Attenuated listeria monocytogenes (LADS)-based cancer vaccines and uses thereof

Abstract

The invention belongs to the field of biotechnology and tumor immunotherapy, and particularly relates to a cancer vaccine based on attenuated listeria monocytogenes (LADS) and application of the cancer vaccine. Specifically, wild listeria monocytogenes (e.g., EGD-e) or attenuated listeria monocytogenes (LADS) are taken as an original strain, and an hly gene sequence or a fragment thereof in the original strain is replaced with polynucleotide of a fusion protein coding sequence containing LLO mutants (LLON478A + V479A) and TT856-1313, so that the recombinant attenuated listeria monocytogenes strain is obtained. Compared with wild listeria monocytogenes, the recombinant attenuated listeria monocytogenes strain has the advantages that the growth activity is not obviously changed, but the toxicity is obviously reduced; the polypeptide has cell infection ability, but has biological safety in a body and tends to colonize tumor tissue. Furthermore, the recombinant attenuated listeria monocytogenes strain can effectively inhibit the growth of cervical cancer and colon cancer, and can significantly induce immune response in a tumor microenvironment (e.g., significantly enhance cytokine expression).

Description

Cancer vaccines based on attenuated Listeria monocytogenes (LADS) and their applications Technical Field

[0001] This invention belongs to the field of biotechnology and tumor immunotherapy, and specifically relates to cancer vaccines based on attenuated Listeria monocytogenes (LADS) and their applications. Background Technology

[0002] With in-depth research into the mechanisms of tumorigenesis, tumor immunotherapy has become one of the important pillars of cancer treatment. Unlike traditional surgery, radiotherapy, and chemotherapy, immunotherapy recognizes and eliminates tumor cells by activating or enhancing the host's own immune system, offering advantages such as high specificity, long-lasting efficacy, and the formation of immune memory. In recent years, immune checkpoint inhibitors (such as anti-PD-1 / PD-L1 and anti-CTLA-4 antibodies) have made breakthrough progress in various solid tumors, significantly prolonging the survival of some patients. However, due to immunosuppression in the tumor microenvironment, low levels of tumor antigen expression, insufficient T cell infiltration, and the lack of effective tumor-specific T cell initiation signals, many patients exhibit primary or secondary resistance to immune checkpoint inhibitors, resulting in limited clinical response rates. Therefore, how to effectively induce potent and specific T cell responses against tumor antigens, especially CD8+, remains a key challenge. + Cytotoxic T lymphocyte (CTL) responses have become key to improving the response rate of immunotherapy.

[0003] Against this backdrop, cancer vaccines, as an active immunization strategy, aim to mimic the pathogen infection process, activate innate immunity, and initiate adaptive immunity, thereby inducing an anti-tumor immune response, and have significant clinical development value. Among these, cancer vaccines, as an active immunization strategy, aim to induce specific T-cell responses and achieve long-term immune memory.

[0004] Listeria monocytogenes (L. monocytogenes) is an intracellular parasite with the natural ability to activate innate and adaptive immunity, and can effectively induce CD8+. +T cell responses are crucial for eliminating tumor cells. L. monocytogenes' hemolysin O (LLO) helps it escape phagosomes and enter the cytoplasm, presenting antigens via the MHC class I pathway to achieve cellular immunity. To overcome the pathogenicity of wild-type Listeria, researchers have developed various attenuated strains, such as mutant strains lacking virulence genes like actA, inlB, and plcB, significantly reducing toxicity while retaining immunogenicity. However, most Listeria-based vaccines currently target only a single tumor-specific antigen, limiting their applicability; and most are concentrated in specific cancer types, lacking broad-spectrum anti-tumor capabilities. Furthermore, challenges remain regarding antigen expression stability, strain safety, and large-scale production. Tumor-associated antigens (TAAs) are specific molecules present on the surface or inside tumor cells, expressed at low levels or only at certain developmental stages in normal tissues. They possess advantages such as tumor targeting and broad-spectrum expression, making them a core target for immunotherapy. Tetanus toxoid (TT) is a non-toxic protein of tetanus toxin, and its Hc fragment is key to TT-induced long-term immune memory. Based on the above research background, there is an urgent need to develop a broad-spectrum vaccine that combines high safety, strong immunogenicity, specific targeting, antigen interchangeability, and broad applicability, in order to achieve efficient immune intervention for multiple tumor types. Summary of the Invention

[0005] Based on existing technologies, this invention uses wild-type Listeria monocytogenes (EGD-e) or attenuated Listeria monocytogenes (LADS) as the starting strain, and modifies the hly gene or a fragment thereof in the starting strain to include an LLO mutant (LLO). N478A+V479A ) and TT 856-1313 The polynucleotide sequence encoding the fusion protein was obtained, thus yielding the recombinant attenuated Listeria monocytogenes strain. This attenuated Listeria monocytogenes was derived from a wild-type Listeria monocytogenes strain whose endogenous hly gene (Locus tag: lmo0202; Gene ID: 987033) was mutated to express the LLO mutant (LLO). N478A+V479A The recombinant attenuated Listeria monocytogenes strain of this invention was obtained by means of [the specific method described]. Verification showed that, compared to wild-type Listeria monocytogenes, the growth activity of this recombinant attenuated Listeria monocytogenes strain remained largely unchanged, but its virulence was significantly reduced; it possessed cellular infectivity but exhibited biocompatibility in vivo and tended to colonize tumor tissue. Further in vivo experiments in mice revealed that the recombinant attenuated Listeria monocytogenes strain of this invention (e.g., in combination with tetanus toxoid protein (TT protein)) effectively inhibited the growth of cervical and colon cancer and significantly induced immune responses in the tumor microenvironment (e.g., significantly enhanced cytokine expression).

[0006] In this regard, the technical solutions of the present invention include, but are not limited to, the following: In one aspect, the present invention provides a recombinant attenuated Listeria monocytogenes, wherein the recombinant attenuated Listeria monocytogenes is formed by replacing the hly gene sequence or a fragment thereof of wild-type Listeria monocytogenes with a sequence containing LLO mutant and TT. 856-1313 The fusion protein is obtained by modifying the hly gene sequence of attenuated Listeria monocytogenes or a fragment thereof to include the LLO mutant and TT. 856-1313 The fusion protein is obtained by transcoding a polynucleotide sequence of the fusion protein to express and secrete the fusion protein, wherein the attenuated Listeria monocytogenes is obtained by mutating the hly gene in wild-type Listeria monocytogenes to express an LLO mutant, the LLO mutant having the following mutations compared to the wild-type LLO protein: N478A and V479A; the amino acid sequence of the wild-type LLO protein is shown in NCBI accession number NP_463733.1, and the TT... 856-1313 The amino acid sequence is shown in SEQ ID NO: 1.

[0007] In one aspect, the present invention also provides a method for preparing recombinant attenuated Listeria monocytogenes, comprising the following steps: (1) constructing a mixture containing LLO mutant and TT... 856-1313 Recombinant plasmids encoding the fusion protein or containing TT 856-1313 (2) Recombinant plasmids containing LLO mutant and TT from step (1); 856-1313 Recombinant plasmids encoding fusion protein sequences are introduced into competent wild-type or attenuated Listeria monocytogenes, or into bacteria containing TT. 856-1313 The recombinant plasmid encoding the protein coding sequence was introduced into attenuated Listeria monocytogenes; and (3) the bacteria obtained in step (2) with the recombinant plasmid introduced were subjected to homologous recombination hybridization culture and passaged until the plasmid was lost to obtain recombinant attenuated Listeria monocytogenes, wherein in step (3), the homologous recombination was carried out with LLO mutant and TT. 856-1313 The fusion protein coding sequence is a polynucleotide substitution of the hly gene sequence of wild-type Listeria monocytogenes or a mutated hly gene sequence of attenuated Listeria monocytogenes, or a fragment thereof; or contains TT. 856-1313 A protein-coding polynucleotide was inserted downstream of the hly gene sequence of a mutant *Listeria monocytogenes* to obtain expression and secretion of LLO mutants and TT mutants. 856-1313A recombinant attenuated Listeria monocytogenes fusion protein; the attenuated Listeria monocytogenes was obtained by mutating the hly gene in wild-type Listeria monocytogenes to express an LLO mutant; the LLO mutant has the following mutations compared to the wild-type LLO protein: N478A and V479A; the amino acid sequence of the wild-type LLO protein is shown in NCBI accession number NP_463733.1, and the TT... 856-1313 The amino acid sequence is shown in SEQ ID NO: 1.

[0008] In one aspect, in step (3) of the present invention, homologous recombination results in an LLO mutant and a TT mutant. 856-1313 The fusion protein coding sequence replaces the hly gene sequence of wild-type Listeria monocytogenes or the hly gene sequence of attenuated Listeria monocytogenes; or TT 856-1313 A protein-coding sequence was inserted downstream of the hly gene sequence of a mutant of attenuated Listeria monocytogenes to obtain expression and secretion of LLO mutants and TT mutants. 856-1313 Recombinant attenuated Listeria monocytogenes containing fusion protein.

[0009] In one aspect, in step (3) of the present invention, homologous recombination is performed as TT. 856-1313 A protein-coding sequence was inserted downstream of the hly gene sequence of a mutant of attenuated Listeria monocytogenes to obtain expression and secretion of LLO mutants and TT mutants. 856-1313 Recombinant attenuated Listeria monocytogenes containing fusion protein.

[0010] In one aspect, the wild-type Listeria monocytogenes of the present invention is an EGD-e strain; and / or, the recombinant attenuated Listeria monocytogenes is composed of a strain containing TT inserted downstream of a mutated hly gene sequence in attenuated Listeria monocytogenes. 856-1313 Obtained by polynucleotides of a protein-coding sequence; and / or, optionally, by LLO mutants and TT mutants in the fusion protein. 856-1313 There are joints between them, and preferably, the joint sequence is GGSGG.

[0011] In one aspect, the wild-type Listeria monocytogenes of the present invention is an EGD-e strain. Preferably, the EGD-e strain is designated ATCC BAA-679.

[0012] In one aspect, the amino acid sequence of the fusion protein of the present invention is shown in SEQ ID NO: 2.

[0013] In one aspect, the recombinant attenuated Listeria monocytogenes of the present invention is derived by modifying the hly gene of attenuated Listeria monocytogenes into a variant containing LLO mutant and TT. 856-1313 The modification was obtained by inserting a polynucleotide containing TT downstream of the hly gene of a fusion protein encoded by *Listeria monocytogenes*.856-1313 Polynucleotides that encode protein sequences.

[0014] In one aspect, the recombinant attenuated Listeria monocytogenes of the present invention is derived by modifying the hly gene of attenuated Listeria monocytogenes into a variant containing LLO mutant and TT. 856-1313 The fusion protein is obtained by expressing and secreting the fusion protein through a polynucleotide encoding sequence, wherein the attenuated Listeria monocytogenes is obtained by mutating the hly gene in wild-type Listeria monocytogenes to express an LLO mutant, the LLO mutant having the following mutations compared to the wild-type LLO protein: N478A and V479A; the amino acid sequence of the wild-type LLO protein is shown in NCBI accession number NP_463733.1, and the TT 856-1313 The amino acid sequence is shown in SEQ ID NO: 1. The modification refers to inserting a gene containing TT downstream of the *hly* gene of a mutated *Listeria monocytogenes*. 856-1313 Polynucleotides that encode protein sequences.

[0015] In one aspect, the LLO mutant of the present invention has the following mutations compared to the wild-type LLO protein: N478A and V479A.

[0016] In one aspect, the coding sequence of the fusion protein of the present invention is shown in SEQ ID NO: 3.

[0017] In one aspect, in step (1) of the present invention, suitable primers are designed for PCR amplification to obtain a sample containing the LLO mutant and TT. 856-1313 The coding sequence of the fusion protein is a polynucleotide, and it will contain LLO mutants and TT. 856-1313 The polynucleotide coding sequence of the fusion protein is transferred into a plasmid to obtain a recombinant plasmid, or a plasmid containing TT. 856-1313 Polynucleotides encoding protein-coding sequences, and those containing TT 856-1313 The protein-coding sequence polynucleotides are transferred into a plasmid to obtain a recombinant plasmid; and / or, in step (2), the plasmid containing LLO mutant and TT is transferred under the conditions of 2000 V-2500 V voltage, resistance above 0 Ω, and power of 25 μF. 856-1313 Recombinant plasmids encoding fusion protein sequences were electroporated into competent wild-type or attenuated Listeria monocytogenes, or plasmids containing TT were transduced into the bacteria. 856-1313 The recombinant plasmid encoding the protein sequence is electroporated into competent attenuated Listeria monocytogenes; and / or, in step (3), the wild-type or attenuated Listeria monocytogenes with the recombinant plasmid introduced in step (2) is subjected to homologous recombination exchange at a constant temperature of 41.5℃-42.5℃ to obtain recombinant attenuated Listeria monocytogenes, and the recombinant attenuated Listeria monocytogenes is passaged until the plasmid is lost.

[0018] In one aspect, in step (1) of the present invention, the plasmid is selected from pKSV7, pAUL-A, pLSV, pIMK, or pPL plasmids. Preferably, the plasmid is pKSV7 plasmid.

[0019] In one aspect, in step (1) of the present invention, suitable primers are designed for PCR amplification to obtain a sample containing TT. 856-1313 Polynucleotides encoding protein-coding sequences, and those containing TT 856-1313 Polynucleotides encoding protein sequences are transferred into plasmids to obtain recombinant plasmids.

[0020] In one aspect, in step (1) of the present invention, using the genomic sequence of attenuated Listeria monocytogenes as a template, the LLO mutant coding sequence fragment is amplified using primers shown in SEQ ID NO: 10 and 11 as the upstream homologous arm; the downstream sequence fragment of the hly gene is amplified using primers shown in SEQ ID NO: 12 and 13 as the downstream homologous arm; and using the TT gene sequence as a template, the TT gene is amplified using primers shown in SEQ ID NO: 14 and 15 as the downstream homologous arm. 856-1313 Protein coding sequence, and upstream homologous arm, TT 856-1313 The protein-coding sequence and downstream homologous arms are sequentially linked together to obtain the target fragment, which is then transferred into a plasmid to obtain a recombinant plasmid.

[0021] In one aspect, in step (2) of the present invention, the component containing TT is subjected to conditions of 2500 V voltage, 200 Ω resistance, and 25 μF power. 856-1313 The recombinant plasmid containing the protein-coding sequence was electroporated into competent attenuated Listeria monocytogenes.

[0022] In one aspect, in step (3) of the present invention, the attenuated Listeria monocytogenes introduced in step (2) is subjected to homologous recombination exchange at a constant temperature of 42°C to obtain recombinant attenuated Listeria monocytogenes, and the recombinant attenuated Listeria monocytogenes is passaged until the plasmid is lost.

[0023] In one aspect, the attenuated Listeria of the present invention may be, for example, a strain constructed by the method disclosed in Chinese patent application CN111269868A, in which the endogenous hly gene is mutated to express an LLO mutant (compared to the wild-type LLO protein, the asparagine at position 478 and the valine at position 479 in the LLO mutant are mutated to alanine, LLO...). N478A+V479A Preferably, the attenuated Listeria strain has the accession number CGMCC 18647, and the depository is the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postcode 100101).

[0024] In one aspect, in step (1) of the present invention, PCR technology (reaction system and reaction procedure are shown in Tables 1 and 2 respectively) is used to amplify the 498bp coding sequence (with the hly stop codon removed) near the 3' end of the mutant hly gene of the attenuated strain LADS genome (the LLO protein mutant it encodes contains amino acid mutation sites N478A and V479A) as the upstream homologous arm, and LLO down homoarm-Pst Ⅰ-fwd / LLO down homoarm-rev primers are used to amplify the downstream 500bp of the hly gene as the downstream homologous arm, and then TT856-1313 is amplified using pSL119 TT-fwd / pSL119 TT-rev primers; SOE-PCR technology (reaction system is shown in Table 3, procedure is shown in Table 4) is used to amplify the 498bp coding sequence (with the hly stop codon removed) of the mutant hly gene of the attenuated strain LADS genome as the upstream homologous arm, and LLO down homoarm-Pst Ⅰ-fwd / L ... The I-fwd primer binds the hly gene coding sequence fragment with the upstream homologous arm to the TT primer. 856-1313 The coding sequence fragment and downstream homologous arm were linked together to obtain the target fragment. The target fragment was introduced into the shuttle plasmid pKSV7. The reaction system is shown in Table 4. The reaction was carried out in a 37°C metal bath for 2-3 h. Enzyme linking was performed according to the enzyme linking system (Table 5) under the conditions of a 16°C metal bath for at least 40 min.

[0025] In one aspect, in step (2) of the present invention, recombinant plasmid DNA is extracted, and 500 ng of plasmid is electroporated and transformed into LADS electrocompetent cells (parameters: 2 mm electroporation cuvette, 2500 V, 200 Ω, 25 μF). The transformation product is added to 1 mL of preheated BHI resuscitation medium, and cultured at 37°C with gentle shaking for 3 h. The culture is then plated on ampicillin-resistant plates, and positive clones are picked for colony PCR verification. The PCR products are initially screened by 1% agarose gel electrophoresis. PCR products with the target band consistent with the expected sequence are selected for sequencing. The sequencing data are compared with the reference sequence using SnapGene software. After confirming that the reading frame is correct and there is no frameshift mutation, the recombinant plasmid is determined to have been successfully transformed into the LADS strain.

[0026] In one aspect, in step (3) of the present invention, the positive clone recombinant attenuated strain obtained by electroporation is inoculated into BHI liquid medium containing chloramphenicol (10 μg / mL) and cultured in a shaker at 37°C (220 rpm) for 2 h. Then it is transferred to a constant temperature shaker at 42°C for continuous subculture (fresh medium is transferred at a ratio of 1:100 every 12 h). After 10 generations of screening, the 10th generation bacterial culture is taken for PCR verification. The verified strain is transferred to antibiotic-free BHI medium and subcultured 10 times at a constant temperature of 30°C to further accelerate the plasmid loss process. Clones that show no colony growth in chloramphenicol-resistant solid medium and normal growth in antibiotic-free solid medium are judged as having successfully lost plasmids. Sequencing is used to screen the target recombinant attenuated Listeria monocytogenes.

[0027] In one aspect, the present invention provides a recombinant attenuated Listeria monocytogenes prepared by the method described in the present invention.

[0028] In another aspect, the present invention provides a biological vaccine against cervical cancer and / or colon cancer, comprising the recombinant attenuated Listeria monocytogenes described herein.

[0029] In another aspect, the present invention provides the use of the recombinant attenuated Listeria monocytogenes or the biological vaccine of the present invention in the preparation of a medicament for treating cervical cancer or colon cancer or inhibiting the growth of cervical cancer or colon cancer tumors.

[0030] In another aspect, the present invention provides a method for treating or inhibiting the growth of cervical or colon cancer tumors, the method comprising: administering to a subject the recombinant attenuated Listeria monocytogenes or the biological vaccine of the present invention.

[0031] In another aspect, the present invention provides the use of the recombinant attenuated Listeria monocytogenes or the biological vaccine of the present invention in the preparation of a medicament, wherein the medicament is used in combination with TT protein for the treatment of cervical cancer or colon cancer or for the inhibition of cervical cancer or colon cancer tumor growth.

[0032] In another aspect, the present invention provides a method for treating or inhibiting the growth of cervical or colon cancer tumors, the method comprising: administering to a subject in combination the recombinant attenuated Listeria monocytogenes described herein or the biological vaccine and TT protein described herein.

[0033] In another aspect, the present invention provides the use of the TT protein described in the present invention and the recombinant attenuated Listeria monocytogenes described in the present invention or the biological vaccine described in the present invention in the preparation of a medicament for treating cervical cancer or colon cancer or inhibiting the growth of cervical cancer or colon cancer tumors.

[0034] In one aspect, in the treatment method or use described in this invention, the subject is first given TT protein to induce an immune response, and then given the subject the recombinant attenuated Listeria monocytogenes or biological vaccine described in this invention.

[0035] In one aspect, the subject is a human or a mammal. Preferably, the mammal is a rodent, more preferably, the mammal is a mouse or a rat, and most preferably, the mammal is a mouse.

[0036] In one aspect, in the treatment methods or uses described in this invention, the drug, biological vaccine, recombinant attenuated Listeria monocytogenes, or TT protein described in this invention is administered intravenously.

[0037] In one aspect, the TT protein (tetanus toxoid protein) described in this invention is its full-length protein. Preferably, the amino acid sequence of the TT protein is shown in NCBI accession number X06214.1.

[0038] In one aspect, the gene sequence of the TT protein described in this invention is shown in NCBI accession number X06214.1.

[0039] The beneficial technical effects achieved by this invention include, but are not limited to: It has been verified that the recombinant attenuated Listeria monocytogenes strain of this invention does not exhibit significant changes in growth activity but its virulence is significantly reduced; it possesses cellular infectivity but exhibits biocompatibility in vivo and tends to colonize tumor tissues. Further in vivo experiments in mice have shown that the recombinant attenuated Listeria monocytogenes strain of this invention can effectively inhibit the growth of cervical and colon cancer and can significantly induce immune responses in the tumor microenvironment (e.g., significantly enhance cytokine expression). Therefore, this invention provides a multivalent tumor vaccine platform based on attenuated Listeria monocytogenes (LADS), which can flexibly carry different tumor-associated or protein antigens to achieve broad-spectrum immune intervention against multiple cancer types, while ensuring good biocompatibility and immunogenicity. Attached Figure Description

[0040] Figure 1. Construction strategy of recombinant strains and analysis of in vitro proliferation capacity and hemolytic activity, where (A) LADS-TT recombinant strain construction strategy; (B) growth curves of different strains in BHI liquid medium; (CD) detection of hemolytic activity of different strains.

[0041] Figure 2 Western blot detection of the expression of TT fragments of different lengths fused with LLO protein. Among them, the secretory and cytoplasmic proteins expressed by the recombinant strains of LADS-TT-L (A), LADS-TT-M (B), and LADS-TT-L (C) were verified by Western blot.

[0042] Figure 3. Virulence analysis of the LADS-TT recombinant strain, where (AB) shows the proliferation of the recombinant strain in RAW264.7 and BMDM macrophages; (CD) shows the intracellular bacterial load and cell death rate of RAW264.7 macrophages infected with the recombinant strain 6 h later, with Mock indicating the group not infected with any bacteria; (EG) shows the mouse survival test of the recombinant strain. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates no significant difference.

[0043] Figure 4. Survival time and tissue colonization of the LADS-TT recombinant strain in mouse blood and its effect on mouse body weight. (A) Bacterial load in blood at different time points; (BD) Bacterial load in liver (B), spleen (C), and tumor tissue (D) at different time points; (E) Changes in mouse body weight. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference.

[0044] Figure 5. Immunotherapy efficacy of LADS-TT recombinant strain against cervical cancer-bearing mice and cytokine levels in mouse tumor tissues. (A) Immunization procedure; (B) TT-specific IgG antibody level detection; (CD) Spleen size and weight; (E) Tumor size changes; (F) mRNA transcription levels of IL-2, IL-12, and IFN-γ genes in mouse tumor tissues. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference.

[0045] Figure 6. Immunotherapy efficacy of LADS-TT-L recombinant strain against cervical cancer-bearing mice. (A) Immunization procedure; (B) Detection of TT-specific IgG antibody levels; (CD) Spleen size and weight; (E) Tumor size changes.

[0046] Figure 7. Immunotherapy efficacy of LADS-TT-L recombinant strain in colon cancer-bearing mice and flow cytometry analysis of activated T cell immune responses. (A) Immunization procedure; (B) TT-specific IgG antibody level detection; (C) Tumor size changes; (D) CD4+ in splenic lymphocytes of mice immunized with LADS and LADS-TT-L. + and CD8 + Flow cytometry results of T cells; (E) CD4 in each group + and CD8 + Statistical analysis of the proportion of T cells in spleen lymphocytes. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates no significant difference. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] This application will encode the LLO protein mutant (LLO) N478A+V479A ) and non-toxic tetanus toxoid protein fragment (TT) 856-1313 The nucleic acid sequence of the fusion protein was cloned into an expression vector containing the hly promoter to construct a recombinant plasmid. This plasmid was then electroporated into competent cells of attenuated Listeria LADS strain, plated on chloramphenicol-containing BHI solid medium plates, and incubated at 37°C for 24 to 48 hours. Single colonies were picked and inoculated into chloramphenicol-containing liquid BHI medium, and cultured for 10 generations to stabilize the plasmid. The bacterial culture was then transferred at a 1:100 ratio to antibiotic-free liquid BHI medium and cultured for another 10 generations to simulate genetic stability under in vivo non-selective pressure. Finally, bacterial genomic DNA was extracted and sequenced to confirm that the antigen gene sequence was intact and without mutations, thus completing the construction of the recombinant LADS-TT vaccine strain.

[0050] Some reagent formulations mentioned in the specific embodiments of this application: The mice used in the following examples were purchased from Zhejiang Academy of Medical Sciences; the study used the mouse lung epithelial TC-1 cell line immortalized with the human papillomavirus 16 (HPV16) E6 / E7 oncogene, which was purchased from the Cancer Cell Center of the Chinese Academy of Medical Sciences and routinely cultured in RPMI 1640 medium (containing 10% FBS) and passaged in a 37°C, 5% CO2 incubator. The CT-26 mouse (Mus musculus) colon adenocarcinoma cell line, derived from Balb / c mice, was purchased from the Cancer Cell Center of the Chinese Academy of Medical Sciences. The cells were cultured in RPMI 1640 (containing 10% fetal bovine serum, FBS) and passaged in a 37°C, 5% CO2 incubator.

[0051] Fetal bovine serum, RPMI 1640 medium, and CO2 cell incubator were purchased from Thermo Fisher Scientific.

[0052] BHI culture medium was purchased from Oxoid, UK.

[0053] The wild-type Listeria monocytogenes strain EGD-e (ATCC BAA-679) was purchased from the ATCC standard strain and can be obtained by the public in accordance with its regulations.

[0054] Example 1, Construction and safety evaluation of LADS-TT recombinant strain 1.1 Attenuated Listeria monocytogenes LADS was constructed according to the method disclosed in Chinese patent application CN111269868A, and genomic DNA was extracted.

[0055] 1.2 Construction of recombinant plasmids: Based on homologous recombination technology, using the temperature-sensitive shuttle plasmid pKSV7 as a vector, the non-toxic tetanus toxoid protein (TT) was transported into the plasmid. 856-1313 ) and its truncated body TT 1042-1171 TT 1115-1315 Directed fusion into the C-terminal domain of the LLO protein mutant of attenuated Listeria monocytogenes LADS strain, including the construction of the recombinant plasmid pSL119 (pKSV7-LLO mutant-TT). 856-1313 pSL120 (pKSV7-LLO mutant-TT) 1042-1171 and pSL124 (pKSV7-LLO mutant-TT) 1115-1315 The recombinant plasmid was then introduced into attenuated Listeria monocytogenes, and the mutated hly gene in the attenuated Listeria monocytogenes was modified using homologous recombination technology so that the modified strain expressed the LLO mutant-TT. 856-1313 LLO mutant-TT 1042-1171 Or LLO mutant-TT 1115-1315 Fusion protein.

[0056] The main steps specifically include: (1) Using PCR technology (the reaction system and reaction procedure are shown in Table 1 and Table 2 respectively), the 498 bp coding sequence (with the hly stop codon removed) of the mutant hly gene (which encodes an LLO protein mutant containing amino acid mutation sites N478A and V479A) of the attenuated strain LADS genome was amplified using LLO up homoarmBamH Ⅰ-fwd / LLO up homoarm-rev primers as the upstream homologous arm, and the downstream 500 bp of the hly gene was cloned using LLO down homoarm-Pst Ⅰ-fwd / LLO down homoarm-rev primers as the downstream homologous arm. Then, the TT gene was amplified using pSL119 TT-fwd / pSL119 TT-rev, pSL120 TT-fwd / pSL120 TT-rev, and pSL124TT-fwd / pSL124 TT-rev primers respectively. 856-1313TT 1042-1171 and TT 1115-1315 Protein-coding fragments. The amplified PCR products were then purified.

[0057] (2) Using SOE-PCR technology (reaction system shown in Table 3, procedure shown in Table 4), the Hly gene coding sequence fragment with the upstream homologous arm was coupled with the truncated TT protein (TT) using LLO up homoarmBamH Ⅰ-fwd / LLO down homoarm-PstⅠ-fwd primers. 856-1313 TT 1042-1171 and TT 1115-1315 The target fragment is obtained by connecting the encoded sequence fragment and the downstream homologous arm together.

[0058] Table 1 PCR reaction system

[0059] Note: Primer-A and Primer-B refer to LLO up homoarmBamHⅠ-fwd and LLO up homoarm-rev, LLO down homoarm-PstⅠ-fwd and LLO down homoarm-rev, pSL119 TT-fwd and pSL119 TT-rev, pSL120 TT-fwd and pSL120 TT-rev, or pSL124TT-fwd and pSL124 TT-rev.

[0060] Table 2 PCR reaction procedure

[0061] Table 3 SOE-PCR Reaction System

[0062] (3) Using BamH Ⅰ and PstⅠ as restriction enzyme sites, the target fragment and Listeria shuttle plasmid pKSV7 were digested. The reaction system is shown in Table 4. The reaction was carried out in a metal bath at 37℃ for 2-3 h. Table 4 Double restriction enzyme digestion system

[0063] (4) After enzyme digestion, purification is performed in the same manner. Enzyme linking is carried out according to the enzyme linking system (Table 5) under the following conditions: 16℃ metal bath, at least 40 min, or overnight; Table 5 Enzyme linking system

[0064] (5) Mix the enzyme-linked product with DH5α competent cells, place in an ice box for 30 min, then in a 42℃ metal bath for 90 s, then in an ice box for 2 min. After that, add 800-1000 μL of LB liquid medium to the mixture, shake and incubate at 37℃ for 50-60 min, centrifuge at 6000 rpm at room temperature for 5 min; discard part of the supernatant so that the remaining liquid is about 150 μL, mix by pipetting and then spread on a spreader onto LB solid medium with ampicillin resistance and incubate at a constant temperature of 37℃ and 200 rpm; (6) After heat shock transformation, screen positive clones into LB liquid medium containing ampicillin (100 μg / mL), and incubate at 37℃ in a shaker (220 rpm) for 12-16 h. Take 1 μL of bacterial solution as a template for colony PCR verification. The primers are covering LLO CDS-fwd / covering LLO CDS-rev. After verifying that the amplified fragments were the same size as expected by 1% agarose gel electrophoresis, they were sent to a sequencing company for paired-end sequencing. The sequencing results were then used for multiple sequence alignment using SnapGene software, which confirmed that the inserted fragment reading frames were intact and without frameshift mutations, indicating that the recombinant plasmids pSL119, pSL120, and pSL124 were successfully constructed.

[0065] 1.3. Recombinant Plasmid Transformation into Attenuated Strains: Recombinant plasmids pSL119, pSL120, and pSL124 (500 ng) were transformed into competent attenuated Listeria monocytogenes LADS via electroporation (parameters: 2 mm electroporation cuvette, 2500 V, 200 Ω, 25 μF). The transformation products were added to 1 mL of preheated BHI resuscitation medium and incubated at 37°C with gentle shaking for 3 h before being plated on ampicillin-resistant plates. Positive clones were picked and colony PCR was performed using Covering LLO CDS-fwd / Covering LLO CDS-rev primers. PCR products were initially screened by 1% agarose gel electrophoresis. PCR products with the expected target band were sequenced. Sequencing data were compared with reference sequences using SnapGene software. After confirming the correct reading frame and the absence of frameshift mutations, the recombinant plasmids were considered successfully transformed into the LADS strain. Positive clones were inoculated into BHI preservation medium containing 15% glycerol and stored at -80°C for later use.

[0066] 1.4 Screening and Validation of Recombinant Attenuated Strains: Positive clones obtained by electroporation were inoculated into BHI liquid medium containing chloramphenicol (10 μg / mL) and cultured at 37°C (220 rpm) for 2 h on a shaker. Subsequently, they were transferred to a 42°C constant-temperature shaker for continuous subculturing (fresh medium was inoculated at a 1:100 ratio every 12 h). After 10 generations of screening, the 10th generation culture was used for PCR validation. Validated strains were transferred to antibiotic-free BHI medium and subcultured 10 times at 30°C to further accelerate plasmid loss. After plasmid loss, the colonies were plated on antibiotic-free BHI solid medium and cultured at 37°C to obtain single colonies. These colonies were inoculated in parallel onto chloramphenicol-resistant solid medium and antibiotic-free solid medium, and cultured at 42°C for 48 h for antibiotic screening. The screening criteria were: clones showing no colony growth on chloramphenicol-resistant solid medium and normal growth on antibiotic-free solid medium were considered to have successfully lost plasmids. The final candidate strain was verified by colony PCR using a flanking primer covering LLOCDS-fwd / covering LLO CDS-rev. Positive samples were sent to a biotechnology company for sequencing. After successful alignment of the sequencing data with the reference sequence using SnapGene software, the recombinant strain LADS-TT-L (expressing the LLO mutant-TT) was identified. 856-1313 fusion protein), LADS-TT-M (expressing LLO mutant-TT) 1042-1171 fusion protein) and LADS-TT-S (expressing LLO mutant-TT) 1115-1315 The fusion protein was successfully constructed and can be used for subsequent in vivo and in vitro functional studies (Figure 1A).

[0067] 1.5 Growth Curve Determination of Recombinant Strains The in vitro proliferation capacity of wild-type EGD-e, attenuated LADS, and recombinant strains LADS-TT-L, LADS-TT-M, and LADS-TT-S was analyzed in a biosafety cabinet. The specific procedure is as follows: The bacterial strains were revived and preserved in glycerol at -80℃, and streaked into BHI solid medium to obtain single colonies. Single colonies were picked and transferred to 5 mL of BHI liquid medium for overnight incubation. The culture was washed twice with 10 mM PBS (5500 rpm, 3 min), and the OD was adjusted. 600nm =0.6, inoculated into BHI liquid medium at a ratio of 1:50, and the absorbance at 600 nm was measured every 1 hour using a multi-functional microplate reader for 12 hours. Finally, a growth curve was plotted based on the experimental data.

[0068] As shown in Figure 1B, the growth curves of the recombinant strains LADS-TT-L, LADS-TT-M, and LADS-TT-S were not significantly different from those of the wild-type strain EGD-e and the attenuated strain LADS, all exhibiting typical bacterial growth curve characteristics. These results indicate that the insertion of TT antigen of different lengths does not affect the in vitro growth ability of the recombinant strains. This provides an important experimental basis for the subsequent application of the recombinant strains in in vivo infection models and their immunogenicity evaluation.

[0069] 1.6. Determination of hemolytic activity of secreted proteins: Prepare a 5% sheep red blood cell suspension, pick a single colony and inoculate it into 5 mL of BHI liquid medium, and culture it in a shaker at 37℃ (200 rpm) for 12-16 h; take 2 mL of bacterial suspension and centrifuge at 5600 rpm for 10 min, and collect the supernatant; incubate the supernatant with PBS containing 1 mM DTT (DTT:PBS = 1:1000) in a 37℃ incubator for 10 min.

[0070] Using a 96-well U-plate, add 200 μL of mixed supernatant to the first well and serially dilute using a multichannel pipette (100 μL / well). Then, add 100 μL of 5% sheep red blood cell suspension (final volume 200 μL / well) sequentially from the back to the front of the 96-well U-plate and incubate at 37°C for 30 min. Finally, centrifuge at 1000 rpm (20°C) for 10 min to form a pellet, and scan the 96-well U-plate to record hemolysis. Transfer 150 μL of supernatant to a flat-bottomed 96-well plate and measure OD. 550nm And calculate the hemolytic activity.

[0071] As shown in Figure 1C, the Δhly deletion strain (loss of hly gene function) completely lost its hemolytic activity, verifying that the hly-edited LLO is the core virulence factor mediating the hemolytic activity of Listeria monocytogenes, further confirming the close relationship between LLO and virulence. The hemolytic activity of the attenuated strain LADS was significantly lower than that of EGD-e, indicating that the LLO mutant, which altered key sites of the virulence gene, effectively weakened the cytotoxicity of LLO while retaining some of its immunostimulatory capacity, achieving a balance between reduced virulence and antigen presentation function. Hemolytic activity analysis of the recombinant strains LADS-TT-L, LADS-TT-M, and LADS-TT-S showed that their hemolytic ability was significantly lower than that of the wild-type strains EGD-e and LADS, also indicating that LADS-TT has good biocompatibility as a vaccine.

[0072] 1.7 Secretory Expression of Fusion Proteins To ensure the stable delivery of TT antigen by recombinant strains LADS-TT-L, LADS-TT-M, and LADS-TT-S, a fusion expression system was constructed using the secretory hemolysin LLO (encoded by the hly gene, theoretical molecular weight 58.8 kDa). Secretory and cytoplasmic proteins from the logarithmic growth phases of the recombinant strains were collected and validated by Western blot. The results are shown in Figure 2A: the secretory and cytoplasmic protein sizes of LADS-TT-L were 111.1 kDa, consistent with the expected protein size. The results are shown in Figure 2B: the secretory and cytoplasmic protein sizes of LADS-TT-M were 82.7 kDa, consistent with the expected protein size. The results are shown in Figure 2C: the secretory and cytoplasmic protein sizes of LADS-TT-S were 73 kDa, consistent with the expected protein size. These results indicate that the designed TT antigen sequences of different lengths were successfully fused with LLO and stably expressed in the strains. This not only verifies the correctness of the fusion expression vector construction but also demonstrates that the LLO-mediated protein secretion mechanism maintains effective function in the recombinant strains.

[0073] Example 2. Safety Evaluation of LADS-TT Recombinant Strains 2.1. Proliferation of LADS-TT Recombinant Strains in Cells Mouse macrophages RAW264.7 and BMDM were cultured in complete medium (DMEM containing 10% FBS) at 37°C and 5% CO2. Six hours after cell infection, the culture supernatant was collected, washed with PBS, scraped off, centrifuged, and a cell suspension was prepared. After appropriate dilution, 0.4% trypan blue solution (final concentration 0.04%) was added, and staining and cell counting were completed within 3 minutes. The number of live and dead cells was counted, and the cell death rate was calculated to evaluate the cytotoxicity of different strains to macrophages.

[0074] Intracellular proliferation assays of macrophages (Figures 3A-B) showed that the recombinant strain had weaker proliferation capacity in RAW264.7 and BMDM compared to the wild-type strain EGD-e, indicating that the vaccinated strain would not proliferate excessively in organs and pose a danger to the body, thus demonstrating its safety. Further investigation of Listeria monocytogenes infectivity was conducted using the RAW264.7 in vitro cell model. The results, shown in Figures 3C-D, indicated that the recombinant strain showed significantly lower infection and mortality rates 6 hours after infection compared to the wild-type strain, but no significant difference compared to the attenuated strain LADS. This suggests that LADS is a qualified vaccine vector with low toxicity, and that the insertion of exogenous antigens does not significantly affect the infectivity of Listeria monocytogenes.

[0075] 2.2 Construction of the Mouse Tumor-Bearing Model: The complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum) and PBS were preheated to 37°C. The supernatant in the TC-1 cell culture flask was discarded, and 5 mL of preheated PBS was added to gently wash the cells twice to remove residual serum components. Then, 2 mL of 0.25% Trypsin-EDTA was added to cover the cell layer, and the cells were digested in a 37°C incubator for approximately 1.5 min. During this time, changes in the intercellular spaces were observed using an inverted microscope. When the intercellular connections became loose, the bottom of the culture flask was gently tapped to help the cells detach. After initial detachment, 3 times the volume of preheated complete culture medium was added to stop the digestion reaction, and the cells were gently pipetted to fully disperse them into a single-cell suspension. 10 μL of the sample was used for cell counting, and the remaining cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 700 rpm for 3 min. The supernatant was discarded. The precipitated cells were resuspended in fresh, preheated culture medium, and the cell concentration was adjusted to 4 × 10⁶ cells / mL. 6 cells / mL. Inject 2 × 10 cells / mL per mouse. 5 SPF-grade C57BL / 6 female mice were immunized with a dose of 1 cell via subcutaneous injection in the abdomen. The injection site was then monitored regularly, and the formation of 1-3 mm tumor nodules was confirmed by palpation, at which point the cervical cancer tumor-bearing model was considered successfully established.

[0076] 2.3. Mouse immunization program (1) When the tumor volume of the mouse cervical cancer model reaches 50 mm 3 LADS, LADS-TT-L, LADS-TT-M and LADS-TT-S were revived from glycerol-preserved strains at -80℃ and streaked into BHI solid medium. Typical single colonies were picked and transferred to 6 mL of BHI liquid medium and incubated at 37℃. (2) Centrifuge at 5500 rpm for 4 min, discard the supernatant in a clean bench and collect the bacterial cells. (3) Resuspend the bacterial cells in sterile PBS, centrifuge at 5500 rpm for 4 min, discard the supernatant, and wash twice with the same parameters. (4) Resuspend in 1 mL of bacterial solution and measure the OD of the bacterial solution using a multi-functional microplate reader. 600nm Value, adjusted to OD using PBS 600nm = 0.6. Take 100 μL of bacterial culture and perform serial dilutions (10... -1 Up to 10 -6 After shaking and mixing, each dilution was inoculated into BHI, and cultured at 37℃ for 24 h. The colony data were then recorded. (5) Tumor-bearing mice were injected via the tail vein using a 1 mL syringe. The immunization dose was 100 μL / mouse (i.e., the injection volume of bacteria was 10 μL). 8(6) Experimental groups (n=3 / group): LADS group, LADS-TT-L group, LADS-TT-M group and LADS-TT-S group (bacterial concentration 1×10⁻⁶ CFU / animal); (7) Experimental groups (n=3 / group): LADS group, LADS-TT-L group, LADS-TT-M group and LADS-TT-S group (bacterial concentration 1×10⁻⁶ CFU / animal); (8) Experimental groups (n=3 / group): LADS group, LADS-TT-L group, LADS-TT-M group and LADS-TT-S group (bacterial concentration 1×10⁻⁶ CFU / animal); (9) Experimental groups (n=3 / group): LADS group, LADS-TT-L group, LADS-TT-M group and LADS-TT-S group (bacterial concentration 1×1 8 (7) On the 7th day after the initial immunization (Day 7), the experimental group was immunized a second time using the same method and dose.

[0077] 2.4 Virulence detection of LADS-TT recombinant strains: Following the experimental procedure in 2.2, recombinant strains LADS-TT-L, LADS-TT-M, and LADS-TT-S were cultured in BHI liquid medium. Different concentration gradients of recombinant strains were inoculated into mice via intraperitoneal injection. Each mouse was inoculated with 100 μL of bacterial solution, and the survival of the mice was observed for 7 days after inoculation.

[0078] The results are shown in Figures 3E-G. When the challenge dose per mouse was 10... 9 At a concentration of CFU / mL bacterial culture, all mice challenged with the recombinant strain survived; when the challenge dose was 10... 9.7 At a concentration of CFU / mL, the survival rate of LADS-TT-L was 50%, while that of LADS-TT-M and LADS-TT-S was 60%. The LD50 of the recombinant strains LADS-TT-L, LADS-TT-M, and LADS-TT-S was calculated. 50 The data are 1×10 9.7 1×10 9.77 1×10 9.77 CFU, compared to 1×10⁻⁶ for wild-type EGD-e as determined in previous studies. 5.25 The virulence of LADS-TT-L was significantly lower than that of LADS compared to wild-type strains, and the virulence was reduced by approximately 4.3 times compared to LADS. These results indicate that the virulence of the recombinant strain was significantly lower than that of the wild-type strain, and also weaker compared to the attenuated strain.

[0079] 2.5 Distribution of LADS-TT recombinant strains in tissues (1) On days 1, 3 and 7 after immunization, liver, spleen and tumor tissue of tumor-bearing mice (n=3 / group) were removed using sterile instruments in a sterile laminar flow hood to ensure that cross-contamination was avoided during the operation; (2) The collected tissue samples were transferred to 2 mL EP tubes (containing sterile grinding steel balls) and the tissues were thoroughly ground in a grinder. The centrifuge tubes were kept on ice throughout the operation; (3) 100 μL of the grinding solution was added to 900 μL of sterile PBS, vortexed and mixed, and then serially diluted. All dilutions were handled with sterile pipettes to avoid contamination; (4) The diluted tissue solution was taken and counted on BHI solid medium. It was incubated at 37℃ for 18-24 h. After the incubation was completed, the colony data were recorded.

[0080] As shown in Figure 4A, 5 minutes after intravenous injection of the recombinant strain, the bacterial load in the peripheral blood of mice in all experimental groups reached its peak, indicating that the strain rapidly entered the bloodstream and began to proliferate within a short period. Over time, the number of bacteria in the blood gradually decreased, until 48 hours later, when the bacterial load in the LADS-TT-S experimental group reached the limit of detection, indicating that the recombinant strain had a short survival time in the blood after tail vein injection. Furthermore, the bacterial load in other groups also decreased significantly within 48 hours, but the clearance rate varied among different strains. As shown in Figures 4B-C, on day 1 after intravenous injection, the bacterial load in the liver and spleen reached its peak, suggesting that the recombinant strain initially accumulated in immune organs after injection. Over time, the bacterial load in the liver and spleen decreased significantly on day 3. By day 7, the bacteria in the liver and spleen were completely cleared, indicating that the strain could be effectively cleared by the immune system in normal tissues. In contrast, the bacterial load in tumor tissue gradually increased over time (Figure 4D), indicating that *Listeria monocytogenes* can clear the virus quickly while maintaining a certain level of biosafety, and can also colonize tumor tissue.

[0081] 2.6 Mouse Weight Measurement: Twenty-four hours before tumor cell inoculation, the initial weight of SPF-grade C57BL / 6 mice in each group was measured using a precision analytical balance, and the data were recorded. After inoculation, mouse weight was continuously measured and recorded every 24 hours to observe physiological changes. All weight data were statistically analyzed using GraphPad Prism software, and weight change curves were plotted to aid in comparing differences between groups. The results are shown in Figure 4E. There was no statistically significant difference in weight change among the groups, indicating that the recombinant strains in different treatment groups did not cause significant weight changes.

[0082] Example 3: Evaluation of the therapeutic effect of LADS-TT based on a mouse cervical cancer model. 3.1 Construction of protein expression plasmid. Using the TT gene (NCBI accession number X06214.1) in the laboratory as a template, upstream and downstream primers pSL386-KpnⅠ-fwd / pSL386-BamHⅠ-rev were designed on both sides of the TT gene using Snapgene software. Restriction sites were added to both ends of the primers. TT was amplified by PCR using these primers and purified. Using the prokaryotic protein expression system pET32a as a vector, the plasmid was heat-transferred into E. coli DH5α competent cells and PCR verification was performed using the pET32a-T7-fwd / pET32a-T7ter-rev primers. After successful verification, samples of the expected size were sent for testing. The successfully sequenced plasmid was named pSL386.

[0083] 3.2 Expression and purification of prokaryotic proteins (1) The successfully constructed protein expression plasmid pSL386 was heat-transferred into BL21 competent cells. After transformation, resistance screening was performed using LB solid plates (ampicillin) and cultured overnight at 37°C. (2) The next day, a single positive clone was picked and inoculated into 5 mL of LB liquid medium containing ampicillin and cultured overnight at 37°C and 220 rpm with shaking. (3) 0.5 mM or 1 mM IPTG was added as an inducer and cultured for 12 h at 16°C and 160 rpm. (4) After induction, the supernatant and precipitate were collected by centrifugation at 5000 rpm for 10 min. The cells were disrupted using a cell disruptor and an SDS-PAGE test was performed to select the optimal induction conditions. (5) According to the optimal induction conditions, 5 mL of bacterial culture was transferred to 500 mL of LB liquid medium containing ampicillin and 1 mM IPTG was used as an inducer. Cultured for 12 h at 16°C and 160 rpm. h; (6) Centrifuge to collect bacterial cells, wash with pre-cooled PBS, resuspend the bacterial cells in 50 mL PBS, break the bacterial cells with a pressure breaker, centrifuge at 5000 rpm for 20 min to collect the supernatant; (7) Mix the supernatant with the pretreated nickel agar gel, and incubate overnight at 4°C with gentle inversion on a horizontal shaker; (8) Slowly pass the overnight protein lysis buffer through the chromatography column, and then perform gradient elution purification with 30 mM and 50 mM imidazole buffers until the nickel column turns sky blue; finally, elute the target protein with 300 mM imidazole; (9) Perform SDS-PAGE experiment. After electrophoresis, cut off the separating gel, add Coomassie Brilliant Blue R-250 staining solution to fully cover it, and stain for 2 h to develop the protein bands; (10) Use destaining solution containing 20% ​​methanol and 10% glacial acetic acid to change and elute multiple times until the background is clear and the protein bands are clearly visible.

[0084] 3.3 Immunization Procedure for Mice 3.3.1 Protein Immunization Procedure: The purified TT protein was adjusted to 2 µg / mL. The protein antigen and Freund's complete adjuvant were mixed at a 1:1 volume ratio for primary immunization. The protein antigen and Freund's incomplete adjuvant were mixed at a 1:1 volume ratio for booster immunization. The syringe was repeatedly injected until stable droplets formed. The skin on the back of the mouse's neck was lifted, and 100 µL / mouse was injected subcutaneously at multiple points. After immunization, the injection sites were observed for redness, swelling, or induration.

[0085] 3.3.2 Antibody level detection (1) The level of TT antibody in mice was determined by indirect ELISA, and the purified TT protein in 3.2 was used as the antigen; (2) The antigen was diluted with freshly prepared coating solution (0.75 g Na2CO3 and 1.46 g NaHCO3 were adjusted to 500 mL with ddH2O) to a final concentration of 5 µg / mL. (2) Antigen diluted 200 µL per well was added to a 96-well plate with a flat bottom. The plate was incubated at 37°C for 2 h. The coating solution was discarded, and the plate was washed 3 times with PBST, with horizontal shaking for 10 min each time. The plate was then patted dry. (3) Blocking: 200 µL of 5% skim milk powder was used for blocking. The plate was incubated at 37°C for 2 h. The blocking solution was discarded, and the plate was washed 3 times with PBST, with horizontal shaking for 10 min each time. The plate was then patted dry. (4) Primary antibody incubation: Mouse serum was diluted 1:100 with TBST. 100 µL of the diluted serum was added to each well of a 96-well plate with a flat bottom. The plate was incubated at 37°C for 1 h. The plate was washed 3 times with PBST, with horizontal shaking for 10 min each time. The plate was then patted dry. (5) Secondary antibody incubation: Goat anti-mouse IgG-HRP was diluted 1:1000 with TBST. 100 µL of the diluted secondary antibody was added to each well. The plate was incubated at 37°C for 1 h. The plate was washed 3 times with PBST, with horizontal shaking for 10 min each time. min, pat dry the liquid; (6) TMB color development: add 100 µL of TMB color development solution to each well, shake horizontally at 37℃ for 10 min, then add 100 µL of 2 M concentrated sulfuric acid to each well to stop the color development, and measure OD. 450 nm .

[0086] ELISA results showed that the level of specific IgG antibodies in the serum of mice immunized with TT protein was significantly higher than that in the control group 14 days after immunization (Figure 5B). This result indicates that TT protein induces a significant antigen-specific immune response.

[0087] 3.4 Construction of mouse cervical cancer model. The specific model construction method is described in 2.2 of Example 2.

[0088] 3.5. General Immunotherapy Procedure in Mice: In the cervical cancer tumor model study, mice were first immunized twice with tetanus toxoid protein (full-length TT protein) to induce pre-existing memory T cells. Subsequently, TC-1 tumor cells were subcutaneously implanted into the abdomen. When the tumor reached the early stage (1-3 mm), single colonies were picked and transferred to BHI for overnight culture; after washing twice with PBS, the bacterial culture was resuspended and the OD was adjusted. 600 nm =0.6. Recombinant or attenuated strains (10) were administered via tail vein injection. 8(CFU / mouse, 6 replicates per group) The recombinant or attenuated bacterial strain was injected again via tail vein one week later. Tumor size was measured every two days, and tumor tissue was isolated at 23-25 ​​days of immunotherapy. Immunotherapy was administered to tumor-bearing mice with pre-existing memory T cells via tail vein injection (Figure 5A) to evaluate the immunotherapy efficacy of the recombinant strain. Tumor growth was significantly slowed in the LADS-TT-L immunized group (Figure 5E), suggesting its potential to inhibit tumor progression.

[0089] Furthermore, as shown in Figures 5C-D, the spleen weight and volume of the immunized mice were significantly increased (the spleen of a normal adult mouse is approximately 0.08-0.12 g), indicating significant lymphocyte proliferation. In summary, LADS-TT-L exhibited certain anti-tumor effects in tumor-bearing models and has the potential to be a candidate tumor vaccine. TT, as an antigen with abundant antigenic epitopes, contains multiple T cell and B cell epitopes. However, LADS-TT-M and LADS-TT-S, which present truncated antigens, may fail to present key epitopes due to the absence of these epitopes, thus failing to activate effective memory T cells.

[0090] The immunotherapy program was optimized by administering a high-dose injection of the recombinant strain followed by a daily booster dose of a low-dose recombinant strain vaccine (10 mg / day). 5 (CFU / mouse, 10 doses in total). The immunization strategy is shown in Figure 6A. This optimized protocol aims to enhance the persistence and stability of the immune response by increasing the number of antigen exposures. Optimizing the immunization procedure not only helps to enhance the activation of naïve T cells, but may also further improve the expansion and persistence of effector T cells, enhancing their ability to recognize and eliminate tumor cells. ELISA results showed that the level of specific antibodies in the serum of mice immunized with tetanus toxoid 14 days later was significantly higher than that in the control group (Figure 6B), confirming that TT successfully induced an antigen-specific immune response. Further, the optimized immunization procedure was administered to tumor-bearing mice with pre-existing memory T cells via tail vein injection. The results showed that the tumor volume growth rate in the LADS-TT-L immunized group was significantly lower than that in the control group (Figure 6E), indicating that the optimized immunization procedure can effectively inhibit tumor growth. Notably, the spleen weight of the immunized group mice was significantly increased compared to normal adult mice (0.08-0.12 g) (Figures 6C-D), and the spleen volume was also significantly increased. As a secondary lymphoid organ, changes in the size and weight of the spleen can often serve as an important indicator of the degree of immune activation. An increase in spleen volume indicates active proliferation of lymphocytes and indirectly reflects the promoting effect of vaccines on systemic immune responses.

[0091] Furthermore, the immunized mice were in good mental condition, with no significant weight loss or behavioral abnormalities, further validating the good safety and feasibility of the optimized protocol. In summary, the LADS-TT-L recombinant vaccine with the optimized immunization schedule demonstrated good anti-tumor efficacy and systemic immune activation in a cervical cancer-bearing mouse model, providing valuable reference for the design of subsequent vaccine immunization strategies.

[0092] 3.6 Cytokine Detection 3.6.1 RNA Extraction and Reverse Transcription Assay RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2. The entire procedure was performed in a clean bench, using RNase-free pipette tips and consumables to prevent RNA degradation. After collecting tissue RNA, the quantitative RNA was reverse transcribed into cDNA using the HiScript IV All-in-One Ultra RT SuperMix for qPCR kit. The cDNA generated by reverse transcription was stored at -80℃ for long-term preservation.

[0093] 3.6.2 Quantitative Real-Time PCR Reaction The quantitative real-time primers for the cytokines detected in this experiment were designed based on relevant literature (see Table 6). The reaction system was prepared in qRT-PCR tubes according to the instructions. The experiment was conducted on ice in the dark, and the qRT-PCR experiment was performed using a quantitative real-time PCR instrument.

[0094] Tumors from mice that had completed the immunotherapy program were collected, and changes in the transcriptional levels of key cytokines were detected using real-time quantitative PCR. The results showed that the mRNA transcriptional levels of IL-2, IL-12, and IFN-γ in tumor tissues of the LADS-TT-L immunized group were significantly upregulated (Figure 5F), suggesting that immunotherapy significantly enhanced the immune response in the tumor microenvironment (TME), promoting the immune system's recognition and clearance of tumors, providing strong evidence for its potential as a tumor vaccine candidate.

[0095] Example 4: Evaluation of the therapeutic effect of LADS-TT based on a mouse colon cancer model 4.1 Immunization procedure for mice 4.1.1 Protein immunization steps refer to 3.3.1 of Example 3.

[0096] 4.1.2 Antibody level detection refers to 3.3.2 of Example 3.

[0097] ELISA results showed that the level of TT-specific IgG antibody in the serum of mice in the experimental group was significantly higher than that in the control group 14 days after immunization with TT protein (Figure 7B), indicating that TT protein successfully induced an antigen-specific humoral immune response.

[0098] 4.2 Construction of a Mouse Colon Cancer Model: The complete culture medium (RPMI 1640 medium supplemented with 10% fetal bovine serum) and PBS were preheated to 37°C. The supernatant in the CT-26 cell culture flask was discarded, and 5 mL of preheated PBS was added to gently wash the cells twice to remove residual serum components. Then, 2 mL of 0.25% Trypsin-EDTA was added to cover the cell layer, and the cells were digested in a 37°C incubator for approximately 1.5 min. During this time, changes in the intercellular spaces were observed using an inverted microscope. When the intercellular connections became loose, the bottom of the culture flask was gently tapped to help the cells detach. After initial detachment, three volumes of preheated complete culture medium were added to stop the digestion reaction, and the cells were gently pipetted to fully disperse them into a single-cell suspension. 10 μL of the sample was used for cell counting, and the remaining cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 700 rpm for 3 min. The supernatant was discarded. The precipitated cells were resuspended in fresh, preheated culture medium, and the cell concentration was adjusted to 6 × 10⁶. 6 cells / mL. Inject 3 × 10 cells / mL per mouse. 5 SPF-grade Balb / c female mice were immunized with a dose of 100 cells via subcutaneous injection in the abdomen. The injection site was then monitored regularly, and the formation of 1-3 mm tumor nodules was confirmed by palpation, at which point the colon cancer tumor-bearing model was considered successfully established.

[0099] 4.3. General Immunotherapy Procedure in Mice: In the study of colon cancer tumor models, mice were first immunized twice with tetanus toxoid protein to induce pre-existing memory T cells. Subsequently, CT-26 tumor cells were subcutaneously implanted in the abdomen. When the tumor reached the early stage (1-3 mm), single colonies were picked and transferred to BHI for overnight culture; after washing twice with PBS, the bacterial culture was resuspended and the OD was adjusted. 600nm =0.6. Recombinant or attenuated strains (10) were administered via tail vein injection. 8 CFU / animal), one week later, re-inject recombinant or attenuated bacterial solution via tail vein, with daily supplemental low-dose recombinant vaccine or attenuated bacterial solution (10 CFU / animal). 5 CFU / tumor, 10 doses in total. Tumor size was measured every two days, and tumor tissue was isolated 23-25 ​​days after immunotherapy.

[0100] Building upon previous studies using cervical cancer models, this study further evaluated the immunotherapeutic effect of LADS-TT-L using a colon cancer model (Figure 7A). Dynamic tumor growth monitoring showed that the tumor volume growth rate in the LADS-TT-L-immunized mice was significantly lower than that in the control group (Figure 7C), suggesting its potential to inhibit tumor progression in colon cancer models as well. This result further confirms the broad applicability of LADS-TT-L in various solid tumor models and its potential as a universal tumor vaccine platform.

[0101] 4.4 Flow Cytometry 4.4.1 Preparation of Spleen Single-Cell Suspension (1) Cut open the skin on the ventral side of the mouse, expose the spleen and carefully remove it, and place it in a culture dish containing RPMI 1640 medium for later use; (2) Transfer the spleen to a well plate with a 75 μm cell sieve, and gently grind the spleen tissue on the sieve using the rubber stopper of a 5 mL syringe with the piston removed, while continuously rinsing the sieve with RPMI 1640 medium to allow the cells to pass through the sieve holes fully and obtain a uniform single-cell suspension; (3) Transfer the obtained cell suspension to a centrifuge tube, centrifuge at low speed for 5 min at room temperature, and discard the supernatant; (4) Add an appropriate amount of red blood cell lysis buffer, gently pipette to disperse the precipitate, lyse at room temperature for 5 min, and then add RPMI 1640 medium to terminate the reaction; (5) Centrifuge again at low speed for 5 min at room temperature. If the cell suspension is still red, repeat the red blood cell lysis step until the red blood cells are basically removed; (6) Centrifuge (1000 rpm, 5 min) (7) Discard the supernatant, add 1 mL of RPMI 1640 medium containing 10% FBS to resuspend the cells, gently disperse the cell clumps, and filter again through a cell sieve to remove undispersed cells; (8) Perform cell counting and adjust the cell concentration to 1×10⁻⁶. 6 Cells / mL, used for subsequent staining procedures.

[0102] 4.4.2 Cell staining (1) Take 1×10 6(1) Centrifuge at 1000 rpm for 5 min with 1000 copies / mL of spleen cell suspension, discard the supernatant, resuspend the cells with Staining Buffer (Wuhan Aibote Biotechnology Co., Ltd.), and aliquot into 1.5 mL centrifuge tubes, adding 100 μL of cell suspension to each tube; (2) Set up control groups for gating and fluorescence compensation regulation, including: ① blank tube (no antibody added), ② only PE anti-mouse CD4 monoclonal antibody added, ③ only ABflo® 488 anti-mouse CD8a rabbit anti-monoclonal antibody added, ④ both antibodies added. Only one set of experimental tubes was set up for the remaining samples (CD4 and CD8a antibodies were added at the same time); all samples were incubated in the dark and on ice for 20 min; (3) Add 500 μL of Staining Buffer to each tube, mix well, centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant, and repeat this step twice to wash thoroughly; (4) Finally, add 150 μL of Staining Buffer to resuspend the cells, and immediately perform the detection. The obtained data were analyzed by FlowJo WorkPlace software.

[0103] The results (Figures 7D-E) showed that, compared with mice immunized with the attenuated LADS strain, the LADS-TT-L immunized group had significantly higher CD4 counts. + The proportion of activated T cells was significantly increased, with a statistically significant difference (P<0.01). Simultaneously, CD8... + The activation level of T cells was also significantly enhanced in the LADS-TT-L group (P<0.05). This indicates that LADS-TT-L can effectively activate T cell-mediated adaptive immune responses by delivering tetanus toxoid antigen.

[0104] Table 6 Primers required in specific implementation methods

[0105] Sequence Listing SEQ ID NO: 1 TT 856-1313Amino acid sequence: STPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTSEQ ID NO: 2 LLO mutant - TT 856-1313Amino acid sequence: DASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNIAAYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIEGGSGGSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTSEQ ID NO: 3 LLO mutant - TT856-13131042-1171 Amino acid sequence: DASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNIAAYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIEGGSGGDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRSEQ ID NO: 5 LLO mutant - TT 1042-11711115-1315 Amino acid sequence: DASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNIAAYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIEGGSGGRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDSEQ ID NO: 7 LLO mutant - TT 1115-1315

Claims

1. A recombinant attenuated Listeria monocytogenes, characterized in that, The recombinant attenuated Listeria monocytogenes is prepared by replacing the hly gene sequence or a fragment thereof of wild-type Listeria monocytogenes with a sequence containing the LLO mutant and TT. 856-1313 The fusion protein coding sequence is obtained by modifying the hly gene sequence or fragment of a mutated Listeria monocytogenes to include the LLO mutant and TT. 856-1313 The fusion protein is obtained by transcoding a polynucleotide sequence of the fusion protein to express and secrete the fusion protein, wherein the attenuated Listeria monocytogenes is obtained by mutating the hly gene in wild-type Listeria monocytogenes to express an LLO mutant, the LLO mutant having the following mutations compared to the wild-type LLO protein: N478A and V479A; the amino acid sequence of the wild-type LLO protein is shown in NCBI accession number NP_463733.1, and the TT... 856-1313 The amino acid sequence is shown in SEQ ID NO:

1.

2. A method for preparing recombinant attenuated Listeria monocytogenes, characterized in that, Includes the following steps: (1) Constructing a system containing LLO mutants and TT mutants 856-1313 Recombinant plasmids encoding fusion protein sequences or containing TT 856-1313 (2) Recombinant plasmids containing LLO mutant and TT from step (1); 856-1313 Recombinant plasmids encoding fusion protein sequences are introduced into competent wild-type or attenuated Listeria monocytogenes, or into bacteria containing TT. 856-1313 Recombinant plasmids encoding protein sequences were introduced into attenuated Listeria monocytogenes. (3) The bacteria obtained in step (2) with the recombinant plasmid introduced are subjected to homologous recombination hybridization culture and passaged until the plasmid is lost to obtain recombinant attenuated Listeria monocytogenes, wherein in step (3), the homologous recombination is a hybridization containing LLO mutant and TT. 856-1313 The fusion protein coding sequence is a polynucleotide substitution of the hly gene sequence of wild-type Listeria monocytogenes or a mutated hly gene sequence of attenuated Listeria monocytogenes, or a fragment thereof; or contains TT. 856-1313 A protein-coding polynucleotide was inserted downstream of the hly gene sequence of a mutant *Listeria monocytogenes* to obtain expression and secretion of LLO mutants and TT mutants. 856-1313 A recombinant attenuated Listeria monocytogenes fusion protein; the attenuated Listeria monocytogenes was obtained by mutating the hly gene in wild-type Listeria monocytogenes to express an LLO mutant; the LLO mutant has the following mutations compared to the wild-type LLO protein: N478A and V479A; the amino acid sequence of the wild-type LLO protein is shown in NCBI accession number NP_463733.1, and the TT... 856-1313 The amino acid sequence is shown in SEQ ID NO:

1.

3. The recombinant attenuated Listeria monocytogenes according to claim 1 or the method according to claim 2, characterized in that, The wild-type Listeria monocytogenes is a wild-type EGD-e strain; and / or, the recombinant attenuated Listeria monocytogenes is composed of a strain containing TT inserted downstream of a mutated hly gene sequence in attenuated Listeria monocytogenes. 856-1313 Obtained by polynucleotides of a protein-coding sequence; and / or, optionally, by LLO mutants and TT mutants in the fusion protein. 856-1313 There are joints between them, and preferably, the joint sequence is GGSGG.

4. The recombinant attenuated Listeria monocytogenes or the method according to claim 3, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

2.

5. The recombinant attenuated Listeria monocytogenes according to claim 1 or the method according to claim 2, characterized in that, The recombinant attenuated Listeria monocytogenes is derived by modifying the hly gene or a fragment thereof of attenuated Listeria monocytogenes to include the LLO mutant and TT. 856-1313 It is obtained by using polynucleotides encoding the fusion protein sequence.

6. The method according to claim 2, characterized in that, In step (1), suitable primers are designed for PCR amplification to obtain the LLO mutant and TT. 856-1313 The coding sequence of the fusion protein is a polynucleotide, and it will contain LLO mutants and TT. 856-1313 The polynucleotide encoding the fusion protein sequence is transferred into a plasmid to obtain a recombinant plasmid, or a plasmid containing TT. 856-1313 Polynucleotides encoding protein-coding sequences, and those containing TT 856-1313 The protein-coding sequence polynucleotides are transferred into a plasmid to obtain a recombinant plasmid; and / or, in step (2), the plasmid containing LLO mutant and TT is transferred under the conditions of 2000 V-2500 V voltage, resistance above 0 Ω, and power of 25 μF. 856-1313 Recombinant plasmids containing multiple nucleotides encoding fusion protein sequences were electroporated into competent wild-type or attenuated Listeria monocytogenes, or plasmids containing TT... 856-1313 The recombinant plasmid of the protein-coding sequence polynucleotide is electroporated into competent attenuated Listeria monocytogenes; and / or, in step (3), the wild-type Listeria monocytogenes or attenuated Listeria monocytogenes introduced with the recombinant plasmid in step (2) is subjected to homologous recombination at a constant temperature of 41.5℃-42.5℃ to obtain recombinant attenuated Listeria monocytogenes, and the recombinant attenuated Listeria monocytogenes is passaged until the plasmid is lost.

7. A biological vaccine against cervical cancer and / or colon cancer, characterized in that, It contains recombinant attenuated Listeria monocytogenes according to any one of claims 1 and 3-5.

8. Use of the recombinant attenuated Listeria monocytogenes according to any one of claims 1 and 3-5 or the biological vaccine according to claim 7 in the preparation of a medicament for treating cervical cancer or colon cancer or inhibiting the growth of cervical cancer or colon cancer tumors.

9. Use of the recombinant attenuated Listeria monocytogenes according to any one of claims 1 and 3-5 or the biological vaccine according to claim 7 in the preparation of a medicament, wherein the medicament is used in combination with TT protein, and the medicament is used to treat cervical cancer or colon cancer or to inhibit the growth of cervical cancer or colon cancer tumors.

10. Use of TT protein and recombinant attenuated Listeria monocytogenes according to any one of claims 1 and 3-5 or the biological vaccine according to claim 6 in the preparation of a medicament for treating cervical cancer or colon cancer or inhibiting the growth of cervical cancer or colon cancer tumors.

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