Listeria monocytogenes strain as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microorganisms and genetic engineering. The invention provides a listeria monocytogenes strain as well as a preparation method and application thereof, the flagellum formation ability of the strain is enhanced, the environmental stress resistance is enhanced, and the immune recognition of host cells on listeria monocytogenes can be enhanced; the strain is subjected to delta yxeA gene deletion mutation; the bacterial strain is named as Listeria monocytogenes Lm-delta lmo2568, the preservation number of the bacterial strain is CGMCC (China General Microbiological Culture Collection Center) No.36001, and the bacterial strain is preserved in the China General Microbiological Culture Collection Center on September 22, 2025. The invention finds that the deletion of Listeria monocytogenes YxeA promotes the formation and expression of flagellum, and the obtained strain can enhance the immune recognition of host cells on Listeria monocytogenes, and can be used for preparing related preparations.

Description

Technical Field

[0001] This invention belongs to the field of microbial and genetic engineering technology, specifically relating to a Listeria monocytogenes strain, its preparation method, and its application. Background Technology

[0002] Listeria monocytogenes, also known as L. monocytogenes, can infect immunocompromised individuals, such as pregnant women and children, causing diseases such as diarrhea, meningitis, and miscarriage. This bacterium has strong environmental adaptability, including tolerance to low temperatures, high salt concentrations, acid and alkali conditions, and ultraviolet radiation. It can survive and proliferate in various extreme environments, causing serious food contamination, especially of livestock by-products such as eggs, posing a serious threat to public health and food safety.

[0003] Currently, research on which genes are involved in controlling Listeria monocytogenes infection of the host is incomplete, limiting the development of corresponding genetically engineered mutant strains. Therefore, there is an urgent need in this field to further investigate the genes involved in controlling Listeria monocytogenes infection of the host and to develop corresponding mutant strains. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a Listeria monocytogenes strain with enhanced flagellar formation ability and resistance to Cu. 2＋ Enhanced environmental stress response can improve the host cell's immune recognition of Listeria monocytogenes; the strain exhibits a ΔyxeA gene deletion mutation.

[0005] Specifically, the strain is named as follows: Listeria monocytogenes Lm-Δ lmo2568 The accession number is CGMCC No.36001, and it is deposited at the China General Microbiological Culture Collection Center on September 22, 2025.

[0006] Specifically, the nucleotide sequence of the yxeA gene is shown in SEQ ID No. 1.

[0007] A method for constructing a Listeria monocytogenes strain, wherein the strain is as described above, and the construction method includes the following steps: (1) Take 463 bp upstream of yxeA as homologous arm A and 475 bp downstream of yxeA as homologous arm B, and design primers to add the selected restriction sites BamHI and KpnI; (2) After ligating the upstream and downstream fragments, the fragments were integrated into the shuttle-type thermosensitive plasmid pKSV7 via enzyme digestion and linking. (3) The obtained plasmid was transformed into the EGD-e competent cells of wild-type Listeria monocytogenes, and homologous recombination was carried out under chloramphenicol resistance conditions and at a temperature of 42°C; (4) After successful homologous recombination, the temperature-sensitive plasmid pKSV7 was discarded at 30°C to obtain the strain.

[0008] Preferably, in step (2), the upstream and downstream homologous arms are amplified by PCR, and the two fragments are fused by overlap PCR; then, restriction endonuclease and high-efficiency ligase are used to ligate the fused homologous arm fragments and the temperature-sensitive plasmid pKSV7, and enzyme digestion is performed.

[0009] Preferably, in step (3), the enzyme-ligated product is added to... E.coli After thorough mixing with DH5α competent bacteria, the mixture was heat-shocked in a metal bath at 42℃ for 90 s, then removed and added to 900 µL of LB medium. The mixture was then incubated at 37℃ and 200 rpm for 45 min. Following this, the mixture was centrifuged at 5000 rpm for 3 min, and the bacterial suspension was resuspended in 100 µL of LB medium. Positive transformants were screened on LB plates containing ampicillin resistance. After verification by PCR and DNA sequencing, the transformed bacteria were purified, and the resulting plasmid was named pSL2190. A single purified colony was then inoculated into 10 mL of medium, and the recombinant plasmid was extracted using the HLingene plasmid extraction kit. The recombinant plasmid was then added to Listeria monocytogenes EGD-e competent bacteria, mixed thoroughly, and incubated on ice for 30 min. Transformation was performed at 2500 V, 200 Ω, 25 µF, and 2 mm. 900 µL of BHI medium containing sucrose was added, and the mixture was incubated statically at 30℃ for 2 h. Finally, the mixture was transformed at 5000 rpm and 300 rpm. After centrifugation, the bacterial suspension was resuspended and spread on BHI plates containing chloramphenicol resistance to screen for positive transformants. After PCR sequencing verification, positive single colonies were picked and inoculated into BHI liquid medium containing resistance. The culture was then passaged at 42℃ and 200rpm, and PCR verification was performed every 5 generations.

[0010] Preferably, the characteristic is that in step (4), after the homologous arm fragments are completely homologously recombined, they are transferred to BHI medium and placed in a shaker at 30°C and 180 rpm for continued subculturing until the pKSV7 plasmid is completely lost.

[0011] 8. The construction method according to claim 7, characterized in that, after step (4), the construction method further includes the following steps: streaking the above bacterial solution on a BHI plate and culturing overnight at 30°C; picking single colonies and picking them on a BHI plate without antibiotic resistance and a BHI plate containing chloramphenicol resistance respectively; placing the picked plates in a 30°C incubator and letting them stand for 24 h; selecting single colonies without chloramphenicol resistance; verifying them by PCR and DNA sequencing; adding 60% sterile glycerol; and storing at -80°C.

[0012] Preferably, the method for preparing the Listeria monocytogenes wild-type strain EGD-e competent cells is as follows: The Listeria monocytogenes wild-type strain to be used to prepare competent cells is revived from a streak plate at -80℃; a single colony is picked and inoculated into a bacterial bottle containing 5 mL of BHI; the culture is incubated overnight at 37℃ with shaking at 200 rpm; the overnight culture is inoculated at a ratio of 1:100 into a conical flask containing 100 mL of BHI liquid medium with a final concentration of 0.5 M sucrose; and the culture is continued under the same conditions until the bacterial culture reaches OD. 600 nm The concentration of the bacteria reached 0.18-0.25. 100 mg / mL penicillin G was added to the conical flask at a ratio of 1:5000, and the mixture was incubated in a shaker at 37°C for 2 h. The conical flask was removed and placed on ice for 10 min. The bacterial suspension was aliquoted into 50 mL centrifuge tubes and centrifuged at 4°C and 6000 rpm for 7 min, discarding the supernatant. The bacterial pellet was gently resuspended in 30 mL of pre-chilled washing buffer (containing 50 mM sucrose and 50 mM HEPEs) at 4°C. The pellet was centrifuged at 4°C and 6000 rpm for 7 min, discarding the supernatant. This washing process was repeated 2-3 times. After discarding the supernatant, 1 mL of HEPEs was added to resuspend the bacterial pellet. The pellet was then aliquoted into 100 µL tubes pre-chilled at 4°C into 1.5 mL EP tubes, yielding the *Listeria monocytogenes* wild-type strain EGD-e competent cells. These cells were stored at -80°C for later use.

[0013] The application of the aforementioned strains in the preparation of formulations that enhance the immune recognition of host cells against Listeria monocytogenes.

[0014] The beneficial effects of this invention are: YxeA protein, a widely distributed accessory protein of the Bce module, plays a crucial role in bacterial defense processes. It assists ABC transporters and the two-component system in recognizing and releasing antimicrobial peptides, while also regulating stress responses. It possesses an N-terminal signaling sequence and a transmembrane region. Currently, functional studies on YxeA protein are limited. It is known that in Bacillus subtilis, YxeA protein influences bacterial cell wall modification and expression, participates in peptidoglycan synthesis, and affects cell membrane permeability; however, its role in bacterial environmental adaptation remains poorly investigated.

[0015] Early studies have reported that in other microorganisms, the YxeA protein participates in peptidoglycan synthesis, affects bacterial cell membrane permeability and cell wall modification, and cell membrane permeability plays an important role in bacterial resistance to external oxidative damage and antibiotic bactericidal effects. Does the YxeA protein in Listeria monocytogenes have the same function, or does it have other biological functions? Therefore, this invention uses the wild-type Listeria monocytogenes strain EGD-e as the research object to explore the biological functions of YxeA and its role in bacterial environmental adaptation.

[0016] This invention discovers that the deletion of YxeA in Listeria monocytogenes actually promotes the formation and expression of flagella, and the resulting strain can enhance the immune recognition of Listeria monocytogenes by host cells and can be used to prepare related preparations. Attached Figure Description

[0017] Figure 1 Listeria monocytogenes yxeA A schematic diagram of the construction of a gene-deleted strain; Figure 2 Listeria monocytogenes yxeA A schematic diagram of the gene complementation strategy; Figure 3 For EGD-e, ΔyxeA, CΔ yxeA Figure showing the experimental results of the growth ability of the Pnative strain; Figure 4 For EGD-e, Δ yxeA and CΔ yxeA Figure 1 shows the results of a Pnative cell membrane permeability assay. Figure 5 For EGD-e, Δ yxeA 、CΔ yxeA Figure 1 shows the experimental results of Pnative growing on plates containing H2O2, MnSO4, ZnSO4, CdCl2, CuSO4 and without BHI. Figure 6 For EGD-e and Δ under 30℃ and 37℃ conditions yxeA Figure 1 shows the experimental results of the diameter of the kinetic circle (parts A and B) and the measurement of athletic ability (part C). Figure 7 For EGD-e and Δ under 30℃ and 37℃ conditions yxeA 、CΔ yxeA Transmission electron microscope image of a flagellum; Figure 8 For EGD-e, yxeA mutant strains and hly Figures showing the experimental results of hemolytic ability determination (Part A) and hemolysis curve (Part B) of the deletion strain; Figure 9 For EGD-e and yxeA Figures showing the results of the mutant strain in the plaque assay (Part A) and the diameter of the formed plaque (Part B); Figure 10 Under conditions of 30℃ (Part A) and 37℃ (Part B), yxeA Figure showing the experimental results of detecting transcriptional levels of virulence genes after deletion; Figure 11 Under conditions of 30℃ (Part A) and 37℃ (Part B), yxeA Figure showing the experimental results of flagellar gene transcription level detection after deletion; Figure 12 Under conditions of 30℃ and 37℃, yxeA Expression results of flagellin FlaA, GamR, and MogR after deletion (Part A) and grayscale analysis (Parts B and C); Figure 13 for yxeA Figure 1. Experimental results of flagellar gene transcription level detection after deletion of infected cells; Figure 14 for yxeA Figure showing the experimental results of detecting the transcriptional level of virulence genes after the absence of infected cells. Detailed Implementation

[0018] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are still within the scope of protection of the present invention.

[0019] In the following examples, the gene, amino acid sequence, and primer information involved are as follows: YxeA (gene number: lmo2568 The nucleotide sequence (324 bp) of the target gene, i.e., SEQ ID No. 1: atgaaaaagggacttttcggtttactggctgcaatttttgtattaacaatggttgtaattgaattttcagcaccaattggatcggcagcaaatacatattacgttacaatcaaaaacgatggaaaagaagttaccaagtcaaaatttaaagggtactcattc cgggaatcgtgtttagataatcaaggaaacagtcacaaagttaacttcatgtcaacaacacaacttaaaaagaataaacagtataaaatcgtcgttcaagacaacgaattactcgtccaagccaaggaaataaacagtgttccacttgcaagtagtaaataa.

[0020] The amino acid sequence corresponding to the YxeA gene is SEQ ID No. 2: MKKGLFGLLAAIFVLTMVVIEFSAPIGSAANTYYVTIKNDGKEVTKSKFKGYSFRESCLDNQGNSHKVNFMSTTQLKKNKQYKIVVQDNELLVQAKEINSVPLASSK*.

[0021] Build yxeA Gene deletion strains and yxeA The primers used for gene replacement strains are shown in Table 1: Table 1

[0022] Example 1

[0023] 1.1 Plasmids, bacterial strains and culture conditions The strains and plasmids used in this experiment were all preserved in the laboratory.

[0024] Strains: including wild-type Listeria monocytogenes EGD-e (ATCC BAA-679) and related mutant strains, as well as Escherichia coli (… Escherichia coli , E . coli DH5α, in which Listeria monocytogenes was cultured in bovine brain heart infusion (BHI); E . coli Cultured in LB basal medium.

[0025] Plasmids: including the shuttle-type temperature-sensitive plasmid pKSV7 and the complement plasmid pIMK2. Add the required antibiotics according to plasmid resistance. Chloramphenicol is stored at a concentration of 25 mg / mL, kanamycin at 50 mg / mL, and ampicillin at 100 mg / mL; their working concentrations are diluted 1:1000 for use.

[0026] The culture conditions were as follows: after strewing solid culture medium, the culture was incubated at 37°C for 10-12 h; after inoculation with liquid culture medium, the culture was placed in a shaker at 37°C and 180 rpm for 8-10 h.

[0027] 1.2 Experimental Reagents 10×CutSmart Buffer, Ligation high Ver.2, Restriction endonuclease: New England Biotechnology Co., Ltd.; 2X MμLtiF Seamless Assembly Mix: Aibotek Inc. DL2000 Plus DNA Maker: Nanjing Novizan Biotechnology Co., Ltd. HLingene PCR product purification and recovery kit, HLingene plasmid extraction kit: Shanghai Huiling Biotechnology Co., Ltd. Disodium hydrogen phosphate, Triton X-100, potassium dihydrogen phosphate: BBI LIFE SCIENCE CORPORATIONS; Hydrazine, copper sulfate, zinc sulfate, manganese sulfate, cadmium chloride, Sigma-Aldrich Trading Co., Ltd. Hydrochloric acid: Xilong Scientific Co., Ltd.; Bovine brain and heart extract (BHI): OXOID; Hydrogen peroxide, yeast extract, chloramphenicol, tryptone, sodium chloride, kanamycin (Kana), agar, agarose, gentamicin, penicillin (Pen), ampicillin (Amp): Shanghai Sangon Biotech.

[0028] Reagent preparation: (1) LB medium: Weigh 4 g tryptone, 4 g NaCl and 2 g yeast extract into a 1000 mL Erlenmeyer flask, dissolve with ddH2O, bring the volume to 400 mL and autoclave to obtain LB liquid medium. If solid medium is required, weigh 6 g agar powder and add it to the Erlenmeyer flask before sterilization. (2) BHI medium: Weigh 7.4 g of BHI powder, dissolve it with ddH2O, bring the volume to 200 mL and autoclave to obtain BHI liquid medium. If solid medium is required, weigh 3 g of agar powder and add it to the conical flask before sterilization. (3) 50× nucleic acid gel electrophoresis buffer TAE: Measure 57 mL of acetic acid and 100 mL of 0.5 M EDTA solution with pH 8.0, mix them, add 242 g of Tris Base powder, dissolve with ddH2O, and bring the volume up to 1000 mL after the powder is completely dissolved. (4) 1% agarose gel: Dilute 50× nucleic acid electrophoresis buffer TAE to its working concentration of 0.5× nucleic acid gel electrophoresis buffer TAE, measure 50 mL, weigh 0.5 g of agarose H, add it and microwave at high temperature to dissolve, shake and mix well, heat until the agarose is completely dissolved in 0.5×TAE, cool at room temperature for 10 min, add 2.5µL of GoldView dye, mix well and pour into the nucleic acid gel tank to solidify; (5) TSA semi-solid culture medium: Weigh 0.3 g NaCl, 0.15 g tryptone and 0.4 g agar powder, add ddH2O to make up to 150 mL, sterilize and set aside; (6) Preparation buffer for competent Escherichia coli: Weigh 1.1 g of calcium chloride, dissolve it with ddH2O and bring the volume to 100 mL to make a 100 mM calcium chloride solution; add glycerol to the 100 mM calcium chloride to make the final concentration 10%, and autoclave for later use. (7) CAMHB medium: Weigh 10 g of CAMHB medium powder, dissolve it with ddH2O and bring the volume to 400 mL, then sterilize and set aside. (8) 1M H2O2 solution: Measure 1 mL of 30% hydrogen peroxide solution, add 9 mL of ddH2O, mix well, filter with a 0.22 μM filter in a clean bench, and prepare fresh before use; (9) 1 M MnSO4 solution: Weigh 1.69 g MnSO4·H2O, make up to 10 mL with ddH2O, the final concentration is 100 mg / mL, sterilize with a 0.22 µm filter, and store at -20℃; (10) 1 M ZnSO4 solution: Weigh 2.87 g ZnSO4·7H2O, make up to 10 mL with ddH2O, the final concentration is 100 mg / mL, sterilize with a 0.22 µm filter, and store at -20℃; (11) 1 M CdCl2 solution: Weigh 1.23 g CdCl2, make up to 10 mL with ddH2O, the final concentration is 100 mg / mL, sterilize with a 0.22 µm filter, and store at -20℃; (12) 1 M CuSO4 solution: Weigh 1.34 g CuSO4, make up to 10 mL with ddH2O, the final concentration is 100 mg / mL, sterilize with a 0.22 µm filter, and store at -20℃; (13) 100 mM PBS solution: Weigh 14.2 g disodium hydrogen phosphate, 2.4 g sodium dihydrogen phosphate, and 8.77 g sodium chloride, add 800 mL ddH2O to dissolve, and adjust the pH to 7.4 using HCl and NaOH, then add ddH2O to bring the volume to 1 L; (14) 10 mM PBS solution: Measure 100 mL of 100 mM PBS solution, add ddH2O to dilute and bring the volume to 1 L; (15) Preparation of ampicillin storage solution: Weigh 1 g of ampicillin powder, add ddH2O to make up to 10 mL, the final concentration is 100 mg / mL, sterilize with a 0.22 µm filter, and store at -20℃; (16) Preparation of kanamycin storage solution: Weigh 0.5 g kanamycin powder, add ddH2O to make up to 10 mL, the final concentration is 100 mg / mL, sterilize with a 0.22 µm filter, and store at -20℃; (17) Preparation of penicillin G stock solution: Weigh 0.2 g penicillin G powder, add ddH2O to make up to 10 mL, the final concentration is 100 mg / mL, sterilize with a 0.22 µm filter, and store at -20℃; (18) Preparation of gentamicin storage solution: Weigh 0.5 g of gentamicin powder, add ddH2O to make up to 10 mL, the final concentration is 100 mg / mL, sterilize with a 0.22 µm filter, and store at -20℃.

[0029] 1.3 Genome extraction of Listeria monocytogenes EGD-e (1) Take out the EGD-e strain stored at -80℃, streak it on a plate, and incubate it at 37℃ until a single colony appears; (2) Pick a single colony of EGD-e and inoculate it into BHI liquid medium, and incubate overnight at 37°C on a shaker; (3) Take 2 mL of overnight bacterial culture into an EP tube, centrifuge at 12,000 rpm for 2 min, take it out, and wash twice with 1 mL of ddH2O; (4) Add 100 µL ddH2O and 100 µL Listeria monocytogenes genome extract to resuspend the precipitate and freeze at -20℃ for 30 min; (5) Boil for 10 minutes, then freeze at -20℃ for 10 minutes; (6) Centrifuge at 4℃ and 12,000 rpm for 10 min. The supernatant is the EGD-e genome. Store at -20℃ for later use.

[0030] 1.4 Preparation of competent Escherichia coli cells (1) Recovery at -80℃ E.coli DH5α was resuscitated on LB plates and incubated overnight at 37°C. (2) Pick a single colony and inoculate it into a bacterial bottle containing LB liquid medium and incubate it at 37°C; (3) Transfer the bacterial culture at a ratio of 1:100 to an Erlenmeyer flask containing 100 mL of LB liquid medium, and incubate with shaking until the bacterial culture reaches OD. 600 nm Close to 0.4; (4) After placing the conical flask on ice for 30 min, centrifuge at 4℃ and 4000 rpm for 8 min, discard the supernatant, add 10 mL of calcium chloride buffer that has been pre-cooled at 4℃, gently resuspend the bacterial pellet, and place it at 4℃ for 30 min. (5) After taking it out, centrifuge at 4℃ and 4000 rpm for 8 min, discard the supernatant, add 10 mL of buffer solution containing 10% glycerol calcium chloride, dispense, and freeze at -80℃ for later use.

[0031] 1.5 PCR reaction system and procedure Table 2 PCR reaction system of 2×Phanta Max Master Mix

[0032] Table 3. PCR reaction program for 2×Phanta Max Master Mix PCR

[0033] Table 4. Taq enzyme verification PCR reaction system

[0034] Table 5 Taq enzyme verification PCR reaction procedure

[0035] 1.6 Purification of PCR Products The PCR products were purified using the HLingene PCR product purification and recovery kit, following the instructions. (1) The PCR product was recovered under UV light and the target band was transferred to an EP tube. 500 µL of GMB binding solution was added and the product was melted in a water bath at 60°C. (2) Add the mixture to the adsorption column, let it stand for 1 min to react, and centrifuge at 12,000 rpm for 30 s; (3) Discard the waste liquid, add WB washing solution and wash twice, centrifuge the empty tube with the waste liquid discarded at 12,000 rpm for 2 min and then place it in a clean bench to dry for 10 min. (4) Place the dried adsorption column into a new EP tube, add 50 µL of EB elution buffer preheated at 60℃, let stand for one minute to dissolve, centrifuge at 12,000 rpm for 30 s to obtain nucleic acid, measure its concentration and purity, and store at -20℃ for later use.

[0036] 1.7 Escherichia coli plasmid extraction Plasmid recovery was performed using the HLingene plasmid extraction kit from Shanghai Huiling Biotechnology Co., Ltd., following the instructions and actual procedures. The specific steps are as follows: (1) Remove bacteria containing the target plasmid from -80℃, streak them on a plate containing the corresponding antibiotic for recovery, inoculate, and incubate overnight at 37℃ and 180 rpm. (2) Add the overnight bacterial culture to an EP tube, centrifuge at 12,000 rpm for 1 min to collect the bacterial precipitate; (3) Add 250 µL of S1 stored at 4℃ to resuspend the precipitate, then add 250 µL of room temperature S2 solution, invert the EP tube 7-8 times, and let it stand at room temperature for 4 min. (4) Add 350 µL of S3 solution, gently invert, centrifuge at 13,000 rpm for 10 min, take the supernatant and add it to the adsorption column, being careful not to take the flocculent material, let it stand for 1 min, and then follow the same steps as in steps (2) to (4) of 1.6.

[0037] 1.8 Listeria monocytogenes yxeA Construction of gene deletion strains 1.8.1 Preparation of Listeria monocytogenes competent cells (1) Recover Listeria monocytogenes that will be made into competent cells from a streak plate at -80℃, pick a single colony and inoculate it into a bacterial bottle containing 5mL BHI, and incubate overnight at 37℃ with shaking at 200 rpm. (2) The overnight culture was inoculated into 100 mL of BHI liquid medium containing 0.5 M sucrose at a ratio of 1:100 into an Erlenmeyer flask, and cultured under the same conditions until the bacterial culture reached OD. 600 nm Reaching 0.18-0.25; (3) Add 100 mg / mL of penicillin G to the conical flask at a ratio of 1:5000, and continue to incubate in a shaker at 37°C for 2 h; (4) Remove the conical flask and place it on an ice bath for 10 min. Then, aliquot the bacterial solution into 50 mL centrifuge tubes and centrifuge at 4°C and 6,000 rpm for 7 min. Discard the supernatant. (5) Add 30 mL of washing solution containing 50 mM sucrose and 50 mM HEPEs pre-cooled at 4℃ and gently resuspend the bacterial precipitate. Centrifuge at 4℃ and 6,000 rpm for 7 min, discard the supernatant, and repeat the washing process 2-3 times. (6) After discarding the supernatant, add 1 mL of HEPEs to resuspend the bacterial cells and dispense 100 µL into 1.5 mL EP tubes that have been pre-cooled at 4°C to obtain Listeria competent cells. Store at -80°C for later use.

[0038] 1.8.2 Listeria monocytogenes yxeA Construction of gene deletion strains Primers containing homologous fragments were designed 500 bp upstream and downstream of the target gene. After ligating the upstream and downstream fragments, they were integrated into the shuttle thermosensitive plasmid pKSV7 via enzyme digestion. Figure 1 The plasmid was transformed into the EGD-e competent cells of the wild-type Listeria monocytogenes strain, allowing homologous recombination to occur under chloramphenicol resistance conditions and at a temperature of 42°C. After successful homologous recombination, the temperature-sensitive plasmid pKSV7 was discarded at 30°C, ultimately yielding the deletion strain. The specific procedure is as follows: (1) Take yxeA The upstream 463 bp was taken as homologous arm A, and the downstream 475 bp of the target gene was taken as homologous arm B. Primers were designed and selected restriction enzyme sites were added. Bam HI and Kpn I; (2) PCR amplification of upstream and downstream homologous arms, and overlap PCR fusion of the two fragments; (3) The homologous arm fragments amplified by fusion and the temperature-sensitive plasmid pKSV7 were digested and ligated using restriction endonucleases and high-efficiency ligases according to the systems and procedures in Tables 6 and 7.

[0039] (4) Add the enzyme-digested ligation product to E.coli After mixing thoroughly, the mixture was heat-shocked in a metal bath at 42°C for 90 s, then removed and added to 900 µL of LB medium. The mixture was then placed in a shaker at 37°C and 200 rpm for 45 min. (5) Centrifuge at 5,000 rpm for 3 min, resuspend the bacterial culture in 100 µL LB, spread it on LB plates containing ampicillin resistance to screen for positive transformants, and purify it after verification by PCR and DNA sequencing. The obtained plasmid was named pSL2190. (6) Collect a purified single colony in 10 mL of culture medium and extract the recombinant plasmid using the Hlingene plasmid extraction kit; (7) Add the recombinant plasmid to Listeria monocytogenes EGD-e competent cells, mix well, incubate on ice for 30 min, transform by electroporation at 2500 V, 200 Ω, 25 µF, 2 mm, add 900 µL of BHI medium containing sucrose, and incubate at 30℃ for 2 h. (8) After centrifugation at 5,000 rpm for 3 min, the bacterial suspension was resuspended and spread on BHI plates containing chloramphenicol resistance to screen for positive transformants. After PCR sequencing verification, positive single colonies were picked and inoculated into BHI liquid medium containing resistance. The culture was passaged at 42℃ and 200 rpm. PCR verification was performed after every 5 generations. (9) After the homologous arm fragments are completely homologously recombined, they are transferred to BHI medium and placed in a shaker at 30°C and 180 rpm for further subculturing until the pKSV7 plasmid is completely lost. (10) The above bacterial culture was streaked on BHI plates and incubated overnight at 30°C. Single colonies were picked and placed on both BHI plates without antibiotic resistance and those containing chloramphenicol resistance. The picked plates were then placed in a 30°C incubator for 24 hours. Single colonies without chloramphenicol resistance were selected, and after PCR and DNA sequencing verification, 60% sterile glycerol was added and the plates were stored at -80°C. The resulting strain was named Δ. yxeA .

[0040] Table 6. Restriction endonuclease digestion reaction system and procedure

[0041] Table 7. High-efficiency ligase reaction system and procedure

[0042] 1.9 Listeria monocytogenes yxeA Construction of gene complementation strains (1) Using bioinformatics websites to... yxeA Promoter regions were predicted, and primers were designed and restriction enzyme sites were added using Snap Gene software. Sac I and Xho I. Using EGD-e strain as a template, amplify the natural promoter region and yxeA Gene fragments; (2) Purify and recover the DNA fragments according to the method steps in 1.7 of this chapter, and use restriction endonucleases and high-efficiency ligases to ligate the DNA fragments containing the promoter region. yxeA Gene fragments and complement plasmid pIMK2 were digested and ligated according to the systems and procedures in Tables 6 and 7. (3) Obtain the recombinant plasmid according to section 1.9.2 (4-5) of this chapter, and name it pSL2191. Figure 2 ); (4) Transform the recombinant plasmid into Listeria monocytogenes Δ following the steps in 1.9.2 (6-8) of this chapter. yxeA In bacterial competent cells, positive transformants were screened from BHI plates containing kanamycin. After verification by PCR and DNA sequencing, they were added to 60% sterile glycerol and stored at -80°C. The resulting strain was named CΔ. yxeA _P native .

[0043] 1.10 Listeria monocytogenes cell membrane permeability assay (1) Adjust the bacterial culture to OD 600nm The value was 0.6, and the mixture was placed in a 37°C incubator and incubated statically for 1 h. (2) Then ethidium bromide was added to a final concentration of 6 μmol / L, and a set of bacterial PBS mixture was set up as a blank control. (3) Use a fluorescence spectrometer to measure the fluorescence values ​​at excitation wavelengths of 545 nm and emission wavelengths of 600 nm. Read the values ​​every 70 s for 26 min.

[0044] 1.11 Antibiotic susceptibility testing of Listeria monocytogenes (1) EGD-e, Δ yxeA 、CΔ yxeA _P native The culture was incubated in BHI liquid medium containing the corresponding resistance until the logarithmic growth phase, and the bacterial concentration was adjusted to 0.5 McFarland units (approximately 1.0 × 10⁻⁶) using a bacterial turbidimeter. 8 (CFU / mL) for later use; (2) Determine the type of antibiotic and set the first well concentration of the antibiotic to be tested according to the antibiotic selection principles recommended by the Clinical and Laboratory Standards Institute (CLSI) and the 2022 Foodborne Disease Surveillance Manual. (3) Add 100 µL of BHI liquid medium to columns 2 to 12 of a 96-well plate. Add 200 µL of antibiotic to column 1 of the 96-well plate and serially dilute it 2-fold to the next well. The concentration of the second well is 16 µg / mL. Continue this process to column 11. Add 100 µL of bacterial culture to columns 1 to 10 and column 12 of the 96-well plate. Add blank medium to the antibiotic in column 11 to set up a negative control well. Add blank medium to the bacterial culture in column 12 to set up a positive control well. (4) Incubate at 37℃ for 14-16 h, and measure the OD of the mixture using a microplate reader. 600nmThe values ​​were compared, and the turbidity of the liquid in the wells was also compared. If the degree of bacterial turbidity was less than or equal to that of the negative control wells, it indicated that the added drug concentration could inhibit bacterial growth in the microwells. The lowest drug concentration used to inhibit bacterial growth is the minimum inhibitory concentration (MIC).

[0045] 1.12 Determination of the antioxidant stress capacity of Listeria monocytogenes (1) Adjust the bacterial culture to OD 600nm The adjusted bacterial solution was serially diluted 6 times to a value of 0.6. (2) Add MnSO4, ZnSO4, CuSO4, CdCl2 and hydrazine to the sterilized BHI solid medium at final concentrations of 1.0 mM, 1.25 mM and 1.5 mM respectively, and H2O2 at final concentrations of 5 mM, 7.5 mM and 10 mM respectively. After mixing, pour into a petri dish and let cool and stand. (3) Add 10 µL of the serially diluted bacterial solution to the solidified plate, let it stand and air dry, and then invert it in a 37℃ constant temperature incubator for 12 h. (4) Take out the plate, observe the colony growth, and use a scanner to scan and record the data.

[0046] 1.13 Determination of Listeria monocytogenes motility (1) Shake the sterilized TSA semi-solid culture medium evenly, pour it into a petri dish, and let it stand and air dry for at least 1 hour before use. (2) Adjust the bacterial culture to OD 600nm The value is 0.6. Use a sterilized toothpick to dip into the bacterial solution and puncture it into the semi-solid culture medium. The puncture depth is about 1 / 3 to 2 / 3 of the depth of the culture medium. (3) Place the plates in constant temperature incubators at 30℃ and 37℃ respectively, and incubate them for 24 h and 48 h in each incubator respectively; (4) Remove the plates at 24 h and 48 h, measure the diameter of the motility zone of each bacterium and scan the plates, use GraphPad to draw diameter bar charts and perform quantitative statistical analysis; (5) EGD-e and Δ were cultured at 37℃ and BHI for 12 h. yxeA 、CΔ yxeA _P native Bacterial flagella growth was observed by staining with 2% uranium acetate and taking transmission electron microscopy.

[0047] 1.14 Listeria monocytogenes flagella growth (1) Listeria monocytogenes was stained using negative staining; EGD-e and Δ yxeAThe strain was streaked on BHI plates for revival, and single colonies were picked and inoculated into two groups of 5 mL BHI liquid medium. One group was placed in 30℃ shaking culture and the other group was placed in 37℃ shaking culture overnight.

[0048] (2) Take a drop of bacterial suspension onto a grid, blot dry, add uranyl acetate for staining, blot dry and observe directly.

[0049] 1.15 Data Statistical Analysis In this embodiment, all experiments were conducted in at least three parallel sets, with data replicated at least three times using GraphPad 8.0 software. t - Test, statistical analysis, data expressed as mean ± SD * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0050] 2. Experimental Results 2.1 Successfully built yxeA Gene deletion strains and complement strains Construct according to the method in step 1.8 yxeA Gene deletion strains, using EGD-e as a template, were amplified by PCR. yxeA The upstream homologous arm is 483 bp and the downstream homologous arm is 495 bp. Overlap PCR was used to ligate the upstream and downstream homologous arms to obtain a 978 bp homologous arm fragment. This fragment and the pKSV7 plasmid were ligated by enzyme digestion to construct a recombinant plasmid. The resulting recombinant plasmid was electroporated into EGD-e competent bacteria. After homologous recombination at 42℃ and plasmid rejection at 30℃, a band 264 bp smaller than the wild-type fragment was obtained. Single colony purification and sequencing verification yielded the deletion strain named Δ. yxeA ; Construct according to the method steps in 1.9 yxeA Gene replacement strains, predicted using bioinformatics websites yxeA Gene promoter region, amplified by PCR yxeA The region and the 147 bp preceding it containing the promoter region were ligated with the empty pIMK2 vector plasmid via enzyme digestion to construct a recombinant plasmid, which was then transformed into a new plasma. [[ID= In competent bacteria, primer verification yielded a fragment indistinguishable from the wild-type strain. After single-colony purification and sequencing verification, the complement strain was named CΔ. ​ _P native .

[0051] 2.2 ​ The growth ability of bacteria remains unchanged even when missing. Determine the OD of bacteria per hour in BHI liquid medium containing the corresponding antibiotics. 600nm The values ​​were measured for a total of 12 hours, and a bacterial growth curve was plotted. The results are as follows: ​ As shown, Δ under a BHI culture environment at 37℃ ​ The growth ability of the deletion strain was no different from that of EGD-e. P >0.05).

[0052] 2.3 ​ The absence does not alter bacterial cell membrane permeability. This invention determines bacterial cell membrane permeability by adding ethidium bromide and further detecting the fluorescence value of the ethidium-nucleic acid complex, as shown in the results. ​ As shown, Δ ​ The cell membrane permeability of the deletion strain was not significantly different from that of EGD-e. P >0.05).

[0053] 2.4 ​ The absence does not change the bacterial antibiotic sensitivity. The interpretation of antibiotic susceptibility results should refer to the CLSI M45 3rd edition test results for Listeria monocytogenes antimicrobial assays to determine the breakpoint. MIC results are shown in Table 7, EGD-e, Δ... ​ The MIC values ​​for ampicillin, penicillin, meropenem, erythromycin, and vancomycin were all within the sensitive range of the judgment criteria. The average MIC value for tetracycline was 64 μg / mL, indicating tetracycline resistance. The MIC value for ciprofloxacin was 2 μg / mL, meaning the sensitivity to ciprofloxacin was intermediate. ​ Gene deletion does not affect the drug sensitivity of bacteria.

[0054] Table 8 ​ The Influence of Genes on Antibiotic Sensitivity

[0055] 2.5 ​ Lack of bacterial resistance to Cu 2＋ Enhanced environmental stress response To further explore ​ The tolerance of bacteria to oxidative stress after gene deletion was assessed using EGD-e and Δ... ​ 、CΔ ​ _P native The experimental strain was used, and its oxidative tolerance under different stress conditions was studied by gradient addition of the strain to BHI plates containing different concentrations of H2O2, MnSO4, ZnSO4, CdCl2, and CuSO4. The results are as follows: ​ As shown, ​The mutant strain showed no significant difference in oxidative tolerance to H2O2, MnSO4, ZnSO4, and CdCl2 compared to the wild-type strain. ​ Compared with EGD-e, YxeA showed a 10-fold increase in resistance to 0.5 mM CuSO4 and 1.0 mM CuSO4, indicating that YxeA is involved in Listeria monocytogenes' resistance to CuSO4 oxidative stress.

[0056] 2.6 ​ Bacterial motility is enhanced after deletion. Determination of EGD-e and Δ in bacterial diffusion capacity in TSA semi-solid medium ​ 、CΔ ​ _P native External motor ability. Results as follows: ​ As shown, Δ after culturing at 30℃ for 24 h and 48 h... ​ The motility of bacteria was significantly enhanced compared to EGD-e, and the bacterial motility diameter was significantly increased (*). P <0.05). After incubation at 37℃ for 24 h and 48 h, Δ was found to be... ​ It possesses a kinetic circle, and the diameter of the kinetic circle is significantly enhanced compared to EGD-e (*** P <0.001). In summary, ​ The deletion resulted in enhanced bacterial motility at 30°C, and the bacteria retained some motility at 37°C. This indicates... ​ Involved in regulating bacterial motility.

[0057] 2.7 ​ The absence of bacterial flagella enhances growth ability This invention uses uranium acetate staining followed by transmission electron microscopy to observe the growth of bacterial flagella. The experimental results are as follows: ​ As shown, bacteria were cultured at 30℃ for 24 h, Δ ​ Transmission electron microscopy (TEM) showed a significant increase in flagella number compared to EGD-e; after 24 h of incubation at 37°C, Δ ​ A certain number of flagella grow, but EGD-e flagella do not grow. In summary, ​ The deletion resulted in an increase in the number of bacterial flagella at 30°C, and flagella growth occurred at 37°C. This indicates... ​ It participates in regulating the growth and expression of bacterial flagella.

[0058] To investigate the biological functions of Listeria monocytogenes YxeA, we successfully constructed [a specific strain] in the examples. ​ The study examined the missing and supplemented plants, and tested their growth and environmental adaptability. The results showed that Δ ​The oxidative tolerance to H2O2, MnSO4, ZnSO4, and CdCl2 showed no significant difference compared to the wild-type strain, while resistance to 0.5 mM CuSO4 and 1.0 mM CuSO4 was 10 times stronger than that of EGD-e. Copper ions, as heavy metal ions, are not only commonly used antibacterial disinfectants in daily life, but also play a crucial role in bacterial infection of host cells. The host's innate immune system utilizes the bactericidal properties of copper ions to create a high-concentration copper ion environment to kill intracellular pathogens. Therefore, bacterial tolerance to copper ions is one of the keys to their virulence within host cells. Copper ion stress enhances virulence-related genes in bacteria, thereby increasing the host's colonization and spread within cells. Furthermore, some bacterial copper resistance genes may be synergistically regulated with lipoprotein biosynthesis, which could enhance the bacteria's ability to evade the host's innate immunity and may also indirectly activate PrfA by affecting glutathione metabolism, thus increasing bacterial pathogenicity.

[0059] YxeA deletion strains do not significantly affect bacterial antibiotic sensitivity or cell membrane permeability, which is consistent with recent findings. ​ The discrepancies in protein-related research records may be due to differences in the genetic background of strains. Strains from different sources may carry additional resistance genes and mutations, masking the effects of the target gene. Specific resistance phenotypes may be influenced by other gene regulation or compensatory mechanisms. Significant differences in resistance patterns exist among different Listeria species, possibly related to genomic background. Differences in serotype and genotype between clinically or food-derived strains may also lead to different resistance phenotypes.

[0060] Bacterial motility in the environment greatly affects their survival. Some bacteria can form flagella to aid in movement. In experiments related to bacterial motility... ​ The impact of the missing element is quite significant, with Δ at 30℃. ​ The diameter of the motility ring was significantly increased compared to the wild-type plant. Transmission electron microscopy revealed results consistent with motility tests. At 30℃, Δ ​ The number and length of flagella were both higher in the wild-type strain, which may alter the bacterial adhesion patterns and invasion efficiency under low-temperature conditions. In addition, flagellar motility may promote early attachment of bacteria to environmental surfaces (such as food processing equipment), thereby forming biofilms, enhancing environmental tolerance and reducing the risk of transmission. ] Bacterial biofilm formation is also an important virulence characteristic. Enhanced flagellated motility helps bacteria survive and spread better in the environment. However, unexpectedly, at 37°C, Δ ​It still possesses some motility, and under transmission electron microscopy, the bacteria exhibit flagella, unlike Listeria monocytogenes under normal conditions. When the bacteria enter the host through the food chain, the expression of flagella is shut down when the temperature rises to 37°C. This reduces the host's immune recognition of the bacteria, thus preventing them from being cleared by the immune system and enhancing their survival ability within the host. The shutdown of flagellar expression allows the bacteria to concentrate their metabolic resources on the expression of other key virulence factors, completing the transition from environmental survival to host infection. Flagella, as the bacterial organ of locomotion, contain a large number of immunogenic molecules (such as flagellin), which can be recognized by the host's immune system (such as the TLR5 receptor) and trigger an inflammatory response. ​ The expression of flagellar genes at 37°C suggests that this gene affects flagellar gene transcription. Does flagellar gene transcription consume certain metabolic resources, affecting virulence gene expression and leading to reduced virulence when bacteria infect host cells? Does the continuous expression of flagella make bacteria more easily recognized by the host immune system? And does the growth of flagella mean that bacteria gain stronger motility within the host, thereby increasing their ability to migrate between cells? This invention focuses on Listeria monocytogenes. ​ The biological functions of genes in vitro, experimental results show Δ ​ Without altering bacterial cell membrane permeability and antibiotic sensitivity, Δ ​ The bacteria exhibited a 10-fold increase in resistance to copper ion stress, and their motility was significantly enhanced at 30°C, with an increased number of flagella observed under transmission electron microscopy. At 37°C, the bacteria retained motility, and flagellated growth was observed under transmission electron microscopy. ​ It participates in the formation of flagella in Listeria monocytogenes.

[0061] Example 2 1. Materials and Methods 1.1 Cells, experimental animals and their culture conditions The L929 cells used in this invention were purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. They are a commercially available, mature standard cell line, stored in liquid nitrogen in our laboratory, and cultured under conditions following the American College of Cell Bank (ATCC) protocol. Specific culture conditions are as follows: (1) Caco-2 cells: human colon cancer epithelial cells, cultured in RPMI 1640 medium containing 10% FBS, in a cell culture incubator at 37°C with 5% CO2, and digested with EDTA when plated; (2) L929 cells: a mouse fibroblast cell line, cultured in DMEM medium containing 10% FBS in a 5% CO2, 37°C cell culture incubator, and digested with EDTA when plated; 1.2 Reagent Ordering and Preparation DMEM cell culture medium, RPMI 1640 cell culture medium, Fetal Bovine Serum (FBS), Trypsin-EDTA (0.25%), phenol red: Thermo Fisher Scientific; Low melting point agarose: Sigma-Aldrich, Inc. Sterile defibrinated sheep blood: Nanjing Senbeijia Biotechnology Co., Ltd.

[0062] Reagent preparation: (1) DMEM medium containing 10% FBS: Mix 5 mL FBS with 45 mL DMEM medium, store at 4℃, and preheat in a 37℃ water bath before use. (2) RPMI 1640 medium containing 10% FBS: Mix 5 mL FBS with 45 mL RPMI 1640 medium, store at 4℃, and preheat in a 37℃ water bath before use. (3) 2.8% low melting point agarose: Weigh 11.2 g of low melting point agarose powder that has been stored at room temperature, make up to 400 mL with ddH2O, autoclave and then place in a 60℃ oven for later use. (4) 0.7% low melting point agar covering solution: Mix 13 mL DMEM with 2 mL FBS and 2 μL gentamicin (10 mg / mL), add 5 mL 2.8% low melting point agarose and mix quickly. Keep warm at 50℃ for later use. (5) Cell lysis buffer: Add 10 mL of 0.25% trypsin to 40 mL of sterile ddH2O and mix well to obtain cell lysis buffer. Store at 4℃ for later use. (6) 5% sheep blood: Take sterile defibrinated sheep blood, mix well and add 1 mL to each sterile EP tube. Centrifuge at 4℃ and 1000 rpm for 12 min. Discard the supernatant, wash 2-3 times with physiological saline, add 800 µL of physiological saline and mix well. Take 400 µL of the washed sheep blood and add 7600 µL of physiological saline to obtain 5% sheep blood. (7) 0.9% NaCl solution: Weigh 0.9 g NaCl, dissolve it with ddH2O and make up to 100 mL; (8) pH=6.5 10 mM PBS solution: Measure 100 mL of 100 mM PBS solution and add 800 mL of ddH2O to mix well. Adjust the pH to 7.4 using HCl and NaOH, and then add ddH2O to make up to 1 L.

[0063] 1.3 Test Instruments Carbon dioxide cell incubator: Thermo Fisher Scientific Electric pipette: Renin Company, USA; Optical inverted microscope: Olympus Corporation, Japan; Biosafety cabinet: Suzhou Jingantai Laboratory Equipment Co., Ltd.; Protein electrophoresis apparatus: Bio-Rad Laboratories; Ice maker: Amasia Corporation, USA.

[0064] 1.4 Bacterial migration assay in L929 cells (1) Resuscitate L929 cells using DMEM medium containing 10% FBS according to step (1) in Chapter 1.4, and then in 6-well plates at 2×10⁻⁶ cells per well. 6 Plating at a density of cells / well, and incubating overnight at 37°C in a 5% CO2 incubator; (2) Adjust the bacterial culture to OD according to step (5) in 1.4 of this chapter. 600nm =0.6, mix the bacterial culture with DMEM medium to make the final bacterial concentration 2×10. 7 CFU / mL; (3) Remove the cell plate, wash it twice with PBS preheated to 37°C, add 2 mL of bacterial solution to each well and infect for 1 h. Remove the cell plate every 15 min and gently shake to mix the culture medium. (4) Remove the cell plate, wash it twice with PBS, add DMEM medium containing 50 µg / mL gentamicin, and put it back into the cell culture incubator to continue culturing for 1 h; (5) Remove the cell plate, wash it twice with PBS, add agar covering solution, wait for the agar to solidify, and invert the six-well plate in the cell culture incubator for 48 h. (6) Add 600 µL of 10% formaldehyde fixative to the sample wells of the cell plate for fixation. After placing it in a 37℃ incubator for 1 h, take it out, invert it to shake off the low melting point agar, and place the cell plate in a 60℃ drying oven for 15 min to remove moisture. (7) Add 300 µL of 1% crystal violet staining solution to each well, gently shake the six-well plate to make it uniform, stain at room temperature for 1 h, rinse with ddH2O, take a picture, import the picture into Photoshop, count the diameter and number of blank spots, use GraphPad to create a diameter and number bar chart, and perform data difference analysis at the same time.

[0065] 1.7 Data Processing and Analysis All experimental data requiring data analysis in this embodiment are analyzed according to the content described in Embodiment 1.

[0066] 2. Experimental Results 2.1 Bacterial absence ​ Enhanced in vitro hemolytic ability after gene therapy This invention is based on ​ The accuracy of the assay for verifying the non-hemolytic ability of the deletion strain was determined by comparing EGD-e, ​ To investigate the destructive ability of mutant strains on sheep blood cells ​ The effect of genes on the in vitro hemolytic ability of Listeria monocytogenes. Results are as follows: ​ As shown, Δ ​ The hemolytic ability of [the substance] differs from that of EGD-e by two gradients, as measured by OD [measurement] in the supernatant. 550nm Plotting the hemolysis curve at the absorbance value at the location revealed Δ ​ The hemolytic activity was significantly higher than that of the wild-type EGD-e (Figure B, ***). P <0.001), indicating ​ The deletion enhanced the hemolytic activity of Listeria monocytogenes.

[0067] 2.2 Bacterial absence ​ The gene enhances migration ability in L929 cells. The intercellular migration ability of Listeria monocytogenes is one of the key factors in its pathogenicity. This invention explores this by comparing the size of plaques formed when L929 cells are infected with the bacteria. ​ The effect of Listeria monocytogenes on cell migration. Results are as follows: ​ As shown, statistical analysis revealed that Δ ​ The diameter of the resulting vacuoles was 0.38 mm larger than that of the wild-type plant (**). P <0.01), indicating ​ Gene deletion leads to enhanced intercellular migration ability of Listeria monocytogenes.

[0068] This invention is to investigate the absence of Listeria monocytogenes. ​ The ability of the gene to escape from phagosomes was tested in vitro, and the results showed that... ​ Gene deletion enhances the in vitro hemolytic activity of Listeria monocytogenes. The main executor of this in vitro hemolytic activity is listeriolysin O (LLO), which directly mediates lysosomal escape and intercellular diffusion of bacteria within host cells. ​ The gene encodes PrfA, which activates the PrfA-box promoter region of the target gene. ​ The expression of hemolysin O plays a role, which is crucial for the virulence of Listeria monocytogenes.

[0069] This invention uses mouse fibroblast L929 cells to observe the ability of bacteria to migrate between cells. The experimental results show that... ​ The deletion significantly increased the intercellular migration ability of bacteria. After Listeria monocytogenes internalized into intestinal epithelial cells, it escaped the phagosome membrane through LLO perforation. In vitro hemolysis assays showed that... ​The absence of LLO (liquid perforin) enhances bacterial perforation, increasing its efficiency in disrupting phagosome membranes and allowing bacteria to escape more quickly into the host cytoplasm. In the cytoplasm, bacteria recruit actin, forming an "actin tail" that propels them through the cytoplasm and diffuses into neighboring cells via membrane protrusions. Since flagella aid bacterial locomotion when other pathogens infect host cells, the inventors hypothesized that Δ... ​ Under these conditions, the bacteria still generate flagella, increasing their motility and thus enhancing the host's ability to migrate between cells. Δ ​ It exhibits enhanced in vitro hemolytic activity and significantly improved intercellular migration ability compared to the wild-type strain.

[0070] Example 3 1.1 Test Reagents Methanol: Xilong Technology Co., Ltd.; Protein maker, β-mercaptoethanol, tricarboxymethylaminomethane: SIGMA-ALDRICH; Sodium dodecyl sulfate (SDS), Tween 20, column-based bacterial total RNA extraction and purification kit, Goat Anti-Mouse lgG (H+L) HRP-tag, Goat Anti-Rabbit lgG (H+L) HRP-tag: Shanghai Bioengineering Co., Ltd. BCA Protein Concentration Assay Kit: Beyotime Biotechnology Co., Ltd. Skim milk powder: Shanghai Yuanye Biotechnology Co., Ltd.; Taq Pro Universal SYBR QPCR Master Mix: TaKaRa.

[0071] Reagent preparation: (1) 10×SDS-PAGE buffer: Weigh 144 g g glycine, 30.3 g Tris and 10 g SDS, and make up to 1000 mL with ddH2O. Dilute with ddH2O before use. (2) 5% blocking solution: Weigh 2 g of skim milk powder, dissolve it in 40 mL of ddH2O, mix well and then use.

[0072] (3) 20 mg / mL lysozyme solution: Weigh 0.14 g of lysozyme powder, add ddH2O to make up to 7 mL, the final concentration is 20 mg / mL, sterilize with a 0.22 µm filter, and store at -20℃; (4) Listeria monocytogenes lysate: Prepare a solution containing 2% TritionX-100, 1% SDS, 100mM sodium chloride, 10mM Tris-HCl and 1mM EDTA with ddH2O, and adjust the pH to 8.0. (5) 10×TBS: Weigh 87.66 g of sodium chloride and 12.114 g of Tris-base, dissolve them in ddH2O, adjust the pH to 8.0, and bring the volume to 500 mL. When using, take 100 mL of the solution, dilute it tenfold, and add 1 mL of Tween 20 to prepare 1×TBST buffer.

[0073] 1.2 Test Instruments Thermostatic water bath: Amasia Inc., USA; Electric pipette: Renin Company, USA; Ultrasonic cell disruptor: Ningbo Xinzhi Bio-instrument Co., Ltd.; Fully automated rapid sample grinding instrument: Shanghai Jingxin Biotechnology Co., Ltd. Blood cell counting chamber: Hangzhou Karl Biotechnology Co., Ltd.

[0074] 1.3 Reaction System Configuration and Procedure Table 9 HiScript III All-in-one RT SuperMix Perfect for qPCR Reaction System and Procedure

[0075] Table 10 2×Taq Pro Universal SYBR qPCR Master Mix Reaction System

[0076] Table 11 2 × Taq Pro Universal SYBR qPCR Master Mix Reaction Program

[0077] 1.4 Determination of transcriptional levels of flagella-related genes in bacteria under different temperature conditions (1) EGD-e, Δ ​ Strand the bacteria on a BHI plate to regenerate them, then inoculate them into BHI liquid medium and incubate at 37°C for 8 hours. After centrifuging 1 mL of the bacterial culture at 5000 rpm for 3 min, wash twice with PBS and adjust the bacterial concentration to OD using PBS. 600 nm It is 0.6. (2) Transfer the above bacterial suspensions at a ratio of 1:100 to two bacterial bottles containing 10 mL of BHI liquid culture medium, and transfer one group of EGD-e and Δ ​ One group was placed in a shaker at 37℃ and 200 rpm, while the other group was placed in a shaker at 30℃ and 200 rpm, and cultured with shaking for 5 h. (3) Pipette 2 mL of bacterial solution into a sterile EP tube, centrifuge at 12,000 rpm for 1 min to collect the bacterial solution, discard the supernatant, and wash 2-3 times with 1 mL of DEPC water. (4) Discard the supernatant, add 120 µL of 20 mg / mL lysozyme, vortex to mix, and let stand at room temperature for 10 min. (5) Add 1 mL of Trizol, invert and shake, and let stand for 10 min after shaking evenly; (6) Add 200 µL of chloroform, shake well, let stand for 3 min, and then centrifuge at 4℃ and 12,000 rpm for 15 min; (7) Carefully aspirate 600 µL of the supernatant after centrifugation and add it to an RNase-free EP tube. Add 500 µL of isopropanol to the tube, let it stand on ice for 10 min, and then centrifuge at 4°C and 12,000 rpm for 10 min. (8) Carefully discard the supernatant, add DEPC water containing 75% ethanol, shake well, and then centrifuge at 4°C and 12,000 rpm for 10 min. Repeat this operation twice. (9) After discarding the supernatant, air dry in a clean bench for 10 min to remove residual ethanol. Add 20 µL of DEPC water to resuspend and shake. The resulting liquid is RNA. Use a multi-functional microplate reader to detect the concentration and purity of RNA. (10) Use the RNA reverse transcription kit to reverse transcribe RNA into cDNA according to the procedure in Table 9, and store the cDNA at -20℃ for later use. (11) Primers for quantitative fluorescence of flagella-related genes were designed using Primer Premier 5.0; (12) Gene transcription levels in cDNA were determined using an Agilent fluorescence quantitative PCR instrument. The results were analyzed using 2... -ΔΔct The calculation was performed using the method described above. GraphPad 8.0 was used to convert the obtained -ΔΔCT values ​​into relative folds, and a histogram of gene transcription levels was plotted.

[0078] 1.5 RNA extraction after bacterial infection of cells (1) Resuscitate Caco-2 cells according to the method steps in 1.4 of Example 2 and culture them to the optimal adherent state, according to 2 × 10⁶ cells per well. 6 The cells were seeded at a density of 100 cells in a six-well plate, shaken to mix, and then incubated overnight at 37°C in a 5% CO2 cell culture incubator. (2) Obtain OD according to the method steps in 1.4(1) of this embodiment. 600 nm The bacterial solution was 0.6. (3) After removing the cell plate, wash it twice with sterile 10 mM PBS, and add 1.9 mL of preheated RPMI 1640 cell culture medium and 100 µL of bacterial solution with adjusted concentration to each well, i.e., infect according to the MOI=100:1 ratio. (4) After 1 h, the culture medium was replaced with RPMI 1640 cell culture medium containing 50 µg / mL gentamicin, and after 2 h, it was replaced with RPMI 1640 cell culture medium containing 5 µg / mL gentamicin. (5) After 4 h, wash twice with DEPC water, add 2 mL of DEPC water pre-cooled at 4℃ to each well, and use a cell scraper to completely scrape off the cells on the cell plate and transfer them to a sterile EP tube; (6) Shake and break the liquid in the EP tube for 3 min. Centrifuge the broken liquid at 4℃ and 600 rpm for 3 min, collect the supernatant, centrifuge at 4℃ and 12,000 rpm for 3 min, and discard the supernatant. (7) Add 100 µL of lysozyme, shake well and let stand at room temperature for 10 min. The subsequent steps are the same as those in 1.4 (5-12) of this chapter.

[0079] 1.6 Extraction of whole bacterial protein after bacterial culture at different temperatures (1) Obtain OD according to the method steps in 1.4(1) of this embodiment. 600 nm The bacterial culture was 0.6; it was then transferred at a ratio of 1:100 to two identical sets of bacterial bottles containing 10 mL of BHI liquid medium. (2) Place one set of bacterial bottles in a shaker at 30℃ and 200 rpm. Place the other set in a shaker at 37℃ and 200 rpm and incubate with shaking for 5 h; (3) Transfer 2 mL of bacterial culture to an EP tube, centrifuge at 12,000 rpm for 1 min, wash twice with 1 mL of 10 mM PBS, add 500 µL of Listeria monocytogenes lysis buffer, and shake well. (4) Ultrasonic disruption: Each tube of bacterial culture is ultrasonically disrupted until it becomes transparent. Each ultrasonication is performed on ice. (5) Centrifuge at 4℃ and 12,000 rpm for 10 min, and collect the supernatant, which is the whole bacterial protein.

[0080] 1.7 Extraction of whole bacterial proteins after bacterial infection of cells (1) Infect and treat cells according to the steps in 1.5 (1-4) of this embodiment; (2) Remove the cell plate, wash twice with 10 mM PBS, add homemade cell lysis buffer, lyse on ice for 10 min, then blow the cells down thoroughly and transfer them to EP tubes, shake to break them up for 3 min. (3) Centrifuge at 4℃ and 600 rpm for 3 min, take the supernatant, and proceed according to step 1.6 (3-5) of this embodiment.

[0081] 1.8 Protein Concentration Determination (1) Weigh 10 mg BSA, add 400 µL of protein standard preparation solution, vortex mix until BSA is completely dissolved to obtain a protein stock solution of 25 mg / mL, and dilute it 50 times with the standard preparation solution to obtain the protein standard. (2) Calculate the required volume of BCA working solution for the protein to be measured, add reagent A and reagent B in a ratio of 50:1, and shake thoroughly to mix. (3) Add 0, 1, 2, 4, 8, 16 and 20 µL of protein standards to the first row of the 96-well plate in sequence. If the amount is less than 20 µL, add 10 mM PBS to make up the difference. (4) Add 2 µL of the protein to be tested to the second row of the 96-well plate, and make up to 20 µL with 18 µL of 10 mM PBS; (5) Add 180 µL of well-mixed BCA working solution and place the 96-well plate in a 60℃ incubator for 15 min. (6) Remove the 96-well plate and measure the OD using a microplate reader. 595nm A standard curve is created using the values ​​obtained from the first row. The values ​​measured from the protein samples in the second row are then substituted into the standard curve for conversion to obtain the sample concentration.

[0082] 1.9 Protein Immunoblotting Assay (1) Take out the protein sample for quantification. Add 4×Protein loading to the quantified target protein at a ratio of 3:1, mix well, and then briefly separate. Place it in boiling water and boil for 10 min. (2) Take out the protein sample, briefly separate it, take out the pre-prepared 12% protein gel, and add the protein sample; (3) Set the protein electrophoresis apparatus voltage to 80 V and run for 30 min. After the samples have run to the separating gel and are level, set the voltage to 120 V and run for 1 h. (4) Use a rapid wet transfer apparatus to perform rapid protein transfer. Place the cut gel into 10 mL of equilibration solution, cut PVDF membranes of the same size into 10 mL of methanol, and immerse the PVDF membranes in equilibration solution when removing them. (5) Open the transfer clamp, place a dry sponge pad inside, first place the activated PVDF film on top of the sponge pad, then place the adhesive on the film, being careful not to create air bubbles; place another dry sponge pad on top of it, and then close the transfer clamp. (6) Place the transfer clamp in the correct lane, ensure sufficient transfer buffer, and set the program to start the transfer. (7) After the transfer is completed, block with TBST solution containing 5% skim milk powder for 2 h; (8) Then wash 3 times with 1×TBST, 5 minutes each time; (9) Incubate the corresponding primary antibody (dilution ratio 1:1000) at 4°C overnight, and repeat step (8); (10) Incubate the corresponding secondary antibody (dilution ratio 1:5000) at room temperature for 45 min, and repeat step (8); (11) HRP chemiluminescent solution was incubated in the dark for 2 min, and immunoblotting was performed using a chemiluminescence imager.

[0083] 1.10 Data Processing and Analysis All experimental data requiring data analysis in this embodiment are analyzed in accordance with the content described in the embodiment.

[0084] 2. Experimental Results 2.1 Missing at 37℃ ​ bacteria ​ and ​ Transcription levels significantly decreased Listeria monocytogenes was measured under the condition of no other external stimuli. ​ Changes in the transcriptional levels of virulence-related genes EGD-e after deletion compared to wild-type strains. Results are as follows: ​ As shown, under the condition of 30℃ ​ After missing ​ and ​ Upregulation of gene transcription (Log2 fold change > 1) at 37°C ​ , ​ , ​ , ​ , ​ , ​ Transcriptional levels were upregulated (Log2 fold change > 1). ​ , ​ Transcription was downregulated 14-16 times. This result suggests... ​ The absence of these substances alters the bacteria's ability to adhere and invade.

[0085] 2.2 Deletion at 30℃ and 37℃ ​ Increased bacterial flagellar gene transcription levels According to the results of Example 1, under 30℃ conditions ​ Gene deletion promotes flagellar growth in Listeria monocytogenes, even at 37°C where flagella should not normally grow. However, it remains unknown which flagellar genes Listeria monocytogenes regulates to promote flagellar expression. Therefore, the inventors investigated the effects of different temperature conditions. ​Changes in the transcriptional levels of flagellar genes after deletion. Results are as follows: ​ As shown, under 30℃ conditions, except for the flagellar negative regulatory factor ​ Except for no significant changes, the transcriptional levels of other flagellated genes were upregulated (Log2 fold change > 1), with the total flagellated promoter showing the most significant changes. ​ Increased by 40 times, related to flagellar matrix ​ and ​ Transcriptional levels were upregulated by 26-fold and 35-fold, respectively. Transcriptional levels of both genes were downregulated at 37°C (Log2 fold change < -2). ​ The transcriptional level of [specific gene name] did not change significantly, while the transcriptional levels of most other flagellated genes were upregulated 4-8 times. These results suggest that culture conditions at 30℃ and 37℃ [are suitable for transcriptional expression]. ​ Deletion of both genes led to increased transcription levels of flagellar genes, which was consistent with the increase in Δ under culture conditions of 30℃ and 37℃. ​ The results were consistent with the enhancement of their external motor abilities.

[0086] 2.3 Under the conditions of 30℃ and 37℃ ​ The absence of expression of bacterial flagella promotes the expression of flagella. Wild plants and Δ were extracted under different temperature conditions ​ The expression of key flagellar genes was observed using a protein immunoblotting assay based on whole bacterial proteins. The results are as follows: ​ As shown, at 30℃, the expression levels of the flagellated negative regulators MogR and GmaR were significantly decreased, and the expression level of FlaA was significantly increased; at 37℃, only the expression level of MogR was significantly decreased. These results suggest that... ​ The transcription and expression of flagellar synthesis-related genes are controlled by positively regulating MogR or interacting with MogR, thereby regulating flagellar formation.

[0087] 2.4 When Caco-2 cells are infected with bacteria ​ Upregulation of flagellar gene transcription levels According to the experimental results in Example 2, ​ The transcriptional levels of adhesion factors InlA and InlB in Listeria monocytogenes were reduced. The inventors speculate that this may be because the expression of flagella at 37°C induces a host innate immune response, or that flagellar expression preempts metabolic resources, leading to the downregulation of key adhesion and invasion factors InlA and InlB. This invention first detects Δ... ​ Changes in the transcriptional level of flagellar genes after bacterial infection of cells, as shown in the following results. ​ As shown, the flagellated negative regulator binds to the promoter region once 37°C is reached. ​ Gene downregulation can interact with ​ Combined with preventing its inhibition of flagellar expression​ Gene and flagellate total promoter ​ Transcription levels were upregulated by about 8-fold.

[0088] 2.5 When Caco-2 cells are infected with bacteria ​ Deletion of virulence gene transcription level downregulation This experiment detects Δ ​ Changes in the transcriptional levels of virulence genes after infection of Caco-2 cells, as shown in the following results. ​ As shown, ​ , ​ , ​ , ​ , ​ , ​ The downregulation of all genes by 2-4 times indicates that the transcriptional level of virulence genes decreases when flagellar genes are expressed in the host. This suggests that in the early stages of infection, the expression of flagellar genes leads to a decrease in the transcriptional level of virulence genes, thereby weakening infectivity.

[0089] According to the results of Examples 1 and 2, Δ ​ In vitro motility and hemolytic activity were significantly enhanced, and the number of flagella formed increased. Enhanced flagellar formation and hemolytic activity are influenced by multiple factors; this embodiment first validates these effects in vitro. ​ Whether genes affect the transcription and expression of Listeria monocytogenes endovirulence genes and flagellar genes was clearly observed in immunofluorescence blotting experiments. At 30°C, the expression level of the flagellar negative regulator MogR decreased, while the expression levels of GmaR and FlaA increased; at 37°C, only the expression level of MogR decreased. Flagellar gene transcription results showed that under culture conditions of 30°C and 37°C... ​ Deletion of both genes led to increased transcription levels of flagellar genes, which was consistent with the increase in Δ under culture conditions of 30℃ and 37℃. ​ The results showed consistent enhancement in in vitro motility and increased flagellar formation. Transcription results of the virulence gene indicated that, under in vitro conditions at 30°C, ​ After missing ​ and ​ The transcriptional level of genes was significantly upregulated at 37°C. ​ , ​ , ​ , ​ , ​ , ​ Transcriptional levels were significantly upregulated. ​ , ​ Transcription was downregulated 14-16 times. This is related to Δ ​ The result corresponds to the enhanced in vitro hemolytic ability.

[0090] In most bacteria, flagellar expression within the host significantly impacts virulence, as motile flagella facilitate bacterial penetration of the host barrier and colonization. However, Listeria monocytogenes infection is influenced by the host's internal environment; it does not express flagella at 37°C. This is a beneficial sacrificial strategy for the bacteria, avoiding a host-induced immune response that kills them and conserving metabolic resources for other virulence factors to function effectively. This example detected Δ... ​ Changes in the transcription levels of bacterial virulence genes and flagellar genes after cell infection. The experimental results showed that... ​ The flagellated negative regulator that binds to the promoter region once the temperature reaches 37°C after deletion. ​ Gene downregulation can interact with ​ Combined with preventing its inhibition of flagellar expression ​ Gene and flagellate total promoter ​ Transcriptional levels were upregulated by approximately 8-fold, indicating that Δ ​ Flagella are expressed within the host. ​ , ​ , ​ , ​ , ​ , ​ The downregulation of all genes by 2-4 times indicates that the transcriptional levels of some virulence genes decrease when flagellar genes are expressed in the host. This may be because flagellar proteins are important factors in triggering the host's innate immune response, and enhanced flagellar expression can promote the recognition of Listeria monocytogenes by host cells. In order to adapt to the intracellular environment, Listeria monocytogenes selectively reduces the expression of related genes such as InlA and InlB.

[0091] In summary, this invention has found that at 37°C, Δ ​ High expression of flagellar genes inhibits core virulence-determining genes such as ​ , ​ , ​ , ​ , ​ , ​ Transcription and expression of [certain substances], however, results from the cell infection model showed that... ​ Gene deletion promotes bacterial intercellular migration and its ability to infect cells. This near-paradoxical result suggests that Δ ​ The infection process relies not only on traditional core virulence factors, but also on flagella, which play a crucial role in the infection process. ​ The resulting flagellar synthesis transforms Listeria monocytogenes from its initial cell-adhering and invasive behavior to its evasion of host cell immune recognition. Specifically, the expression of flagellar Δ... ​The mutant can enhance the host cell's immune recognition of Listeria monocytogenes. This result suggests that antibacterial infection can be achieved by modifying bacterial flagella formation. This lays the research foundation for subsequent development of antimicrobial peptides and other formulations. It is known that... ​ At 30°C in vitro, the transcription and expression levels of flagellar gene positive regulators were significantly upregulated, while the transcription levels of virulence genes showed no significant difference. At 37°C in vitro, flagellar gene transcription was significantly upregulated, MogR expression was downregulated, and virulence gene expression was significantly downregulated. ​ , ​ Transcription downregulated 14-16 times; under intracellular conditions of 37°C, ​ , ​ Transcription levels were upregulated 8-10 times, while transcription levels of virulence genes decreased.

Claims

1. A Listeria monocytogenes strain, characterized in that, The strain exhibits enhanced flagellar formation ability and resistance to Cu. 2+ Enhanced environmental stress response can improve the host cell's immune recognition of Listeria monocytogenes; the strain exhibits a ΔyxeA gene deletion mutation.

2. The Listeria monocytogenes strain according to claim 1, characterized in that, The strain is named Listeria monocytogenes Lm-Δlmo2568, with accession number CGMCC No.36001, and is deposited at the China General Microbiological Culture Collection Center on September 22, 2025.

3. The Listeria monocytogenes strain according to claim 2, characterized in that, The nucleotide sequence of the yxeA gene is shown in SEQ ID No.

1.

4. A method for constructing a single Listeria monocytogenes strain, characterized in that, The strain is the strain according to any one of claims 1 to 3, and the construction method includes the following methods: (1) Take 463bp upstream of yxeA as homologous arm A and 475bp downstream of yxeA as homologous arm B. Design primers to add the selected restriction sites BamHI and KpnI. (2) After linking the upstream and downstream fragments, the fragments were integrated into the shuttle thermosensitive plasmid pKSV7 via enzyme digestion and linking. (3) The obtained plasmid was transformed into the EGD-e competent cells of wild-type Listeria monocytogenes, and homologous recombination was carried out under chloramphenicol resistance conditions and at a temperature of 42°C. (4) After successful homologous recombination, the temperature-sensitive plasmid pKSV7 was discarded at a temperature of 30°C, and the strain was finally obtained.

5. The construction method according to claim 4, characterized in that, In step (2), the upstream and downstream homologous arms are amplified by PCR, and the two fragments are fused by overlap PCR. Then, restriction endonucleases and high-efficiency ligases are used to ligate the fused homologous arm fragments and the temperature-sensitive plasmid pKSV7.

6. The construction method according to claim 5, characterized in that, In step (3), the enzyme-ligated product is added to E. coli DH5α competent cells, mixed thoroughly, and then heat-shocked in a metal bath at 42°C for 90 seconds. Afterward, 900 μL of medium is added, and the mixture is incubated at 37°C and 200 rpm for 45 minutes. Then, the mixture is centrifuged at 5000 rpm for 3 minutes, resuspended in 100 μL of medium, and plated on LB agar plates containing ampicillin resistance to screen for positive transformants. After verification by PCR and DNA sequencing, the transformed cells are purified, and the resulting plasmid is named pSL2190. A single purified colony is then inoculated into 10 mL of medium, and HLingene plasmid extraction is performed. The recombinant plasmid was extracted using a kit; then, the recombinant plasmid was added to Listeria monocytogenes EGD-e competent cells, mixed thoroughly, and incubated on ice for 30 min. Transformation was performed using an electric shock at 2500 V, 200 Ω, 25 μF, and 2 mm. 900 μL of BHI medium containing sucrose was added, and the mixture was incubated at 30 °C for 2 h. Finally, the bacterial suspension was centrifuged at 5000 rpm for 3 min, resuspended, and plated on BHI plates containing chloramphenicol resistance to screen for positive transformants. After PCR sequencing verification, positive single colonies were picked and inoculated into BHI liquid medium containing resistance. The culture was then passaged at 42 °C and 200 rpm, with PCR verification performed every 5 generations.

7. The construction method according to any one of claims 4 to 6, characterized in that, In step (4), after the homologous arm fragments are completely homologously recombined, they are transferred to BHI medium and placed in a shaker at 30°C and 180 rpm for further subculturing until the pKSV7 plasmid is completely lost.

8. The construction method according to claim 7, characterized in that, The construction method, after step (4), further includes the following steps: streaking the above bacterial solution on a BHI plate and culturing it overnight at 30°C; picking single colonies and picking them on a BHI plate without antibiotic resistance and a BHI plate containing chloramphenicol resistance respectively; placing the picked plates in a 30°C incubator and letting them stand for 24 hours; selecting single colonies without chloramphenicol resistance; verifying them by PCR and DNA sequencing; adding 60% sterile glycerol; and storing at -80°C.

9. The construction method according to claim 8, characterized in that, The method for preparing the EGD-e competent cells of the wild-type Listeria monocytogenes strain is as follows: The wild-type Listeria monocytogenes strain to be used for competent cell preparation is recovered from a streak plate at -80℃. A single colony is picked and inoculated into a bacterial bottle containing 5 mL of BHI, and cultured overnight at 37℃ with shaking at 200 rpm. The overnight culture is then inoculated at a ratio of 1:100 into a conical flask containing 100 mL of BHI liquid medium with a final concentration of 0.5 M sucrose, and cultured under the same conditions until the bacterial culture reaches OD. 600nm The concentration of the bacteria reached 0.18-0.

25. 100 mg / mL penicillin G was added to the conical flask at a ratio of 1:5000, and the mixture was incubated in a shaker at 37°C for 2 hours. The conical flask was removed and placed on ice for 10 minutes. The bacterial suspension was aliquoted into 50 mL centrifuge tubes and centrifuged at 4°C and 6000 rpm for 7 minutes, discarding the supernatant. The bacterial pellet was gently resuspended in 30 mL of pre-chilled washing buffer containing 50 mM sucrose and 50 mM HEPEs at 4°C. The pellet was centrifuged at 4°C and 6000 rpm for 7 minutes, discarding the supernatant. This washing process was repeated 2-3 times. After discarding the supernatant, 1 mL of HEPEs was added to resuspend the bacterial pellet. The pellet was then aliquoted into 100 μL tubes pre-chilled at 4°C into 1.5 mL EP tubes to obtain the wild-type Listeria monocytogenes EGD-e competent cells. These cells were stored at -80°C for later use.

10. The use of the strain according to any one of claims 1 to 3 or the strain constructed by the construction method according to claims 4 to 9 in the preparation of an agent that enhances the immune recognition of host cells against Listeria monocytogenes.

Images