Homologous recombination and traceless deletion detection primer for polar flagellin gene locus of vibrio parahaemolyticus and application of homologous recombination and traceless deletion detection primer

Abstract

The invention discloses a primer for homologous recombination and traceless deletion detection of vibrio parahaemolyticus polar flagellin gene loci, the vibrio parahaemolyticus polar flagellin gene loci comprise flaA, flaB, flaC, flaD, flaE and flaF genes, and the primer is specifically shown as a table 1; the invention also discloses a traceless deletion construction method of the vibrio parahaemolyticus polar flagellin gene locus, and application of the traceless deletion vibrio parahaemolyticus polar flagellin gene locus constructed by the method in regulation and control of vibrio parahaemolyticus motility, biofilm formation, adhesion capability, toxicity and immunogenicity. According to the primer provided by the invention, efficient traceless genetic manipulation on the polar flagellin gene locus of the vibrio parahaemolyticus is realized; the traceless deletion polar flagellin gene locus provided by the invention obviously influences the motility, biofilm formation, adhesion capacity, toxicity and immunogenicity of the vibrio parahaemolyticus, and has an application prospect in prevention and control of vibrio parahaemolyticus diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry and molecular biology, and in particular relates to a homologous recombination and traceless deletion detection primer for a polar flagellin gene locus of Vibrio parahaemolyticus and an application thereof. Background Art

[0002] Vibrio parahaemolyticus, a member of the Vibrionaceae family and the genus Vibrio, is a Gram-negative halophilic bacterium that is widely distributed in oceans, estuaries and coastal areas. Its harm to humans and aquatic organisms has caused a huge burden on global public health and fishery economy. Its virulence factors mainly include hemolysin, adhesin, quorum sensing system, secretion system effector and flagella system.

[0003] Successful pathogens need to repeat the process of gravitating to host cells / infection sites, successfully adhering to colonization and defeating the host's immune system, maintaining growth and replication, and finally gravitating to the next host cell / pathogenesis site. Flagella, as one of the most complex molecular machines in bacteria, plays different roles in these processes. In addition to being essential for bacterial movement, flagella also play a key role in colonization, biofilm formation, penetration of the host mucosal layer, cell adhesion and invasion.

[0004] The study of flagellin is of great significance for studying pathogenic mechanisms and the development and application of pathogenic microorganism vaccines. The use of bacterial flagellin components alone in vaccines can activate innate immunity and adaptive immunity against vaccine antigens by inducing the production of proinflammatory cytokines, making flagellin a potential vaccine adjuvant as a pattern recognition receptor activator.

[0005] Most of the existing technologies use the transposon insertion method to mutate the entire polar flagellin locus of Vibrio parahaemolyticus. Compared with homologous recombination, the transposon insertion method has higher randomness and lower efficiency, and introduces exogenous sequences into the genome. Summary of the invention

[0006] The purpose of the present invention is to provide a primer for detecting homologous recombination and traceless deletion of the polar flagellin gene locus of Vibrio parahaemolyticus.

[0007] The present invention also aims to provide a method for constructing a seamless deletion of the polar flagellin gene locus of Vibrio parahaemolyticus using the above primers.

[0008] The last object of the present invention is to provide the use of the seamlessly deleted polar flagellin gene locus of Vibrio parahaemolyticus constructed by the above method in regulating the motility, biofilm formation, adhesion ability, virulence and immunogenicity of Vibrio parahaemolyticus.

[0009] The first object of the present invention can be achieved by the following technical scheme: a primer for homologous recombination and traceless deletion detection of the polar flagellin gene locus of Vibrio parahaemolyticus, wherein the polar flagellin gene locus of Vibrio parahaemolyticus includes flaA, flaB, flaC, flaD, flaE and flaF genes, and the primers are specifically shown in Table 1 below:

[0010] Table 1 Primers used for homologous recombination and scarless deletion detection of the polar flagellin locus of Vibrio parahaemolyticus

[0011]

[0012]

[0013] in:

[0014] flaA-up / flaA-down, flaB-up / flaB-down, flaC-up / flaC-down, flaD-up / flaD-down, flaE-up / flaE-down, and flaF-up / flaF-down are primer pairs for amplifying the left and right homology arms of flaA, flaB, flaC, flaD, flaE, and flaF in the polar flagellin locus, respectively. TA, flaB-up-F / flaB-down-R, flaC-up-F / flaC-down-R, flaD-up-F / flaD-down-R, flaE-up-F / flaE-down-R, and flaF-up-F / flaF-down-R are primer pairs for detecting traceless deletions of flaA, flaB, flaC, flaD, flaE, and flaF in the polar flagellin locus, respectively.

[0015] Preferably, the flaF gene, flaB gene, and flaA gene are located within the base range of 912795-916746 of chromosome 1 of Vibrio parahaemolyticus RIMD 2210633 strain, the flaC gene, flaD gene, and flaE gene are located within the base range of 2461519-2465722 of chromosome 1 of Vibrio parahaemolyticus RIMD 2210633 strain, and the NCBI reference sequence of chromosome 1 of Vibrio parahaemolyticus RIMD 2210633 strain is GCF_000196095.1.

[0016] The second object of the present invention can be achieved by the following technical solution: A method for constructing a seamless deletion of the polar flagellin gene locus of Vibrio parahaemolyticus, comprising the following steps:

[0017] (1) Extracting Vibrio parahaemolyticus genomic DNA;

[0018] (2) performing PCR amplification on the extracted DNA using the above-mentioned left and right homology arm amplification primer pairs to obtain an amplification product;

[0019] (3) homologously recombining the amplified product in step (2) with the suicide vector after enzyme digestion and introducing it into the Vibrio parahaemolyticus genome through conjugation transfer;

[0020] (4) Two rounds of homologous recombination were performed and verified using the above-mentioned primer pair for detecting scarless deletion to obtain a scarless deletion of the polar flagellin gene locus of Vibrio parahaemolyticus.

[0021] In the prior art, the entire polar flagellin gene locus of Vibrio parahaemolyticus was mutated by using the transposon insertion method. Compared with homologous recombination, the transposon insertion method has higher randomness and lower efficiency, and introduces exogenous sequences into the genome. The homologous homogroup method and primers used in the present invention utilize the upstream and downstream homologous arms of the known polar flagellin encoding gene. Therefore, compared with the existing methods, the targeted deletion of nucleotide sequences can be efficiently achieved, and the deletion will not introduce other nucleotide sequences into the genome.

[0022] Method for constructing a scarless deletion in the polar flagellin locus of Vibrio parahaemolyticus:

[0023] Preferably, the suicide vector in step (3) is suicide plasmid pDS132.

[0024] Preferably, during the conjugation transfer in step (3), the reaction is carried out at 37° C. for 30 minutes.

[0025] Preferably, the seamless deletion of the polar flagellin gene locus of Vibrio parahaemolyticus obtained in step (4) includes the polar flagellin gene deletion strains of Vibrio parahaemolyticus with successful gene knockout of △flaA, △flaB, △flaC, △flaD, △flaE, and △flaF.

[0026] The last object of the present invention can be achieved by the following technical scheme: application of the traceless deletion of the polar flagellin locus of Vibrio parahaemolyticus constructed by the above method in regulating the motility, biofilm formation, adhesion ability, virulence and immunogenicity of Vibrio parahaemolyticus.

[0027] The present invention has the following advantages:

[0028] (1) The primers provided by the present invention realize efficient and traceless genetic manipulation of the polar flagellin locus of Vibrio parahaemolyticus;

[0029] (2) The traceless deletion of the polar flagellin locus provided by the present invention significantly affects the motility, biofilm formation, adhesion ability, virulence and immunogenicity of Vibrio parahaemolyticus, and has application prospects in the prevention and control of Vibrio parahaemolyticus diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the PCR amplification product of the left and right homology arm fragments of the polar flagellin gene in Example 1, M: DS2000 marker; 1-12: the left homology arm and right homology arm of flaA, the left homology arm and right homology arm of flaB, the left homology arm and right homology arm of flaC, the left homology arm and right homology arm of flaD, the left homology arm and right homology arm of flaE, and the left homology arm and right homology arm of flaF;

[0031] Figure 2 is the PCR amplification product of S17-1-λpir carrying the recombinant vector in Example 1, M: DS2000: 1-6 are the amplification products of pDS132-flaA, pDS132-flaB, pDS132-flaC, pDS132-flaD, pDS132-flaE, and pDS132-flaF, respectively;

[0032] Figure 3 PCR amplification products of the wild strain and the deletion strain in Example 1, PCR amplification products of the wild strain and the deletion strain M: Marker 3; 1, 3, 5, 7, 9, 11: PCR amplification products of the wild strain; 2, 4, 6, 8, 10, 12: PCR amplification products of △flaA, △flaB, △flaC, △flaD, △flaE, △flaF;

[0033] Figure 4 The swimming ability and swarming ability of the wild-type strain and each deletion strain in Example 2. A, B: swimming plate and colony diameter measurement results; C, D: swarming plate and colony diameter measurement results; error bars represent the mean ± standard error of 3 replicates; *P<0.05, **P<0.01;

[0034] Figure 5 The biofilm formation ability of the wild-type strain and each deletion strain in Example 2. The error bars represent the mean ± standard error of 3 replicates; *P<0.05, ***P<0.001;

[0035] Figure 6 The adhesion ability of the wild-type strain and each deletion strain to HeLa cells in Example 2. The error bars represent the mean ± standard error of 3 replicates; ****P<0.0001;

[0036] Figure 7The toxicity of the wild-type strain and each deletion strain to HeLa cells in Example 2. The error bars represent the mean ± standard error of 3 replicates; **P<0.01, ****P<0.0001;

[0037] Figure 8 The effect of infection with the wild-type strain and each deletion strain on the cytokine expression of GS cells in Example 2. The asterisks on the correlation lines and columns respectively represent the results of the significance analysis comparison between the MLB group and each strain group, and the WT group and each deletion strain group; the error bars represent the mean ± standard error of 3 replicates; **P<0.01, ****P<0.0001. DETAILED DESCRIPTION

[0038] Example 1

[0039] The technical scheme of the present invention is described in detail below in conjunction with specific embodiments, so that those skilled in the art can better understand and implement the technical scheme of the present invention. The following examples and accompanying drawings are only used for exemplary description and cannot be construed as limiting the present invention. The reagents or materials used in the embodiments, if not otherwise specified, are all derived from commercial channels. Unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.

[0040] To illustrate the present invention in more detail, the following examples are provided. It should be emphasized that the following examples are only for explaining the present invention and are not intended to limit the essential scope or content of the present invention.

[0041] Example 1: Traceless genetic manipulation of the polar flagellin locus of Vibrio parahaemolyticus

[0042] The specific steps are as follows:

[0043] (1) The primers used for homologous recombination and knockout verification are shown in Table 1 below:

[0044]

[0045]

[0046] Note: flaA-up / flaA-down, flaB-up / flaB-down, flaC-up / flaC-down, flaD-up / flaD-down, flaE-up / flaE-down, and flaF-up / flaF-down are primer pairs for amplifying the left and right homology arms of flaA, flaB, flaC, flaD, flaE, and flaF in the polar flagellin locus, respectively. TA, flaB-up-F / flaB-down-R, flaC-up-F / flaC-down-R, flaD-up-F / flaD-down-R, flaE-up-F / flaE-down-R, and flaF-up-F / flaF-down-R are primer pairs for detecting seamless deletions of flaA, flaB, flaC, flaD, flaE, and flaF in the polar flagellin locus, respectively.

[0047] (2) The genomic DNA of Vibrio parahaemolyticus RIMD2210633 strain was extracted using Rapid Bacterial Genomic DNA Isolation Kit. Max Super-Fidelity DNA Polymerase amplifies the homology arms of about 500 bp on the left and right sides; reaction conditions: use a pipette to gently mix, and centrifuge briefly to collect the reaction solution to the bottom of the tube. 95℃, 3min; (95℃, 15s; 63℃, 15s; 72℃, 30s)×35cycles; 72℃, 5min. After the reaction is completed, the amplification product is electrophoresed in 1% agarose gel to detect the results and perform gel recovery steps.

[0048] The results are as follows Figure 1 As shown:

[0049] Figure 1 The homology arm amplified fragments with lengths of about 500 bp on the left and right sides, where M: DS2000 marker; 1-12: the left homology arm and right homology arm of flaA in the polar flagellin gene locus, the left homology arm and right homology arm of flaB, the left homology arm and right homology arm of flaC, the left homology arm and right homology arm of flaD, the left homology arm and right homology arm of flaE, the left homology arm and right homology arm of flaF, and the theoretical sizes of the homology arms are 533, 529, 537, 525, 521, 541, 529, 533, 534, 528, 530, and 532 bp, respectively.

[0050] (3) XbaⅠ single enzyme digestion or SphⅠ-HF and SacⅠ-HF double enzyme digestion to linearize 1 μg suicide plasmid pDS132. Reaction conditions: Use a pipette to gently mix, and briefly centrifuge to collect the reaction solution to the bottom of the tube. React at 37℃ for 1 hour. After the reaction is completed, the enzyme digestion products are electrophoresed in 1% agarose gel to detect the results.

[0051] (4)Use MultiS One Step Cloning Kit was used to connect 0.01 ng of homology arms obtained in step (2) with 0.1 μg of linearized pDS132 plasmid obtained in step (3). Reaction conditions: Use a pipette to gently mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube. React at 37°C for 30 min; cool to 4°C or immediately place on ice.

[0052] (5) Take 10 μL of the above recombinant product and add it to 100 μL of E. coli S17-1λpir competent cells, mix gently, and place on ice for 30 min; heat shock in a 42°C water bath for 45 s, and then place on ice for 3 min; add 900 μL of LB liquid medium and culture at 37°C, 250 rpm for 1.5 h. Centrifuge at 5000 rpm for 5 min at room temperature, discard 900 μL of supernatant, resuspend the cells in the remaining medium, and spread on LB plates (containing 30 μg / mL of chloramphenicol), and culture in a 37°C incubator for 16 h.

[0053] (6) Colony verification was performed using 2×T5 Super PCR Mix (Colony). Each pair of homology arm fragments obtained was connected to the linearized pDS132 vector and transformed into E. coli S17-1-λpir. The bacterial solution was then used as a template and amplified using pDS132 universal primers to obtain fragments of 1329 bp in size, indicating that each pair of homology arm fragments had been inserted into the suicide vector and successfully transformed. Six strains of S17-1-λpir carrying the recombinant suicide vectors pDS132-△flaA, pDS132-△flaB, pDS132-△flaC, pDS132-△flaD, pDS132-△flaE, and pDS132-△flaF were obtained. The electrophoresis results are shown in Figure 2. Figure 2 shown.

[0054] Reaction conditions: Use a pipette to gently mix, and centrifuge briefly to collect the reaction solution to the bottom of the tube. 98℃, 3min; (98℃, 10s; 56℃, 10s; 72℃, 30s)×35cycles; 72℃, 2min. After the reaction is completed, the amplification product is electrophoresed in 1% agarose gel to detect the results. 20% glycerol is frozen in a -80℃ ultra-low temperature refrigerator.

[0055] The pDS132 universal primers are as follows in Table 2:

[0056] Table 2 Universal primers for pDS132

[0057]

[0058]

[0059] (7) Inoculate E. coli S17-1-λpir△ into 10 mL of liquid LB medium (containing 30 μg / mL of chloramphenicol), and at the same time inoculate wild-type Vibrio parahaemolyticus RIMD 2210633 into 10 mL of MLB medium, and culture at 37°C, 250 rpm, for 8 h; transfer 1 mL of E. coli S17-1-λpir△ culture medium into a 1.5 mL centrifuge tube, centrifuge at 4000 rpm at room temperature for 5 min, discard the supernatant, rinse the cells twice with 1 mL of liquid LB medium (without chloramphenicol), centrifuge at 4000 rpm at room temperature for 5 min, and discard the supernatant.

[0060] (8) Take 1 mL of the culture medium of Vibrio parahaemolyticus and transfer it to a 1.5 mL centrifuge tube. Centrifuge at 4000 rpm for 5 min at room temperature and discard the supernatant. Rinse the cells twice with 1 mL of MLB medium and centrifuge at 4000 rpm for 5 min at room temperature and discard the supernatant. Finally, mix and resuspend the two cells with 100 μL of MLB medium respectively. Drop the resuspended liquid on a 0.45 μm filter placed on LB agar medium, air-dry and invert for 12 h.

[0061] (9) Use sterile tweezers to pick up the filter membrane and place it in a 50 mL centrifuge tube containing 5 mL of MLB medium. Place it in a 37°C incubator for 1 h, and then culture it at 37°C and 250 rpm for 1 h. Then, dilute the culture solution to 10 million times and spread 100 μL of each on 10 MLB plates (containing 5 μg / mL chloramphenicol and 100 μg / mL ampicillin). Culture it at 37°C for several days until a single colony is formed (in most cases, it grows into a bacterial lawn). Single colonies or bacterial lawns are isolated and purified more than three times on MLB plates (containing 5 μg / mL chloramphenicol and 100 μg / mL ampicillin) by streaking to obtain purified Vibrio parahaemolyticus (pDS132△) that has undergone the first homologous recombination, and freeze it in a -80°C ultra-low temperature refrigerator with 20% glycerol.

[0062] (10) Inoculate the first batch of Vibrio parahaemolyticus (pDS132△) into a 50-mL centrifuge tube containing 15 mL of liquid LB medium (containing 100 μg / mL ampicillin and 20% sucrose). When the culture medium becomes slightly turbid, dilute it by appropriate multiples and spread 100 μL of each dilution on a TCBS plate (containing 20% ​​sucrose).

[0063] (11) Use an inoculating loop to tap a single colony on a TCBS plate, and then tap a TCBS plate (containing 20% ​​sucrose) and an MLB plate (containing 5 μg / mL chloramphenicol and 100 μg / mL ampicillin) in sequence, and incubate the plate upside down at 37°C for 6 h.

[0064] (12) The clones that can grow on TCBS plates (containing 20% ​​sucrose) but not on MLB plates (containing 5 μg / mL chloramphenicol and 100 μg / mL ampicillin) were selected and placed in 1 mL of liquid MLB medium. After culturing at 37°C and 250 rpm for 2.5 h, the colonies were verified using the primers listed in Table 1. The reaction conditions were as follows: Use a pipette to gently mix, and briefly centrifuge to collect the reaction solution to the bottom of the tube. 98°C, 3 min; (98°C, 10 s; 66°C, 10 s; 72°C, 30 s) × 35 cycles; 72°C, 2 min. After the reaction was completed, the amplification product was electrophoresed in 1% agarose gel to detect the results. The polar flagellar protein gene deletion strains of Vibrio parahaemolyticus △flaA, △flaB, △flaC, △flaD, △flaE, and △flaF that had undergone the second homologous recombination, i.e., the gene was successfully knocked out, were frozen in 20% glycerol and stored in a -80°C ultra-low temperature freezer.

[0065] The PCR amplification products of the wild-type strain and the polar flagellin gene locus deletion strain are shown in Figure 3 As shown:

[0066] Figure 3 It is the PCR amplification product of the wild strain and the polar flagellin gene locus deletion strain, wherein the peripheral primers of the knocked-out gene (for flaA, TA-F / TA-R are used, and the outermost primer pairs constructed by homologous arms are used for other genes) are used to perform colony verification on the Vp wild-type strain and the corresponding deletion strain, respectively, and fragments of sizes of 1576, 445 bp (flaA length is 1131 bp), 2199, 1062 bp (flaB length is 1137 bp), 2217, 1062 bp (flaC length is 1155 bp), 2199, 1062 bp (flaD length is 1137 bp), 2187, 1062 bp (flaE length is 1125 bp), and 2196, 1062 bp (flaF length is 1134 bp) are obtained, respectively.

[0067] Example 2: Detection of the polar flagellin locus regulating motility, biofilm formation, adhesion ability, virulence and immunogenicity of Vibrio parahaemolyticus

[0068] (1) Motility evaluation: The wild-type strain of Vibrio parahaemolyticus and the strain with a scarless deletion of the polar flagellin gene locus constructed in Example 1 were revived and inoculated into new MLB medium at a ratio of 1:100 to culture until the early logarithmic phase. The culture was diluted until OD600 = 0.5, and 2.5 μL of the bacterial solution was vertically dripped onto the plate in triplicate. The movement diameter was measured and photographed and saved under a gel imaging analysis system. The analysis results are shown in FIG. Figure 4 shown.

[0069] The results of studies on the regulation of motility by the polar flagellin locus are as follows Figure 4 As shown, flaC significantly enhanced the swimming ability, and flaD, flaE, and flaF significantly enhanced the clustering ability.

[0070] (2) Biofilm assay: measuring the OD of overnight cultures 600 and use the formula 0.5 × 0.05 / OD 600 Calculate the final OD of 0.5 mL of culture medium 600 The required culture volume is 0.05. Transfer this volume to a 24-well plate containing 0.5 mL of MLB medium in triplicate. Incubate statically at 37°C for 12 h, 24 h, 36 h, and 48 h, respectively. Discard the culture medium, add 500 μL of PBS to each well to rinse twice, discard the PBS, add 500 μL of 1% crystal violet solution to each well, and discard the dye after staining for 30 minutes. Slowly wash the well wall with running water until the running water is completely colorless and transparent. Then invert it on filter paper and place it in a 65°C oven to dry for 30 minutes. After drying, add 1 mL of 33% glacial acetic acid solution to each well, gently blow and elute the biofilm, and place it in a 37°C oven to dissolve for 30 minutes. Measure the absorbance at a wavelength of 590 nm, and the analysis results are as follows. Figure 5 shown.

[0071] Figure 5 The results of the study on the regulation of biofilm formation ability by the polar flagellin locus showed that at 12h, there was no significant difference in the biofilm formation ability of each strain; at 24h, △flaE decreased significantly; at 36h, △flaA and △flaD increased significantly; at 48h, △flaB, △flaC and △flaD all decreased significantly. There was no significant difference between the other deletion strains and the wild-type strain at the corresponding time points.

[0072] (3) Cell adhesion assay: HeLa cells were plated at 5×10 4 cells / mL in a 96-well plate containing 100 μL of DMEM containing 10% FBS; measure the OD of the overnight culture. 600, and diluted to 1 using MLB liquid culture medium. Wash Hela cells twice with PBS, add 100μL DMEM containing 10% FBS, and transfer 1μL bacterial solution (i.e., the multiplicity of infection MOI is 10) to each corresponding well. Centrifuge at 200×g for 5min at room temperature to synchronize infection, and incubate at 37°C for 1h. Wash the cells twice with PBS, add 100μL 1% Triton X-100, and lyse at 37°C for 10min to obtain adherent bacteria; dilute the cell lysate continuously, spread it on an MLB plate, and culture it at 37°C for 12h. Count the number of adherent bacteria, and analyze the results as shown in the figure. Figure 6 shown.

[0073] Figure 6 The results of the study on the regulation of adhesion ability by the polar flagellin locus showed that △flaE and △flaF significantly enhanced the adhesion ability of cells.

[0074] (4) Cytotoxicity assay: refer to Invitrogen CyQUANT TM LDH Cytotoxicity Assay Kit Instructions: Spontaneous LDH activity group, experimental group, and maximum LDH activity group were set up. HeLa cells were cultured at 5×10 4 cells / mL in a 96-well plate containing 100 μL of phenol red-free DMEM containing 10% FBS; the OD of the overnight culture was measured. 600 , and diluted to 1 using MLB liquid culture medium. Wash Hela cells twice with PBS, add 100μL of new phenol red-free DMEM, transfer 1μL of bacterial solution and 9μL of ultrapure water to each corresponding well, transfer 10μL of ultrapure water to the spontaneous LDH activity well, and transfer 10μL of 10x Lysis Buffer to the maximum LDH activity well; centrifuge at 200×g for 5min at room temperature to synchronize infection, and incubate at 37°C for 1h; transfer 50μL from each well to a new 96-well plate, add 50μL of Reaction Mixture to each, and incubate at room temperature in the dark for 30min. Add 50μL Top Solution to each well and mix gently; measure the absorbance at wavelengths of 490nm and 680nm, and calculate the percentage of cytotoxicity. The analysis results are as follows: Figure 7 shown.

[0075] Figure 7 The results of the study on the regulation of cytotoxicity by the polar flagellin gene locus showed that the toxicity of △flaC to cells was significantly reduced, while the toxicity of △flaD was significantly increased.

[0076] (5) Immunogenicity assay: Grouper spleen GS cells were cultured at 5×10 4cells / mL in a 96-well plate containing 100 μL of L15 containing 10% FBS; the OD of the overnight culture was measured. 600 , and diluted to 1 with MLB liquid medium. Wash GS cells twice with PBS, add 100μL L15 containing 10% FBS, transfer 1μL bacterial solution (i.e., MOI of 10) to each corresponding well; centrifuge at 200×g for 5min at room temperature to synchronize infection, and incubate at 37°C for 1h; discard the medium, wash GS cells twice with PBS, add 200μL L15 containing 10% FBS and 100ug / mL gentamicin, and incubate for 2h. Discard the medium and add 100μL PBS to each well to wash the cells. Discard PBS and add TOYOBO 49.7μL Lysis Solution and 0.3μL gDNARemover of Cell Lysis&RT Kit for qPCR were incubated at room temperature for 5min. 9.5μL Top Solution and 0.5μL Rnase Inhibitor were added to each well and incubated at room temperature for 2min. 8μL cell lysis solution, 8μL 5×RT Master Mix and 12μL ddH2O were mixed and placed in a PCR instrument to complete the reverse transcription reaction. The reaction conditions were: 37℃, 15min; 50℃, 5min; 98℃, 5min. After the reaction was completed, it was stored at -20℃ for later use. Fluorescence quantitative PCR was performed, and the reaction procedure was: 95℃, 60s; (95℃, 15s; 56℃, 15s; 72℃, 45s)×50cycles; significance analysis was performed using one-way analysis of variance and Dunnett's multiple t-test for post hoc comparison. The analysis results are shown in the figure. Figure 8 shown.

[0077] Figure 8 The results of the study on the regulation of immunogenicity by the flagellin locus at the central pole showed that WT, △flaB, △flaE, and △flaF significantly upregulated the expression of IL-6 and IL-1β mRNA, △flaA, △flaB, △flaC, △flaD, and △flaE significantly downregulated the expression of IL-6 mRNA, △flaA, △flaC, and △flaD significantly downregulated the expression of IL-1β mRNA, and △flaF significantly upregulated the expression of IL-6 and IL-1β mRNA, and the absence of FlaF led to a significant upregulation of the expression of IFN-γ2 mRNA.

[0078] The above research results show that each polar flagellin plays a role in the movement, growth, biofilm formation, adhesion to host cells and virulence during infection, as well as the activation of immune response of Vibrio parahaemolyticus to varying degrees. This polar flagellin locus has application prospects in the prevention and control of Vibrio parahaemolyticus diseases.

[0079] The above embodiments are only used to illustrate the present invention, and the protection scope of the present invention is not limited to the above embodiments. A person skilled in the art can achieve the purpose of the present invention based on the above disclosure of the present invention, and any improvement and deformation based on the concept of the present invention shall fall within the protection scope of the present invention, and the specific protection scope shall be subject to the claims.

Claims

1. A primer for homologous recombination and scarless deletion detection of the polar flagellin gene locus of Vibrio parahaemolyticus, It is characterized in that The polar flagellin gene locus of Vibrio parahaemolyticus includes flaA, flaB, flaC, flaD, flaE and flaF genes, and the primers are specifically shown in Table 1 below: Table 1 Primers used for homologous recombination and scarless deletion detection of the polar flagellin locus of Vibrio parahaemolyticus in: flaA-up / flaA-down, flaB-up / flaB-down, flaC-up / flaC-down, flaD-up / flaD-down, flaE-up / flaE-down, and flaF-up / flaF-down are primer pairs for amplifying the left and right homology arms of flaA, flaB, flaC, flaD, flaE, and flaF in the polar flagellin locus, respectively. TA, flaB-up-F / flaB-down-R, flaC-up-F / flaC-down-R, flaD-up-F / flaD-down-R, flaE-up-F / flaE-down-R, and flaF-up-F / flaF-down-R are primer pairs for detecting traceless deletions of flaA, flaB, flaC, flaD, flaE, and flaF in the polar flagellin locus, respectively.

2. A method for constructing a scarless deletion of the polar flagellin gene locus of Vibrio parahaemolyticus, It is characterized in that The following steps are involved: (1) Extracting Vibrio parahaemolyticus genomic DNA; (2) performing PCR amplification on the extracted DNA using the left and right homologous arm amplification primer pairs in claim 1 to obtain an amplified product; (3) homologously recombining the amplified product in step (2) with the suicide vector after enzyme digestion and introducing it into the Vibrio parahaemolyticus genome through conjugation transfer; (4) performing two rounds of homologous recombination and verifying using the primer pair for detecting the scarless deletion in claim 1 to obtain a scarless deletion of the polar flagellin gene locus of Vibrio parahaemolyticus.

3. The method for constructing a seamless deletion of the polar flagellin gene locus of Vibrio parahaemolyticus according to claim 2, It is characterized in that The suicide vector described in step (3) is suicide plasmid pDS132.

4. The method for constructing a seamless deletion of the polar flagellin gene locus of Vibrio parahaemolyticus according to claim 2, It is characterized in that During the ligation transfer in step (3), the reaction was carried out at 37°C for 30 minutes.

5. The method for constructing a seamless deletion of the polar flagellin gene locus of Vibrio parahaemolyticus according to claim 2, It is characterized in that The traceless deletion of the polar flagellin gene locus of Vibrio parahaemolyticus obtained in step (4) includes the polar flagellin gene deletion strains of Vibrio parahaemolyticus with successful gene knockout of △flaA, △flaB, △flaC, △flaD, △flaE, and △flaF.

6. Use of the seamlessly deleted polar flagellin gene locus of Vibrio parahaemolyticus constructed by the method according to any one of claims 2 to 5 in regulating the motility, biofilm formation, adhesion ability, virulence and immunogenicity of Vibrio parahaemolyticus.

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