (1) to (5):

[0051] (1) Tumor targeting;

[0052] (2) Induces pyroptosis;

[0053] (3) Drug delivery;

[0054] (4) In vivo tumor imaging;

[0055] (5) Prevention and treatment of tumors.

[0056] Preferably, the tumor is at least one of colorectal cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, breast cancer, prostate cancer, gastrointestinal cancer, peritoneal cancer, melanoma, endometrial cancer, ovarian cancer, cervical cancer, uterine cancer, bladder cancer, glioblastoma, brain metastases, salivary gland cancer, thyroid cancer, brain cancer, lymphoma, myeloma, and head and neck cancer.

[0057] A sixth aspect of the present invention provides a product comprising the penetrating peptide of the first aspect of the present invention, the complex of the second aspect of the present invention, or the bio-related material of the third aspect of the present invention.

[0058] Preferably, the product is a drug.

[0059] Preferably, the drug may also contain pharmaceutically acceptable excipients.

[0060] The beneficial effects of this invention are:

[0061] This invention provides a cell-penetrating peptide discovered in human proteins. This peptide is screened from gasdermin family proteins, which possess cell-penetrating capabilities. Addressing the issue of immunogenicity associated with previous cell-penetrating peptides used in humans, this invention seeks a human-derived protein polypeptide with cell-penetrating properties. Specifically, a short peptide GCP (gasderminC cell-penetratinig peptide, GCP) of only 30 amino acids was obtained by optimizing and truncating the membrane-penetrating functional segment of gasderminC. Compared to the widely accepted cell-penetrating peptide H16, it exhibits superior cell penetration rate and permeability; it can be fused with green fluorescent protein (EGFP) for expression and internalize EGFP, which cannot independently penetrate the cell membrane; and it is non-cytotoxic. Most importantly, this CCP is derived from a human-derived protein and is immunogenic, making it suitable for human imaging or treatment. This expands the applicability of existing cell-penetrating peptides, enabling efficient drug delivery to cells without significant cytotoxicity and / or immunogenicity, providing a new approach to drug delivery and holding significant importance in the discovery and treatment of diseases such as tumors.

[0062] This invention also discovered that the cell-penetrating peptide GCP can be fused with the lysin-like protein element SUMO and the pyroptosis element GSDME. This invention is the first to design and construct a fusion protein with tumor cell penetration and tumor environment-specific activation targeting the anti-tumor immune protein GSDME, providing a new approach for the development of anti-cancer targeted drugs. Compared with the technology of finding and using small molecules to activate GSDME, the fusion protein provided by this invention is simple to synthesize, can be expressed in prokaryotes or eukaryotes, making production convenient, highly efficient, and easier to mass-produce and commercialize. Attached Figure Description

[0063] Figure 1 This is a graph showing the protein purification results after the selected transmembrane peptide was fused with EGFP.

[0064] Figure 2 This is a graph showing the cell penetration rate of cell-penetrating peptides from the gasdermin protein family screened by computer: [Image showing cell penetration rate of these peptides] Figure 2 The figure shows the fluorescence microscopy results of A-EGFP, E-EGFP, C1-EGFP, C2-EGFP and H16-EGFP molecules compared with H16-EGFP molecules in pancreatic cancer cells SW1990. Figure 2 Figure B shows the flow cytometry results of the fluorescence intensity of A-EGFP, E-EGFP, C1-EGFP, C2-EGFP and H16-EGFP molecules in pancreatic cancer cells SW1990. Figure 2 The figure shows the fluorescence microscopy results of the A-EGFP, E-EGFP, C1-EGFP, C2-EGFP and H16-EGFP molecules compared with H16-EGFP molecules in nasopharyngeal carcinoma SUNE2. Figure 2 Figure D shows the flow cytometry results of the fluorescence intensity of A-EGFP, E-EGFP, C1-EGFP, C2-EGFP and H16-EGFP molecules in SUNE2 nasopharyngeal carcinoma cells.

[0065] Figure 3 It is the optimized sequence of the C2 penetrant peptide.

[0066] Figure 4 This is a diagram showing the purification results of EGFP protein after the C2 penetrant peptide was truncated and linked.

[0067] Figure 5 This is a flow cytometry result of the C2 penetrant truncated and linked to EGFP protein: [Image of the flow cytometry results]. Figure 5 Figure A shows the fluorescence intensity of the molecule formed by fusing C2 truncated peptide with EGFP protein in non-small cell lung cancer H358 cells by flow cytometry. Figure 5Figure B shows the fluorescence intensity of the molecule formed by fusing the C2 truncated peptide with EGFP protein in RKO colorectal cancer cells by flow cytometry. Figure 5 The graph shows the fluorescence intensity of the molecule formed by fusing the C2 truncated peptide with EGFP protein in normal human renal epithelial HEK-293T cells by flow cytometry.

[0068] Figure 6 This is a graph showing the cytotoxicity detection results of GCP-penetrating peptide-linked EGFP protein in HepG2 liver cancer cells and HCT116 colorectal cancer cells.

[0069] Figure 7 This is an in vivo imaging result of GCP-EGFP protein injected subcutaneously into a mouse tumor.

[0070] Figure 8 This is a schematic diagram of the construction of the TRAP2 system and a schematic diagram of the process of inducing pyroptosis.

[0071] Figure 9 This is a graph showing the purification results of GCP-GSDME MMP11 CA-SUMO protein. The bands within the boxes are the target protein bands.

[0072] Figure 10 This is an image showing the effect of GCP-GSDME MMP11 CA-SUMO inducing pyroptosis in HepG2 and HCT116 cells. The white arrows point to the pyroptotic cells. Detailed Implementation

[0073] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0074] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0075] Example 1: Verification of GCP-EGFP membrane penetration effect

[0076] This example demonstrates the feasibility and advantages of the present invention by constructing a recombinant cell-penetrating peptide-green fluorescent protein (EGFP) system for cell membrane penetration and drug delivery. EGFP protein is a fluorescent protein that cannot actively penetrate the cell membrane; therefore, the effect of the cell-penetrating peptide can be evaluated by observing the quantity and intensity of intracellular fluorescence. However, cell-penetrating peptides generally exhibit cytotoxicity and immunogenicity, and no such peptide has yet been approved for clinical use.

[0077] I. CPP Screening and Optimization

[0078] 1. Screening of human gasdermin family protein transmembrane sequences

[0079] In the early stages of this embodiment, computer simulation software was used to calculate four polypeptides derived from gasdermin family proteins that may have cell membrane penetration function, based on algorithms for sequence length, net charge, average hydrophobicity, and other physicochemical properties of membrane-penetrating peptides. The sequences are shown in Table 1.

[0080]

[0081] 2. Sequence optimization of SEQ ID NO.14

[0082] The amino acid sequence of SEQ ID NO.14 was truncated four times, starting from the N-terminus, by shortening it by 3 amino acids at a time; the C-terminus was also truncated four times, starting from the N-terminus, by shortening it by 3 amino acids at a time, resulting in a total of 8 truncated sequences, such as... Figure 3 As shown.

[0083] II. Preparation of the fusion protein CPP-EGFP

[0084] 1. Construction of CPP-EGFP expression vector

[0085] DNA sequences containing the screened and optimized CPP and EGFP molecular elements were synthesized and cloned into the prokaryotic expression vector pET28a (plasmid synthesized and constructed by Jiangsu Saisofe Biotechnology Co., Ltd.). The synthesized pET28a-CPP-EGFP plasmid was transformed into E. coli BL (Rosetta) competent cells. Positive recombinants were selected using kanamycin-resistant plates, and their verification was performed by colony PCR and DNA sequencing. Sequencing results were analyzed by NCBI BLAST alignment. The plasmid was stored at -20℃ for long-term storage, while correctly sequenced strains were stored at -80℃ with 25% glycerol. The pET28a vector carries a 6*His tag and can be used for the expression and purification of His proteins.

[0086] 2. Expression and purification of CPP-EGFP system

[0087] (1) CPP-EGFP was induced to express using isopropyl thiogalactoside (IPTG), and different IPTG concentrations, temperatures, and times were used to explore the optimal expression conditions. SDS-PAGE and Coomassie Brilliant Blue assays were then used to determine the optimal expression conditions.

[0088] In this embodiment, the correctly sequenced CPP-EGFP was inoculated into TB medium containing kanamycin (50 ng / μL) and cultured overnight at 37°C with shaking. The next day, the activated bacterial culture was inoculated into fresh medium at a ratio of 1:100 for expansion. When the OD600 reached 0.6 at 37°C with shaking at 150 rpm, the temperature was lowered to 18°C ​​and cultured for another hour. Then, isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and expression was induced for approximately 18 hours under the same conditions. The bacterial cells were then collected, centrifuged at 6000 rpm at 4°C for 10 min, resuspended in 1×PBS buffer, and centrifuged again. The collected bacterial cells were resuspended in 1×PBS buffer to a 10% concentration and then hyperbarically disrupted at 700 Pa at 4°C for 2 min. The cells were then centrifuged at 20000 rpm at 4°C for 1 h, the precipitate was discarded, and the supernatant was filtered through a 0.22 mm pore size filter membrane for subsequent purification.

[0089] (2) Purification of CPP-EGFP: The supernatant obtained above was resuspended in Ni-NTA agarose gel and incubated at 4℃ for 2 h (5 g of bacterial cells: 1 mL of 50% Ni-NTA agarose gel). During incubation, the Ni-NTA agarose gel changed color from blue to brown. The gel was then passed through a column, and 20 μL of the column buffer was collected for subsequent purification verification. The gel was washed three times with 1×PBS buffer containing 30 mM imidazole, twice the column volume each time. 20 μL of the final wash was collected. Elution was then performed with 1×PBS buffer containing 500 mM imidazole. If the Ni-NTA agarose gel turned blue again, the elution was considered complete. 20 μL of the eluent was retained as a sample. The purification efficiency of the fusion protein was determined by 10% SDS-PAGE. The fusion protein was concentrated using a 10KD ultrafiltration tube (Millipore, catalog number: UFC900396), centrifuged at 3900g, and ultrafiltered to 500μL at 4℃. The concentrate was then washed three times with 1×PBS, and centrifuged three times in total. The concentration of the obtained concentrated protein was determined using a BCA protein concentration assay kit (Thermo Fisher, catalog number: 23227) and stored at -80℃.

[0090] The purification effect of CPP-EGFP is as follows: Figure 1 and Figure 4 As shown, the purity of all samples reached over 90%, which can be used for subsequent experimental verification.

[0091] The amino acid sequence of the EGFP molecule is: MSRVSKGEELFTGVVPILFELDGDVNGHKFSVSGE GEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNIL GHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK(SEQ ID NO.2).

[0092] The amino acid sequence of the H16-EGFP molecule is as follows: HHHHHHHHHHHHH MSRVSKGEELFTGV VPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO. 5) (wherein, the underlined part is the amino acid sequence of the cell-penetrating peptide H16, which is recognized in the art, SEQ ID NO. 6).

[0093] The amino acid sequence of the A-EGFP molecule is as follows: FKRFHPFCLVLRKRKSTLFWGARYVRT MSRVSKG EELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO.7) (where the underlined part is the amino acid sequence of the gasderminA transmembrane peptide, SEQ ID NO.8).

[0094] The amino acid sequence of the E-EGFP molecule is as follows: KLQLLSLVTKKKRFWCWQRPKYQFLSLTL MSRVS KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO. 9) (where the underlined part is the amino acid sequence of the gasderminE transmembrane peptide, SEQ ID NO. 10).

[0095] The amino acid sequence of the C1-EGFP molecule is as follows: PVKYLLSATKLRQFVILRKKK MSRVSKGEELFT GVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO. 11) (where the underlined part is the amino acid sequence of gasderminC transmembrane peptide 1, SEQ ID NO. 12).

[0096] The amino acid sequence of the C2-EGFP molecule is as follows: LRVKKKALTLQKGMVMAYKRKQLVIKEKAILISMSRVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO.13) (where the underlined part is the amino acid sequence of gasderminC transmembrane peptide 2, SEQ ID NO.14).

[0097] The amino acid sequence of the C2-2-EGFP (i.e., GCP-EGFP) molecule is as follows: KKKALTLQKGMVMAYKRK QLVIKEKAI LIS MSRVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO.3) (wherein, the underlined part is the amino acid sequence of the truncated peptide GCP of SEQ ID NO.14, SEQ ID NO.1).

[0098] The amino acid sequence of the C2-3-EGFP molecule is as follows: ALTLQKGMVMAYKRKQLVIKEKAILISMSRVS KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO.15) (wherein, the underlined part is the amino acid sequence of the truncated peptide 3 of SEQ ID NO.14, SEQ ID NO.16).

[0099] The amino acid sequence of the C2-4-EGFP molecule is as follows: LQKGMVMAYKRKQLVIKEKAILIS MSRVSKGE ELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO.17) (wherein, the underlined part is the amino acid sequence of the truncated peptide 14 of SEQ ID NO.4, SEQ ID NO.18).

[0100] The amino acid sequence of the C2-5-EGFP molecule is as follows: GMVMAYKRKQLVIKEKAILISMSRVSKGEELF TGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK(SEQ ID NO.19) (where the underlined part is the amino acid sequence of the truncated peptide 5 of SEQ ID NO.14, SEQ ID NO.20).

[0101] The amino acid sequence of the C2-6-EGFP molecule is as follows: LRVKKKALTLQKGMVMAYKRKQLVIKEKAI MSRVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO.21) (where the underlined part is the amino acid sequence of the truncated peptide 6 of SEQ ID NO.14, SEQ ID NO.22).

[0102] The amino acid sequence of the C2-7-EGFP molecule is as follows: LRVKKKALTLQKGMVMAYKRKQLVIKEMSR VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO.23) (where the underlined part is the amino acid sequence of the truncated peptide 7 of SEQ ID NO.14, SEQ ID NO.24).

[0103] The amino acid sequence of the C2-8-EGFP molecule is as follows: LRVKKKALTLQKGMVMAYKRKQLV MSRVSK GEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO.25) (where the underlined part is the amino acid sequence of the truncated peptide 8 of SEQ ID NO.14, SEQ ID NO.26).

[0104] The amino acid sequence of the C2-9-EGFP molecule is as follows: LRVKKKALTLQKGMVMAYKRKMSRVSKGEE LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO.27) (where the underlined part is the amino acid sequence of the truncated peptide 9 of SEQ ID NO.14, SEQ ID NO.28).

[0105] III. Verification of the membrane-penetrating function of CPP-EGFP protein

[0106] This embodiment first compares the membrane-penetrating abilities of four membrane-penetrating peptides derived from gasderminA, gasderminE, and gasderminC, selected by computer screening, with those of the H16 membrane-penetrating peptide, which is recognized in the art. The membrane-penetrating peptide with stronger membrane-penetrating ability than H16 is then selected and truncated for optimization. In vitro cell experiments were conducted using purified protein fused with EGFP, and the membrane-penetrating function of the peptides was detected using fluorescence microscopy and flow cytometry.

[0107] 1. Comparison of membrane penetration ability of A-EGFP, E-EGFP, C1-EGFP, C2-EGFP and H16-EGFP molecules

[0108] (1) Cell source and culture: Human pancreatic cancer cells SW1990 were obtained from the American Type Culture Collection (ATCC), catalog number CRL-2172; human nasopharyngeal cancer cells SUNE2 were constructed in the applicant's team laboratory (doi:10.5732 / cjc.011.10317). All cell lines were cultured in DMEM medium (Gbico) containing 10% fetal bovine serum (Invitrogen); the culture conditions were 5% CO2, 37℃.

[0109] (2) SW1990 and SUNE2 were seeded in 96-well plates, with 10,000 cells per well; each was divided into 7 groups. In the experimental group, 10 μM of EGFP, A-EGFP, E-EGFP, C1-EGFP, C2-EGFP and H16-EGFP molecules were added to the culture wells, and in the control group, PBS was used instead of the above molecules for the same treatment; after mixing, the cells were cultured at 37°C for 4 hours.

[0110] (3) Wash cells twice with PBS, add 50 μL of PBS to each well, and place in an Incucyte to photograph live cells under white light and green fluorescence. Results are as follows: Figure 2 As shown in A and 2C, the C2-EGFP molecule contains more green fluorescence in the two cell types than the H16-EGFP molecule.

[0111] (4) Remove PBS, add 50 μL of 0.25% trypsin to each well to digest the cells and detach them from the culture dish wall; then stop digestion with 100 μL of complete culture medium per well, collect the cell suspension into a 1.5 mL EP tube, centrifuge at 200 g speed at room temperature for 3 minutes, collect the cells and resuspend them with 1 mL of PBS, and repeat the centrifugation once.

[0112] (5) After removing the supernatant, the cells were resuspended in 500 μL PBS, and the green fluorescence intensity was detected by flow cytometry. Results are as follows: Figure 2 As shown in B and 2D, the green fluorescence intensity of the C2-EGFP molecule is stronger than that of the H16-EGFP molecule in both cell types, indicating that the C2 penetrating peptide has a stronger cell penetration ability than H16.

[0113] 2. Optimization of C2 penetrating peptide truncation

[0114] (1) According to Figure 3 The C2 penetrating peptide was truncated and fused with EGFP protein for purification. The purification results are as follows: Figure 4 As shown, the purity of all samples reached over 90%, which can be used for subsequent experimental verification.

[0115] (2) Cell source and culture: Human non-small cell lung cancer cells H358, human colon cancer cells RKO, and normal human renal epithelial cells HEK-293T were obtained from the American Type Culture Collection (ATCC) (catalog numbers: CRL-5807; CRL-2577; CRL-3216). H358 and RKO cells were cultured in 1640 medium; HEK-293T cells were cultured in DMEM medium; all cells were supplemented with 10% fetal bovine serum (Invitrogen); the culture conditions were 5% CO2, 37°C.

[0116] (3) The cells were seeded into 96-well plates, with 10,000 cells per well; each well was divided into 11 groups. In the experimental group, 10 μM of C2-EGFP, C2-2-EGFP, C2-3-EGFP, C2-4-EGFP, C2-5-EGFP, C2-6-EGFP, C2-7-EGFP, C2-8-EGFP, C2-9-EGFP and H16-EGFP molecules were added to the culture wells, respectively. In the control group, EGFP was used instead of the above molecules for the same treatment. The cells were mixed and cultured at 37°C for 4 hours.

[0117] (4) Wash the cells twice with PBS, remove the PBS, add 50 μL of 0.25% trypsin to each well to digest the cells and detach them from the culture dish wall; then stop the digestion with 100 μL of complete culture medium per well, collect the cell suspension into a 1.5 mL EP tube, centrifuge at 200 g speed at room temperature for 3 minutes, collect the cells and resuspend them with 1 mL of PBS, and repeat the centrifugation once.

[0118] (5) After removing the supernatant, the cells were resuspended in 500 μL PBS, and the green fluorescence intensity was detected by flow cytometry. Results are as follows: Figure 5 As shown, the green fluorescence intensity of the C2-2-EGFP molecule was stronger than that of H16-EGFP and C2-EGFP molecules in the three cell types, indicating that the C2-2 penetrating peptide has a stronger cell penetration ability than H16 and C2. The C2-2 peptide with the strongest cell penetration ability was named GCP penetrating peptide and used for subsequent experimental verification.

[0119] IV. Cytotoxicity Verification of the Fusion Protein GCP-EGFP

[0120] Many CPPs have serious side effects on the cells to which they are applied because, in fact, most proteins derived from most CPPs can act as antimicrobial substances or toxins. Therefore, the CCK-8 assay demonstrated that GCP-EGFP is not cytotoxic.

[0121] 1. Logarithmic growth phase colorectal cancer cells HCT116 and human hepatocellular carcinoma cells HepG2 (derived from ATCC, catalog number HB-8065) ​​were collected, digested with 0.25% trypsin, counted, and seeded into 96-well culture plates. Each cell type was divided into 7 groups, with 4 accessory wells per group. 100 μL of cell suspension was added to each well, with a concentration of 1×10⁻⁶. 4 Cells / ml. At the same time, add 100μL of phosphate-buffered saline (PBS) around the perimeter of the 96-well cell culture plate to prevent the cell culture medium from evaporating. Incubate at 37°C in a 5% CO2 incubator for 12 hours to allow the cells to adhere to the plate.

[0122] 2. After rinsing three times with PBS, replace the medium with different concentrations of GCP-EGFP (0μM, 1μM, 10μM, 20μM, 40μM, 80μM, 160μM) and continue culturing for 24 hours (0μM concentration is replaced with an equal volume of PBS). Discard the culture medium, wash three times with PBS, and add 100μL of CCK-8 dilution buffer (CCK-8: medium = 1:10). Incubate at 37℃ with 5% CO2 for 2 hours, and then use an enzyme-linked immunosorbent assay (ELISA) reader to measure the corresponding OD value at a wavelength of 450nm.

[0123] 3. Use GraphPad Prism8 software to create graphs and perform statistical analysis. The results are as follows: Figure 6As shown, the CCK-8 cell proliferation assay confirmed that GCP-EGFP has an IC50 > 160 μM on cells, which can be considered as having almost no cytotoxicity.

[0124] V. Validation of in vivo delivery function of the fusion protein GCP-EGFP

[0125] 1. Two female Balb / c nude mice, aged 4-5 weeks, were used in this experiment. They were purchased from the Guangdong Provincial Experimental Animal Center and housed at the Experimental Animal Center of Sun Yat-sen University.

[0126] 2. HCT116 cells in the logarithmic growth phase were digested with trypsin. After digestion was terminated with complete culture medium, cell counting was performed. 10 million HCT116 cells were collected and washed twice with PBS. After each wash, the cells were centrifuged at 200g for 5 minutes and collected.

[0127] 3. Prepare a cell dilution buffer to resuspend the cells. The buffer composition is PBS:Matrix gel (Corning) = 1:1. Then, resuspend each HCT116 cell in 1 mL of the buffer and place them on ice for later use.

[0128] 4. Subcutaneous tumorigenesis experiment: 100 μL (500,000 cells) of HCT116 cell suspension was injected subcutaneously into the right back of mice, and imaging experiments were started after one week of feeding.

[0129] 5. GCP-EGFP was administered via orthotopic injection into the tumor, with a single injection volume of 50 μL (100 μg). In vivo imaging of mice was performed at 0 h, 0.5 h, 5 h, and 24 h post-injection to observe the distribution of GCP-EGFP in the mice. The experimental results are as follows: Figure 7 As shown, GCP-EGFP can continuously emit light in tumors for at least 24 hours, indicating that GCP can serve as an in vivo drug delivery carrier.

[0130] Example 2: Construction and Effect Verification of the TRAP2 System

[0131] This example uses the TRAP2 system designed and constructed to induce pyroptosis as an example to demonstrate and confirm the feasibility and advantages of the technical solution of this invention. GSDME, a protein with a pore-forming effect, is a tumor suppressor gene involved in anti-tumor immunity. Therefore, designing a transmembrane peptide to bring the N-terminus of the GSDME protein into cells to induce pyroptosis is of great significance for the clinical treatment of tumors. However, to date, there are no clinical therapeutic drugs targeting GSDME.

[0132] I. Preparation of the fusion protein TRAP2 molecule

[0133] (I) Design and construction of TRAP2 molecular tools

[0134] A DNA sequence of GSDME containing GCP cell-penetrating peptide molecular elements, SUMO protein, and other solubilizing protein sequences was synthesized, with the restriction enzyme sites mutated to MMP cleavage sites and the cysteine ​​residue mutated to alanine. This sequence was then cloned into the prokaryotic expression vector pET28a (plasmid synthesized and constructed by Jiangsu Saisofe Biotechnology Co., Ltd.). The synthesized pET28a-SUMO-GCP-GSDMEMMP11CA plasmid was transformed into E. coli BL (Rosetta) competent cells. Positive recombinants were screened using Kansas resistance plates, and their colony PCR and DNA sequencing were performed for verification. The sequencing results were analyzed by NCBI BLAST alignment. The plasmid was stored at -20℃ for long-term storage, while correctly sequenced strains were stored at -80℃ with 15% glycerol. The vector pET28a carries a 6*His tag and can be used for the expression and purification of His protein. A schematic diagram of the construction and cleavage process of the TRAP2 molecule is shown below. Figure 8 As shown.

[0135] (II) Expression and purification of the TRAP2 system

[0136] 1. TRAP2 was induced to express using isopropyl thiogalactoside (IPTG). Different IPTG concentrations, temperatures, and durations were used to explore optimal expression conditions. SDS-PAGE and Coomassie Brilliant Blue assays were then used to determine the best expression conditions. In this example, the correctly sequenced bacterial culture was inoculated into TB medium containing kanamycin (50 ng / μL) and cultured overnight at 37°C with shaking. The next day, the activated bacterial culture was inoculated into fresh medium at a 1:100 ratio for expansion. The culture was then incubated at 37°C with shaking at 180 rpm until OD600 = 0.6. The temperature was then lowered to 18°C, and isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.5 mM. Expression was induced for approximately 18 hours under the same conditions. The cells were then collected, centrifuged at 5000 rpm at 4°C for 30 min, resuspended in 1×PBS buffer, and centrifuged again to collect the cells. The collected bacterial cells were resuspended in 1×PBS buffer to a 10% bacterial suspension. The suspension was then subjected to three freeze-thaw cycles at 37°C and liquid nitrogen, followed by sonication on ice for 30 minutes (5 seconds on, 5 seconds off) until the bacterial suspension was completely lysed (or directly using a high-pressure homogenizer at 900 Pa and 4°C for 3 minutes). The suspension was centrifuged at 10,000 rpm for 1 hour at 4°C, the precipitate was discarded, and the supernatant was filtered through a 0.22 mm pore size filter membrane. The supernatant was used for subsequent purification.

[0137] 2. Resuspend the supernatant obtained above in Ni-NTA agarose gel and incubate at 4°C for 2 hours (5g of bacterial cells: 1mL of 50% Ni-NTA agarose gel). During incubation, the Ni-NTA agarose gel changes color from blue to brown. Centrifuge at 1000g, 4°C for 1 minute, and collect 20µL of supernatant for subsequent purification effect verification. Discard most of the supernatant, retaining only a small amount for resuspending the Ni-NTA agarose gel. Load the gel onto a column and wash three times with 1×PBS buffer containing 20mM imidazole, twice the column volume each time. Collect 20µL of the last wash buffer. Then elute with 1×PBS buffer containing 300mM imidazole. If the Ni-NTA agarose gel turns blue again, it indicates that the elution is basically complete. Keep 20µL of the eluent sample. Analyze the purification efficiency of the fusion protein using 10% SDS-PAGE. The eluent was centrifuged at 10000g for 10 min. The supernatant was concentrated into a fusion protein using a 10KD ultrafiltration tube (Millipore, catalog number: UFC900396), centrifuged at 4500g, and ultrafiltered to 500 μL at 4°C. The concentrate was then washed three times with 1×PBS, and centrifuged three times in total. The concentration of the obtained concentrated protein was determined using a BCA protein concentration assay kit (Thermo Fisher, catalog number: 23227) and stored at -80°C.

[0138] The purification effect of TRAP2 is as follows Figure 9 As shown, the purity reaches over 90%, which is sufficient for subsequent experimental verification. The amino acid sequence of the SUMO-GCP-GSDME MMP11 CA molecule is as follows:

[0139] (SEQ ID NO.4) (wherein, the single underline below is the SUMO tag sequence, with emphasis on the amino acid sequence of the penetrant peptide GCP; the double underline is the amino acid sequence of GSDME).

[0140] II. Verification of TRAP2's effect on inducing pyroptosis in tumor cells

[0141] This embodiment verifies the effect of TRAP2 in inducing pyroptosis in tumor cells.

[0142] (1) HepG2 liver cancer cells and HCT116 colon cancer cells (from the American Type Culture Collection (ATCC), catalog number: HB-8065, CCL-247) were pre-caught in 96-well cell culture dishes, and 100 μg / mL of SUMO-GCP-GSDME MMP11 CA and its control solvent PBS were added respectively.

[0143] Cells were cultured at 37°C for 6 hours using the IncuCyte live cell analysis system, and the results are as follows: Figure 10 As shown, SUMO-GCP-GSDME MMP11 CA has the effect of targeting tumor cells to induce pyroptosis.

[0144] The above data indicate that the truncated, optimized membrane-penetrating segment of gasderminC yields a short peptide GCP of only 30 amino acids, exhibiting superior cell penetration and permeability compared to the widely recognized cell-penetrating peptide H16. It can be fused with green fluorescent protein (EGFP) for expression and internalize EGFP, which cannot independently penetrate the cell membrane, into the cell; it also lacks cytotoxicity. Most importantly, this CPP is derived from the human body's own protein, is non-immunogenic, and can be used for human imaging or therapy. This has significant implications for the application of cell-penetrating peptides in drug delivery and in vivo imaging.

[0145] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A cell-penetrating peptide, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. A complex comprising the cell-penetrating peptide of claim 1 and a cargo molecule, said cargo molecule comprising a pharmaceutically active molecule, a tag protein, and a recombinant drug delivery system 2; The active drug molecule is GSDME, the tag protein is SUMO, and the recombinant drug delivery system 2 is TRAP2; The amino acid sequence of the complex is shown in SEQ ID NO.

4.

3. Protein-related biological materials, wherein the related biological materials are any one of the following (B1) to (B8): (B1) A nucleic acid molecule encoding the penetrating peptide of claim 1 or the complex of claim 2; (B2) An expression cassette containing the nucleic acid molecule described in (B1); (B3) A recombinant vector containing the nucleic acid molecule described in (B1); (B4) A recombinant vector containing the expression cassette described in (B2); (B5) Recombinant microorganisms containing the nucleic acid molecules described in (B1); (B6) Recombinant microorganisms containing the expression cassette described in (B2); (B7) Recombinant microorganisms containing the recombinant vector described in (B3); (B8) Recombinant microorganisms containing the recombinant vector described in (B4).

4. The application of the penetrating peptide of claim 1, the complex of claim 2, or the related biomaterial of claim 3 in the preparation of the product; wherein the product has the function of at least one of (1) to (5): (1) Tumor targeting; (2) Induces pyroptosis; (3) Drug delivery; (4) In vivo tumor imaging; (5) Prevention and treatment of tumors; The tumors mentioned in (1), (4) and (5) are liver cancer or colorectal cancer; The cells mentioned in (2) are liver cancer cells or colorectal cancer cells; The drug delivery described in (3) is for liver cancer or colorectal cancer; The product in question is a medicine.

5. A product comprising the penetrating peptide of claim 1, the complex of claim 2, or the related biomaterial of claim 3.

6. The product according to claim 5, characterized in that, The product in question is a medicine.

7. The product according to claim 6, characterized in that, The drug also contains pharmaceutically acceptable excipients.

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