Detection primer for homologous recombination and scarless deletion of vibrio parahaemolyticus polar flagellum protein gene locus and application thereof

Abstract

The application discloses a primer for homologous recombination and scarless deletion detection of Vibrio parahaemolyticus flagellum protein loci, wherein the Vibrio parahaemolyticus flagellum protein loci comprises flaA, flaB, flaC, flaD, flaE and flaF genes, and the primer is specifically shown in Table 1; a scarless deletion construction method of the Vibrio parahaemolyticus flagellum protein loci is also disclosed, and application of the scarless deletion Vibrio parahaemolyticus flagellum protein loci constructed by the method in regulating motility, biofilm formation, adhesion capacity, virulence and immunogenicity of Vibrio parahaemolyticus is disclosed. The primer provided by the application realizes efficient scarless genetic operation of the Vibrio parahaemolyticus flagellum protein loci; the scarless deletion flagellum protein loci provided by the application significantly affect the motility, biofilm formation, adhesion capacity, virulence and immunogenicity of Vibrio parahaemolyticus, and have application prospects in Vibrio parahaemolyticus disease prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and molecular biology, specifically relating to a primer for detecting homologous recombination and traceless deletion of the polar flagellin locus of Vibrio parahaemolyticus and its application. Background Technology

[0002] Vibrio parahaemolyticus, a member of the family Vibrionaceae and the genus Vibrio, is a Gram-negative halophilic bacterium widely distributed in marine, estuarine, and coastal areas. Its toxicity to humans and aquatic organisms places a significant burden on global public health and fisheries economies. Its virulence factors mainly include hemolysins, adhesins, quorum sensing systems, secretory effectors, and flagellated systems.

[0003] Successful pathogens require a repeated process of approaching host cells / infection sites, successfully adhering and colonizing, overcoming the host's immune system, maintaining growth and replication, and ultimately approaching the next host cell / pathogenesis site. Flagella, as one of the most complex molecular machines in bacteria, play different roles in these processes. In addition to being essential for bacterial motility, they also play a crucial role in colonization, biofilm formation, penetration of the host mucosal layer, cell adhesion, and invasion.

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

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

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

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

[0008] The final objective of this invention is to provide the application of the Vibrio parahaemolyticus polar flagellin locus constructed by the above method in regulating the motility, biofilm formation, adhesion, virulence and immunogenicity of Vibrio parahaemolyticus.

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

[0010] Table 1 Primers for detecting homologous recombination and traceless deletion at 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 used for amplifying the left and right homologous arms of flaA, flaB, flaC, flaD, flaE, and flaF at 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 used for detecting the seamless deletion of flaA, flaB, flaC, flaD, flaE, and flaF at the polar flagellin locus, respectively.

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

[0016] The second objective of this invention can be achieved through the following technical solution: a method for the scarless deletion construction of the Vibrio parahaemolyticus polar flagellin gene locus, comprising the following steps:

[0017] (1) Extraction of Vibrio parahaemolyticus genomic DNA;

[0018] (2) The extracted DNA was amplified by PCR using the primer pairs for amplification of the left and right homologous arms described above to obtain the amplification product;

[0019] (3) The amplification product from step (2) is ligated with the enzyme-digested suicide vector via homologous recombination and introduced into the Vibrio parahaemolyticus genome via conjugation transfer;

[0020] (4) Two rounds of homologous recombination were performed, and the above-mentioned primer pairs for the traceless deletion detection were used for verification to obtain the traceless deletion of the Vibrio parahaemolyticus polar flagellin locus.

[0021] In existing technologies, transposon insertion is used to mutate the entire polar flagellin locus of Vibrio parahaemolyticus. Compared with homologous recombination, transposon insertion has high randomness and low efficiency, and introduces exogenous sequences into the genome. However, the homologous homologous method and primers used in this invention utilize the upstream and downstream homologous arms of the known polar flagellin coding gene. Therefore, compared with existing methods, it can efficiently achieve targeted deletion of nucleotide sequences, and the deletion will not introduce other nucleotide sequences into the genome.

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

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

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

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

[0026] The last objective of the present invention can be achieved by the following technical solution: the application of the scarless deletion of the Vibrio parahaemolyticus polar flagellin gene locus 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 this invention enable efficient and traceless genetic manipulation of the polar flagellin locus of Vibrio parahaemolyticus;

[0029] (2) The scarless deletion 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. Attached Figure Description

[0030] Figure 1 The images are PCR amplification products of the left and right homologous arm fragments of the polar flagellin gene in Example 1. M: DS2000 marker; 1-12: are the left and right homologous arms of flaA, flaB, flaC, flaD, flaE, and flaF, respectively.

[0031] Figure 2 The PCR amplification products of S17-1-λpir carrying the recombinant vector in Example 1 are shown. 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 The PCR amplification products of the wild-type strain and the deletion strain in Example 1 are shown below. M: Marker 3; 1, 3, 5, 7, 9, 11: PCR amplification products of the wild-type strain; 2, 4, 6, 8, 10, 12: PCR amplification products of ΔflaA, ΔflaB, ΔflaC, ΔflaD, ΔflaE, ΔflaF.

[0033] Figure 4 The swimming and clustering abilities of the wild-type strain and each deletion strain in Example 2 are shown. A, B: Results of swimming plates and colony diameter measurements; C, D: Results of clustering plates and colony diameter measurements; Error bars are represented as the mean ± standard error of three replicates; *P<0.05, **P<0.01;

[0034] Figure 5 The biofilm formation capacity of the wild-type strain and each deletion strain in Example 2 is given. Error bars are expressed as the mean ± standard error of three 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 is shown. Error bars are expressed as the mean ± standard error of three replicates; ****P<0.0001;

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

[0037] Figure 8 This section describes the effects of wild-type strains and various deletion strains on cytokine expression in GS cells in Example 2. The correlation lines and asterisks on the bars represent the significance comparison results between the MLB group and each strain group, and between the WT group and each deletion strain group, respectively; the error bars represent the mean ± standard error of three replicates; **P<0.01, ****P<0.0001. Detailed Implementation

[0038] Example 1

[0039] The technical solution of the present invention will be described in detail below with reference to specific embodiments, so that those skilled in the art can better understand and implement the technical solution of the present invention. The following embodiments and drawings are for illustrative purposes only and should not be construed as limiting the present invention. Unless otherwise specified, the reagents or materials used in the embodiments are all from commercial sources. Unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.

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

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

[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 used for amplification of the left and right homologous arms of flaA, flaB, flaC, flaD, flaE, and flaF at 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 used for the detection of seamless deletion of flaA, flaB, flaC, flaD, flaE, and flaF at the polar flagellin locus, respectively.

[0047] (2) Genomic DNA of Vibrio parahaemolyticus strain RIMD2210633 was extracted using the Rapid Bacterial Genomic DNA Isolation Kit. Max Super-Fidelity DNA Polymerase amplifies the left and right homologous arms, each approximately 500 bp in length. Reaction conditions: Gently pipette and mix; briefly centrifuge to collect the reaction solution at the bottom of the tube. 95℃, 3 min; (95℃, 15 s; 63℃, 15 s; 72℃, 30 s) × 35 cycles; 72℃, 5 min. After the reaction is complete, the amplified products are analyzed by electrophoresis on a 1% agarose gel and then recovered from the gel.

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

[0049] Figure 1 The amplified fragments of homologous arms, approximately 500 bp in length, are located on the left and right sides of the central flagellin locus. M represents the DS2000 marker; 1-12 represent the left and right homologous arms of flaA, flaB, flaC, flaD, flaE, and flaF, respectively. The theoretical values ​​of the homologous arms are 533, 529, 537, 525, 521, 541, 529, 533, 534, 528, 530, and 532 bp.

[0050] (3) XbaⅠ single enzyme digestion or SphⅠ-HF and SacⅠ-HF double enzyme digestion to linearize 1 μg of suicide plasmid pDS132. Reaction conditions: Gently pipette and mix well, then briefly centrifuge to collect the reaction solution to the bottom of the tube. React at 37℃ for 1 h. After the reaction is complete, the enzyme digestion products are detected by electrophoresis on a 1% agarose gel.

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

[0052] (5) Take 10 μL of the above recombinant product and add it to 100 μL of Escherichia coli S17-1λpir competent cells. Mix gently and incubate on ice for 30 min. Heat shock in a 42℃ water bath for 45 s, then place on ice for 3 min. Add 900 μL of LB liquid medium and incubate at 37℃ and 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, spread on LB plates (containing 30 μg / mL chloramphenicol), and incubate at 37℃ for 16 h.

[0053] (6) Colony verification was performed using 2×T5 Super PCR Mix (Colony). Each pair of homologous arm fragments was ligated into the linearized pDS132 vector and transformed into *E. coli* S17-1-λpir. Using the bacterial culture as a template, amplification was performed using universal pDS132 primers, yielding fragments of 1329 bp each. This indicated that each pair of homologous arm fragments had been inserted into the suicide vector and successfully transformed. Six *E. coli* S17-1-λpir strains carrying the recombinant suicide vectors pDS132-△flaA, pDS132-△flaB, pDS132-△flaC, pDS132-△flaD, pDS132-△flaE, and pDS132-△flaF were obtained. Electrophoresis results are shown below. Figure 2 As shown.

[0054] Reaction conditions: Gently pipette and mix well, then briefly centrifuge to collect the reaction solution at the bottom of the tube. Incubate at 98℃ for 3 min; (98℃, 10 s; 56℃, 10 s; 72℃, 30 s) × 35 cycles; then at 72℃ for 2 min. After the reaction is complete, detect the amplified products by electrophoresis on a 1% agarose gel. Freeze correctly sequenced transformants in 20% glycerol at -80℃.

[0055] The universal primers for pDS132 are shown in Table 2 below:

[0056] Table 2 pDS132 universal primers

[0057]

[0058]

[0059] (7) Inoculate E. coli S17-1-λpir△ into 10 mL of liquid LB medium (containing chloramphenicol 30 μg / mL), and simultaneously inoculate wild-type Vibrio parahaemolyticus RIMD 2210633 into 10 mL of MLB medium. Incubate at 37℃ and 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 for 5 min at room temperature, discard the supernatant, rinse the bacterial cells twice with 1 mL of liquid LB medium (without chloramphenicol), centrifuge at 4000 rpm for 5 min at room temperature, and discard the supernatant.

[0060] (8) Transfer 1 mL of Vibrio parahaemolyticus culture medium into a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min at room temperature, and discard the supernatant. Wash the bacterial cells twice with 1 mL of MLB medium, centrifuge at 4000 rpm for 5 min at room temperature, and discard the supernatant. Finally, mix the two bacterial cells with 100 μL of MLB medium and resuspend them. Drop the resuspended solution onto a 0.45 μm filter membrane placed on LB agar medium, air dry, and incubate upside down for 12 h.

[0061] (9) Use sterile forceps to pick up the filter membrane and put it into a 50 mL centrifuge tube containing 5 mL of MLB medium. Incubate at 37°C for 1 h, then incubate at 37°C and 250 rpm for 1 h. Then, serially dilute the culture solution to tens of millions of times and take 100 μL of each solution and spread it on 10 MLB plates (containing 5 μg / mL chloramphenicol and 100 μg / mL ampicillin). Incubate at 37°C for several days until single colonies are formed (in most cases, bacterial moss grows). The single colonies or bacterial moss are isolated and purified more than three times by streaking on MLB plates (containing 5 μg / mL chloramphenicol and 100 μg / mL ampicillin) to obtain purified Vibrio parahaemolyticus (pDS132△) that has undergone the first homologous recombination. Store in 20% glycerol at -80°C.

[0062] (10) Inoculate Vibrio parahaemolyticus (pDS132△) for the first time 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 serially to the appropriate multiples and take 100 μL of each and spread it on TCBS plates (containing 20% ​​sucrose).

[0063] (11) Use an inoculation loop to lightly spot a single colony on a TCBS plate, and then lightly spot the TCBS plate (containing 20% ​​sucrose) and MLB plate (containing 5 μg / mL chloramphenicol and 100 μg / mL ampicillin) in sequence. Incubate in an inverted incubator at 37°C for 6 h.

[0064] (12) Select 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) and incubate them in 1 mL of liquid MLB medium. Incubate at 37℃ and 250 rpm for 2.5 h. Then verify the colonies using the primers listed in Table 1. Reaction conditions: Gently pipette and mix, then briefly centrifuge to collect the reaction solution to the bottom of the tube. 98℃, 3 min; (98℃, 10 s; 66℃, 10 s; 72℃, 30 s) × 35 cycles; 72℃, 2 min. After the reaction is complete, detect the results by electrophoresis of the amplified products on a 1% agarose gel. The Vibrio parahaemolyticus polar flagellin gene deletion strains △flaA, △flaB, △flaC, △flaD, △flaE, and △flaF, which underwent a second homologous recombination (i.e., gene knockout was successful), were frozen in 20% glycerol to -80℃ ultra-low temperature freezer.

[0065] PCR amplification products of wild-type strain and polar flagellin locus deletion strain, such as Figure 3 As shown:

[0066] Figure 3 The PCR amplification products were obtained from the wild-type strain and the polar flagellin locus deletion strain. The outer primers of the knocked-out gene (TA-F / TA-R for flaA, and the outermost primer pairs constructed from homologous arms for other genes) were used to verify the colonies of the Vp wild-type strain and the corresponding deletion strain, respectively. The fragments were 1576 and 445 bp (flaA length is 1131 bp), 2199 and 1062 bp (flaB length is 1137 bp), 2217 and 1062 bp (flaC length is 1155 bp), 2199 and 1062 bp (flaD length is 1137 bp), 2187 and 1062 bp (flaE length is 1125 bp), and 2196 and 1062 bp (flaF length is 1134 bp).

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

[0068] (1) Motility assessment: The wild-type Vibrio parahaemolyticus strain and the scarless deletion strain of the polar flagellin locus constructed in Example 1 were inoculated 1:100 into fresh MLB medium and cultured until the prelog phase. The medium was then diluted until OD600 = 0.5. 2.5 μL of the bacterial suspension was vertically dropped into three aliquots. The motility diameter was measured and images were captured and saved using a gel imaging analysis system. The analysis results are as follows: Figure 4 As shown.

[0069] The results of research on the regulation of motility by polar flagellin loci are as follows: Figure 4 As shown, flaC significantly enhances swimming ability, while flaD, flaE, and flaF significantly enhance swarming ability.

[0070] (2) Biofilm assay: OD of overnight cultures was measured. 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. This volume is transferred to triplicate of 24-well plates containing 0.5 mL of MLB medium. Static incubation at 37°C for 12 h, 24 h, 36 h, and 48 h, respectively. Discard the medium, add 500 μL PBS to each well and wash twice. Discard the PBS, add 500 μL 1% crystal violet solution to each well and stain for 30 min. Discard the stain and gently rinse the well walls with running water until the water is completely colorless and transparent. Then, invert the plates onto filter paper and dry in a 65°C oven for 30 min. After drying, add 1 mL of 33% glacial acetic acid solution to each well, gently elute the biofilm by pipetting, and thaw in a 37°C oven for 30 min. Measure the absorbance at 590 nm. Analyze the results as follows: Figure 5 As shown.

[0071] Figure 5 The results of the study on the biofilm formation ability regulated by the polar flagellin locus showed that at 12 h, there was no significant difference in biofilm formation ability among the strains; at 24 h, ΔflaE decreased significantly; at 36 h, ΔflaA and ΔflaD increased significantly; and at 48 h, ΔflaB, ΔflaC, and ΔflaD all decreased significantly. Other deletion strains showed no significant difference compared to wild-type strains at the corresponding time points.

[0072] (3) Cell adhesion assay: HeLa cells were subjected to a 5×10⁻⁶ m² / h²· ... 4 Cells / mL were seeded in 96-well plates containing 100 μL of DMEM containing 10% FBS; the OD of the overnight culture was measured. 600The cells were diluted to 1% using MLB liquid medium. HeLa cells were washed twice with PBS, and 100 μL of DMEM containing 10% FBS was added. 1 μL of bacterial culture (i.e., MOI of 10) was transferred to each well. Cells were centrifuged at 200 × g for 5 min at room temperature for simultaneous infection and incubated at 37°C for 1 h. Cells were washed twice with PBS, and 100 μL of 1% Triton X-100 was added. The cells were lysed at 37°C for 10 min to obtain adherent bacteria. Cell lysates were serially diluted and plated on MLB plates, incubated at 37°C for 12 h, and the number of adherent bacteria was counted. The results were analyzed as follows: Figure 6 As shown.

[0073] Figure 6 The results of the study on the regulation of adhesion ability by polar flagellin loci show that ΔflaE and ΔflaF significantly enhance cell adhesion.

[0074] (4) Cytotoxicity assay: Refer to Invitrogen CyQUANT TM The LDH Cytotoxicity Assay Kit instructions were followed to determine cytotoxicity: Spontaneous LDH activity group, experimental group, and maximum LDH activity group were established. HeLa cells were cultured at 5 × 10⁻⁶ cells / day. 4 Cells / mL were seeded in 96-well plates containing 100 μL of phenol red-free DMEM containing 10% FBS; the OD of the overnight culture was measured. 600 The cells were diluted to 1% using MLB liquid medium. HeLa cells were washed twice with PBS, and 100 μL of fresh phenol red-free DMEM was added. 1 μL of bacterial culture and 9 μL of ultrapure water were transferred to each well, 10 μL of ultrapure water was transferred to the spontaneous LDH-active well, and 10 μL of 10x Lysis Buffer was transferred to the maximum LDH-active well. The cells were centrifuged at 200×g for 5 min at room temperature for simultaneous infection and incubated at 37°C for 1 h. 50 μL of each well was transferred to a new 96-well plate, and 50 μL of Reaction Mixture was added to each well. The plates were incubated at room temperature in the dark for 30 min. 50 μL of Top Solution was added to each well and gently mixed. The absorbance at 490 nm and 680 nm was measured, and the percentage of cytotoxicity was calculated. The results are analyzed as follows: Figure 7 As shown.

[0075] Figure 7 The results of the study on the regulation of cytotoxicity by the polar flagellin locus show that ΔflaC significantly reduces cytotoxicity, while ΔflaD significantly increases cytotoxicity.

[0076] (5) Immunogenicity assay: GS cells from the spleen of grouper were subjected to immunogenicity assay at 5 × 10⁻⁶ m³ / h. 4Cells / mL were seeded in 96-well L15 plates containing 100 μL of 10% FBS; the OD of the overnight culture was measured. 600 Diluted to 1% using MLB liquid medium. Wash GS cells twice with PBS, add 100 μL of L15 containing 10% FBS, and transfer 1 μL of bacterial culture (i.e., MOI of 10) to each well; centrifuge at 200×g for 5 min at room temperature for simultaneous infection, and incubate at 37°C for 1 h; discard the medium, wash GS cells twice with PBS, add 200 μL of L15 containing 10% FBS and 100 μg / mL gentamicin, and incubate for 2 h. Discard the medium, add 100 μL of PBS to each well to wash cells. Discard the PBS, add TOYOBO to each well. 49.7 μL of Lysis Solution and 0.3 μL of gDNARemover were added to the Cell Lysis & RT Kit for qPCR and incubated at room temperature for 5 min. 9.5 μL of Top Solution and 0.5 μL of Lnase Inhibitor were added to each well and incubated at room temperature for 2 min. 8 μL of cell lysis buffer, 8 μL of 5×RT Master Mix, and 12 μL of ddH2O were mixed and placed in a PCR instrument to complete the reverse transcription reaction. The reaction conditions were: 37℃, 15 min; 50℃, 5 min; 98℃, 5 min. After the reaction was complete, the mixture was stored at -20℃ for later use. Quantitative real-time PCR was performed using the following reaction program: 95℃, 60 s; (95℃, 15 s; 56℃, 15 s; 72℃, 45 s) × 50 cycles. One-way ANOVA and Dunnett's multiple t-test for post-hoc comparisons were used for significance analysis. The results are shown below. Figure 8 As shown.

[0077] Figure 8 The results of the study on the regulation of immunogenicity by the polar flagellin gene locus showed that WT, ΔflaB, ΔflaE, and ΔflaF significantly upregulated the expression of IL-6 and IL-1β mRNA, ΔflaA, ΔflaB, ΔflaC, ΔflaD, and ΔflaE significantly downregulated the expression of IL-6 mRNA, ΔflaA, ΔflaC, and ΔflaD significantly downregulated the expression of IL-1β mRNA, while ΔflaF significantly upregulated the expression of IL-6 and IL-1β mRNA. Furthermore, the absence of FlaF led to a significant upregulation of IFN-γ2 mRNA expression.

[0078] The above results indicate that flagellin proteins at different poles play varying roles in the motility, growth, biofilm formation, adhesion to host cells and virulence during infection of Vibrio parahaemolyticus, as well as in the activation of the immune response. This flagellin locus shows promise for application in the prevention and control of Vibrio parahaemolyticus diseases.

[0079] The above embodiments are only used to illustrate the present invention, and the scope of protection of the present invention is not limited to the above embodiments. Those skilled in the art can achieve the purpose of the present invention based on the above disclosure. Any improvements and modifications made based on the concept of the present invention fall within the scope of protection of the present invention, and the specific scope of protection is determined by the claims.

Claims

1. Primers for detecting homologous recombination and traceless deletion at the polar flagellin locus of Vibrio parahaemolyticus, characterized in that, The Vibrio parahaemolyticus polar flagellin gene locus includes the flaA, flaB, flaC, flaD, flaE, and flaF genes, and the primers are shown in Table 1 below: Table 1 Primers for detecting homologous recombination and traceless deletion at 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 used for amplifying the left and right homologous arms of flaA, flaB, flaC, flaD, flaE, and flaF at 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 used for detecting the seamless deletion of flaA, flaB, flaC, flaD, flaE, and flaF at the polar flagellin locus, respectively.

2. A method for the scarless deletion construction of the polar flagellin gene locus of Vibrio parahaemolyticus, characterized in that, Includes the following steps: (1) Extraction of Vibrio parahaemolyticus genomic DNA; (2) The extracted DNA was amplified by PCR using the primer pair for amplification of the left and right homologous arms in claim 1 to obtain the amplification product; (3) The amplification product from step (2) is ligated with the enzyme-digested suicide vector via homologous recombination and introduced into the Vibrio parahaemolyticus genome via conjugation transfer; (4) Perform two rounds of homologous recombination and verify the result using the primer pair for the traceless deletion detection in claim 1 to obtain the traceless deletion of the Vibrio parahaemolyticus polar flagellin locus.

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

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

5. The method for seamless deletion construction of the Vibrio parahaemolyticus polar flagellin gene locus according to claim 2, characterized in that, Step (4) yields the scarless deletion of the Vibrio parahaemolyticus polar flagellin gene locus, including the Vibrio parahaemolyticus polar flagellin gene deletion strains △flaA, △flaB, △flaC, △flaD, △flaE, and △flaF, which were successfully knocked out.

6. The use of the scarless deletion of the Vibrio parahaemolyticus polar flagellin gene locus constructed by the method of any one of claims 2-5 in regulating the motility, biofilm formation, adhesion ability, virulence and immunogenicity of Vibrio parahaemolyticus.

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