Construction method and application of vibrio parahaemolyticus hfq gene deleted strain

Abstract

The invention discloses a vibrio parahaemolyticus hfq gene deleted strain as well as a construction method and application thereof. The invention provides a deletion strain, a construction method of the deletion strain and application of the deletion strain in construction of an enrofloxacin drug resistance model. The invention finds that after the hfq gene is deleted, the drug resistance to the enrofloxacin is obviously increased. It is further proved that the vibrio parahaemolyticus ATCC33846 delta hfq deletion strain enhances the drug resistance to enrofloxacin by improving the excretion of rhodamine 6G. The vibrio parahaemolyticus deletion strain is constructed by utilizing high efficiency and accuracy of homologous recombination, and the important effect of the hfq gene on the drug resistance of the vibrio parahaemolyticus is proved by comparing the drug resistance of the deletion strain and a wild strain on enrofloxacin. The compound can be used for developing a new therapy for drug-resistant bacteria or improving an existing antibacterial strategy.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and biotechnology, and more particularly to a method for constructing a Vibrio parahaemolyticus hfq gene deleted strain and its application. Background Art

[0002] With the increasing prevalence of seafood consumption, Vibrio parahaemolyticus, a common pathogen, poses a serious threat to human health. To control its harm, people use a large amount of antibiotics for sterilization. However, this abuse has led to significant changes in the drug resistance of V. parahaemolyticus, especially the increasing resistance to commonly used antimicrobial drugs such as fluoroquinolones (e.g., enrofloxacin), posing a serious challenge to public health and safety. Therefore, in-depth research on the resistance mechanisms of V. parahaemolyticus and the development of effective antimicrobial strategies are of great significance for controlling infections caused by this bacterium.

[0003] The hfq gene encodes a small RNA-binding protein that plays an important regulatory role in a variety of bacteria. The hfq protein can bind to small RNA (sRNA) and regulate gene expression by affecting sRNA stability or its interaction with target mRNA. In some bacteria, hfq has been shown to be involved in various biological processes, including responses to environmental stress and expression of virulence factors. However, the specific mechanism of action of the hfq gene in enrofloxacin resistance in Vibrio parahaemolyticus remains unclear.

[0004] Therefore, providing a method for constructing a hfq gene-deficient strain of Vibrio parahaemolyticus and its application is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for constructing an hfq gene-deficient strain of Vibrio parahaemolyticus and its application, aiming to reveal the potential role of the hfq gene in the bacterial resistance mechanism and provide a theoretical basis for the development of new antibacterial strategies.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A hfq gene deleted strain of Vibrio parahaemolyticus.

[0008] The present invention also provides a method for constructing the above-mentioned Vibrio parahaemolyticus hfq gene deletion strain, comprising the following steps:

[0009] (1) Overlapping arm construction: Using the genomic DNA of Vibrio parahaemolyticus ATCC33846 as a template, primers U-hfq-F / U-hfq-R, D-hfq-F / D-hfq-R, and U-Gm-F / D-Gm-R were used to amplify the upstream and downstream homologous arms of the target gene hfq and the gentamicin resistance fragment, respectively. Using the above three fragments as templates, primers U-hfq-F / D-hfq-R were used for amplification, and the three gene segments were spliced ​​to obtain the overlapping arm UP-LoxL-Gm-LoxR-DOWN;

[0010] (2) Construction of recombinant plasmid: The overlapping arms UP-LoxL-Gm-LoxR-DOWN were integrated into the pDS132 plasmid to obtain the recombinant plasmid pDS132-hfq;

[0011] (3) Construction of recombinant bacteria:

[0012] 31) Transform the recombinant plasmid pDS132-hfq into E. coli CC118 competent cells to obtain CC118 / pDS-hfq recombinant strain;

[0013] 32) Extract the pDS132-hfq plasmid and transform it into E. coli S17-1 competent cells to obtain the S17-1 / pDS-hfq recombinant strain;

[0014] 33) S17-1 / pDS-hfq was conjugated with ATCC33846 to obtain the recombinant bacteria ATCC33846 / pDS132-hfq containing the recombinant plasmid pDS132-hfq after one round of recombination. The recombinant bacteria were inoculated into sucrose medium for reverse screening and completed the second round of recombination to obtain Vibrio parahaemolyticus ATCC33846Δhfq with the target gene knocked out.

[0015] Preferably: the nucleotide sequence of U-hfq-F in step (1) is as shown in SEQ ID No. 1;

[0016] The nucleotide sequence of U-hfq-R is shown in SEQ ID No. 2;

[0017] The nucleotide sequence of D-hfq-F is shown in SEQ ID No. 3;

[0018] The nucleotide sequence of D-hfq-R is shown in SEQ ID No. 4;

[0019] The nucleotide sequence of U-Gm-F is shown in SEQ ID No. 5;

[0020] The nucleotide sequence of D-Gm-R is shown in SEQ ID No. 6;

[0021] The nucleotide sequence of the hfq upstream homology arm fragment is shown in SEQ ID No. 7;

[0022] The nucleotide sequence of the hfq downstream homology arm fragment is shown in SEQ ID No. 8;

[0023] The nucleotide sequence of the gentamicin resistance fragment is shown in SEQ ID No. 9;

[0024] The nucleotide sequence of the UP-LoxL-Gm-LoxR-DOWN overlapping arms is shown in SEQ ID No.10.

[0025] The present invention also provides the use of a Vibrio parahaemolyticus hfq gene deletion strain in constructing an enrofloxacin resistance model.

[0026] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for constructing a hfq gene deleted strain of Vibrio parahaemolyticus and its application, and the technical effects achieved are:

[0027] By measuring the minimum inhibitory concentration (MIC) values ​​of ATCC33846 wild strain and ATCC33846Δhfq deletion strain to enrofloxacin, it was found that the resistance to enrofloxacin was significantly increased after the hfq gene was deleted.

[0028] Furthermore, compared with the wild-type strain of Vibrio parahaemolyticus ATCC33846, the ATCC33846Δhfq deletion strain increased the efflux of rhodamine 6G and enhanced its resistance to enrofloxacin.

[0029] Therefore, the present invention utilizes the high efficiency and accuracy of homologous recombination to construct a deletion strain of Vibrio parahaemolyticus, and compares the drug resistance of the deletion strain with that of the wild-type strain to enrofloxacin to demonstrate the important role of the hfq gene in drug resistance of Vibrio parahaemolyticus.

[0030] This study focuses on an hfq gene-deficient strain of Vibrio parahaemolyticus. The construction of this specific strain provides a more defined research model for investigating enrofloxacin resistance in this bacterium, potentially enabling the development of new therapies for drug-resistant bacteria or improvements to existing antibacterial strategies. The construction of this enrofloxacin-resistant model not only provides a deeper understanding of the function of the hfq gene but also provides direct scientific evidence for addressing the global health issue of antibiotic resistance, providing an important theoretical foundation for developing new treatment strategies and controlling the spread of drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0032] Figure 1 The accompanying figure is a PCR verification diagram for knocking out the hfq gene of Vibrio parahaemolyticus by the homologous recombination method provided by the present invention, wherein M: DNA marker, Line 1: hfq gene knockout genome PCR verification, Line 2: pDS132-hfq plasmid verification, Line 3: ATCC33846 genome fragment PCR verification.

[0033] Figure 2 The accompanying drawings are scanning electron microscopy analyses of the morphological changes of the cell membrane surfaces of ATCC33846 and ATCC33846Δhfq provided by the present invention.

[0034] Figure 3 The accompanying drawing is a graph showing the MIC values ​​of ATCC33846 and ATCC33846Δhfq to enrofloxacin provided by the present invention.

[0035] Figure 4 The accompanying drawings are experimental diagrams of the efflux capacity of Vibrio parahaemolyticus ATCC33846 and ATCC33846Δhfq for rhodamine 6G provided by the present invention, wherein the working concentrations of rhodamine 6G are 0, 2, 6, and 8 mg / mL from top to bottom, respectively. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] The embodiment of the present invention discloses a method for constructing a Vibrio parahaemolyticus hfq gene deleted strain and its application.

[0038] The examples are described using only Vibrio parahaemolyticus ATCC33846 and ATCC33846Δhfq as examples, but the method of the present invention is suitable for all Vibrio parahaemolyticus.

[0039] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the present technical field, and the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0040] The primer sequences involved in the examples are shown in Table 1:

[0041] Table 1

[0042]

[0043]

[0044] Among them, the underlined sequence in U-Gm-F is the additional terminal primer that is reverse complementary to U-hfq-R. Therefore, the front end of the gentamicin box resistance fragment has a gene fragment that is complementary to the end of the upstream homologous arm fragment of hfq, and splicing is performed through the overlapping gene fragments.

[0045] The underlined sequence in D-Gm-R is the additional terminal primer that is reverse complementary to D-hfq-F. Therefore, the rear end of the gentamicin box resistance fragment has a gene fragment that is complementary to the front end of the hfq downstream homology arm fragment, and splicing is performed through the overlapping gene fragments.

[0046] The sources of the strains and plasmids involved in the following examples are shown in Table 2:

[0047] Table 2

[0048]

[0049]

[0050] Example 1

[0051] Construction of strain S17-1 / pDS-hfq:

[0052] (1) The hfq upstream and downstream homology arm gene fragments were amplified from the genome of Vibrio parahaemolyticus ATCC33846 (DNA as template) using primers U-hfq-F, U-hfq-R and D-hfq-F, D-hfq-R. The PCR products were recovered using a PCR product recovery kit.

[0053] The loxL-Gm-loxR fragment was amplified from the pWJW101 plasmid using primers U-Gm-F and D-Gm-R, and the PCR product was recovered using a PCR product recovery kit.

[0054] Using primers U-hfq-F and D-hfq-R, the upstream and downstream homologous arms of the hfq gene and the gentamicin resistance fragment were used as templates to splice the three gene segments to obtain overlapping arms UP-LoxL-Gm-LoxR-DOWN.

[0055] Reaction system: Taq enzyme 25 μL, primers U-hfq-F and D-hfq-R 1 μL each, genomic template 1 μL, ddH2O 22 μL.

[0056] Reaction program: pre-denaturation temperature 94℃ for 2 min, denaturation temperature 94℃ for 30 s, annealing temperature 58℃ for 30 s, extension temperature 72℃ for 1 min 40 s, and final extension at 72℃ for 2 min.

[0057] The PCR products were recovered using a PCR product recovery kit, and the overlapping arms and the pDS132 plasmid were digested with XbaI and SphI.

[0058] Enzyme digestion system: 10 μL of PCR product, 2 μL of 10× Fast Digest Buffer, 1 μL of XbaI enzyme, 1 μL of SphI enzyme, add nuclease-free water to 20 μL, and incubate at 37°C for 30 min.

[0059] The overlapping arms after enzyme digestion and the pDS132 plasmid were ligated with T4 DNA ligase to construct the recombinant plasmid pDS132-hfq.

[0060] Ligation system: 8 μL of a 3:1 mixture of fragment and vector, 1 μL of 10×T4 DNA Ligase Buffer and 1 μL of T4 DNA Ligase, and add nuclease-free water to 20 μL. Incubate at 4°C overnight.

[0061] Nucleotide sequence of the upstream homologous arm fragment of hfq: ATCTCTAGActcttcctgctgcaaatcctgaaattcgtcagca aattgaacaagaagcattgactaaaggttggtcagtgttgcacgatgaactgaaagaaattgatcctgtatcagctgcgagaatacatccaaatgatccccagcgtttatctagggcgttggaagtgtatcgtatttcaggtaaaactcttactgagttgactcaaactaaaggtgaaagcctgccgtaccgagttaaacagtttgcaatagctcccaaggatagggcagaacttcaccgtcgcattgaactgcgtttcgacaaaatgatggaagcgggttttgaagaggaaatgaaagcgttgtatgcgcgcaaagatcttcatccggatcttccctctattcgttgcgttggttaccgacagatgtgggagtatttagatggggagtgcacacgtgatgaagctgtatttcgtggcgtatgtgcgactcgtcaattggccaagcgtcaaatcacctggttacgcagctgggatgacttaacttggttggatagcgataacattgagcaggcactcgaaaccatgtcagaagcaatagcatctgattgagatagctgtgtataatgtgatcgtttttgcgtgaacgcactctcaaaggatctgttgttggcagtttttatactatgctgctaattacttagagtgcatttttgattcgtttagctcagatgcaaaagcagtactttttgcattgcaccactagctaaataattacaactacaaattaaataaggaaaataaaatggctaaggggcaatCTCTACAAGACCCATTTCCGTAATACGACTCACTATAGGGC, as shown in SEQ ID NO.7.

[0062] Nucleotide sequence of the hfq downstream homologous arm fragment: CTTTAGTGAGGGTTAATTGCgcgtccacaagaga aatcagaagattaattcttcatcgagtatttttaaaataaggagttgattgcttgtttgaccgttatgaatccggtgagcgagccgtacttgttcatatcaacttcacgcaagaaggtgaatgggaagatctggctgagttcgagatgctggtttcttccgctggggttgagacgctgcaagtggtaactggtagtcgtcaatccccacacccgaaatactatgtcggagagggtaaggcccaagaaattgctactacggtacagttgactggtgctgaaattgtgatttttaatcactccctctctcctgcccaagaacgtaacctcgaagcactgtgcaaatgtcgagttctcgaccgcactggtctcatcctcgatatctttgctcaacgagcacgaacacacgaaggtaagctacaggtcgagctggcccaacttcgacacatttctactcgtttaattcgtggttggacgcaccttgaaaggcagaaaggggggatcggtttacgtggcccgggtgaaacccagctggaaaccgaccgtcgtttattgcgtgaacgaatcaaagccatcctgcgccgcctagaaaaggtagccaagcagcgtgaacaaggacgccgtgcccgaaatcgagctgaaatcccaacaatttctctcgtgggttacaccaacgcgggtaagtcgacactcttcaaccgaataaccgaagctggcgtgtatgccgctgaccaattgtttgctactttggatcctacactacgtaagatggagttagcggatgttggcccagccatactggcagatacggttggttttattcgacatttgcctcacgacttggttgctgccttcaaagcgaccttgcaggaaacgcaagaagctgacattttgttacatgttgttgatgccagtgatgagcgttttcgtgagaatattcaagctgttGCATGCGGT, as shown in SEQ ID NO.8.

[0063]

[0064]

[0065] (2) Thawed E. coli CC118 (λpir) competent cells were mixed with 10 μL pDS132-hfq (1:10) and placed on ice for 30 minutes. After heat shock at 42°C for 90 seconds, pre-cooled LB medium was immediately added and cultured at 37°C and 100 rpm for 1 hour. Centrifuge at 12,000 rpm for 1 minute and spread the bacterial suspension on an LB plate containing 50 mg / L chloramphenicol. The correct bacteria were selected by (conventional) colony PCR and cultured at 37°C for 24 hours. The plasmid was extracted to verify its correctness and named pDS132-hfq, thus obtaining the CC118 / pDS-hfq recombinant strain.

[0066] (3) Extract the pDS132-hfq plasmid (Plasmid Miniprep Kit, Beijing Solebeau Technology Co., Ltd.), mix the thawed Escherichia coli S17-1 (λpir) electroporated competent cells and the pDS132-hfq plasmid, transfer to a pre-cooled electroporation cup, and place on ice for 30 minutes. Immediately after 1.8 kV electroporation, add 1 mL of pre-cooled LB medium. Resuscitate at 37°C and 100 rpm for 2 hours. Centrifuge at 12,000 rpm for 1 minute, spread the bacterial suspension on a plate containing 50 mg / L chloramphenicol, verify by (conventional) colony PCR, pick the correct bacteria and culture at 37°C for 24 hours. After the colony is verified, pick the correct transformant and culture at 37°C for 12 hours. Take 800 μL of the bacterial solution and add it to a glycerol tube and store it at -70°C to obtain the S17-1 / pDS-hfq strain.

[0067] 2. A round of restructuring:

[0068] Press OD 600 S17-1 / pDS-hfq and ATCC33846 were inoculated into a test tube and cultured at 37°C and 200 rpm until the OD 600 The value was between 0.8-1.2. 1 mL of bacterial solution was washed twice with LB liquid medium containing 0.1 mol / L magnesium sulfate, and then co-suspended with 100 μL LB liquid medium containing 0.1 mol / L magnesium sulfate, spotted on LB plates, and cultured at 37°C overnight.

[0069] Use a sterile cotton swab to scrape the bacterial moss and suspend it in 1 mL of LB. Wash it once with LB medium containing 0.1 mol / L magnesium sulfate, and spread it on an LB plate containing 10 mg / mL chloramphenicol and 30 mg / L ampicillin after gradient dilution. Pick single colonies on the above plates for (conventional) colony PCR verification. The correct colony has two bands, one is the length of the overlapping arm fragment 1973 bp, and the other is the length of the gene fragment on the genome 2801 bp.

[0070] The recombinant bacteria ATCC33846 / pDS132-hfq containing the recombinant plasmid pDS132-hfq were obtained.

[0071] 3. Second round of restructuring:

[0072] The strain verified correctly by PCR in the previous step was inoculated into a medium containing 50 mg / mL gentamicin and 10% sucrose for reverse screening to complete the second round of recombination. A single colony was selected for PCR verification using U-hfq-F and D-hfq-R primers, and the genome was extracted for sequencing.

[0073] The target gene was knocked out of Vibrio parahaemolyticus ATCC33846Δhfq.

[0074] PCR verification results Figure 1 shown.

[0075] The ATCC33846Δhfq deletion strain was successfully obtained.

[0076] Example 2

[0077] Scanning electron microscopy analysis of morphological changes in the cell membrane surface of ATCC33846 and ATCC33846Δhfq:

[0078] Adjust the overnight cultured bacterial solution to OD 600 The sample was fixed with a pH of 0.8 and then washed three times with 0.1 M phosphate buffer. The bacterial solution was fixed with 4% glutaraldehyde solution and incubated overnight. The sample was dehydrated in a gradient of 50%, 70%, 80%, 90%, and 100% ethanol. The sample was freeze-dried for 48 hours, then gold-sprayed and observed using a scanning electron microscope.

[0079] Scanning electron microscopy results Figure 2 As shown: The left image shows that ATCC33846 cells are intact, exhibiting a relatively regular rod-shaped structure with a smooth surface and relatively uniform morphology. The right image shows that ATCC33846Δhfq cells show significant changes, with a rougher cell surface, obvious wrinkles, and increased volume.

[0080] Example 3

[0081] Determination of the MIC values ​​of ATCC33846 and ATCC33846Δhfq against enrofloxacin:

[0082] ATCC33846 and ATCC33846Δhfq cultured overnight at 37°C were adjusted to OD 600 = 0.5, and 10 5cfu of bacteria. Different concentrations of enrofloxacin were added to each well (2-fold serial dilution range: 128 μg / mL to 0.000244141 μg / mL). After incubation at 37°C for 16 h, the OD 600 value.

[0083] The results are as follows Figure 3 As shown, the MIC value of ATCC33846 to enrofloxacin is 0.25 μg / mL, and the MIC of ATCC33846Δhfq to enrofloxacin is 0.50 μg / mL. This shows that the deletion of the hfq gene significantly enhances the resistance of ATCC33846 to enrofloxacin.

[0084] Example 4

[0085] Determination of the efflux capacity of Vibrio parahaemolyticus ATCC33846 and ATCC33846Δhfq to rhodamine 6G: 10 5 cfu bacterial solution and incubate at 37°C for 20 h to observe the staining degree of Vibrio parahaemolyticus cells.

[0086] The staining results are as follows Figure 4 As shown, the working concentrations of rhodamine 6G (from top to bottom) are 0, 2, 6, and 8 mg / mL. Drug accumulation in ATCC33846 and ATCC33846Δhfq gradually increases with increasing concentration. At 8 mg / mL rhodamine 6G, ATCC33846 exhibits a deep red color, while ATCC33846Δhfq exhibits a light pink color. This indicates that compared to ATCC33846, the ATCC33846Δhfq strain accumulates less drug and exhibits stronger drug efflux activity, indicating strong resistance to enrofloxacin.

[0087] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0088] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A hfq gene deleted strain of Vibrio parahaemolyticus.

2. The method for constructing the hfq gene deleted strain of Vibrio parahaemolyticus according to claim 1, characterized in that: The following steps are involved: (1) Overlapping arm construction: Using the genomic DNA of Vibrio parahaemolyticus ATCC33846 as a template, primers U-hfq-F / U-hfq-R, D-hfq-F / D-hfq-R, and U-Gm-F / D-Gm-R were used to amplify the upstream and downstream homologous arms of the target gene hfq and the gentamicin resistance fragment, respectively. Using the above three fragments as templates, primers U-hfq-F / D-hfq-R were used for amplification, and the three gene segments were spliced ​​to obtain the overlapping arm UP-LoxL-Gm-LoxR-DOWN; (2) Construction of recombinant plasmid: The overlapping arms UP-LoxL-Gm-LoxR-DOWN were integrated into the pDS132 plasmid to obtain the recombinant plasmid pDS132-hfq; (3) Construction of recombinant bacteria: 31) Transform the recombinant plasmid pDS132-hfq into E. coli CC118 competent cells to obtain CC118 / pDS-hfq recombinant strain; 32) Extract the pDS132-hfq plasmid and transform it into E. coli S17-1 competent cells to obtain the S17-1 / pDS-hfq recombinant strain; 33) S17-1 / pDS-hfq was conjugated with ATCC33846 to generate the recombinant bacteria ATCC33846 / pDS132-hfq containing the recombinant plasmid pDS132-hfq after one round of recombination. The recombinant bacteria were inoculated into sucrose medium for reverse screening and completed the second round of recombination to obtain Vibrio parahaemolyticus ATCC33846Δhfq with the target gene knocked out.

3. The construction method according to claim 2, wherein: The nucleotide sequence of U-hfq-F in step (1) is shown in SEQ ID No. 1; The nucleotide sequence of U-hfq-R is shown in SEQ ID No. 2; The nucleotide sequence of D-hfq-F is shown in SEQ ID No. 3; The nucleotide sequence of D-hfq-R is shown in SEQ ID No. 4; The nucleotide sequence of U-Gm-F is shown in SEQ ID No. 5; The nucleotide sequence of D-Gm-R is shown in SEQ ID No. 6; The nucleotide sequence of the hfq upstream homology arm fragment is shown in SEQ ID No. 7; The nucleotide sequence of the hfq downstream homology arm fragment is shown in SEQ ID No. 8; The nucleotide sequence of the gentamicin resistance fragment is shown in SEQ ID No. 9; The nucleotide sequence of the UP-LoxL-Gm-LoxR-DOWN overlapping arms is shown in SEQ ID No.

10.

4. Application of hfq gene deletion strain of Vibrio parahaemolyticus in constructing enrofloxacin resistance model.

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