Recombinant plasmid for expressing recombinant pyroptosis protein, engineering strain, preparation method of recombinant plasmid and engineering strain, and tumor vaccine

Abstract

The invention discloses a recombinant plasmid for expressing recombinant pyroptosis protein, an engineering strain, a preparation method of the engineering strain and a tumor vaccine. The recombinant plasmid comprises an expression vector and a coding sequence which is inserted into the expression vector and is used for coding pyroptosis protein, the expression vector comprises pCDFDuet and pExoS54F, and the expression vector comprises pCDFDuet and pExoS54F; the coding sequence is added with a connection switch disulfide bond linker responding to glutathione in a tumor microenvironment. The recombinant plasmid constructed by the invention can be delivered through bacteria with tumor targeting, can stably express tumor microenvironment response recombinant pyroptosis protein, reduces damage to normal cells, and is a safe and efficient protein drug delivery technology.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a recombinant plasmid expressing recombinant pyroptosis protein, an engineered bacterial strain and its preparation method, and a tumor vaccine. Background Technology

[0002] In situ tumor vaccines utilize tumor antigens present at the tumor site to induce a tumor-specific adaptive immune response, showing great potential for the treatment of many solid tumors. The concept of in situ tumor vaccines was first proposed in the 1890s, when research revealed that injecting heat-inactivated bacteria into tumors could elicit a strong anti-tumor immune response. Subsequently, BCG vaccine (a live attenuated vaccine form of *Bifidobacterium bovis*) was approved for the treatment of bladder cancer. Furthermore, an oncolytic virus that preferentially infects tumor cells and causes tumor cell lysis has been introduced into clinical trials as an in situ vaccine formulation. These clinical and pre-clinical studies all demonstrate the significant potential of in situ tumor vaccines in anti-tumor therapy.

[0003] Pyroptosis is a type of programmed cell death dependent on an inflammatory response. Its defining characteristic is the disruption of cell membrane integrity, resulting in the production of numerous pyroptotic vesicles accompanied by the release of inflammatory cytokines and danger signaling molecules. These natural tumor-specific antigens and damage-associated molecular patterns can trigger a robust anti-tumor immune response, potentially leading to long-term immune memory and providing anti-tumor immune protection. However, current research on the specific induction of tumor cell pyroptosis faces numerous challenges, one of the most critical being the achievement of highly specific activation of pyroptosis proteins in the tumor microenvironment. Because pyroptosis possesses strong inflammatory characteristics, accidental activation in non-target tissues can lead to severe side effects.

[0004] Therefore, the existing technology still needs further research and improvement. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a recombinant plasmid expressing recombinant pyroptosis protein, an engineered bacterial strain, its preparation method, and a tumor vaccine. The aim is to solve the problem of how to specifically initiate pyroptosis in tumor cells to increase the number and activity of infiltrating cytotoxic T cells, thereby triggering long-term immune protection and enhancing the efficacy of tumor immunotherapy. Specifically:

[0006] In a first aspect, embodiments of the present invention provide a recombinant plasmid expressing a recombinant pyroptosis protein, comprising: an expression vector and a coding sequence encoding the pyroptosis protein inserted in the expression vector; the expression vector comprising: pCDFDuet, pExoS 54 F; The coding sequence contains a disulfide linker that is a linker for glutathione in response to the tumor microenvironment.

[0007] As a preferred technical solution, in the recombinant plasmid expressing recombinant pyroptosis protein, the linker disulfide linker is added at the N-domain end of the coding sequence or between the C-domain and N-domain of the coding sequence.

[0008] As a preferred technical solution, the recombinant plasmid expressing recombinant pyroptosis protein, wherein the promoter sequence of pCDFDuet is shown in SEQ ID NO.1.

[0009] As a preferred technical solution, the recombinant plasmid expressing recombinant pyroptosis protein, wherein the pExoS 54 The activation sequence of F is shown in SEQ ID NO.2.

[0010] As a preferred technical solution, the recombinant plasmid expressing recombinant pyroptosis proteins includes GSDMA, GSDMB, GSDMC, GSDME, and GSDMD.

[0011] This invention utilizes genetic engineering techniques to construct recombinant plasmids expressing recombinant pyroptosis proteins. By transforming these plasmids into expression vectors (pCDFDuet, pExoS54F), the efficient release of the pyroptosis protein into tumor cells is achieved, triggering pyroptosis and thus inducing a robust anti-tumor immune response. This invention constructs different recombinant plasmids by adding different sequences encoding recombinant pyroptosis proteins to the expression vector plasmid backbone sequence.

[0012] Specifically, the pyroptosis protein gene expression sequence is introduced into a plasmid vector, and a glutathione linker disulfide bond (SS bond) responding to the tumor microenvironment is added at 5' to 3'. This SS bond sequence contains 134 base pairs, and the encoded linker region can be added at the N-domain end of the aforementioned pyroptosis protein gene sequence or at the junction between its C-domain and N-domain, thereby constructing different recombinant plasmids. For example:

[0013] (1) Added to the left of the PSAFEKVVKNVIKEVSGSRGDLI (SS linker added at Ile-23) domain of GSDMD-NT (referencing mouse GSDMD protein, UniProt ID: P57764); (2) Added between the LLSDGID (SS linker added at Asp-275 cleavage site) domain of GSDMD-NT. The addition methods are: adding the SS linker at position (1) alone; position (2); or adding the SS linker at both positions (1) and (2). The sequence of the SS linker is shown in SEQ ID NO.3.

[0014] In a second aspect, there is an engineered bacterial strain, wherein the engineered bacterial strain contains the recombinant plasmid described in the first aspect.

[0015] As a preferred technical solution, the engineered strain wherein the bacterial host into which the recombinant plasmid is transferred is *Escherichia coli* that is induced by arabinose to express recombinant proteins or *Pseudomonas aeruginosa* with a protein secretion system.

[0016] Thirdly, a method for constructing an engineered bacterial strain, comprising: transferring the recombinant plasmid described in the first aspect into *Escherichia coli* or attenuated *Pseudomonas aeruginosa* to obtain the engineered bacterial strain.

[0017] In this invention, the recombinant plasmid was transformed into a strain of *E. coli* to obtain an engineered strain with exogenous initiation capable of intracellular delivery of GSDMD protein. Furthermore, similar genetic engineering was performed on the DNA sequence expressing pyroptosis protein, adding a tumor microenvironment-responsive linker disulfide bond element (SS bond) to the N-domain end of this sequence. This SS bond sequence contains 134 base pairs. The resulting sequence was encoded into a plasmid vector (pExoS) with a complete type III secretion system. 54 The recombinant plasmid obtained at F) was electroporated into an attenuated strain of *Pseudomonas aeruginosa*, thus constructing an engineered strain that endogenously responds to the tumor microenvironment and can deliver GSDMD protein intracellularly. Both delivery vector engineered bacteria in the above embodiments possess tumor cell targeting capabilities and can overexpress recombinant pyroptosis protein in the tumor microenvironment in response to different microenvironments, releasing cytotoxic N-terminal pyroptosis protein, thereby triggering tumor cell pyroptosis.

[0018] The constructed plasmid expressing recombinant pyroptosis protein was transformed into tumor-targeting bacteria. The delivery bacteria were either *Escherichia coli* or a live attenuated *Pseudomonas aeruginosa*. The former was the probiotic *E. coli* Nissle 1917 (EcN); the latter is characterized by its live attenuation and limited survival time in vivo due to nutritional deficiencies. Both are facultative anaerobes with tumor hypoxia tropism and high biosafety. After delivering recombinant pyroptosis protein to tumor cells via these bacteria, the expression level of cytotoxic N-terminal pyroptosis protein significantly increased in tumor cells in response to arabinose or the tumor microenvironment.

[0019] In this invention, the two vectors mentioned above were chosen primarily because they are more likely to stably express and deliver pyroptosis proteins into tumor cells in specific bacteria, while also facilitating the identification and purification of the target protein. The choice of plasmid vectors is generally more conducive to the stable and efficient expression of the target protein. Two recombinant plasmid vectors (pCDFDuet and pExoS54F) and two vectors (Escherichia coli (ECN) and attenuated Pseudomonas aeruginosa (Δ6)) can be sequenced to form various engineered bacteria (taking GSDMD (GD) as an example: Escherichia coli: pCDFDuet-PGase-GSDMD (PGase-GSDMD); engineered Pseudomonas aeruginosa: pExoS...). 54 F-GSDMD(Δ6-GD), pExoS 54 F-SS-GSDMD(Δ6-S-GD),

[0020] Fourthly, a tumor vaccine, wherein the tumor vaccine comprises the engineered strains described above.

[0021] As a preferred technical solution, the tumor vaccine is applicable to breast cancer, pancreatic cancer, melanoma, lung cancer, and glioma.

[0022] In this invention, taking attenuated Pseudomonas aeruginosa engineered bacteria as an example, the recombinant plasmid stably expresses tumor microenvironment-responsive recombinant pyroptosis protein in the delivery vector engineered bacteria. In response to high glutathione concentration, the SS bond breaks, and the concentration of intracellular cytotoxic N-terminal pyroptosis protein increases. It can efficiently induce tumor cell pyroptosis in vivo and in vitro, and increase the release of lactate dehydrogenase (LDH) and inflammatory factors (IL-1β and IL-18).

[0023] Taking engineered Escherichia coli as an example, by injecting this engineered bacterium into a subcutaneous tumor model, its overexpressed recombinant pyroptosis protein can significantly kill tumor cells, not only significantly inhibiting tumor growth, but also showing a significant tumor-suppressing effect in distant tumor suppression challenges.

[0024] Taking the attenuated Pseudomonas aeruginosa engineered bacteria as an example, the tumor microenvironment-responsive recombinant pyroptosis protein GSDMD delivered into tumor cells can significantly trigger pyroptosis, increasing the number and activity of T cells. More importantly, the tumor cell immunogenic cell death induced by this engineered bacteria can serve as a tumor vaccine, activating anti-tumor immune protection and effectively inhibiting the occurrence and development of distant tumors.

[0025] Beneficial effects: Compared with the prior art, the embodiments of the present invention have the following advantages:

[0026] The recombinant plasmid expressing a response-initiated recombinant pyroptosis protein constructed in this invention can not only deliver pyroptosis protein with high specificity in the tumor microenvironment, but also, as a delivery vector, trigger tumor cell pyroptosis simultaneously with intracellular injection of the recombinant pyroptosis protein. This can efficiently induce tumor cell pyroptosis, improve the tumor immune microenvironment, activate systemic anti-tumor protective effects, and effectively inhibit tumor development and progression. The recombinant plasmid constructed in this invention can be delivered via tumor-targeting bacteria and can stably express tumor microenvironment-responsive recombinant pyroptosis protein, reducing damage to normal cells. This represents a safe and efficient protein drug delivery technology. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The attached figure shows the construction diagram of the recombinant plasmid expression vector encoding recombinant pyroptosis protein provided by the present invention;

[0029] Figure 2 The attached figure shows the construction diagram of the recombinant plasmid expression vector encoding recombinant pyroptosis protein in response to the tumor microenvironment provided by the present invention;

[0030] Figure 3 The attached image is an agarose gel electrophoresis diagram used to preliminarily confirm the successful transfer of the recombinant plasmid encoding the recombinant pyroptosis protein that responds to the tumor microenvironment.

[0031] Figure 4 The attached figure shows the use of Western blotting to demonstrate that the recombinant plasmid encoding the tumor microenvironment-responsive recombinant pyroptosis protein stably expresses the tumor microenvironment-responsive recombinant pyroptosis protein in engineered Pseudomonas aeruginosa.

[0032] Figure 5 The attached figure shows an in vitro cell experiment demonstrating that engineered *Pterocarya aeruginosa* delivers recombinant pyroptosis protein into triple-negative breast cancer cells (4T1) via the pyroptosis pathway, activating intracellular caspase-1p20, effectively triggering tumor cell pyroptosis, and releasing cytotoxic N-pyroptosis protein.

[0033] Figure 6 The attached figure shows in vitro cell experiments confirming from cell morphology that the recombinant pyroptosis protein expressed by engineered *Aureobasidium aeruginosa* can induce pyroptosis in 4T1 cells;

[0034] Figure 7 The attached figure shows an in vitro cell experiment demonstrating that recombinant pyroptosis protein expressed by engineered *Aureobasidium aeruginosa* can induce the release of lactate dehydrogenase (LDH) in 4T1 cells through cell membrane damage.

[0035] Figure 8 The attached figure shows an in vivo animal experiment demonstrating that the recombinant pyroptosis protein expressed by engineered Pseudomonas aeruginosa can inhibit tumor (4T1 tumor) proliferation by observing changes in tumor size.

[0036] Figure 9 The attached figure shows an in vivo animal experiment demonstrating, through changes in the tumor microenvironment, that recombinant pyroptosis protein expressed by engineered *Pterocaryonium aeruginosa* can activate the number of infiltrating T cells within tumor cells;

[0037] Figure 10 The attached figure shows in vivo animal experiments demonstrating that recombinant pyroptosis protein expressed by engineered Escherichia coli can inhibit the proliferation of bilateral tumors, based on changes in tumor size.

[0038] Figure 11 The attached figure shows in vivo animal experiments demonstrating that the recombinant pyroptosis protein expressed by engineered Pseudomonas aeruginosa can serve as an in situ tumor vaccine to inhibit the proliferation of distant tumors, based on changes in tumor size.

[0039] Figure 12 The attached figure shows the biosafety evaluation of engineered Pterospermum aeruginosa for in vivo tumor therapy; Detailed Implementation

[0040] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] The delivery strains and plasmid vectors used in this invention are as follows:

[0042] One bacterial species is the probiotic *E. coli* Nissle 1917 (EcN). Another engineered bacterium is an attenuated auxotrophic *Pseudomonas aeruginosa* (Δ6). The delivery vector for this bacterium utilizes genetic engineering to obtain a D-glutamate auxotrophic attenuated *Pseudomonas aeruginosa* Δ6, whose sequence is shown in SEQ ID NO.4. This bacterium exhibits weak cytotoxicity to mammalian cells and higher biocompatibility. Its reproductive capacity is weak without the addition of exogenous D-glutamate, and its proliferation can be inhibited through D-glutamate auxotrophy. The Δ6 strain possesses a fully functional protein secretion system, enabling rapid transfection and delivery of proteins to mammalian cells.

[0043] The tumor cells used in this invention are 4T1 and B16F10 cells.

[0044] Experimental animals used in this invention

[0045] Female BALB / c mice (6-8 weeks old) were purchased from Guangdong Yaokang Biotechnology Co., Ltd.; female C57BL / 6 mice (6-8 weeks old) were purchased from Zhuhai Baishitong Biotechnology Co., Ltd. A 12-hour light-dark cycle environment was guaranteed, and all procedures and experiments followed internationally accepted standards for the breeding and use of laboratory animals.

[0046] The reagents used in this invention

[0047] PrimeSTAR HSDNA polymerase, 2×PrimeSTAR GC Buffer, dNTP Mixture, and restriction endonucleases were purchased from Takara Bio Inc.; DNA Marker, SanTaq Plus PCR Mix, 4S GelRed nucleic acid dye, 6X DNA Loading Dye, T4 DNA ligase (Promega), ethylene glycol di(2-aminoethyl ether)tetraacetic acid, ciprofloxacin hydrochloride, carbenicillin disodium, kanamycin, lactate dehydrogenase (LDH) activity assay kit, and tumor immunohistochemistry-related antibodies (CD8a Monoclonal Antibody, CD4 Monoclonal Antibody, CD3 Monoclonal Antibody, FOXP3 Monoclonal Antibody) were all purchased from Thermo Fisher Scientific China Co., Ltd.; sucrose was a product of Beijing Solarbio Science & Technology Co., Ltd.; technical agar powder (Bacto-agar), LB broth, and tryptone soybean broth were purchased from Guangdong Huankai Microbial Technology Co., Ltd.; agarose was purchased from BIOWEST; and plasmid mini-prep kit (Plasmid Mini) was used. The Prep Kit is a product of Tiangen Biotech (Beijing) Co., Ltd.; the DNA Purification Kit (DNA Clean & Concentrator) and the DNA Gel Recovery Kit (Zymoclean Gel DNA Recovery Kit) are products of Zymo Research, Inc. (USA); the Anti-GAPDH antibody is a product of Abcam, Inc. (UK); the Anti-E. coli RNA Polymerase α antibody is a product of Biolegend, Inc. (USA); the Anti-Flag antibody and the Anti-mouse secondary antibody are products of Sigma-Aldrich, Inc. (USA); the Anti-Gasdermin D antibody, the Anti-Cleaved Gasdermin D antibody, the Anti-Cleaved caspase-1 antibody, and the Anti-rabbit HRP-linked secondary antibody are products of Cell Signaling Technology, Inc. (USA); the Stripping Buffer for protein blot membrane regeneration is a product of Kangwei Century Biotechnology Co., Ltd.; and the Dimethyl Sulfoxide (DMSO), SDS-PAGE Protein Loading Buffer, Western Primary Antibody Diluent, and Western Secondary Antibody Diluent are products of Beyotime Biotechnology Co., Ltd. (Shanghai).

[0048] Example 1

[0049] Construction of engineered bacteria expressing recombinant pyroptosis protein

[0050] The plasmid pCDFDuet-PGase-GSDMD (its sequence is shown in SEQ ID NO.3) used in this implementation case was constructed by Shanghai Sangon Biotech Co., Ltd. Its construction diagram can be found in [reference needed]. Figure 1 The plasmid was transformed into *E. coli* DH5α to construct the engineered bacteria FEP. The specific transformation steps are as follows: The bacterial culture was taken out of the -80℃ freezer, streaked on an LB agar plate, and placed in a 37℃ incubator for recovery; the next day, a single colony from the fresh streaked culture was picked, inoculated into LB medium, and cultured overnight in a 37℃ constant temperature shaker at 180 rpm; on the third day, the overnight culture was inoculated into antibiotic-free LB medium, and cultured at 37℃ and 200 rpm for 2-3 hours. When the OD600 of the bacterial culture reached 0.4-0.6, the culture was stopped. Then, the plasmid was electroporated into *E. coli* DH5α using an electroporator. After recovery and plating, positive colonies were screened in agar plates containing the corresponding antibiotics and the bacterial culture was further preserved to obtain the engineered bacteria expressing recombinant pyroptosis protein.

[0051] Example 2

[0052] Construction of recombinant plasmids expressing recombinant pyroptosis protein in response to tumor microenvironment

[0053] The experimental steps for constructing a recombinant plasmid expressing a tumor microenvironment response recombinant pyroptosis protein are as follows: (1) The pyroptosis protein gene expression sequence is introduced into the plasmid vector, and Sac I and Hind III restriction sites are added at the 5' and 3' ends, respectively. At the same time, a stop codon is added. (2) Primers for full-length amplification of the target gene are designed. (3) A tumor microenvironment response linker disulfide bond element (SS bond) is added to the N-domain end of the pyroptosis protein sequence. The SS bond sequence contains 134 base pairs. (4) Gene synthesis is performed using the optimized edited gene sequence and the designed primer sequence. (5) After receiving the recombinant plasmid storage bacteria, the plasmid is extracted, and the target fragment is amplified by PCR using the designed primers. The size and approximate concentration of the PCR product are detected by electrophoresis in a 1.0% agarose gel. (6) The above PCR product is digested with appropriate restriction endonucleases. The target fragment is ligated to pExoS with corresponding sticky ends by double digestion. 54 (6) The ligation product was transformed into DH5α competent cells, positive selection was performed, transformants were obtained, and PCR and plasmid enzyme digestion were used to identify the transformants. After positive identification, the plasmid was amplified; (7) The plasmid was extracted and further electroporated into Δ6 cells to construct an engineered bacterium expressing recombinant pyroptosis protein in response to the tumor microenvironment and to perform verification experiments such as PCR verification, protein verification, and sequencing identification. Its construction map can be referred to Figure 2 .

[0054] Example 3

[0055] Construction of engineered bacteria expressing recombinant pyroptosis protein in response to tumor microenvironment

[0056] PCR amplification of the tumor microenvironment-responsive recombinant pyroptosis protein gene and cloning it into the expression vector pExoS 54 In this process, corresponding recombinant plasmids were constructed. These recombinant plasmids were then electroporated into Δ6 strain using an electroporator. Positive colonies were screened on plates containing the corresponding antibiotics and further cultured to obtain engineered delivery bacteria expressing tumor microenvironment-responsive recombinant pyroptosis protein. Upon contact with mammalian cells, these engineered bacteria, induced by their unique protein secretion system, expressed a large amount of tumor microenvironment-responsive recombinant pyroptosis protein. In this embodiment, the recombinant pyroptosis protein interacts with the secretion signal ExoS of the protein secretion system on the expression vector. 54 The fusion, with the addition of a GSH-responsive SS bond element, allows for the release of cytotoxic N-terminal pyroptosis protein in response to GSH after injection into tumor cells via the T3SS secretion system. This release is independent of tumor-specific limitations, activating caspase-1 and releasing cytotoxic N-terminal pyroptosis protein, thereby initiating the pyroptosis pathway within tumor cells to inhibit tumor growth. Preliminary validation of successful construction can be found in [link to documentation]. Figure 3 Its successful expression of the deliverable protein can be seen in [the following text is incomplete and requires further context]. Figure 4 .

[0057] Example 4

[0058] Western blot analysis of the expression of recombinant pyroptosis protein in tumor cells induced by engineered bacteria.

[0059] Pick a single fresh streaked colony and inoculate it into 5 mL of L-Broth medium containing an appropriate antibiotic. Incubate overnight at 37°C with shaking at 180 rpm. The next day, inoculate the colony at a 1:50 ratio into L-Broth medium containing the corresponding antibiotic and 10 mM D-Glu. Incubate at 37°C with shaking at 180 rpm until OD500 is reached. 600The concentration was approximately 1.0. 1 mL of bacterial culture was placed in a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 1 min at room temperature to collect the cells. The supernatant was discarded, and the cells were washed twice with 1×PBS. The cells were resuspended in DMEM medium containing 5% FBS and no antibiotics and inoculated into tumor cells in 6-well plates with an MOI of 100. After co-culturing for 12 h, the bacteria were discarded, and the cells were washed three times with PBS. Cell lysis buffer containing a protease inhibitor (PMSF) was added, and the cells were lysed on ice. Protein samples were prepared and subjected to SDS-PAGE gel electrophoresis. The successfully constructed engineered bacteria expressed and secreted recombinant pyroptosis protein. Western blotting was used to verify the expression using corresponding specific antibodies. β-actin was used as an intracellular reference to assess the expression level of recombinant pyroptosis protein. Activation of the intracellular pyroptosis signaling pathway is described in the appendix. Figure 5 .

[0060] Example 5

[0061] Engineered bacteria expressing recombinant pyroptosis protein induce pyroptosis in tumor cells

[0062] The bacterial culture obtained from the above treatment was used to inoculate 4T1 cells in confocal microscopy dishes with an MOI of 100. After co-culturing for 4 hours, staining was performed according to the instructions of the apoptosis detection kit (Annexin-V FITC / PI kit). Finally, the morphological characteristics of pyroptosis in tumor cells were observed using confocal fluorescence microscopy. The actual experimental results are shown in [link to experimental results]. Figure 6 .

[0063] Lactate dehydrogenase (LDH) release characterization was used to evaluate the in vitro toxicity of engineered bacteria.

[0064] The bacterial culture obtained after the above treatment was inoculated into 4T1 cells in 96-well plates with an MOI of 100. After co-culturing for 12 hours, the cells were cultured according to the lactate dehydrogenase assay kit (CyQUANT). TM The experiment was conducted according to the instructions of the LDH Cytotoxicity Assay Kit to detect the LDH release level from cells in different groups. The actual experimental results are shown in [link to kit]. Figure 7 .

[0065] Evaluation of engineered bacteria for tumor suppression in vivo (4T1 model of triple-negative breast cancer)

[0066] 100 μL of a healthy 4T1 suspension (cell count 5 x 10) was subcutaneously injected into the right posterior back of healthy female BALB / c mice (6-8 weeks old). 6 / mL), observe and measure every two days, until the tumor volume grows to 50-100 mm. 3Intratumoral administration was performed. Freshly streaked single colonies (Δ6, Δ6-GD, and Δ6-S-GD) were picked and inoculated into 5 mL of L-Broth medium containing an appropriate antibiotic, and cultured overnight at 37°C with shaking at 180 rpm. The next day, at a ratio of 1:50, the inoculation was performed into L-Broth medium containing the corresponding antibiotic and 10 mM D-Glu, and cultured at 37°C with shaking at 180 rpm until OD... 600 The value is approximately 1.0. Take 1 mL of bacterial culture and place it in a 1.5 mL centrifuge tube. Centrifuge at 12000 rpm for 1 min at room temperature to collect the bacterial cells. Discard the supernatant, wash twice with 1×PBS, and resuspend in L-Broth medium containing 10 mM D-Glu. Use PBS, Δ6, Δ6-GD, and Δ6-S-GD at a concentration of 1×10⁻⁶. 8 CFU was injected subcutaneously into the tumor, followed by intratumoral injection of engineered bacteria for three consecutive days. Tumor volume changes were observed in mice, and tumor growth curves were plotted. Tumor-bearing mice were euthanized after treatment, and tumor tissue was fixed and used for immunohistochemical analysis of T cell infiltration (CD4, CD8, and FOXP3). Actual experimental results are shown below. Figure 8 ,9.

[0067] Evaluation of engineered bacteria in an in vivo bilateral tumor model (triple-negative breast cancer 4T1 model)

[0068] 100 μL of a healthy 4T1 suspension (5 × 10⁶ cells) was subcutaneously injected into the right posterior back of healthy female BALB / c mice (6-8 weeks old). 7 / mL), observe and measure every two days, until the tumor volume grows to 50-100 mm. 3 Intratumoral administration of bacteria was then performed. Finally, the length and width of the subcutaneous tumor were measured the following day, based on the formula: Tumor volume = length × (width) 2 / 2 Calculate tumor volume. Induce overnight induction culture of EcN and PGase-GSDMD in LB medium containing 1 g / L. When the tumor volume is 30-150 mm², calculate the tumor volume. 3 Mice bearing tumors were divided into PBS, EcN, and PGase-GSDMD groups based on tumor volume. On day 1 of the experiment, 100 μL of a well-growing 4T1 suspension (cell count 5 × 10⁶) was subcutaneously injected into the contralateral back of the mice. 7 / mL). On days 0, 2, 4, and 6 of the experiment, 50 μL of a 2×10⁻⁶ concentration was injected into the right tumor of mice in the EcN and PGase-GSDMD groups using a 1 mL syringe. 9 CFU / mL EcN or PGase-GSDMD. Changes in bilateral tumor volume in mice were observed starting on day 0 of the experiment, and tumor growth curves were plotted for subsequent analysis. The actual experimental results are shown below. Figure 10 .

[0069] Evaluation of engineered bacteria used as an in situ tumor vaccine to inhibit tumor progression (B16F10 melanoma model).

[0070] Before the contralateral subcutaneous tumor challenge, as previously mentioned, suspensions of Δ6 and engineered bacteria (Δ6-S-GD) were obtained. Pyroptosis of B16F10 cells was induced using Δ6 and the engineered bacteria (Δ6-S-GD). After 12 hours of co-incubation, the two groups of treated, dying tumor cells were digested and seeded subcutaneously on the right side of mice to activate the immune response. Seven days later, B16F10 cells were seeded subcutaneously on the left posterior side of the mouse abdomen. The growth size of the left-sided tumor and changes in mouse body weight were then observed and recorded daily. The actual experimental results are detailed below. Figure 11 .

[0071] Example 6

[0072] In vivo biosafety evaluation of engineered bacteria and their expression of recombinant pyroptosis protein

[0073] As previously mentioned, suspensions of Δ6 and engineered bacteria (Δ6-S-GD) were obtained using PBS, Δ6, Δ6-GD, and Δ6-S-GD at a concentration of 1 × 10⁻⁶. 8 CFU was injected subcutaneously into the right posterior back of healthy BALB / c mice for three consecutive days. Changes in body weight and behavior were observed. After 14 days of observation, mice were euthanized by enucleation to collect blood samples for biochemical analysis. H&E staining analysis was performed on major organs to assess biosafety. Actual experimental results are shown in [link to experimental results]. Figure 12 .

[0074] It should be understood that the present invention is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of the invention is limited only by the appended claims.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant plasmid expressing recombinant pyroptosis protein, characterized in that, include: An expression vector and a coding sequence for a pyroptosis protein inserted in the expression vector; the expression vector includes: pCDFDuet, pExoS 54 F; The coding sequence contains a disulfide linker that is a linker for glutathione in response to the tumor microenvironment.

2. The recombinant plasmid expressing recombinant pyroptosis protein according to claim 1, characterized in that, The connecting switch disulfide bond connector is added to the N-terminal of the encoded sequence or between the C-terminal and N-terminal of the encoded sequence.

3. The recombinant plasmid expressing recombinant pyroptosis protein according to claim 1, characterized in that, The startup sequence of pCDFDuet is shown in SEQ ID NO.

1.

4. The recombinant plasmid expressing recombinant pyroptosis protein according to claim 1, characterized in that, pExoS 54 The activation sequence of F is shown in SEQ ID NO.

2.

5. The recombinant plasmid expressing recombinant pyroptosis protein according to claim 1, characterized in that, Pyroplasin proteins include: GSDMA, GSDMB, GSDMC, GSDME, and GSDMD.

6. An engineered bacterial strain, characterized in that, The engineered strain contains the recombinant plasmid as described in claim 1.

7. The engineered strain according to claim 6, characterized in that, The bacterial host into which the recombinant plasmid is transferred is either *Escherichia coli* that is induced by arabinose to express recombinant proteins or *Pseudomonas aeruginosa* with a protein secretion system.

8. A method for constructing an engineered bacterial strain, characterized in that, include: The recombinant plasmid described in claim 1 is transferred into Escherichia coli or attenuated Pseudomonas aeruginosa to obtain the engineered strain.

9. A tumor vaccine, characterized in that, The tumor vaccine comprises the engineered strain described in claim 6.

10. The tumor vaccine according to claim 9, characterized in that, The tumor vaccine is applicable to breast cancer, pancreatic cancer, melanoma, lung cancer, and glioma.

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