Wild-type ST87 listeria monocytogenes deleted strain and preparation method thereof

Abstract

The invention discloses a wild type ST87 listeria monocytogenes gene deletion strain and a preparation method thereof, relates to the technical field of gene knockout, and particularly discloses a construction method of four gene knockout strains of wild type ST87 listeria monocytogenes, and the four gene knockout strains are respectively delta actA, delta hly, delta inlA and delta inlP mutant strains; by constructing the gene knockout strain, the difference between the wild strain and the knockout strain in the infection process can be compared, so that the specific effect of the gene in the listeria monocytogenes infection process is determined.

Description

Technical Field

[0001] The present invention relates to the technical field of gene knockout, and particularly relates to a wild-type ST87 Listeria monocytogenes gene knockout bacterium and a preparation method thereof. Background Art

[0002] Listeria monocytogenes, simply referred to as L. monocytogenes, is a typical foodborne pathogenic bacterium with frequent global hazard events. The main disease manifestations are meningitis, septicemia, abortion, monocytosis and other symptoms, especially posing serious hazards to pregnant women, the elderly, children, newborns and immunocompromised patients. From 1964 to 2010 for more than 40 years, a total of 147 cases of L. monocytogenes infection were reported in China. However, only from 2011 to 2017, 562 cases of infection were reported, far higher than the number of cases reported in the past forty years, and the mortality rate was as high as over 20%. From the analysis of clinical outbreak data, the isolation proportion of L. monocytogenes ST87 (15.38%) is relatively high. Therefore, from the perspective of China's epidemiological data, L. monocytogenes has become a major hidden danger threatening people's lives and health.

[0003] Pathogenic L. monocytogenes can cross the intestinal barrier, blood-brain barrier and placental barrier, and then infect host cells. Each step of L. monocytogenes infecting the host is regulated by specific virulence factors. InlA is a unique virulence factor that can mediate the adhesion and entry of bacteria into host cells during the infection process of L. monocytogenes. InlP is a secreted protein in the internalin family of L. monocytogenes and has a unique crystal structure. Current studies have shown that it is related to the invasion of the placenta. Listeriolysin O (LLO) is a member of the cholesterol-dependent cytolysins (CDCs) family, encoded by the hly gene, which can promote the formation of primary phagosomes and plays an important role in L. monocytogenes escaping into the cytoplasm. In addition, L. monocytogenes can mediate its migration within and between cells by using ActA. Therefore, studying the gene knockout L. monocytogenes can better explore the roles played by these virulence factors in the process of L. monocytogenes infecting the host.

[0004] The methods for bacterial gene knockout mainly include the following: 1. Gene editing techniques (such as CRISPR / Cas9): Using specific enzymes to cut and modify DNA sequences to delete or modify target genes. 2. Homologous recombination: Constructing the upstream and downstream homologous arms of the target gene and a selection marker gene into a suitable vector, and then introducing the vector into the bacterial cell to replace or delete the target gene. 3. Chemical mutagenesis: Treating bacteria with chemical substances to cause gene mutations and thus disrupt the function of the gene. 4. Transposon: Using transposon elements to insert foreign DNA into the host genome to disrupt the function of the gene. 5. Mutation accumulation: Culturing bacteria under a series of conditions to allow the bacteria to mutate spontaneously, and finally screening out strains with deletion or variation of the target gene. Among them, homologous recombination is a technique for directly constructing knockout or site-directed mutations of target genes and is also the main method for most current studies to construct bacterial knockout strains.

[0005] At present, the construction of Listeria monocytogenes gene deletion strains using homologous recombination technology has been relatively mature, but there are still some problems. 1. The genome of Listeria monocytogenes is relatively large and complex, and the frequency of homologous recombination may be relatively low, resulting in low efficiency in obtaining gene knockout strains. Often, a large number of transformants need to be screened to find the correct gene knockout mutants, which increases the workload and time cost of the experiment. 2. During homologous recombination, two homologous recombination events are required to achieve precise gene knockout and remove the selection marker. However, the probability of the second recombination event usually is lower than the first, making it difficult to obtain a completely marker-free gene knockout strain. 3. The length and sequence of the homologous arms are crucial for the efficiency and accuracy of homologous recombination. If the homologous arms are too short, it may lead to low recombination efficiency or non-specific recombination; while too long homologous arms may increase the difficulty and cost of constructing recombinant vectors. In addition, the sequence selection of the homologous arms also needs to avoid high similarity with other regions in the genome to prevent mismatching and unexpected recombination. 4. There may be certain differences in the genomic sequences of different Listeria monocytogenes strains. Even for the same gene, the sequences and regulatory elements may vary in different strains. Therefore, the homologous arms designed for a certain strain may not work well in other strains, and need to be optimized and adjusted for different strains. 5. During homologous recombination, the knockout of certain genes may affect the normal growth and reproduction of bacteria, and may even change the virulence or growth characteristics of Listeria monocytogenes, bringing difficulties to the cultivation and preservation of gene knockout strains, and may also affect the accurate evaluation of their phenotypes and functions. 6. Even if a gene knockout strain is successfully obtained, during subsequent cultivation and passage, there may be a phenomenon of reversion of the gene knockout mutation, that is, the knocked-out gene regains or partially restores its function. This may be caused by genomic instability, residual gene fragments that have not been completely removed, or other unknown factors, posing challenges to the reliability and stability of experimental results. 7. Gene knockout may cause changes in other regions of the Listeria monocytogenes genome, such as changes in chromosome structure, gene expression regulation, etc. These changes may gradually accumulate with passage, leading to changes in the genetic background of the strain, and thus affecting the accurate judgment of gene knockout effects and gene functions. Summary of the Invention

[0006] In view of this, the main object of the present invention is to provide a method for constructing a wild-type ST87 Listeria monocytogenes gene deletion strain to solve the problems existing in the prior art.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] A method for constructing a wild-type ST87 Listeria monocytogenes gene deletion strain, comprising the following steps:

[0009] S1. Using the genomic DNA of wild-type ST87 Listeria monocytogenes as a template, amplify the upstream homologous arm fragment and downstream homologous arm fragment of the deleted gene; the deleted genes include the actA gene, hly gene, inlA gene, and inlP gene; among them, the wild-type ST87 Listeria monocytogenes is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, classified as Listeria monocytogenes, with the preservation number CGMCC No. 33595 and the preservation date of March 5, 2025;

[0010] S2. Connect and fuse the upstream homologous arm fragment and downstream homologous arm fragment of the corresponding deleted gene to obtain a fused fragment of the upstream and downstream homologous arms;

[0011] S3. Using the fused fragment as a template, amplify the fused fragment with the vector sequence;

[0012] S4. Perform double digestion on the pLR16-pheS* plasmid with kpnI and XhoI restriction endonucleases;

[0013] S5. Connect the fused fragment obtained in step S3 with the vector pLR16-pheS*, then transfer it into Escherichia coli DH5α competent cells, coat it on an LB-ChL plate (chloramphenicol ChL is 25 μg / ml) for culture and screening. After sequencing and comparison, obtain the recombinant plasmid of the corresponding deleted gene;

[0014] S6. Mix the prepared recombinant plasmid with the thawed Listeria monocytogenes competent cells. After electroporation treatment, add BHI-sucrose solution and incubate on ice for 20 - 40 min to obtain the electroporated solution;

[0015] S7. Incubate the solution obtained in step S6 at 25 - 35 °C for 2.5 - 3.5 h, centrifuge to remove the upper layer solution, mix the remaining bacterial solution, and coat and inoculate it on a BHI-Chl solid medium, that is, a sterilized BHI solid medium containing chloramphenicol (10 μg / mL). After culturing at 25 - 35 °C for 40 - 60 h, obtain single colonies;

[0016] S8. Perform PCR verification on the obtained single colonies to obtain positive transformants, inoculate them into preheated BHI-Chl liquid medium, and repeat continuous passage culture at 42 °C for 2 - 3 times;

[0017] S9. Inoculate the cultured bacterial solution into BHI liquid medium and culture it overnight at 30 °C; then dilute it and spread it on a BHI-chlorobenzene plate, culture it overnight at 37 °C, and select multiple single colonies on the BHI-chlorobenzene plate for detection to obtain the wild-type ST87 Listeria monocytogenes gene deletion strain.

[0018] According to steps S1 - S9, four gene - knockout strains can be prepared correspondingly: ΔactA, Δhly, ΔinlA and ΔinlP mutants. By constructing gene - knockout strains, the differences between wild - type strains and knockout strains during the infection process can be compared, so as to clarify the specific role of genes during the infection process of Listeria monocytogenes.

[0019] The ST87 - type gene - knockout strains constructed by this method can be stably inherited and do not affect bacterial growth and other characteristics.

[0020] Furthermore, in step S1,

[0021] When the deleted gene is the actA gene, the primers for amplifying the upstream homologous - arm fragment of the actA gene are up - actA.F and up - actA.R. The nucleotide sequence of up - actA.F is as shown in SEQ ID NO: 1, and the nucleotide sequence of up - actA.R is as shown in SEQ ID NO: 2. The primers for amplifying the downstream homologous - arm fragment of the actA gene are down - actA.F and down - actA.R. The nucleotide sequence of down - actA.F is as shown in SEQ ID NO: 3, and the nucleotide sequence of down - actA.R is as shown in SEQ ID NO: 4;

[0022] And / or when the deleted gene is the hly gene, the primers for amplifying the upstream homologous - arm fragment of the hly gene are up - hly.F and up - hly.R. The nucleotide sequence of up - hly.F is as shown in SEQ ID NO: 5, and the nucleotide sequence of up - hly.R is as shown in SEQ ID NO: 6. The primers for amplifying the downstream homologous - arm fragment of the hly gene are down - hly.F and down - hly.R. The nucleotide sequence of down - hly.F is as shown in SEQ ID NO: 7, and the nucleotide sequence of down - hly.R is as shown in SEQ ID NO: 8;

[0023] And / or when the deleted gene is the inlA gene, the primers for amplifying the upstream homologous - arm fragment of the inlA gene are up - inlA.F and up - inlA.R. The nucleotide sequence of up - inlA.F is as shown in SEQ ID NO: 9, and the nucleotide sequence of up - inlA.R is as shown in SEQ ID NO: 10. The primers for amplifying the downstream homologous - arm fragment of the inlA gene are down - inlA.F and down - inlA.R. The nucleotide sequence of down - inlA.F is as shown in SEQ ID NO: 11, and the nucleotide sequence of down - inlA.R is as shown in SEQ ID NO: 12;

[0024] When the missing gene is the inlP gene, the primers for amplifying the upstream homologous arm fragment of the inlP gene are up-inlP.F and up-inlP.R. The nucleotide sequence of up-inlP.F is shown in SEQ ID NO: 13, and the nucleotide sequence of up-inlP.R is shown in SEQ ID NO: 14. The primers for amplifying the downstream homologous arm fragment of the inlP gene are down-inlP.F and down-inlP.R. The nucleotide sequence of down-inlP.F is shown in SEQ ID NO: 15, and the nucleotide sequence of down-inlP.R is shown in SEQ ID NO: 16.

[0025] Furthermore, in step S2,

[0026] When the missing gene is the actA gene, the primers for ligation and fusion include 2up-actA.F, 2up-actA.R, 2down-actA.F, and 2down-actA.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 17-20;

[0027] When the missing gene is the hly gene, the primers for ligation and fusion include 2up-hly.F, 2up-hly.R, 2down-hly.F, and 2down-hly.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 21-24;

[0028] When the missing gene is the inlA gene, the primers for ligation and fusion include 2up-inlA.F, 2up-inlA.R, 2down-inlA.F, and 2down-inlA.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 25-28;

[0029] When the gene is the inlP gene, the primers for ligation and fusion include 2up-inlP.F, 2up-inlP.R, 2down-inlP.F, and 2down-inlP.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 29-32.

[0030] Furthermore, in step S3,

[0031] When the missing gene is the actA gene, the primers for amplifying the fusion fragment with the vector sequence are actA-16.F and actA-16.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 33-34;

[0032] When the missing gene is the hly gene, the primers for amplifying the fusion fragment with the vector sequence are hly-16.F and hly-16.R, and the corresponding nucleotide sequences are shown in SEQ ID NO: 35-36;

[0033] When the missing gene is the inlA gene, the primers for amplifying the fusion fragment with the vector sequence are inlA-16.F and inlA-16.R, and the corresponding nucleotide sequences are shown in SEQ ID NO: 37-38;

[0034] When the missing gene is the inlP gene, the primers for amplifying the fusion fragment with the vector sequence are inlP-16.F and inlP-16.R, and the corresponding nucleotide sequences are shown in SEQ ID NO: 39-40.

[0035] Further, in step S5, in the LB-ChL plate, the chloramphenicol content is 25 μg / mL;

[0036] In step S6, the mixing ratio of the recombinant plasmid to the competent cells of Listeria monocytogenes is 1:8 - 12. After mixing, perform ice bath for 5 - 10 min, and then perform electrotransformation; when performing electrotransformation, the voltage is 2.5 kV and the electrotransformation time is 5 ms.

[0037] Further, in step S6,

[0038] The preparation of the competent cells of Listeria monocytogenes includes the following steps:

[0039] Inoculate the cryopreserved (-80 °C) Listeria monocytogenes strain by streaking on a sterilized BHI solid medium, and place it in a constant temperature incubator at 37 °C overnight;

[0040] Pick a single colony from the medium into 50 mL of sterile BHI liquid medium, and culture it at 37 °C, 180 rpm / min until OD 600 = 0.6;

[0041] Absorb the above bacterial solution and add it to the BHI-sucrose solution, and culture it in a shaker at 37 °C for 4 - 5 h. When the OD 600 value is 0.18 - 0.25, add penicillin G solution to the bacterial solution, and culture it in a shaker at 37 °C for 2 h; the mixing ratio of the bacterial solution to the BHI-sucrose solution is 1:45 - 55, the mixing ratio of the bacterial solution to the penicillin G solution is 1 mL:15 - 25 μL, and the concentration of the penicillin G solution is 50 mg / mL;

[0042] When the OD 600 value reaches 0.5 - 0.6, place it on ice and add it to a sterilized centrifuge tube, and centrifuge at 4 °C, 8000 rpm / min for 10 min;

[0043] Aspirate the upper layer solution, wash the precipitate with HEPES-sucrose solution 2-3 times, and centrifuge at 8000 rpm / min at 4°C for 10 min;

[0044] Aspirate the upper layer solution, resuspend the precipitate with HEPES-sucrose solution, mix with lysozyme solution, and place in a constant temperature water bath at 37°C for 20 min;

[0045] Centrifuge at 8000 rpm / min at 4°C for 10 min;

[0046] Aspirate the upper layer solution, wash the precipitate with HEPES-sucrose solution twice, and centrifuge at 8000 rpm / min at 4°C for 10 min;

[0047] Aspirate the upper layer solution, add HEPES-sucrose solution to resuspend the bacterial cell precipitate, transfer it to a sterilized cryopreservation tube, and store it at -80°C for later use.

[0048] Further, in step S9,

[0049] When the deleted gene is the actA gene, the primers for detecting single colonies include up-actA.F, down-actA.R, actA.F, and actA.R. The nucleotide sequence of actA.F is shown in SEQ ID NO: 41, and the nucleotide sequence of actA.R is shown in SEQ ID NO: 42;

[0050] When the deleted gene is the hly gene, the primers for detecting single colonies include up-hly.F, down-hly.R, hly.F, and hly.R. The nucleotide sequence of hly.F is shown in SEQ ID NO: 43, and the nucleotide sequence of hly.R is shown in SEQ ID NO: 44;

[0051] When the deleted gene is the inlA gene, the primers for detecting single colonies include up-inlA.F, down-inlA.R, inlA.F, and inlA.R. The nucleotide sequence of inlA.F is shown in SEQ ID NO: 45, and the nucleotide sequence of inlA.R is shown in SEQ ID NO: 46;

[0052] When the deleted gene is the inlP gene, the primers for detecting single colonies include up-inlP.F, down-inlP.R, inlP.F, and inlP.R. The nucleotide sequence of inlP.F is shown in SEQ ID NO: 47, and the nucleotide sequence of inlP.R is shown in SEQ ID NO: 48.

[0053] Specifically, the present invention provides a method for constructing a wild-type ST87 Listeria monocytogenes actA gene deletion strain:

[0054] (1) Select conventional antibiotics, such as chloramphenicol, ampicillin, etc., to conduct drug sensitivity tests on wild-type Listeria monocytogenes strains to determine the sensitivity of wild-type Listeria monocytogenes to different antibiotics, so as to select appropriate antibiotics for subsequent screening steps. In the present invention, it is verified that chloramphenicol is used for subsequent screening;

[0055] (2) Using wild-type Listeria monocytogenes genomic DNA as a template, construct the gene recombinant plasmid pLR16-pheS*-ΔactA:

[0056] Using wild-type Listeria monocytogenes genomic DNA as a template, amplify the upstream and downstream homologous arm fragments of the actA gene with primers up-actA.F / up-actA.R and down-actA.F / down-actA.R;

[0057] Using the upstream and downstream homologous arm fragments of the actA gene as templates, amplify the upstream and downstream fragments with about 20 bp homologous arms with primers 2up-actA.F / 2up-actA.R and 2down-actA.F / 2down-actA.R respectively, and then use this as a template for Overlap PCR to amplify the fusion fragment of the upstream and downstream homologous arms of the actA gene;

[0058] Using the fusion fragment AB as a template, amplify the fusion fragment 2AB with about 20 bp homologous arms of the vector with primers AB-16.F / R;

[0059] Use kpnI and XhoI restriction endonucleases to perform double digestion on the pLR16-pheS* plasmid;

[0060] Use a seamless cloning kit to ligate the fusion fragment 2AB with the vector pLR16-pheS*, and then transfer it into Escherichia coli DH5α competent cells;

[0061] The transformed cells are spread on an LB-ChL25ug / mL plate for culture and screening. After sequencing and comparison, the recombinant plasmid pLR16-pheS*-ΔactA is obtained;

[0062] (3) Prepare the gene knockout strain ΔactA:

[0063] Take the cryopreserved Listeria monocytogenes competent cells and place them on an ice box for slow thawing;

[0064] After complete thawing, take about 10 μL (1 - 2 μg) of pLR16 - pheS* - ΔactA and transfer it into 100 μL of competent cells, gently and slowly mix them, and incubate on ice for 5 - 10 min;

[0065] Transfer it to a 2 - mm electroporation cuvette using a pipette, apply a voltage of 2.5 kV for 5 ms, immediately add 1 mL of BHI - sucrose solution under sterile conditions after electroporation, and incubate on ice for 30 min;

[0066] Transfer the solution after electroporation into a sterilized 2 - mL centrifuge tube, incubate at 30 °C for 3 h, then centrifuge at 8000 rpm / min for 10 min, aspirate about 800 μL of the supernatant, gently and slowly mix the remaining bacterial solution, and evenly spread and inoculate it onto a sterilized BHI solid medium containing chloramphenicol (10 μg / mL). After culturing at about 30 °C for about 48 h, single colonies are obtained;

[0067] Use primers 2up - actA.F / 2down - actA.R to verify the transformants by colony PCR. After obtaining positive transformants, inoculate them into a pre - warmed 10 - mL BHI - Chl 10 μg / mL liquid medium, culture at 42 °C until the strain reaches the stationary phase, then dilute it 1 / 1000 with 10 mL of pre - warmed BHI - Chl 10, and reach the stationary phase at 42 °C. Repeat continuous sub - culture 2 - 3 times;

[0068] For each sub - culture, Listeria monocytogenes is cultured on a pre - warmed (42 °C) BHI - Chl 10 agar plate and incubated at 42 °C.

[0069] Inoculate the above bacterial solution into 3 mL of BHI liquid medium and culture overnight at 30 °C to eliminate the exogenous plasmid;

[0070] Dilute the culture to 10 -4 , spread 100 μL on each of the BHI - chlorobenzene, BHI + Chl 10, and BHI plates, and culture overnight at 37 °C; after culturing, the colonies on the BHI plate are significantly more than those on the BHI - chlorobenzene plate, and there is no colony growth on the BHI + Chl 10 plate;

[0071] Select 8 single colonies from the BHI - chlorobenzene plate, use primers up - actA.F / down - actA.R and primers actA.F / R to detect the upstream and downstream sequences and the target respectively, and use the wild - type as a control to obtain the deletion strain ΔactA.

[0072] The present invention also provides a method for testing the genetic stability of the deletion strain ΔactA, including:

[0073] The deletion strain ΔactA was inoculated into BHI medium and continuously passaged for about ten generations. The wild strain and the deletion strain were identified by PCR using the primer pairs up-actA.F / down-actA.R and actA.F / R to determine the genetic stability of ΔactA.

[0074] It can be understood that according to the above steps, other gene deletion strains can be correspondingly obtained. Of course, the corresponding primers can be adjusted.

[0075] The primer sequences used in the present invention are shown in Table 1 below:

[0076]

[0077]

[0078]

[0079] The second object of the present invention is to provide the gene recombinant plasmids obtained by any of the above construction methods. The gene recombinant plasmids include pLR16-pheS*-ΔactA, pLR16-pheS*-Δhly, pLR16-pheS*-ΔinlA, and pLR16-pheS*-ΔinlP.

[0080] The third object of the present invention is to provide the Listeria monocytogenes gene deletion strains obtained by any of the above construction methods. The Listeria monocytogenes gene deletion strains include ΔactA, Δhly, ΔinlA, and ΔinlP mutant strains.

[0081] The present invention also aims to provide the application of the above Listeria monocytogenes gene deletion strains in studying the infection of Listeria monocytogenes to hosts.

[0082] In the present invention, Escherichia coli DH5α was cultured in LB medium, and Listeria monocytogenes was cultured in BHI medium. The use concentrations of chloramphenicol were 25 μg / mL and 10 μg / mL respectively.

[0083] LB medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride 10 g / L.

[0084] LB solid medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride 10 g / L, Agar 15 g / L.

[0085] BHI medium: Peptone 10.0 g / L, Dehydrated calf brain infusion powder 12.5 g / L, Dehydrated beef heart infusion powder 5.0 g / L, Sodium chloride 5.0 g / L, Glucose 2.0 g / L, Disodium hydrogen phosphate 2.5 g / L, pH value 7.4 ± 0.2.

[0086] BHI solid medium: Peptone 10.0 g / L, dehydrated calf brain extract powder 12.5 g / L, dehydrated beef heart extract powder 5.0 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L, agar 15 g / L, pH value 7.4 ± 0.2.

[0087] Penicillin G solution (50 mg / mL): Accurately weigh 0.05 g of sodium penicillin G and dissolve it in 1 mL of ddH2O. After complete mixing, filter it using a filter (0.22 μm) under sterile conditions, and dispense it into 2 mL sterilized EP tubes. Note that it needs to be prepared and used immediately.

[0088] HEPES-sucrose solution: Accurately weigh 0.0714 g of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and 51.3 g of sucrose and dissolve them in 300 mL of sterilized ddH2O. Completely dissolve and mix, adjust the pH value to approximately 7.0, filter it using a filter (0.22 μm), and store it in a 4°C refrigerator for later use.

[0089] The beneficial effects of the present invention at least include:

[0090] (1) The length and sequence of the homologous arms of the corresponding gene designed in the present invention have high homologous recombination efficiency and accuracy, which can prevent non-specific recombination of the wild-type ST87 strain, and prevent mismatching and unexpected recombination.

[0091] (2) The recombinant plasmid of the corresponding gene prepared and synthesized in the present invention can better provide homologous sequences for the corresponding gene of the wild-type ST87 strain, guide the plasmid and the host bacterium genome to undergo homologous recombination at the corresponding position, making the subsequent gene knockout process have targeting and specificity. At the same time, the resistance gene of the wild-type ST87 strain is also marked, which greatly improves the screening efficiency and facilitates quickly finding the strains that have undergone homologous recombination from a large number of host bacteria.

[0092] (3) The plasmid of the corresponding gene constructed in the present invention is electrotransformed into the prepared competent cells of wild-type ST87 Listeria monocytogenes. The electrotransformation conditions are optimized, and antibiotic plate screening and memory PCR verification are used to prepare the corresponding gene deletion strain. The corresponding gene deletion strain prepared by this method can grow and reproduce normally without affecting its growth characteristics. In addition, during the subsequent culture and passage processes, there will be no phenomenon of back-complementation of gene knockout mutations. Description of the Drawings

[0093] Figure 1Recombinant plasmid map of the corresponding genes of wild-type ST87 Listeria monocytogenes constructed for the present invention; (A) pLR16-pheS*-ΔactA; (B) pLR16-pheS*-Δhly; (C) pLR16-pheS*-ΔinlA; (D) pLR16-pheS*-ΔinlP.

[0094] Figure 2 Agarose gel electrophoresis map of PCR products of gene deletion strains ΔactA (A), Δhly (B), ΔinlA (C), and ΔinlP (D) of wild-type ST87 Listeria monocytogenes. Note: M: DL5000 Marker, lane 1 is the PCR amplification product of the original strain, lanes 2-3 are the PCR amplification products of the deletion strains, and lane 4 is negative.

[0095] Figure 3 Agarose gel electrophoresis map of upstream and downstream PCR products after the gene deletion strains ΔactA (A), Δhly (B), ΔinlA (C), and ΔinlP (D) of wild-type ST87 Listeria monocytogenes were continuously passaged for dozens of generations. Note: M: DL5000 Marker, lane 1 is the upstream and downstream PCR amplification products of the original strain, the last lane in each figure is negative, and the middle lanes are the upstream and downstream PCR amplification products of the deletion strains. Detailed implementation manners

[0096] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0097] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement it. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0098] Unless otherwise specified, the materials and reagents used in the present invention can be obtained from commercial products in the art. The wild-type ST87 Listeria monocytogenes used in the present invention was isolated from the laboratory of the inventor, and it is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC No. 33595 and the preservation date of March 5, 2025; the used Escherichia coli DH5α competent cells and plasmid pLR16-pheS* were purchased from Ningbo Taisituo Biotechnology Co., Ltd.

[0099] The following specifically illustrates the solutions proposed by the present invention through specific examples:

[0100] Construction of the corresponding gene deletion strain of wild-type ST87 Listeria monocytogenes in Example 1

[0101] 1. Construction of recombinant plasmid

[0102] Using the genomic DNA of wild-type ST87 Listeria monocytogenes as a template, amplify the upstream and downstream homologous arm fragments of the corresponding gene, and use this as a template for Overlap PCR to amplify the fusion fragment of the upstream and downstream homologous arms of the corresponding gene. Using the fusion fragment AB as a template, amplify the fusion fragment 2AB with homologous arms of about 20 bp of the vector. The primers used are shown in Table 1.

[0103] Double digest the pLR16-pheS* plasmid with kpnI and XhoI restriction endonucleases, ligate the fusion fragment 2AB with the vector pLR16-pheS* using a seamless cloning kit, and then transfer it into competent Escherichia coli DH5α cells. The transformed cells are spread on an LB-ChL25ug / ml plate for culture and screening. After sequencing and comparison, the recombinant plasmid is obtained.

[0104] The specific experimental steps are as follows:

[0105] 1.1 Construction of pLR16-pheS*-ΔactA

[0106] 1) Using the genomic DNA of wild-type Listeria monocytogenes as a template, amplify the upstream and downstream homologous arm fragments of the actA gene with primers up-actA.F / up-actA.R and down-actA.F / down-actA.R.

[0107] 2) Using the upstream and downstream homologous arm fragments of the actA gene as templates, amplify the upstream and downstream fragments with homologous arms of about 20 bp with primers 2up-actA.F / 2up-actA.R and 2down-actA.F / 2down-actA.R respectively, and then use this as a template for Overlap PCR to amplify the fusion fragment AB of the upstream and downstream homologous arms of the actA gene.

[0108] 3) Using the fusion fragment AB as a template, amplify the fusion fragment 2AB with homologous arms of about 20 bp of the vector with primers actA-16.F / R.

[0109] 4) Double digest the pLR16-pheS* plasmid with kpnI and XhoI restriction endonucleases.

[0110] 5) Ligate the fusion fragment 2AB with the vector pLR16-pheS* using a seamless cloning kit, and then transfer it into competent Escherichia coli DH5α cells.

[0111] 6) The transformed cells were spread on an LB - ChL 25 μg / mL plate for culture and screening. After being sent for sequencing and comparison, the recombinant plasmid pLR16 - pheS* - ΔactA was obtained.

[0112] 1.2 Construction of pLR16 - pheS* - Δhly

[0113] 1) Using the wild - type genomic DNA of Listeria monocytogenes as a template, the upstream and downstream homologous arm fragments of the hly gene were amplified with primers up - hly.F / up - hly.R and down - hly.F / down - hly.R.

[0114] 2) Using the upstream and downstream homologous arm fragments of the hly gene as templates, the upstream and downstream fragments with approximately 20 bp homologous arms were amplified with primers 2up - hly.F / 2up - hly.R and 2down - hly.F / 2down - hly.R respectively. Then, using these as templates, an overlapping (Overlap) PCR was performed to amplify the fused fragment AB of the upstream and downstream homologous arms of the hly gene.

[0115] 3) Using the fused fragment AB as a template, the fused fragment 2AB with homologous arms of approximately 20 bp of the vector was amplified with primers hly - 16.F / R.

[0116] 4) The pLR16 - pheS* plasmid was double - digested with restriction enzymes kpnI and XhoI.

[0117] 5) The fused fragment 2AB was ligated to the vector pLR16 - pheS* using a seamless cloning kit, and then transferred into Escherichia coli DH5α competent cells.

[0118] 6) The transformed cells were spread on an LB - ChL 25 μg / mL plate for culture and screening. After being sent for sequencing and comparison, the recombinant plasmid pLR16 - pheS* - Δhly was obtained.

[0119] 1.3 Construction of pLR16 - pheS* - ΔinlA

[0120] 1) Using the wild - type genomic DNA of Listeria monocytogenes as a template, the upstream and downstream homologous arm fragments of the inlA gene were amplified with primers up - inlA.F / up - inlA.R and down - inlA.F / down - inlA.R.

[0121] 2) Using the upstream and downstream homologous arm fragments of the inlA gene as templates, amplify the upstream and downstream fragments with approximately 20 bp homologous arms using primers 2up-inlA.F / 2up-inlA.R and 2down-inlA.F / 2down-inlA.R respectively. Then, use this as a template for overlap PCR to amplify the fusion fragment AB of the upstream and downstream homologous arms of the inlA gene.

[0122] 3) Using the fusion fragment AB as a template, amplify the fusion fragment 2AB with approximately 20 bp homologous arms of the vector using primers inlA-16.F / R.

[0123] 4) Double digest the pLR16-pheS* plasmid with the restriction enzymes kpnI and XhoI.

[0124] 5) Use a seamless cloning kit to ligate the fusion fragment 2AB with the vector pLR16-pheS*, and then transform it into competent Escherichia coli DH5α cells.

[0125] 6) Spread the transformed cells on an LB-ChL25ug / ml plate for culture and screening. After sequencing and comparison by sending for detection, obtain the recombinant plasmid pLR16-pheS*-ΔinlA.

[0126] 1.4 Construction of pLR16-pheS*-ΔinlP

[0127] 1) Using the wild-type genomic DNA of Listeria monocytogenes as a template, amplify the upstream and downstream homologous arm fragments of the inlP gene using primers up-inlP.F / up-inlP.R and down-inlP.F / down-inlP.R.

[0128] 2) Using the upstream and downstream homologous arm fragments of the inlP gene as templates, amplify the upstream and downstream fragments with approximately 20 bp homologous arms using primers 2up-inlP.F / 2up-inlP.R and 2down-inlP.F / 2down-inlP.R respectively. Then, use this as a template for overlap PCR to amplify the fusion fragment AB of the upstream and downstream homologous arms of the inlP gene.

[0129] 3) Using the fusion fragment AB as a template, amplify the fusion fragment 2AB with approximately 20 bp homologous arms of the vector using primers inlP-16.F / R.

[0130] 4) Double digest the pLR16-pheS* plasmid with the restriction enzymes kpnI and XhoI.

[0131] 5) The fusion fragment 2AB was ligated to the vector pLR16-pheS* using a seamless cloning kit and then transformed into competent Escherichia coli DH5α cells.

[0132] 6) The transformed cells were spread on an LB-ChL 25 μg / ml plate for culture and screening. After sequencing and comparison by sending for detection, the recombinant plasmid pLR16-pheS*-ΔinlP was obtained.

[0133] 1.5 Sequencing results of the recombinant plasmid

[0134] The constructed plasmid pLR16-pheS*-ΔactA was 9030 bp in size, pLR16-pheS*-Δhly was 8980 bp in size, pLR16-pheS*-ΔinlA was 9123 bp in size, and the constructed plasmid pLR16-pheS*-ΔinlP was 8945 bp in size. It contained a chloramphenicol resistance gene and could be used for the screening of subsequent transformants. The maps of all recombinant plasmids are shown in Figure 1 .

[0135] 2. Construction of gene deletion strains

[0136] Investigation index: The agarose gel electrophoresis pattern of the PCR product showed that the wild-type ST87 strain had the corresponding gene band while the gene deletion strain did not.

[0137] 2.1 Experimental design

[0138] 2.1.1 Preparation of competent cells of wild-type ST87 Listeria monocytogenes

[0139] The Listeria monocytogenes strain stored at -80 °C was streaked onto a sterilized BHI solid medium and incubated at 37 °C overnight. Pipette 1 mL of the cultured bacterial solution with OD 600 = 0.6 and add it to 50 mL of BHI-sucrose solution, and culture it in a shaker at 37 °C for 4 - 5 h. When the OD 600 value was about 0.18 - 0.25 (not greater than 0.25), add 20 μL of penicillin G solution (50 mg / mL) to the bacterial solution and culture it in a shaker at 37 °C for about 2 h. When the OD 600When the value is about 0.5 - 0.6, place it in ice and add it separately to 50 mL sterilized centrifuge tubes, and centrifuge at 4°C, 8000 rpm / min for 10 min. Aspirate the upper layer solution, wash the precipitate once with 50 mL and 25 mL of HEPES - sucrose solution respectively, and centrifuge at 4°C, 8000 rpm / min for 10 min. Aspirate the upper layer solution, gently and slowly resuspend the precipitate with 5 mL of HEPES - sucrose solution, add 15 μL of lysozyme solution and mix, and place it in a 37°C constant temperature water bath for 20 min. Centrifuge at 4°C, 8000 rpm / min for 10 min. Aspirate the upper layer solution, wash the precipitate twice with 30 mL of HEPES - sucrose solution respectively, and centrifuge at 4°C, 8000 rpm / min for 10 min. Aspirate the upper layer solution, add 1 mL of HEPES - sucrose solution again and slowly and fully resuspend the cell precipitate, and transfer it into 2 mL sterilized cryotubes respectively, and store it at -80°C for later use.

[0140] 2.1.2 Preparation and screening of gene - deleted transformants

[0141] Take the frozen - preserved competent cells of Listeria monocytogenes and thaw them slowly on an ice box. After complete thawing, transfer about 10 μL (1 - 2 μg) of the corresponding gene recombinant plasmid into 100 μL of competent cells; gently and slowly mix during the process and incubate on ice for 5 - 10 min. Use a pipette to transfer it into a 2 mm electroporation cuvette, apply a voltage of 2.5 kV, and electroporate for 5 ms. Immediately after electroporation, add 1 mL of BHI - sucrose solution under sterile conditions and incubate on ice for 30 min. Transfer the electroporated solution into a sterilized 2 mL centrifuge tube, incubate at 30°C for 3 h, then centrifuge at 8000 rpm / min for 10 min, aspirate about 800 μL of the upper layer solution, gently and slightly mix the remaining bacterial solution, and evenly spread and inoculate it on a sterilized BHI solid medium containing chloramphenicol (10 μg / mL). Incubate at about 30°C for about 48 h to obtain single colonies. Use primer pairs to verify the transformants by colony PCR to obtain positive transformants.

[0142] 2.1.3 Elimination of foreign plasmids

[0143] Inoculate the positive transformants into a pre - warmed 10 mL BHI - chl 10 μg / mL liquid medium, culture at 42°C until the strain reaches the stationary phase, then dilute it 1000 - fold with 10 mL of pre - warmed BHI - Chl10, reach the stationary phase at 42°C, and repeat the continuous sub - culture 3 times. For each sub - culture of Listeria monocytogenes, plate it on a pre - warmed (42°C) BHI - Chl10 agar plate and incubate at 42°C. Inoculate the above bacterial solution into 3 mL of BHI liquid medium and culture overnight at 30°C to eliminate foreign plasmids.

[0144] 2.1.4 Preparation of gene deletion strain

[0145] Dilute the cell concentration of the culture by 10 -4 times, and spread 100 μL on each of the BHI-chlorobenzene, BHI+Chl10 and BHI plates, and culture overnight at 37 °C. After culturing, the colonies on the BHI plate were significantly more than those on the BHI-chlorobenzene plate, and no colonies grew on the BHI+Chl10 plate. Select 8 single colonies on the BHI-chlorobenzene plate, and use the corresponding primers (see Table 1) to detect the upstream and downstream sequences and the target gene respectively, with the wild type as a control. The agarose gel electrophoresis pattern of the PCR products showed that the wild-type ST87 strain had the corresponding gene band while the gene deletion strain did not, indicating that the gene deletion strain was successfully constructed. The agarose gel electrophoresis pattern of the PCR products of the wild-type ST87 Listeria monocytogenes and the corresponding gene deletion strain is shown in Figure 2 . The agarose gel electrophoresis pattern of the PCR products showed that the wild-type ST87 strain had the corresponding gene band while the gene deletion strain did not, indicating that the corresponding gene deletion strain was successfully prepared in this example.

[0146] 3. Identification of genetic stability of gene deletion strain

[0147] Investigation index: The agarose gel electrophoresis pattern of the PCR products showed that the corresponding gene band was missing after the deletion strain was passaged in culture.

[0148] 3.1 Experimental design

[0149] Inoculate the deletion strain into BHI medium, and continuously passage and culture for ten generations. Use the corresponding primers (see Table 1) to perform PCR identification on the wild strain and the deletion strain. The agarose gel electrophoresis pattern of the PCR products showed that the wild-type ST87 strain had the corresponding gene band while the gene deletion strain did not, indicating that the gene deletion strain had genetic stability and no reversion phenomenon was found. The agarose gel electrophoresis pattern of the upstream and downstream PCR products after continuous passage for ten generations is shown in Figure 3 , and the agarose gel electrophoresis pattern of the PCR products after the deletion strain was passaged in culture showed that the corresponding gene band was missing, further indicating that the gene deletion strain had excellent genetic stability and no reversion phenomenon was found.

[0150] Comparative Example 1

[0151] The difference between this example and Example 1 is as follows: 1. In the step of plasmid construction, the plasmid used is pskv7, and the rest remains the same.

[0152] In this example, four gene deletion strains were successfully constructed using the same steps. However, in the step of identifying the genetic stability of the gene deletion strains, after the four constructed gene deletion strains ΔactA, Δhly, ΔinlA, and ΔinlP were continuously passaged for ten generations, the wild-type strain and the deletion strains were identified by PCR using the corresponding primers (see Table 1). The agarose gel electrophoresis pattern of the PCR products showed that bands were produced in all four gene deletion strains, proving that gene reversion occurred in the four gene deletion strains. Therefore, the pLR16-pheS* plasmid was re-selected for the preparation of the deletion strains and the preparation was successfully completed after verification.

[0153] Comparative Example 2

[0154] The difference between this example and Example 1 lies in that: in the step of preparing and screening the gene deletion transformants in step 2.1.2, the voltage during electroporation was 2.0 kv and the electroporation time was 3 ms; the rest remained the same.

[0155] In this example, the frozen Listeria monocytogenes competent cells were slowly thawed on an ice box. After complete thawing, about 10 μL (1 - 2 μg) of the corresponding gene recombinant plasmid was transferred into 100 μL of the competent cells; during the process, it was gently and slowly mixed, and ice-bathed for 5 - 10 min. It was transferred to a 2 mm electroporation cuvette using a pipette, and electroporated at a voltage of 2.0 kv for 3 ms. Immediately after electroporation, 1 mL of BHI-sucrose solution was added under sterile conditions, and ice-bathed for 30 min. The solution after electroporation was transferred to a sterilized 2 mL centrifuge tube, incubated at 30 °C for 3 h, then centrifuged at 8000 rpm / min for 10 min, about 800 μL of the upper solution was aspirated, the remaining bacterial solution was gently and slowly mixed, and evenly spread and inoculated onto a sterilized BHI solid medium containing chloramphenicol (10 μg / mL). After culturing at about 30 °C for about 48 h, single colonies were obtained. The transformants were verified by colony PCR using primers. No positive transformants were obtained for all four genes. The electroporation conditions were optimized, the electroporation voltage was adjusted to 2.5 kv, and the electroporation time was 3 ms. After the above steps, the transformants were verified by colony PCR using primers. No positive transformants were obtained for all four genes. Therefore, the electroporation time was adjusted to 5 ms. After the above steps, the transformants were verified by colony PCR using primers, and positive transformants were obtained for all four genes. Subsequently, the subsequent experiments of eliminating the exogenous plasmid and preparing the gene deletion strains were continued.

[0156] It should be noted that, in this document, the terms "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or apparatus. Without further limitation, an element defined by the statement "comprising one..." does not exclude the presence of additional identical elements in the process, method, article or apparatus comprising such element.

[0157] The serial numbers of the embodiments of the present invention above are only for description and do not represent the superiority or inferiority of the embodiments.

[0158] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for constructing a wild-type ST87 Listeria monocytogenes gene-deficient strain, characterized in that: The following steps are involved: S1. Using wild-type ST87 Listeria monocytogenes genomic DNA as a template, amplifying the upstream homology arm fragment and the downstream homology arm fragment of the deleted gene; the deleted genes include actA gene, hly gene, inlA gene and inlP gene; wherein the wild-type ST87 Listeria monocytogenes is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.33595 and a deposit date of March 5, 2025; S2, connecting and fusing the upstream homology arm fragment and the downstream homology arm fragment corresponding to the deleted gene to obtain a fusion fragment of the upstream and downstream homology arms; S3, using the fusion fragment as a template, amplifying the fusion fragment with the vector sequence; S4, double digest the pLR16-pheS* plasmid with kpnI and XhoI restriction endonucleases; S5, connecting the fusion fragment obtained in step S3 with the vector pLR16-pheS*, and then transferring it into Escherichia coli DH5α competent cells, coating it on LB-ChL plates for culture and screening, and obtaining the recombinant plasmid corresponding to the deleted gene after sequencing and comparison; S6, mixing the prepared recombinant plasmid with the thawed Listeria monocytogenes competent cells, adding BHI-sucrose solution after electroporation, and ice bathing for 20-40 minutes to obtain an electroporated solution; S7, incubate the solution obtained in step S6 at 25-35° C. for 2.5-3.5 h, remove the upper solution by centrifugation, mix the remaining bacterial solution, spread it on BHI-Chl solid culture medium, and culture it at 25-35° C. for 40-60 h to obtain a single colony; S8. Perform PCR verification on the obtained single colony to obtain the positive transformant, inoculate it into preheated BHI-Chl liquid medium, and repeat continuous subculture 2-3 times at 42°C; S9. Inoculate the bacterial liquid obtained after culture into BHI liquid culture medium and culture it at 30°C overnight; then spread it on BHI-chlorobenzene plate after dilution, culture it at 37°C overnight, select multiple single colonies on the BHI-chlorobenzene plate for detection, and obtain the wild-type ST87 Listeria monocytogenes gene deletion strain.

2. The construction method according to claim 1, characterized in that: In step S1, When the deleted gene is the actA gene, the primers for amplifying the upstream homology arm fragment of the actA gene are up-actA.F and up-actA.R, the nucleotide sequence of the up-actA.F is shown in SEQ ID NO: 1, and the nucleotide sequence of the up-actA.R is shown in SEQ ID NO: 2; the primers for amplifying the downstream homology arm fragment of the actA gene are down-actA.F and down-actA.R, the nucleotide sequence of the down-actA.F is shown in SEQ ID NO: 3, and the nucleotide sequence of the down-actA.R is shown in SEQ ID NO: 4; and / or when the deleted gene is the hly gene, the primers for amplifying the upstream homology arm fragment of the hly gene are up-hly.F and up-hly.R, the nucleotide sequence of up-hly.F is shown in SEQ ID NO: 5, the nucleotide sequence of up-hly.R is shown in SEQ ID NO: 6, and the primers for amplifying the downstream homology arm fragment of the hly gene are down-hly.F and down-hly.R, the nucleotide sequence of down-hly.F is shown in SEQ ID NO: 7, and the nucleotide sequence of down-hly.R is shown in SEQ ID NO: 8; and / or when the deleted gene is the inlA gene, the primers for amplifying the upstream homologous arm fragment of the inlA gene are up-inlA.F and up-inlA.R, the nucleotide sequence of up-inlA.F is shown in SEQ ID NO: 9, the nucleotide sequence of up-inlA.R is shown in SEQ ID NO: 10, and the primers for amplifying the downstream homologous arm fragment of the inlA gene are down-inlA.F and down-inlA.R, the nucleotide sequence of down-inlA.F is shown in SEQ ID NO: 11, and the nucleotide sequence of down-inlA.R is shown in SEQ ID NO: 12; And / or when the deleted gene is the inlP gene, the primers for amplifying the upstream homologous arm fragment of the inlP gene are up-inlP.F and up-inlP.R, the nucleotide sequence of the up-inlP.F is shown in SEQ ID NO: 13, the nucleotide sequence of the up-inlP.R is shown in SEQ ID NO: 14, and the primers for amplifying the downstream homologous arm fragment of the inlP gene are down-inlP.F and down-inlP.R, the nucleotide sequence of the down-inlP.F is shown in SEQ ID NO: 15, and the nucleotide sequence of the down-inlP.R is shown in SEQ ID NO:

16.

3. The construction method according to claim 1, characterized in that: In step S2, When the deleted gene is the actA gene, the primers used for connection and fusion include 2up-actA.F, 2up-actA.R, 2down-actA.F and 2down-actA.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 17-20; When the deleted gene is the hly gene, the primers used for connection and fusion include 2up-hly.F, 2up-hly.R, 2down-hly.F and 2down-hly.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 21-24; When the deleted gene is the inlA gene, the primers used for connection and fusion include 2up-inlA.F, 2up-inlA.R, 2down-inlA.F and 2down-inlA.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 25-28; When the gene is the inlP gene, the primers used for connection and fusion include 2up-inlP.F, 2up-inlP.R, 2down-inlP.F and 2down-inlP.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 29-32.

4. The construction method according to claim 1, characterized in that: In step S3, When the deleted gene is the actA gene, the primers for amplifying the fusion fragment with the vector sequence are actA-16.F and actA-16.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 33-34; When the deleted gene is the hly gene, the primers for amplifying the fusion fragment with the vector sequence are hly-16.F and hly-16.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 35-36; When the deleted gene is the inlA gene, the primers for amplifying the fusion fragment with the vector sequence are inlA-16.F and inlA-16.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 37-38; When the deleted gene is the inlP gene, the primers for amplifying the fusion fragment with the vector sequence are inlP-16.F and inlP-16.R, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 39-40.

5. The construction method according to claim 1, characterized in that: In step S5, the chloramphenicol content in the LB-ChL plate is 25ug / mL; In step S6, the mixing ratio of the recombinant plasmid and the Listeria monocytogenes competent cells is 1:8-12, and after mixing, the mixture is placed in an ice bath for 5-10 minutes, and then subjected to electric shock treatment; the voltage during the electric shock treatment is 2.5 kV, and the electric shock time is 5 ms.

6. The construction method according to claim 1, characterized in that: In step S6, The preparation of the Listeria monocytogenes competent cells comprises the following steps: The stored Listeria monocytogenes strain was streaked into sterilized BHI solid medium and cultured at 37°C overnight; Pick a single colony from the culture medium into 50 mL of sterile BHI liquid medium and culture at 37°C, 180 rpm / min until OD 600 =0.6; The above bacterial solution was added to the BHI-sucrose solution and cultured in a shaker at 37°C for 4-5 hours. 600 When the value is 0.18-0.25, add penicillin G solution to the bacterial solution and culture it in a shaker at 37°C for 2 hours; the mixing ratio of the bacterial solution to the BHI-sucrose solution is 1:45-55, the mixing ratio of the bacterial solution to the penicillin G solution is 1ml:15-25μL, and the concentration of the penicillin G solution is 50mg / mL; Waiting for OD 600 When the value reaches 0.5-0.6, add it to a sterilized centrifuge tube in ice and centrifuge at 4°C, 8000 rpm / min for 10 min. The upper solution was aspirated, and the precipitate was washed 2-3 times with HEPES-sucrose solution, and centrifuged at 4°C, 8000 rpm / min for 10 min; The upper layer solution was aspirated, and the precipitate was re-blown with HEPES-sucrose solution, and then the lysozyme solution was added to mix, and the mixture was placed in a constant temperature water bath at 37°C for 20 min. By centrifugation at 4°C, 8000 rpm / min for 10 min; The upper solution was aspirated, and the precipitate was washed twice with HEPES-sucrose solution and centrifuged at 4°C, 8000 rpm / min for 10 min; The upper solution was aspirated, and HEPES sucrose solution was added to blow up the bacterial precipitate, which was then placed into sterilized cryopreservation tubes and stored at -80°C for later use.

7. The construction method according to claim 1, characterized in that: In step S9, When the deleted gene is the actA gene, the primers for detecting a single colony include up-actA.F, down-actA.R, actA.F and actA.R, the nucleotide sequence of the actA.F is shown in SEQ ID NO: 41, and the nucleotide sequence of the actA.R is shown in SEQ ID NO: 42; When the deleted gene is the hly gene, the primers for detecting a single colony include up-hly.F, down-hly.R, hly.F and hly.R, the nucleotide sequence of hly.F is shown in SEQ ID NO: 43, and the nucleotide sequence of hly.R is shown in SEQ ID NO: 44; When the deleted gene is the inlA gene, the primers for detecting a single colony include up-inlA.F, down-inlA.R, inlA.F and inlA.R, the nucleotide sequence of inlA.F is shown in SEQ ID NO: 45, and the nucleotide sequence of inlA.R is shown in SEQ ID NO: 46; When the deleted gene is the inlP gene, the primers for detecting a single colony include up-inlP.F, down-inlP.R, inlP.F and inlP.R, the nucleotide sequence of the inlP.F is shown in SEQ ID NO: 47, and the nucleotide sequence of the inlP.R is shown in SEQ ID NO:

48.

8. A genetic recombinant plasmid constructed by any one of the methods of claims 1 to 5, wherein the genetic recombinant plasmid comprises pLR16-pheS*-ΔactA, pLR16-pheS*-Δhly, pLR16-pheS*-ΔinlA and pLR16-pheS*-ΔinlP.

9. A Listeria monocytogenes gene-deficient strain constructed by any one of the methods of claims 1-7, wherein the Listeria monocytogenes gene-deficient strain comprises ΔactA, Δhly, ΔinlA and ΔinlP mutants.

10. Use of the Listeria monocytogenes gene-deficient strain according to claim 9 in studying hosts infected by Listeria monocytogenes.

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