Function identification of a novel virulence factor of cell and application thereof

Abstract

The application provides a functional identification of a novel virulence factor of a cell and an application thereof. Based on the research on the novel virulence factor of the cell, the application further provides a polypeptide delivery system and an application thereof. The polypeptide delivery system comprises a protein complex and a signal peptide, the signal peptide comprises at least 20 amino acids at the N-terminus of a PagT1 protein or a homologous protein thereof, the signal peptide is used for guiding the polypeptide to be loaded into the protein complex, and guiding the polypeptide to be positioned to the nucleus of a eukaryotic cell. The research of the application provides an important basis for constructing a safe and efficient novel tumor treatment system, and provides an innovative solution for the treatment of cancer and other major diseases.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more specifically, to a polypeptide delivery system and its applications. Background Technology

[0002] Immunotoxins (ITs), also known as biological missiles, are a highly attractive approach to cancer therapy, designed to specifically deliver highly potent cytotoxic protein toxins to tumor cells. The mechanism of action of immunotoxins involves internalizing the toxin into the cytosol through endocytosis mediated by targeting cell-specific receptors, subsequently killing the designated tumor cells through the toxin's cytotoxic effects. Immunotoxins are chemically coupled or recombinantly fused with a targeting domain (antibody, cytokine, or other cell-binding protein) and a highly cytotoxic payload (toxin, including bacterial and plant toxins or human cytotoxic proteins).

[0003] Immunotoxins utilize a variety of cytotoxic agents from diverse sources, including bacterial toxins, plant toxins, and engineered molecules. Each toxin possesses a unique mechanism and therapeutic potential, highlighting the versatility of immunotoxin technology in targeted cancer therapy and the treatment of other diseases. While numerous in vivo and in vitro studies have been conducted on bacterial toxins with highly efficient cell-killing capabilities, research exploring the use of immunotoxins for cancer treatment remains relatively limited. In fact, the U.S. Food and Drug Administration (FDA) has approved only three immunotoxins that specifically recognize tumors for clinical use: Denileukin Diftitox (Ontak), Moxetumomab Pasudotox (Lumoxiti), and Tagraxofus (Elzonris).

[0004] In addition, the application of immunotoxins also has some limitations:

[0005] (i) Off-target and targeted toxicity

[0006] Non-human (bacterial and plant) toxins have evolved the ability to enter a wide range of cell types by binding to host receptors (such as glycoproteins and glycolipids) via cell-binding domains or motifs. These receptors are widely expressed in most cells and tissues. Therefore, although their specific receptors may appear different, toxins achieve internalization by binding to a broad range of carbohydrate or lipid portions of the host receptor, enabling them to act on a wide range of cell types. In this context, many immunotoxins containing whole or truncated toxins have shown poor targeting specificity in preclinical and clinical trials, leading to significant off-target systemic toxicity, primarily vascular leakage syndrome (VLS).

[0007] Despite the use of tumor-specific targeting fractions to selectively deliver toxin payloads to tumor cells, the clinical development of immunotoxins remains limited by a narrow therapeutic window, primarily due to off-target and targeted toxicities. For example, the therapeutic dose of Denileukin Diftitox is 9 or 18 μg / kg, while the maximum tolerated dose in humans is 27 μg / kg. Even small increases in dose can lead to significant adverse events. Therefore, minimizing off-target and targeted toxicities of immunotoxins is crucial for broadening the therapeutic window, particularly for the treatment of solid tumors.

[0008] (ii) Immunogenicity

[0009] The clinical development of immunotoxins is often hampered by their induction of immune responses, a characteristic known as immunogenicity. Immunotoxins carrying heterogeneous bacterial and plant-derived toxins, in particular, are highly immunogenic, inducing T-cell-dependent and B-cell-mediated antidrug antibodies (ADAs) against the exogenous toxin fraction in patients with healthy immune systems. These antibodies neutralize the immunotoxin, rendering it ineffective and accelerating its clearance from the body, while also causing serious immune system-related adverse reactions, thus hindering long-term or repeated dosing in clinical trials. Since immunotoxins typically require 2–5 cycles of dosing to achieve a significant antitumor response, immunogenicity remains a critical issue.

[0010] (iii) Cytoplasmic delivery efficiency

[0011] The toxic portion of an immunotoxin needs to be internalized and transported into the cytoplasm of tumor cells to exert its lethal effect before lysosomal degradation. Therefore, the potency of an immunotoxin is closely related to its ability to deliver its toxic load to the cytoplasm. However, most internalized immunotoxins are trapped in endocytic vesicles and degraded in lysosomes without effectively escaping into the cytoplasm. Although cytoplasmic entry of immunotoxins can be influenced by a variety of factors, including the rate of antigen internalization, antigen affinity, and subcellular transport, the most rate-limiting step is the escape of internalized endoplasmic reticulum into the cytoplasm.

[0012] In summary, despite the enormous potential shown by immunotoxins, their clinical application still faces numerous limitations. Given the limited variety of protein toxins currently available as mature immunotoxins, and the challenges they face such as in vivo degradation, immunogenicity, severe systemic toxicity, adverse accumulation, and difficulty in entering tumor cells, there is an urgent need to develop novel protein toxins. Simultaneously, there is a need to develop delivery vectors that can improve protein stability, avoid harmful immune stimulation, and deliver specific proteins into specific cell types. Only through the complementary development of these two approaches can precision medicine be applied to diseases such as cancer. Summary of the Invention

[0013] In view of the problems existing in the prior art, the purpose of this application is to provide a polypeptide delivery system and its application.

[0014] Specifically, this application relates to the following aspects:

[0015] 1. A signal peptide for guiding the loading of a polypeptide into a protein complex and guiding the polypeptide to be localized to the nucleus of a eukaryotic cell, wherein the signal peptide comprises at least 20 amino acids at the N-terminus of PagT1 protein or a homolog thereof.

[0016] 2. The signal peptide according to claim 1, wherein the protein complex comprises a non-symbiotic luminescent bacterium virulence box (PVC), AFP, or a metamorphosis-associated stretching structure (MAC);

[0017] Preferably, the protein complex comprises a non-symbiotic luminescent bacillus virulence box (PVC).

[0018] 3. The signal peptide according to claim 1 or 2, wherein the signal peptide comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40 or 20-30 amino acids at the N-terminus of PagT1 protein or its homologous protein.

[0019] 4. The signal peptide according to any one of claims 1-3, wherein the signal peptide comprises an amino acid sequence as shown in any one of SEQ ID NO: 20-30, or comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 20-30.

[0020] 5. The signal peptide according to any one of claims 1-4, wherein the polypeptide comprises any one or more of signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone-regulating molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene-editing proteins.

[0021] 6. A nuclear localization peptide for localizing a polypeptide linked thereto to the nucleus of a eukaryotic cell, wherein the nuclear localization peptide comprises a nuclear localization sequence comprising at least 20 amino acids at the N-terminus and / or C-terminus of PagT1 protein or its homologs;

[0022] Preferably, the nuclear localization sequence comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40 or 20-30 amino acids from the N-terminus and / or C-terminus of the PagT1 protein or its homologous protein.

[0023] 7. The nuclear localization peptide according to claim 6, wherein the nuclear localization peptide comprises an amino acid sequence as shown in any one of SEQ ID NO: 20-30, or comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 20-30.

[0024] 8. The nuclear localization peptide according to claim 6 or 7, wherein the polypeptide comprises any one or more of signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone regulatory molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene editing proteins.

[0025] 9. A fusion protein comprising a nuclear localization peptide as described in any one of items 6-8 and a polypeptide linked thereto, said linkage being covalent or non-covalent.

[0026] 10. A protein toxin, which is PagT1 protein or its homolog, comprising the amino acid sequence shown in SEQ ID NO: 1, or comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0027] 11. The protein toxin according to claim 10, wherein the protein toxin is conserved relative to a reference sequence at aspartic acid at position 217 and glutamic acid at position 219, the reference sequence being shown in SEQ ID NO: 1.

[0028] 12. A conjugate comprising the protein toxin described in item 10 or 11.

[0029] 13. A fusion protein comprising the protein toxin described in item 10 or 11.

[0030] 14. An apparatus or article comprising the protein toxin described in item 10 or 11.

[0031] 15. A complex comprising a protein toxin as described in item 10 or 11 or its encoded nucleic acid.

[0032] 16. The complex according to claim 15, wherein the complex comprises a component for delivering the protein toxin or its encoded nucleic acid into cells, optionally the component comprising lipids, proteins or nucleic acids, optionally the complex being a lipid nanoparticle (LNP), a nucleic acid-protein complex or a protein complex.

[0033] 17. A nucleic acid encoding a signal peptide as described in any one of items 1-5.

[0034] 18. An expression vector comprising the nucleic acid as described in item 17.

[0035] 19. A host cell comprising the nucleic acid as described in item 17 or the expression vector as described in item 18.

[0036] 20. A polypeptide delivery system comprising a protein complex and a signal peptide as described in any one of items 1-5;

[0037] Preferably, the polypeptide delivery system is an extracellular retractable injection system (eCIS).

[0038] 21. The polypeptide delivery system according to claim 20, wherein the protein complex comprises a non-symbiotic luminescent bacterium virulence box (PVC), AFP, or a metamorphosis-associated stretching structure (MAC);

[0039] Preferably, the protein complex comprises a non-symbiotic luminescent bacillus virulence box (PVC).

[0040] 22. The polypeptide delivery system according to claim 20 or 21, wherein the signal peptide is covalently linked to the polypeptide;

[0041] Preferably, the signal peptide is covalently linked to the N-terminus of the polypeptide.

[0042] 23. The polypeptide delivery system according to claim 22, wherein the polypeptide comprises any one or more of the following: signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone-regulating molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene-editing proteins.

[0043] 24. The polypeptide delivery system according to claim 20 or 21, wherein the polypeptide is a PagT1 protein or a homolog thereof, optionally, the PagT1 protein or a homolog thereof comprises an amino acid sequence as shown in SEQ ID NO: 1, or comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1;

[0044] Optionally, the PagT1 protein or its homolog is conserved relative to a reference sequence at aspartic acid position 217 and glutamate position 219, as shown in SEQ ID NO: 1;

[0045] Specifically, the amino acid sequence of the PagT1 protein is shown in SEQ ID NO: 1, and the amino acid sequence of the signal peptide is shown in any one of SEQ ID NO: 20-30.

[0046] 25. The polypeptide delivery system according to any one of claims 20-24, wherein the protein complex is a PVC-V complex;

[0047] Preferably, the structural protein Pvc13 in the PVC-V complex is modified to recognize cell surface molecules;

[0048] More preferably, the structural protein Pvc13 in the PVC-V complex is modified to recognize tumor-specific antigens or tumor-associated antigens on the cell surface.

[0049] 26. The peptide delivery system according to claim 25, wherein the structural protein Pvc13 in the PVC-V complex is modified to specifically recognize HER2.

[0050] 27. The polypeptide delivery system according to item 25 or 26, wherein a protein that recognizes cell surface molecules is inserted into the receptor-binding domain of the structural protein Pvc13, optionally, a protein that recognizes cell surface tumor-specific antigens or tumor-associated antigens is inserted, optionally, DARPin that specifically recognizes HER2 is inserted.

[0051] 28. The polypeptide delivery system according to claim 26 or 27, wherein the amino acid sequence of the signal peptide is as shown in any one of SEQ ID NO: 20-30, and a DARPin that specifically recognizes HER2 is inserted into the receptor-binding domain of the structural protein Pvc13 in the PVC-V complex.

[0052] 29. The polypeptide delivery system according to any one of claims 26-28, wherein the polypeptide comprises any one or more of signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone regulatory molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene editing proteins;

[0053] Optionally, the polypeptide is PagT1 protein or its homologous protein. Optionally, the PagT1 protein or its homologous protein comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0054] Optionally, the PagT1 protein or its homolog is conserved relative to a reference sequence at aspartic acid position 217 and glutamate position 219, as shown in SEQ ID NO: 1;

[0055] Specifically, the amino acid sequence of the PagT1 protein is shown in SEQ ID NO: 1.

[0056] 30. A nucleic acid encoding a polypeptide delivery system as described in any one of items 20-29.

[0057] 31. The nucleic acid according to claim 30, wherein the nucleic acid comprises one or more of the following:

[0058] (i) The nucleotide sequence encoding the protein complex in the polypeptide delivery system;

[0059] (ii) The nucleotide sequence encoding the signal peptide in the polypeptide delivery system;

[0060] (iii) The nucleotide sequence encoding the polypeptide.

[0061] 32. An expression vector comprising the nucleic acid as described in item 30 or 31.

[0062] 33. A host cell comprising a polypeptide delivery system as described in any one of items 20-29, a nucleic acid as described in item 30 or 31, or an expression vector as described in item 32.

[0063] 34. A method for preparing a polypeptide delivery system as described in any one of items 20-29, comprising culturing the host cells described in item 33 to obtain the polypeptide delivery system.

[0064] 35. A method for transferring a polypeptide into target cells, comprising contacting the polypeptide delivery system of any one of claims 20-29 with the target cells, thereby causing the polypeptide delivery system to deliver the polypeptide into the target cells;

[0065] Optionally, the target cells are eukaryotic cells;

[0066] Preferably, the eukaryotic cell is a yeast cell, insect cell, mammalian cell, plant cell, or fungal cell;

[0067] More preferably, the eukaryotic cells are human cells;

[0068] More preferably, the eukaryotic cells are human tumor cells.

[0069] 36. A method of killing target cells, comprising contacting the peptide delivery system of any one of claims 20-29 with the target cells such that the peptide delivery system delivers the peptide into the target cells, thereby killing the target cells;

[0070] Optionally, the target cells are eukaryotic cells;

[0071] Preferably, the eukaryotic cells are human tumor cells;

[0072] More preferably, the eukaryotic cells are HER2-positive human tumor cells.

[0073] 37. Use of the signal peptide as described in any one of items 1-5, the polypeptide delivery system as described in any one of items 20-29, the nucleic acid as described in item 30 or 31, the expression vector as described in item 32, or the host cell as described in item 33 in the preparation of a drug, reagent, or kit for delivering polypeptides.

[0074] 38. The use according to item 37, wherein the drug, reagent or kit is a drug, reagent or kit for treating tumors.

[0075] 39. The use according to item 38, wherein the tumor is a HER2-positive tumor.

[0076] 40. The use of the PagT1 gene or the protein encoded therein in the preparation of a medicament for treating tumors, wherein the amino acid sequence of the protein encoded by the PagT1 gene is as shown in SEQ ID NO: 1, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 1.

[0077] Beneficial effects:

[0078] This application is the first to discover a novel protein toxin, PagT1, that can be loaded into the virulence box (PVC) of non-symbiotic luminescent bacteria, and identifies its toxicity mechanism. Furthermore, this application verifies that the N-terminal signal peptide of PagT1 has a dual function: guiding peptide loading into PVC and directing the peptide to the nucleus after delivery to eukaryotic cells. This research not only provides a theoretical basis for elucidating the pathogenic mechanism of luminescent bacteria and developing clinical treatment strategies for bacterial infectious diseases, but also screens for a suitable virulence effector molecule and a bifunctional signal peptide for PVC, laying a crucial research foundation for its application in the development of therapeutic tools for diseases such as cancer.

[0079] This application describes the delivery of PagT1 to tumor cells and tumor organoids via PVC. The results show that PagT1 effectively kills tumor cells. This discovery provides important evidence for constructing a safe and efficient new tumor treatment system and offers an innovative solution for the treatment of major diseases such as cancer. Attached Figure Description

[0080] Figures 1A-1C show the distribution of PagT1 and its homologous proteins, as well as the results of cytotoxicity assays. Figure 1A is a schematic diagram of the PVC gene cluster and the location of the PAU_02097 (PagT1) gene; Figure 1B is an evolutionary diagram of the relationship between PagT1 and its homologous proteins; Figure 1C shows the cell viability of 293T cells transfected with PagT1, its homologous proteins, and the control vector pEGFP for 24 hours. Pal: Pseudomonas alkylphenolica; Dba: Deltaproteobacteria bacterium; Phe: Photorhabdus heterorhabditis; Pau: Photorhabdus australis; Bub: Burkholderiaubonensis; Sav: Streptomyces avermitilis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

[0081] Figures 2A-2D show the detection results of PVC-PagT1 and PVCR7-PagT1 assembly, as well as the results of eukaryotic cell cytotoxicity detection. Figure 2A shows the results of Western blot analysis of PagT loading in purified PVC-PagT1 and PVCR7-PagT1. Pvc16 antibody was used as an internal control to detect PVC / PVCR7 structural proteins. Figure 2B shows the cell viability of J774A.1 cells 24 hours after PagT1 delivery with PVC-PagT1. Empty PVC and PBS were used as controls, and the PVC concentration was 15 μg / mL. Figure 2C shows the cell viability of THP-1 cells 24 hours after PagT1 delivery with PVCR7-PagT1. Empty PVCR7 and PBS were used as controls, and the PVCR7 concentration was 15 μg / mL. Figure 2D shows the cell viability of BMDM cells 24 hours after PagT1 delivery with PVCR7-PagT1. Empty PVCR7 and PBS were used as controls, and the PVCR7 concentration was 15 μg / mL. **p<0.01, ***p<0.001, ****p<0.0001.

[0082] Figure 3 The results are from DeepLoc 2.0's subcellular localization analysis of the PagT1 amino acid sequence.

[0083] Figures 4A-4D show the detection results of PagT1 localization in the cell nucleus. Figure 4A shows the results of confocal microscopy observation of A549 cells transfected with wild-type PagT1 (WT), PagT1 NLS mutant, and control empty vector pEGFP, respectively, scale bar = 10 μm; Figure 4B shows the results of immunofluorescence detection of A549 cells 18 h after PagT1 delivery via PVCR7, with cell morphology captured by bright field imaging and drawn with dashed lines, scale bar = 10 μm; Figure 4C shows the schematic diagram of the NLS position of wild-type (WT) PagT1 and the schematic diagram of the NLS mutant (Del-N-NLS: PagT1 with N-terminal NLS removed, for delivery with PVC-loaded signal peptide SP (Pnf) added to the N-terminus; Del-C-NLS: PagT1 with C-terminal NLS removed; Del-N,C-NLS: PagT1 with both N-terminal and C-terminal NLS removed, for delivery with PVC-loaded signal peptide SP (Pnf) added to the N-terminus); Figure 4D shows the results of Western blotting. Blot analysis was performed on the Tcst (with a flag tag) fused to express different lengths of PagT1 N-terminal peptides in the purified PVC complex. Pvc16 antibody was used to detect PVC structural proteins as an internal control. The amino acid sequence of Pnf-N50 is shown in SEQ ID NO: 38.

[0084] Figures 5A-5B show the results of PagT1 cytotoxicity assays dependent on its nuclear localization. Figure 5A shows the cell viability of 293T cells 24 hours after transfection with wild-type PagT1 (WT) and its NLS mutant, with empty pEGFP vector as a control. Figure 5B shows the cell viability of THP-1 cells 24 hours after delivery of wild-type PagT1 (WT) and its NLS mutant via PVCR7, with empty PVC and PBS as controls. *p<0.05, ***p<0.001, ****p <0.0001, ns indicates no significant difference.

[0085] Figures 6A-6D show the detection results of PagT1-induced genome damage in eukaryotic cells. Specifically, Figure 6A shows the results of Western blot detection of phosphorylated histone H2AX (phosphorylated S139) expression in J774A.1, THP-1, and BMDM cells 24 h after PagT1 delivery via PVC / PVCR7, with empty PVC / PVCR7 and PBS as controls, and GAPDH as an internal control; Figure 6B shows the results of immunofluorescence detection of phosphorylated histone H2AX spots in J774A.1, THP-1, and BMDM cells 18 h after PagT1 delivery via PVC, with empty PVC and PBS as controls, scale bar = 5 μm; Figure 6C shows the results of comet electrophoresis detection of genome damage in THP-1 cells 24 h after PagT1 delivery via PVCR7, with empty PVCR7 and PBS as controls; Figure 6D shows the statistical results of tail moments (the product of tail length and tail DNA percentage) in the comet electrophoresis experiment. ***p<0.001, ns indicates no significant difference.

[0086] Figures 7A-7G show the predicted results of PagT1 active sites. Specifically, Figure 7A shows the results of AlphaFold3's prediction of the three-dimensional structure of PagT1; Figure 7B shows the results of Foldseek's search for proteins with similar three-dimensional structures to PagT1 in the PDB database; Figure 7C shows the spatial structure diagram of the proteins selected in Figure 7B; Figure 7D shows the results of DoGSiteScorer's prediction of possible active pockets for PagT1; Figure 7E shows a schematic diagram of the spatial locations of D217 and E219; Figure 7F shows the results of PagT1 amino acid sequence conservation analysis; and Figure 7G shows the cell viability of 293T cells transfected with PagT1, the mutant 217219 with mutations at positions A in PagT1 D217 and E219, and the empty vector pEGFP for 24 hours. *p<0.05, ***p<0.001.

[0087] Figures 8A-8E show the detection results of cell Parthanatos-related indicators caused by PagT1 delivery. Figure 8A shows the results of Western blot detection of PARP1 expression in THP-1 cells 24 hours after PagT1 delivery via PVCR7. Empty PVCR7, PVCR7-217219 (PVCR7 loaded with mutant 217219), and PBS were used as controls, and GAPDH was used as an internal control. Figure 8B shows the cell viability of J774A.1 cells 24 hours after PagT1 delivery via PVCR7 after the addition of NAD+. Figure 8C shows the cell viability of THP-1 cells 24 hours after PagT1 delivery via PVCR7 after the addition of NAD+. Figure 8D shows the cell viability of J774A.1 cells 24 hours after PagT1 delivery via PVCR7 after the addition of the PARP1 inhibitor Rucaparib. Figure 8E shows the cell viability of THP-1 cells 24 hours after PagT1 delivery via PVCR7 after the addition of the PARP1 inhibitor Rucaparib. *p<0.05, **p <0.01, ***p<0.001, ****p<0.0001, ns indicates no significant difference.

[0088] Figures 9A-B show the results of immunofluorescence detection of pADPr expression in J774A.1 and THP-1 cells 18 h after PagT1 delivery via PVC / PVCR7. Figure 9A shows the detection results of pADPr expression in J774A.1 cells; Figure 9B shows the detection results of pADPr expression in THP-1 cells. Scale bar = 10 μm.

[0089] Figure 10 This is a violin plot of gene beta scores. A positive beta score indicates that the gene was positively selected, and a negative beta score indicates that the gene was negatively selected. This application uses positive selection, and the degree of gene change is obtained by subtracting the beta scores of the experimental group from those of the control group.

[0090] Figure 11 This is a distribution chart of gene beta score differences in positive selection. The top ten genes are marked in the chart.

[0091] Figure 12 KEGG and GO enrichment analysis plots for genes selected for positive screening.

[0092] Figure 13The results show the cell viability of J774A.1 cells (shIrf9, shSTAT1, shTYK2) after stable knockdown of Irf9, STAT1, and TYK2 genes, delivered with PagT1 via PVC for 24 hours. shNC was the control shRNA group. *p<0.05, **p<0.01. Results are statistical analyses of three independent experiments.

[0093] Figures 14A-14D show the cell viability of J774A.1 and THP-1 cells after PagT1 delivery via PVC / PVCR7 following the addition of the JAK inhibitor Ruxolitinib or the STAT1 inhibitor SH-4-54. Specifically, Figure 14A shows the cell viability of J774A.1 cells 24 hours after PagT1 delivery via PVC following the addition of the JAK inhibitor Ruxolitinib; Figure 14B shows the cell viability of THP-1 cells 24 hours after PagT1 delivery via PVCR7 following the addition of the JAK inhibitor Ruxolitinib; Figure 14C shows the cell viability of J774A.1 cells 24 hours after PagT1 delivery via PVC following the addition of the STAT1 inhibitor SH-4-54; and Figure 14D shows the cell viability of THP-1 cells 24 hours after PagT1 delivery via PVCR7 following the addition of the STAT1 inhibitor SH-4-54. *p<0.05, **p<0.01, ns indicates no significant difference.

[0094] Figure 15 Immunofluorescence assay of Irf9 cellular sublocalization in BMDM cells 18 h after PagT1 delivery via PVCR7. Scale bar = 10 μm.

[0095] Figure 16 RNA-seq was used to detect changes in the transcriptome of J774A.1 cells compared to the empty PVC group 12 h after PagT1 delivery via PVC.

[0096] Figures 17A-17D show the results of qRT-PCR detection of IFN-β, IFN-κ, IFN-α, and IFN-ε gene transcription levels in THP-1 cells 12 h after PagT1 delivery via PVCR7. Specifically, Figure 17A shows the IFN-β gene transcription level; Figure 17B shows the IFN-κ gene transcription level; Figure 17C shows the IFN-α gene transcription level; and Figure 17D shows the IFN-ε gene transcription level. **p < 0.01, ****p < 0.0001, ns indicates no significant difference.

[0097] Figure 18Results of qRT-PCR detection of IFN-β gene transcription levels in BMDM cells after PagT1 delivery via PVCR7. ****p<0.0001, ns indicates no significant difference.

[0098] Figure 19 Results of Lucia gene expression detection in THP1-Dual cells after treatment with PBS, PVCR7, PVCR7-PagT1, or PVCR7-217219 for 24 h. *p<0.05, ns indicates no significant difference.

[0099] Figure 20 The results of p-STAT1 expression in J774A.1, THP-1 and BMDM cells were obtained by Western blot analysis 24 h after PagT1 delivery via PVC / PVCR7. Empty PVC / PVCR7 and PBS were used as controls, and GAPDH was used as an internal control.

[0100] Figure 21 The results of p-STAT1 expression in THP-1 cells were detected by Western blot 24 h after PagT1 was delivered via PVC with the PARP1 inhibitor Rucaparib. Empty PVCR7 and PBS were used as controls, and GAPDH was used as an internal control.

[0101] Figures 22A-22D show the detection results of the pan-toxicity of PagT1 to eukaryotic cells. Specifically, Figure 22A shows the cell viability of HeLa cells 24 hours after PagT1 delivery via PVCR7; Figure 22B shows the cell viability of A549 cells 24 hours after PagT1 delivery via PVCR7; Figure 22C shows the cell viability of Hep G2 cells 24 hours after PagT1 delivery via PVCR7; and Figure 22D shows the cell viability of WEHI-Rb-1 cells 24 hours after PagT1 delivery via PVCR7. Empty PVCR7 and PBS served as controls. *p<0.05, ****p<0.0001, ns indicates no significant difference.

[0102] Figures 23A-23D show the construction and purification results of PVCHER2. Specifically, Figure 23A is a schematic diagram of the Pvc13-targeted HER2 modification of PVCHER2; Figure 23B shows the results of Western blot analysis of the purified PVCHER2-PagT1 loading; Figure 23C is a transmission electron microscope (TEM) image of negatively stained empty PVCHER2 (scale bar = 100 nm); and Figure 23D is a TEM image of negatively stained PVCHER2-PagT1 (scale bar = 100 nm).

[0103] Figures 24A-24C show the cell viability of T98G, U251, and U87MG cells 24 hours after the addition of PVCHER2-PagT1 and PVCHER2-217219 (PVCHER2 loaded with mutant 217219). Specifically, Figure 24A shows the cell viability of T98G cells; Figure 24B shows the cell viability of U251 cells; and Figure 24C shows the cell viability of U87MG cells. **p<0.01, ***p<0.001, ns indicates no significant difference.

[0104] Figures 25A and 25B show the cell viability of BIU87 and T24 cells after 24 hours of treatment with PVCHER2-PagT1 and PVCHER2-217219. Figure 25A shows the cell viability of BIU87 cells; Figure 25B shows the cell viability of T24 cells. **p<0.01, ***p<0.001.

[0105] Figure 26 The results of Western blot analysis of PARP1 expression in BIU87 cells 24 h after PagT1 delivery via PVCHER2 were obtained. Empty PVCHER2 and PBS were used as controls, and GAPDH was used as an internal control.

[0106] Figure 27 The results show the cellular sublocalization of AIF in BIU87 cells detected by immunofluorescence 18 h after PagT1 delivery via PVCHER2. Scale bar = 10 μm.

[0107] Figure 28 Results of immunohistochemical and immunofluorescence detection of HER2 expression in a bladder cancer organoid model. Scale bar = 20 μm.

[0108] Figures 29A-29C show the results of detecting the organ-killing effect of PVCHER2-PagT1 on bladder cancer organoids. Specifically, Figure 29A shows the results of ATP assay for bladder cancer organoid killing after adding empty PVCHER2 to the organoid culture medium for 120 h; Figure 29B shows the results of ATP assay for bladder cancer organoid killing after adding PVCHER2-PagT1 to the organoid culture medium for 120 h; and Figure 29C shows the results of ATP assay for bladder cancer organoid killing after adding PVCHER2-217219 to the organoid culture medium for 120 h.

[0109] Figure 30 The results show the expression of luciferase in U251-Luci cells, where U251 represents wild-type U251 cells.

[0110] Figures 31A-31E illustrate the efficacy evaluation of PVCHER2-PagT1. Figure 31A shows in vivo imaging of mice; Figure 31B is a statistical graph of Luci signal intensity in in vivo mouse imaging, with PBS as the control group for analysis of variance; Figure 31C is a magnetic resonance imaging (MRI) image of the mouse brain, with tumor sites indicated by arrows; Figure 31D shows the results of Kaplan-Meier survival analysis of mice, with PBS as the control group for analysis of variance; Figure 31E is a graph showing the change in mouse body weight. *p<0.05. Detailed Implementation

[0111] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0112] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0113] definition

[0114] The term “contractile injection system (CIS)” as used in this paper refers to a diverse range of evolutionarily related macromolecular devices that utilize retractable sheaths to deliver nucleic acids and proteins. Contractile injection systems can help microorganisms transport a variety of effector molecules extracellularly to gain a survival advantage (NMITaylor, MJvan Raaij, and PGLeiman, Contractile injection systems of bacteria and related systems. Mol Microbiol, 2018.108(1):p.6-15.). Typical CISs include the retractable tails of bacteriophages T4, P2, and Mu. Besides retractable bacteriophages, retractable injection systems similar to bacteriophage tails are also prevalent in bacteria and archaea. For example, the type VI secretion system (T6SS) is used to mediate intercellular communication and exert cellular defense functions (NMITaylor, MJvan Raaij, and PGLeiman, Contractile injection systems of bacteriophages and related systems. Mol Microbiol, 2018, 108(1):p.6-15.). Contractile injection systems also include extracellular contractile injection systems (eCIS).

[0115] The term “extracellular retractable injection system” or “eCIS” as used in this article refers to a retractable injection system that can be released extracellularly and attack target cells from external space. Extracellular contractile injection systems include bacterial tailocin / pyocin, as well as the virulence cassette (PVC) found in the genus *Photorhabdus* (G. Yang, et al., *Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth. J Bacteriol, 2006, 188(6): p. 2254-61.), antifeeding prophage (AFP) (A. Desfosses, H. Venugopal, T. Joshi, J. Felix, M. Jessop, H. Jeong, J. Hyun, JB Heymann, MRH Hurst, I. Gutsche, and A.K. Mitra, *Atomic structures of an entire contractile injection system in both the extended and contracted states. Nature Microbiology*, 2019.4(11):p.1885-1894.) and metamorphosis-associated contractile structure (MAC) (NJShikuma et al., Marinetubeworm metamorphosis induced by arrays of bacterial phage tail-likestructures.Science, 2014.343(6170):p.529-33.), etc.

[0116] The term “Photorhabdus asymbiotica” as used in this article belongs to the genus Photorhabdus. While bacteria of the genus Photorhabdus are generally considered insect pathogens, Photorhabdus asymbiotica can infect humans (P. Wilkinson et al., Comparative genomics of the emerging human pathogen Photorhabdus asymbiotica with the insect pathogen Photorhabdus luminescens. BMC Genomics, 2009.10:p.302).

[0117] In this paper, the term "PVC" generally refers to a retractable injection system produced by the genus *Photorhabdus*. The PVC of the non-symbiotic *Photorhabdus asymbiotica* ATCC43949 is a protein complex device exceeding 10 MDa, structurally similar to a simplified T4 phage tail. It comprises a hexagonal baseplate complex with six fibers and a 117 nm long sheath trunk with a cap structure containing an inner tube in which effector proteins are loaded. PVC can be released extracellularly by the bacteria to exert its effects. PVC is generally considered to be toxic to eukaryotic cells because it can transfer effector proteins into insect hemocytes and promote actin aggregation (G. Yang et al., *Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth. J Bacteriol, 2006, 188(6): p. 2254-61.). Specifically, in the context of this application, PVC refers to a retractable injection system isolated from the non-symbiotic luminescent bacterium Photorhabdus asymbiotica ATCC43949 or the host cells described herein, capable of penetrating the human cell membrane to deliver polypeptides or proteins within the sheath of the PVC into the cytoplasm of human cells. There are five types of PVCs among the non-symbiotic luminescent bacterium: PVC-I, PVC-II, PVC-III, PVC-IV, and PVC-V.

[0118] In this paper, the term "PVC gene cluster" refers to a cluster of genes encoding multiple structural proteins of PVC that exist in the genome of non-symbiotic luminescent bacterium *Photorhabdus asymbiotica* (e.g., *Photorhabdus asymbiotica* ATCC43949). There are five PVC gene clusters in the genome of non-symbiotic luminescent bacterium: PVC-I, PVC-II, PVC-III, PVC-IV, and PVC-V. Each PVC gene cluster also contains one or more genes downstream that potentially encode effector factors.

[0119] As used herein, the term "signal peptide" generally refers to a peptide chain that guides the transfer of a synthesized polypeptide or protein toward a target. In the context of this application, a "signal peptide" is capable of guiding a polypeptide or protein to be delivered into the lumen of a PVC sheath.

[0120] In this paper, the term "effect factor" refers to bacterial secretory proteins produced by bacteria and transported into plant or animal cells via the secretory system, which play a role in recognition or pathogenicity. Structurally, effector factors can be divided into signaling regions and functional regions.

[0121] As used in this paper, the term "virulence factor" refers to the property (i.e., genetic product) that enables a microorganism to establish itself on or within a host of a particular species and enhance its potential to cause disease. Virulence factors include bacterial toxins, cell surface proteins that mediate bacterial attachment, cell surface carbohydrates and proteins that protect bacteria, and hydrolytic enzymes that may contribute to bacterial pathogenicity.

[0122] As used herein, the term "immunotoxin" refers to a bifunctional molecule comprising a targeting portion for delivery and a toxic portion for cytotoxicity. Immunotoxins can be used to kill cells expressing a specific target (i.e., recognized by the targeting portion).

[0123] As used in this article, the term "protein toxin" refers to a class of toxic protein molecules produced by organisms (such as bacteria, fungi, plants, or animals) that can damage cells, tissues, or other organisms. These toxins exert their toxic effects by interfering with normal cellular physiological functions, potentially leading to infection, disease, or even death.

[0124] The term “nuclear localization sequence” or “NLS” as used in this article refers to an amino acid sequence that facilitates protein importation into the cell nucleus (e.g., via nuclear transport).

[0125] As used herein, the term “amino acid” or “amino acid sequence” refers to an oligopeptide, peptide, polypeptide, or protein sequence, or any fragment thereof, and refers to a naturally occurring or synthetic molecule. When “amino acid sequence” is described herein as referring to the amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and similar terms are not intended to limit the amino acid sequence to the complete naturally occurring amino acid sequence associated with the described protein molecule.

[0126] The term “amino acid” as used in this article may be referred to by its name, its commonly known three-letter symbol, or a single-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0127] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” “polynucleotide,” “polynucleotide sequence,” “RNA sequence,” or “DNA sequence” refer to oligonucleotides, nucleotides, or polynucleotides, and fragments or portions thereof, and refer to DNA or RNA of genotype or synthetic origin, which may be single-stranded or double-stranded and represent sense or antisense strands. Sequences may be non-coding sequences, coding sequences, or mixtures of both. The nucleic acid sequences of this application may be prepared using standard techniques well known to those skilled in the art.

[0128] The percentage of "identity" used herein, such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, and 99.5%, refers to the degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment, specifically 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, and 99.5%. For example, it is the percentage of positions with identical bases or amino acid residues relative to the total number of positions after two sequences have as many identical residues as possible by introducing vacancies, etc. The percentage of "identity" can be determined using software programs known in the art. It is preferred to use default parameters for alignment. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0129] The term "PVC structural protein" as used in this article refers to the monomeric proteins that make up the PVC structure.

[0130] As used herein, the term "expression vector" refers to a linear or circular DNA molecule containing a polynucleotide encoding a polypeptide, which is effectively linked to a control sequence for its expression.

[0131] As used herein, the term “host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, and eukaryotic cells such as microbial cells, fungal cells, animal cells, and plant cells.

[0132] Those skilled in the art will understand that the design of expression vectors can depend on factors such as the selection of host cells to be transformed and the desired expression level.

[0133] As used in this article, the term "cancer" or "tumor" generally refers to a physiological condition in mammals characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, lymphomas (such as Hodgkin's and non-Hodgkin's lymphomas), blastomas, sarcomas, and leukemias. More specific examples of cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, leukemia, and other lymphoproliferative disorders, as well as various types of head and neck cancers.

[0134] The term "tumor-specific antigen" as used in this article refers to novel antigens that are unique to tumor cells or exist only in certain tumor cells and not in normal cells. These antigens are confirmed through tumor transplantation between syngeneic animals and are therefore also called tumor-specific transplantation antigens (TSTA) or tumor rejection antigens (TRA).

[0135] The term "tumor-associated antigen (TAA)" as used in this article refers to antigens that are not specific to tumor cells but are also present in normal cells and other tissues, although their levels increase significantly during cell carcinogenesis. These antigens only exhibit quantitative changes and do not have strict tumor specificity.

[0136] As used herein, the term "complex" refers to a combination of two or more molecules. In some embodiments, complexes include polypeptide and nucleic acid molecules that interact with each other (e.g., bind, contact, adhere).

[0137] As used in this paper, the term "fusion protein" refers to a hybrid protein expressed by a nucleic acid molecule containing nucleotide sequences of at least two genes.

[0138] As used herein, the term "conjugate" refers to a molecule obtained by conjugating one molecule with another. In this application, the conjugate refers to a conjugate obtained by conjugating the protein toxin described in this application with another molecule. This application does not impose any limitations on the other molecule, and those skilled in the art can make conventional selections as needed. In some embodiments, conjugation is performed using chemical methods.

[0139] The term “codon optimization” as used in this paper refers to the configuration of the nucleotide sequence encoding a polypeptide to contain codons preferred by the host cell or organism in order to improve gene expression and translation efficiency in the host cell or organism.

[0140] As used herein, the term "homological protein" refers to a protein whose amino acid sequence has significant similarity and performs the same or similar functions in different organisms or within the same organism. The protein sequences for which homology analysis is performed can be newly discovered or obtained from public databases such as NCBI's GenBank and UniProt. Bioinformatics tools known in the art can be selected for homology searches; BLAST (Basic Local Alignment Search Tool) is one of the most widely used tools. By analyzing the alignment results, the similarity and possible evolutionary relationships between sequences are assessed, and based on the alignment results, the function of the target protein can be predicted, and its evolutionary relationship can be inferred. Homology analysis allows for the prediction of the function of unknown proteins, especially when they are similar to protein sequences with known functions. In one embodiment, homologous proteins with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% homology can be selected.

[0141] signal peptide

[0142] This application discovers that the PagT1 protein can load itself onto PVC without the need for an additional signal peptide at its N-terminus, and that the PagT1 protein can be localized to the cell nucleus.

[0143] In this application, the amino acid sequence of the PagT1 protein is as shown in SEQ ID NO: 1, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 1.

[0144] The amino acid sequence shown in SEQ ID NO: 1 is a protein encoded by the PagT1 gene (PAU_02097) located downstream of the PVC-II structural gene (Photorhabdusasymbiotica ATCC43949).

[0145] Those skilled in the art will understand that the PagT1 protein is not limited to the specific sequences listed above. The PagT1 protein encompasses sequences containing one, two, or three or more nucleotide mutations compared to the sequence shown in SEQ ID NO: 1, but still substantially functionally identical, and also includes sequences having at least 80%, 85%, 96%, 97%, 98%, or 99% sequence identity compared to the sequence shown in SEQ ID NO: 1. Further, the PagT1 protein should be conserved relative to a reference sequence at positions 217 (aspartic acid D) and 219 (glutamic acid E), as shown in SEQ ID NO: 1.

[0146] In this application, the above-mentioned sites are counted starting from the N-end. The term "relative to" has the meaning commonly understood by those skilled in the art. Specifically, "relative to" refers to the position in one sequence corresponding to a specified position in another sequence after homology or sequence identity alignment.

[0147] Based on this, this application provides a signal peptide for guiding the loading of a polypeptide into a protein complex and guiding the polypeptide to be localized to the nucleus of a eukaryotic cell, wherein the signal peptide comprises at least 20 amino acids at the N-terminus of PagT1 protein or its homologous protein.

[0148] Those skilled in the art will understand that proteins with a significantly similar amino acid sequence to the PagT1 protein, and which perform the same or similar functions in different organisms or the same organism, should also be within the scope of protection of this application, as long as their N-terminus retains the function of the signal peptide.

[0149] Homologous proteins typically share a high degree of sequence conservation, such as at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence conservation, and a high degree of sequence identity, such as at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the homologous protein is a homologous protein of PagT1 derived from Pseudomonas alkylphenolica, Deltaproteobacteria bacterium, Photorhabdusheterorhabditis, Photorhabdus australis, or Burkholderia ubonensis.

[0150] In some embodiments, the protein complex comprises a non-symbiotic luminescent bacterium virulence box (PVC), AFP, or a metamorphosis-associated stretching structure (MAC).

[0151] This application is not intended to limit the specific type of the protein complex. As long as the selected protein complex can recognize and effectively load the signal peptide and achieve the correct assembly and delivery of the polypeptide, it meets the requirements of this application.

[0152] In some embodiments, the protein complex comprises a non-symbiotic luminescent bacterium virulence box (PVC). In some embodiments, the PVC is derived from the non-symbiotic luminescent bacterium Photorhabdus asymbiotica ATCC43949.

[0153] The genome of the non-symbiotic luminescent bacterium *Photorhabdus asymbiotica* contains five luminescent bacterium virulence cassette gene clusters: PVC-I, PVC-II, PVC-III, PVC-IV, and PVC-V. The PVC-V structural proteins include Pvc1, Pvc2, Pvc3, Pvc4, Pvc5, Pvc6, Pvc7, Pvc8, Pvc9, ​​Pvc10, Pvc11, Pvc12, Pvc13, Pvc14, Pvc15, and Pvc16. In some embodiments, the protein complex is a PVC-V complex, which includes PVC-V structural proteins Pvc1, Pvc2, Pvc3, Pvc4, Pvc5, Pvc6, Pvc7, Pvc8, Pvc9, ​​Pvc10, Pvc11, Pvc12, Pvc13, Pvc14, Pvc15, and Pvc16, wherein the PVC-V structural proteins are derived from the non-symbiotic luminescent bacterium Photorhabdusasymbiotica ATCC43949.

[0154] In some embodiments, the signal peptide comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30 amino acids at the N-terminus of the PagT1 protein or its homologs. In some embodiments, the signal peptide comprises 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acids at the N-terminus of the PagT1 protein or its homologs.

[0155] In some embodiments, the signal peptide comprises an amino acid sequence as shown in any one of SEQ ID NO: 20-30, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence as shown in any one of SEQ ID NO: 20-30.

[0156] In some embodiments, the signal peptide comprises an amino acid sequence having 1 to 5, for example 1, 2 or 3 amino acid substitutions compared to the amino acid sequence shown in any of SEQ ID NO: 20-30.

[0157] In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 20. In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 21. In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 22. In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 23. In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 24. In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 25. In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 26. In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 27. In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 28. In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 29. In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 30.

[0158] In some embodiments, the polypeptide comprises any one or more of the following: signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone-regulating molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene-editing proteins.

[0159] In this application, the term "cytotoxin" refers to a polypeptide or protein that has a toxic effect on specific cells and can cause cell damage or death.

[0160] In this application, the term "antimicrobial peptide" refers to a peptide with broad-spectrum antipathogenic activity that can rapidly kill pathogens. Antimicrobial peptides typically consist of 20-60 amino acid residues. Targets of antimicrobial peptides include Gram-negative bacteria, Gram-positive bacteria, fungi, parasites, tumor cells, etc. Depending on their origin, antimicrobial peptides can include insect antimicrobial peptides, mammalian antimicrobial peptides, amphibian antimicrobial peptides, antimicrobial peptides derived from fish, mollusks, crustaceans, plants, and bacteria. The antimicrobial peptides can be defensins, Cecropin A and its analogues, Magainins, Melitiin, Cecropins, cathelicidin, apidaecins, drosocin, coleoptericin, hemipteracin, bactenecin, Cecropin, etc.

[0161] In some embodiments, the polypeptide is a hormone or a hormone-regulating molecule that can regulate the expression and / or secretion of a hormone.

[0162] In some embodiments, the polypeptide is a cytotoxin, such as a protein toxin. The cytotoxin can be fused with the signal peptide to be loaded into the PVC-V complex (specifically, into the lumen of the PVC-V complex sheath) and delivered by the PVC-V complex to the nucleus of a eukaryotic cell, thereby exerting cytotoxic and killing effects on the cell. In some embodiments, the polypeptide is a TcsT protein (Trichosanthes kirilowii pollen protein, TcsT).

[0163] In some embodiments, the polypeptide is an anti-angiogenic inhibitor.

[0164] In some embodiments, the polypeptide is a pathogen-specific antigen. The pathogen-specific antigen may be a pathogen-specific antigen of bacteria, viruses, fungi, mycoplasma, chlamydia, parasites, or other pathogens.

[0165] In some embodiments, the polypeptide is a malaria-specific antigen, such as the Plasmodium falciparum-specific antigens PfSir2a and PfRH5.

[0166] In some embodiments, the polypeptide is a specific antigen or related antigen of a certain type of cell in the organism itself. In some embodiments, the polypeptide is a tumor-specific antigen or tumor-associated antigen.

[0167] In some embodiments, the polypeptide is an antigen derived from a protein that causes or progresses a disease. In some embodiments, the polypeptide is GSDMD (gasdermin D) or GSDMD-NT (obtained by cleaving GSDMD after Asp276 and Asp275), a protein involved in pyroptosis (or inflammatory apoptosis) that causes renal tubular damage. In some embodiments, the polypeptide is an antigen derived from a protein that causes or progresses a tumor. For example, RhoA can regulate actin polymerization, cell adhesion, cell transformation, and cell movement, proliferation, and migration closely related to tumor cell invasion and metastasis.

[0168] In some embodiments, the polypeptide is a tag protein or a reporter protein. For example, the tag protein or reporter protein may be BlaM, luciferase, CyaA, β-galactosidase, chloramphenicol acetyltransferase (CAT), secretory phosphatase (SEAP), fluorescent protein, etc. In some embodiments, the tag protein or reporter protein is, for example, BlaM, Renilla Luciferase, or mRFP.

[0169] In some embodiments, the polypeptide is an antimicrobial peptide. For example, the antimicrobial peptide may be defensins, cecropin A and its analogues, magainins, melittin, cecropins, cathelicidin, apidaecins, drosocin, coleoptericin, hemipteracin, bactenecin, cecropin, human centrifugal peptide-1 (HNP-1), hBD-1, hepcidin25, E50-52, PK34, etc.

[0170] In some embodiments, the polypeptide is an enzyme. In some embodiments, the enzyme is an enzyme involved in cellular metabolism. In some embodiments, the enzyme may be selected from Aritilysin, recombinant phage lysin LysSAP26, ribonuclease RNase 3, and RNase 7.

[0171] In some embodiments, the polypeptide is a gene-editing protein. The gene-editing protein may be, for example, a zinc finger nuclease, a TALEN nuclease, Cas9, Cas12 (formerly known as Cpf1), Cas12a, Cas13, Cas13a, Cas13b, etc.

[0172] In some embodiments, the polypeptide is an effector of the bacterial secretion system. In some embodiments, the polypeptide is a T3SS effector, such as EcEspN, EcCif, PaExoU, EcNleC, SfOspB, SfOspF, YpYopT. In some embodiments, the polypeptide is a T4SS effector, such as LpAnkB, BaBspB, HpCagA. In some embodiments, the polypeptide is a T6SS effector, such as EtEvpP, BcTecA, PpTge2, YpYezP, PaTse1, PaTse3, PaPldA, PaPldB.

[0173] This application also provides a nucleic acid that encodes any of the signal peptides described above.

[0174] The nucleic acids include sequences that have been isolated from their natural environment, isolates of recombinant or cloned (e.g., DNA), chemically synthesized analogs, or analogs biosynthesized in heterologous systems.

[0175] The nucleic acid can be prepared by any method known in the art. For example, it can be produced by replication and / or expression in a suitable host cell. Typically, a natural or synthetic DNA fragment encoding the desired segment is incorporated into a recombinant nucleic acid construct (usually a DNA construct) capable of being introduced into and replicated in prokaryotic or eukaryotic cells. Typically, the DNA construct will be adapted for autonomous replication in a single-celled host (such as yeast or bacteria), but can also be used to introduce and integrate into the genome of cultured bacteria, insects, mammals, plants, or other eukaryotic cell lines. The nucleic acid can also be produced by chemical synthesis.

[0176] This application also provides an expression vector comprising any of the aforementioned nucleic acids.

[0177] In some embodiments, the expression vector is a plasmid, granule, bacteriophage, or viral vector, preferably a plasmid.

[0178] This application also provides a host cell comprising any of the above-described nucleic acids or any of the above-described expression vectors.

[0179] In some embodiments, the host cell may be a mammalian cell, insect cell, yeast cell, bacterial cell (e.g., Escherichia coli), or plant cell. In some embodiments, the host cell is a bacterial cell (preferably Escherichia coli).

[0180] Nuclear localization peptides

[0181] This application discovers that the PagT1 protein has a nuclear localization sequence at its N-terminus and C-terminus, which can serve as a nuclear localization signal to guide the polypeptide to the cell nucleus.

[0182] Based on this, this application provides a nuclear localization peptide for localizing a polypeptide linked thereto into the nucleus of a eukaryotic cell, wherein the nuclear localization peptide contains a nuclear localization sequence comprising at least 20 amino acids at the N-terminus and / or C-terminus of PagT1 protein or its homologous protein.

[0183] Those skilled in the art will understand that proteins with a significantly similar amino acid sequence to the PagT1 protein, and which perform the same or similar functions in different organisms or the same organism, should also be within the scope of protection of this application, as long as their N-terminus and / or C-terminus retain the function of the nuclear localization peptide.

[0184] In some embodiments, the nuclear localization sequence comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30 amino acids from the N-terminus and / or C-terminus of the PagT1 protein or its homologs. In some embodiments, the nuclear localization sequence comprises 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 100 amino acids from the N-terminus and C-terminus of the PagT1 protein or its homologs. In some embodiments, the nuclear localization sequence comprises 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 100 amino acids from the N-terminus of the PagT1 protein or its homologs. In some embodiments, the nuclear localization sequence comprises 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 100 amino acids from the C-terminus of the PagT1 protein or its homologs.

[0185] In some embodiments, the nuclear localization sequence comprises the N-terminal and C-terminal 20 amino acids of the PagT1 protein or its homologs. In some embodiments, the nuclear localization sequence comprises the N-terminal 20 amino acids of the PagT1 protein or its homologs. In some embodiments, the nuclear localization sequence comprises the C-terminal 20 amino acids of the PagT1 protein or its homologs.

[0186] In some embodiments, the nuclear localization peptide comprises an amino acid sequence as shown in any one of SEQ ID NO: 20-30, or comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 20-30.

[0187] In some embodiments, the nuclear localization peptide comprises an amino acid sequence having 1 to 5, for example 1, 2 or 3 amino acid substitutions compared to the amino acid sequence shown in any of SEQ ID NO: 20-30.

[0188] In some embodiments, the amino acid sequence of the nuclear localization peptide is shown in SEQ ID NO: 20. In some embodiments, the amino acid sequence of the nuclear localization peptide is shown in SEQ ID NO: 21. In some embodiments, the amino acid sequence of the nuclear localization peptide is shown in SEQ ID NO: 22. In some embodiments, the amino acid sequence of the nuclear localization peptide is shown in SEQ ID NO: 23. In some embodiments, the amino acid sequence of the nuclear localization peptide is shown in SEQ ID NO: 24. In some embodiments, the amino acid sequence of the nuclear localization peptide is shown in SEQ ID NO: 25. In some embodiments, the amino acid sequence of the nuclear localization peptide is shown in SEQ ID NO: 26. In some embodiments, the amino acid sequence of the nuclear localization peptide is shown in SEQ ID NO: 27. In some embodiments, the amino acid sequence of the nuclear localization peptide is shown in SEQ ID NO: 28. In some embodiments, the amino acid sequence of the nuclear localization peptide is shown in SEQ ID NO: 29. In some embodiments, the amino acid sequence of the nuclear localization peptide is shown in SEQ ID NO: 30.

[0189] This application also provides a fusion protein comprising any of the above-mentioned nuclear localization peptides and a polypeptide linked thereto, wherein the linkage is covalent or non-covalent.

[0190] In some implementations, the connection is a covalent connection.

[0191] In some embodiments, the polypeptide in the fusion protein can be loaded into the PVC-V complex via the nuclear localization peptide and delivered to the nucleus of a eukaryotic cell via the PVC-V complex.

[0192] In some embodiments, the fusion protein is not naturally occurring, meaning that the fusion protein formed by the fusion of nuclear localization peptides and polypeptides as described in this application does not exist in nature under natural conditions; that is, the fusion protein is artificially synthesized through recombinant technology.

[0193] In some embodiments, the polypeptide in the fusion protein comprises any one or more of the following: signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone-regulating molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene-editing proteins.

[0194] In some embodiments, the polypeptide in the fusion protein is the PagT1 protein.

[0195] In some embodiments, the polypeptide is a hormone or a hormone-regulating molecule that can regulate the expression and / or secretion of a hormone.

[0196] In some embodiments, the polypeptide in the fusion protein is a cytotoxin. In some embodiments, the polypeptide is a TcsT protein.

[0197] In some embodiments, the polypeptide in the fusion protein is an anti-angiogenic inhibitor.

[0198] In some embodiments, the polypeptide in the fusion protein is a pathogen-specific antigen. The pathogen-specific antigen can be a pathogen-specific antigen of bacteria, viruses, fungi, mycoplasma, chlamydia, parasites, or other pathogens.

[0199] In some embodiments, the polypeptide in the fusion protein is a malaria-specific antigen, such as the Plasmodium falciparum-specific antigens PfSir2a and PfRH5.

[0200] In some embodiments, the polypeptide in the fusion protein is a specific antigen or related antigen of a certain type of cell in the organism. In some embodiments, the polypeptide in the fusion protein is a tumor-specific antigen or tumor-associated antigen.

[0201] In some embodiments, the polypeptide in the fusion protein is an antigen derived from a protein that causes or progresses a disease. In some embodiments, the polypeptide in the fusion protein is GSDMD (gasdermin D), a protein involved in pyroptosis (or inflammatory apoptosis) that causes renal tubular damage, or GSDMD-NT (obtained by cleaving GSDMD after Asp276 and Asp275). In some embodiments, the polypeptide in the fusion protein is an antigen derived from a protein that causes or progresses tumors. For example, RhoA can regulate actin polymerization, cell adhesion, cell transformation, and is involved in cell movement, proliferation, and migration closely related to tumor cell invasion and metastasis.

[0202] In some embodiments, the polypeptide is a tag protein or a reporter protein. For example, the tag protein or reporter protein may be BlaM, luciferase, CyaA, β-galactosidase, chloramphenicol acetyltransferase (CAT), secretory phosphatase (SEAP), fluorescent protein, etc. In some embodiments, the tag protein or reporter protein is, for example, BlaM, Renilla Luciferase, or mRFP.

[0203] In some embodiments, the polypeptide in the fusion protein is an antimicrobial peptide. For example, the antimicrobial peptide may be defensins, cecropin A and its analogues, magazineins, melittin, cecropins, cathelicidin, apidaecins, drosocin, coleoptericin, hemipteracin, bactenecin, cecropin, human centrifugal peptide-1 (HNP-1), hBD-1, hepcidin25, E50-52, PK34, etc.

[0204] In some embodiments, the polypeptide in the fusion protein is an enzyme. In some embodiments, the enzyme is an enzyme involved in cellular metabolism. In some embodiments, the enzyme may be selected from Aritilysin, recombinant phage lysin LysSAP26, ribonuclease RNase 3, and RNase 7.

[0205] In some embodiments, the polypeptide in the fusion protein is a gene-editing protein. The gene-editing protein may be, for example, a zinc finger nuclease, a TALEN nuclease, Cas9, Cas12 (formerly known as Cpf1), Cas12a, Cas13, Cas13a, Cas13b, etc.

[0206] In some embodiments, the peptide in the fusion protein is an effector of the bacterial secretion system. In some embodiments, the peptide is a T3SS effector, such as EcEspN, EcCif, PaExoU, EcNleC, SfOspB, SfOspF, or YpYopT. In some embodiments, the peptide is a T4SS effector, such as LpAnkB, BaBspB, or HpCagA. In some embodiments, the peptide is a T6SS effector, such as EtEvpP, BcTecA, PpTge2, YpYezP, PaTse1, PaTse3, PaPldA, or PaPldB.

[0207] protein toxins

[0208] This application is the first to discover a novel protein toxin, PagT1, which can cause DNA damage in host cells and lead to PARP1-dependent cell death in eukaryotic cells, specifically Parthanatos.

[0209] Based on this, this application provides a protein toxin, which is PagT1 protein or its homologous protein, the protein toxin comprising the amino acid sequence shown in SEQ ID NO: 1, or comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0210] Those skilled in the art will understand that proteins with a significantly similar amino acid sequence to the PagT1 protein, and which perform the same or similar functions in different organisms or the same organism, should also be within the scope of protection of this application, as long as they retain the function of the protein toxin.

[0211] In some embodiments, the protein toxin is conserved relative to a reference sequence at aspartic acid at position 217 and glutamic acid at position 219, the reference sequence being shown in SEQ ID NO: 1.

[0212] In some embodiments, the amino acid sequence of the protein toxin is as shown in SEQ ID NO: 1, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 1.

[0213] This application also provides a conjugate comprising any of the aforementioned protein toxins.

[0214] This application also provides a fusion protein comprising any of the aforementioned protein toxins.

[0215] This application also provides an apparatus or article comprising any of the aforementioned protein toxins.

[0216] This application also provides a complex comprising any of the aforementioned protein toxins.

[0217] In some embodiments, the complex includes components for delivering the protein toxin or its encoded nucleic acid into cells. These components may include, for example, lipids, proteins, or nucleic acids; the complex may be, for example, lipid nanoparticles (LNPs), nucleic acid-protein complexes, or protein complexes.

[0218] Peptide delivery system

[0219] This application provides a peptide delivery system comprising a protein complex and any of the aforementioned signal peptides. The peptide delivery system is, for example, an extracellular retractable injection system.

[0220] The polypeptide is loaded into the protein complex under the guidance of the signal peptide and delivered to the nucleus of a eukaryotic cell via the protein complex.

[0221] In some embodiments, the signal peptide is covalently linked to the polypeptide. In some embodiments, the signal peptide is covalently linked to the N-terminus of the polypeptide. In some embodiments, a linker sequence is further included between the signal peptide and the polypeptide.

[0222] In this application, the term "linker" can refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that connects two molecules or parts (e.g., two components of a protein complex), such as two domains of a fusion protein. A linker can be located between or on either side of two groups, molecules, or other parts and is connected to each of them via a covalent bond or non-covalent interaction, thereby connecting the two. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can be one or more amino acids (e.g., a peptide or protein). In some embodiments, the length of the linker can be about 5 to 100 amino acids, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 amino acids. In some embodiments, the length of the linker can be about 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, or 450 to 500 amino acids. Longer or shorter linkers are also possible.

[0223] In some embodiments, the molecular weight of the polypeptide is 5kDa-200kDa, for example 10kDa-200kDa, 15kDa-200kDa, 20kDa-200kDa, 25kDa-200kDa, 30kDa-200kDa, 40kDa-200kDa, 5kDa-180kDa, 10kDa-180kDa, 15kDa -180kDa, 20kDa-180kDa, 25kDa-180kDa, 30kDa-180kDa, 40kDa-180kDa, 5kDa-160kDa, 10kDa-160k Da, 15kDa-160kDa, 20kDa-160kDa, 25kDa-160kDa, 30kDa-160kDa, 40kDa-160kDa, 5kDa-150kDa, 10k Da-150kDa, 15kDa-150kDa, 20kDa-150kDa, 25kDa-150kDa, 30kDa-150kDa, 40kDa-150kDa, 5kDa-140 kDa, 10kDa-140kDa, 15kDa-140kDa, 20kDa-140kDa, 25kDa-140kDa, 30kDa-140kDa, or 40kDa-140kDa.

[0224] In some embodiments, the isoelectric point of the polypeptide is 2-12, for example 2-11.5, 2-11, 2-10.5, 2-10, 2-9.5, 2-9.10, 2.5-11.5, 2.5-11, 2.5-10.5, 2.5-10, 2.5-9.5, 2.5-9.10, 3-11.5, 3-11, 3-10.5, 3-10, 3-9.5, 3-9.10, 3.5-11.5, 3.5-11, 3. 5-10.5, 3.5-10, 3.5-9.5, 3.5-9.10, 4-11.5, 4-11, 4-10.5, 4-10, 4-9.5, 4-9.10, 4.5-11.5, 4.5-11, 4.5-10.5, 4.5-10, 4.5-9.5, 4.5-9.10, 4.6-11.5, 4.6-11, 4.6-10.5, 4.6-10, 4.6-9.5, or 4.6-9.10.

[0225] In some embodiments, the polypeptide comprises any one or more of the following: signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone-regulating molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene-editing proteins.

[0226] In some embodiments, the polypeptide is a PagT1 protein or a homolog thereof, which is loaded into the protein complex under the guidance of its own N-terminal signal peptide. The PagT1 protein or its homolog comprises the amino acid sequence shown in SEQ ID NO:1, or comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the PagT1 protein or its homolog is conserved relative to a reference sequence at positions 217 (aspartic acid) and 219 (glutamic acid), the reference sequence being shown in SEQ ID NO:1. In some embodiments, the amino acid sequence of the PagT1 protein is as shown in SEQ ID NO:1, and the amino acid sequence of the signal peptide is as shown in any one of SEQ ID NO: 20-30.

[0227] In some embodiments, the polypeptide is a hormone or a hormone-regulating molecule that can regulate the expression and / or secretion of a hormone.

[0228] In some embodiments, the polypeptide is a cytotoxin, such as a protein toxin. In some embodiments, the polypeptide is a TcsT protein.

[0229] In some embodiments, the polypeptide in the fusion protein is an anti-angiogenic inhibitor.

[0230] In some embodiments, the polypeptide is a pathogen-specific antigen. The pathogen-specific antigen can be a specific antigen of bacteria, viruses, fungi, mycoplasma, chlamydia, parasites, or other pathogens. The polypeptide delivery system of this application can be used to incubate with cells in vitro to deliver the pathogen-specific antigen in the polypeptide delivery system into the cells, thereby producing a vaccine corresponding to the pathogen-specific antigen. After administration to a subject, the vaccine can be used to induce an immune response against the pathogen-specific antigen polypeptide in the subject, thereby preventing and / or treating infection with the pathogen and related diseases.

[0231] In some embodiments, the polypeptide is a malaria-specific antigen, such as the Plasmodium falciparum-specific antigens PfSir2a and PfRH5.

[0232] In some embodiments, the polypeptide is a specific antigen or related antigen of a certain type of cell in the organism itself. In some embodiments, the polypeptide is a tumor-specific antigen or tumor-associated antigen. The polypeptide delivery system of this application can be used to incubate with cells in vitro to deliver the tumor-specific antigen or tumor-associated antigen in the polypeptide delivery system into the cells, thereby producing a vaccine against a tumor corresponding to the tumor-specific antigen or tumor-associated antigen. After administration to a subject, the vaccine can induce an immune response against the polypeptide in the subject, thereby preventing and / or treating the related tumor. The cells may be human embryonic kidney cells HEK293, antigen-presenting cells (e.g., dendritic cells), etc.

[0233] In some embodiments, the polypeptide is an antigen derived from a protein that causes or progresses a disease. This antigen can be delivered to antigen-presenting cells using the polypeptide delivery system of this application, thereby creating a vaccine. Upon administration to a subject, the vaccine can elicit an immune response against the protein that causes or progresses the disease. In some embodiments, the polypeptide is GSDMD (gasdermin D) or GSDMD-NT (obtained by cleaving GSDMD after Asp276 and Asp275), a protein involved in pyroptosis (or inflammatory apoptosis) that causes renal tubular damage. In some embodiments, the polypeptide is an antigen derived from a protein that causes or progresses tumors. For example, RhoA, which can regulate actin polymerization, cell adhesion, cell transformation, and cell movement, proliferation, and migration closely related to tumor cell invasion and metastasis.

[0234] In some embodiments, the polypeptide is a tag protein or a reporter protein. The tag protein or reporter protein can be delivered to cells using the polypeptide delivery system of this application, and the cells can be characterized by detecting the signal of the tag protein or reporter protein. For example, the tag protein or reporter protein may be BlaM, luciferase, CyaA, β-galactosidase, chloramphenicol acetyltransferase (CAT), secretory phosphatase (SEAP), fluorescent protein, etc. In some embodiments, the tag protein or reporter protein is, for example, BlaM, Renilla Luciferase, or mRFP.

[0235] In some embodiments, the polypeptide is an antimicrobial peptide. The antimicrobial peptide can be delivered into cells by the polypeptide delivery system of this application, thereby exerting an antipathogenic effect within the cells. For example, the antimicrobial peptide may be defensins, cecropin A and its analogues, magainins, melittin, cecropins, cathelicidin, apidaecins, drosocin, coleoptericin, hemipteracin, bactenecin, cecropin, human neutrophil peptide-1 (HNP-1), hBD-1, hepcidin25, E50-52, PK34, etc.

[0236] In some embodiments, the polypeptide is an enzyme. The enzyme can be delivered into cells by the polypeptide delivery system of this application, thereby exerting a corresponding catalytic effect in the cells. In some embodiments, the enzyme is an enzyme involved in cellular metabolism. In some embodiments, the enzyme may be selected from Aritilysin, recombinant phage lyase LysSAP26, ribonuclease RNase 3, and RNase 7.

[0237] In some embodiments, the polypeptide is a gene-editing protein. The gene-editing protein can be delivered into cells using the polypeptide delivery system of this application, thereby exerting gene-editing functions within the cells. The gene-editing protein may be, for example, a zinc finger nuclease, a TALEN nuclease, Cas9, Cas12 (formerly known as Cpf1), Cas12a, Cas13, Cas13a, Cas13b, etc.

[0238] In some embodiments, the polypeptide is an effector of the bacterial secretion system. In some embodiments, the polypeptide is a T3SS effector, such as EcEspN, EcCif, PaExoU, EcNleC, SfOspB, SfOspF, YpYopT. In some embodiments, the polypeptide is a T4SS effector, such as LpAnkB, BaBspB, HpCagA. In some embodiments, the polypeptide is a T6SS effector, such as EtEvpP, BcTecA, PpTge2, YpYezP, PaTse1, PaTse3, PaPldA, PaPldB.

[0239] In some embodiments, the protein complex comprises a non-symbiotic luminescent bacterium virulence box (PVC), AFP, or a metamorphosis-associated stretching structure (MAC).

[0240] This application is not intended to limit the specific type of the protein complex. As long as the selected protein complex can recognize and effectively load the signal peptide and achieve the correct assembly and delivery of the polypeptide, it meets the requirements of this application.

[0241] In some embodiments, the protein complex comprises a non-symbiotic luminescent bacterium virulence box (PVC). In some embodiments, the PVC is derived from the non-symbiotic luminescent bacterium Photorhabdus asymbiotica ATCC43949.

[0242] In some embodiments, the protein complex is a PVC-V complex, which includes PVC-V structural proteins Pvc1, Pvc2, Pvc3, Pvc4, Pvc5, Pvc6, Pvc7, Pvc8, Pvc9, ​​Pvc10, Pvc11, Pvc12, Pvc13, Pvc14, Pvc15, and Pvc16, wherein the PVC-V structural proteins are derived from the non-symbiotic luminescent bacterium Photorhabdusasymbiotica ATCC43949.

[0243] In some embodiments, the structural protein Pvc13 in the PVC-V complex is modified to recognize cell surface molecules. For example, a protein that recognizes surface molecules is inserted into the receptor-binding domain of the structural protein Pvc13.

[0244] In some embodiments, the structural protein Pvc13 in the PVC-V complex is modified to recognize cell surface tumor-specific antigens or tumor-associated antigens. In some embodiments, the structural protein Pvc13 in the PVC-V complex is modified to specifically recognize HER2.

[0245] Specifically, the PVC-V complex is engineered to specifically target HER2-positive cells by inserting DARPin, which specifically recognizes HER2, into the receptor-binding domain of the structural protein Pvc13.

[0246] More specifically, the PVC-V complex is engineered to specifically target HER2-positive cells by inserting a HER2-specific DARPin into the receptor-binding domain of the structural protein Pvc13 and linking the DARPin to Pvc13 using a adapter. The adapter can be a commonly used adapter and can be attached to the N-terminus and / or C-terminus of the DARPin; for example, the adapter is GGSGGGGSGG (SEQ ID NO: 9). In some embodiments, the nucleotide sequence of the HER2-specific DARPin containing the adapter is shown in SEQ ID NO: 10.

[0247] In some embodiments, the polypeptide is loaded into the PVC-V complex under the guidance of the signal peptide and delivered to the nucleus of a eukaryotic cell via the PVC-V complex. Specifically, the polypeptide is loaded into the sheath lumen of the PVC-V complex under the guidance of the signal peptide.

[0248] In some embodiments, the polypeptide loaded into the PVC-V complex is PagT1 protein or a homolog thereof. The PagT1 protein or its homolog comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the PagT1 protein or its homolog is conserved relative to a reference sequence at positions 217 (aspartic acid) and 219 (glutamic acid), the reference sequence being shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the PagT1 protein is as shown in SEQ ID NO: 1, and the amino acid sequence of the signal peptide is as shown in any one of SEQ ID NO: 20-30.

[0249] This application also provides a nucleic acid that encodes any of the aforementioned polypeptide delivery systems.

[0250] In some embodiments, the nucleic acid comprises one or more of the following:

[0251] (i) The nucleotide sequence encoding the protein complex in the polypeptide delivery system;

[0252] (ii) The nucleotide sequence encoding the signal peptide in the polypeptide delivery system;

[0253] (iii) The nucleotide sequence encoding the polypeptide.

[0254] In some implementations, the nucleotide sequence encoding the peptide delivery system is codon-optimized. This type of optimization may require mutations in the nucleotide sequence encoding the protein delivery system to mimic the codon preferences of the intended host organism or cell while simultaneously encoding the same protein.

[0255] This application also provides an expression vector comprising one or more of the following: (i) a nucleotide sequence encoding a protein complex in the polypeptide delivery system;

[0256] (ii) The nucleotide sequence encoding the signal peptide in the polypeptide delivery system;

[0257] (iii) The nucleotide sequence encoding the polypeptide.

[0258] In some embodiments, any two or all three of (i) to (iii) above can be in the same expression vector. In some embodiments, any two or all three of (i) to (iii) above can be in different expression vectors.

[0259] In some embodiments, the expression vector is a plasmid, granule, bacteriophage, or viral vector, preferably a plasmid.

[0260] This application also provides a host cell comprising any of the above-described polypeptide delivery systems, any of the above-described nucleic acids, or any of the above-described expression vectors.

[0261] In some embodiments, the host cell may be a mammalian cell, insect cell, yeast cell, bacterial cell (e.g., Escherichia coli), or plant cell. In some embodiments, the host cell is a bacterial cell (preferably Escherichia coli, such as Escherichia coli EP1300 strain cells).

[0262] In some embodiments, the host cell comprises an expression vector containing a nucleotide sequence encoding a PVC-V structural protein. The PVC-V structural protein includes Pvc1, Pvc2, Pvc3, Pvc4, Pvc5, Pvc6, Pvc7, Pvc8, Pvc9, ​​Pvc10, Pvc11, Pvc12, Pvc13, Pvc14, Pvc15, and Pvc16. The host cell is capable of expressing the PVC-V structural protein and assembling it into a complete PVC-V complex.

[0263] In some embodiments, the expression vector further comprises a nucleotide sequence encoding a polypeptide and a signal peptide in the polypeptide delivery system. In some embodiments, the signal peptide is covalently linked to the polypeptide; preferably, the signal peptide is linked to the N-terminus of the polypeptide; more preferably, a linker sequence is further included between the signal peptide and the polypeptide.

[0264] In some embodiments, the polypeptide comprises any one or more of the following: signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone-regulating molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene-editing proteins.

[0265] In some embodiments, the polypeptide is a PagT1 protein or a homologous protein thereof, the PagT1 protein or a homologous protein comprising the amino acid sequence shown in SEQ ID NO: 1, or comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the PagT1 protein or a homologous protein is conserved relative to a reference sequence at positions 217 (aspartic acid) and 219 (glutamic acid), the reference sequence being shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the PagT1 protein is as shown in SEQ ID NO: 1, and the amino acid sequence of the signal peptide is as shown in any one of SEQ ID NO: 20-30.

[0266] In some embodiments, the host cell is also able to express the LysR regulator.

[0267] In some embodiments, the host cell comprises:

[0268] (i) A first vector containing a gene encoding a LysR regulator, preferably under the control of its natural promoter, the first vector being preferably a plasmid, more preferably pBR60;

[0269] (ii) A second vector comprising a nucleotide sequence encoding a PVC-V structural protein, wherein the vector is preferably a plasmid, more preferably a pCNM3 plasmid, and the PVC-V structural protein comprises Pvc1, Pvc2, Pvc3, Pvc4, Pvc5, Pvc6, Pvc7, Pvc8, Pvc9, ​​Pvc10, Pvc11, Pvc12, Pvc13, Pvc14, Pvc15 and Pvc16;

[0270] (iii) A third carrier comprising the nucleotide sequence encoding a polypeptide and a signal peptide in the polypeptide delivery system described in this application.

[0271] This application also provides a method for preparing any of the above-mentioned polypeptide delivery systems, which includes culturing any of the above-mentioned host cells to obtain the polypeptide delivery system.

[0272] In some embodiments, the method further includes the step of isolating proteins from cultured host cells. In some embodiments, the method further includes the steps of purifying the isolated proteins and removing endotoxins.

[0273] Applications of peptide delivery systems

[0274] This application provides a method for transferring a polypeptide into a target cell, comprising contacting any of the above-described polypeptide delivery systems with the target cell, thereby causing the polypeptide delivery system to deliver the polypeptide into the target cell.

[0275] In some embodiments, the polypeptide comprises any one or more of the following: signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone-regulating molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene-editing proteins.

[0276] This application provides a method for killing target cells, comprising contacting any of the above-described peptide delivery systems with the target cells, such that the peptide delivery system delivers the peptide into the target cells, thereby killing the target cells.

[0277] In some embodiments, the polypeptide is a cytotoxin.

[0278] In some embodiments, the polypeptide is a PagT1 protein or a homologous protein thereof, the PagT1 protein or a homologous protein comprising the amino acid sequence shown in SEQ ID NO: 1, or comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the PagT1 protein or a homologous protein is conserved relative to a reference sequence at positions 217 (aspartic acid) and 219 (glutamic acid), the reference sequence being shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the PagT1 protein is as shown in SEQ ID NO: 1, and the amino acid sequence of the signal peptide is as shown in any one of SEQ ID NO: 20-30.

[0279] In some embodiments, in the peptide delivery system, the protein complex is a PVC-V complex, wherein the structural protein Pvc13 in the PVC-V complex is modified to specifically recognize HER2.

[0280] This application provides a method for regulating cell signal transduction, the method comprising contacting any of the above-described peptide delivery systems with target cells, thereby causing the peptide delivery system to deliver the peptide into the target cells. The peptide is a signaling pathway regulatory protein.

[0281] This application provides a method for regulating cell metabolism, the method comprising contacting any of the above-described peptide delivery systems with target cells, thereby causing the peptide delivery system to deliver the peptide into the target cells. The peptide is an enzyme involved in cell metabolism.

[0282] This application provides a method for regulating molecular transport and / or secretion in cells, the method comprising contacting any of the above-described peptide delivery systems with target cells, thereby causing the peptide delivery system to deliver the peptide into the target cells. The peptide is a transport protein.

[0283] This application provides a method for gene editing of cells, the method comprising contacting any of the above-described polypeptide delivery systems with target cells, thereby causing the polypeptide delivery system to deliver the polypeptide into the target cells. The polypeptide is a gene-editing protein.

[0284] This application provides a method for labeling cells, the method comprising contacting any of the above-described peptide delivery systems with target cells, thereby causing the peptide delivery system to deliver the peptide into the target cells. The peptide is a tag protein or a reporter protein.

[0285] This application provides a method for enhancing the antipathogenic ability of cells, the method comprising contacting any of the above-described peptide delivery systems with target cells, thereby causing the peptide delivery system to deliver the peptide into the target cells. The peptide is an antimicrobial peptide.

[0286] This application provides a method for preparing a cell-based vaccine, the method comprising contacting any of the above-described peptide delivery systems with target cells, thereby causing the peptide delivery system to deliver the peptide into the target cells. The peptide is an antigen or immunogen.

[0287] The target cells mentioned above are eukaryotic cells, such as yeast cells, insect cells, mammalian cells, plant cells, or fungal cells. In some embodiments, the eukaryotic cells are human cells. In some embodiments, the eukaryotic cells are human tumor cells, such as HER2-positive human tumor cells.

[0288] This application provides a method for treating and / or preventing a disease, comprising administering to a subject any of the aforementioned peptide delivery systems, wherein the peptide delivery system is loaded with a therapeutically effective amount of a biomolecule. The disease can be determined based on the function of the biomolecule used.

[0289] This application provides an immunotoxin comprising any of the aforementioned polypeptide delivery systems.

[0290] This application provides a pharmaceutical composition comprising any of the above-described polypeptide delivery systems.

[0291] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0292] The term "pharmaceutical acceptable" means that when the molecular basis and the composition are properly administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions.

[0293] Examples of substances that can serve as pharmaceutically acceptable carriers or components thereof include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions and phosphate buffers, etc.

[0294] In some embodiments, the pharmaceutical composition may be formulated into various dosage forms as needed.

[0295] This application provides the use of any of the above-mentioned signal peptides, any of the above-mentioned polypeptide delivery systems, any of the above-mentioned nucleic acids, any of the above-mentioned expression vectors, or any of the above-mentioned host cells in the preparation of drugs, reagents, or kits for delivering polypeptides.

[0296] In some embodiments, the drug, reagent, or kit is a drug, reagent, or kit for treating tumors.

[0297] The tumor is, for example, a solid tumor.

[0298] The tumors mentioned include, for example, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, leukemia, and other lymphoproliferative diseases and various types of head and neck cancers.

[0299] In some embodiments, the tumor is a HER2-positive tumor.

[0300] Applications of the PagT1 gene and its encoded protein

[0301] This application also provides the use of the PagT1 gene or the protein it encodes in the preparation of drugs for treating tumors.

[0302] The amino acid sequence of the protein encoded by the PagT1 gene is as shown in SEQ ID NO: 1, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 1.

[0303] Those skilled in the art will understand that the PagT1 protein is not limited to the specific sequences listed above. The PagT1 protein encompasses sequences containing one, two, or three or more nucleotide mutations compared to the sequence shown in SEQ ID NO: 1, but still substantially functionally identical, and also includes sequences having at least 80%, 85%, 96%, 97%, 98%, or 99% sequence identity compared to the sequence shown in SEQ ID NO: 1. Further, the PagT1 protein should be conserved relative to a reference sequence at positions 217 (aspartic acid D) and 219 (glutamic acid E), as shown in SEQ ID NO: 1.

[0304] The tumor is, for example, a solid tumor.

[0305] The tumors mentioned include, for example, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, leukemia, and other lymphoproliferative diseases and various types of head and neck cancers.

[0306] In some embodiments, the tumor is a HER2-positive tumor.

[0307] This application identifies a novel protein toxin, PagT1, for the first time, and no homologous proteins have been found to date. This discovery suggests that PagT1 has a novel mechanism of action, different from the protein toxin mechanisms of action involved in existing immunotoxins. Research on the virulence mechanism of PagT1 broadens our understanding of PVC virulence factors and lays the foundation for research on similar novel virulence factors. It also provides important reference for the pathogenicity of *C. luminifera* and the treatment of clinical bacterial infectious diseases.

[0308] Unlike existing immunotoxins that use protein toxins, PagT1 has the advantage of being able to be loaded onto PVC and can serve as a highly cytotoxic payload to co-form a novel "immunodelivery toxin" with PVC, providing a new therapeutic tool for targeted therapy of diseases such as tumors. Furthermore, this application finds that the PVC in this novel "immunodelivery toxin" has the following advantages compared to existing immunotoxins:

[0309] (1) PVC can be obtained from a large number of cultured Escherichia coli. The preparation process is simple and the cost is low. Furthermore, the function of the extracted PVC does not depend on the presence of bacteria. It is free from bacterial pyrogens and is relatively safe.

[0310] (2) Unlike the existing immunotoxin-dependent cell endocytosis process, PVC delivers the loaded toxic payload to the cell through the energy stored in the exoskeleton protein. This has a huge advantage for tumor-targeted killing, which can make the toxic payload exert its toxic effect more effectively and avoid the degradation of the toxic payload and the triggering of human immune response.

[0311] (3) This application has obtained a PVC that can recognize tumor-associated antigens / tumor-specific antigens by modifying the tail filament protein Pvc13 of the PVC delivery carrier. This discovery provides the possibility for targeting solid tumors.

[0312] In summary, this application not only reveals a novel protein toxin, PagT1, with a unique mechanism of action, but also demonstrates its potential for development into novel "immunodelivery toxins" when combined with PVC. This discovery not only advances our understanding of the pathogenic mechanisms of *Cyclophorus*, but also provides new ideas and technical support for developing safer and more effective targeted cancer therapies, possessing significant scientific value and broad application prospects.

[0313] Example

[0314] The following description, in conjunction with specific embodiments, illustrates the content of this application, but the scope of this application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following embodiments are all conventional reagents and instruments in the art and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described schemes and obtain corresponding results based on the embodiments.

[0315] The main experimental materials and reagents involved in the examples are shown in Table 1 below.

[0316] Table 1. Main experimental materials and reagents involved in the examples

[0317] ;

[0318] ;

[0319]

[0320] The plasmid information involved in the examples is shown in Table 2 below.

[0321] Table 2. Plasmid information involved in the examples

[0322] ;

[0323]

[0324] The antibody information involved in the examples is shown in Table 3 below.

[0325] Table 3. Antibody information involved in the examples.

[0326]

[0327] Cell lines and cell cultures involved in the examples

[0328] Human embryonic kidney 293 cells (293T cell line) were purchased from the American College of Cell Bank (ATCC) (CRL-3216).

[0329] The mouse monocyte-macrophage-like cell line J774A.1 was purchased from the American ATCC cell bank (TIB-67).

[0330] The human monocytic leukemia cell line THP-1 was purchased from the American ATCC cell bank (TIB-202).

[0331] Human alveolar adenocarcinoma basal epithelial cells (A549 cell line) were purchased from the American ATCC cell bank (CCL-185).

[0332] Human cervical tumor cells (Henrietta Lacks cells) Hela cell line were purchased from the American ATCC Cell Bank (CCL-2);

[0333] Human hepatocarcinoma cells (Hep G2 cell line) were purchased from the American ATCC cell bank (HB-8065).

[0334] Human glioma cells U251 were purchased from the National Biomedical Experimental Cell Resource Bank (1101HUM-PUMC000058).

[0335] The human retinal glioma cell line WERI-Rb-1 is preserved in our laboratory.

[0336] Human cervical tumor cells (Henrietta Lacks cells) Hela cell line were purchased from the American ATCC Cell Bank (CCL-2);

[0337] Human glioblastoma cells (T98G) were purchased from the National Biomedical Experimental Cell Resource Bank (3101HUMSCSP5274).

[0338] Human astrocytic glioblastoma cells U87MG were purchased from the National Biomedical Experimental Cell Resource Bank (1101HUM-PUMC000208).

[0339] The human bladder transitional cell carcinoma cell line T24 is preserved in our laboratory;

[0340] The human bladder transitional cell carcinoma cell line BIU87 is preserved in our laboratory.

[0341] All cells were identified by STR (Short Tandem Repeat). 293T, J774A.1, Hep G2, and Hela cells were cultured in high-glucose DMEM medium; THP-1, A549, and U87MG cells were cultured in RPMI 1640 medium; U251 and T98G cells were cultured in MEM medium; BIU87 and T24 cells were cultured in McCoy's 5A medium; and WERI-Rb-1 cells were cultured in IMDM medium. All media were supplemented with 10% FBS and 1× penicillin-streptomycin (100 μg / mL streptomycin and 100 U / mL penicillin), and cultured under stable conditions of 37°C, 5% CO2, and saturated humidity.

[0342] The main instruments and equipment involved in the embodiments are shown in Table 4 below.

[0343] Table 4. Main instruments and equipment involved in the embodiments

[0344]

[0345] The specific steps of some of the experimental operations involved in the embodiment are as follows:

[0346] Construction of gene expression vectors

[0347] The following proteins were synthesized via gene synthesis: Photorhabdus asymbiotica WP_015834233.1 (PagT1) protein (amino acid sequence shown in SEQ ID NO: 1), Pseudomonas alkylphenolica WP_051939377.1 protein C-terminus protein (amino acid sequence shown in SEQ ID NO: 2), Deltaproteobacteria bacterium MCE9576703.1 protein C-terminus protein (amino acid sequence shown in SEQ ID NO: 3), Okeania WP_293063881.1 protein (amino acid sequence shown in SEQ ID NO: 4), Photorhabdus heterorhabditis WP_172908611.1 protein (amino acid sequence shown in SEQ ID NO: 5), Photorhabdus australis WP_065822174.1 protein (amino acid sequence shown in SEQ ID NO: 6), and Burkholderia ubonensis. The C-terminus of KVU23967.1 protein (amino acid sequence as shown in SEQ ID NO: 7) and the sequence of Streptomyces avermitilis WP_037649420.1 protein (amino acid sequence as shown in SEQ ID NO: 8) were inserted into the pEGFP-C1 vector to construct a eukaryotic expression vector.

[0348] Nucleotide sequences expressing the above proteins were synthesized to construct the pEGFP-C1 expression vector. Codon optimization was performed during synthesis to adapt them for expression in human cells.

[0349] The sequence encoding the Photorhabdus asymbiotica WP_015834233.1 (PagT1) protein was inserted into the pBBRN vector to construct the PVC loading vector.

[0350] A nucleotide sequence (SEQ ID NO: 10) of DARPin binding to HER2 was synthesized by gene synthesis, with KpnI and HindIII restriction sites and a linker GGSGGGGSGG (SEQ ID NO: 9) at both ends. After KpnI and HindIII restriction, the sequence was inserted into the pRK404-PVC vector for PVC (PVCHER2) expression targeting HER2.

[0351] Primer design and synthesis

[0352] The names and sequences of the PCR primers involved in the examples are shown in Table 5 below, where F is the forward primer and R is the reverse primer.

[0353] Table 5. PCR primer names and sequences

[0354]

[0355] PCR system and procedure for mutation

[0356] The PCR systems used for mutations in the examples are shown in Table 6 below.

[0357] Table 6 PCR system for mutation

[0358]

[0359] After mixing, centrifuge and amplify on a PCR instrument, as shown in Table 7 below.

[0360] Table 7 PCR Procedures for Mutation

[0361]

[0362] The annealing temperature is determined based on the primer's Tm value, and is usually 2-3℃ lower than the Tm value; while the extension time is determined according to the length of the amplified fragment (1 min per kb).

[0363] After the reaction was complete, add 1 μL of DpnI, mix thoroughly, and incubate at 37°C for 5 min.

[0364] The gene knockdown shRNA sequences involved in the embodiments are shown in Table 8 below.

[0365] Table 8 shRNA sequence information

[0366]

[0367] plasmid extraction

[0368] plasmid small-scale extraction

[0369] Take 1 mL of the cultured bacterial solution and place it in a 1.5 mL centrifuge tube. Centrifuge at 12000×g for 1 min and discard the supernatant. Follow the instructions of the plasmid small-scale extraction kit (Beijing Qingke Biotechnology Co., Ltd.) and use a spectrophotometer to measure the concentration of the obtained plasmid.

[0370] Large-scale plasmid extraction

[0371] Add 100-200 mL of LB culture medium containing antibiotics to a 1 L bacterial culture flask; then inoculate the primary bacterial culture into the culture flask at a ratio of 0.1% and incubate at 37℃ for 16-18 h; follow the instructions of the plasmid large-scale extraction kit (Beijing Qingke Biotechnology Co., Ltd.) and measure the concentration of the obtained plasmid using a spectrophotometer.

[0372] Constructing a phylogenetic tree of PagT1 and its homologous proteins

[0373] The NCBI (https: / / www.ncbi.nlm.nih.gov) database was used to search for proteins homologous to the PagT1 amino acid sequence using the BLAST function, and their FASTA format sequences were downloaded. The sequences were aligned using MEGA software and ClustalW. A phylogenetic tree was generated using the maximum likelihood method and then beautified using iTOL (https: / / itol.embl.de / ).

[0374] Extraction and culture of primary mouse bone marrow-derived macrophages (BMDM)

[0375] Mice were euthanized by cervical dislocation, and the tibia and femur were then separated, soaked in 75% ethanol, and transferred to PBS. The ends of the tibia and femur were cut open, and bone marrow cells were flushed from one end into a 50 mL sterile centrifuge tube using a 1 mL syringe of complete culture medium. This process was repeated multiple times until the bone turned white. Red blood cells were removed according to the instructions for using red blood cell lysis buffer. The cells were resuspended in DMEM complete culture medium containing 10 ng / mL M-CSF, counted, and then adjusted to 1×10⁶ cells / mL with DMEM complete culture medium. 6 Cells / mL were seeded into culture dishes and incubated statically at 37°C and 5% CO2.

[0376] Cell passage and transfection

[0377] Cell passage

[0378] a. 293T cells and A549 cells:

[0379] When cells reach approximately 90% confluence in 10 cm culture dishes, aspirate the culture medium from the dish. Rinse the dish with 1 mL of 0.25% trypsin, then discard the trypsin solution. Add another 1 mL of 0.25% trypsin and place the dish in an incubator until the cells are digested and detached. Add 1 mL of culture medium containing 10% FBS to stop the digestion. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 800 rpm for 5 minutes, discarding the supernatant. Add 1 mL of culture medium to the centrifuge tube and gently resuspend the cells. Pipette 150 μL of the cell suspension from the centrifuge tube into a culture dish containing 10 mL of culture medium, or into a six-well plate containing 1.5 mL of culture medium per well. Shake well after adding the medium and incubate statically at 37°C and 5% CO2.

[0380] b. For J774A.1 cells and BMDM cells:

[0381] The cells were cultured in a 10 cm culture dish until they reached a density of approximately 90%. The culture medium was then aspirated from the dish. 3 mL of cell culture medium was added to the dish, and the cells were gently scraped off in one direction using a cell scraper. The scraped cells were transferred to a 15 mL centrifuge tube and centrifuged at 800 rpm for 5 min. The supernatant was discarded. 3 mL of culture medium was added, the cells were resuspended, and then counted.

[0382] Different handling scenarios: For cell passage, take 150 μL from the resuspended and counted cell suspension and drop it into a culture dish containing 10 mL of culture medium; when seeding 96-well plates, seed 1 × 10⁶ cells per well. 4 To calculate the required cell volume, aspirate the appropriate number of cells, resuspend them in a suitable culture medium, and then transfer them to 96-well plates for incubation. For 12-well plates, seed the cells at a density of 1 × 10⁶ cells per well. 5 Calculate the cell usage for each cell, take the required amount of cells, resuspend them in the corresponding culture medium, and then transfer them to a 12-well plate for static culture at 37°C and 5% CO2.

[0383] c. For THP-1 cells:

[0384] The cells were cultured in T75 cell culture flasks. After 2 days, 10 mL of cell suspension was aspirated from the original culture flask and transferred to a new T75 cell culture flask. Then, 30 mL of fresh RPMI 1640 medium (containing 1% penicillin-streptomycin and 10% fetal bovine serum) was added to the new culture flask. The culture flask was then placed in a static culture environment at 37°C and 5% CO2.

[0385] Induction of THP-1 differentiation into macrophages: After 2 days of cell culture, the cell suspension was transferred to a 50 mL centrifuge tube, centrifuged at 800 rpm for 5 min, and the supernatant was discarded; cell culture medium containing 100 ng / mL PMA, 1% penicillin-streptomycin, and 10% fetal bovine serum was added, and the cells were gently resuspended; for 96-well plates, 1 × 10⁶ cells were seeded per well. 4 To determine the standard for cell culture, resuspended cells were seeded into 96-well plates, which were then incubated statically at 37°C and 5% CO2. For 12-well plates, 1 × 10⁶ cells were seeded per well. 5 To determine the standard for individual cells, the resuspended cells were seeded into 12-well plates and incubated statically at 37°C and 5% CO2.

[0386] Cell transfection

[0387] Cells were evenly seeded in six-well plates. When the cell density reached approximately 70%, transfection was performed. 1 μg of plasmid was dissolved in 100 μL of FBS-free medium, and 2 μL of PolyJet transfection reagent was also dissolved in 100 μL of the same FBS-free medium. The medium containing PolyJet was added to the medium containing the plasmid, and the mixture was thoroughly mixed and incubated at room temperature for 15 min. The mixture was then added dropwise to the cell-containing medium. Six hours after transfection, the cell culture medium was replaced with fresh medium.

[0388] Cell viability assay

[0389] Aspirate the cell culture supernatant and rinse the cells with 1×PBS; prepare the CCK-8 working solution according to the reagent manufacturer's instructions; add the CCK-8 working solution to the cells in the 96-well plate and incubate in a cell culture incubator for 1 hour;

[0390] The absorbance at 450 nm was measured using an ELISA reader.

[0391] Western blot assay for proteins

[0392] SDS-PAGE was placed in an electrophoresis tank, and 1× electrophoresis buffer was added. 5 μL of protein molecular weight marker and 20 μL of protein sample were added to the corresponding wells. The constant voltage was set to 120 V, and the electrophoresis time was 90 min. The PVDF membrane was activated with anhydrous methanol. After activation, the PVDF membrane and the gel after electrophoresis were clamped together in a transfer clamp. The transfer clamp was placed in the transfer tank, and 1× transfer buffer was added. The constant current was set to 300 mA, and the transfer time was 60 min. After transfer, the membrane was blocked with 5% skim milk powder or 5% BSA at room temperature for 1 h or overnight at 4°C. The primary antibody was incubated with the membrane at room temperature for 1 h or overnight at 4°C. After thoroughly washing away the antibody on the membrane with TBST, ECL developing solution was applied, and development was performed using a high-sensitivity chemiluminescence imaging system.

[0393] The production of PVC

[0394] PVCR7 is derived from previously published literature (Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F. Programmable protein delivery with a bacterial contractile injection system[J]. Nature. 2023; 616:357).

[0395] The generation, extraction, and purification of empty PVC vectors, empty PVCR7 vectors, empty PVCHER2 vectors, and PVC complexes were performed in accordance with previously published literature (Purification of Photorhabdus Virulence Cassette (PVC) Protein Complexes from Escherichia coli for Artificial Translocation of Heterologous Cargo Proteins. Bio-protocol 14(7): e4966.). Specifically, the PVC-V complex (i.e., the empty PVC vector, derived from Photorhabdus asymbiotica ATCC43949) was generated using the methods disclosed in the literature. The empty PVCR7 vector and the empty PVCHER2 vector were obtained by modifying the tail filament protein Pvc13 of the PVC-V complex.

[0396] In the embodiments of this application, the PVC complex refers to the complex formed by the empty PVC / PVCR7 / PVCHER2 carrier and the loaded protein.

[0397] Extraction and purification of PVC complex

[0398] Select a single colony and place it in a container containing 2-4 mL of liquid LB medium supplemented with the appropriate antibiotic; place the container on a shaker and incubate overnight at 37°C and 220 rpm / min; after incubation, transfer the bacterial culture to a container containing 200 mL of liquid LB medium supplemented with the appropriate antibiotic at a ratio greater than 1:100; place the container on a shaker and incubate for 24 h at 30°C and 220 rpm / min; after incubation, centrifuge to collect the bacterial cells; resuspend in 30 mL of PBS, centrifuge again to collect the bacterial sludge; store the bacterial sludge at -80°C for at least overnight.

[0399] Prepare a bacterial lysis buffer with the following formula: 25 mM Tris (pH adjusted to 7.4), 140 mM NaCl, 3 mM KCl, 200 μg / ml lysozyme, 50 μg / ml deoxyribonuclease I, 0.5% Triton X-100, 5 mM MgCl2, and 1× protease inhibitor. For every 200 ml of bacterial sludge collected from LB liquid medium, add 10 mL of bacterial lysis buffer, resuspend thoroughly, and mix well. Incubate the mixture at 37°C for 30 min to allow bacterial lysis. After lysis, centrifuge at 4°C and 12000 rpm / min for 10 min to remove unlysed bacteria and bacterial fragments.

[0400] Transfer the supernatant after bacterial lysis and centrifugation to an ultracentrifuge tube and centrifuge at 200,000 g / min for 1 h at 4 °C. Discard the supernatant and resuspend the precipitate in 1 mL PBS. Centrifuge at 14,000 rpm / min for 10 min at 4 °C. Transfer the supernatant to an ultracentrifuge tube again and centrifuge at 200,000 g / min for 1 h at 4 °C. Discard the supernatant and resuspend the precipitate in 200 μL PBS. Centrifuge at 14,000 rpm / min for 10 min at 4 °C. Collect the supernatant at this point. This is the PVC complex solution obtained after extraction and purification. This solution can be temporarily stored at 4 °C.

[0401] Western blot detection of PVC complex: The loading of the protein was detected using a flag antibody, while the total amount of the complex was detected using a PVC structural protein 16 antibody.

[0402] Removal of endotoxins from purified PVC compounds

[0403] Pre-cool the PVC sample at 4℃. Prepare Triton X-114 with a final concentration of 1-2% (W / V) and also pre-cooled at 4℃. Add it to the pre-cooled PVC sample. After addition, mix thoroughly by inverting the container. Then, incubate the sample in an ice bath for 15 min, inverting the container multiple times during the ice bath to ensure that the sample is always in a uniformly mixed state. After the ice bath, incubate in a water bath at 37℃ for 5 min. After incubation, centrifuge at 37℃ and 12000g for 5 min to completely separate Triton X-114 from the aqueous solution and discard the supernatant. Repeat the above steps 2-3 times.

[0404] Endotoxin detection in PVC composites

[0405] Sample pretreatment and reagent preparation: Endotoxin standards are dissolved according to the labeled potency (P). For P≤20 EU / vial, 10 mL of pyrogen-free water is added to prepare a 10 EU / mL stock solution. The stock solution is then serially diluted with pyrogen-free water (single dilution factor ≤10, vortex mixing ≥30s) to the target concentration (0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1 EU / mL), and prepared fresh each time. When dissolving Limulus Amebocyte Lysate (LAL), 1.7 mL of pyrogen-free water is added, and the solution is gently swirled and mixed. The solution is allowed to stand for 30s and should be used within 10 minutes after dissolution. The chromogenic substrate and stop solution are dissolved according to the instructions and stored in the dark at 2-8℃.

[0406] Standard curve construction: Mix 100 μL of a series of concentration standards (covering at least the range of 0.01-1 EU / mL, including 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, and 1 EU / mL) with an equal volume of LAL reagent and incubate at 37±1℃ (T1 time for the low concentration range of 0.01-0.1 EU / mL and T2 time for the high concentration range of 0.1-1 EU / mL; refer to the kit label for specific times). After incubation, add 100 μL of chromogenic substrate (incubate at 37℃ for 6 min) and 500 μL of stop solution. Measure the absorbance at 545 nm using a microplate reader. Construct a standard curve by plotting the endotoxin concentration of the standards (EU / mL) on the x-axis and Δabsorbance (sample absorbance - blank absorbance) on the y-axis, using linear regression analysis. The goodness of fit should be R0. 2 ≥0.96.

[0407] Protein sample detection: Dilute protein samples with pyrogen-free water to the detection range (dilution factor not exceeding the maximum effective dilution factor MVD); if the sample pH deviates from 6.0-8.0, adjust to neutral using 0.1 M NaOH or HCl; mix 100 μL of the diluted sample with an equal volume of LAL reagent, and incubate and perform the colorimetric reaction according to the standard curve conditions. Set up 3 technical replicates for each sample, and perform the negative control simultaneously using pyrogen-free water instead of the sample.

[0408] Data analysis and calculation: The endotoxin concentration in the sample was calculated based on the standard curve equation, and the concentration of the original solution was converted by the dilution factor to calculate the endotoxin content.

[0409] PVC-treated cells

[0410] After 24 hours of cell culture, empty PVC vector, empty PVCR7 vector, empty PVCHER2 vector, or PVC complex were added to the cell culture medium at a concentration of 15 μg / mL, unless otherwise specified. After 24 hours of addition, cell viability was detected using CCK-8 according to the reagent manufacturer's instructions. Immunofluorescence was performed 18 hours after addition.

[0411] Bioinformatics analysis of PagT1 protein

[0412] Cellular sublocalization analysis of the PagT1 amino acid sequence was performed using the DeepLoc 2.0 protein language model (https: / / services.healthtech.dtu.dk / services / DeepLoc-2.0 / ). Its three-dimensional structure was predicted using AlphaFold3 (https: / / deepmind.google / technologies / alphafold / alphafold-server / ), the potential presence of an active pocket was predicted using DoGSiteScorer (https: / / proteins.plus / ), and proteins with similar three-dimensional structures to PagT1 were searched using Foldseek (https: / / search.foldseek.com / search).

[0413] Confocal microscopy observation of cellular sublocalization of PagT1 during transient expression in A549 cells

[0414] Cells were seeded evenly in glass dishes, transfected after 12 hours, replaced with fresh culture medium after 6 hours, and observed and photographed using a laser confocal microscope after 18 hours.

[0415] Immunofluorescence staining

[0416] Cells were evenly seeded onto 20 mm cell spreaders in 12-well plates. After 24 h, empty PVC vector, empty PVCR7 vector, empty PVCHER2 vector, or PVC / PVCR7 / PVCHER2 loaded with PagT1 were added to the wells for 18 h. After treatment, the culture medium was aspirated, and the cells were fixed with methanol for 3 min. 1 mL of 5% BSA was added to each well, and the cells were blocked at 37 °C for 1 h. 100 μL of the corresponding primary antibody diluted with 1×PBS was added to each well (antibody information is shown in Table 3), and the cells were incubated at 37 °C for 1 h. 100 μL of the corresponding fluorescent secondary antibody diluted with 1×PBS was added to each well (antibody information is shown in Table 3), and the cells were incubated at 37 °C for 1 h. The cell spreaders were inverted onto glass slides containing mounting medium (containing DAPI), and observed and photographed using a laser confocal microscope.

[0417] comet electrophoresis experiment

[0418] After 24 hours of cell treatment, comet electrophoresis was performed using a comet electrophoresis kit (Shanghai Beyotime Biotechnology Co., Ltd.). Propidium iodide staining resulted in red fluorescence, with an excitation wavelength (Ex) / emission wavelength (Em) of 535 / 617 nm. Tail moments were statistically analyzed; the tail moment was calculated as the product of tail length and the percentage of DNA in the tail.

[0419] CRISPR / Cas9 sgRNA library screening

[0420] (1) J774A.1 cells were transduced with LentiCas9 Blast lentivirus and screened with Blastidin.

[0421] (2) sgRNA library lentiviral packaging

[0422] Prepare 200 μg of Mouse Brie CRISPR knockout pooled library plasmid (https: / / www.addgene.org / pooled-library / broadgpp-mouse-knockout-brie / ) with an endotoxin content ≤0.01 EU / μg. The quality of the library is verified by NGS. The library contains at least 99% sgRNA, and the Gini coefficient of sgRNA distribution must be less than 0.1.

[0423] Lentiviral packaging plasmids and sgRNA library plasmids were transfected into 293T cells. Lentiviral virus was harvested 72 h after transfection and its titer was determined.

[0424] (3) Screening for the optimal transduction cell density

[0425] Puromycin killing curves were determined in Cas9-overexpressing J774A.1 cells. J774A.1 cells were transduced with different volumes of Brie library virus to select appropriate puromycin concentrations and optimal seeding densities.

[0426] (4) Lentiviral Brie library transduction and cell library generation

[0427] Cas9-overexpressing J774A.1 cells were transduced with a mixed Brie lentiviral library with an MOI of approximately 0.3 and screened with appropriate antibiotics. Cells were then analyzed by NGS.

[0428] (5) Determine the optimal screening conditions for PVC

[0429] By adding different concentrations of PVC-PagT1, the concentration (30 μg / mL) that caused approximately 80% death in Cas9-overexpressing J774A.1 cells was selected as the screening criterion.

[0430] (6) The cell library was screened three times using 30 μg / mL PVC-PagT1 and empty vector PVC.

[0431] (7) After screening, the genome is extracted and an NGS sequencing library is constructed through DNA fragmentation, PCR enrichment and fragment screening.

[0432] (8) NGS experimental procedure and data analysis

[0433] a. Library construction and sequencing

[0434] NGS sequencing libraries were constructed through steps such as PCR enrichment and fragment screening. After the libraries passed the quality test and qPCR quantification test by Agilent 2100 Bioanalyzer, they were sequenced on the Illumina platform.

[0435] PCR enrichment was performed by adding adapters containing P5, P7, and index in a PCR reaction.

[0436] The constructed library products were purified using a PCR product purification kit;

[0437] The fragment distribution of the library was detected using an Agilent 2100 Bioanalyzer, and the library was quantified using qPCR.

[0438] b. Data Analysis

[0439] The raw data after the test is processed by bcl2fastq and split according to the sample index to obtain the corresponding data for the sample;

[0440] Based on the library construction method, the original sequences were deconnected to remove low-quality reads, resulting in clean data. R1 and R2 were then merged based on overlap to obtain the data used for analysis.

[0441] The sgRNA sequences were identified using MAGeCK (version 0.5.9.6) software, and the abundance of sgRNA sequences was statistically analyzed.

[0442] Using MAGeCK software, based on the aligned sgRNA and gene counts, the counting results were first standardized, and then the differences in sgRNA between samples were compared. Differential genes between samples were analyzed, and database annotations and visualizations were performed.

[0443] Gene knockdown was performed on J774A.1 cells using a lentiviral shRNA vector.

[0444] (1) Lentiviral packaging and production

[0445] 24 hours before plasmid transfection, 293T cells were digested with trypsin (log phase), followed by cell counting, and the cell density was set at 6 × 10⁶ cells / year. 5 10 cells / mL were seeded in 10 cm cell culture dishes and cultured at 37°C in a 5% CO2 incubator for 24 h. When the cell confluence reached 70%-80%, the cells were used for transfection. The culture medium was replaced with basal medium 2 h before transfection.

[0446] 293T cells were co-transfected with the shRNA vector pTSB-U6-PGK-Fluor-2A-ARGs required for lentiviral packaging and the helper plasmids pSPAX2 and pMD2.G. After 8 hours, the culture medium was aspirated, the cells were washed with PBS, the supernatant was discarded, and fresh culture medium was replaced. The cells were then incubated at 37°C and 5% CO2 for another 48 hours.

[0447] Collect the supernatant into centrifuge tubes, centrifuge at 2000 rpm for 10 min to remove the precipitate, ultrafilter and determine the titer, then aliquot and freeze at -80℃.

[0448] (2) Construction of stable strains by lentiviral infection of J774A.1 cells

[0449] 18-24 hours before lentivirus transfection, J774A.1 cells were inoculated at a dose of 1×10⁻⁶. 5 Seed cells per well into a 24-well plate, replace with serum-free medium, add 8 μg / mL polybrene, add lentivirus, and incubate at 37°C and 5% CO2 for 6 h. After incubation, replace with fresh medium containing serum and continue culturing for 48 h, then observe fluorescence expression. Add puromycin to screen for stable knockdown cells. qRT-PCR is used to detect the gene knockdown efficiency of stable knockdown cells.

[0450] Cells treated with NAD+ and inhibitors

[0451] Cells were seeded in 96-well or 24-well plates and cultured for 24 h. Then, NAD+, PARP1 inhibitor Rucaparib, JAK inhibitor Ruxolitinib, or STAT inhibitor SH-4-54 were added to the culture medium. After 3 h, empty PVC vector, empty PVCR7 vector, or PVC / PVCR7 loaded with PagT1 were added to the culture medium and treated for 24 h. Cell viability was then detected or cell lysates were collected for Western blot analysis.

[0452] Transmission electron microscopy observation of PVC composites

[0453] (1) Network preprocessing

[0454] The carrier mesh was hydrophilicated using a glow discharge apparatus. A 200-mesh carbon-coated copper mesh was placed face up on filter paper and then placed in the glow discharge apparatus. The parameters were set to negative electrode, current 15 mA, and discharge for 45 s to enhance the hydrophilicity of the carbon film.

[0455] (2) Protein sample adsorption

[0456] The extracted PVCHER2, PVCHER2-PagT1 and other protein samples were diluted to 0.3 mg / mL using PBS (based on Nanodrop assay). 10 μL of the protein solution was dropped onto the hydrophilicated grid and allowed to stand for 2 min. Excess liquid was gently absorbed from the edge of the grid with filter paper moistened with distilled water, avoiding contact with the carbon film.

[0457] Place 100 μL of distilled water onto the sealing film. Hold the edge of the screen with tweezers, invert the screen so that the front of the screen contacts the surface of the water droplet, gently shake the tweezers, and clean the screen for 15 seconds. Use filter paper moistened with distilled water to gently absorb excess liquid from the edge of the screen, avoiding contact with the carbon film. Also, be careful to avoid contact between the back of the screen and the liquid.

[0458] (3) Negative staining of samples

[0459] Place three drops of 100 μL 1% uranium acetate staining solution onto the sealing film. Hold the edge of the screen with tweezers, invert the screen so that the front of the screen contacts the surface of the first staining solution drop. Gently shake the tweezers to clean the screen for 15 seconds. Repeat the steps for the second and third staining solutions. Let the screen stand and continue staining for 1 minute through the staining solution adsorbed on the surface. Gently absorb excess liquid from the edge of the screen with filter paper moistened with distilled water, avoiding contact with the carbon film. During the entire staining process, avoid contact between the back of the screen and the liquid. After letting the screen dry at room temperature for 10 minutes, return it to the sample box for storage.

[0460] (4) Observation by transmission electron microscopy (TEM)

[0461] Select a 120 kV transmission electron microscope, place the grid face up into the TEM sample holder for observation, look for areas where protein particles are evenly distributed, and adjust the focus for imaging.

[0462] Bladder cancer organoid sample collection

[0463] Bladder cancer organoids were obtained from Beijing Ketu Medical Technology Co., Ltd. Frozen bladder cancer organoids were thawed and cultured. When the organoids reached a passageable state, they were thawed at 4°C using a Pasteur pipette. The cultured organoids were then collected using OrganoPro and added to the culture. TM The passage enzyme in the bladder cancer organoid culture kit is used to enzymatically digest bladder cancer organoids to obtain a single-cell suspension; after cell counting, OrganoPro is used... TM The cell concentration in the bladder cancer organoid culture medium was adjusted to 1.0 × 10⁻⁶ cells. 6 Collect cells / mL and take an appropriate amount of cell suspension and place it on ice for later use; at the same time, prepare the matrix gel mixture on ice for later use; mix the cell suspension with matrix gel, add 200 μL of the mixture to each well of a 24-well plate, incubate at 37°C for 30 min, and then add 200 μL of OrganoPro to each well. TM Bladder cancer organoid culture medium, after static culture for 2 days, at which time the formation and growth of bladder cancer organoids can be observed.

[0464] When the diameter of the bladder cancer organoids reaches approximately 200 μm or larger, collect the cultured bladder cancer organoids into a 15 mL centrifuge tube using a Pasteur pipette, centrifuge at 300 g for 5 min, discard the supernatant, resuspend the precipitate in PBS, wash by pipetting, centrifuge at 300 g for 5 min, discard the PBS, and collect the organoid precipitate; cut off the tip of a 1 mL pipette tip, take 500 μL of 4% paraformaldehyde tissue cell fixation solution, mix it with the organoids by pipetting, and transfer it into a 1.5 mL centrifuge tube; then, using the cut-off pipette tip, transfer the organoids into the 1.5 mL centrifuge tube by pipetting, and store at 4°C.

[0465] Immunohistochemistry of bladder cancer organoids

[0466] After the samples were fixed in formalin, they were subjected to immunohistochemical analysis. The specific information on the primary and secondary antibodies is shown in Table 3. Hematoxylin was used to stain the cell nuclei.

[0467] Immunofluorescence of bladder cancer organoids

[0468] After the samples were fixed in formalin, they were subjected to immunofluorescence staining. The specific information on the primary and secondary antibodies is shown in Table 3. DAPI was used to stain the cell nuclei.

[0469] bladder cancer organoid drug efficacy testing

[0470] Organoid seeding: When the bladder cancer organoids reach a passageable state, melt the Matrigel at 4°C for later use; collect the cultured bladder cancer organoids and add OrganoPro. TM The passage enzyme digest in the bladder cancer organoid culture kit is used to enzymatically digest bladder cancer organoids to prepare a single-cell suspension; after cell counting, the cell concentration is adjusted to 1.0 × 10⁶ cells / year using Matrigel. 6 Cells / mL; mix the cell pellet with matrix gel thoroughly, add 4 μL of the mixture to each well of a 96-well plate, incubate at 37°C for 30 min, then add 86 μL of OrganoPro to each well. TM Bladder cancer organoid culture medium was used to incubate the bladder cancer organoids for 2 days to observe their initial formation and growth.

[0471] Preparation and dilution of empty PVCHER2 vector, PVCHER2 loaded with PagT1 (PVCHER2-PagT1), and PVCHER2 (PVCHER2-217219) with PagT1 D217 and E219 mutations to A: serial dilutions were performed using PBS in a biosafety cabinet.

[0472] Add the test drug: After observing the formation and growth of bladder cancer organoids, add different concentrations of freshly prepared empty PVCHER2 vector, PVCHER2-PagT1, and PVCHER2-217219 in sequence, and incubate in an incubator for 120 hours.

[0473] Chemiluminescence assay: After culture, add 50 μL of cell viability fluorescence detection reagent and measure the fluorescence value.

[0474] U251-Luci cell construction

[0475] U251 cells were infected with a lentivirus containing the Ubi-MCS-firefly_Luciferase-IRES-Puromycin element; positive cells were selected after 24 hours by adding puromycin; and luciferase expression was detected by the Bright-Lite luciferase detection system (Vazyme, DD1204-02).

[0476] mice

[0477] The 6-week-old male NTG mice used in this application were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. All SPF-grade animals were housed in a temperature and humidity-controlled environment with a 12h / 12h light / dark cycle, and were provided with standardized rodent food and drinking water.

[0478] Orthotopic brain tumor xenograft and PVC therapy

[0479] NTG mice were anesthetized with 100 mg / kg ketamine and 10 mg / kg thiazide; after disinfection and skin preparation, the mice were fixed on a stereotaxic apparatus to expose the scalp and skin; U251-luci cells (2×10⁻⁶) were then injected. 4 One cell line (dissolved in 2 μL PBS) was injected into the brain at an injection site 1 mm lateral to the posterior fontanelle, 1 mm anteriorly, and 2 mm deep. Tumor-bearing mice were randomly divided into 4 groups. On day 10 post-transplantation, the lateral ventricles of each group were injected with PBS, empty PVCHER2 vector (control), low-dose PVCHER2-PagT1 (15 μg), and high-dose PVCHER2-PagT1 (30 μg), respectively. The injection site was 1 mm lateral to the posterior fontanelle, 0.46 mm posteriorly, and 2.2 mm deep. The mice were euthanized on day 35 post-inoculation.

[0480] Bioluminescence imaging (BMI)

[0481] On days 7, 21, and 35 post-inoculation, patients received intraperitoneal injections of 150 mg / kg d-fluorescein (PerkinElmer, 122799) and were placed in an IVIS Lumina III (PerkinElmer) for bioluminescence imaging of in situ gliomas. Bioluminescence signals were continuously acquired using a 1 / 8-inch filter with an exposure time of 1 min. Results were analyzed using Living Image 4.2 software (Caliper Life Sciences).

[0482] Magnetic resonance imaging (MRI)

[0483] In vivo MRI was performed using a 7.0 T small animal MRI system. Thirty minutes prior to the MRI examination, mice were injected via the tail vein with 0.1 mmol / kg Magnevist (gadolinium-DTPA). T1-weighted MRI images of the mouse brain in the coronal plane were acquired using spin-echo (SEMS) sequences with the following parameters: field of view (FOV) = 20 × 20 mm; slice thickness = 1 mm (zero slice gap); repetition time (TR) = 400 ms; echo time (TE) = 18.0 ms; image matrix = 256 × 256; mean = 10. Regions of interest (tumors in 7-day and 35-day mouse models) were manually plotted on the images to determine changes in tumor volume.

[0484] Statistical analysis

[0485] Data analysis was performed using GraphPad Prism 6 software. All data were presented as a mean and the standard error of the mean (SEM). For comparisons between two groups of variables, Student's t-test was used; when comparing differences between three or more groups of variables, ANOVA (Analysis of Variance) was used, followed by Bonferroni's post-hoc analysis. To determine statistical significance, a p-value less than 0.05 was considered statistically significant; a p-value less than 0.01 was considered statistically significant; and a p-value less than 0.001 was considered highly statistically significant.

[0486] Example 1: Functional identification of the virulence factor PagT1

[0487] 1.1 Effects of PagT1 on host cell viability

[0488] The PagT1 gene (PAU_02097, whose nucleotide sequence is shown in SEQ ID NO: 39) is located downstream of the PVC-II structural gene (Photorhabdus asymbiotica ATCC43949) (Figure 1A). The two genes before and after it (MARTX and YopT) are virulence factors, suggesting that PagT1 may be a potential virulence factor of the non-symbiotic luminescent bacterium (Photorhabdus asymbiotica).

[0489] The inventors of this application searched for homologous proteins of PagT1 using the BLAST function in the NCBI database and found that most of them were distributed in the genus *Photorhabdus*, while they were also found in genera such as *Okeania*, *Pseudomonas alkylphenolica*, *Deltaproteobacteria bacterium*, *Photorhabdus heterorhabditis*, *Photorhabdus australis*, *Burkholderia ubonensis*, and *Streptomyces avermitilis*. Figure 1BThe inventors of this application selected homologous proteins of PagT1 from different bacterial genera, constructed them into the eukaryotic expression vector pEGFP-C1, and transiently expressed them in 293T cells. Cell viability was then detected by CCK-8 assay, and the results are shown in Figure 1C. It was found that PagT1 could cause the strongest cytotoxicity compared with homologous proteins.

[0490] Transforming the pBBRN-PagT1-flag plasmid into a PVC expression strain and purifying it yielded the PVC complex (PVC-PagT1). Western blot analysis detected the flag-tagged loading protein PagT1 and the final component of the PVC complex assembly, Pvc16, indicating successful assembly of PVC-PagT1 (Figure 2A). This result confirms that PagT1 can be loaded into PVC without the need for an additional loading signal peptide at the N-terminus, suggesting that PagT1 is a natural effector of PVC.

[0491] Furthermore, when PVC-PagT1 was added to J774A.1 cells, cell viability was detected by CCK-8 assay. It was found that J774A.1 cells showed significant cell death, while empty PVC did not have a significant effect on the cells (Figure 2B), indicating that PVC-PagT1 can target mouse macrophages to deliver PagT1.

[0492] Furthermore, PVCR7-PagT1 was obtained by loading PagT1 into PVC-Ad5 (RGD / PK7) (Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F. Programmable protein delivery with a bacterial contractile injection system[J]. Nature. 2023; 616:357) (hereinafter referred to as PVCR7). PVCR7 possesses targeting similar to adenovirus Ad5 (Figure 2 A). When the purified PVCR7-PagT1 was added to cells, it was found to cause significant death in THP-1 cells (Figure 2 C) and BMDM cells (Figure 2 D).

[0493] The above results indicate that Pagt1 is a virulence factor of PVC, can be loaded into PVC, and can cause cell death in mice and humans.

[0494] 1.2 PagT1 is localized in the nucleus of eukaryotic cells and causes DNA damage in host cells.

[0495] To explore the eukaryotic cytotoxic mechanism of PagT1, this example investigated the sublocalization of PagT1 in cells. The amino acid sequence of PagT1 was analyzed for sublocalization using the DeepLoc 2.0 protein language model for predicting sublocalization. The results are as follows: Figure 3 As shown, the results indicate that the most likely cellular sublocality of PagT1 is the cell nucleus, and that there are nuclear localization signals (NLS) at its N-terminus and C-terminus, respectively.

[0496] Next, experiments were conducted to verify whether PagT1 would localize to the cell nucleus. First, PagT1 labeled with GFP was transiently expressed in A549 cells. After 18 hours, confocal microscopy revealed that PagT1 co-localized with the cell nucleus (Figure 4A), confirming the predicted nuclear localization of PagT1. Furthermore, in this embodiment, PagT1 labeled with a flag was directly delivered to A549 cells via PVCR7, and immunofluorescence detection using a flag antibody was performed, again observing the nuclear localization of PagT1 (Figure 4B).

[0497] By constructing mutants of PagT1 with the N-terminal and C-terminal NLS removed separately and simultaneously (Figure 4C), the importance of NLS for PagT1 nuclear localization was investigated. After transient expression of A549, confocal microscopy revealed that the removal of N-terminal (Del-N-NLS, SEQ ID NO:31) or C-terminal (Del-C-NLS, SEQ ID NO:32) NLS did not affect the nuclear localization of PagT1; only the simultaneous removal of N-terminal and C-terminal (Del-N, C-NLS, SEQ ID NO: 33) NLS caused PagT1 to lose its nuclear localization (Figure 4A).

[0498] The above results indicate that this application has identified a "dual-function" signal peptide, which can both guide the loading of effector proteins into the PVC protein complex and serve as a nuclear localization signal to guide the localization of effector proteins to the cell nucleus.

[0499] To further verify this, different lengths of the N-terminus of PagT1 were extracted and fused with Tcst protein (Trichosanthin, TcsT from Trichosanthes kirilowii) for expression. It was found that peptides of different lengths of the N-terminus of PagT1 (PagT1-N15 to PagT1-N70, SEQ ID NO: 19-30) (except for PagT1-N15, all of which contain the complete N-terminal NLS of PagT1) could all act as signal peptides to guide Tcst loading into PVC (Figure 4 D). This demonstrates the bifunctionality of the PagT1 N-terminal signal peptide.

[0500] To investigate the mechanism of PagT1's cytotoxicity, this application verified whether PagT1's cytotoxicity is related to its localization in the eukaryotic nucleus by transient expression in eukaryotic cells and by delivery of wild-type PagT1 (WT) and its NLS mutant using PVCR7. The results are shown in Figures 5A-5B. After transient expression in 293T cells (Figure 5A) and delivery of PVCR7 to THP-1 cells (Figure 5B), CCK-8 assays of cell viability revealed that simultaneous removal of the N-terminal and C-terminal NLS of PagT1 significantly reduced PagT1-induced cell death, while removal of the N-terminal and C-terminal NLS separately did not. This confirms the importance of PagT1's nuclear localization for its cytotoxicity.

[0501] Furthermore, nuclear damage in cells following PagT1 delivery with PVC was examined. Twenty-four hours after PagT1 delivery to mouse and human macrophages using PVC or PVCR7, Western blot detected the production of phosphorylated histone H2AX (phosphorylated S139), a significant marker of nuclear damage, in J774A.1, THP-1, and BMDM cells (Fig. 6A); and immunofluorescence also revealed the formation of more phosphorylated histone H2AX spots in the nuclei (Fig. 6B). Additionally, comet electrophoresis showed tailing in THP-1 cells after PagT1 delivery (Fig. 6C), with the tail distance in the PagT1 group significantly higher than that in the PBS and empty PVCR7 groups (Fig. 6D). These results indicate that PagT1 can cause damage to the eukaryotic genome and is a genotoxin.

[0502] 1.3 Determination of PagT1 active sites

[0503] Since PagT1 can cause genome damage in eukaryotic cells, its potential enzymatic activity was investigated to determine its specific mechanism. The three-dimensional structure of PagT1 was predicted using AlphaFold3 (Figure 7A), and the active pocket was predicted using DoGSiteScorer (Figure 7D). Using Foldseek to search for proteins with similar three-dimensional structures to PagT1, a search in the PDB database revealed ADP-ribotransferases with spatial structures similar to those near the active pocket of PagT1 (Figures 7B-7C), suggesting that PagT1 may also be an ADP-ribotransferase. Sequence alignment with its homologous proteins showed that the conserved amino acid residues in PagT1 are D217 and E219, which are also present in the PagT1 active pocket (Figures 7E-7F).

[0504] The above results suggest that D217 and E219 may be the active sites of PagT1. Mutating these two sites to A created the PagT1 mutant 217219 (SEQ ID NO: 34). After transient expression in 293T cells, mutant 217219 lost the cytotoxicity of PagT1 and had virtually no impact on cell viability (Figure 7 G), indicating that D217 and E219 are the active sites of PagT1.

[0505] 1.4 PagT1 induces PARP1-dependent cell death in eukaryotic cells (Parthanatos).

[0506] PARP-1 is a key protein of Parthanatos and also a DNA repair protein. First, we examined the expression of PARP-1 in THP-1 cells after PagT1 was delivered via PVCR7. Western blot results showed that PARP1 expression in THP-1 cells was significantly increased after PagT1 delivery (Figure 8A).

[0507] Since Parthanatos involves nicotinamide adenine dinucleotide (NAD+) depletion, to determine whether PagT1-induced cell death was caused by Parthanatos, NAD+ was added to the cell culture medium before delivering PagT1 to the cells. This ultimately rescued PagT1-induced cell death in J774A.1 (Fig. 8 B) and THP-1 (Fig. 8 C) cells. Furthermore, inhibiting the poly-ADP-ribose polymerase activity of PARP1 using the inhibitor Rucaparib also inhibited PagT1-induced cell death (Fig. 8 E) in J774A.1 (Fig. 8 D) and THP-1 cells.

[0508] Furthermore, pADPr in J774A.1 and THP-1 cells was detected after PagT1 was delivered via PVC or PVCR7. Immunofluorescence detected that more pADPr was produced in J774A.1 (Fig. 9A) and THP-1 (Fig. 9B) cells.

[0509] The above results indicate that PagT1 induces cell death via Parthanatos.

[0510] 1.5 PagT1 mediates the effects of type I interferon-JAK-STAT signaling in eukaryotic Parthanatos cells.

[0511] To identify the signaling pathways involved in Parthanatos in eukaryotic cells after PagT1 delivery, CRISPR / Cas9 sgRNA library screening technology was used for analysis. Based on a forward screening strategy, the abundance of sgRNA in pre-screening cell libraries, empty PVC control groups, and PVC-delivered PagT1 experimental groups was compared using the MAGeCK MLE analysis method to screen genes and perform gene function enrichment annotation. Large-scale screening was performed on J774A.1 cells using the Mouse Brie CRISPR knockout pooled library to identify key genes involved in Parthanatos in J774A.1 cells after PVC delivery of PagT1.

[0512] The results of positive selection are used to calculate the "beta score" of each target gene using the MLE (maximum likelihood estimation) algorithm to measure the degree of selection during gene perturbation. The distribution of the beta scores of the selected genes is as follows: Figure 10 As shown.

[0513] The differential distribution of gene beta scores in positive selection is as follows: Figure 11 As shown in Table 9, the top three genes were Irf9, STAT1, and TYK2, which are key genes in the JAK-STAT signaling pathway. KEGG enrichment of the positively selected genes also enriched JAK-STAT and type I interferon signaling pathways. Figure 12 ).

[0514] Table 9 Top 10 genes selected by positive screening

[0515] Gene EntrezID symbol beta score difference Ranking Irf9 16391 Irf9 17.01579 1 STAT1 20846 Stat1 16.15256 2 TYK2 54721 Tyk2 14.43434 3 Rab9 56382 Rab9 13.94099 4 E230019M04Rik 331537 E230019M04Rik 13.02971 5 Olfr1126 258834 Olfr1126 10.75014 6 Smad4 17128 Smad4 10.72474 7 Nrp2 18187 Nrp2 10.60565 8 Eif2d 16865 Eif2d 10.42453 9 Slc17a5 235504 Slc17a5 7.947586 10

[0516] In the classical pathway, the type I IFN receptors IFNAR1 / 2 interact, inducing conformational changes that activate the receptor-associated JAK (Janus kinase) family (JAK1, JAK2, and TYK2). This activation promotes the recruitment and phosphorylation of signal transduction and transcriptional activation (STAT) proteins. Phosphorylated STATs then dimerize and bind to Irf9 to form the IFN-stimulated gene factor 3 (ISGF3) complex. After translocating to the nucleus, this complex acts as a transcription factor, regulating the expression of IFN-stimulated genes (ISGs). Therefore, the PagT1-induced Parthanatos process may be involved in the type I interferon-JAK-STAT signaling pathway.

[0517] The roles of Irf9, STAT1, and TYK2 genes, as well as the type I interferon-JAK-STAT signaling pathway, in PagT1-induced parthanatos were further verified. Irf9, STAT1, and TYK2 genes in J774A.1 cells were stably knocked down using an shRNA lentiviral vector, followed by PagT1 delivery via PVC. The results showed that, compared with the control shRNA group, knockdown of Irf9, STAT1, and TYK2 genes significantly reduced cell death induced by PVC delivery of PagT1. Figure 13 ).

[0518] In addition, JAK and STAT1 were inhibited by the JAK inhibitor Ruxolitinib and the STAT1 inhibitor SH-4-54, respectively. The results showed that Ruxolitinib and SH-4-54 could significantly improve cell death induced by PagT1 delivery to J774A.1 and THP-1 cells via PVC / PVCR7 (Figure 14A-Figure 14D).

[0519] Next, we analyzed Irf9, another key protein in the type I interferon-JAK-STAT signaling pathway. In the classic type I interferon-JAK-STAT signaling pathway, Irf9 translocates to the cell nucleus. Therefore, we examined the cellular sublocalization of Irf9 in BMDM cells after PagT1 delivery via PVCR7. Immunofluorescence showed that after PagT1 delivery, Irf9 was localized in the cell nucleus, while in the empty PVC and mutant PVCR7-217219 groups, Irf9 was distributed throughout the entire cell. Figure 15 ).

[0520] In summary, the above results demonstrate the crucial roles of type I interferon-JAK-STAT signaling pathway-related proteins Irf9, STAT1, TYK2, and JAK in PagT1-induced cellular parthanatos.

[0521] 1.6 IFN-β-induced STAT1 phosphorylation is essential for PagT1-induced Parthanatos.

[0522] To further confirm that the JAK-STAT signaling pathway is activated by type I interferon during PagT1-induced cellular parthanatos, this example used RNA-seq to detect changes in the transcriptome of J774A.1 cells after PagT1 delivery via PVC compared to the empty PVC group. The results are as follows: Figure 16 As shown, the transcriptional level of the interferon IFN-β gene increased after PagT1 delivery.

[0523] IFN-β is a type I interferon. qRT-PCR analysis of the transcriptional levels of IFN-β and other type I interferons (IFN-κ, IFN-α, and IFN-ε) revealed that the most significant increase in IFN-β transcriptional levels occurred in THP-1 cells after PagT1 delivery via PVCR7 (Figures 17A-17D), suggesting that activation of the JAK-STAT signaling pathway during cellular Parthanatos may be induced by IFN-β. Furthermore, validation in BMDM cells also showed a significant increase in IFN-β gene transcriptional levels after PagT1 delivery. Figure 18 ).

[0524] To further verify whether PagT1 activates the host cell interferon signaling pathway, THP1-Dual cells (containing the secretory luciferase reporter gene Lucia driven by the ISG54 minimal promoter with five interferon-stimulating response elements) were used. Interferon signaling pathway activation was detected by Lucia gene expression. After treating cells with PBS, empty PVCR7, PVCR7-PagT1, and PVCR7-217219, a higher Lucia gene expression level was observed in the PVCR7-PagT1 group. Figure 19 This indicates that the IRF signaling pathway was significantly activated, meaning that the host cell interferon signaling pathway was activated.

[0525] This embodiment further confirmed the activation of the JAK-STAT signaling pathway by detecting STAT1 phosphorylation. The results showed that after J774A.1, THP-1, and BMDM cells were delivered PagT1 via PVC / PVCR7, Western blot detected significant phosphorylated STAT1 (phosphorylated Y701, p-STAT1), while after cell treatment with empty PVC and PVCR7-217219, p-STAT1 showed no significant change compared to the PBS group. Figure 20 ).

[0526] Furthermore, to investigate the relationship between PagT1-activated PARP1 and the type I interferon signaling pathway, PARP1 activity in THP-1 cells was inhibited by the PARP1 inhibitor Rucaparib. It was found that after PVR7 delivered PagT1, the phosphorylation level of STAT1 in THP-1 cells was significantly reduced compared to the DMSO control group. Figure 21 This indicates that PagT1 activation of the type I interferon signaling pathway is caused by PARP1 activation.

[0527] In summary, the results in Examples 1.1-1.6 of Example 1 indicate that PagT1 is a virulence factor of PVC, with its active sites being D217 and E219. When PagT1 is loaded into PVC and delivered to eukaryotic cells, it localizes to the cell nucleus using its own nuclear localization signal. During this process, PagT1 consumes NAD+ through ADP-ribotransferase activity, leading to NAD+ depletion. This affects the PARP1 repair process for genomic damage, thereby inducing cellular genome damage and resulting in PARP1-dependent parthanatos. Subsequently, cell death is ultimately caused through the type I interferon-JAK-STAT signaling pathway.

[0528] Example 2: Engineered targeted PVC delivery of PagT1 for precise tumor killing

[0529] 2.1 PagT1 exhibits pan-toxicity to eukaryotic cells

[0530] After loading PagT1 with PVCR7, it was found that PVCR7-PagT1 could significantly kill HeLa, A549, HepG2 and WEHI-Rb-1 cells, while loading the mutant 217219 did not significantly affect the viability of these tumor cells (Figure 22 A-Figure 22 D). This indicates that PagT1 has pan-toxicity to eukaryotic cells and is cytotoxic to a variety of human tumor cells in addition to mouse and human macrophages.

[0531] 2.2 Construction of targeted PVCs targeting tumor-associated antigens such as HER2

[0532] By modifying the PVC delivery carrier tail filament protein Pvc13, a targeted PVC that specifically targets tumor antigens such as Her2 was obtained. By loading PagT1, a precise killing tool targeting tumor cells was finally obtained.

[0533] HER2 is an important target for tumor targeted therapy. Studies have shown that over 30% of human tumors exhibit HER2 gene overexpression. This embodiment utilizes a validated, designed ankylosing squaring protein (DARPin) that specifically binds to HER2 on the cell membrane. By fusing DARPin to Pvc13 (replacing the Pvc13 receptor-binding domain with a HER2-DARPin linker, its nucleotide sequence as shown in SEQ ID NO: 10), PVC is targeted to obtain HER2-targeting PVC—Pvc13-HER2-DARPin (PVCHER2). Figure 23A ).

[0534] After loading PagT1, PVCHER2-PagT1 was purified. Western blot detected flag-tagged PagT1 and Pvc16, indicating successful assembly of the PVC complex (Fig. 23 B). In addition, PVCHER2 and PVCHER2-PagT1 were also observed by negative staining (Fig. 23 C-Fig. 23 D).

[0535] 2.3 Cell-level detection of PVCHER2 targeting and delivery efficiency

[0536] The targeting of PVCHER2 and the delivery efficiency of PagT1 were tested using U251 (HER2-positive), T98G (HER2-positive), and U87MG (HER2-negative) cells. The results, shown in Figures 24A-24C, indicate that after loading PagT1, PVCHER2-PagT1 caused significant cell death in T98G and U251 cells, while no significant cell death was observed in U87MG cells. Empty PVCHER2 and PVCHER2-217219 did not significantly affect the cell viability of T98G and U251 cells, suggesting that targeted PVCHER2 can effectively deliver the virulence factor PagT1, and that the delivery process is highly specific.

[0537] Furthermore, this embodiment tested whether PVCHER2-PagT1 could recognize and kill human bladder tumor cells. The results are shown in Figures 25A-25B. Cell viability was assessed 24 hours after the addition of empty PVCHER2, PVCHER2-PagT1, and PVCHER2-217219. The results showed that PVCHER2-PagT1 caused significant cell death in BIU87 and T24 cells, while empty PVCHER2 and PVCHER2-217219 did not significantly affect the viability of BIU87 and T24 cells.

[0538] 2.4 Targeted PVCHER2 delivery of PagT1 leads to Parthanatos in tumor cells.

[0539] This embodiment further verified whether PagT1 delivery via targeted PVCHER2 would induce Parthanatos in BIU87 cells. Western blot results showed that after PagT1 delivery via PVCHER2, PARP1 expression in BIU87 cells was significantly increased, while the expression of PARP1 in the empty PVCHER2 and PVCHER2-217219 groups was not significantly different from that in the PBS group. Figure 26 ).

[0540] The core mechanism of Parthanatos is that PARP1 activation leads to the accumulation of PAR polymers, which in turn triggers the release and translocation of AIF from mitochondria to the nucleus. Therefore, we further examined the cellular sublocalization of AIF in BIU87 cells after PVCHER2 delivery of PagT1. Immunofluorescence results showed that after PVCHER2 delivery of PagT1, AIF in BIU87 cells partially translocated to the nucleus and incompletely co-localized with mitochondria. Compared with the PBS group, the empty PVCHER2 and PVCHER2-217219 groups showed no significant change in the cellular sublocalization of AIF, and continued to co-localize with mitochondria. Figure 27 ).

[0541] The above results demonstrate at the cellular level that the HER2-targeting delivery vector PVCHER2-PagT1, loaded with the virulence factor PagT1, can specifically induce parthanatos in glioma cells and bladder tumor cells, and PVCHER2-PagT1 has the potential to be used as a precision oncology drug.

[0542] 2.5 PVCHER2-PagT1 significantly kills organoids from bladder cancer.

[0543] Tissue samples obtained from a 78-year-old male patient with invasive urothelial carcinoma of the left renal pelvis that had metastasized to the bladder, after undergoing transurethral resection of the bladder, were used to establish an organoid model. Immunohistochemistry and immunofluorescence detected HER2 expression in the bladder cancer organoid model. Figure 28 ).

[0544] Empty PVCHER2, PVCHER2-PagT1, and PVCHER2-217219 were added to organoid culture medium to detect the killing effect of PVCHER2-PagT1 on bladder cancer organoids. The results are shown in Figures 29A-29C. PVCHER2-PagT1 can effectively kill bladder cancer organoids with an IC50 of approximately 0.792 μg / mL, while empty PVCHER2 and PVCHER2-217219 have no significant effect on bladder cancer organoids.

[0545] The above results demonstrate at the organoid level that the HER2-targeting delivery vector PVCHER2-PagT1, loaded with the virulence factor PagT1, can effectively kill bladder cancer organoids, which further confirms the feasibility of PVCHER2-PagT1 as a precision oncology drug.

[0546] 2.6 Targeted therapy of mouse orthotopic gliomas using PVCHER2 loaded with PagT1

[0547] To further verify the therapeutic effect of PVCHER2-PagT1 on tumors in vivo, a mouse orthotopic glioma model was constructed for testing.

[0548] U251-Luci cells expressing firefly luciferase (Luci) were constructed using a lentiviral vector, and the successful cell construction was verified by detecting luciferase levels. Figure 30 ).

[0549] Take U251-Luci cells (2×10⁻⁶) 4 Mice were in situ inoculated with cells / 2 μL. Starting from day 7 post-inoculation, in vivo imaging was performed every 7 days to monitor tumor development. On day 8 and day 35 post-inoculation, magnetic resonance imaging was performed to monitor brain tumor status. On day 10 post-inoculation, intraventricular administration was performed according to the group, with an administration volume of 2 μL. The dosage was 15 μg for the low-dose group, 30 μg for the high-dose group, and 30 μg for the empty vector group (control).

[0550] The results of in vivo imaging monitoring are shown in Figures 31A-31B. In vivo imaging detected gliomas in the mouse brain on day 7 post-inoculation. In vivo imaging on days 21 and 35 showed a significant decrease in tumor signal intensity in both the low-dose and high-dose groups compared to day 7 (Figure 31A). Statistical analysis of the Luci signal intensity in in vivo imaging revealed that the tumor signal intensity in the low-dose and high-dose groups was also significantly lower than that in the PBS and empty vector groups (Figure 31B).

[0551] The magnetic resonance imaging results are shown in Figure 31 C. On day 7 after inoculation, in situ gliomas in the mouse brain could also be detected by magnetic resonance imaging. On day 35, the tumor signal in both the low-dose and high-dose groups was significantly weakened compared to day 7.

[0552] The mouse survival curve results and mouse weight changes are shown in Figures 31D-31E. The survival time of the high-dose group was significantly longer than that of the PBS group (Figure 31D); there was no significant difference in mouse weight changes (Figure 31E).

[0553] The above results demonstrate that the PVCHER2-PagT1 delivery vector, which loads the virulence factor PagT1 and targets HER2, can effectively kill mouse orthotopic gliomas.

[0554] In summary, the results in Examples 2.1-2.6 demonstrate that replacing the PVC tail filament protein's receptor-specific domain with DARPin, which can bind to the tumor-associated antigen HER2, and loading it with PagT1, can effectively kill glioma cells, bladder tumor cells, bladder cancer organoids, and mouse orthotopic gliomas. This confirms the feasibility of targeted modification and PagT1-loaded PVC as a precision oncology drug.

[0555] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

[0556] The sequences involved in this application are as follows:

[0557] Amino acid sequence of Photorhabdus asymbiotica WP_015834233.1 (PagT1) protein

[0558] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVASNKRDPITKDQVSVPPDGNLFTELKDKHLDRAAEYKKLKTWPTHASIIATSPSANTPIAQHVSGDDALSPYISTGDKPGAVQNTVRNWNGIGPASERRLRPEKTWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTTNSDIAQNAFADKEVLISPEIPGLAITRVINDPEEIKQIANLNPSQSLIEKKNTIPEEKIIFEEKKSVPIHDSDADIPSSSFVFPKRKKPRNIRSRTDS (SEQ ID NO: 1);

[0559] Amino acid sequence at the C-terminus of Pseudomonas alkylphenolica WP_051939377.1 protein

[0560] DENGFSPDVDILEKFESPGAFYERTGREFLYRAIRTMESEKLHPETGDKGKLPPDGDAFPELKDKHLERAAKYRSMKTWEGGDIVSTGPELKRLPLQHVSGDNQQSQYISTGTRGAAAYNTVYNWNSKGVGKERRKRPEKDWDPIIKIDVKKLDSGTKIYDMQQEGLKDRDRSDIGQLAEADAEVLISNKINASAIVSVFTNSADYNKFLEEYFPPAKSDGAAASPLVNKIKRSERGGSRRTVSESVADAPVEVFKKRAPIKPQSFRKR (SEQ ID NO: 2);

[0561] Amino acid sequence at the C-terminus of Deltaproteobacteria bacterium MCE9576703.1 protein:

[0562] DVFETPAQYLTRTGKHLLYRAVRMDGTSKKVFSGAGSTSSADGGSVGVAPDGNLFAGIPQKEREARYRALAAWPADHPITAAAPQATADPLVQISGSNYATQYISTGAAIHGAAHNTVRSWNSPGAPGDRPIRRPQSWAPVITIDVRRLAPQTRVYDTQRPDVNYMYREDALGMMAGLSAADAEVLLTGEIPAAAIVGIMTQPSAIAEAAQVTIAELL(SEQ ID NO: 3);

[0563] Amino acid sequence of the C-terminus of Okeania WP_293063881.1 protein:

[0564] IVKISVDNHKQINLDENCHQFETVNEYVERTGVRTLYRSIRSKDVDKDKFELNQKVSVPAEIKLPTSPGERFKELKNLHRHPMESHESPKRTKITNEIELIPGDSMAQLLPADQVSGSFATQFISTSSNPLGAIVNTVKNWDSGSNRTRMRPLRFWDPVVKIDLTYLLNHQDIRIYDMKADYIEWPNLNTTGVGVKANAEVDNEILISGKLPPQAV (SEQID NO: 4);

[0565] Amino acid sequence of Photorhabdus heterorhabditis WP_172908611.1 protein:

[0566] MVYEYGKTNDRKRKRSTQSDNYEEKLFAPVLDLPRNNQNTPNMEDEYETPKDFINRTGREKLFRAIRMIASDKRDPITQDQVSVPPDGNLFTELEKTHQGRADEYKKLKEWPTHASIIATSPNANIPIAQHVSGDNILSPYISTGDEPGAAQNTVRSWNQAGPVSERRLRPEKNWSPIIEIDVNKLPDTTKIFDLNKPSNTFFSTANSDISQNAFADKEVLISPEIPGHAITRVINDPKEIKQIANLNPSQSPIEKKNIIPEEKIIFEEKKSAPIHESDANLPSSSFIFPKRKKPRNIRSRTDS (SEQ ID NO: 5);

[0567] The amino acid sequence of the protein Photorhabdus australis WP_065822174.1:

[0568] MVYEYGKTNDRKRKRSTQSDDYEEKPFSPVLDLSRNNQNTPNMEDEYETPKDFINRTGREKLFRAIRMMASDKQDPITKYQVSVSPDGNLFTELKDKHLDRAAEYKKLKEWPTHVPIMATSLSTNIPIAQHVSGDNILSPYISTGDAPGATQ NTVRSWNQAGPISERRLRPEKDWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTDNSDIAQNAFADKEVLISPEIPGHAITRVINDPKEIKQIAKLNPSQSSIEKKNTMPEKKITFEEKKSIPIHDSDANLPSSSFVFPKRKKPRNIRSRTDSW (SEQ ID NO: 6);

[0569] The amino acid sequence at the C-terminus of the Burkholderia ubonensis KVU23967.1 protein:

[0570] NPTNIAGVESEVPYHSPSTFIAATGRRYLYRALNAIQDSALGKSGNVPSDFEIAATNPDANYSPVVHVGSSDYEGGTQYISTGDASGAARNTVLNWNAGVERRLRPSRLWNRIVEVDVAKLPDDTRLYNVGKDWNLYSDEKKNPIHSEVGNNAFSDQEVLIQGRINGSAIRDV (SEQ ID NO: 7);

[0571] The amino acid sequence of the protein Streptomyces avermitilis WP_037649420.1:

[0572] QTVEEYRASGGPSKLYRSIRIESQLKFRADRATVTAEADGNLGAGMPAKEVSRIYQTLKEWPRTKNIEPVDANAAFTVAHHVGGDNYGTQYISFSPDYGRAADYAQHDFKLGPAENLEGARKPRSVRKWAPVIEIDIAKLGPGNRLVNLGNPNIAGLTNLKEVTDIASMASNDSEVLIKGTIPAGAVTKVYGVEDAVKSISLEARKNLIEQ (SEQ IDNO: 8);

[0573] The nucleotide sequence of DARPin binding to HER2, with KpnI and HindIII restriction sites and the linker GGSGGGGSGG (SEQ ID NO: 9) at both ends:

[0574] GGTTGGGCGTTATGTGATGGCAATAATGGTACACCAAATTTAATCGATCGATTTATTTTAGGTGGGAAAGGTACCGATATTAATGGAGTGAGTACTAATACAGCTTCAGGTACTAAAAATAGTAAGTTATTCGATTTCAGTTCTGATGAAGCTACATTAACTATTGATGGTAAAACACTGGGGAGAggtggctctggcggtggtggatctggtggcCGCGGCAGCCATCATCATCATCATCATGGCAGCGATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCGGGCCAGGATGATGAAGTGCGCATTCTGATGGCGAACGGCGCGGATGTGAACGCGCATGATTTTTATGGCATTACCCCGCTGCATCTGGCGGCGAACTTTGGCCATCTGGAAATTGTGGAAGTGCTGCTGAAACATGGCGCGGATGTGAACGCGTTTGATTATGATAACACCCCGCTGCATCTGGCGGCGGATGCGGGCCATCTGGAAATTGTGGAAGTGCTGCTGAAATATGGCGCGGATGTGAACGCGAGCGATCGCGATGGCCATACCCCGCTGCATCTGGCGGCGCGCGAAGGCCATCTGGAAATTGTGGAAGTGCTGCTGAAAAACGGCGCGGATGTGAACGCGCAGGATAAATTTGGCAAAACCCCGTTTGATCTGGCGATTGATAACGGCAACGAAGATATTGCGGAAGTGCTGCAGAAAGCGGCGAAACTGAACggtggttcaggtggtggtggtagtggtggcCACGATCATGATATTAAGATAACGGGCACAGGAAAACATTCTCACAAAAACAAAGTAACAGTCCCTTATTATATTCTGGCTTTCATCATAAAGCTTTAATATATATGAAAAATTGAAAATATAAATTATCCATTAATAATAAAGAGGAT(SEQ ID NO: 10);

[0575] Forward primer sequence of N-NLS-F:

[0576] ATTTCACACAGGAAACATATGTTAAAATATGCTAATCCTCAGACCG (SEQ ID NO: 11);

[0577] N-NLS-R reverse primer sequence:

[0578] AATGATTTTTCTTCATAATTGATTTTCCTAATCTTATGGCCTTCG (SE Q ID NO: 12);

[0579] C-NLS-F forward primer sequence:

[0580] TACCGTCAGACTACAAGGACGATGATGACAAGTAA (SEQ ID NO: 13)

[0581] C-NLS-R reverse primer sequence:

[0582] CCTTGTAGTCTGACGGTATATCGGCGTCACTGT (SEQ ID NO: 14);

[0583] 217219-F forward primer sequence:

[0584] TGCAGCAAAAGCAGTGCTAATTTCACCAGAAATTCCA (SEQ ID NO: 15);

[0585] 217219-R reverse primer sequence:

[0586] GCACTGCTTTTGCTGCAAAGGCGTTTTGAGCA (SEQ ID NO: 16);

[0587] Vector-F forward primer sequence:

[0588] AATTATGAAGAAAAAATCATTTTCTCCC (SEQ ID NO: 17);

[0589] Vector-R reverse primer sequence:

[0590] ATGTGTTTCCTGTGTGAAATTGTTATC (SEQ ID NO: 18);

[0591] The amino acid sequence of PagT1-N15:

[0592] MVYEYAKTNDRKRKL (SEQ ID NO: 19);

[0593] The amino acid sequence of PagT1-N20:

[0594] MVYEYAKTNDRKRKLSTQSD (SEQ ID NO: 20);

[0595] The amino acid sequence of PagT1-N25:

[0596] MVYEYAKTNDRKRKLSTQSDNYEEK (SEQ ID NO: 21);

[0597] The amino acid sequence of PagT1-N30:

[0598] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPV (SEQ ID NO: 22);

[0599] The amino acid sequence of PagT1-N35:

[0600] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSR (SEQ ID NO: 23);

[0601] The amino acid sequence of PagT1-N40:

[0602] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNT (SEQ ID NO: 24);

[0603] The amino acid sequence of PagT1-N45:

[0604] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMED (SEQ ID NO: 25);

[0605] The amino acid sequence of PagT1-N50:

[0606] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETP (SEQ ID NO: 26);

[0607] The amino acid sequence of PagT1-N55:

[0608] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFIN (SEQ ID NO:27);

[0609] The amino acid sequence of PagT1-N60:

[0610] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGRE (SEQ IDNO: 28);

[0611] The amino acid sequence of PagT1-N65:

[0612] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRA(SEQ ID NO: 29);

[0613] The amino acid sequence of PagT1-N70:

[0614] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVA (SEQ ID NO: 30);

[0615] PagT1 removes the N-terminal Del-N-NLS amino acid sequence.

[0616] NYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVASNKRDPITKDQVSVPPDGNLFTELKDKHLDRAAEYKKLKTWPTHASIIATSPSANTPIAQHVSGDDALSPYISTGDKPGAVQNTVRNWNGIG PASERRLRPEKTWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTTNSDIAQNAFADKEVLISPEIPGLAITRVINDPEEIKQIANLNPSQSLIEKKNTIPEEKIIFEEKKSVPIHDSDADIPSSSFVFPKRKKPRNIRSRTDS (SEQ ID NO:31);

[0617] PagT1 removes the C-terminal Del-C-NLS amino acid sequence:

[0618] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVASNKRDPITKDQVSVPPDGNLFTELKDKHLDRAAEYKKLKTWPTHASIIATSPSANTPIAQHVSGDDALSPYISTGDKPGAVQNTVRNWNGIGPASERRLRPEKTWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTTNSDIAQNAFADKEVLISPEIPGLAITRVINDPEEIKQIANLNPSQSLIEKKNTIPEEKIIFEEKKSVPIHDSDADIPS (SEQ ID NO:32);

[0619] The amino acid sequence of PagT1 with both the N-terminal Del-N and C-terminal C-NLS removed:

[0620] NYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVASNKRDPITKDQVSVPPDGNLFTELKDKHLDRAAEYKKLKTWPTHASIIATSPSANTPIAQHVSGDDALSPYISTGDKPGAVQNTVRNWNGIGPASERRLRPEKTWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTTNSDIAQNAFADKEVLISPEIPGLAITRVINDPEEIKQIANLNPSQSLIEKKNTIPEEKIIFEEKKSVPIHDSDADIPS (SEQ ID NO: 33);

[0621] The amino acid sequence of PagT1 mutant 217219:

[0622] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVASNKRDPITKDQVSVPPDGNLFTELKDKHLDRAAEYKKLKTWPTHASIIATSPSANTPIAQHVSGDDALSPYISTGDKPGAVQ NTVRNWNGIGPASERRLRPEKTWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTTNSDIAQNAFAAKAVLISPEIPGLAITRVINDPEEIKQIANLNPSQSLIEKKNTIPEEKIIFEEKKSVPIHDSDADIPSSSFVFPKRKKPRNIRSRTDS (SE Q ID NO: 34);

[0623] Knockdown of Irf9 shRNA sequence:

[0624] CCCTACAAAGTATATCGAATA (SEQ ID NO: 35);

[0625] Knockdown of STAT1 shRNA sequence:

[0626] CCGAAGAACTTCACTCTCTTA (SEQ ID NO: 36);

[0627] Knockdown of TYK2 shRNA sequence:

[0628] GCGCATACCTGTGTGTCATCT (SEQ ID NO: 37);

[0629] The amino acid sequence of Pnf-N50:

[0630] MLKYANPQTVATQRTKNTAKKPPSSTSFDGHLELSNGENQPYEGHKIRKI (SEQ ID NO: 38);

[0631] The nucleotide sequence of the PagT1 gene:

[0632] ATGGTGTATGAATACGCTAAAACCAATGATAGAAAAGAAAACTCTCAACACACAGTCAGATAATTATGAAGAAAAATCATTTTCTCCCGTATTAGATTTATCCAGAAACAATCAAAATACGCCTAATATGGAAGATGAATATGAAACACCGCAGAATTTTATTAATAGAACTGGTCGAGAAAAACTATTCCGTGCAATCCGTATGGTAGCCTCGAATAAACGCGATCCC ATTACAAAAGATCAAGTATCCGTGCCACCTGATGGTAATCTATTTACCGAACTTAAAGATAAACATCTGGATAGAGCCGCGGAATATAAAAAATTAAAAACATGGCCAACACATGCTTCAATTATAGCAACCTCTCCCAGTGCTAATACCCCTATTGCACAACAACATGTTTCCGGTGATGATGCATTAAGCCCTTATATCTCTACAGGTGACAAACCAGGGGCCGTGCAA AATACGGTAAGGAATTGGAATGGGATTGGACCCGCATCAGAAAGAAGACTGAGACCAGAAAAAACATGGTCTCCAATAATAGAGATCGACGTTAATAAGCTGCCTGACACCACTAAAATCTTCGATCTGAATAAACCAAATAATACCTTCTTTAGTACCACCAATAGCGACATTGCTCAAAACGCCTTTGCAGACAAAGAAGTGCTAATTTCACCAGAAATTCCAGGCCTCGCTATTACACGTTGTAATTAATGATCCAGAAGAGATCAAACAGATTGCTAATCTTAATCCAAGCCAATCGCTTATAGAGAAAAAGAACACCATACCAGAAGAAAAAATAATCTTTGAAGAAAAAAAATCGGTGCCAATACACGACAGTGACGCCGATATACCGTCATCATCATTTGTTTTTCCAAAACGCAAGAAACCAAGAAATATAAGATCTCGTACTACTGATAGT (SEQ ID NO: 39)。

Claims

1. A signal peptide for guiding the loading of a polypeptide into a protein complex and guiding the polypeptide to localize to the nucleus of a eukaryotic cell, wherein the signal peptide is a peptide consisting of the first 20 to 70 amino acids from the N-terminus of a PagT1 protein, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The signal peptide of claim 1, wherein the protein complex comprises a non-symbiotic luminescent bacterium virulence box (PVC), AFP, or a metamorphosis-associated stretching structure (MAC).

3. The signal peptide according to claim 1 or 2, wherein the amino acid sequence of the signal peptide is shown in any one of SEQ ID NO:20-30.

4. The signal peptide according to claim 3, wherein the polypeptide comprises any one or more of the following: signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone regulatory molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene editing proteins.

5. A nuclear localization peptide for localizing a polypeptide linked thereto into the nucleus of a eukaryotic cell, wherein the nuclear localization peptide is a peptide composed of the first 20 to 70 amino acids from the N-terminus of a PagT1 protein and / or the last 20 amino acids from the C-terminus of a PagT1 protein, the amino acid sequence of which is shown in SEQ ID NO:

1.

6. The nuclear localization peptide according to claim 5, wherein the amino acid sequence of the nuclear localization peptide is shown in any one of SEQ ID NO: 20-30.

7. The nuclear localization peptide according to claim 5 or 6, wherein the polypeptide comprises any one or more of the following: signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone regulatory molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene editing proteins.

8. Use of PagT1 protein in the preparation of a drug as a protein toxin, wherein the amino acid sequence of said PagT1 protein is shown in SEQ ID NO:

1.

9. A nucleic acid encoding a signal peptide as described in any one of claims 1-4.

10. An expression vector comprising the nucleic acid as described in claim 9.

11. A host cell comprising the nucleic acid as claimed in claim 9 or the expression vector as claimed in claim 10.

12. A polypeptide delivery system comprising a protein complex and a signal peptide according to any one of claims 1-4.

13. The polypeptide delivery system of claim 12, wherein the protein complex comprises a non-symbiotic luminescent bacterium virulence box (PVC), AFP, or a metamorphosis-associated stretching structure (MAC).

14. The polypeptide delivery system of claim 12 or 13, wherein the signal peptide is covalently linked to the polypeptide.

15. The polypeptide delivery system of claim 14, wherein the polypeptide comprises any one or more of the following: signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone-regulating molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene-editing proteins.

16. The polypeptide delivery system according to claim 12 or 13, wherein the polypeptide is PagT1 protein.

17. The polypeptide delivery system according to claim 12 or 13, wherein the protein complex is a PVC-V complex.

18. The peptide delivery system of claim 17, wherein the structural protein Pvc13 in the PVC-V complex is modified to specifically recognize HER2.

19. The polypeptide delivery system of claim 18, wherein a protein that recognizes cell surface molecules is inserted into the receptor-binding domain of the structural protein Pvc13.

20. The polypeptide delivery system of claim 19, wherein the amino acid sequence of the signal peptide is as shown in any one of SEQ ID NO: 20-30, and wherein a DARPin that specifically recognizes HER2 is inserted into the receptor-binding domain of the structural protein Pvc13 in the PVC-V complex.

21. The polypeptide delivery system of claim 20, wherein the polypeptide comprises any one or more of the following: signaling pathway regulatory proteins, structural proteins, transport proteins, hormones or hormone-regulating molecules, cytotoxins, antigens or immunogens, antibody proteins or fragments thereof, tag proteins or reporter proteins, antimicrobial peptides, enzymes involved in cell metabolism, and gene-editing proteins.

22. A nucleic acid encoding a polypeptide delivery system as described in any one of claims 12-21.

23. The nucleic acid according to claim 22, wherein, The nucleic acid includes one or more of the following: (i) The nucleotide sequence encoding the protein complex in the polypeptide delivery system; (ii) The nucleotide sequence encoding the signal peptide in the polypeptide delivery system; (iii) The nucleotide sequence encoding the polypeptide.

24. An expression vector comprising the nucleic acid as described in claim 22 or 23.

25. A host cell comprising a polypeptide delivery system as described in any one of claims 12-21, a nucleic acid as described in claim 22 or 23, or an expression vector as described in claim 24.

26. A method for preparing a polypeptide delivery system as described in any one of claims 12-21, comprising culturing the host cells of claim 25 to obtain the polypeptide delivery system.

27. Use of the signal peptide of any one of claims 1-4, the polypeptide delivery system of any one of claims 12-21, the nucleic acid of any one of claims 22 or 23, the expression vector of any one of claims 24, or the host cell of any one of claims 25 in the preparation of a drug, reagent, or kit for delivering a polypeptide.

28. The use according to claim 27, wherein the drug, reagent, or kit is a drug, reagent, or kit for treating tumors.

29. The use according to claim 28, wherein the tumor is a HER2-positive tumor.

30. The use of the PagT1 gene or the protein it encodes in the preparation of a drug for treating HER2-positive tumors, wherein the amino acid sequence of the PagT1 protein is shown in SEQ ID NO: 1.

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