Multi-gene-deleted salmonella typhimurium as well as construction method and application thereof

Abstract

The invention provides salmonella typhimurium with multiple gene deletion, wherein two or more of an invF gene, a spaL gene, a sipC gene and a prgH gene are deleted at the same time. The invention also discloses a construction method of the polygene-deleted salmonella typhimurium and an application of the polygene-deleted salmonella typhimurium in preparation of a live attenuated vaccine of the salmonella typhimurium. Compared with wild type salmonella typhimurium, the virulence of the salmonella typhimurium deleted strain with simultaneous deletion of four genes provided by the invention is obviously reduced, the colonization time in a host body is obviously shortened, no adverse reaction is caused to a mouse after inoculation, a good protection effect is provided for high-virulence salmonella typhimurium, and infection of high-virulence strains can be effectively eliminated; the salmonella typhimurium attenuated live vaccine has good immunogenicity when being applied to the salmonella typhimurium attenuated live vaccine, can provide a firm immune protection effect on mice, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a Salmonella typhimurium with multiple gene deletions, a construction method thereof, and an application thereof. Background Art

[0002] Salmonella is an important zoonotic pathogen, including more than 2,600 serotypes. Among them, Salmonella Typhimurium has become an important threat in the global public health field due to its wide host range and diverse transmission routes (gastrointestinal tract, contact, and vertical transmission). This bacterium mediates host cell invasion through the type III secretion system (T3SS), causing enteritis, septicemia, and persistent infections, resulting in approximately 150 million human infection cases globally each year, with more than 60% in developing countries. China adopts a strategy of "prevention first, comprehensive prevention and control" for salmonellosis, mainly including: (1) strengthening the biosafety of farms and implementing all-in-all-out management; (2) carrying out antibiotic susceptibility monitoring; (3) promoting vaccination. Research shows that in areas where salmonellosis is prevalent, vaccination can reduce the incidence rate of the animal population by more than 85%, which is significantly better than the simple antibiotic intervention plan.

[0003] Existing Salmonella vaccines mainly include inactivated vaccines, subunit vaccines, and live attenuated vaccines. Among them, the live attenuated vaccine is recognized as having the most application potential because it can simultaneously induce humoral immunity (IgG titer ≥ 1:32000) and cellular immunity (the secretion amount of IFN-γ increases by 8.2 times), and the immune protection period can reach more than 180 days. Constructing a multi-gene deletion strain through genetic technology can significantly improve the safety and immunogenicity of the attenuated strain.

[0004] Some studies have shown that the spaL gene is involved in the assembly and effector protein transport of Salmonella pathogenicity island (SPI-1), and the spaL mutation can reduce the survival rate in macrophages to 15% of the wild strain. Research shows that the spaL gene affects the invasion efficiency of bacteria by regulating the stability of the T3SS needle complex. For example, the invasion efficiency of the spaL deletion strain in the mouse model decreases by 98%, and at the same time, it significantly reduces the inflammatory response caused by endotoxin release. In addition, the spaL gene is also involved in regulating the biofilm formation of Salmonella, and its deletion can lead to a reduction in biofilm thickness by more than 50%, significantly reducing the survival ability of bacteria in the environment.

[0005] The sipC gene can directly mediate the perforation of the host cell membrane and the injection of effector proteins. The colonization amount of the sipC deletion strain decreases by three orders of magnitude in the mouse model. The latest research finds that the sipC gene affects the intracellular movement and immune escape of bacteria by regulating the actin reorganization of host cells. For example, the survival rate of the sipC deletion strain in macrophages drops to 20% of the wild strain, and at the same time, it induces a stronger Th17-type immune response (the level of IL-17 increases by 3 times). In addition, the sipC gene is also involved in regulating the metabolic adaptability of Salmonella, and its deletion can lead to the survival rate of bacteria in a low-nutrient environment dropping to 40% of the wild strain.

[0006] The prgH gene is a core component of the T3SS needle complex. Inactivation of prgH can reduce the bacterial invasion efficiency by 98% and at the same time reduce the inflammatory response caused by the release of endotoxin. Research shows that the prgH gene affects the inflammatory response and immune escape of host cells by regulating the secretion of T3SS effector proteins. For example, the invasion efficiency of the prgH deletion strain in the mouse model drops by 98%, and at the same time, it significantly reduces the inflammatory response caused by the release of endotoxin. In addition, the prgH gene is also involved in regulating the biofilm formation of Salmonella, and its deletion can lead to a reduction in biofilm thickness by more than 50%, significantly reducing the survival ability of bacteria in the environment.

[0007] The live attenuated vaccine with multiple gene deletions can mimic the natural infection process and replicate limitedly in the host, thereby inducing a comprehensive immune response, which is significantly higher than that of inactivated vaccines and subunit vaccines. The live attenuated Salmonella vaccine has become the preferred strategy for the prevention and control of Salmonella diseases due to its excellent immunogenicity, long-lasting protective effect, and controllable safety. With the progress of gene editing technology, the new attenuated strains constructed by the strategy of coordinated deletion of multiple genes will further optimize the vaccine performance and provide a more effective solution for the prevention and control of Salmonella diseases. The development of attenuated vaccines requires more abundant alternative target genes for better selection. Summary of the Invention

[0008] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a Salmonella typhimurium with multiple gene deletions, which simultaneously deletes two or more of the invF gene, spaL gene, sipC gene, and prgH gene. It has three characteristics: high efficiency of attenuation, strong immunogenicity, and high protection efficiency. It is suitable as a candidate for the genetically engineered attenuated vaccine of Salmonella typhimurium, providing more possibilities for the development of live attenuated vaccines; at the same time, the present invention will also provide a method for constructing a Salmonella typhimurium with multiple gene deletions; in addition, the present invention will also provide the application of the Salmonella typhimurium with multiple gene deletions.

[0009] To achieve the above object and other related objects, the present invention adopts the following technical solutions: In a first aspect of the present invention, there is provided a Salmonella typhimurium with multiple gene deletions, wherein the Salmonella typhimurium with multiple gene deletions simultaneously deletes two or more of the invF gene, spaL gene, sipC gene, and prgH gene.

[0010] Furthermore, the Salmonella typhimurium with multiple gene deletions simultaneously deletes the invF gene, spaL gene, sipC gene, and prgH gene.

[0011] Furthermore, the nucleotide sequence of the invF gene is as shown in SEQ ID NO.1, the nucleotide sequence of the spaL gene is as shown in SEQ ID NO.2, the nucleotide sequence of the sipC gene is as shown in SEQ ID NO.3, and the nucleotide sequence of the prgH gene is as shown in SEQ ID NO.4.

[0012] In a second aspect of the present invention, there is provided a method for constructing the above-mentioned Salmonella typhimurium with multiple gene deletions, in which two or more of the invF gene, spaL gene, sipC gene, and prgH gene of Salmonella typhimurium SL1344 are knocked out.

[0013] Furthermore, the method for knocking out genes uses homologous recombination to knock out genes.

[0014] Furthermore, the method for knocking out genes uses a suicide plasmid to knock out genes.

[0015] Furthermore, it specifically includes the following steps: S1. According to the genomic sequence of Salmonella typhimurium SL1344, primers are designed for amplifying the upstream and downstream homologous arm sequences of two or more of the invF gene, spaL gene, sipC gene, and prgH gene respectively; S2. The genome of Salmonella typhimurium SL1344 is extracted as a PCR amplification template, and the upstream and downstream fragments of the target gene are amplified respectively using the primers in step S1. After the PCR products are verified successfully by electrophoresis, they are purified; S3. The pDM4 plasmid is extracted and recovered after double digestion with Sal Ⅰ and Sac Ⅰ; S4. The upstream and downstream fragments in step S2 and the pDM4 digested product in step S3 are ligated by one-step method. The ligation product is transformed into competent cells, and positive clones are picked and verified by PCR to obtain the conjugative transfer donor bacteria; S5. Conduct conjugation transfer of the conjugation transfer donor bacteria with SL1344, pick positive clones, and identify them by PCR to obtain single exchange strains; culture the single exchange strains, pick positive clones, and identify them by PCR to obtain Salmonella typhimurium with deletion of two or more of the invF gene, spaL gene, sipC gene, and prgH gene.

[0016] Further, in step S1, a pair of primers is designed respectively for the upstream and downstream of each gene.

[0017] Further, in step S1, the primers for amplifying the upstream and downstream homologous arm sequences of the invF gene are shown in SEQ ID NO.5 - SEQ ID NO.8; the primers for amplifying the upstream and downstream homologous arm sequences of the spaL gene are shown in SEQ ID NO.9 - SEQ ID NO.12; the primers for amplifying the upstream and downstream homologous arm sequences of the sipC gene are shown in SEQ ID NO.13 - SEQ ID NO.16; the primers for amplifying the upstream and downstream homologous arm sequences of the prgH gene are shown in SEQ ID NO.17 - SEQ ID NO.20.

[0018] In the third aspect of the present invention, there is provided the use of the above-mentioned Salmonella typhimurium with multiple gene deletions in the preparation of a live attenuated vaccine against Salmonella typhimurium.

[0019] In the fourth aspect of the present invention, there is provided a live attenuated vaccine against Salmonella typhimurium, which contains any one of the above-mentioned Salmonella typhimurium with multiple gene deletions.

[0020] As described above, the Salmonella typhimurium with multiple gene deletions of the present invention, its construction method and application have the following beneficial effects: The deletion strain of Salmonella typhimurium with four genes deleted provided by the present invention has a significantly reduced virulence compared with the wild-type Salmonella typhimurium, a significantly shortened colonization time in the host, no adverse reactions in mice after inoculation, provides good protection against highly virulent Salmonella typhimurium, and can effectively clear the infection of highly virulent strains; when applied to the live attenuated vaccine against Salmonella typhimurium, it has good immunogenicity, can provide solid immune protection for mice, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 For the scanning electron micrographs of the Salmonella typhimurium four-gene deletion strain SL1344Δ invF Δ spaL Δ sipC Δ prgH and SL1344.

[0022] Figure 2 For SL1344Δ invF Δ spaL ΔsipC Delta prgH and Cytotoxicity test results of SL1344 on J774.1 cells.

[0023] Figure 3 For SL1344 Delta invF Delta spaL Delta sipC Delta prgH and Death curve of C57BL / 6 mice challenged with SL1344.

[0024] Figure 4 For C57BL / 6 mice challenged with SL1344 Delta invF Delta spaL Delta sipC Delta prgH and the results of bacterial load in organs after challenge with SL1344.

[0025] Figure 5 For C57BL / 6 mice challenged with SL1344 Delta invF Delta spaL Delta sipC Delta prgH and the results of average body weight change after challenge with SL1344.

[0026] Figure 6 For C57BL / 6 mice inoculated with SL1344 Delta invF Delta spaL Delta sipC Delta prgH and the detection results of serum antibody IgG after challenge with SL1344.

[0027] Figure 7 For the vaccine candidate strain SL1344 Delta invF Delta spaL Delta sipC Delta prgH Pathological changes of the cecum of mice challenged with SL1344 after immunizing C57BL / 6 mice. Detailed implementation method

[0028] The following specific embodiments illustrate the implementation manner of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0029] Example 1 Construction of a quadruple gene deletion strain of Salmonella typhimurium: In this example, the wild-type strain of Salmonella typhimurium SL1344 (a commercially available standard strain) was used as the basic strain, and a Salmonella typhimurium strain simultaneously deleted for the invF gene, spaL gene, sipC gene, and prgH gene was constructed by homologous recombination mediated by the suicide plasmid pDM4, named SL1344 Delta invF DeltaspaL Δ sipC Δ prgH, The deletion strain is also simply named SL1344Δ4. The specific steps are as follows: S1. Mutation primer design: According to the genomic sequence of Salmonella typhimurium SL1344, primer pairs were designed using the primer design software Primer 5.0 to amplify the upstream and downstream homologous arm sequences of the invF gene, spaL gene, sipC gene, and prgH gene respectively. The primer sequences are shown in Table 1: Table 1. Primer sequences for PCR amplification

[0030] Note: The underlined part is the complementary pairing fragment of the pDM4 restriction enzyme cleavage site flanking sequence.

[0031] S2. PCR amplification and purification of the upstream and downstream homologous fragments of the invF, spaL, sipC, and prgH genes: The genome of the wild-type Salmonella typhimurium SL1344 was extracted and used as the template for PCR amplification. Using the above-mentioned Up-F / R and Down-F / R as primers, the upstream and downstream fragments of the target genes were amplified respectively. The amplification reaction system is shown in Table 2 below. After the PCR products were verified successfully by electrophoresis, they were purified.

[0032] Table 2. PCR reaction system and reaction conditions

[0033] 2.1 The steps for extracting the genome of the wild-type Salmonella typhimurium SL1344 are as follows: (1) Inoculate the bacteria into LB liquid medium and culture overnight at 37°C with 180 r / min; (2) Take 1 - 5 mL of the bacterial solution, centrifuge at 10000 r / min for 1 min, and aspirate the supernatant; (3) Add 200 μL of buffer GA to the bacterial cells and shake to suspend the cells; (4) Add 20 μL of proteinase K solution and mix well; (5) Add 220 μL of buffer GB, shake for 15 sec, place at 70°C for 10 min, and briefly centrifuge to remove water droplets; (6) Add 220 μL of absolute ethanol, shake for 15 sec to mix well, and briefly centrifuge to remove water droplets; (7) Transfer the solution and floccules to the adsorption column CB3, centrifuge at 12000 r / min for 30 sec, and discard the waste liquid; (8) Add 500 μL of buffer GD to the column and repeat step 7; (9) Add 600 μL of wash buffer PW and repeat step 7; (10) Repeat step 9; (11) Place the column back into the collection tube, centrifuge at 12,000 r / min for 2 min to discard the waste liquid, and air-dry the residual liquid at room temperature; (12) Transfer the column to a new centrifuge tube, add 50 - 200 μL of TE buffer, incubate at room temperature for 2 - 5 min, and centrifuge at 12,000 r / min for 2 min to collect the solution; (13) Use NanoDrop TM One to measure the genomic concentration and quality.

[0034] 2.2 Recovery and purification of the upstream and downstream fragments of the target gene: (1) Prepare an agarose gel using TAE buffer, and then perform gel electrophoresis to separate the target DNA; (2) Cut out the gel region containing the target DNA fragment under ultraviolet light, minimize the gel volume as much as possible, and blot the liquid on the gel surface with a tissue paper; (3) Cut the excised gel block into small pieces, weigh the gel, and calculate the gel volume at a ratio of 1 mg = 1 μL; (4) Add 3 volumes of gel dissolution buffer Buffer GM to the gel block; (5) After thorough mixing, intermittently shake to dissolve the gel block at room temperature; (6) When the gel is completely dissolved and the solution color changes from yellow to orange - red, add 10 μL of 3M sodium acetate solution and mix until the solution returns to yellow; (7) Place the Spin Column into the Collection Tube; (8) Transfer the solubilized solution to the Spin Column, centrifuge at 12,000 r / min for 1 min, and discard the filtrate; (9) Add 700 μL of Buffer WB to the Spin Column, centrifuge at 12,000 r / min for 30 sec at room temperature, and discard the filtrate; (10) Repeat step 9; (11) Place the Spin Column into the Collection Tube and spin at 12,000 r / min for 1 min at room temperature without sample; (12) Transfer the Spin Column to a new 1.5 mL centrifuge tube, add 30 μL of elution buffer pre - heated to 60°C to the center of the membrane, and let it stand at room temperature for 1 min; (13) Centrifuge at 12,000 r / min for 1 min at room temperature to elute the DNA; (14) Determine the concentration and purity of the DNA fragment on the NanoDropTM One; (15) Recover the purified DNA fragment and directly use it for downstream experiments or store it at -20 °C.

[0035] S3. Extraction and digestion of the pDM4 plasmid: Extract the pDM4 plasmid and, after Sal I and Sac I double digestion, recover it. The digestion system and conditions are shown in Table 3 below.

[0036] Table 3. Digestion system

[0037] 3.1 The steps for extracting the pDM4 plasmid are as follows: (1) Pick a single colony and inoculate it into LB liquid medium. Culture overnight at 37 °C with a rotation speed of 180 r / min; (2) Add 500 μL of equilibration buffer BL to the adsorption column CP4, centrifuge at 12000 r / min for 1 min, and discard the waste liquid; (3) Take 5 - 15 mL of the overnight bacterial culture, centrifuge at 12000 r / min for 1 min, and aspirate the supernatant; (4) Add 500 μL of solution P1 to the pellet; (5) Add 500 μL of solution P2 and invert the tube 6 - 8 times to lyse the cells; (6) Add 700 μL of solution P3, immediately invert the tube 6 - 8 times to mix well, and centrifuge at 12000 r / min for 10 min; (7) Add the supernatant to the adsorption column CP4 in portions without aspirating the pellet. Centrifuge at 12000 r / min for 1 min and discard the waste liquid; (8) Add 500 μL of protein removal solution PD to the column, centrifuge at 12000 r / min for 1 min, and discard the waste liquid; (9) Add 600 μL of washing buffer PW to the column, centrifuge at 12000 r / min for 1 min, and discard the waste liquid; (10) Repeat step 8; (11) Centrifuge at 12000 r / min for 2 min; (12) Transfer the column to a new centrifuge tube, add 100 - 300 μL of elution buffer EB, incubate at room temperature for 2 - 5 min, and centrifuge at 12000 r / min for 2 min to collect the plasmid solution; (13) Use NanoDrop TM One to determine the plasmid concentration and quality.

[0038] S4. Ligate the digested product of the pDM4 plasmid with the upstream and downstream fragments recovered in Step 2 by one-step ligation. Transform the product into X7213 competent cells, pick positive clones, and obtain the conjugation transfer donor bacteria through PCR verification.

[0039] Among them, the sequences of the verification primers are as shown in SEQ ID NO.21 to SEQ ID NO.22; and the PCR system is shown in Table 4.

[0040] Table 4. PCR reaction system and reaction conditions

[0041] 4.1 The X7213 competent cells are prepared by the following steps: (1) Inoculate the bacteria into LB liquid medium and culture at 37°C and 250 r / min for 12 - 16 h; (2) Take 0.5 mL of the bacterial solution and inoculate it into 50 mL of BT Media, and culture with shaking at 37°C until OD600 = 0.5 - 0.6; (3) Transfer the bacterial solution to a 50 mL plastic centrifuge tube and place it on ice for 5 - 10 min; (4) Centrifuge at 4°C and 5000 r / min for 5 min to collect the bacterial cell precipitate; (5) Resuspend every 50 mL of the bacterial cells with 4 mL of pre-cooled BT Buffer B, and aliquot into pre-cooled 1.5 mL centrifuge tubes, 100 μL per tube; (6) The bacterial cells can be directly used for transformation or stored at -70°C.

[0042] 4.2 The plasmid transformation steps are as follows: (1) Take out the competent bacteria from the ultra-low temperature freezer and place them on ice to thaw; (2) Add 100 pg - 10 ng of the DNA to be transformed or the ligation product to each tube of the competent bacteria, mix gently, and let it stand in an ice bath for 30 min; (3) Transfer the centrifuge tube to a 42°C water bath for a short incubation of 45 sec, and immediately cool it in an ice bath for 3 min after taking it out; (4) Add 900 µL of pre-warmed LB liquid medium without resistance at 37°C, and invert and mix well; (5) Incubate with shaking at 37°C and 180 r / min for 60 min; (6) Take an appropriate amount of the bacterial solution and spread it on an LB plate containing the resistance corresponding to the target plasmid, and place it in an inverted position in a 37°C incubator overnight; (7) Select positive clone strains and identify them by PCR method.

[0043] 4.3 One-step ligation of upstream and downstream fragments with pDM4 vector: (1)Calculation of the amounts of vector and fragment used: Optimal amount of cloning vector used = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol) Optimal amount of inserted fragment used = [0.04 × number of base pairs of inserted fragment] ng (0.06 pmol) (2)According to the above calculation results, prepare the reaction system in an ice bath: 5 μL of 2 × ClonExpress Mix, add the required cloning vector and inserted fragment, and make up to 10 μL with ddH2O; (3)Gently pipette and mix the reaction solution, and briefly centrifuge to collect the reaction solution at the bottom of the tube; (4)Incubate the reaction solution at 50 °C for 15 min; (5)Immediately cool it in an ice bath after incubation; (6)The recombinant DNA can be directly used for subsequent experiments or stored at -20 °C.

[0044] S5. Drop the conjugation transfer donor bacteria and SL1344 onto a hydrophilic filter membrane, place the hydrophilic filter membrane on an LB solid plate medium, and statically culture at 37 °C for 12 h for conjugation transfer. Pick positive clones. The single crossover strains are identified by PCR using two pairs of primers combined with the SEQ ID NO.21 primer and the OUT-F and OUT-R primers of each gene (Table 5). If a band can be amplified by any one set of primers and no band can be amplified by the other set, it is confirmed as a single crossover strain; Culture the single crossover strains to the logarithmic phase, expand the culture in LB liquid containing sucrose, pick positive clones, and verify by PCR using the inner and outer primers of the four genes. The PCR verification primers are shown in Table 5, and the conditions are shown in Table 6.

[0045] Among them, the amplified band of the outer primer gene deletion strain is smaller than that of the wild strain, while the inner primer gene deletion strain has no amplified band, and the wild strain has an amplified band. The strain with the correct PCR band is the gene deletion strain SL1344Δ invF Δ spaL Δ sipC Δ prgH .

[0046] Table 5. Sequences of PCR verification primers

[0047] Table 6. PCR reaction system and reaction conditions

[0048] S6. Biochemical identification Pick the pure cultured SL1344Δ invF Δ spaL Δ sipC Δ prgH and SL1344 single colonies, and identify their biochemical characteristics with a Gram-negative biochemical identification card. The results are shown in Table 7 below.

[0049] Table 7. Identification of Biochemical Characteristics

[0050] Note: "+" represents positive; "-" represents negative Experimental conclusion: The biochemical results show that SL1344Δ invF Δ spaL Δ sipC Δ prgH has no difference from the wild strain.

[0051] S7. Scanning Electron Microscope Observation of Gene Deletion Strains (1) Inoculate SL1344Δ invF Δ spaL Δ sipC Δ prgH and SL1344 in LB liquid medium, and shake culture overnight at 37°C and 180 r / min; (3) Wash the cultured bacteria three times with sterile water, and centrifuge at 8000 × g for 10 min at 4°C; (4) Fix with 2.5% (w / v) glutaraldehyde at 4°C for 12 h; (5) Wash the bacteria three times in sterile PBS buffer; (6) Dehydrate in gradient ethanol concentrations (25%, 50%, 70%, 80%, 90%) for 15 min each; (7) Dry the sample at its isoelectric point, coat it with a layer of metal, and observe using GeminiSEM 300.

[0052] Experimental conclusion: The scanning electron microscope results are as Figure 1 shown, and there are no obvious morphological differences between the deletion strain and the wild strain.

[0053] Example 2. Safety Evaluation of Salmonella typhimurium SL1344Δ invF Δ spaL Δ sipC Δ prgH Safety evaluation: 1. Cytotoxicity determination of wild strain and deletion strain (1) Seed 2 × 105 J774.1 cells in a 24-well plate and incubate overnight in a 37°C, 5% CO2 incubator; (2) Select SL1344 and SL1344Δ respectively invF Δ spaL Δ sipC Δ prgH A single colony was inoculated into LB liquid medium and incubated at 37°C and 180 rpm overnight; (3) Expand the cultured strain into fresh LB liquid medium at a ratio of 1:100 and incubate at 37°C for 3 h. (4) Centrifuge the cultured bacteria at 4°C, 5000 rpm for 4 min, remove the supernatant, wash twice with sterile PBS, resuspend in Opti medium, and adjust OD600 to 1.0; (5) Aspirate the original cell culture medium and rinse once with DPBS. Replace with fresh Opti culture medium and divide the culture wells into cell-free control wells, untreated cell control wells, maximum enzyme activity control wells, and strain-treated cell wells; (6) Add 1 mL of Opti medium containing bacterial solution (MOI = 100) to the strain treatment well, centrifuge at 23°C, 1000 rpm for 10 min, and continue conventional culture for 2 h. (7) 1 h before the scheduled detection time, remove the cell plate from the incubator, add 10% of the original culture volume of LDH releaser to the maximum enzyme activity control well, mix thoroughly and continue incubation; (8) After 2 h, centrifuge at 400 × g for 5 min in a multi-well plate centrifuge, transfer 120 μL of the supernatant from each well to a new 96-well plate, and test immediately. (9) Add 60 μL of LDH detection working solution to each well, mix well, and incubate at room temperature in the dark for 30 min. Measure the absorbance at 490 nm, using 600 nm as the reference wavelength for dual wavelength determination; (10) Cytotoxicity (%) = (absorbance of treated sample - absorbance of sample control) / (absorbance of maximum enzyme activity - absorbance of sample control) × 100%.

[0054] Experimental conclusion: Cytotoxicity results are as follows Figure 2 As shown in the figure, the deletion strain can significantly reduce the cytotoxicity to J774.1 cells compared with the wild-type strain, indicating that SL1344Δ invF Δ spaL Δ sipC Δ prgH It has the potential to prepare live attenuated Salmonella typhimurium vaccine.

[0055] 2. Survival curve determination of C57BL / 6 mice (1) Raise female C57BL / 6 mice to 6-8 weeks of age; (2) Select SL1344 and SL1344Δ invFDelta spaL Delta sipC Delta prgH Inoculate into LB liquid medium and culture overnight at 37 °C with 180 r / min; (3) Expand the culture of the overnight culture by 1:100 and culture statically at 37 °C for 3 h; (4) Centrifuge the well-cultured secondary bacterial solution at 4 °C and 8000 r / min for 2 min, discard the supernatant, wash twice with sterile PBS, resuspend the bacterial cells with PBS, and adjust the OD 600 to 1.0 and dilute 100 times; (5) Intraperitoneally inject 100 μL of the mixed bacterial suspension into each mouse, and the infection dose is 3×105 CFU / mouse. Inject 100 μL of sterile PBS into the abdominal cavity of the control group mice; (6) Keep the mice in an environmental isolator and record the death of each group of mice every day.

[0056] Experimental conclusion: The survival curve results are as Figure 3 shown. The results show that the deletion strain can significantly reduce the mortality of mice compared with the wild strain. On the 18th day after challenge, no death was found in the mice of the deletion strain group, while the mortality of the wild strain group was 100%. It is shown that SL1344Δ invF Delta spaL Delta sipC Delta prgH has good safety as a live attenuated vaccine of Salmonella typhimurium.

[0057] Example 3 Colonization test of Salmonella typhimurium SL1344Δ invF Delta spaL Delta sipC Delta prgH in the host: 1. Detection of Salmonella typhimurium colonization in mouse organs (1) Raise female C57BL / 6 mice to 6 - 8 weeks old; (2) Pick SL1344Δ invF Delta spaL Delta sipC Delta prgH Inoculate into LB liquid medium and culture overnight at 37 °C with 180 r / min; (3) Expand the culture of the overnight culture by 1:100 and culture statically at 37 °C for 3 h; (4) Centrifuge the well-cultured secondary bacterial solution at 4 °C and 8000 r / min for 2 min, discard the supernatant, wash twice with sterile PBS, resuspend the bacterial cells with PBS, and adjust the OD 600 to 1.0 and dilute 100 times; (5) Inject 100 μL of the mixed bacterial suspension into the peritoneal cavity of each mouse, with an infection dose of 3×10³ CFU / mouse. Inject 100 μL of sterile PBS into the peritoneal cavity of the control group mice. (6) Place 3 ceramic grinding balls with a diameter of 3 mm into a 2 mL grinding tube and sterilize them under high pressure. (7) Euthanize the mice infected for 1 day, 3 days, 5 days, 7 days, and 14 days, and soak them in 75% ethanol for disinfection. (8) Dissect the mice in a biosafety cabinet. Under sterile conditions, take a small amount of spleen, liver, and cecum tissues and put them into the grinding tube. After weighing, add 1 mL of sterile PBS. (9) Place it in a tissue grinder and grind at 4°C and 65 Hz for 120 sec to grind the tissue into a homogeneous slurry. (10) After mixing, make 10-fold serial dilutions with sterile PBS. (11) Take 100 μL of the appropriately diluted solution and spread it on three XLT4 (Cabr, Kmr) selective plates, with 3 replicates for each dilution. (12) Invert the plates and incubate them in an incubator at 37°C for 12 - 16 h. (13) Count the bacterial colonies and calculate the Salmonella load in each organ of the mice.

[0058] The results of the colonization experiment are as Figure 4 shown. The results show that after the mice were challenged with the deletion strain, the deletion strain could be isolated from the liver and spleen on the 1st day, 3rd day, and 5th day. In the cecum, the deletion strain could still be isolated on the 7th day, but not on the 14th day. This indicates that the clearance period of the deletion strain in the mouse body is 14 days.

[0059] 2. Analysis of body weight differences For the mice infected for 14 days, observe their body weight changes. The results are as Figure 5 shown. Compared with the blank control group, there was no significant difference in the body weight of the mice infected with SL1344Δ invF Δ spaL Δ sipC Δ prgH group, indicating that the infection with the gene deletion strain does not affect the growth of mice.

[0060] Example 4 Immunoprotection test of Salmonella typhimurium SL1344Δ invF Δ spaL Δ sipC Δ prgH : 1. Immunogenicity test: IgG antibody detection Detect the IgG titer of mouse serum by indirect ELISA (1) Treat the ELISA strip with 5% glutaraldehyde, add it to the 96-well ELISA plate at a dose of 100 μL per well, incubate at 37°C for 2 h, wash three times with distilled water, pat dry, add 50 μL of SL1344 bacterial solution, and place it in an oven at 56°C to dry; (2) Add 100 μL of pre-cooled absolute methanol, let it act at room temperature for 15 min, wash three times with distilled water, and pat dry; (3) Add 200 μl of ELISA blocking solution and block at 37°C for 1 h; (4) Discard the blocking solution, wash 3 times with PBST, and pat dry; (5) Add the primary antibody dilution solution to the 96-well ELISA plate at a dose of 100 μL per well, dilute the serum to be detected with the primary antibody dilution solution to the initial concentration, add 100 μL of the serum to be detected to the initial well of the ELSIA plate, perform serial dilution to the required concentration, and incubate at 37°C for 2 h; (6) Discard the serum, wash 5 times with PBST, and pat dry; (7) Add goat anti-mouse IgG-HRP enzyme-labeled antibody (diluted 1:5000), incubate at 37°C in the dark for 1 h; (8) Discard the enzyme-labeled antibody, wash 7 times with PBST, and pat dry; (9) Add 100 μL of single-component TMB chromogenic solution and incubate at 37°C in the dark for 10 min; (10) Add 50 μL of H2SO4 termination solution (2N), measure the absorbance at 450 nm, and measure with a correction wavelength of 540 nm; (11) Calculate the P / N value and calculate the antibody titer in each sample.

[0061] The results are as Figure 6 shown. Inoculate C57BL / 6 mice with SL1344 and SL1344Δ 3 Δ invF Δ spaL Δ sipC Δ prgH at a concentration of 3×10

[0062] CFU / mouse. Perform aseptic blood collection on the immunized mice on the 3rd, 5th, 7th, 14th, and 21st days, and detect the serum IgG antibody titer. The results show that 7 days after immunization, Salmonella typhimurium IgG antibodies can be detected, and the antibody levels of the wild strain and the deletion strain are comparable, indicating that the deletion strain has good immunogenicity in mice. invF Δ spaL Δ sipC Δ prgH Protection test (1)Raise female C57BL / 6 mice to 6 - 8 weeks old; (2)Pick SL1344Δ invF Δ spaL Δ sipC Δ prgH Inoculate it into LB liquid medium and culture overnight at 37°C with 180 r / min; (3)Enlarge - culture the overnight - cultured bacterial liquid at a ratio of 1:100 and static - culture it at 37°C for 3 h; (4)Centrifuge the well - cultured secondary bacterial liquid at 4°C and 8000 r / min for 2 min, discard the supernatant, wash it twice with sterile PBS, resuspend the bacteria with PBS, and adjust the OD 600 to 1.0 and dilute it 100 times; (5)Immunize mice with doses of 1×10 5 CFU / mouse, 1×10 6 CFU / mouse, 1×10 7 CFU / mouse respectively. Inject 100 μL of sterile PBS into the abdominal cavity of the control - group mice; (6)14 days after immunization, challenge the mice with SL1344 at a dose of 1×10 9 CFU / mouse, and observe the vaccine protection rate (Vaccine protection rate = (Mortality rate of control group - Mortality rate of immunized group) / Mortality rate of control group × 100%).

[0063] The results are shown in Table 8. The results show that when immunized with 1×10 6 SL1344Δ invF Δ spaL Δ sipC Δ prgH , the mice can obtain 100% immune protection efficiency, indicating that SL1344Δ invF Δ spaL Δ sipC Δ prgH has good protective ability.

[0064] Table 8. Immune protection efficiency of different immunization doses of SL1344Δ invF Δ spaL Δ sipC Δ prgH Immune protection efficiency of different immunization doses

[0065] 3. Analysis of mouse cecum pathological sections (1)On the third day after challenge, quickly dissect the fresh intestinal tissues of the mice in the mutant - strain immunized and challenged group and the wild - strain challenged group, and immediately immerse them in the tissue fixative for fixation for more than 24 h; (2) Remove the tissue from the fixative, use a scalpel to trim the required area on a ventilated operating table, and place the processed tissue and corresponding labels into the embedding box; (3) Dehydration in a dehydrator according to the alcohol gradient: 75% alcohol for 4 h, 85% alcohol for 2 h, 90% alcohol for 2 h, 95% alcohol for 1 h, anhydrous ethanol for 30 min, alcohol-benzene 1:1 mixture for 10 min, xylene for 10 min, paraffin melted at 65°C for 1 h; (4) Place the wax-impregnated tissue into the embedding machine, first inject the molten paraffin into the embedding box, and place the tissue in the embedding box as required before the wax solidifies and label it. Place the wax block on a -20°C cold plate to solidify it, then take it out and trim it; (5) Place the trimmed wax block into a paraffin slicer and cut it into 4 μm thick slices. Float the slices on 40°C warm water and flatten them. Pick them up with a glass slide, dry them in a 60°C oven, and store them at room temperature for later use. (6) Place the dried sections into the molten paraffin in a wax melter for a few seconds, remove them and allow them to cool and dry, so that the slides and tissues are completely covered with paraffin film; (7) Soak the sections in dewaxing solution for 20 min, anhydrous ethanol for 5 min, 75% alcohol for 5 min, and rinse with running water; (8) The sections were treated in high-definition staining pretreatment solution for 1 min; (9) The sections were stained with hematoxylin solution for 3-5 min, differentiated with differentiation solution, blued with bluing solution, and rinsed with running water; (10) After dehydration in 95% alcohol for 1 min, the sections were stained in eosin solution for 15 sec; (11) The sections were sequentially immersed in anhydrous ethanol for 2 min, n-butanol for 2 min, and xylene for 2 min, and finally sealed with a neutral resin sealing medium; (12) Observe under a microscope and take pictures for image analysis.

[0066] Pathological section results Figure 7 As shown in the figure, the intestinal tissue of the mice in the challenge group after immunization showed that the intestinal villi were well-developed and abundant, the intestinal villus epithelium was intact, no obvious degeneration and necrosis were observed, there were a large number of intestinal glands in the lamina propria, which were straight tubular and densely arranged, the submucosal connective tissue was loose, the muscular layer structure was clear, and no obvious pathological changes were observed. The intestinal epithelium of the mice in the wild strain challenge group was separated from the lamina propria, the number of goblet cells decreased, the mucosal layer was necrotic in a small area, the intestinal villi and intestinal gland structure disappeared, and there was a small amount of lymphocyte infiltration.

[0067] In summary, the four-gene deletion strain of Salmonella typhimurium constructed in the present invention has attenuated virulence compared to the wild strain, with good safety and immunogenicity. At the same time, it provided 100% protective efficacy in the immunoprotection test, reducing the mortality rate of mice after challenge and alleviating intestinal lesions. It has the potential to prepare a live attenuated vaccine against Salmonella typhimurium, is an excellent vaccine candidate strain, and has broad application prospects. Therefore, the present invention effectively overcomes various drawbacks in the prior art and has high industrial utilization value.

[0068] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A multi-gene-deficient Salmonella typhimurium, characterized in that: The multi-gene-deficient Salmonella typhimurium simultaneously lacks two or more of the invF gene, the spaL gene, the sipC gene, and the prgH gene.

2. The multi-gene deleted Salmonella typhimurium according to claim 1, characterized in that: The multi-gene deleted Salmonella typhimurium simultaneously lacks the invF gene, the spaL gene, the sipC gene and the prgH gene.

3. The multi-gene deleted Salmonella typhimurium according to claim 1 or 2, characterized in that: The nucleotide sequence of the invF gene is shown in SEQ ID NO.1, the nucleotide sequence of the spaL gene is shown in SEQ ID NO.2, the nucleotide sequence of the sipC gene is shown in SEQ ID NO.3, and the nucleotide sequence of the prgH gene is shown in SEQ ID NO.

4.

4. The method for constructing a multi-gene deleted Salmonella typhimurium according to any one of claims 1 to 3, characterized in that: Two or more of the invF gene, spaL gene, sipC gene and prgH gene of Salmonella typhimurium SL1344 are knocked out.

5. The construction method according to claim 4, characterized in that: The method for knocking out genes uses a homologous recombination method to knock out genes.

6. The construction method according to claim 4, characterized in that: The method for knocking out genes uses a suicide plasmid method to knock out genes.

7. The construction method according to claim 4, characterized in that: The specific steps include: S1. According to the genome sequence of Salmonella typhimurium SL1344, primers are designed for amplifying two or more upstream and downstream homology arm sequences in the invF gene, spaL gene, sipC gene and prgH gene respectively; S2, extracting the genome of Salmonella typhimurium SL1344, using it as a template for PCR amplification, using the primers in step S1 to amplify the upstream and downstream fragments of the target gene respectively, and purifying the PCR product after successful verification by electrophoresis; S3, extract pDM4 plasmid, Sal Ⅰ and Sac Ⅰ Recovery after double enzyme digestion; S4, one-step ligation of the upstream and downstream fragments of step S2 and the pDM4 digestion product of step S3, transforming the ligation product into competent cells, picking positive clones, and performing PCR verification to obtain the conjugative transfer donor bacteria; S5. Perform conjugation transfer on the conjugation transfer donor bacteria and SL1344, pick positive clones, and identify by PCR to obtain single-exchange strains; culture the single-exchange strains, pick positive clones, and identify by PCR to obtain Salmonella typhimurium that simultaneously lacks two or more of the invF gene, spaL gene, sipC gene, and prgH gene.

8. The construction method according to claim 7, characterized in that: In step S1, the primers used to amplify the upstream and downstream homology arm sequences of the invF gene are shown in SEQ ID NO.5 to SEQ ID NO.8; The primers used to amplify the upstream and downstream homology arm sequences of the spaL gene are shown in SEQ ID NO.9 to SEQ ID NO.12; the primers used to amplify the upstream and downstream homology arm sequences of the sipC gene are shown in SEQ ID NO.13 to SEQ ID NO.16; The primers used to amplify the upstream and downstream homology arm sequences of the prgH gene are shown in SEQ ID NO.17 to SEQ ID NO.

20.

9. Use of the multi-gene deleted Salmonella typhimurium according to any one of claims 1 to 3 in the preparation of a live attenuated Salmonella typhimurium vaccine.

10. A live attenuated Salmonella typhimurium vaccine comprising the multi-gene-deficient Salmonella typhimurium according to any one of claims 1 to 3.

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