Application of yncJ gene in regulation and control of virulence of salmonella typhimurium

Abstract

The invention discloses application of a yncJ gene in regulation and control of toxicity of salmonella typhimurium, and belongs to the technical field of biology. According to the invention, the function of the yncJ gene is studied by constructing the yncJ gene deleted strain of salmonella typhimurium, and it is found that after the yncJ gene is deleted, the multiplication capacity of the strain in macrophages is significantly reduced; infected mouse experiments show that the lethal ability of the yncJ gene deleted strain to mice is remarkably reduced. Moreover, the conventional biochemical characteristics and the in-vitro growth speed of the yncJ gene deletion strain are not obviously changed, so that the yncJ gene deletion strain has an application prospect in production of attenuated vaccines for preventing and treating salmonella typhimurium.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of the yncJ gene in regulating the virulence of Salmonella typhimurium. Background Art

[0002] Salmonella enterica subsp. enterica serovarTyphimurium (STM) is one of the most important strains of Salmonella, which is distributed worldwide with a high isolation rate. The bacteria are mostly found in the digestive tract, muscles, blood and other tissues of humans and various animals, and are also common in animal products such as dairy products, meat and eggs. As an intracellular parasitic bacterium, Salmonella typhimurium mainly causes paratyphoid fever in the host, manifested as gastroenteritis or sepsis, which seriously threatens human health.

[0003] The pathogenic process of Salmonella is divided into two stages: intestinal infection and intracellular infection. The bacteria invade the host through the fecal-oral route, enter the intestine through the stomach, and invade the intestinal epithelial cells that have no active phagocytic ability through endocytosis, which is the intestinal infection stage. Once the barrier of the intestinal epithelial cells is destroyed, the bacteria invade the macrophages and then replicate inside the cells, which is the intracellular infection stage. Once intracellular infection occurs, the macrophages will cause cell necrosis or apoptosis due to infection. After the infected cells become necrotic, they will release the replicated and proliferated bacteria into the extracellular space, which will then be transferred through the blood to infect different organs or tissues, while releasing toxins, causing systemic infection.

[0004] Therefore, for the prevention and treatment of Salmonella typhimurium, blocking its replication in macrophages is the key to avoid systemic infection. The ability of Salmonella typhimurium to survive, reproduce and spread in the host is related to a variety of virulence factors. The currently known virulence genes of Salmonella mainly involve fimbriae adhesion, non-fimbriae adhesion, secretion system, Mg 2+ absorption or transport, etc.; however, there are few reports on virulence genes related to the replication of Salmonella in macrophages. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide the application of the yncJ gene in regulating the virulence of Salmonella typhimurium. The present invention studies the function of the yncJ gene by constructing a yncJ gene-deficient strain of Salmonella typhimurium, and finds that after the yncJ gene is deleted, the proliferation ability of the strain in macrophages is significantly reduced; through the infection mouse experiment, it is found that the lethality of the yncJ gene-deficient strain to mice is significantly reduced. Moreover, the conventional biochemical characteristics and in vitro growth rate of the yncJ gene-deficient strain have no significant changes. Therefore, the yncJ gene-deficient strain has the application prospect of producing attenuated vaccines for the prevention and treatment of Salmonella typhimurium.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, the use of the yncJ gene as a target gene in at least one of the following (1)-(3) is provided:

[0008] (1) Regulate the proliferation of Salmonella typhimurium in macrophages;

[0009] (2) regulating the virulence of Salmonella typhimurium;

[0010] (3) Preparation of attenuated Salmonella typhimurium vaccine;

[0011] The yncJ gene is a nucleic acid molecule as shown in the following i) or ii):

[0012] i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID No.1;

[0013] ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 2 other than i).

[0014] The yncJ gene has GeneID: 1253121 in NCBI. It is a hypothetical periplasmic protein with unknown function. The nucleotide sequence of its CDS region is shown in SEQ ID No. 1, as follows:

[0015] atgcttaca aaaacgttat cagtagtttt actgacctgt gcgctgtttt caggccagttactggcgaaa cagcaagacc acgcatttcgt atggtttgcg acgggagggc accagcttag acacgaagccgacagcgatg aattacgggc ggcagcggaa gaatccgcgg aaggcttgcg cgagcaccac aactggcagaaatcgcgcaa accagaaagc tattttcgtt aa.

[0016] The amino acid sequence of the encoded protein is shown in SEQ ID No. 2, which is as follows:

[0017] MLTKTLSVVLLTCALFSGQLLAKQQDHAFVWFATGGHQLRHEADSDELRAAESAEGLREHHNWQKSRKPESYFR.

[0018] Taking the yncJ gene as the target gene, the proliferation ability and virulence of Salmonella typhimurium in macrophages can be reduced by knocking out the yncJ gene or downregulating the expression of the yncJ gene.

[0019] The second aspect of the present invention provides the use of a protein encoded by the yncJ gene as a target in the following (1) or (2):

[0020] (1) Regulate the proliferation of Salmonella typhimurium in macrophages;

[0021] (2) Regulate the virulence of Salmonella typhimurium.

[0022] In the above application, the amino acid sequence of the protein encoded by the yncJ gene is shown in SEQ ID No.2.

[0023] By taking the protein encoded by the yncJ gene as the target and regulating the activity of the protein, the proliferation ability and virulence of Salmonella typhimurium in macrophages can be regulated accordingly.

[0024] The third aspect of the present invention provides the use of a substance for inhibiting yncJ gene expression in at least one of the following (1)-(3):

[0025] (1) Preparing a product that reduces the proliferation of Salmonella typhimurium in macrophages;

[0026] (2) preparing products that reduce the virulence of Salmonella typhimurium;

[0027] (3) Preparation of attenuated Salmonella typhimurium vaccine.

[0028] In the above application, the substance that inhibits the expression of yncJ gene can be selected from: suicide plasmid, siRNA, shRNA and CRISPi, etc. Among them:

[0029] Suicide plasmids have the characteristics of a wide host range and conjugative transfer genes. Their replication requires a special protein that most bacteria do not produce. Therefore, when entering the host cell, they either cannot replicate and are eliminated, or are integrated into the chromosome and replicated together with the chromosome. Taking advantage of this feature of suicide plasmids, gene-deficient DNA fragments constructed by genetic engineering technology are cloned into suicide plasmids, and the integration site of suicide plasmids is located using homologous fragments at both ends of the deleted gene. The principle that homologous DNA fragments can undergo recombination is used to construct precise gene-deficient strains.

[0030] Therefore, the substance that inhibits the expression of the yncJ gene is preferably a suicide plasmid pRE112 carrying upstream and downstream fragments of the yncJ gene.

[0031] The nucleotide sequence of the upstream fragment of the yncJ gene is shown in SEQ ID No.3, and is as follows:

[0032] 5'-GAGGTACCGGTCATGATCACCAAACACTGCCTGCGTTTTGCGTTCAACCTTT GTCCAAAGCAGGCGAAAGGAAATATTAAGAGCTGGAAAGCCACGCCGATGCAGTTGGTGCATGGCGATGAGGTACTGACGCTAAAATTCGACTGCCGCCCTTGCGAAATGCATGTCATTGGCAAAATTAAAAACCACATC TTAAAATGCCCCAGCCCGGCAGCGTTGTCGCTTCAGTGAGCCCTGAAGCGCTGATGAAAACGCTGCCGAAGCGCAGGGGCGTTTAATTAACGAAAATAGCTTTCTGGTTTGCGCGATTTCTGCCAGTTGTGGTGCT-3'.

[0033] The nucleotide sequence of the downstream fragment of the yncJ gene is shown in SEQ ID No.4, and is as follows:

[0034] 5'-CATGCCACCTTCCAGTTACGTCGTGAGGCTATGATGCCAATAATTTCTTTGA -3'.

[0035] A fourth aspect of the present invention provides an attenuated Salmonella typhimurium vaccine, wherein the attenuated Salmonella typhimurium vaccine uses a yncJ gene-deficient strain of Salmonella typhimurium as an active ingredient.

[0036] Preferably, the yncJ gene-deficient strain of Salmonella typhimurium is constructed by the following method:

[0037] The upstream and downstream fragments of the yncJ gene were cloned into the suicide plasmid pRE112 respectively to obtain the recombinant plasmid pRE-ΔyncJ; then the recombinant plasmid pRE-ΔyncJ was transformed into Escherichia coli χ7213, and the Escherichia coli χ7213 carrying the recombinant plasmid pRE-ΔyncJ was used as the donor bacteria and the wild-type Salmonella typhimurium was used as the recipient bacteria for conjugation transfer, and the yncJ gene-deficient strain was screened by the two-step allelic exchange technology.

[0038] Beneficial effects of the present invention:

[0039] The present invention uses suicide plasmid to construct the yncJ gene deletion strain STM-ΔyncJ of STM. Through comparison of biological characteristics, it is found that the conventional biochemical characteristics of STM-ΔyncJ are consistent with those of STM-WT, and the in vitro growth rate has no significant change; it is determined that both STM-ΔyncJ and STM-WT have swimming mobility, but the swimming shape is different from that of STM-WT; the effect of yncJ knockout on the structural morphology of STM is observed by scanning electron microscopy, and it can be seen that the extracellular matrix components of STM-ΔyncJ are significantly increased. Through in vitro cell infection experiments, the survival and proliferation abilities of STM-ΔyncJ and STM-WT in macrophage RAW 264.7 are compared and analyzed, and the proliferation ability of STM-ΔyncJ in macrophages is significantly reduced compared with STM-WT. Through the infection of BALB / c mice, the lethality of STM-ΔyncJ to mice is significantly reduced compared with STM-WT. This proves that the yncJ gene is a new virulence gene of STM, which is of great significance for the prevention and treatment of STM and the development of attenuated vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : Schematic diagram of homologous recombination; M13:5'-AACAGCTATGACCATGA-3'.

[0041] Figure 2 : Colony PCR identification results of STM-ΔyncJ; in the figure, 1 and 2 are the identification results of the gene-deficient strain STM-ΔyncJ; 3 is the identification result of the wild strain without deletion.

[0042] Figure 3 : In vitro growth curve of STM-ΔyncJ.

[0043] Figure 4 : Results of bacterial motility experiments.

[0044] Figure 5 :Scanning electron microscopy image of STM-ΔyncJ bacterial morphology.

[0045] Figure 6 :The results of intracellular proliferation ability detection of STM-ΔyncJ and STM-WT in macrophages (P<0.01).

[0046] Figure 7 :STM-ΔyncJ lethality detection results (P<0.001). DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0048] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0049] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out in accordance with conventional test methods or the operating instructions recommended by the supplier. Among them:

[0050] The standard strain S. Typhimurium (ATCC 14028) and Escherichia coli TOP10 were purchased from Shanghai Angyu Biotechnology Co., Ltd.; Escherichia coli χ7232 and χ7213 were recorded in the literature (Roland K, Curtiss R3rd, Sizemore D. Construction and evaluation of a delta cya delta crpSalmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens. Avian Dis. 1999 Jul-Sep; 43(3): 429-41.); the pRE112 suicide plasmid was recorded in the literature (Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression, doi: 10.1016 / s0378-1119(97)00619-7.); pBluescript II SK(+) plasmid template was obtained from Shanghai Shenggong Bioengineering Technology Service Co., Ltd.; RAW264.7 cells were maintained by our laboratory; 4-6 week old BALB / c mice were purchased from the Experimental Animal Center of Shandong University.

[0051] Salmonella biochemical identification tubes were purchased from Hangzhou Tianhe Microbiological Reagent Company, dNTPs, Taq enzyme, PCR product purification kit, DNA Marker, high-fidelity enzyme (PRIMER STAR); genome extraction kit was purchased from Beijing Biotech Biotechnology Co., Ltd.; homologous recombination enzyme (Exnase multis) was purchased from Nanjing Novozyme Biotechnology Co., Ltd.; DNA gel recovery kit and plasmid extraction kit were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; total RNA extraction kit was purchased from Shanghai Promega Biological Products Co., Ltd.; liquid paraffin was purchased from Zhengzhou Zhongtian Experimental Instrument Co., Ltd.

[0052] DMEM, trypsin, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Inc. Cell screens, ELISA plates, and 12-well cell culture plates (TCP011012) were purchased from Guangzhou Jiete Biofiltration Co., Ltd.

[0053] The sequences of the PCR amplification primers designed in the embodiments of the present invention are as follows:

[0054]

[0055]

[0056] Note: The underlined part is the overlapping sequence of the homology arm of pBlue; the primers were based on S.Typhimurium (Salmonellaenterica subsp.

[0057] enterica serovar Typhimurium str.LT2), whose full sequence can be downloaded from GenBank with the accession number NC_003197.2.

[0058] Example 1: Construction of a yncJ gene-deficient strain of Salmonella typhimurium (STM-ΔyncJ)

[0059] Primers were designed with reference to the yncJ gene sequence of the STM LT2 strain from NCBI. The genome of STM ATCC 14028 (STM-WT) was used as a template to amplify the upstream and downstream fragments of the yncJ gene, respectively. The upstream and downstream fragments of the yncJ gene were cloned into the pBlue vector using a three-fragment homologous recombination method to obtain the pSK-ΔyncJ plasmid, which was sequenced for verification. Then, the upstream and downstream fragments of the yncJ gene were amplified from the pSK-ΔyncJ plasmid using a high-fidelity DNA polymerase and homologously recombined with the backbone fragment of the suicide plasmid pRE112 ( Figure 1Schematic diagram of homologous recombination), and then the recombinant suicide plasmid pRE-ΔyncJ was transformed into E. coli χ7213 cells. E. coli χ7213 carrying the recombinant suicide plasmid pRE-ΔyncJ was used as the donor bacteria, and STM-WT was used as the recipient bacteria for conjugation transfer, and the yncJ deletion strain was screened by the two-step allelic exchange technology. The specific process is as follows:

[0060] 1. Construction of recombinant suicide plasmid:

[0061] (1) PCR amplification of target band:

[0062] The pBlue vector fragment was amplified using the pBluescript II SK(+) plasmid as a template using the pBlue-F / pBlue-R primers; the upstream homology arm fragment and the downstream homology arm fragment of the yncJ gene were amplified using the yncJ-FU / yncJ-FL and yncJ-RU / yncJ-RL primers using the STM genome as a template, respectively. The pRE112 vector fragment was amplified using the pRE112-F / pRE112-R primers using the pRE112 plasmid as a template.

[0063] The PCR reaction program is shown in Table 1:

[0064] Table 1: PCR reaction program

[0065]

[0066] (2) Connection and transformation of target fragments

[0067] According to the instructions of the homologous recombinase (Exnase multis), the recovered pRE112 vector fragment was connected to the upper and lower arms of the yncJ gene homologous arm using the multi-fragment homologous recombination method.

[0068] The ligation product was transformed into competent cells (Escherichia coli TOP10), positive bacteria were screened, plasmids were extracted, and recombinant plasmid pSK-ΔyncJ was prepared.

[0069] According to the instructions of the homologous recombinase (Exnase multis), the recovered pRE112 vector fragment was connected with the upper arm + lower arm fragment of the yncJ gene homologous arm by using the multi-fragment homologous recombination method.

[0070] The ligation product was transformed into Escherichia coliχ7232 competent cells, positive bacteria were screened, plasmids were extracted, and recombinant suicide plasmid pRE-ΔyncJ was prepared.

[0071] 2. Construction of STM-ΔyncJ:

[0072] The recombinant suicide plasmid pRE-ΔyncJ was transformed into strain χ7213, and the donor strain χ7213-pRE-ΔyncJ was obtained by screening and identification.

[0073] Inoculate χ7213-pRE-ΔyncJ in 5 mL of LB (Cm, 25 μg / mL; DAP, 50 μg / mL) liquid culture medium; inoculate STM-WT in 5 mL of antibiotic-free LB liquid culture medium and culture overnight in a constant temperature horizontal shaker at 37°C and 220 rpm; the next day, take 1 mL of χ7213-pRE-ΔyncJ bacterial solution, centrifuge at 5,000 rpm for 5 min, discard the supernatant, and add 500 μL of STM-WT, after mixing, drop it on LB (DAP, 50μg / mL) solid medium, slowly shake the bacterial solution, carefully place it flat in a 37℃ incubator, and culture it statically to ensure the formation of bacterial lawns to facilitate the conjugation and transfer of plasmids; on the clean bench, use a sterile inoculation loop to carefully pick up a single colony, and streak it on the XLD (Cm, 25μg / mL) solid medium, and invert it in a 37℃ incubator for incubation for about 12h; in the clean bench, use a sterile inoculation loop to accurately pick up a single colony, and streak it on the LB solid medium containing Cm resistance, and then invert it in a 37℃ incubator for incubation for about 12h; pick up several single colonies with a sterile white gun tip, inoculate them in several EP tubes containing LB, label them, and culture them in a horizontal shaker at 37℃ and 220rpm for about 3-4h until the bacterial solution is slightly mixed (OD 600 About 0.35); Gradient dilution with appropriate multiples, take about 90 μL and spread it on LB solid medium containing 5% Suc and no NaCl, and culture it flat at room temperature for 48 to 60 hours; Patch the single clone on LB and LB (Cm, 25 μg / mL) solid medium at the same time, and culture the medium upside down in a 37°C incubator for 10 to 12 hours, select the colonies that grow on the LB solid medium without resistance but not on the LB solid medium containing chloramphenicol resistance, and use yncJ-U / yncJ-L as primers for PCR identification.

[0074] Theoretically, STM-WT can amplify a band of 1494 bp, while STM-ΔyncJ can only amplify a band of about 1251 bp. Agarose electrophoresis showed that the size of the fragment amplified by the picked positive colony was consistent with the theoretical value of the deletion strain ( Figure 2 ),

[0075] The PCR product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing, and compared and analyzed with the gene sequence of S. Typhimurium published in GenBank. Based on the above results, it was proved that the gene deletion strain STM-ΔyncJ was successfully constructed.

[0076] Example 2: In vitro growth and physiological and biochemical properties of the yncJ gene-deficient strain of Salmonella typhimurium (STM-ΔyncJ)

[0077] 1. In vitro growth curve of STM-ΔyncJ:

[0078] In a clean bench, single colonies of STM-ΔyncJ and STM-WT were picked up with a sterile white pipette tip, inoculated into EP tubes containing antibiotic-free LB liquid culture medium, and cultured overnight in a horizontal shaker at 37°C and 220 rpm. The next day, the colonies were inoculated into a conical flask containing antibiotic-free LB culture medium at a dilution of 1:1000, and the overnight shaken bacterial solution was repeated three times, and cultured in a constant temperature shaker at 37°C and 220 rpm to obtain the best culture effect. Samples were taken every 1 hour and OD was measured using a UV spectrophotometer. 600 , until the bacteria grow to the stationary phase, and then the OD 600 Draw a growth curve over time.

[0079] The results are as follows Figure 3 As shown, the results showed that the deletion of yncJ gene in STM did not affect the growth rate of bacteria.

[0080] 2. Biochemical characterization of STM-ΔyncJ:

[0081] STM-ΔyncJ was subjected to biochemical identification test, STM-WT was used as positive control, and blank control was set up at the same time. Biochemical identification included lysine decarboxylation broth with liquid paraffin covering the liquid surface, mannitol, dulcitol semisolid, sorbitol, salicin, amino acid decarboxylation control with liquid paraffin covering the liquid surface, malonate, ONPG, tryptophan broth with indigo matrix reagent, urease, potassium cyanide experimental biochemical tube, and potassium cyanide experimental control. The specific steps are as follows:

[0082] (1) In order to conduct the lysine decarboxylase test (STM is positive), we need to inoculate the colonies to be tested into the control tube: the amino acid decarboxylase biochemical tube, and the test tube lysine decarboxylase broth tube. In order to ensure that the pH value of the culture medium is within a reasonable range, we also need to cover the surface of the culture medium with a half-centimeter thick sterile paraffin oil, which can effectively help the bacteria consume the dissolved oxygen in the culture medium. After about one day of cultivation at 37°C, the results showed that the lysine decarboxylase broth showed a distinct yellow color, indicating that it was negative; while the lysine decarboxylase broth showed a distinct purple color, and the amino acid decarboxylase showed a distinct positive color.

[0083] (2) Amino acid decarboxylase test (STM is positive): Inject the colony to be tested into the amino acid decarboxylase test tube and the control tube respectively, and cover the colony with sterile paraffin oil so that the colony height is not less than 0.5 cm. Then, place the colony at a temperature of about 37°C and culture it for 1-4 days to observe the growth of the colony. To ensure the accuracy and reliability of the experimental results. To observe the changes in the colony. The test tube is purple with bromocresol purple indicator, indicating a positive reaction, while yellow indicates a negative reaction.

[0084] (3) ONPG test (STM is negative): Pick a colony and inoculate it into ONPG broth. Culture it at about 37°C for about one day. After observation, when the solution turns yellow, it indicates a positive reaction; otherwise, it indicates a negative reaction.

[0085] (4) Urease test (STM is positive): Select the colonies to be tested and inoculate them into urease broth respectively, and culture them at about 37°C for about one day. The results show that red indicates a positive reaction, while colorless or slightly yellow indicates a negative reaction.

[0086] (5) Under the environment of 36±1℃, select the colonies to be tested, inoculate them into the protein indole water culture medium, and culture them in this environment for 18-24 hours. Use the indole matrix test (STM is negative) to detect the presence of the colonies. Then add a small amount of indole reagent to the culture medium and observe the results.

[0087] The results are shown in Table 2.

[0088] Table 2: Biochemical characterization results of STM-ΔyncJ

[0089]

[0090] The results showed that the biochemical characteristics of the gene-deficient strain STM-ΔyncJ were the same as those of the reference strain, indicating that the deletion of the yncJ gene did not affect the biochemical characteristics of STM.

[0091] 3. Bacterial motility test

[0092] The swimming ability of Salmonella is essential for them to obtain nutrients and infect host cells. In order to detect this ability, we used a semi-solid culture medium, namely a 0.3% agar solution. By observing the chemotaxis zone of bacteria, we found that the larger the chemotaxis zone, the stronger their swimming ability, and vice versa, the weaker their swimming ability. In this experiment, we took STM-ΔyncJ and STM-WT as research objects. Pick a single colony and inoculate it into an EP tube containing LB. The next day, inoculate the two strains of bacteria into new LB at a ratio of 1:10, and culture them in a 37°C shaker. Adjust the OD of the two bacterial strains. 600 When both are 1.0, 2 μL of bacterial solution was inoculated on LB plates containing 0.3% and 1.5% agar (the bacterial solution was slowly dripped onto the solid culture medium), placed in a 37°C incubator, and observed and photographed at 3h and 4h.

[0093] The results are as follows Figure 4 As shown, both STM-WT and STM-ΔyncJ have swimming mobility, but the swimming shape of STM-ΔyncJ is observed to be different from that of STM-WT in the swimming medium.

[0094] 4. Scanning electron microscopy observation of STM-ΔyncJ bacterial morphology

[0095] SEM pre-treatment: After inoculating single colonies of STM-WT and STM-ΔyncJ overnight, the next day the overnight bacteria were transferred to new culture medium at a ratio of 1:10 and shaken to the logarithmic growth phase. The bacteria were cultured to OD 600 About 0.6; then dilute with sterile PBS, and when diluted to the appropriate multiple, spread the diluted bacterial solution on LB solid culture medium at the same time, after 12h of culture. Take out the solid culture medium, rinse the colonies on the solid culture medium with sterile PBS, blow off the colonies and collect them, and then rinse them with sterile PBS 5 times, 20 minutes each time. After adding PBS each time, blow and mix with a gun tip. Starve overnight, rinse with PBS 5 times the next day, 20 minutes each time, dehydrate with 45% ethanol, 55% ethanol, 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol gradient, and then change to 100% ethanol for dehydration twice, treat with isoamyl acetate, and continue to treat with isoamyl acetate the next day. Scanning electron microscope critical point drying, scanning electron microscope ion spraying, and finally scanning electron microscope observation (Zeiss field emission).

[0096] The results are as follows Figure 5 As shown, the results showed that the extracellular secretion of STM-ΔyncJ was significantly increased compared with STM-WT.

[0097] Example 3: Investigation of the virulence of the yncJ gene-deficient strain of Salmonella typhimurium (STM-ΔyncJ)

[0098] 1. Investigation of the proliferation ability of STM-ΔyncJ in macrophages:

[0099] Single colonies of STM-WT and STM-ΔyncJ carrying green fluorescent plasmid (pTurboGFP-B) were picked and cultured overnight. The next day, the bacterial solution was transferred to fresh LB (Cm, 100 μg / mL) medium at a ratio of 1:100. The bacteria grown to the logarithmic phase were centrifuged at 2,000 rpm for 5 min, washed twice with PBS, and the OD was adjusted. 600 =0.6, then the number of bacteria is 5×10 8 CFU / mL, digest and count one well of the plated macrophages (RAW264.7 cells), calculate the number of cells in one well, add bacterial cells to the cell monolayer at MOI=10, centrifuge the cell plate at 100g for 5min, and incubate at 37°C, 5% CO2 for 30min. Wash 3 times with sterile PBS solution at 37°C, then gently blow, and finally add DMEM medium containing gentamicin (100μg / mL) to the infected cells and incubate for 30min to effectively eliminate extracellular bacteria. After the 30min incubation process, we added DMEM (containing 10%) medium with a concentration of 20μg / mL of gentamicin to the infected cells at a concentration of 20μg / mL per mL for further study. Incubate for another 1h to inhibit extracellular bacterial growth. At 2h, 6h, and 10h after infection, the cells were washed three times with a sterile PBS solution (pH=7.4) preheated at 37°C to remove uninvaded bacteria. 200 μL of trypsin was added to each well, and then 400 μL of culture medium with serum was added to neutralize the trypsin. The mean fluorescence intensity was measured by upflow. Three independent biological replicates were performed for each group of experiments.

[0100] The results are as follows Figure 6 As shown in the figure, the intracellular proliferation ability of STM-ΔyncJ in Raw 264.7 cells was significantly lower than that of STM-WT (P<0.01); According to the above results, the intracellular proliferation ability of STM-ΔyncJ in macrophages was significantly lower than that of STM-WT. This shows that the yncJ gene plays an important role in the intracellular proliferation of STM, and the loss of the yncJ gene significantly reduces the proliferation of STM in macrophages.

[0101] 2. Detection of pathogenicity of STM-ΔyncJ to mice:

[0102] Select a single colony from each of STM-WT and STM-ΔyncJ, place it in LB liquid medium, and shake it at 37°C and 220 rpm. The next day, transfer the overnight bacteria to new medium and shake it until the logarithmic growth phase. Cultivate the bacteria until OD 600The concentration of the bacterial solution was about 1.0; the cells were washed twice with sterile PBS, and then diluted with PBS in multiple ratios to adjust the concentration of the bacterial solution to 5 × 10 2 CFU / mL, 200 μL / mouse, i.e. 100 CFU / mouse, were intraperitoneally injected into BALB / c mice. Ten mice were inoculated with each bacterium. 200 μL / mouse of sterile PBS solution was used as negative control. The mice were observed and recorded daily to evaluate their survival status, and the results were plotted into a survival curve.

[0103] The results are as follows Figure 7 As shown, the results showed that all mice injected with wild-type STM-WT died within 6 days, while mice injected intraperitoneally with STM-ΔyncJ maintained a high survival rate 10 days after injection. The statistical analysis results (Logrank) of the survival curve showed that STM-ΔyncJ had a reduced lethality to mice compared with STM-WT.

[0104] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of the yncJ gene as a target gene in at least one of the following (1)-(3): (1) Regulate the proliferation of Salmonella typhimurium in macrophages; (2) regulating the virulence of Salmonella typhimurium; (3) Preparation of attenuated Salmonella typhimurium vaccine; The yncJ gene is a nucleic acid molecule as shown in the following i) or ii): i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID No.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID No. 2 other than i).

2. Use of the protein encoded by the yncJ gene as a target in the following (1) or (2): (1) Regulate the proliferation of Salmonella typhimurium in macrophages; (2) Regulate the virulence of Salmonella typhimurium.

3. The use according to claim 2, characterized in that: The amino acid sequence of the protein encoded by the yncJ gene is shown in SEQ ID No.

2.

4. Use of a substance for inhibiting yncJ gene expression in at least one of the following (1)-(3): (1) Preparing a product that reduces the proliferation of Salmonella typhimurium in macrophages; (2) preparing products that reduce the virulence of Salmonella typhimurium; (3) Preparation of attenuated Salmonella typhimurium vaccine.

5. The use according to claim 4, characterized in that: The substance that inhibits the expression of yncJ gene is selected from: suicide plasmid, siRNA, shRNA and / or CRISPi.

6. The use according to claim 4 or 5, characterized in that: The substance that inhibits the expression of the yncJ gene is a suicide plasmid pRE112 carrying upstream and downstream fragments of the yncJ gene.

7. An attenuated Salmonella typhimurium vaccine, characterized in that: The attenuated Salmonella typhimurium vaccine uses the yncJ gene-deficient strain of Salmonella typhimurium as an active ingredient.

8. The attenuated Salmonella typhimurium vaccine according to claim 7, characterized in that The yncJ gene-deficient strain of Salmonella typhimurium is constructed by the following method: The upstream and downstream fragments of the yncJ gene were cloned into the suicide plasmid pRE112 respectively to obtain the recombinant plasmid pRE-ΔyncJ; then the recombinant plasmid pRE-ΔyncJ was transformed into Escherichia coli χ7213, and the Escherichia coli χ7213 carrying the recombinant plasmid pRE-ΔyncJ was used as the donor bacteria and the wild-type Salmonella typhimurium was used as the recipient bacteria for conjugation transfer, and the yncJ gene-deficient strain was screened by the two-step allelic exchange technology.

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