Functional identification of novel cell virulence factor and application of novel cell virulence factor

Abstract

The invention provides functional identification and application of a novel cell virulence factor. Based on the research on the novel virulence factor of the cell, the invention also provides a polypeptide delivery system and application thereof. Wherein the polypeptide delivery system comprises a protein complex and a signal peptide, the signal peptide comprises at least 20 amino acids at the N-terminal of PagT1 protein or homologous protein thereof, and 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 eukaryotic cells. The research of the invention provides an important basis for constructing a safe and efficient novel tumor treatment system, and provides an innovative solution for treating serious diseases such as cancers.

Description

Technical Field

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

[0002] Immunotoxins (ITs), also known as biological missiles, are an attractive anti-cancer treatment modality aimed at specifically delivering highly cytotoxic protein toxins to tumor cells. The mechanism of action of immunotoxins is to internalize the toxin into the cytosol through receptor-mediated endocytosis targeted to cell-specific receptors, followed by killing the designated tumor cells through the cytotoxic effects of the toxin. Immunotoxins are chemically conjugated or recombinantly fused from 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, etc.).

[0003] Immunotoxins utilize a variety of cytotoxic agents from different sources, including bacterial toxins, plant toxins, and engineered molecules. Each toxin has its unique mechanism and therapeutic potential, highlighting the diversity of immunotoxin technology in targeted cancer therapy and other disease treatments. Although numerous in vivo and in vitro studies have been conducted on bacterial toxins with high cell-killing ability, the research on using immunotoxins for cancer treatment is still relatively scarce. In fact, the US Food and Drug Administration (FDA) has only approved three tumor-specific immunotoxins for clinical use: Denileukin Diftitox (Ontak), Moxetumomab Pasudotox (Lumoxiti), and Tagraxofus (Elzonris).

[0004] In addition, there are also some limitations in the application of immunotoxins:

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

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

[0007] Despite the use of tumor - specific targeting moieties to selectively deliver toxin payloads to tumor cells, the clinical development of immunotoxins is still limited by a narrow therapeutic window, mainly due to off - target and on - target 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. A slight increase in the dose can lead to significant adverse events. Given this, minimizing the off - target and on - target toxicities of immunotoxins is crucial for broadening the therapeutic window, especially for the treatment of solid tumors.

[0008] (ii) Immunogenicity

[0009] The clinical development of immunotoxins is often hindered by the induction of immune responses, a property known as immunogenicity. In particular, immunotoxins carrying heterologous bacterial and plant - derived toxins are highly immunogenic and can trigger T - cell - dependent and B - cell - mediated anti - drug antibodies (ADAs) against the exogenous toxin moiety in immunocompetent patients. These antibodies neutralize the immunotoxin, rendering it ineffective, and accelerate its clearance from the body, while causing severe immune system - related adverse reactions, thus hindering long - term or repeated dosing in clinical trials. Since immunotoxins usually require 2 - 5 cycles of administration to achieve a significant anti - tumor response, immunogenicity is a key issue.

[0010] (iii) Cytoplasmic delivery efficiency

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

[0012] In summary, despite the great potential shown by immunotoxins, they still face many limitations in clinical applications. Given the limited number of protein toxins in currently mature immunotoxins, as well as problems such as in - vivo degradation, immunogenicity, severe systemic toxicity, adverse accumulation, and difficulty in entering tumor cells faced by immunotoxins, there is an urgent need to develop novel protein toxins and also delivery carriers that can improve protein stability, avoid harmful immune stimulation, and have the ability to deliver specific proteins into specific cell types. The two complement each other to be used for the precise treatment of diseases such as tumors. Summary of the Invention

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

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

[0015] 1. A signal peptide, which is used to direct the loading of a polypeptide into a protein complex and direct the localization of the polypeptide to the nucleus of a eukaryotic cell, wherein the signal peptide comprises at least 20 amino acids at the N-terminus of the PagT1 protein or its homologous protein.

[0016] 2. The signal peptide according to item 1, wherein the protein complex comprises a non-symbiotic Photorhabdus virulence cassette (PVC), AFP or a contractile structure associated with metamorphosis (MAC);

[0017] Preferably, the protein complex comprises a non-symbiotic Photorhabdus virulence cassette (PVC).

[0018] 3. The signal peptide according to item 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 the PagT1 protein or its homologous protein.

[0019] 4. The signal peptide according to any one of items 1-3, wherein the signal peptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 20-30, or comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in any one of SEQ ID NOs: 20-30.

[0020] 5. The signal peptide according to any one of items 1-4, wherein the polypeptide comprises any one or more of a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein.

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

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

[0023] 7. The nuclear localization peptide according to item 6, wherein the nuclear localization peptide comprises the amino acid sequence 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%, 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 20-30.

[0024] 8. The nuclear localization peptide according to item 6 or 7, wherein the polypeptide comprises any one or more of a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein.

[0025] 9. A fusion protein comprising the nuclear localization peptide according to any one of items 6-8 and a polypeptide linked thereto, wherein the linkage is a covalent or non-covalent linkage.

[0026] 10. A protein toxin, which is 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.

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

[0028] 12. A conjugate comprising the protein toxin according to item 10 or 11.

[0029] 13. A fusion protein comprising the protein toxin according to item 10 or 11.

[0030] 14. A device or article comprising the protein toxin according to item 10 or 11.

[0031] 15. A complex comprising the protein toxin according to item 10 or 11 or its coding nucleic acid.

[0032] 16. The complex according to item 15, wherein the complex comprises a component for delivering the protein toxin or its coding nucleic acid into the cell, optionally, the component comprises a lipid, a protein or a nucleic acid, optionally, the complex is a lipid nanoparticle (LNP), a nucleic acid-protein complex or a protein complex.

[0033] 17. A nucleic acid encoding the signal peptide according to any one of items 1-5.

[0034] 18. An expression vector comprising the nucleic acid according to item 17.

[0035] 19. A host cell comprising the nucleic acid according to item 17 or the expression vector according to item 18.

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

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

[0038] 21. The polypeptide delivery system according to item 20, wherein the protein complex comprises a Photorhabdus virulence cassette (PVC), an AFP or a Metamorphosis Associated Contractile structure (MAC);

[0039] Preferably, the protein complex comprises a Photorhabdus virulence cassette (PVC).

[0040] 22. The polypeptide delivery system according to item 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 item 22, wherein the polypeptide comprises any one or more of a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein.

[0043] 24. The polypeptide delivery system according to item 20 or 21, wherein the polypeptide is the 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 to the amino acid sequence shown in SEQ ID NO: 1;

[0044] Optionally, the PagT1 protein or its homologous protein is conserved at aspartic acid at position 217 and glutamic acid at position 219 relative to the reference sequence 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 NOs: 20 - 30.

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

[0047] Preferably, the structural protein Pvc13 in the PVC-V complex is modified into a protein that recognizes cell surface molecules;

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

[0049] 26. The polypeptide delivery system according to item 25, wherein the structural protein Pvc13 in the PVC-V complex is modified into a protein that specifically recognizes 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 tumor-specific antigens or tumor-associated antigens on the cell surface is inserted, and optionally, a DARPin that specifically recognizes HER2 is inserted.

[0051] 28. The polypeptide delivery system according to item 26 or 27, wherein the amino acid sequence of the signal peptide is shown in any one of SEQ ID NOs: 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 items 26-28, wherein the polypeptide comprises any one or more of a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein;

[0053] Optionally, the polypeptide is the PagT1 protein or a homologous protein thereof. Optionally, the PagT1 protein or a homologous protein thereof 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 to the amino acid sequence shown in SEQ ID NO: 1;

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

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

[0056] 30. A nucleic acid encoding the polypeptide delivery system according to any one of items 20-29.

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

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

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

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

[0061] 32. An expression vector comprising the nucleic acid according to item 30 or 31.

[0062] 33. A host cell comprising the polypeptide delivery system according to any one of items 20-29, the nucleic acid according to item 30 or 31, or the expression vector according to item 32.

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

[0064] 35. A method for transferring a polypeptide into a target cell, comprising contacting the polypeptide delivery system according to any one of items 20 - 29 with the target cell, such that the polypeptide delivery system delivers the polypeptide into the target cell;

[0065] Optionally, the target cell is a eukaryotic cell;

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

[0067] More preferably, the eukaryotic cell is a human cell;

[0068] Even more preferably, the eukaryotic cell is a human tumor cell.

[0069] 36. A method for killing a target cell, comprising contacting the polypeptide delivery system according to any one of items 20 - 29 with the target cell, such that the polypeptide delivery system delivers the polypeptide into the target cell, thereby killing the target cell;

[0070] Optionally, the target cell is a eukaryotic cell;

[0071] Preferably, the eukaryotic cell is a human tumor cell;

[0072] Even more preferably, the eukaryotic cell is a HER2 - positive human tumor cell.

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

[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. Application of the PagT1 gene or the protein encoded thereby in the preparation of a drug 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%, 99% identity with the sequence shown in SEQ ID NO: 1.

[0077] Beneficial effects:

[0078] This application first discovered a novel protein toxin PagT1 that can be loaded into the Photorhabdus virulence cassette (PVC), and identified its toxicity mechanism. In addition, this application also verified that the N-terminal signal peptide of PagT1 has dual functions, namely guiding the polypeptide to be loaded into the PVC and directing the polypeptide to localize in the nucleus after eukaryotic cell delivery. The research of this application not only provides a theoretical basis for analyzing the pathogenic mechanism of Photorhabdus and developing clinical treatment strategies for bacterial infectious diseases; at the same time, it also screens an adapted virulence effector molecule and a bifunctional signal peptide for the PVC, laying a key research foundation for the development of tools for treating diseases such as tumors.

[0079] This application delivered PagT1 to tumor cells and tumor organoids through PVC, and the detection results showed that PagT1 could effectively kill tumor cells. This discovery provides an important basis for constructing a safe and efficient novel tumor treatment system, and provides an innovative solution for the treatment of major diseases such as cancer. Brief Description of the Drawings

[0080] Figures 1A - 1C show the distribution of PagT1 and its homologous proteins and the results of cytotoxicity detection. Among them, Figure 1A is a schematic diagram of the PVC gene cluster and the location of the PAU_02097 (PagT1) gene; Figure 1B is a phylogenetic relationship diagram between PagT1 and its homologous proteins; Figure 1C shows the results of the detection of the cell viability of 293T cells after transfection with PagT1, 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 the assembly of PVC-PagT1 and PVCR7-PagT1, as well as the results of eukaryotic cell cytotoxicity detection. Among them, Figure 2A shows the loading results of PagT in purified PVC-PagT1 and PVCR7-PagT1 detected by Western blot. The Pvc16 antibody was used to detect the PVC / PVCR7 structural protein as an internal control; Figure 2B shows the detection results of the cell viability of J774A.1 cells 24 hours after the delivery of PagT1 by PVC-PagT1. Empty PVC and PBS were used as controls, and the PVC concentration was 15 μg / mL; Figure 2C shows the detection results of the cell viability of THP-1 cells 24 hours after the delivery of PagT1 by PVCR7-PagT1. Empty PVCR7 and PBS were used as controls, and the PVCR7 concentration was 15 μg / mL; Figure 2D shows the detection results of the cell viability of BMDM cells 24 hours after the delivery of PagT1 by 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 of the subcellular localization analysis of the PagT1 amino acid sequence by DeepLoc 2.0.

[0083] Figures 4A-4D show the detection results of PagT1 localized in the nucleus. Among them, Figure 4A shows the results of confocal microscopy observation of A549 cells transfected with wild-type PagT1 (WT), the NLS mutant of PagT1, and the control empty vector pEGFP. Scale bar = 10 μm; Figure 4B shows the results of immunofluorescence detection of A549 cells 18 hours after the delivery of PagT1 by PVCR7. The cell morphology was photographed by bright field and outlined by a dotted line. Scale bar = 10 μm; Figure 4C shows the schematic diagram of the NLS position of wild-type (WT) PagT1 and the NLS mutant schematic diagram (Del-N-NLS: PagT1 with the N-terminal NLS removed, and the PVC loading signal peptide SP (Pnf) was added to the N-terminal during delivery; Del-C-NLS: PagT1 with the C-terminal NLS removed; Del-N,C-NLS: PagT1 with both the N-terminal and C-terminal NLS removed, and the PVC loading signal peptide SP (Pnf) was added to the N-terminal during delivery); Figure 4D shows the loading results of Tcst (with a flag tag) fused with different lengths of polypeptides at the N-terminal of PagT1 in the purified PVC complex detected by Western blot. The Pvc16 antibody was used to detect the PVC structural protein as an internal control. The amino acid sequence of Pnf-N50 is shown in SEQ ID NO: 38.

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

[0085] Figures 6A-6D show the detection results that PagT1 causes genomic damage in eukaryotic cells. Among them, Figure 6A shows the results of Western blot detection of the expression of phosphorylated histone H2AX (phosphorylated at S139) in J774A.1, THP-1, and BMDM cells after delivery of PagT1 by PVC / PVCR7 for 24 h, with empty PVC / PVCR7 and PBS as the controls and GAPDH as the internal reference; Figure 6B shows the results of immunofluorescence detection of the formation of phosphorylated histone H2AX foci in J774A.1, THP-1, and BMDM cells after delivery of PagT1 by PVC for 18 h, with empty PVC and PBS as the controls, scale bar = 5 μm; Figure 6C shows the results of comet assay for detecting genomic damage in THP-1 cells after delivery of PagT1 by PVCR7 for 24 h, with empty PVCR7 and PBS as the controls; Figure 6D shows the statistical results of the tail moment (the product of the tail length and the percentage of DNA in the tail) in the comet assay. ***p<0.001, ns indicates no significant difference.

[0086] Figures 7A-7G show the prediction results of the active site of PagT1. Among them, Figure 7A shows the results of three-dimensional structure prediction of PagT1 by AlphaFold3; Figure 7B shows the results of Foldseek searching for proteins with three-dimensional structures similar to PagT1 in the PDB database; Figure 7C shows the spatial structure diagram of the protein selected in the box in Figure 7B; Figure 7D shows the results of DoGSiteScorer predicting the possible active pockets of PagT1; Figure 7E shows the schematic diagram of the spatial positions of D217 and E219; Figure 7F shows the results of the conservative analysis of the PagT1 amino acid sequence; Figure 7G shows the detection results of the cell viability of 293T cells after transfection with PagT1, the mutant 217219 with D217 and E219 at the A position, and the empty vector pEGFP for 24 h. *p<0.05, ***p<0.001.

[0087] Figures 8A-8E show the detection results of cell Parthanatos-related indicators caused by PagT1 delivery. Among them, Figure 8A shows the results of detecting the expression of PARP1 in THP-1 cells by Western blot 24 hours after delivering PagT1 via PVCR7. Empty PVCR7, PVCR7-217219 (PVCR7 loaded with mutant 217219), and PBS were used as controls, and GAPDH was used as an internal reference. Figure 8B shows the detection results of the cell viability of J774A.1 cells after delivering PagT1 via PVC for 24 hours after adding NAD+. Figure 8C shows the detection results of the cell viability of THP-1 cells after delivering PagT1 via PVCR7 for 24 hours after adding NAD+. Figure 8D shows the detection results of the cell viability of J774A.1 cells after delivering PagT1 via PVC for 24 hours after adding the PARP1 inhibitor Rucaparib. Figure 8E shows the detection results of the cell viability of THP-1 cells after delivering PagT1 via PVCR7 for 24 hours after adding 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 hours after delivering PagT1 via PVC / PVCR7. Among them, 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 It is a violin plot of gene beta scores. A positive beta score indicates that the gene is positively selected, and a negative beta score indicates that the gene is negatively selected. In this application, positive screening was performed. The beta scores of the experimental group and the control group were subtracted to obtain the degree of gene change.

[0090] Figure 11 It is a distribution map of the differences in gene beta scores for positive screening. The top ten genes are marked in the figure.

[0091] Figure 12 It is a KEGG and GO enrichment analysis map of the genes for positive screening.

[0092] Figure 13Detection results of cell viability of J774A.1 cells (shIrf9, shSTAT1, shTYK2) after 24 h of PagT1 delivery by PVC after stable knockdown of Irf9, STAT1, and TYK2 genes. shNC is the control shRNA group. *p < 0.05, **p < 0.01. The results are the statistics of 3 independent experiments.

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

[0094] Figure 15 Results of immunofluorescence detection of subcellular localization of Irf9 in BMDM cells after 18 h of PagT1 delivery by PVCR7. Scale bar = 10 μm.

[0095] Figure 16 Changes in RNA-seq detection of J774A.1 cells after 12 h of PagT1 delivery by PVC compared with the transcriptome of the empty PVC group.

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

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

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

[0099] Figure 20 Results of Western blot detection of p-STAT1 expression in J774A.1, THP-1 and BMDM cells after delivery of PagT1 via PVC / PVCR7 for 24 h, with empty PVC / PVCR7 and PBS as controls and GAPDH as internal reference.

[0100] Figure 21 Results of Western blot detection of p-STAT1 expression in THP-1 cells after delivery of PagT1 via PVC for 24 h after addition of PARP1 inhibitor Rucaparib, with empty PVCR7 and PBS as controls and GAPDH as internal reference.

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

[0102] Figures 23 A - 23 D show the results of the construction of PVCHER2 and the detection of PVCHER2 after purification. Among them, Figure 23 A is a schematic diagram of the modification of PVCHER2 with Pvc13 targeting HER2; Figure 23 B shows the results of Western blot detection of the loading of purified PVCHER2-PagT1; Figure 23 C is a transmission electron micrograph of empty PVCHER2 after negative staining, scale bar = 100 nm; Figure 23 D is a transmission electron micrograph of PVCHER2-PagT1 after negative staining, scale bar = 100 nm.

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

[0104] Figures 25A - 25B show the results of detecting the cell viability of BIU87 and T24 cells 24 h after adding PVCHER2-PagT1 and PVCHER2-217219. Among them, Figure 25A shows the result of detecting the cell viability of BIU87 cells; Figure 25B shows the result of detecting the cell viability of T24 cells. **p < 0.01, ***p < 0.001.

[0105] Figure 26 It shows the result of detecting the expression of PARP1 in BIU87 cells by Western blot 24 h after delivering PagT1 via PVCHER2. Empty PVCHER2 and PBS were used as controls, and GAPDH was used as an internal reference.

[0106] Figure 27 It shows the result of immunofluorescence detecting the subcellular localization of AIF in BIU87 cells 18 h after delivering PagT1 via PVCHER2. Scale bar = 10 μm.

[0107] Figure 28 It shows the results of immunohistochemistry and immunofluorescence detecting the expression of HER2 in a bladder cancer organoid model. Scale bar = 20 μm.

[0108] Figures 29A - 29C show the results of detecting the killing effect of PVCHER2-PagT1 on bladder cancer organoids. Among them, Figure 29A shows the result of detecting the killing effect of bladder cancer organoids by ATP measurement 120 h after adding empty PVCHER2 to the organoid medium; Figure 29B shows the result of detecting the killing effect of bladder cancer organoids by ATP measurement 120 h after adding PVCHER2-PagT1 to the organoid medium; Figure 29C shows the result of detecting the killing effect of bladder cancer organoids by ATP measurement 120 h after adding PVCHER2-217219 to the organoid medium.

[0109] Figure 30 It shows the result of detecting the luciferase expression of U251-Luci cells, where U251 represents wild-type U251 cells.

[0110] Figures 31A - 31E show the efficacy evaluation of PVCHER2 - PagT1. Among them, Figure 31A is the in - vivo imaging of mice; Figure 31B is the statistical chart of Luci signal intensity in the in - vivo imaging of mice, and PBS is used as the control group for variance analysis; Figure 31C is the magnetic resonance imaging of the mouse brain, and the tumor site is indicated by an arrow; Figure 31D is the result of Kaplan - Meier survival analysis of mice, and PBS is used as the control group for variance analysis; Figure 31E is the curve graph of the body weight change of mice. *p < 0.05. Detailed implementation manners

[0111] The present application will be further described 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 used to limit the present application.

[0112] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein can be used in experiments or practical applications, the materials and methods are described below. In case of conflict, this specification, including its definitions, shall prevail. Additionally, the materials, methods, and examples are for illustrative purposes only and are not restrictive. The following is a further description of the present application with specific embodiments, but it is not used to limit the scope of the present application.

[0113] Definition

[0114] As used herein, the term "Contractile injection system (CIS)" refers to a diverse series of evolutionarily related macromolecular devices that utilize contractile sheaths to deliver nucleic acids and proteins. Contractile injection systems can assist microorganisms in transporting various effector molecules extracellularly to gain a survival advantage (N.M.I. Taylor, M.J. van Raaij, and P.G. Leiman, Contractile injection systems of bacteriophages and related systems. Mol Microbiol, 2018. 108(1): p.6-15.). Contractile injection systems include typical CISs (including the contractile tails of bacteriophages T4, P2, and Mu). In addition to contractile bacteriophages, contractile injection systems similar to bacteriophage tails are also widespread in bacteria and archaea. For example, the type VI secretion system (T6SS), which is used to mediate intercellular communication and play a role in cellular defense (N.M.I. Taylor, M.J. van Raaij, and P.G. Leiman, 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 (eCISs).

[0115] As used herein, the term "extracellular contractile injection system" or "eCIS" is a contractile injection system that can be released extracellularly and attack target cells from the external space. Extracellular contractile injection systems include bacterial tailocin / pyocin, as well as the Photorhabdus virulence cassette (PVC) found in Photorhabdus (abbreviated as PVC) (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, J. B. Heymann, M. R. H. 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 the metamorphosis-associated contractile structure (MAC) (N. J. Shikuma et al., Marine tube worm metamorphosis induced by arrays of bacterial phage tail-like structures. Science, 2014. 343(6170): p. 529-33.), etc.

[0116] The term "Photorhabdus asymbiotica" as used herein belongs to the genus Photorhabdus. Generally, bacteria of the genus Photorhabdus are considered pathogens of insects, but 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] The term "PVC" as used herein generally refers to a contractile injection system produced by the genus Photorhabdus. The PVC of Photorhabdus asymbiotica ATCC 43949 is a protein complex device with a molecular weight of over 10 - MDa, whose structure is similar to a simplified T4 phage tail, including a hexagonal baseplate complex with six fibers and an 117 - nm - long sheath trunk with a cap structure. The inner cavity of the sheath contains an inner tube, and effector proteins are loaded into the inner cavity. The PVC can be released by bacteria extracellularly to function. The PVC is generally considered to be toxic to eukaryotic cells because it can transfer effector proteins into insect blood cells and promote the aggregation of actin (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 the present application, the PVC refers to a contractile injection system isolated from Photorhabdus asymbiotica ATCC 43949 or the host cells described herein, which can penetrate the cell membrane of humans and deliver polypeptides or proteins in the sheath of the PVC into the cytoplasm of human cells. There are five types of PVCs in Photorhabdus asymbiotica, namely PVC - I, PVC - II, PVC - III, PVC - IV, and PVC - V.

[0118] As used herein, the term "PVC gene cluster" refers to a gene cluster encoding multiple structural proteins of PVC present in the genome of Photorhabdus asymbiotica (e.g., Photorhabdus asymbiotica ATCC43949). There are a total of five PVC gene clusters in the genome of Photorhabdus asymbiotica, namely PVC-I, PVC-II, PVC-III, PVC-IV, and PVC-V. One or more potential effector-encoding genes also exist downstream of each PVC gene cluster.

[0119] As used herein, the term "signal peptide" generally refers to a peptide chain that directs the transfer of a synthesized polypeptide or protein to a target. In the context of the present application, the "signal peptide" can direct the polypeptide or protein to be delivered into the lumen of the sheath of PVC.

[0120] As used herein, the term "effector" refers to a bacterial secreted protein produced by bacteria that is transported into plant or animal cells through a secretion system and plays a role in recognition or pathogenesis. Effectors can be structurally divided into a signal region and a functional region.

[0121] As used herein, the term "virulence factor" refers to a characteristic (i.e., gene product) that enables a microorganism to establish itself in or on 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 hydrolases that may contribute to bacterial pathogenicity.

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

[0123] As used herein, the term "protein toxin" refers to a class of toxic protein molecules produced by organisms (such as bacteria, fungi, plants, or animals) that are capable of causing harm to cells, tissues, or other organisms. These toxins exert their toxic effects by interfering with the normal physiological functions of cells and may lead to infection, disease, or even death.

[0124] As used herein, the term "nuclear localization sequence" or "NLS" refers to an amino acid sequence that promotes the import of a protein into the nucleus (e.g., through nuclear transport).

[0125] As used herein, the term "amino acid" or "amino acid sequence" refers to oligopeptides, peptides, polypeptides, or protein sequences, or fragments of any of these, and refers to naturally occurring or synthetic molecules. When an "amino acid sequence" is recited herein as referring to the amino acid sequence of a naturally occurring protein molecule, the "amino acid sequence" and like terms are not intended to limit the amino acid sequence to the full native amino acid sequence associated with the recited protein molecule.

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

[0127] The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence", "polynucleotide", "polynucleotide sequence", "RNA sequence", or "DNA sequence" as used herein refer to oligonucleotides, nucleotides, or polynucleotides and fragments and portions thereof, and refer to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and represent the sense or antisense strand. The sequence may be a non-coding sequence, a coding sequence, or a mixture of both. The nucleic acid sequences of the present application can be prepared using standard techniques well known to those skilled in the art.

[0128] The term "percentage of identity" as used herein, such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to the degree of similarity between amino acid sequences or nucleotide sequences, determined by sequence alignment, which is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%. For example, after introducing gaps and other means to make the two sequences have the same residues at as many positions as possible, the proportion of the number of positions with the same bases or amino acid residues to the total number of positions is determined. The "percentage of identity" can be determined using software programs known in the art. Preferably, the alignment is performed using default parameters. 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 herein 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 that contains a polynucleotide encoding a polypeptide, and the polynucleotide is operably linked to control sequences for its expression.

[0131] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, 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 an expression vector can depend on factors such as the choice of host cell to be transformed and the desired level of expression.

[0133] As used herein, the term "cancer" or "tumor" generally refers to a physiological condition in mammals characterized by uncontrolled cell growth / proliferation. Examples of cancers include but are not limited to lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, 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, liver cancer, leukemia, and other lymphoproliferative diseases and various types of head and neck cancers.

[0134] As used herein, the term "tumor-specific antigen" refers to a neoantigen that is unique to tumor cells or present only in certain tumor cells and not in normal cells. Such antigens are demonstrated by transplantation of tumors between syngeneic animals and are thus also referred to as tumor-specific transplantation antigen (TSTA) or tumor rejection antigen (TRA).

[0135] As used herein, the term "tumor-associated antigen (TAA)" refers to an antigen that is not unique to tumor cells and is also present on normal cells and other tissues, but its content is significantly increased during cell carcinogenesis. Such antigens only show 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, the complex includes polypeptide and nucleic acid molecules that interact with each other (e.g., bind, contact, adhere).

[0137] As used herein, the term "fusion protein" refers to a hybrid protein expressed from a nucleic acid molecule comprising a nucleotide sequence of at least two genes.

[0138] As used herein, the term "conjugate" refers to a molecule conjugated to another molecule. In the present application, the conjugate refers to a conjugate obtained by conjugating the protein toxin described in the present application with another molecule, and no limitation is imposed on the other molecule, and those skilled in the art can make a conventional selection according to needs. In some embodiments, the conjugation is carried out by a chemical method.

[0139] As used herein, the term "codon optimization" means that the nucleotide sequence encoding a polypeptide has been configured to contain the codons preferred by a host cell or organism to improve gene expression in the host cell or organism and to increase translation efficiency.

[0140] As used herein, the term "homologous 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 homology analysis can be newly discovered or can be obtained from public databases such as GenBank of NCBI, UniProt, etc. One can choose bioinformatics tools known in the art for homology search, and 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 evaluated, and based on the alignment results, the function of the target protein can be predicted and its evolutionary relationship can be speculated. Through homology analysis, the function of an unknown protein can be predicted, especially when it is similar to the protein sequence with a known function. 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] The present application discovers that the PagT1 protein can achieve its own loading into PVC without the need to additionally add a signal peptide at its N-terminus, and the PagT1 protein can localize to the nucleus.

[0143] In the present 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%, 99% identity with the sequence shown in SEQ ID NO: 1.

[0144] Among them, 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 (Photorhabdus asymbiotica ATCC43949).

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

[0146] In this application, the above positions are counted from the N-terminus. The "relative to" has the meaning commonly understood by those of ordinary skill in the art. Specifically, "relative to" means the position corresponding to a specified position in one sequence after homology or sequence identity alignment with another sequence.

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

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

[0149] Homologous proteins generally 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, Photorhabdus heterorhabditis, Photorhabdus australis, or Burkholderia ubonensis.

[0150] In some embodiments, the protein complex comprises an aposymbiotic Photorhabdus virulence cassette (PVC), an AFP, or a metamorphosis-associated contractile 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, which meets the requirements of this application.

[0152] In some embodiments, the protein complex comprises an aposymbiotic Photorhabdus virulence cassette (PVC). In some embodiments, the PVC is derived from Photorhabdus asymbiotica ATCC43949.

[0153] The genome of Photorhabdus asymbiotica contains five Photorhabdus virulence cassette gene clusters, namely 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, and the PVC-V complex includes the PVC-V structural proteins Pvc1, Pvc2, Pvc3, Pvc4, Pvc5, Pvc6, Pvc7, Pvc8, Pvc9, Pvc10, Pvc11, Pvc12, Pvc13, Pvc14, Pvc15 and Pvc16, and the PVC-V structural proteins are derived from Photorhabdus asymbiotica ATCC43949.

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

[0155] In some embodiments, the signal peptide comprises an amino acid sequence 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%, 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 20-30.

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

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

[0158] In some embodiments, the polypeptide comprises any one or more of a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein.

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

[0160] In the present application, the term "antimicrobial peptide" refers to a peptide having broad-spectrum anti-pathogen activity and capable of rapidly killing pathogens. Antimicrobial peptides are usually composed of 20-60 amino acid residues. The targets of antimicrobial peptides are Gram-negative bacteria, Gram-positive bacteria, fungi, parasites, tumor cells, etc. According to their sources, antimicrobial peptides can include insect antimicrobial peptides, mammalian antimicrobial peptides, amphibian antimicrobial peptides, fish mollusks, antimicrobial peptides from crustaceans, plant antimicrobial peptides, bacterial antimicrobial peptides. The antimicrobial peptides can be defensins, Cecropin A and its analogs, magainins, Melitiin, cecropins, cathelicidin, apidaecins, drosocin, coleoptericin, hemiptericin, bactenecin, Cecropin, etc.

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

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

[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 can be a pathogen-specific antigen of bacteria, viruses, fungi, mycoplasmas, chlamydias, parasites, etc.

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

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

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

[0168] In some embodiments, the polypeptide is a tag protein or a reporter protein. For example, the tag protein or reporter protein can be BlaM, luciferase, CyaA, β-galactosidase, chloramphenicol acetyltransferase (CAT), secreted alkaline 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 can be Defensins, Cecropin A and its analogs, Magainins, Melitiin, Cecropins, cathelicidin, apidaecins, drosocin, coleoptericin and hemiptericin, bactenecin, Cecropin, human neutrophil 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 cell metabolism. In some embodiments, the enzyme can be selected from Aritilysin, recombinant phage lyase LysSAP26, ribonuclease RNase 3 and RNase 7.

[0171] In some embodiments, the polypeptide is a gene editing protein. The gene editing protein can be, for example, zinc finger nuclease, 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] The present application also provides a nucleic acid encoding any one of the above signal peptides.

[0174] The nucleic acid includes a sequence that has been isolated from its naturally occurring environment, an isolate that has been recombinantly or cloned (e.g., DNA), a chemically synthesized analogue, or an analogue biosynthesized in a heterologous system.

[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. Usually, a natural or synthetic DNA fragment encoding the desired fragment is incorporated into a recombinant nucleic acid construct (usually a DNA construct) capable of being introduced into and replicating in a prokaryotic or eukaryotic cell. Generally, the DNA construct will be suitable for autonomous replication in a unicellular host such as yeast or bacteria, but can also be used to introduce and integrate into the genome of cultured bacterial, insect, mammalian, plant, 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 one of the above nucleic acids.

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

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

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

[0180] Nuclear localization peptide

[0181] This application discovers that the PagT1 protein has a nuclear localization sequence at its N-terminus and C-terminus respectively, and this sequence can act as a nuclear localization signal to direct the polypeptide to localize to the nucleus.

[0182] Based on this, this application provides 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, and the nuclear localization sequence comprises at least 20 amino acids at the N-terminus and / or C-terminus of the PagT1 protein or its homologous protein.

[0183] Those skilled in the art can understand that proteins that have significant similarity to the amino acid sequence of the PagT1 protein and perform the same or similar functions in different organisms or within the same organism should also be within the protection scope 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 at the N - terminus and / or C - terminus of the PagT1 protein or its homologous protein. 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 at the N - terminus and C - terminus of the PagT1 protein or its homologous protein. 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 at the N - terminus of the PagT1 protein or its homologous protein. 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 at the C - terminus of the PagT1 protein or its homologous protein.

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

[0186] In some embodiments, the nuclear localization peptide comprises an amino acid sequence 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%, 99% identity with the 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 - 5, such as 1, 2, or 3 amino acid substitutions compared with the amino acid sequence shown in any one of SEQ ID NO: 20 - 30.

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

[0189] The present application also provides a fusion protein, which comprises any one of the above nuclear localization peptides and a polypeptide linked thereto, and the linkage is a covalent or non-covalent linkage.

[0190] In some embodiments, the linkage is a covalent linkage.

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

[0192] In some embodiments, the fusion protein is non-naturally occurring, that is, in the natural state, there is no fusion protein formed by the fusion of the nuclear localization peptide and the polypeptide as described in the present application in nature, that is, the fusion protein is artificially synthesized by recombinant technology.

[0193] In some embodiments, the polypeptide in the fusion protein comprises any one or more of a signaling pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme participating in cell metabolism, and a gene editing protein.

[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 regulatory molecule, and the hormone regulatory molecule can regulate the expression and / or secretion of hormones.

[0196] In some embodiments, the polypeptide in the fusion protein is a cytotoxin. In some embodiments, the polypeptide is the 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, mycoplasmas, chlamydias, parasites, etc.

[0199] In some embodiments, the polypeptide in the fusion protein is a Plasmodium-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 of the organism itself. In some embodiments, the polypeptide in the fusion protein is a tumor-specific antigen or a tumor-associated antigen.

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

[0202] In some embodiments, the polypeptide is a tag protein or a reporter protein. For example, the tag protein or reporter protein can be BlaM, luciferase, CyaA, β-galactosidase, chloramphenicol acetyltransferase (CAT), secreted alkaline 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 can be Defensins, Cecropin A and its analogs, Magainins, Melitiin, Cecropins, cathelicidin, apidaecins, drosocin, coleoptericin, hemiptericin, bactenecin, Cecropin, human neutrophil 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 cell metabolism. In some embodiments, the enzyme can be selected from Aritilysin, recombinant phage lyase 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 can be, for example, zinc finger nuclease, TALEN nuclease, Cas9, Cas12 (previously known as Cpf1), Cas12a, Cas13, Cas13a, Cas13b, etc.

[0206] In some embodiments, the polypeptide in the fusion protein 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.

[0207] Protein toxin

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

[0209] Based on this, the present application provides a protein toxin, which is PagT1 protein or its homologous protein. The protein toxin 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.

[0210] Those skilled in the art can understand that proteins with obvious similarity to the amino acid sequence of PagT1 protein and performing the same or similar functions in different organisms or within the same organism should also be within the protection scope of the present application as long as they retain the function of the protein toxin.

[0211] In some embodiments, the protein toxin is conserved at aspartic acid at position 217 and glutamic acid at position 219 relative to the reference sequence 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%, 99% identity with the sequence shown in SEQ ID NO: 1.

[0213] The present application also provides a conjugate, which comprises any one of the above protein toxins.

[0214] The present application also provides a fusion protein, which comprises any one of the above protein toxins.

[0215] The present application also provides a device or article, which comprises any one of the above protein toxins.

[0216] The present application also provides a complex, which comprises any one of the above protein toxins.

[0217] In some embodiments, the complex comprises components for delivering the protein toxin or its coding nucleic acid into cells. Among them, the components include, for example, lipids, proteins or nucleic acids; the complex is, for example, a lipid nanoparticle (LNP), a nucleic acid-protein complex or a protein complex.

[0218] Polypeptide delivery system

[0219] The present application provides a polypeptide delivery system, which comprises a protein complex and any one of the above signal peptides. The polypeptide delivery system is, for example, an extracellular contractile 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 through 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 the present application, the term "linker" may refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that links two molecules or moieties (e.g., two components of a protein complex), such as two domains of a fusion protein. The linker can be located between or on both sides of two groups, molecules, or other moieties and is connected to each by covalent bonds or non-covalent interactions, thereby linking 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 amino acid or multiple amino acids (e.g., a peptide or a protein). In some embodiments, the length of the linker can be about 5 to 100 amino acids, such as 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 length. 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 can also be considered.

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

[0224] In some embodiments, the isoelectric point of the polypeptide is 2 - 12, such as 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 a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein.

[0226] In some embodiments, the polypeptide is a PagT1 protein or a homologous protein thereof, which is loaded into the protein complex under the guidance of its own N-terminal signal peptide. Among them, the PagT1 protein or a homologous protein thereof contains the amino acid sequence shown in SEQ ID NO:1, or contains 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 thereof is conserved at aspartic acid at position 217 and glutamic acid at position 219 relative to the reference sequence as 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 NOs: 20-30.

[0227] In some embodiments, the polypeptide is a hormone or a hormone regulatory molecule, and the hormone regulatory molecule 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 pathogen-specific antigen such as bacteria, viruses, fungi, mycoplasmas, chlamydias, parasites, etc. The polypeptide delivery system of the present 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 preparing a vaccine corresponding to the pathogen-specific antigen. After being administered 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 the pathogen infection and its related diseases.

[0231] In some embodiments, the polypeptide is a Plasmodium-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 an organism itself. In some embodiments, the polypeptide is a tumor-specific antigen or tumor-related antigen. The polypeptide delivery system of the present application can be used to incubate with cells in vitro to deliver the tumor-specific antigen or tumor-related antigen in the polypeptide delivery system into the cells, thereby preparing a vaccine against the tumor corresponding to the tumor-specific antigen or tumor-related antigen. After being administered to a subject, the vaccine can induce an immune response against the polypeptide in the subject's body, thereby preventing and / or treating related tumors. The cells can be human embryonic kidney cells HEK293, antigen-presenting cells (such as dendritic cells), etc.

[0233] In some embodiments, the polypeptide is an antigen derived from a protein that causes the formation or progression of a certain disease. This antigen can be delivered into antigen-presenting cells through the polypeptide delivery system of the present application, thereby preparing a vaccine. After being administered to a subject, the vaccine can elicit an immune response against the protein that causes the formation or progression of a certain disease. In some embodiments, the polypeptide is the protein GSDMD (gasdermin D) or GSDMD-NT (obtained by cleaving GSDMD after Asp276 and Asp275) that participates in pyroptosis (or inflammatory apoptosis) leading to renal tubular injury. In some embodiments, the polypeptide is an antigen derived from a protein that causes the formation or progression of a tumor. For example, RhoA, which can regulate actin polymerization, cell adhesion, cell transformation, and participate in cell movement, proliferation, and migration that are closely related to the invasion and metastasis of tumor cells.

[0234] In some embodiments, the polypeptide is a tag protein or a reporter protein. The tag protein or reporter protein can be delivered into cells by the polypeptide delivery system of the present application, and the cell can be characterized by detecting the signal of the tag protein or reporter protein. For example, the tag protein or reporter protein can be BlaM, luciferase, CyaA, β-galactosidase, chloramphenicol acetyltransferase (CAT), secreted alkaline 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 the present application, so as to play an anti-pathogen role in the cells. For example, the antimicrobial peptide can be Defensins, Cecropin A and its analogs, Magainins, Melitiin, Cecropins, cathelicidin, apidaecins, drosocin, coleoptericin, hemiptericin, 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 the present application, so as to play a corresponding catalytic role in the cells. In some embodiments, the enzyme is an enzyme involved in cell metabolism. In some embodiments, the enzyme can 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 by the polypeptide delivery system of the present application, so as to play a gene editing function in the cells. The gene editing protein can be, for example, zinc finger nuclease, TALEN nuclease, Cas9, Cas12 (previously 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 Photorhabdus virulence cassette (PVC), AFP, or a contractile structure associated with metamorphosis (MAC).

[0240] The present 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, which meets the requirements of the present application.

[0241] In some embodiments, the protein complex comprises a Photorhabdus virulence cassette (PVC). In some embodiments, the PVC is derived from Photorhabdus asymbiotica ATCC43949.

[0242] In some embodiments, the protein complex is a PVC-V complex, and the PVC-V complex comprises PVC-V structural proteins Pvc1, Pvc2, Pvc3, Pvc4, Pvc5, Pvc6, Pvc7, Pvc8, Pvc9, Pvc10, Pvc11, Pvc12, Pvc13, Pvc14, Pvc15 and Pvc16, and the PVC-V structural proteins are derived from Photorhabdus asymbiotica ATCC43949.

[0243] In some embodiments, the structural protein Pvc13 in the PVC-V complex is modified into a protein that recognizes 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 into a protein that recognizes tumor-specific antigens or tumor-associated antigens on the cell surface. In some embodiments, the structural protein Pvc13 in the PVC-V complex is modified into a protein that specifically recognizes HER2.

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

[0246] More specifically, by inserting a DARPin that specifically recognizes HER2 into the receptor-binding domain of the structural protein Pvc13 and using a linker to connect the DARPin to Pvc13, the PVC-V complex is modified to specifically target HER2-positive cells. The linker can be a common linker and can be connected to the N-terminus and / or C-terminus of the DARPin. The linker is, for example, GGSGGGGSGG (SEQ ID NO: 9). In some embodiments, the nucleotide sequence of the DARPin that specifically recognizes HER2 and contains a linker is as 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 eukaryotic cells through the PVC-V complex. Specifically, the polypeptide is loaded into the lumen of the sheath 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 its homologous protein. Among them, the PagT1 protein or its homologous protein contains the amino acid sequence shown in SEQ ID NO: 1, or contains 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 homologous protein is conserved at aspartic acid at position 217 and glutamic acid at position 219 relative to the reference sequence shown in SEQ ID NO: 1. In some embodiments, 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 NOs: 20-30.

[0249] The present application also provides a nucleic acid encoding any one of the above polypeptide delivery systems.

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

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

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

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

[0254] In some embodiments, the nucleotide sequence encoding the polypeptide 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 preference of the expected host organism or cell while encoding the same protein.

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

[0256] (ii) a 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 the above (i)-(iii) can be in the same expression vector. In some embodiments, any two or all three of the above (i)-(iii) can be in different expression vectors.

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

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

[0261] In some embodiments, the host cell can be a mammalian cell, insect cell, yeast cell, bacterial cell (such as 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, and the expression vector comprises a nucleotide sequence encoding the PVC-V structural protein. Among them, 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 into a complete PVC-V complex.

[0263] In some embodiments, the expression vector further comprises a nucleotide sequence encoding the polypeptide and the 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, and further preferably, there is also a linker sequence between the signal peptide and the polypeptide.

[0264] In some embodiments, the polypeptide comprises any one or more of a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein.

[0265] In some embodiments, the polypeptide is PagT1 protein or its homologous protein, and 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 to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the PagT1 protein or its homologous protein is conserved at aspartic acid at position 217 and glutamic acid at position 219 relative to the reference sequence shown in SEQ ID NO: 1. In some embodiments, 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.

[0266] In some embodiments, the host cell is also capable of expressing the LysR regulator.

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

[0268] (i). A first vector, the first vector comprises a gene encoding the LysR regulator, preferably the gene is under the control of its native promoter, the first vector is preferably a plasmid, more preferably pBR60;

[0269] (ii) A second vector, the second vector comprises a nucleotide sequence encoding the PVC-V structural protein, the vector is preferably a plasmid, more preferably 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 vector, the third vector comprises a nucleotide sequence encoding the polypeptide and the signal peptide in the polypeptide delivery system of the present application.

[0271] The present application also provides a method for preparing any one of the above polypeptide delivery systems, which comprises culturing any one of the above host cells to obtain the polypeptide delivery system.

[0272] In some embodiments, the method further comprises the step of separating the protein from the cultured host cells. In some embodiments, the method further comprises the steps of purifying the separated protein and removing endotoxin.

[0273] Application of polypeptide delivery system

[0274] The present application provides a method for transferring a polypeptide into a target cell, which includes contacting any one of the above polypeptide delivery systems with the target cell, so that the polypeptide delivery system delivers the polypeptide into the target cell.

[0275] In some embodiments, the polypeptide includes any one or more of a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein.

[0276] The present application provides a method for killing a target cell, which includes contacting any one of the above polypeptide delivery systems with the target cell, so that the polypeptide delivery system delivers the polypeptide into the target cell, thereby killing the target cell.

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

[0278] In some embodiments, the polypeptide is the PagT1 protein or a homologous protein thereof. The PagT1 protein or a homologous protein thereof includes the amino acid sequence shown in SEQ ID NO: 1, or includes 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 thereof is conserved at aspartic acid at position 217 and glutamic acid at position 219 relative to a reference sequence, and the reference sequence is shown in SEQ ID NO: 1. In some embodiments, 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 NOs: 20-30.

[0279] In some embodiments, in the polypeptide delivery system, the protein complex is a PVC-V complex, and the structural protein Pvc13 in the PVC-V complex is modified into a protein that specifically recognizes HER2.

[0280] The present application provides a method for regulating cell signal transduction. The method includes contacting any one of the above polypeptide delivery systems with a target cell, so that the polypeptide delivery system delivers the polypeptide into the target cell. The polypeptide is a signal pathway regulatory protein.

[0281] The present application provides a method for regulating cell metabolism, the method comprising contacting any one of the above polypeptide delivery systems with a target cell, such that the polypeptide delivery system delivers the polypeptide into the target cell. The polypeptide is an enzyme involved in cell metabolism.

[0282] The present application provides a method for regulating molecular transport and / or secretion in cells, the method comprising contacting any one of the above polypeptide delivery systems with a target cell, such that the polypeptide delivery system delivers the polypeptide into the target cell. The polypeptide is a transporter protein.

[0283] The present application provides a method for gene editing of cells, the method comprising contacting any one of the above polypeptide delivery systems with a target cell, such that the polypeptide delivery system delivers the polypeptide into the target cell. The polypeptide is a gene editing protein.

[0284] The present application provides a method for labeling cells, the method comprising contacting any one of the above polypeptide delivery systems with a target cell, such that the polypeptide delivery system delivers the polypeptide into the target cell. The polypeptide is a tag protein or a reporter protein.

[0285] The present application provides a method for enhancing the pathogen resistance of cells, the method comprising contacting any one of the above polypeptide delivery systems with a target cell, such that the polypeptide delivery system delivers the polypeptide into the target cell. The polypeptide is an antimicrobial peptide.

[0286] The present application provides a method for preparing a cell vaccine, the method comprising contacting any one of the above polypeptide delivery systems with a target cell, such that the polypeptide delivery system delivers the polypeptide into the target cell. The polypeptide is an antigen or an immunogen.

[0287] Wherein, the above target cell is a eukaryotic cell, and the eukaryotic cell is, for example, a yeast cell, an insect cell, a mammalian cell, a plant cell or a fungal cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is a human tumor cell, such as a HER2-positive human tumor cell.

[0288] The present application provides a method for treating and / or preventing a disease, which comprises administering to a subject any one of the above polypeptide delivery systems, the polypeptide delivery system being loaded with a therapeutically effective amount of a biomacromolecule. The disease can be determined according to the function of the biomacromolecule used.

[0289] The present application provides an immunotoxin, which comprises any one of the above polypeptide delivery systems.

[0290] The present application provides a pharmaceutical composition, which comprises any one of the above polypeptide delivery systems.

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

[0292] The term "pharmaceutically acceptable" means that when the molecular entity and the composition are appropriately administered to an animal or a human, they do not produce adverse, allergic, or other untoward reactions.

[0293] Exemplarily, some substances that can serve as pharmaceutically acceptable carriers or their components are saccharides such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and methyl cellulose; tragacanth 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, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifying agents such as wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions, and phosphate buffer solutions, etc.

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

[0295] The present application provides the use of any one of the above signal peptides, any one of the above polypeptide delivery systems, any one of the above nucleic acids, any one of the above expression vectors, or any one of the above host cells in the preparation of a drug, reagent, or kit for delivering a polypeptide.

[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 are, for example, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, 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, lymphoproliferative diseases such as leukemia, and various types of head and neck cancers.

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

[0300] Application of PagT1 gene and its encoded protein

[0301] The present application also provides the use of the PagT1 gene or the protein encoded thereby in the preparation of a drug 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%, 99% identity with the sequence shown in SEQ ID NO: 1.

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

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

[0305] The tumor is, for example, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, 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, lymphoproliferative diseases such as leukemia, and various types of head and neck cancers.

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

[0307] In this application, a completely new protein toxin PagT1 was first identified, and no related homologous proteins have been found so far. This discovery suggests that PagT1 has a relatively novel mode of action, which is different from the protein toxin action mechanisms involved in existing immunotoxins. The study of the virulence mechanism of PagT1 broadens the understanding of PVC virulence factors, also lays a foundation for the study of widespread similar novel virulence factors, and at the same time provides an important reference for the pathogenicity of Photorhabdus and the treatment of clinical bacterial infectious diseases.

[0308] Different from the protein toxins used in existing immunotoxins, the advantage of PagT1 is that it can be loaded into PVC and can be used as a highly cytotoxic payload to jointly form a novel "immune delivery toxin" with PVC, providing a completely new treatment tool for the targeted treatment of diseases such as tumors. Further, this application discovers that PVC in this novel "immune delivery 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 relatively low. Moreover, the function of the extracted PVC does not depend on the presence of bacteria. It is free from bacterial heat sources and is relatively safe.

[0310] (2) Different from the existing intracellular endocytosis process dependent on immunotoxins, PVC delivers the loaded toxic payload to cells through the energy stored in the outer sheath protein. This has great advantages for tumor-targeted killing, enabling the toxic payload to exert its toxic effect more effectively and avoiding the degradation of the toxic payload and triggering the human immune response.

[0311] (3) In this application, by modifying the tail fiber protein Pvc13 of the PVC delivery vector, a PVC that can recognize tumor-associated antigens / tumor-specific antigens is obtained. 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 the potential of combining it with PVC for the development of a new type of "immune delivery toxin". This discovery not only promotes the understanding of the pathogenic mechanism of Photorhabdus luminescens, but also provides new ideas and technical support for the development of safer and more effective tumor-targeted treatment methods, with important scientific significance and broad application prospects.

[0313] Examples

[0314] The following will illustrate the content of this application with specific examples, but the scope of this application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following examples are conventional reagents and instruments in the art and can be obtained through commercial purchase. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described solutions and obtain corresponding results according to the content of the examples.

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

[0318] Table 2 Plasmid information involved in the examples ;

[0319] The antibody information involved in the examples is shown in Table 3 below.

[0320] Table 3 Antibody Information Involved in the Examples

[0321] Cell Lines and Cell Cultures Involved in the Examples

[0322] The 293T cell line of human embryonic kidney cells (Human embryonic kidney 293 cells) was purchased from the American Type Culture Collection (ATCC) cell bank (CRL-3216);

[0323] The J774A.1 cell line of murine macrophage-like cells (Murine macrophage-like cell line) was purchased from the American Type Culture Collection (ATCC) cell bank (TIB-67);

[0324] The THP-1 cell line of human monocytic leukaemia cells (Human monocytic leukaemia cell line) was purchased from the American Type Culture Collection (ATCC) cell bank (TIB-202);

[0325] The A549 cell line of alveolar basal epithelial cells (Alveolar basal epithelial cells) was purchased from the American Type Culture Collection (ATCC) cell bank (CCL-185);

[0326] The Hela cell line of human cervical tumour cells (Henrietta Lacks cells) was purchased from the American Type Culture Collection (ATCC) cell bank (CCL-2);

[0327] The Hep G2 cell line of human hepatocarcinoma cells (Human hepatocarcinoma cells) was purchased from the American Type Culture Collection (ATCC) cell bank (HB-8065);

[0328] The U251 cell line of human glioma cells (Human glioma cells) was purchased from the National Resource Bank of Biomedical Experimental Cells (1101HUM-PUMC000058);

[0329] The WERI-Rb-1 cell line of human retinal glioma cells (Human retinal glioma cells) was preserved in this laboratory;

[0330] The Hela cell line of human cervical tumour cells (Henrietta Lacks cells) was purchased from the American Type Culture Collection (ATCC) cell bank (CCL-2);

[0331] The human glioblastoma cells T98G cell line was purchased from the National Cell Bank of Biomedical Sciences (3101HUMSCSP5274);

[0332] The human astrocytic glioblastoma cells U87MG cell line was purchased from the National Cell Bank of Biomedical Sciences (1101HUM-PUMC000208);

[0333] The human bladder transitional cell carcinoma cells T24 cell line was preserved in our laboratory;

[0334] The human bladder transitional cell carcinoma cells BIU87 cell line was preserved in our laboratory;

[0335] All cells were identified by STR (Short tandem repeat). The culture media for 293T, J774A.1, Hep G2, and Hela cells were high-glucose DMEM media; the culture media for THP-1, A549, and U87MG cells were RPMI1640 media; the culture media for U251 and T98G cells were MEM media, the culture media for BIU87 and T24 cells were McCoy’s 5A media, and the culture media for WERI-Rb-1 cells were IMDM media; all media were supplemented with 10% FBS, 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.

[0336] The main instruments and equipment involved in the examples are shown in Table 4 below.

[0337] Table 4 Main instruments and equipment involved in the examples

[0338] The specific steps of some experimental operations involved in the examples are as follows:

[0339] Construction of gene expression vectors

[0340] Synthesize the sequences encoding Photorhabdus asymbiotica WP_015834233.1 (PagT1) protein (amino acid sequence shown in SEQ ID NO: 1), the C-terminus of Pseudomonas alkylphenolica WP_051939377.1 protein (amino acid sequence shown in SEQ ID NO: 2), the C-terminus of Deltaproteobacteria bacterium MCE9576703.1 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), the C-terminus of Burkholderia ubonensis KVU23967.1 protein (amino acid sequence shown in SEQ ID NO: 7) and Streptomyces avermitilis WP_037649420.1 protein (amino acid sequence shown in SEQ ID NO: 8) by gene synthesis, and insert them into the pEGFP-C1 vector to construct a eukaryotic expression vector.

[0341] Synthesize the nucleotide sequences encoding the above proteins for constructing the pEGFP-C1 expression vector, and codon optimization is performed during synthesis to adapt to expression in human cells.

[0342] Insert the sequence encoding Photorhabdus asymbiotica WP_015834233.1 (PagT1) protein into the pBBRN vector to construct a PVC loading vector.

[0343] Synthesize the nucleotide sequence (SEQ ID NO: 10) of the DARPin that binds to HER2 with KpnI and HindIII restriction sites and linker GGSGGGGSGG (SEQ ID NO: 9) at both ends by gene synthesis. After digestion with KpnI and HindIII, insert it into the pRK404-PVC vector for targeted HER2 PVC (PVCHER2) expression.

[0344] Primer design and synthesis

[0345] 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.

[0346] Table 5 PCR Primer Names and Sequences

[0347] PCR System and Procedure for Mutation

[0348] The PCR system for mutation involved in the examples is shown in Table 6 below.

[0349] Table 6 PCR System for Mutation

[0350] After mixing, centrifuge and perform amplification on a PCR instrument. The procedure is shown in Table 7 below.

[0351] Table 7 PCR Procedure for Mutation

[0352] The annealing temperature is determined based on the Tm value of the primer, usually 2 - 3°C lower than the Tm value; while the extension time is determined according to the length of the amplified fragment (1 min per kb).

[0353] After the reaction, add 1 μL of DpnI, mix well, and incubate at 37°C for 5 min.

[0354] The shRNA sequences for gene knockdown involved in the examples are shown in Table 8 below

[0355] Table 8 shRNA Sequence Information

[0356] Plasmid Extraction

[0357] Small-scale Plasmid Extraction

[0358] Take 1 mL of the cultured bacterial solution and place it in a 1.5 mL centrifuge tube. Centrifuge at 12,000×g for 1 min and discard the supernatant; operate according to the instructions of the small-scale plasmid extraction kit (Beijing Tsingke Biotechnology Co., Ltd.) and measure the concentration of the obtained plasmid using a spectrophotometer.

[0359] Large-scale Plasmid Extraction

[0360] Add 100 - 200 mL of LB culture medium containing antibiotics to a 1 L bacterial culture flask; then inoculate the primary bacterial solution into the culture flask at a ratio of 0.1% and culture at 37°C for 16 - 18 h; operate according to the instructions of the large-scale plasmid extraction kit (Beijing Tsingke Biotechnology Co., Ltd.) and measure the concentration of the obtained plasmid using a spectrophotometer.

[0361] Construct the phylogenetic tree of PagT1 and its homologous proteins

[0362] Search for proteins homologous to the PagT1 amino acid sequence through the BLAST function of the NCBI (https: / / www.ncbi.nlm.nih.gov) database and download their sequences in fasta format; use MEGA software to perform sequence alignment through ClustalW; generate a phylogenetic tree by the maximum likelihood method and beautify the phylogenetic tree through iTOL (https: / / itol.embl.de / ).

[0363] Extraction and culture of primary mouse bone marrow-derived macrophages (BMDMs)

[0364] The mice were sacrificed by cervical dislocation, and then the tibias and femurs of the mice were separated, soaked in 75% alcohol and transferred to PBS; the two ends of the tibias and femurs were cut open, and a 1 mL syringe was used to aspirate the complete medium, and the bone marrow cells were rinsed from one end into a 50 mL sterile centrifuge tube, and repeated several times until the bones turned white; operate according to the instructions of the red blood cell lysate to remove red blood cells; resuspend the cells with DMEM complete medium containing 10 ng / mL M-CSF, adjust to 1×10 6 cells / mL after cell counting, inoculate into culture dishes, and statically culture under the conditions of 37 °C and 5% CO2.

[0365] Cell passage and transfection

[0366] Cell passage

[0367] a. 293T cells and A549 cells:

[0368] When the cells were cultured in a 10 cm culture dish and their density reached about 90%, the medium in the culture dish was aspirated; first, take 1 mL of 0.25% trypsin to rinse the culture dish, and then aspirate and discard the trypsin; then add 1 mL of 0.25% trypsin again, put the culture dish into the incubator until the cells were digested to the detached state; add 1 mL of medium containing 10% FBS to it to terminate the digestion; transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 800 rpm / min for 5 min, discard the supernatant; add 1 mL of medium to the centrifuge tube and gently resuspend the cells; aspirate 150 μL of the cell suspension from the centrifuge tube and drop it into a culture dish containing 10 mL of medium, or drop it into a six-well plate with 1.5 mL of medium in each well, shake well after dropping, and statically culture under the conditions of 37 °C and 5% CO2.

[0369] b. For J774A.1 cells and BMDM cells:

[0370] The cells were cultured in a 10 cm culture dish. When the cell density reached approximately 90%, the culture medium in the dish was aspirated; 3 mL of cell culture medium was added to the dish, and then a cell scraper was used to gently scrape the cells off in a single direction; the scraped cells were transferred to a 15 mL centrifuge tube and centrifuged at 800 rpm / min for 5 min, and the supernatant was discarded; 3 mL of medium was added, and the cells were resuspended and counted after that.

[0371] Treatment of different operation scenarios: For cell passage, 150 μL was aspirated from the resuspended and counted cell suspension and dropped into a culture dish containing 10 mL of medium; when plating in a 96-well plate, the required cell amount was calculated according to 1×10 4 cells per well, the corresponding amount of cells was aspirated, resuspended in the medium suitable for the culture of this cell, and then transferred to a 96-well plate and placed in an incubator for culture; for plating in a 12-well plate, the cell usage amount was calculated according to 1×10 5 cells per well, the required amount of cells was aspirated, resuspended in the medium required for the corresponding cell culture, and then transferred to a 12-well plate and statically cultured under the conditions of 37°C and 5% CO2.

[0372] c. For THP-1 cells:

[0373] The cells were cultured in a T75 cell culture flask. 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 RPMI1640 medium (containing 1% penicillin-streptomycin and 10% fetal bovine serum) was added to the new flask; the flask was placed in an incubator and statically cultured under the conditions of 37°C and 5% CO2.

[0374] Inducing THP-1 to differentiate into macrophages: After 2 days of cell culture, the cell suspension was transferred to a 50 mL centrifuge tube and centrifuged at 800 rpm / min 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 plating in a 96-well plate, according to the standard of inoculating 1×10 4 cells per well, the resuspended cells were inoculated into a 96-well plate, and then the 96-well plate was placed in an incubator and statically cultured under the conditions of 37°C and 5% CO2; for plating in a 12-well plate, according to the standard of inoculating 1×10 5 cells per well, the resuspended cells were inoculated into a 12-well plate and also placed in an incubator and statically cultured under the conditions of 37°C and 5% CO2.

[0375] Cell transfection

[0376] Cells were evenly seeded in six-well plates. When the cell density reached approximately 70%, transfection was performed. Take 1 μg of plasmid and dissolve it in 100 μL of medium without FBS. At the same time, take another 2 μL of transfection reagent PolyJet and also dissolve it in 100 μL of the same medium without FBS. Add the medium containing the transfection reagent PolyJet to the medium containing the plasmid, mix well, and let it stand at room temperature for 15 min. Then, drop the mixture into the medium containing the cells drop by drop. 6 h after the transfection operation, replace the cell medium with fresh medium.

[0377] Cell viability detection

[0378] Aspirate and discard 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 place it in the cell culture incubator for 1 h.

[0379] Measure the absorbance at 450 nm using a microplate reader.

[0380] Protein immunoblotting experiment (Western blot)

[0381] Place the SDS-PAGE in the electrophoresis tank and add 1× electrophoresis buffer. Add 5 μL of protein molecular weight marker and 20 μL of protein sample to the corresponding loading wells respectively. Set the constant voltage to 120 V and the electrophoresis time to 90 min. Activate the PVDF membrane with anhydrous methanol. After activation, sandwich the PVDF membrane and the gel after electrophoresis into the transfer clip. Place the transfer clip in the transfer tank and add 1× transfer buffer. Set the constant current to 300 mA and the transfer time to 60 min. After the transfer is completed, choose 5% non-fat milk powder or 5% BSA to block the membrane. The blocking condition is to block at room temperature for 1 h or block overnight at 4°C. Incubate the primary antibody with the membrane at room temperature for 1 h or overnight at 4°C. After thoroughly washing the antibody on the membrane with TBST, lay flat the ECL developing solution and use an ultrasensitive chemiluminescence imaging instrument for imaging.

[0382] Production of PVC

[0383] PVCR7 is derived from a previously published literature (Kreitz J, Friedrich M J, Guru A, Lash B, Saito M, Macrae R K, Zhang F. Programmable protein delivery with a bacterial contractile injection system[J]. Nature. 2023; 616:357).

[0384] The generation, extraction, and purification of the empty PVC vector, empty PVCR7 vector, empty PVCHER2 vector, and PVC complex were carried out with reference to a 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 by the method disclosed in the literature, and the empty PVCR7 vector and empty PVCHER2 vector were obtained by modification on the tail fiber protein Pvc13 of the PVC-V complex.

[0385] In the examples of this application, the PVC complex refers to the complex formed by the empty PVC / PVCR7 / PVCHER2 vector and the loaded protein.

[0386] Extraction and Purification of PVC Complex

[0387] Pick a single colony and place it in a container containing 2 - 4 mL of liquid LB medium supplemented with the corresponding antibiotic; place the container on a shaker and incubate overnight at 37°C and 220 rpm / min; after the incubation, transfer the bacterial solution to a container containing 200 mL of liquid LB medium supplemented with the corresponding antibiotic at a ratio greater than 1:100; place the container on a shaker and incubate at 30°C and 220 rpm / min for 24 h; after the incubation, centrifuge to collect the bacterial cells; resuspend with 30 mL of PBS and centrifuge again to collect the bacterial pellet; store the bacterial pellet at -80°C for at least overnight.

[0388] Prepare a bacterial lysis buffer with the following formulation: 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 the bacterial pellet collected from every 200 ml of LB liquid medium, add 10 mL of the bacterial lysis buffer, resuspend it thoroughly and mix evenly; incubate the mixture at 37°C for 30 min to lyse the bacteria; after lysis is completed, centrifuge at 4°C, 12000 rpm / min for 10 min to remove unlysed bacteria and cell debris.

[0389] Transfer the supernatant after bacterial lysis centrifugation to an ultracentrifuge tube, and centrifuge at 4°C, 200000 g / min for 1 h; discard the supernatant, resuspend the pellet with 1 mL of PBS; centrifuge at 4°C, 14000 rpm / min for 10 min; transfer the supernatant to the ultracentrifuge tube again, and centrifuge at 4°C, 200000 g / min for 1 h; discard the supernatant, resuspend the pellet with 200 μL of PBS; centrifuge at 4°C, 14000 rpm / min for 10 min; at this time, collect the supernatant, which is the PVC complex solution obtained after extraction and purification, and this solution can be stored temporarily at 4°C.

[0390] Western blot detection of the PVC complex: Use the flag antibody to detect the protein loading, and at the same time use the PVC structural protein 16 antibody to detect the total amount of the complex.

[0391] Removal of endotoxin from the purified PVC complex

[0392] Pre-cool the PVC sample at 4°C, prepare Triton X-114 with a final concentration of 1-2% (W / V) and also pre-cooled at 4°C, add it to the pre-cooled PVC sample, after addition, mix well by inverting the container, then ice-bath the sample for 15 min, and invert the container multiple times during the ice-bath to ensure that the sample is always in a homogeneous mixture state; after the ice-bath ends, incubate in a 37°C water bath for 5 min; after the incubation ends, centrifuge at 37°C, 12000 g for 5 min to completely separate Trion X-114 from the aqueous solution, and discard the supernatant; repeat the above steps 2-3 times.

[0393] Endotoxin detection in the PVC complex

[0394] Sample pretreatment and reagent preparation: Endotoxin standards were dissolved according to the label titer (P). For P≤20EU / bottle, 10mL of pyrogen-free water was added to prepare a 10 EU / mL stock solution. The stock solution was diluted step by step 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 before use. When dissolving the horseshoe crab reagent (LAL), 1.7 mL of pyrogen-free water was added, the mixture was gently inverted and mixed, and then allowed to stand for 30s. It must be used within 10 min after dissolution. The chromogenic substrate and stop solution were dissolved according to the instructions and stored at 2-8°C in the dark.

[0395] Construction of standard curve: 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) were mixed with an equal volume of LAL reagent and incubated in a constant temperature water bath at 37±1°C (T1 time was used for the low concentration range of 0.01-0.1 EU / mL, and T2 time was used for the high concentration range of 0.1-1 EU / mL. For specific time, refer to the kit label); after incubation, 100 μL of colorimetric substrate (incubated at 37°C for 6 min) and 500 μL of stop solution were added in sequence; the absorbance at 545 nm was measured using an ELISA reader, with the standard endotoxin concentration (EU / mL) as the horizontal axis and Δ absorbance (sample absorbance - blank absorbance) as the vertical axis. The standard curve was constructed by linear regression analysis, and the fit requirement was R 2 ≥0.96.

[0396] Protein sample detection: The protein sample is diluted with pyrogen-free water to within the detection range (the dilution factor does not exceed the maximum effective dilution factor MVD); if the sample pH deviates from 6.0-8.0, adjust it to neutral with 0.1 M NaOH or HCl; take 100 μL of the diluted sample and mix it with an equal volume of LAL reagent, and incubate and color develop according to the standard curve conditions. Set up 3 technical replicates for each sample, and the negative control is operated synchronously with pyrogen-free water instead of the sample.

[0397] Data analysis and calculation: Calculate the sample endotoxin concentration according to the standard curve equation, convert the original solution concentration based on the dilution multiple, and calculate the endotoxin content.

[0398] PVC treated cells

[0399] After 24 hours of cell culture, empty PVC vector, empty PVCR7 vector, empty PVCHER2 vector or PVC complex was added to the cell culture medium at a concentration of 15 μg / mL unless otherwise specified; CCK-8 was used to detect cell viability according to the instructions of the reagent manufacturer 24 hours after addition unless otherwise specified; immunofluorescence detection was performed 18 hours after addition.

[0400] Bioinformatics Analysis of PagT1 Protein

[0401] The cell sublocalization of the PagT1 amino acid sequence was analyzed by the protein language model DeepLoc 2.0 for cell sublocalization prediction (https: / / services.healthtech.dtu.dk / services / DeepLoc-2.0 / ); its three-dimensional structure was predicted by AlphaFold3 (https: / / deepmind.google / technologies / alphafold / alphafold-server / ), its possible active pocket was predicted by DoGSiteScorer (https: / / proteins.plus / ), and proteins with a three-dimensional structure similar to PagT1 were searched by Foldseek (https: / / search.foldseek.com / search).

[0402] Confocal Microscopy Observation of the Cell Sublocalization of PagT1 during Transient Expression in A549 Cells

[0403] Cells were evenly seeded in a glass-bottom dish, transfected after 12 h, the fresh medium was changed after 6 h, and observation and photography were performed using a laser confocal microscope after 18 h.

[0404] Immunofluorescence Staining

[0405] Cells were evenly seeded on 20 mm cell slides in a 12-well plate; after 24 h, empty PVC vector, empty PVCR7 vector, empty PVCHER2 vector, or PVC / PVCR7 / PVCHER2 loaded with PagT1 was added to the wells for 18 h; after the treatment, the medium was aspirated and the cells were fixed with methanol for 3 min; 1 mL of 5% BSA was added to each well and blocked at 37 °C for 1 h; 100 μL of the corresponding primary antibody diluted with 1×PBS (antibody information is shown in Table 3) was added to each well and incubated at 37 °C for 1 h; 100 μL of the corresponding fluorescent secondary antibody diluted with 1×PBS (antibody information is shown in Table 3) was added to each well and incubated at 37 °C for 1 h; the cell slides were inverted onto a glass slide with a drop of mounting medium (containing DAPI), and observation and photography were performed using a laser confocal microscope.

[0406] Comet Assay

[0407] After 24 hours of cell treatment, comet electrophoresis was detected with reference to the Comet Assay Kit (Shanghai Beyotime Biotechnology Co., Ltd.). After propidium iodide staining, red fluorescence would appear, and its excitation wavelength (Ex) / emission wavelength (Em) was 535 / 617 nm; the tail moment was statistically analyzed, and the calculation method of the tail moment was the product of the tail length and the percentage of DNA in the tail.

[0408] CRISPR / Cas9 sgRNA library screening

[0409] (1) Transduce J774A.1 cells with LentiCas9 Blast lentivirus and screen with Blasticidin.

[0410] (2) sgRNA library lentivirus packaging

[0411] Prepare 200 μg of the Mouse Brie CRISPR knockout pooled library (https: / / www.addgene.org / pooled-library / broadgpp-mouse-knockout-brie / ) whole-genome library plasmid with endotoxin content ≤0.01 EU / μg. The quality of the library was verified by NGS. The library contained at least 99% of sgRNAs, and the Gini coefficient of the sgRNA distribution was less than 0.1;

[0412] Transfect the lentivirus packaging plasmid and the sgRNA library plasmid into 293T cells. Harvest the lentivirus 72 hours after transfection and measure the titer.

[0413] (3) Screen for the optimal transduction cell density

[0414] Determine the puromycin killing curve in Cas9-overexpressing J774A.1 cells. Transduce J774A.1 cells with different volumes of the Brie library virus, and select the appropriate puromycin concentration and the optimal seeding density of the transduced cells.

[0415] (4) Lentivirus Brie library transduction and cell library generation

[0416] Transduce Cas9-overexpressing J774A.1 cells with a mixed Brie lentivirus library with an MOI of approximately 0.3 and screen with appropriate antibiotics. Detect the cells by NGS.

[0417] (5) Determine the optimal screening conditions for PVC

[0418] By adding different concentrations of PVC-PagT1, the concentration (30 μg / mL) that can cause about 80% death of Cas9-overexpressing J774A.1 cells was selected as the screening condition.

[0419] (6) The cell library was screened three times using 30 μg / mL of PVC-PagT1 and the empty vector PVC.

[0420] (7) After screening, the genome was extracted, and an NGS sequencing library was constructed through steps such as DNA fragmentation, PCR enrichment, and fragment screening.

[0421] (8) NGS experimental procedures and data analysis

[0422] a. Library construction and sequencing

[0423] An NGS sequencing library was constructed through steps such as PCR enrichment and fragment screening. After the library passed the quality detection by Agilent 2100 Bioanalyzer and the quantitative detection by qPCR, it was sequenced on the illumina platform device;

[0424] PCR enrichment was performed by adding adapters containing P5, P7, and index through PCR reactions;

[0425] The constructed library products were purified using a PCR product purification kit;

[0426] The fragment distribution of the library was detected using Agilent 2100 Bioanalyzer, and the library was quantified using qPCR;

[0427] b. Data analysis

[0428] The raw data downloaded from the machine was processed by bcl2fastq and split according to the sample Index to obtain the corresponding data for the samples;

[0429] According to the library construction method, the adapters were removed from the original sequences, and the low-quality reads were removed to obtain clean data. R1 and R2 were merged based on overlap to obtain the data for analysis;

[0430] The MAGeCK (version 0.5.9.6) software was used to identify the sgRNA sequences and perform the abundance statistics of the sgRNA sequences;

[0431] With the help of the MAGeCK software, based on the aligned sgRNA and gene count situations, the count results were first standardized, and then the differences in sgRNA between various samples were compared, and the differential genes between samples were analyzed, annotated in the database, and visually displayed.

[0432] Gene knockdown in J774A.1 cells using lentiviral shRNA vectors

[0433] (1) Lentivirus packaging and production

[0434] Twenty-four hours before plasmid transfection, trypsinize 293T cells (logarithmic phase), then perform cell counting. Set the cell density at 6×10 5 cells / mL, seed them in a 10 cm cell culture dish, and culture them in an incubator at 37°C and 5% CO2 for 24 h. When the cell confluence reaches 70%-80%, use them for transfection. Replace the medium with basal medium 2 h before transfection;

[0435] Co-transfect 293T cells with the shRNA vector pTSB-U6-PGK-Fluor-2A-ARGs required for lentivirus packaging and the helper plasmids pSPAX2 and pMD2.G. After 8 h, aspirate the medium, wash with PBS, discard the supernatant, replace with fresh medium, and continue culturing in an incubator at 37°C and 5% CO2 for 48 h;

[0436] Collect the supernatant into a centrifuge tube, centrifuge at 2000 rpm for 10 min to remove the precipitate, perform ultrafiltration, then measure the titer, and finally aliquot and store at -80°C.

[0437] (2) Construction of stable cell lines by lentivirus infection of J774A.1 cells

[0438] Eighteen to twenty-four hours before lentivirus transfection, seed J774A.1 cells at 1×10 5 cells / 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 normal fresh serum-containing medium and continue culturing for 48 h, then observe fluorescence expression; add puromycin to screen for stably knocked-down cells; detect the gene knockdown efficiency of stably knocked-down cells by qRT-PCR.

[0439] Treatment of cells with NAD+ and inhibitors

[0440] After culturing cells seeded in 96-well or 24-well plates for 24 h, add NAD+, the PARP1 inhibitor Rucaparib, the JAK inhibitor Ruxolitinib, or the STAT inhibitor SH-4-54. After 3 h, add an empty PVC vector, an empty PVCR7 vector, or a PVC / PVCR7 loaded with PagT1 to the medium and incubate for 24 h, then detect cell viability or collect cell lysates for Western blot analysis.

[0441] Observation of PVC complexes by transmission electron microscopy

[0442] (1) Pre-treatment of the carrier grid

[0443] The carrier grid is subjected to hydrophilic treatment using a glow discharge instrument. Place the 200-mesh carbon-coated support film copper grid face-up on a filter paper and put it into the glow discharge instrument. Set the parameters as the negative electrode, current 15 mA, and discharge for 45 s to enhance the hydrophilicity of the carbon film.

[0444] (2) Adsorption of protein samples

[0445] Dilute the extracted protein samples such as PVCHER2 and PVCHER2-PagT1 to 0.3 mg / mL using PBS (subject to Nanodrop measurement). Take 10 μL of the protein solution and drop it on the hydrophilized carrier grid. Let it stand for 2 min. Gently absorb the excess liquid from the edge of the carrier grid with a filter paper moistened with distilled water, and avoid touching the carbon film.

[0446] Drop 100 μL of distilled water on the sealing film. Hold the edge of the carrier grid with forceps, invert the carrier grid so that the front of the carrier grid contacts the surface of the water droplet. Gently shake the forceps to wash the carrier grid for 15 s. Gently absorb the excess liquid from the edge of the carrier grid with a filter paper moistened with distilled water, and avoid touching the carbon film. At the same time, pay attention to preventing the back of the carrier grid from contacting the liquid.

[0447] (3) Negative staining of samples

[0448] Drop three drops of 100 μL of 1% uranyl acetate staining solution on the sealing film. Hold the edge of the carrier grid with forceps, invert the carrier grid so that the front of the carrier grid contacts the surface of the first drop of staining solution. Gently shake the forceps to wash the carrier grid for 15 s; repeat the steps for the second and third drops of staining solution; let the carrier grid stand, and continue to stain for 1 min through the adsorbed staining solution on the surface; gently absorb the excess liquid from the edge of the carrier grid with a filter paper moistened with distilled water, and avoid touching the carbon film. During the entire staining process, pay attention to preventing the back of the carrier grid from contacting the liquid; after leaving the carrier grid to stand at room temperature and dry for 10 min, put it back into the sample box for storage.

[0449] (4) Observation with a transmission electron microscope (TEM)

[0450] Select a transmission electron microscope with a voltage of 120 kV. Place the carrier grid face-up in the sample holder of the TEM for observation. Find the area where the protein particles are evenly distributed and adjust the focus for imaging.

[0451] Collection of bladder cancer organoid samples

[0452] The bladder cancer organoids are from Beijing Ketu Medical Technology Co., Ltd. Take out the cryopreserved bladder cancer organoids for resuscitation culture. When the bladder cancer organoids reach the passageable state, place the Matrigel at 4°C to melt it; collect the cultured bladder cancer organoids using a Pasteur pipette, and then add OrganoProTM 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 bladder cancer organoid culture medium is used to adjust the cell concentration to 1.0×10 6 cells / mL. An appropriate amount of the cell suspension is taken out and placed on ice for later use. At the same time, a Matrigel mixture is prepared on ice for later use. The cell suspension is mixed with Matrigel, and 200 μL of the mixture is added to each well of a 24-well plate and incubated at 37°C for 30 min. Then, 200 μL of OrganoPro TM bladder cancer organoid culture medium is added, and the cells are statically cultured for 2 days. At this time, it can be observed that bladder cancer organoids begin to form and grow.

[0453] When the diameter of the bladder cancer organoids reaches about 200 μm or larger, the cultured bladder cancer organoids are collected into a 15 mL centrifuge tube using a Pasteur pipette, centrifuged at 300 g for 5 min, the upper-layer culture medium is discarded, the precipitate is resuspended with PBS, washed by pipetting, centrifuged at 300 g for 5 min, the PBS is discarded, and the organoid precipitate is collected; the tip part of a 1 mL pipette tip is cut off with scissors, 500 μL of 4% paraformaldehyde tissue cell fixative is aspirated, mixed with the organoids by pipetting, and then aspirated into a 1.5 mL centrifuge tube; 500 μL of 4% paraformaldehyde tissue cell fixative is aspirated into a 1.5 mL centrifuge tube, and then the organoids are transferred to the 1.5 mL centrifuge tube by pipetting using the pipette tip with the cut-off tip, and stored at 4°C.

[0454] Immunohistochemistry of bladder cancer organoids

[0455] The samples are subjected to immunohistochemical detection after being fixed with formalin-fixed tissue samples. The specific primary and secondary antibody information is shown in Table 3, and hematoxylin is used to stain the cell nuclei.

[0456] Immunofluorescence of bladder cancer organoids

[0457] The samples are subjected to immunofluorescence staining after being fixed with formalin-fixed tissue samples. The specific primary and secondary antibody information is shown in Table 3, and DAPI is used to stain the cell nuclei.

[0458] Pharmacodynamic detection of bladder cancer organoids

[0459] Organoid seeding: When the bladder cancer organoids reach the subculture state, the Matrigel is melted at 4°C for later use; the cultured bladder cancer organoids are collected, and the passage enzymatic digestion solution in the OrganoPro TM bladder cancer organoid culture kit is used to enzymatically digest the bladder cancer organoids to prepare a single-cell suspension. After cell counting, the cell concentration is adjusted to 1.0×10 6cells / mL; Mix the cell pellet with Matrigel evenly, add 4 μL of the mixture to each well of a 96-well plate, incubate at 37 °C for 30 min, and then add 86 μL of OrganoPro to each well. TM Bladder cancer organoid medium, culture in an incubator for 2 days, and observe the initial formation and growth of bladder cancer organoids.

[0460] Preparation and dilution of empty PVCHER2 vector, PVCHER2 loaded with PagT1 (PVCHER2-PagT1), and PVCHER2 loaded with a mutant with mutations at positions D217 and E219 of PagT1 mutated to A (PVCHER2-217219): Gradient dilute them with PBS in a biosafety cabinet.

[0461] Add the drug to be tested: After observing the formation and growth of bladder cancer organoids, sequentially add freshly prepared empty PVCHER2 vector, PVCHER2-PagT1, and PVCHER2-217219 at different concentrations on the same day, and incubate in an incubator for 120 h.

[0462] Chemiluminescence assay: After the culture is completed, add 50 μL of cell viability fluorescence detection reagent and measure the fluorescence value.

[0463] Construction of U251-Luci cells

[0464] Infect U251 cells with lentivirus containing Ubi-MCS-firefly_Luciferase-IRES-Puromycin element; Add puromycin to screen for positive cells 24 h later; Detect the expression of luciferase by Bright-Lite luciferase detection system (Vazyme, DD1204-02).

[0465] Mice

[0466] The 6-week-old male NTG mice used in this application were purchased from Beijing Sparf Bioscience Co., Ltd. All SPF-grade animals were housed in a constant temperature and humidity breeding room, maintained on a 12 h / 12 h light / dark cycle, and provided with standardized mouse food and drinking water.

[0467] Orthotopic brain tumor xenograft and PVC treatment

[0468] NTG mice were anesthetized with 100 mg / kg ketamine and 10 mg / kg xylazine; After disinfection and hair removal, the mice were fixed on a stereotaxic apparatus for the brain, and the cranial skin was exposed; U251-luci cells (2×10 4Cells (dissolved in 2 μL PBS) were injected into the brain. The injection site was determined as 1 mm lateral, 1 mm anterior, and 2 mm deep from the outer side of the cerebral meninges. The tumor-bearing mice were randomly and evenly divided into 4 groups. On the 10th day after transplantation, PBS, empty PVCHER2 vector (control), low-dose PVCHER2-PagT1 (15 μg), and high-dose PVCHER2-PagT1 (30 μg) were respectively injected into the lateral ventricles of each group. The injection site was determined as 1 mm lateral, 0.46 mm posterior, and 2.2 mm deep from the lambda. The mice were euthanized on the 35th day after inoculation.

[0469] Bioluminescence imaging (BMI)

[0470] On the 7th, 21st, and 35th days after inoculation, 150 mg / kg d-luciferin (PerkinElmer, 122799) was intraperitoneally injected, and in vivo glioma bioluminescence imaging was performed using IVIS Lumina III (PerkinElmer). Bioluminescence signals were continuously collected using a 1 / 8 filter, and the exposure time was set to 1 min. The results were analyzed using Living Image 4.2 software (Caliper Life Sciences).

[0471] Magnetic resonance imaging (MRI)

[0472] In vivo MRI was performed using a 7.0 T small animal MRI system. 30 minutes before the MRI examination, 0.1 mmol / kg Magnevist (gadolinium-DTPA) was injected into the tail vein of the mice. Spin echo (sems) sequence was used to collect the T1-weighted MRI images of the coronal plane of the mouse brain. The parameters were as follows: field of view (FOV) = 20×20 mm; slice thickness = 1 mm (zero slice gap); TR (repetition time) = 400 ms; TE (echo time) = 18.0 ms; image matrix = 256×256; average number = 10. Regions of interest (tumors of the mouse models on the 7th and 35th days) were manually drawn on the images to determine the changes in tumor volume.

[0473] Statistical analysis

[0474] The analysis of the data was completed with the aid of GraphPad Prism 6 software, and all data were presented in the form of mean and standard error of the mean (SEM). For the comparison of differences between two sets of variables, Student’s t-test was used; while when comparing the differences of three or more sets of variables, ANOVA (Analysis of Variance) was adopted, followed by Bonferroni’s post hoc analysis. In determining statistical significance, if the p-value was less than 0.05, it was considered that there was a statistical difference; when the p-value was less than 0.01, it was considered that there was a significant statistical difference; if the p-value was less than 0.001, it was considered that there was an extremely significant statistical difference.

[0475] Example 1 Functional identification of virulence factor PagT1

[0476] 1.1 Effect of PagT1 on host cell viability

[0477] 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 1 A). The two genes (MARTX and YopT) before and after this gene are virulence factors, indicating that PagT1 may be a potential virulence factor of Photorhabdus asymbiotica.

[0478] The inventors of the present application searched for homologous proteins of PagT1 through the BLAST function of the NCBI database and found that most of them were distributed in the genus Photorhabdus, and they were also distributed in genera such as Okeania, Pseudomonas alkylphenolica, Deltaproteobacteria bacterium, Photorhabdus heterorhabditis, Photorhabdus australis, Burkholderia ubonensis, Streptomyces avermitilis, etc. ( Figure 1B ). The inventors of the present application selected the homologous proteins of PagT1 from these different genera, constructed them into the eukaryotic expression vector pEGFP-C1, and after transient expression in 293T cells, the cell viability was detected by CCK-8. The results are shown in Figure 1 C. It was found that compared with the homologous proteins, PagT1 could cause the strongest cytotoxicity.

[0479] By transforming the pBBRN-PagT1-flag plasmid into the PVC expression strain, the PVC complex (PVC-PagT1) was obtained after purification. The loading protein PagT1 labeled with the flag tag and the final component Pvc16 in the PVC complex assembly process were detected by Western blot, indicating the successful assembly of PVC-PagT1 (Figure 2A). This result confirmed that PagT1 could be loaded into PVC without the need to add an additional loading signal peptide at the N-terminus, and PagT1 is a natural effector of PVC.

[0480] Furthermore, PVC-PagT1 was added to J774A.1 cells, and cell viability was detected by CCK-8. It was found that obvious cell death occurred in J774A.1 cells, while empty PVC had no obvious effect on the cells (Figure 2B), indicating that PVC-PagT1 could target the delivery of PagT1 to mouse macrophages.

[0481] In addition, PagT1 was also loaded into PVC-Ad5 (RGD / PK7) (Kreitz J, Friedrich M J, Guru A, Lash B, Saito M, Macrae R K, Zhang F. Programmable protein delivery with a bacterial contractile injection system[J]. Nature. 2023; 616: 357) (abbreviated as PVCR7 in this application) to obtain PVCR7-PagT1, where PVCR7 has a targeting property similar to that of adenovirus Ad5 (Figure 2A). After adding the purified PVCR7-PagT1 to cells, obvious cell death was also found in THP-1 cells (Figure 2C) and BMDM cells (Figure 2D).

[0482] The above results indicated that Pagt1 is a virulence factor of PVC, can be loaded into PVC, and can cause the death of mouse and human cells.

[0483] 1.2 PagT1 is localized in the eukaryotic nucleus and causes host cell DNA damage

[0484] To explore the cytotoxic mechanism of PagT1 in eukaryotic cells, the subcellular localization of PagT1 in cells was investigated in this example. The amino acid sequence of PagT1 was analyzed for subcellular localization by the cell sublocalization prediction protein language model DeepLoc 2.0, and the results were as Figure 3As shown, the results indicate that the most likely subcellular localization of PagT1 is the nucleus, and there is a nuclear localization signal (NLS) at its N-terminus and C-terminus respectively.

[0485] Next, it was experimentally verified whether PagT1 would localize to the nucleus. First, PagT1 tagged with GFP was transiently expressed in A549 cells. After 18 h, confocal microscopy observation revealed that PagT1 co-localized with the nucleus (Figure 4A), which confirmed the predicted nuclear localization of PagT1. Further, in this example, PVCR7 was directly used to deliver PagT1 with a flag tag to A549 cells, and immunofluorescence detection was performed using a flag antibody. The nuclear localization of PagT1 was also observed (Figure 4B).

[0486] By constructing mutants with the N-terminal and C-terminal NLSs of PagT1 removed separately and simultaneously (Figure 4C), the importance of NLS for the nuclear localization of PagT1 was explored. After transient expression in A549, confocal microscopy observation showed that removing the N-terminal (Del-N-NLS, SEQ ID NO: 31) or C-terminal (Del-C-NLS, SEQ ID NO: 32) NLS separately did not affect the nuclear localization of PagT1; only when the N-terminal and C-terminal NLSs were removed simultaneously (Del-N, C-NLS, SEQ ID NO: 33), PagT1 lost its nuclear localization (Figure 4A).

[0487] The above results indicate that a "dual-functional" signal peptide has been identified in this application, that is, this signal peptide can not only direct the effector protein to be loaded into the PVC protein complex, but also serve as a nuclear localization signal to direct the effector protein to localize to the nucleus.

[0488] For further verification, different lengths of the N-terminus of PagT1 were truncated and fused with the Tcst protein (Trichosanthin (TcsT) from Trichosanthes kirilowii). It was found that polypeptides of different lengths at the N-terminus of PagT1 (PagT1-N15 to PagT1-N70, SEQ ID NO: 19 - 30) (except PagT1-N15, all contain the complete N-terminal NLS of PagT1) could serve as signal peptides to direct the loading of Tcst into PVC (Figure 4D), which proved the dual function of the N-terminal signal peptide of PagT1.

[0489] To investigate the mechanism by which PagT1 exerts cytotoxicity, in this application, eukaryotic transient expression and the use of PVCR7 to deliver wild-type PagT1 (WT) and its NLS mutants were respectively employed to verify whether the cytotoxicity of PagT1 is related to its localization in the eukaryotic nucleus. As 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 assay for cell viability showed that simultaneous removal of the NLS at both the N-terminus and C-terminus of PagT1 could significantly improve the cell death caused by PagT1, while separate removal of the NLS at the N-terminus and C-terminus could not, which confirmed the importance of the nuclear localization of PagT1 for its cytotoxicity.

[0490] Furthermore, nuclear damage of cells after delivery of PagT1 by PVC was detected. After delivery of PagT1 to mouse and human macrophages using PVC or PVCR7 for 24 h, Western blot detected the production of the obvious nuclear damage marker phosphorylated histone H2AX (phosphorylated at S139) in J774A.1, THP-1, and BMDM cells (Figure 6A); moreover, more phosphorylated histone H2AX foci were also seen in the nucleus by immunofluorescence (Figure 6B). In addition, comet electrophoresis assay also observed the tailing of THP-1 cells after delivery of PagT1 (Figure 6C), and the tail moment statistics of the PagT1 group were also significantly higher than those of the PBS group and the empty PVCR7 group (Figure 6D). These results indicate that PagT1 can cause damage to the eukaryotic cell genome and is a genotoxin.

[0491] 1.3 Determination of the active site of PagT1

[0492] Since PagT1 can cause damage to the eukaryotic cell genome, to determine its specific mechanism, the possible enzymatic activity of PagT1 was explored. The three-dimensional structure of PagT1 was predicted by AlphaFold3 (Figure 7A), and the active pocket was predicted by DoGSiteScorer (Figure 7D). Foldseek was used to search for proteins with a three-dimensional structure similar to that of PagT1, and proteins with a spatial structure similar to that near the active pocket of PagT1 were found to be ADP ribosyltransferases in the PDB database (Figures 7B - 7C), suggesting that PagT1 may also be an ADP ribosyltransferase. By alignment with its homologous protein sequences, the conserved amino acid residues of PagT1 are D217 and E219, and this site is also present in the active pocket of PagT1 (Figures 7E - 7F).

[0493] The above results suggest that D217 and E219 may be the active centers of PagT1. These two sites were mutated to A to construct the PagT1 mutant 217219 (SEQ ID NO: 34). After transient expression in 293T cells, the mutant 217219 lost the cytotoxicity of PagT1 and basically did not affect cell viability (Figure 7G), indicating that D217 and E219 are the active centers of PagT1.

[0494] 1.4 PagT1 causes PARP1-dependent eukaryotic cell death, Parthanatos

[0495] PARP-1, as a key protein of Parthanatos and also a DNA repair protein, first detected the expression of PARP1 in THP-1 cells after delivering PagT1 through PVCR7. The Western blot results showed that the expression of PARP1 in THP-1 cells increased significantly after delivering PagT1 (Figure 8A).

[0496] Since Parthanatos involves the depletion of nicotinamide adenine dinucleotide (NAD+), to determine whether the cell death caused by PagT1 is Parthanatos, NAD+ was added to the cell culture medium, and then PagT1 was delivered to the cells. Finally, it was found that the cell death caused by PagT1 in J774A.1 (Figure 8B) and THP-1 (Figure 8C) cells could be rescued. After inhibiting the poly ADP-ribose polymerase activity of PARP1 using the inhibitor Rucaparib, the cell death caused by PagT1 in J774A.1 (Figure 8D) and THP-1 cells (Figure 8E) could also be inhibited.

[0497] Furthermore, the pADPr in J774A.1 and THP-1 cells was detected after delivering PagT1 through PVC or PVCR7. Immunofluorescence detected more pADPr in J774A.1 (Figure 9A) and THP-1 (Figure 9B) cells.

[0498] The above results indicate that the mode of cell death caused by PagT1 is Parthanatos.

[0499] 1.5 PagT1 mediates eukaryotic cell Parthanatos through the type I interferon-JAK-STAT signaling pathway

[0500] To determine the signaling pathways involved in Parthanatos in eukaryotic cells after delivering PagT1, CRISPR / Cas9 sgRNA library screening technology was used for analysis. Based on the forward screening strategy, the MAGeCK MLE analysis method was used to compare the sgRNA abundances of the cell library before screening, the empty PVC control group, and the PVC-delivered PagT1 experimental group to screen for genes and annotate gene function enrichment. The Mouse Brie CRISPR knockout pooled library whole-genome library was used for large-scale screening on J774A.1 cells to determine the key genes involved in Parthanatos in J774A.1 cells after delivering PagT1 via PVC.

[0501] The results of the forward screening were calculated for 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 screened genes is as Figure 10 shown.

[0502] The differential distribution of the beta scores of the forward-screened genes is as Figure 11 and Table 9 shown. The results showed that the top three genes were Irf9, STAT1, and TYK2, which are key genes in the JAK-STAT signaling pathway. The KEGG enrichment of the forward-screened genes was also enriched in the JAK-STAT and type I interferon signaling pathways ( Figure 12 ).

[0503] Table 9 Top 10 genes of forward screening

[0504] Gene EntrezID Symbol Beta score difference Rank 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

[0505] In the classical pathway, the type I IFN receptors IFNAR1 / 2 interact, inducing conformational changes, which activate the JAK (Janus kinase) family (JAK1, JAK2, and TYK2) associated with the receptor. This activation promotes the recruitment and phosphorylation of signal transducer and activator of transcription (STAT) proteins. The phosphorylated STAT then dimerizes and binds to Irf9 to form the IFN-stimulated gene factor 3 (ISGF3) complex. After translocating to the nucleus, this complex acts as a transcription factor to regulate the expression of IFN-stimulated genes (ISGs). Therefore, the Parthanatos process induced by PagT1 may be related to the type I interferon-JAK-STAT signaling pathway.

[0506] Further verified the roles of Irf9, STAT1, and TYK2 genes, as well as the type I interferon-JAK-STAT signaling pathway in PagT1-induced Parthanatos. Stable knockdown of Irf9, STAT1, and TYK2 genes in J774A.1 cells was performed using shRNA lentiviral vectors, and then PagT1 was delivered via PVC. The results showed that compared with the control shRNA group, knockdown of Irf9, STAT1, and TYK2 genes significantly improved cell death induced by PagT1 delivery via PVC( Figure 13 ).

[0507] In addition, the JAK inhibitor Ruxolitinib and the STAT1 inhibitor SH-4-54 were used to inhibit JAK and STAT1, respectively. The results showed that both Ruxolitinib and SH-4-54 significantly improved cell death induced by PagT1 delivery via PVC / PVCR7 in J774A.1 and THP-1 cells (Figure 14 A - Figure 14 D).

[0508] Next, another key protein Irf9 in the type I interferon-JAK-STAT signaling pathway was analyzed. In the classical type I interferon-JAK-STAT signaling pathway, Irf9 translocates to the nucleus. Therefore, the subcellular localization of Irf9 in BMDM cells after PagT1 delivery via PVCR7 was detected. Immunofluorescence showed that after PagT1 delivery, Irf9 was localized in the nucleus, while Irf9 was distributed throughout the cell in the empty PVC and mutant PVCR7-217219 groups( Figure 15 ).

[0509] In summary, the above results demonstrated the crucial roles of the type I interferon-JAK-STAT signaling pathway-related proteins Irf9, STAT1, TYK2, and JAK in PagT1-induced cellular Parthanatos.

[0510] 1.6 STAT1 phosphorylation induced by IFN-β is essential for PagT1-induced Parthanatos

[0511] To further confirm that the JAK-STAT signaling pathway is activated by type I interferon during PagT1-induced cellular Parthanatos, in this example, RNA-seq was used to detect the transcriptional changes in J774A.1 cells after PagT1 delivery via PVC compared with the empty PVC group. The results were as Figure 16 shown. After PagT1 delivery, the transcriptional level of the interferon IFN-β gene increased.

[0512] IFN-β belongs to type I interferons. By detecting the transcriptional levels of IFN-β and other type I interferons (IFN-κ, IFN-α, and IFN-ε) through qRT-PCR, it was found that after THP-1 cells were transfected with PagT1 via PVCR7, the transcriptional level of IFN-β increased most significantly (Figure 17 A - Figure 17 D), suggesting that during the process of cellular Parthanatos, the activation of the JAK-STAT signaling pathway might be caused by IFN-β. In addition, verification was carried out in BMDM cells, and it was also found that after transfection with PagT1, the transcriptional level of the IFN-β gene increased significantly( Figure 18 ).

[0513] To further verify whether PagT1 would cause the activation of the host cell interferon signaling pathway, THP1-Dual cells (containing the secreted luciferase reporter gene Lucia driven by the ISG54 minimal promoter with five interferon-stimulated response elements) were used to detect the activation of the interferon signaling pathway through the expression of the Lucia gene. After treating the cells with PBS, empty PVCR7, PVCR7-PagT1, and PVCR7-217219, a higher level of Lucia gene expression was detected in the PVCR7-PagT1 group( Figure 19 ), indicating that the IRF signaling pathway was significantly activated, that is, the host cell interferon signaling pathway was activated.

[0514] In this example, the activation of the JAK-STAT signaling pathway was further confirmed by detecting the phosphorylation of STAT1. The results showed that after J774A.1, THP-1, and BMDM cells were transfected with PagT1 via PVC / PVCR7, obvious phosphorylated STAT1 (phosphorylated Y701, p-STAT1) could be detected by Western blot, while after treating the cells with empty PVC and PVCR7-217219, there was no obvious change in p-STAT1 compared with the PBS group( Figure 20 ).

[0515] In addition, to explore the relationship between the PARP1 and type I interferon signaling pathways activated by PagT1, after inhibiting the PARP1 activity of THP-1 cells with the PARP1 inhibitor Rucaparib, it was found that after THP-1 cells were transfected with PagT1 via PVCR7, the phosphorylation level of STAT1 in THP-1 cells was significantly weakened compared with the DMSO control group( Figure 21 ), indicating that the activation of the type I interferon signaling pathway by PagT1 was caused by the activation of PARP1.

[0516] In summary, the results of 1.1 - 1.6 in Example 1 indicate that PagT1 belongs to the virulence factors of PVC, and its active sites are D217 and E219. When PagT1 is loaded into PVC and delivered to eukaryotic cells, it localizes to the nucleus by virtue of its own nuclear localization signal. During this process, PagT1 consumes NAD+ with the help of ADP - ribosyltransferase activity, resulting in NAD+ depletion. This situation affects the repair process of genomic damage by PARP1, thereby causing damage to the cell genome and leading to PARP1 - dependent cell death (Parthanatos). Subsequently, it ultimately causes cell death through the type I interferon - JAK - STAT signaling pathway.

[0517] Example 2 Engineering - modified targeted PVC for delivering PagT1 for precise tumor killing

[0518] 2.1 PagT1 has pan - toxicity to eukaryotic cells

[0519] After loading PagT1 using PVCR7, it was detected that PVCR7 - PagT1 could significantly kill Hela, A549, HepG2, and WEHI - Rb - 1 cells, while loading the mutant 217219 had no obvious effect on the viability of these tumor cells (Figure 22 A - Figure 22 D), indicating 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.

[0520] 2.2 Construction of targeted PVCs targeting tumor - related antigens such as HER2

[0521] By modifying the tail fiber protein Pvc13 of the PVC delivery vector, targeted PVCs that specifically target tumor antigens such as Her2 were obtained. And by loading PagT1, a precise killing tool targeting tumor cells was ultimately obtained.

[0522] HER2 is an important target for the selection of tumor - targeted therapeutic drugs. Studies have shown that overexpression of the HER2 gene exists in more than 30% of human tumors. In this example, a validated designed ankyrin repeat protein (DARPin) (which can specifically bind to HER2 on the cell membrane) was used. By fusing DARPin to Pvc13 (replacing the receptor - binding domain of Pvc13 with HER2 - DARPin with a linker, and its nucleotide sequence is as shown in SEQ ID NO: 10), the targeting modification of PVC was carried out to obtain PVC targeting HER2 - Pvc13 - HER2 - DARPin (PVCHER2) ( Figure 23A ).

[0523] After loading PagT1, PVCHER2-PagT1 was purified. Western blot detected the PagT1 and Pvc16 labeled with flag tags, indicating the successful assembly of the PVC complex (Figure 23 B). In addition, negative staining also observed PVCHER2 and PVCHER2-PagT1 (Figure 23 C - Figure 23 D).

[0524] 2.3 Detection of the targeting and delivery efficiency of PVCHER2 at the cellular level

[0525] U251 (HER2 positive), T98G (HER2 positive), and U87MG (HER2 negative) cells were used to detect the targeting of PVCHER2 and the delivery efficiency of PagT1. The results are shown in Figure 24 A - Figure 24 C. After loading PagT1, PVCHER2-PagT1 could cause obvious cell death in T98G and U251 cells, while there was no obvious death in U87MG cells; empty PVCHER2 and PVCHER2-217219 also did not significantly affect the cell viability of T98G and U251 cells, indicating that the targeted PVCHER2 could effectively deliver the virulence factor PagT1, and the delivery process was highly specific.

[0526] Furthermore, in this example, it was detected whether PVCHER2-PagT1 could recognize and kill human bladder tumor cells. The results are shown in Figure 25 A - Figure 25 B. After adding empty PVCHER2, PVCHER2-PagT1, and PVCHER2-217219 for 24 h, the cell viability was detected, and it was found that PVCHER2-PagT1 could cause obvious cell death in BIU87 and T24 cells, while empty PVCHER2 and PVCHER2-217219 did not significantly affect the viability of BIU87 and T24 cells.

[0527] 2.4 Targeted PVCHER2 delivery of PagT1 leads to Parthanatos in tumor cells

[0528] In this example, it was further verified whether the delivery of PagT1 through targeted PVCHER2 would lead to Parthanatos in BIU87 cells. The Western blot detection results showed that after the delivery of PagT1 through PVCHER2, the expression of PARP1 in BIU87 cells increased significantly, while there was no significant change in the expression of PARP1 in the empty PVCHER2 and PVCHER2-217219 groups compared with the PBS group ( Figure 26 ).

[0529] The core mechanism of parthanatos is that PARP1 activation leads to the accumulation of PAR polymers, which in turn triggers the release of AIF from mitochondria and its translocation to the nucleus. Therefore, the subcellular localization of AIF in BIU87 cells after the delivery of PagT1 by PVCHER2 was further detected. Immunofluorescence results showed that after the delivery of PagT1 by PVCHER2, AIF in BIU87 cells was partially translocated to the nucleus and did not completely co-localize with mitochondria. In contrast, compared with the PBS group, there was no obvious change in the subcellular localization of AIF in the empty PVCHER2 and PVCHER2-217219 groups, and it co-localized with mitochondria( Figure 27 ).

[0530] The above results demonstrated at the cellular level that the HER2-targeted delivery vector PVCHER2-PagT1 loaded with the virulence factor PagT1 could specifically induce parthanatos in glioma cells and bladder tumor cells, and PVCHER2-PagT1 has the potential to be a precision tumor therapeutic drug.

[0531] 2.5 PVCHER2-PagT1 causes significant killing of bladder cancer organoids

[0532] Tissue samples obtained from a 78-year-old male patient with left renal pelvic invasive urothelial carcinoma after transurethral resection of the bladder for bladder metastasis were used to establish an organoid model. The expression of HER2 in the bladder cancer organoid model could be detected by immunohistochemistry and immunofluorescence( Figure 28 ).

[0533] Empty PVCHER2, PVCHER2-PagT1, and PVCHER2-217219 were added to the organoid medium respectively to detect the killing effect of PVCHER2-PagT1 on bladder cancer organoids. The results are shown in Figures 29A - 29C. PVCHER2-PagT1 could effectively kill bladder cancer organoids, with an IC50 of approximately 0.792 μg / mL, while empty PVCHER2 and PVCHER2-217219 had no obvious effect on bladder cancer organoids.

[0534] The above results demonstrated at the organoid level that the HER2-targeted delivery vector PVCHER2-PagT1 loaded with the virulence factor PagT1 could effectively kill bladder cancer organoids, which further confirmed the feasibility of PVCHER2-PagT1 as a precision tumor therapeutic drug.

[0535] 2.6 PVCHER2 loaded with PagT1 targets the treatment of mouse orthotopic glioma

[0536] To further verify the therapeutic effect of PVCHER2-PagT1 on tumors in vivo, a mouse orthotopic glioma model was constructed for detection.

[0537] Based on the lentiviral vector, U251-Luci cells expressing Firefly luciferase (Luci) were constructed, and the successful construction of the cells was verified by detecting luciferase ( Figure 30 ).

[0538] Take U251-Luci cells (2×10 4 cells / 2 μL) for orthotopic inoculation in the mouse brain. From the 7th day after inoculation, in vivo imaging was performed every 7 days to monitor tumor development; magnetic resonance imaging was performed on the 8th and 35th days after inoculation to monitor the brain tumors; on the 10th day after inoculation, ventricular administration was performed according to the grouping, with a administration volume of 2 μL, a dosage of 15 μg in the low-dose group, a dosage of 30 μg in the high-dose group, and a dosage of 30 μg in the empty vector group (control).

[0539] The results of in vivo imaging monitoring are shown in Figures 31A - 31B. Orthotopic gliomas in the mouse brain could be detected by in vivo imaging on the 7th day after inoculation. On the 21st and 35th days, it was observed that the tumor signals in the low-dose and high-dose groups were significantly weaker compared to the 7th day (Figure 31A). By statistically analyzing the Luci signal intensity of in vivo imaging, it was found that the tumor signals in the low-dose and high-dose groups were also significantly lower than those in the PBS group and the empty vector group (Figure 31B).

[0540] The results of magnetic resonance imaging are shown in Figure 31C. Orthotopic gliomas in the mouse brain could also be detected by magnetic resonance imaging on the 7th day after inoculation. On the 35th day, it was also observed that the tumor signals in the low-dose and high-dose groups were significantly weaker compared to the 7th day.

[0541] The results of the mouse survival curve and the changes in mouse body weight are shown in Figures 31D - 31E. The survival period of the high-dose group was significantly longer than that of the PBS group (Figure 31D); there was no significant difference in the changes in mouse body weight (Figure 31E).

[0542] The above results demonstrated that the HER2-targeted delivery vector PVCHER2-PagT1 loaded with the virulence factor PagT1 could effectively kill mouse orthotopic gliomas.

[0543] In summary, the results of 2.1 - 2.6 in Example 2 show that by replacing the domain of PVC tail fiber protein that specifically recognizes cell receptors with DARPin that can bind to tumor - associated antigen HER2 and loading PagT1, glioma cells, bladder tumor cells, bladder cancer organoids, and mouse orthotopic gliomas can be effectively killed. This confirms the feasibility of targeted - modified and PagT1 - loaded PVC as a precision tumor - treatment drug.

[0544] The above - mentioned are only the preferred embodiments of this application, and it is not intended to limit this application in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change, and modification made to the above - mentioned embodiments based on the technical essence of this application without departing from the technical solution content of this application still fall within the protection scope of the technical solution of this application.

[0545] The sequences involved in this application are as follows:

[0546] The amino - acid sequence of Photorhabdus asymbiotica WP_015834233.1 (PagT1) protein

[0547] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVASNKRDPITKDQVSVPPDGNLFTELKDKHLDRAAEYKKLKTWPTHASIIATSPSANTPIAQHVSGDDALSPYISTGDKPGAVQNTVRNWNGIGPASERRLRPEKTWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTTNSDIAQNAFADKEVLISPEIPGLAITRVINDPEEIKQIANLNPSQSLIEKKNTIPEEKIIFEEKKSVPIHDSDADIPSSSFVFPKRKKPRNIRSRTDS (SEQ ID NO: 1);

[0548] The amino - acid sequence at the C - terminus of Pseudomonas alkylphenolica WP_051939377.1 protein

[0549] DENGFSPDVDILEKFESPGAFYERTGREFLYRAIRTMESEKLHPETGDKGKLPPDGDAFPELKDKHLERAAKYRSMKTWEGGDIVSTGPELKRLPLQHVSGDNQQSQYISTGTRGAAAYNTVYNWNSKGVGKERRKRPEKDWDPIIKIDVKKLDSGTKIYDMQQEGLKDRDRSDIGQLAEADAEVLISNKINASAIVSVFTNSADYNKFLEEYFPPAKSDGAAASPLVNKIKRSERGGSRRTVSESVADAPVEVFKKRAPIKPQSFRKR (SEQ ID NO: 2);

[0550] Amino acid sequence at the C-terminus of the protein of Deltaproteobacteria bacterium MCE9576703.1:

[0551] DVFETPAQYLTRTGKHLLYRAVRMDGTSKKVFSGAGSTSSADGGSVGVAPDGNLFAGIPQKEREARYRALAAWPADHPITAAAPQATADPLVQISGSNYATQYISTGAAIHGAAHNTVRSWNSPGAPGDRPIRRPQSWAPVITIDVRRLAPQTRVYDTQRPDVNYMYREDALGMMAGLSAADAEVLLTGEIPAAAIVGIMTQPSAIAEAAQVTIAELL(SEQ ID NO: 3);

[0552] Amino acid sequence at the C-terminus of the protein of Okeania WP_293063881.1:

[0553] IVKISVDNHKQINLDENCHQFETVNEYVERTGVRTLYRSIRSKDVDKDKFELNQKVSVPAEIKLPTSPGERFKELKNLHRHPMESHESPKRTKITNEIELIPGDSMAQLLPADQVSGSFATQFISTSSNPLGAIVNTVKNWDSGSNRTRMRPLRFWDPVVKIDLTYLLNHQDIRIYDMKADYIEWPNLNTTGVGVKANAEVDNEILISGKLPPQAV (SEQID NO: 4);

[0554] Amino acid sequence of Photorhabdus heterorhabditis WP_172908611.1 protein:

[0555] MVYEYGKTNDRKRKRSTQSDNYEEKLFAPVLDLPRNNQNTPNMEDEYETPKDFINRTGREKLFRAIRMIASDKRDPITQDQVSVPPDGNLFTELEKTHQGRADEYKKLKEWPTHASIIATSPNANIPIAQHVSGDNILSPYISTGDEPGAAQNTVRSWNQAGPVSERRLRPEKNWSPIIEIDVNKLPDTTKIFDLNKPSNTFFSTANSDISQNAFADKEVLISPEIPGHAITRVINDPKEIKQIANLNPSQSPIEKKNIIPEEKIIFEEKKSAPIHESDANLPSSSFIFPKRKKPRNIRSRTDS (SEQ ID NO: 5);

[0556] Amino acid sequence of Photorhabdus australis WP_065822174.1 protein:

[0557] MVYEYGKTNDRKRKRSTQSDDYEEKPFSPVLDLSRNNQNTPNMEDEYETPKDFINRTGREKLFRAIRMMASDKQDPITKYQVSVSPDGNLFTELKDKHLDRAAEYKKLKEWPTHVPIMATSLSTNIPIAQHVSGDNILSPYISTGDAPGATQNTVRSWNQAGPISERRLRPEKDWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTDNSDIAQNAFADKEVLISPEIPGHAITRVINDPKEIKQIAKLNPSQSSIEKKNTMPEKKITFEEKKSIPIHDSDANLPSSSFVFPKRKKPRNIRSRTDSW (SEQ ID NO: 6);

[0558] Amino acid sequence of the C-terminus of Burkholderia ubonensis KVU23967.1 protein:

[0559] NPTNIAGVESEVPYHSPSTFIAATGRRYLYRALNAIQDSALGKSGNVPSDFEIAATNPDANYSPVVHVGSSDYEGGTQYISTGDASGAARNTVLNWNAGVERRLRPSRLWNRIVEVDVAKLPDDTRLYNVGKDWNLYSDEKKNPIHSEVGNNAFSDQEVLIQGRINGSAIRDV (SEQ ID NO: 7);

[0560] Amino acid sequence of the Streptomyces avermitilis WP_037649420.1 protein:

[0561] QTVEEYRASGGPSKLYRSIRIESQLKFRADRATVTAEADGNLGAGMPAKEVSRIYQTLKEWPRTKNIEPVDANAAFTVAHHVGGDNYGTQYISFSPDYGRAADYAQHDFKLGPAENLEGARKPRSVRKWAPVIEIDIAKLGPGNRLVNLGNPNIAGLTNLKEVTDIASMASNDSEVLIKGTIPAGAVTKVYGVEDAVKSISLEARKNLIEQ (SEQ ID NO: 8);

[0562] Nucleotide sequence of the DARPin binding HER2 with KpnI and HindIII restriction sites and linker GGSGGGGSGG (SEQ ID NO: 9) at both ends:

[0563] GGTTGGGCGTTATGTGATGGCAATAATGGTACACCAAATTTAATCGATCGATTTATTTTAGGTGGGAAAGGTACCGATATTAATGGAGTGAGTACTAATACAGCTTCAGGTACTAAAAATAGTAAGTTATTCGATTTCAGTTCTGATGAAGCTACATTAACTATTGATGGTAAAACACTGGGGAGAggtggctctggcggtggtggatctggtggcCGCGGCAGCCATCATCATCATCATCATGGCAGCGATCTGGGCAAAAAACTGCTGGAAGCGGCGCGCGCGGGCCAGGATGATGAAGTGCGCATTCTGATGGCGAACGGCGCGGATGTGAACGCGCATGATTTTTATGGCATTACCCCGCTGCATCTGGCGGCGAACTTTGGCCATCTGGAAATTGTGGAAGTGCTGCTGAAACATGGCGCGGATGTGAACGCGTTTGATTATGATAACACCCCGCTGCATCTGGCGGCGGATGCGGGCCATCTGGAAATTGTGGAAGTGCTGCTGAAATATGGCGCGGATGTGAACGCGAGCGATCGCGATGGCCATACCCCGCTGCATCTGGCGGCGCGCGAAGGCCATCTGGAAATTGTGGAAGTGCTGCTGAAAAACGGCGCGGATGTGAACGCGCAGGATAAATTTGGCAAAACCCCGTTTGATCTGGCGATTGATAACGGCAACGAAGATATTGCGGAAGTGCTGCAGAAAGCGGCGAAACTGAACggtggttcaggtggtggtggtagtggtggcCACGATCATGATATTAAGATAACGGGCACAGGAAAACATTCTCACAAAAACAAAGTAACAGTCCCTTATTATATTCTGGCTTTCATCATAAAGCTTTAATATATATGAAAAATTGAAAATATAAATTATCCATTAATAATAAAGAGGAT(SEQ ID NO: 10);

[0564] Forward primer sequence of N-NLS-F:

[0565] ATTTCACACAGGAAACACATATGTTAAAATATGCTAATCCTCAGACCG (SEQ ID NO: 11);

[0566] N-NLS-R reverse primer sequence:

[0567] AATGATTTTTCTTCATAATTGATTTTCCTAATCTTATGGCCTTCG (SEQ ID NO: 12);

[0568] C-NLS-F forward primer sequence:

[0569] TACCGTCAGACTACAAGGACGATGATGACAAGTAA (SEQ ID NO: 13)

[0570] C-NLS-R reverse primer sequence:

[0571] CCTTGTAGTCTGACGGTATATCGGCGTCACTGT (SEQ ID NO: 14);

[0572] 217219-F forward primer sequence:

[0573] TGCAGCAAAAGCAGTGCTAATTTCACCAGAAATTCCA (SEQ ID NO: 15);

[0574] 217219-R reverse primer sequence:

[0575] GCACTGCTTTTGCTGCAAAGGCGTTTTGAGCA (SEQ ID NO: 16);

[0576] Vector-F forward primer sequence:

[0577] AATTATGAAGAAAAATCATTTTCTCCC (SEQ ID NO: 17);

[0578] Vector-R reverse primer sequence:

[0579] ATGTGTTTCCTGTGTGAAATTGTTATC (SEQ ID NO: 18);

[0580] Amino acid sequence of PagT1-N15:

[0581] MVYEYAKTNDRKRKL (SEQ ID NO: 19);

[0582] Amino acid sequence of PagT1-N20:

[0583] MVYEYAKTNDRKRKLSTQSD (SEQ ID NO: 20);

[0584] Amino acid sequence of PagT1-N25:

[0585] MVYEYAKTNDRKRKLSTQSDNYEEK (SEQ ID NO: 21);

[0586] Amino acid sequence of PagT1-N30:

[0587] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPV (SEQ ID NO: 22);

[0588] Amino acid sequence of PagT1-N35:

[0589] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSR (SEQ ID NO: 23);

[0590] Amino acid sequence of PagT1-N40:

[0591] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNT (SEQ ID NO: 24);

[0592] Amino acid sequence of PagT1-N45:

[0593] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMED (SEQ ID NO: 25);

[0594] Amino acid sequence of PagT1-N50:

[0595] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETP (SEQ ID NO: 26);

[0596] Amino acid sequence of PagT1-N55:

[0597] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFIN (SEQ ID NO:27);

[0598] Amino acid sequence of PagT1-N60:

[0599] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGRE (SEQ ID NO: 28);

[0600] Amino acid sequence of PagT1-N65:

[0601] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRA(SEQ ID NO: 29);

[0602] Amino acid sequence of PagT1-N70:

[0603] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVA (SEQ ID NO: 30);

[0604] Amino acid sequence of Del-N-NLS with the N-terminus removed from PagT1

[0605] NYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVASNKRDPITKDQVSVPPDGNLFTELKDKHLDRAAEYKKLKTWPTHASIIATSPSANTPIAQHVSGDDALSPYISTGDKPGAVQNTVRNWNGIGPASERRLRPEKTWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTTNSDIAQNAFADKEVLISPEIPGLAITRVINDPEEIKQIANLNPSQSLIEKKNTIPEEKIIFEEKKSVPIHDSDADIPSSSFVFPKRKKPRNIRSRTDS (SEQ ID NO:31);

[0606] Amino acid sequence of Del-C-NLS with the C-terminus removed from PagT1:

[0607] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVASNKRDPITKDQVSVPPDGNLFTELKDKHLDRAAEYKKLKTWPTHASIIATSPSANTPIAQHVSGDDALSPYISTGDKPGAVQNTVRNWNGIGPASERRLRPEKTWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTTNSDIAQNAFADKEVLISPEIPGLAITRVINDPEEIKQIANLNPSQSLIEKKNTIPEEKIIFEEKKSVPIHDSDADIPS (SEQ ID NO:32);

[0608] The amino acid sequence of PagT1 with Del-N and C-NLS removed from both the N-terminus and C-terminus:

[0609] NYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVASNKRDPITKDQVSVPPDGNLFTELKDKHLDRAAEYKKLKTWPTHASIIATSPSANTPIAQHVSGDDALSPYISTGDKPGAVQNTVRNWNGIGPASERRLRPEKTWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTTNSDIAQNAFADKEVLISPEIPGLAITRVINDPEEIKQIANLNPSQSLIEKKNTIPEEKIIFEEKKSVPIHDSDADIPS (SEQ ID NO: 33);

[0610] The amino acid sequence of the PagT1 mutant 217219:

[0611] MVYEYAKTNDRKRKLSTQSDNYEEKSFSPVLDLSRNNQNTPNMEDEYETPQNFINRTGREKLFRAIRMVASNKRDPITKDQVSVPPDGNLFTELKDKHLDRAAEYKKLKTWPTHASIIATSPSANTPIAQHVSGDDALSPYISTGDKPGAVQNTVRNWNGIGPASERRLRPEKTWSPIIEIDVNKLPDTTKIFDLNKPNNTFFSTTNSDIAQNAFAAKAVLISPEIPGLAITRVINDPEEIKQIANLNPSQSLIEKKNTIPEEKIIFEEKKSVPIHDSDADIPSSSFVFPKRKKPRNIRSRTDS (SE Q ID NO: 34);

[0612] shRNA sequence for knocking down Irf9:

[0613] CCCTACAAAGTATATCGAATA (SEQ ID NO: 35);

[0614] shRNA sequence for knocking down STAT1:

[0615] CCGAAGAACTTCACTCTCTTA (SEQ ID NO: 36);

[0616] shRNA sequence for knocking down TYK2:

[0617] GCGCATACCTGTGTGTCATCT (SEQ ID NO: 37);

[0618] Amino acid sequence of Pnf-N50:

[0619] MLKYANPQTVATQRTKNTAKKPPSSTSFDGHLELSNGENQPYEGHKIRKI (SEQ ID NO: 38);

[0620] Nucleotide sequence of PagT1 gene:

[0621] ATGGTGTATGAATACGCTAAAACCAATGATAGAAAAAGAAAACTCTCAACACAGTCAGATAATTATGAAGAAAAATCATTTTCTCCCGTATTAGATTTATCCAGAAACAATCAAAATACGCCTAATATGGAAGATGAATATGAAACACCGCAGAATTTTATTAATAGAACTGGTCGAGAAAAACTATTCCGTGCAATCCGTATGGTAGCCTCGAATAAACGCGATCCCATTACAAAAGATCAAGTATCCGTGCCACCTGATGGTAATCTATTTACCGAACTTAAAGATAAACATCTGGATAGAGCCGCGGAATATAAAAAATTAAAAACATGGCCAACACATGCTTCAATTATAGCAACCTCTCCCAGTGCTAATACCCCTATTGCACAACATGTTTCCGGTGATGATGCATTAAGCCCTTATATCTCTACAGGTGACAAACCAGGGGCCGTGCAAAATACGGTAAGGAATTGGAATGGGATTGGACCCGCATCAGAAAGAAGACTGAGACCAGAAAAAACATGGTCTCCAATAATAGAGATCGACGTTAATAAGCTGCCTGACACCACTAAAATCTTCGATCTGAATAAACCAAATAATACCTTCTTTAGTACCACCAATAGCGACATTGCTCAAAACGCCTTTGCAGACAAAGAAGTGCTAATTTCACCAGAAATTCCAGGCCTCGCTATTACACGTGTAATTAATGATCCAGAAGAGATCAAACAGATTGCTAATCTTAATCCAAGCCAATCGCTTATAGAGAAAAAGAACACCATACCAGAAGAAAAAATAATCTTTGAAGAAAAAAAATCGGTGCCAATACACGACAGTGACGCCGATATACCGTCATCATCATTTGTTTTTCCAAAACGCAAGAAACCAAGAAATATAAGATCTCGTACTGATAGT (SEQ ID NO: 39).

Claims

1. A signal peptide that is used to direct a polypeptide to be loaded into a protein complex and to direct the polypeptide to localize to the nucleus of a eukaryotic cell, wherein the signal peptide comprises at least 20 amino acids at the N-terminus of the PagT1 protein or its homologous protein.

2. The signal peptide according to claim 1, wherein the protein complex comprises a non-symbiotic Photorhabdus virulence cassette (PVC), AFP, or a morphogenesis-associated contractile structure (MAC).

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 the PagT1 protein or its homologous protein.

4. The signal peptide according to claim 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%, 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 20-30.

5. The signal peptide according to claim 4, wherein the polypeptide comprises any one or more of a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein.

6. A nuclear localization peptide that is used to localize a polypeptide linked thereto to the nucleus of a eukaryotic cell, wherein the nuclear localization peptide comprises a nuclear localization sequence, and the nuclear localization sequence comprises at least 20 amino acids at the N-terminus and / or C-terminus of the PagT1 protein or its homologous protein.

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%, 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 20-30.

8. The nuclear localization peptide according to claim 6 or 7, wherein the polypeptide comprises any one or more of a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein.

9. A fusion protein that comprises the nuclear localization peptide according to any one of claims 6-8 and a polypeptide linked thereto, and the linkage is a covalent or non-covalent linkage.

10. A protein toxin, which is 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 to the amino acid sequence shown in SEQ ID NO:

1.

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

1.

12. A conjugate, which comprises the protein toxin according to claim 10 or 11.

13. A fusion protein, which comprises the protein toxin according to claim 10 or 11.

14. A device, which comprises the protein toxin according to claim 10 or 11.

15. An article, which comprises the protein toxin according to claim 10 or 11.

16. A complex, which comprises the protein toxin according to claim 10 or 11 or its encoding nucleic acid.

17. The complex according to claim 16, wherein the complex comprises a component for delivering the protein toxin or its encoding nucleic acid into cells.

18. A nucleic acid, which encodes the signal peptide according to any one of claims 1-5.

19. An expression vector, which comprises the nucleic acid according to claim 18.

20. A host cell, which comprises the nucleic acid according to claim 18 or the expression vector according to claim 19.

21. A polypeptide delivery system, which comprises a protein complex and the signal peptide according to any one of claims 1-5.

22. The polypeptide delivery system according to claim 21, wherein the protein complex comprises a Photorhabdus asymbiotica virulence cassette (PVC), AFP or a metamorphosis-associated contractile structure (MAC).

23. The polypeptide delivery system according to claim 21 or 22, wherein the signal peptide is covalently linked to the polypeptide.

24. The polypeptide delivery system according to claim 23, wherein the polypeptide comprises any one or more of a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein.

25. The polypeptide delivery system according to claim 21 or 22, wherein the polypeptide is PagT1 protein or its homologous protein.

26. The polypeptide delivery system according to claim 21 or 22, wherein the protein complex is a PVC-V complex.

27. The polypeptide delivery system according to claim 26, wherein the structural protein Pvc13 in the PVC-V complex is engineered to be a protein that specifically recognizes HER2.

28. The polypeptide delivery system according to claim 27, wherein a protein that recognizes a cell surface molecule is inserted into the receptor-binding domain of the structural protein Pvc13.

29. The polypeptide delivery system according to claim 28, wherein the amino acid sequence of the signal peptide is 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.

30. The polypeptide delivery system according to claim 29, wherein the polypeptide comprises any one or more of a signal pathway regulatory protein, a structural protein, a transport protein, a hormone or a hormone regulatory molecule, a cytotoxin, an antigen or an immunogen, an antibody protein or a fragment thereof, a tag protein or a reporter protein, an antimicrobial peptide, an enzyme involved in cell metabolism, and a gene editing protein.

31. A nucleic acid encoding the polypeptide delivery system according to any one of claims 21-30.

32. The nucleic acid according to claim 31, wherein, The nucleic acid comprises one or more of the following: (i) a nucleotide sequence encoding the protein complex in the polypeptide delivery system; (ii) a nucleotide sequence encoding the signal peptide in the polypeptide delivery system; (iii) a nucleotide sequence encoding the polypeptide.

33. An expression vector comprising the nucleic acid according to claim 31 or 32.

34. A host cell comprising the polypeptide delivery system according to any one of claims 21-30, the nucleic acid according to claim 31 or 32, or the expression vector according to claim 33.

35. A method for preparing the polypeptide delivery system according to any one of claims 21-30, comprising culturing the host cell according to claim 34 to obtain the polypeptide delivery system.

36. A method for transferring a polypeptide into a target cell, comprising contacting the polypeptide delivery system according to any one of claims 21-30 with the target cell, so that the polypeptide delivery system delivers the polypeptide into the target cell.

37. A method for killing a target cell, comprising contacting the polypeptide delivery system according to any one of claims 21-30 with the target cell, so that the polypeptide delivery system delivers the polypeptide into the target cell, thereby killing the target cell.

38. Use of the signal peptide according to any one of claims 1-5, the polypeptide delivery system according to any one of claims 21-30, the nucleic acid according to claim 31 or 32, the expression vector according to claim 33, or the host cell according to claim 34 in the preparation of a drug, reagent or kit for delivering a polypeptide.

39. The use according to claim 38, wherein the drug, reagent or kit is a drug, reagent or kit for treating tumors.

40. The use according to claim 39, wherein the tumor is a HER2-positive tumor.

41. Use of the PagT1 gene or the protein encoded thereby 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%, 99% identity with the sequence shown in SEQ ID NO: 1.

Images