DC (Dendritic Cell) capture type engineering bacterial vaccine for realizing cytoplasm antigen transfer by utilizing gap connection

Abstract

The invention discloses a DC (dendritic cell) capture type engineering bacterial vaccine for realizing cytoplasm antigen transfer by utilizing gap connection, which is characterized in that attenuated salmonella VNP20009 is utilized to simultaneously express Antigen 4T1-M8 and DC capture peptide CBP-12 to increase contact so as to enhance formation of gap connection between tumor cells and DC and promote delivery of antigen to DC cytoplasm, so that MHC-I mediated cross presentation is enhanced. In addition, as an intracellular bacterium, the VNP20009 can infect tumor cells and colonize in the tumor cells. Therefore, the tumor antigen carried by the VNP20009 can be expressed in cells, so that the tumor antigen can be presented to the surfaces of tumor cells by MHC-I as an endogenous antigen to be recognized by specific CD8 + T cells.

Description

Technical Field

[0001] This invention belongs to the fields of biology and medicine and pharmaceutical formulation. This invention relates to a novel vaccine and its preparation method that promotes the formation of gap junctions in tumor-capturing dendritic cells (DCs) and increases antigen delivery to DC cytoplasm. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Antigen-specific T cells are key effector cells for the efficacy of immunotherapy. The initiation of cytotoxic T cells requires antigen presentation via the major histocompatibility complex I (MHC-I) pathway on the surface of antigen-presenting cells (APCs). Normally, exogenous antigens taken up by dendritic cells (DCs) are presented via the MHC-II pathway after lysosomal degradation. Therefore, effectively increasing antigen presentation via the MHC-I pathway is a crucial and rate-limiting step in the generation of specific cytotoxic T cells. Promoting cytoplasmic delivery of antigens is an effective means to achieve MHC-I presentation of exogenous antigens. Much research has focused on designing vectors to promote lysosomal escape of antigens. However, this may increase the risk of digestive enzyme leakage from the endosomal lysosomes, leading to cell necrosis.

[0004] Gap junctions are crucial pathways for cargo exchange between neighboring cells, enabling the cytoplasmic delivery of both large and small molecules. Antigens can be directly delivered to dendritic cells (DCs) cytoplasm via gap junctions. Intratumoral microbes play a vital role in promoting tumor progression and activating the immune system. Utilizing the natural affinity between bacteria and tumor tissue, bacteria have been widely applied in drug delivery. Once bacteria reach the tumor site, they tend to colonize intracellularly rather than extracellularly. Simultaneously, the expression level of the key gap junction protein Cx43 is significantly increased on the surface of tumor cells infected with bacteria, facilitating the formation of gap junctions and material exchange between tumor cells and neighboring cells. However, the tumor microenvironment contains diverse cell types. To improve the effective formation of gap junctions between tumor cells and DCs, it is necessary to enhance the contact between tumor cells and DCs and achieve tumor capture of DCs. Summary of the Invention

[0005] To address the aforementioned challenges in antigen delivery, designing a system that facilitates antigen delivery to dendritic (DC) cytoplasm is crucial. Intratumoral microorganisms can achieve intracellular delivery of tumor antigens and induce the formation of gap junctions in infected cells. Simultaneously, to improve the efficiency of direct gap junction formation between tumor cells and DCs, it is necessary to increase the contact between them. This is expected to improve antigen presentation efficiency, effectively initiate cytotoxic T cell-specific killing of tumors, and enhance the antitumor therapeutic efficacy of tumor vaccines.

[0006] The technical solution adopted in this invention is as follows: In a first aspect of the invention, a DC-capturing engineered bacterium, VNP-Apep, is provided that utilizes gap junctions to achieve cytoplasmic antigen delivery. The engineered bacterium VNP-Apep comprises the following gene fragments: The tumor antigen (Antigen 4T1-M8) gene is used to express the target tumor antigen. The DC capture peptide (DCpep) gene is used to express DCpep; and before and after the DCpep gene are the IL-2 signal peptide gene and the platelet-derived growth factor receptor (PDGFR signal peptide) gene, respectively, which are used to promote the secretion and membrane anchoring of DCpep.

[0007] In one or more embodiments of the present invention, the engineered bacterium VNP-Apep comprises a plasmid containing a tumor antigen (Antigen 4T1-M8) gene and a plasmid containing a DC capture peptide (DCpep) gene, wherein the DC capture peptide (DCpep) gene is preceded and followed by an IL-2 signal peptide gene and a PDGFR signal peptide gene, respectively.

[0008] In a second aspect of the invention, a method for constructing the DC-captured engineered bacterium VNP-Apep, which utilizes gap junctions to achieve cytoplasmic antigen delivery, is provided, the method comprising the following steps: Using attenuated Salmonella VNP20009 as the starting strain, target gene 1 and target gene 2 were carried; The target gene is the tumor antigen (Antigen 4T1-M8) gene; The second target gene is the DC capture peptide (DCpep) gene, with the IL-2 signal peptide gene and the PDGFR signal peptide gene inserted before and after the DCpep gene, respectively.

[0009] In one or more embodiments of the present invention, the construction method specifically includes the following steps: plasmid construction, preparation of VNP20009 competent cells, and electroporation of VNP-Apep. Those skilled in the art can perform conventional construction based on the combination of gene sequences according to the present invention. The plasmid construction method includes, but is not limited to, the following: An embodiment of the present invention provides a method for constructing a dual-plasmid co-expression system, specifically including the following steps: inserting the Antigen 4T1-M8 gene sequence into the multiple cloning site of the pBAD plasmid to obtain the pBAD-Antigen plasmid; inserting the gene sequence containing the IL-2-sig-DC capture peptide-PDGFR-sig into the multiple cloning site of the pBAD plasmid to obtain the pBAD-IL-2-sig-DC capture peptide-PDGFR-sig plasmid; and preparing a plasmid solution using sterile, enzyme-free water. Further, introducing two plasmids containing the target gene into VNP20009 cells to obtain VNP-Apep.

[0010] Furthermore, VNP-Apep was obtained by introducing two plasmids containing the target gene into VNP20009 using electroporation.

[0011] In one or more embodiments of the present invention, Antigen 4T1-M8 refers to a tumor-associated antigen expressed by 4T1M-8, a highly metastatic cell subline selected from the mouse breast cancer cell line 4T1, which is a conventional technique.

[0012] Furthermore, the amino acid sequence of the tumor antigen (Antigen 4T1-M8) is QGVTVLAVSAVYDIFVFHRLKM-KQILP, as shown in SEQ ID No. 1; The nucleotide sequences that can encode its amino acids include, but are not limited to, those that... CAGGGCGTGACCGTGCTGGCGGTGAGCGCGGTGTATGATATTTTTGTGTTTCATCGCCTGAAAATGAAACAGATTCTGCCG, as shown in SEQ ID No. 2.

[0013] In one or more embodiments of the present invention, the DC-capturing peptide includes, but is not limited to, the 12-meric peptide CBP-12 that targets the C-type lectin receptor on DCs, synthetic polymannose (pMAM) with affinity for mannose receptors, polydextrose (pMAG), short peptides that target other DC receptors, etc.

[0014] Furthermore, the amino acid sequence of the CBP-12 is WPRFHSSVFHTH, as shown in SEQ ID No. 3; The nucleotide sequences that can encode its amino acids include, but are not limited to, AGTGGCCGCGCTTTCATAGCAGCGTGCGCCATACCCAT, as shown in SEQ ID No. 4.

[0015] In one or more embodiments of the present invention, the amino acid sequence of the IL-2 signal peptide is YRMQLLSCIALSLALVTNS, as shown in SEQ ID No. 5; The nucleotide sequences that can encode its amino acids include, but are not limited to, TACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG, as shown in SEQ ID No. 6.

[0016] In one or more embodiments of the present invention, the amino acid sequence of the PDGFR signal peptide is AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR, as shown in SEQ ID No. 7. The nucleotide sequences that can encode its amino acids include, but are not limited to, GCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGCCCTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGT, as shown in SEQ ID No. 8.

[0017] In a third aspect of the invention, the use of the VNP-Apep in the preparation of a cytoplasmic delivery vaccine that promotes tumor antigen delivery is provided.

[0018] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) The present invention prepares a novel engineered bacterial vaccine that integrates antigen expression and delivery to DC cytoplasm, and the preparation process is simple.

[0019] (2) This novel engineered bacteria can effectively express DC capture peptides and increase the expression of Cx43 protein in tumor cells, thereby promoting the formation of direct gap junctions between tumors and DCs, which improves the efficiency of antigen delivery to DC cytoplasm compared with traditional tumor vaccines.

[0020] (3) This invention uses attenuated Salmonella as an antigen delivery vector, which has an innate tumor targeting ability and deletes some toxic genes to improve safety.

[0021] (4) The engineered bacterial vaccine prepared by the present invention can effectively promote antigen presentation, initiate the specific response of cytotoxic T cells to tumors, and the intracellular bacterial characteristics can realize the intracellular expression of tumor antigens, so that tumor antigens can be presented to the surface of tumor cells by MHC-I as endogenous antigens for recognition by specific CD8 T cells, and simultaneously realize the initiation and recognition of tumor-specific killing. Attached Figure Description

[0022] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0023] Figure 1 A schematic diagram of the structure of VNP-Apep.

[0024] Figure 2 Anchoring of DC-captured peptides on the surface of tumor cells: a. Schematic diagram of DC-captured peptides on the surface of tumor cells; b. Display of DC-captured peptides on the surface of tumors after different treatments; c. Quantitative results of DC-captured peptides on tumors (n=3; ****p<0.0001).

[0025] Figure 3 Affinity assessment of the capture peptide to DC: a. Schematic diagram of the binding experiment between the capture peptide and DC in free DC; b. Quantitative results of the binding of the free capture peptide to DC (n=3; p<0.0001); c. Confocal image of DC capture by tumor; d. Quantitative results of DC capture by tumor (n=5).

[0026] Figure 4 Antigen presentation on DC: a. Confocal image of antigen presentation on DC; b. Quantitative results of OVA-MHCI complex on DC (n=3; ****p<0.0001). Detailed Implementation

[0027] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] This invention designs a novel engineered oncolytic bacterial tumor neoantigen vaccine (VNP-Apep), utilizing the highly expressed C-type lectin domain family 9 member a (Clec9a) on the dendritic cell (DC) surface as a viable target. This invention utilizes the attenuated Salmonella VNP20009 to simultaneously express the tumor antigen (Antigen 4T1-M8) and the DC-capturing peptide CBP-12 to increase contact, thereby enhancing the formation of gap junctions between tumor cells and DCs, promoting antigen delivery to the DC cytoplasm, and thus enhancing MHC-I-mediated cross-presentation. Furthermore, VNP20009, as an intracellular bacterium, can infect and colonize tumor cells. Therefore, the tumor antigen carried by VNP20009 can be expressed intracellularly, enabling it to be presented as an endogenous antigen to the tumor cell surface by MHC-I for recognition by specific CD8+ T cells.

[0029] The composition of VNP-Apep in this invention is as follows: Figure 1 The gene containing the tumor antigen (Antigen 4T1-M8) and the DC capture peptide (DCpep) was added before and after the DC capture peptide to promote the secretion and membrane anchoring of DCpep. VNP-Apep was obtained by introducing the two plasmids containing the target genes into bacteria by electroporation.

[0030] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0031] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0032] Example 1 Experimental materials: Attenuated Salmonella VNP20009 was obtained from Qilu Hospital of Shandong University.

[0033] Mouse breast cancer cells (4T1) were purchased from the Chinese Academy of Sciences and cultured in RPMI-1640 medium containing a mixture of 10% fetal bovine serum and 1% penicillin and streptomycin.

[0034] The gene and amino acid sequences used in this embodiment are as follows: Tumor antigen (Antigen 4T1-M8), DNA sequence: CAGGGCGTGACCGTGCTGGCGGTGAGCGCGGTGTATGATATTTTTGTGTTTCATCGCCTGAAAATGAAACAGATTCTGCCG, as shown in SEQ ID No. 2.

[0035] Amino acid sequence: QGVTVLAVSAVYDIFVFHRLKM-KQILP, as shown in SEQ ID No. 1.

[0036] IL-2-sig: DNA sequence: TACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG, as shown in SEQ ID No. 4.

[0037] Amino acid sequence: YRMQLLSCIALSLALVTNS, as shown in SEQ ID No. 3.

[0038] DC-capturing peptide: DNA sequence: ATGTGGCCGCGCTTTCATAGCAGCGTGCGCCATACCCAT, as shown in SEQ ID No. 6.

[0039] Amino acid sequence: WPRFHSSVFHTH, as shown in SEQ ID No. 5.

[0040] PDGFR-sig: DNA sequence: GCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGCCCTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGT, as shown in SEQ ID No. 8.

[0041] amino acid sequence: AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR, as shown in SEQ ID No. 7.

[0042] VNP-Apep construction methods include: Step 1: Plasmid Construction The pBAD-Antigen plasmid was obtained by inserting the gene sequence containing the tumor antigen (Antigen 4T1-M8) into the multiple cloning site of the pBAD plasmid, and the pBAD-IL-2-sig+DC capture peptide+PDGFR-sig plasmid was obtained by inserting the gene sequence containing IL-2-sig+DC capture peptide+PDGFR-sig into the multiple cloning site of the pBAD plasmid. Both plasmids were prepared into a 200 ng / μL plasmid solution using sterile, enzyme-free water.

[0043] Step 2: Preparation of VNP20009 competent cells Remove VNP20009 bacterial culture from the -80℃ freezer and streak it onto LB agar plates, incubating overnight at 37℃. Select healthy single colonies and inoculate them into 2 mL of LB liquid medium, incubating overnight at 37℃ and 220 rpm. Add 0.5 mL of the overnight culture to 50 mL of LB medium and incubate at 37℃ and 220 rpm until the OD value reaches 0.5. Remove the shake flask from the shaker and place it in an ice bath for 30 min. Pour 25 mL of bacterial culture into a pre-chilled 50 mL round-bottom centrifuge tube and centrifuge at 1000g for 15 min at 4℃ to precipitate the bacteria, discarding the supernatant. Resuspend the bacterial pellet in 10 mL of ice-cold 10% glycerol-water solution and centrifuge at 1000g for 15 min at 4℃ to precipitate the bacteria, discarding the supernatant. Wash the bacterial pellet again with 10 mL of ice-cold 10% glycerol-water solution, retaining the bacterial pellet. Finally, the bacterial pellet was fully resuspended in 50 μL of 10% glycerol and transferred to a pre-cooled 0.5 mL centrifuge tube, which yielded usable electrocompetent cells.

[0044] Step 3: Electroconversion of VNP-Apep Take the VNP20009 competent cells obtained in the above steps, add 0.5 μL of pBAD-Antigen plasmid solution and pBAD-IL-2-sig+DC capture peptide+PDGFR-sig plasmid solution respectively, mix gently, place on ice for 1 min, transfer to a pre-chilled electroporation cuvette, wipe the surface of the cuvette dry, and place the electrode for electroporation transformation. (Voltage: 1800V, Capacitance: 25 μF, Resistance: 200Ω) Immediately after electroporation, add 1 mL of antibiotic-free LB liquid medium to the cuvette, gently pipette to suspend, and transfer all to a new sterile 1.5 mL centrifuge tube. Incubate at 37℃ and 160 rpm for 1.5 h-3 h for recovery. Take 200 μL and plate on an LB agar plate for single-clone culture to finally obtain the target strain VNP-Apep. VNP-A was constructed according to the above method; it is an engineered bacterium expressing only Antigen 4T1-M8.

[0045] Example 2: Anchoring of VNP-Apep-expressed DCpep on the surface of tumor cells: To assess the expression of DC-capturing peptides on the surface of tumor cells, 1×10⁻⁶ cells were used. 5 Four T1 cells per dish were seeded in confocal microscopy dishes and cultured overnight until adherence. The culture medium was then replaced with fresh medium supplemented with VNP-A and VNP-Apep, respectively, and 0.2% arabinose inducer was added for 6 h of co-incubation. The culture medium was then replaced with fresh medium supplemented with 0.2% arabinose inducer and cultured overnight to induce intracellular VNP-Apep expression of the DC-capturing peptide. Primary antibody (Flag-tagged antibody) and Alexa Fluor 488-conjugated secondary antibody were added sequentially, and each was incubated with the cells for 1 h. After washing with PBS, the cells were fixed with 4% tissue fixative for 10 min. After washing with PBS, the cell membrane was labeled with Dil (10 μm), and the cell nucleus was labeled with DAPI. Cells were then observed under a confocal microscope or directly collected for flow cytometry to detect the quantitative expression of the DC-capturing peptide. Figure 2 a). Confocal results show ( Figure 2 (b) Obvious green fluorescence was observed on the surface of tumor cells in the VNP-Apep group, overlapping with the orange fluorescence of the cell membrane, indicating that VNP-Apep successfully expressed the capture peptide and that the capture peptide was able to anchor to the tumor cell membrane surface. Quantitative flow cytometry data showed ( Figure 2 c), the VNP-Apep group (8.48±0.51%) was significantly lower than that of the 4T1 group (0.47±0.05%). p <0.0001) More capture peptides are produced.

[0046] Example 3: Investigation of tumor cells' ability to capture dendritic cells (DCs) To test the affinity of the DC-capturing peptide expressed by VNP-Apep for DCs, VNP-Apep was seeded and cultured to the logarithmic growth phase (OD600 approximately 0.4-0.6), then cultured overnight with 0.2% arabinose inducer, followed by centrifugation to collect the supernatant. 1×10⁻⁶... 5 DC cells were seeded in 12-well plates and cultured overnight until adherence. The cell culture medium was replaced with different bacterial culture supernatants. After 6 h of incubation, cells were collected and then incubated with primary antibody (Flag-tagged antibody) and secondary antibody (Alexa Fluor 488-conjugated), respectively, for 1 h each. After washing with PBS, flow cytometry was performed to analyze the content of DC-capturing peptides on the DCs. Subsequently, to investigate whether DC-capturing peptides on the tumor surface could attract more DC cells, 1×10⁶ DC cells were seeded in each well. 5Four T1 cells per dish were seeded into confocal culture dishes and co-incubated with VNP-Apep for 6 h followed by overnight culture. VNP-A-treated tumor cells and untreated tumor cells were used as controls. Dil-labeled DC cells were co-cultured with 4T1 cells for 30 s or 30 min, washed with PBS, and then photographed and counted using confocal imaging.

[0047] Experimental results: VNP-Apep culture supernatant was used to co-incubate DC cells ( Figure 3 a) Flow cytometry was used to analyze the affinity of free DC-capturing peptides for DCs. For example... Figure 3 As shown in b, the percentage of DCpep-positive DC cells in the VNP-Apep group (8.48±0.63%) was significantly higher than that in the Control group (0.47±0.06%, p<0.0001). VNP-Apep-treated 4T1 cells were briefly co-incubated with Dil-stained DC cells to detect tumor cell capture of DCs; the results are shown in Figure b. Figure 3 As shown in c, after 30 s of incubation, no significant DC binding was observed on the surface of any of the three groups of tumor cells. After 30 min of incubation, a greater number of DC bindings were observed in the VNP-Apep group. The counting results ( Figure 3 d) also showed that the VNP-Apep group had the highest DC binding. These results indicate that the capture peptide can increase the affinity of tumor cells for DCs.

[0048] Example 4: Investigation of antigen cross-presentation ability in DCs To evaluate the ability of VNP-Apep to promote antigen cross-presentation in dendritic cells (DCs), an oral antigen assay (OVA) was used as a model antigen. 1×10⁻⁶ cells were used. 5DC cells were seeded in confocal microscopy dishes and cultured overnight until adherent. After staining with Dil and washing with PBS, 4T1 tumor cells treated with different methods (untreated 4T1, VNP+4T1, VNP-A+4T1, VNP-Apep+4T1, VNP-Apep+4T1+heptanol) were added and co-incubated for 4 h. Non-adherent tumor cells were washed away, and OVA-H-2Kb-conjugated primary antibody and Alexa Fluor 488-conjugated secondary antibody were added sequentially. After incubation with each for 1 hour, the cells were fixed with 4% tissue fixative, stained with DAPI, and then imaged under a confocal microscope. To quantify antigen cross-presentation in dendritic cells (DCs), the above-described method was used to co-incubate DCs with tumor cells. DCs were then collected, and cells were labeled with FITC anti-mouse CD11c and PE anti-mouse H-2Kb bound to SIINFEKL antibodies. The proportion of DCs containing the OVA-MHCI complex was analyzed by flow cytometry. Figure 4 a. The green fluorescence of the VNP-Apep and VNP-A groups, representing OVA-MHCI, was significantly higher than that of the other three groups. However, no green fluorescence was observed after adding heptanol to block the gap junctions, demonstrating that the antigen is transferred to the dendritic cells (DCs) via gap junctions and subsequently presented by the DCs. Flow cytometry results showed ( Figure 4 b) The proportion of DC cells containing OVA-MHCI in the VNP-Apep group (17.27±0.62%) was significantly higher than that in the VNP-A group (9.45±1.48%). p The result (<0.0001) indicates that the DC-capturing peptide can increase the contact between tumor cells and DCs, promote the formation of gap junctions, and improve the efficiency of antigen delivery to DCs. These results demonstrate that VNP-Apep and VNP-A antigens are successfully expressed and promote gap junction formation; the capture peptide in VNP-Apep can further promote antigen cross-presentation.

[0049] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A DC-capture engineered bacterium, VNP-Apep, utilizing gap junctions to achieve cytoplasmic antigen transfer, characterized in that... The engineered bacterium VNP-Apep contains the following target genes: The tumor antigen (Antigen 4T1-M8) gene is used to express the target tumor antigen. The DC capture peptide (DCpep) gene is used to express DCpep; and before and after the DCpep gene are IL-2 signal peptide genes and PDGFR signal peptide genes, respectively, which are used to promote the secretion and membrane anchoring of DCpep.

2. The DC-captured engineered bacterium VNP-Apep utilizing gap junctions to achieve cytoplasmic antigen transfer as described in claim 1, characterized in that, The engineered bacteria VNP-Apep contains a plasmid containing the tumor antigen (Antigen 4T1-M8) gene and a plasmid containing the DC capture peptide (DCpep) gene, wherein the DC capture peptide (DCpep) gene is preceded and followed by the IL-2 signal peptide gene and the PDGFR signal peptide gene, respectively.

3. The DC-captured engineered bacterium VNP-Apep that utilizes gap junctions to achieve cytoplasmic antigen transfer as described in claim 1, characterized in that, DC-capturing peptide genes are CBP-12 genes, pMAM genes, pMAG genes, or short peptide genes that express other DC receptors.

4. The DC-captured engineered bacterium VNP-Apep that utilizes gap junctions to achieve cytoplasmic antigen transfer as described in claim 3, characterized in that, The DC-capturing peptide gene is the CBP-12 gene.

5. A method for constructing VNP-Apep, a DC-captured engineered bacterium utilizing gap junctions to achieve cytoplasmic antigen transfer, as described in any one of claims 1 to 4, characterized in that... The method includes the following steps: Using attenuated Salmonella VNP20009 as the starting strain, target gene 1 and target gene 2 were carried, wherein target gene 1 is Antigen 4T1-M8 gene; The second target gene is the DC capture peptide (DCpep) gene, with the IL-2 signal peptide gene and the PDGFR signal peptide gene inserted before and after the DCpep gene, respectively.

6. The method for constructing VNP-Apep, a DC-captured engineered bacterium utilizing gap junctions to achieve cytoplasmic antigen transfer, as described in claim 5, is characterized in that... The construction method includes the following steps: plasmid construction, preparation of VNP20009 competent cells, and electroporation of VNP-Apep.

7. The method for constructing VNP-Apep, a DC-captured engineered bacterium utilizing gap junctions to achieve cytoplasmic antigen transfer, as described in claim 5, is characterized in that... in, The plasmid construction method specifically includes the following steps: inserting the Antigen 4T1-M8 gene sequence into the multiple cloning site of the pBAD plasmid to obtain the pBAD-Antigen plasmid; inserting the gene sequence containing the IL-2-sig-DC capture peptide-PDGFR-sig into the multiple cloning site of the pBAD plasmid to obtain the pBAD-IL-2-sig-DC capture peptide-PDGFR-sig plasmid; and preparing a plasmid solution using sterile, enzyme-free water.

8. The method for constructing VNP-Apep, a DC-captured engineered bacterium utilizing gap junctions to achieve cytoplasmic antigen transfer, as described in claim 5, is characterized in that... DC-capturing peptide genes are CBP-12 genes, pMAM genes, pMAG genes, or short peptide genes that express other DC receptors.

9. The method for constructing VNP-Apep, a DC-captured engineered bacterium utilizing gap junctions to achieve cytoplasmic antigen transfer, as described in claim 5, is characterized in that... VNP-Apep was obtained by introducing two plasmids containing the target gene into VNP20009.

10. The use of the engineered bacteria VNP-Apep according to any one of claims 1 to 4, or the engineered bacteria VNP-Apep prepared by the method according to any one of claims 5 to 9, in the preparation of a cytoplasmic delivery vaccine that promotes tumor antigen delivery.

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