Phage protein Gp61 and its application in preparing preparations for inhibiting Vibrio cholerae colonization and toxin secretion

Abstract

The present invention discloses a bacteriophage protein Gp61 and its use in preparing a preparation that inhibits Vibrio cholerae colonization and toxin secretion. The present invention screens a library of phage genes specifically targeting Vibrio cholerae, searching for proteins that can inhibit Vibrio cholerae colonization and toxin secretion during interaction with the host. The discovery and identification of a novel regulatory protein, Gp61, that inhibits Vibrio cholerae colonization or virulence not only supplements the functional information of unknown phage proteins but also clarifies their primary biological functions influencing the host, providing a foundation for the prevention, control, and virulence control of Vibrio cholerae.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a bacteriophage protein Gp61 and an application thereof in preparing a preparation for inhibiting Vibrio cholerae colonization and toxin secretion. Background Art

[0002] Cholera is an acute intestinal infectious disease caused by the non-invasive bacterium Vibrio cholerae, which colonizes the intestinal mucosa. It is estimated that Vibrio cholerae causes 3 to 5 million cases of cholera annually and over 100,000 deaths. Because cholera prevalence is closely linked to sanitation, sewage systems, and socioeconomic conditions, Vibrio cholerae remains a key pathogen under surveillance in economically underdeveloped countries or regions.

[0003] Vibrio cholerae ( Vibrio cholerae The prerequisite for causing human disease is successful colonization in the host's small intestine. The pathogenic factors that affect Vibrio cholerae colonization are tcpA After successful colonization, bacteria begin to multiply and start expressing a series of virulence factors, the most important of which are ctxAB Gene-encoded cholera toxin CTX. Cholera toxin CTX enters the small intestinal epithelial cells, causing a large amount of electrolytes and water to be secreted into the lumen, and the patient experiences diarrhea. CTX and TCP synthesis are regulated by a unique, cascade regulatory system, with upstream regulatory proteins including ToxT, TcpP, and ToxR. tcpP The expression of phage is controlled by several regulatory factors: Fur, AphA, AphB, OhrR tcpP Direct activation of the promoter by binding tcpP expression, while HapR tcpP Direct inhibition of promoter binding tcpP In addition to the proteins mentioned above that regulate the virulence and colonization phenotypes of V. cholerae, many other regulatory proteins are involved. Proteins regulating these two pathogenic genes are still being added and discovered. Especially with the emergence of drug resistance in V. cholerae, new regulatory proteins that inhibit colonization or virulence are urgently needed.

[0004] Bacteriophages can specifically target and lyse pathogens, making phage gene libraries an excellent source for screening proteins that regulate killing, growth inhibition, or virulence. As vast amounts of phage genomic information are mined, the functions of over two-thirds of phage genes remain unannotated. Screening for proteins with unknown phage functions that interact with host virulence regulatory proteins is a new research approach and direction. A small number of studies have been reported in this area, examining different types of phage proteins targeting Pseudomonas aeruginosa. These studies have revealed that these phage-encoded proteins of unknown function play important roles in host interactions. For example, phage-encoded Dap1, the novel phage protein Gp21 encoded by the virulent phage vB_Pae_QDWS, and the phage protein SrpA all play important biological roles in influencing the virulence of Pseudomonas aeruginosa, as well as influencing host metabolism, growth, and division. A peptide derived from the Tip protein of the Pseudomonas aeruginosa phage can specifically inhibit the assembly of type IV pili (T4P), which are crucial for bacterial adhesion, biofilm formation, and reducing bacterial survival and spread in the host. This peptide has potential for antibacterial treatment against drug-resistant strains and may provide a new strategy for clinical treatment.

[0005] Discovering new members that inhibit the colonization or virulence of Vibrio cholerae from the phage gene library that specifically targets Vibrio cholerae can further clarify the pathogenic mechanism of Vibrio cholerae and the complex regulatory network of its regulatory proteins, providing a research basis for targeted inhibition of its virulence and reduction of its transmission hazards in the future. Summary of the Invention

[0006] The purpose of the present invention is to provide a bacteriophage protein Gp61 and its application in preparing a preparation for inhibiting Vibrio cholerae colonization and toxin secretion.

[0007] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a new phage protein Gp61, which is derived from phage VP1 and is:

[0008] (a) a protein or polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; or

[0009] (b) A protein or polypeptide derived from (a) with equivalent function, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO: 1.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the bacteriophage protein Gp61.

[0011] In a third aspect, the present invention provides a biological material containing the nucleic acid molecule, wherein the biological material includes but is not limited to an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

[0012] In a fourth aspect, the present invention provides the use of bacteriophage protein Gp61 in the preparation of a preparation for inhibiting Vibrio cholerae colonization and toxin secretion.

[0013] In a fifth aspect, the present invention provides bacteriophage protein Gp61 as a ctxAB Application in gene inhibitors;

[0014] described ctxAB Genes are made up of genes ctxA and ctxB Gene clusters, genes ctxA and ctxB Genes located in the same operon ctxA and ctxB The reference sequence numbers in GenBank are DQ774432.1 and DQ774431.1 respectively.

[0015] In a sixth aspect, the present invention provides a bacteriophage protein Gp61 as a tcpA Application in gene inhibitors;

[0016] described tcpA The gene encodes TcpA protein, and the reference sequence number of the gene in GenBank is DQ773719.1.

[0017] In a seventh aspect, the present invention provides a bacteriophage protein Gp61 as an inhibitor of genes related to colonization and virulence in the virulence regulatory pathway of Vibrio cholerae, as well as genes in other branch regulatory pathways related to these genes ( Figure 11 ) in the application of inhibitors.

[0018] The genes associated with colonization and virulence include but are not limited to genes tcpP, toxR, toxT , and their reference sequence numbers in GenBank are DQ773718.1, DQ774024.1, and DQ773728.1, respectively.

[0019] In an eighth aspect, the present invention provides a method for identifying that the bacteriophage protein Gp61 inhibits the colonization and toxin secretion of Vibrio cholerae (including non-disease diagnosis and treatment purposes), wherein the gene encoding the bacteriophage protein Gp61 is introduced into Vibrio cholerae via a plasmid to obtain a recombinant Vibrio cholerae expressing the bacteriophage protein Gp61, and then in vivo and in vitro experiments are performed to detect the virulence and colonization phenotype of the recombinant Vibrio cholerae.

[0020] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0021] (1) This invention addresses the current problem of serious drug resistance in pathogenic bacteria and the urgent need for new antibiotic alternatives. By screening a library of phage genes specifically targeting Vibrio cholerae, the authors seek proteins that can inhibit Vibrio cholerae colonization and toxin secretion during their interactions with the host. This not only provides information on the functions of unknown phage proteins but also clarifies their primary biological functions in the host, providing a foundation for the prevention, control, and virulence control of Vibrio cholerae.

[0022] (2) The present invention uses phage gene libraries as screening targets, greatly increasing the likelihood of obtaining positive results. The characteristics of specific targeting, inherent bactericidal and antibacterial effects, and the large number of candidate unknown proteins are also significant.

[0023] (3) Phage proteins have short-term and efficient gene transcription during the interaction between phage and host. Their proteins are not only easy to express in the host body, but also relatively easy to construct plasmids and express proteins in Escherichia coli in vitro, which is conducive to phenotypic and functional analysis and identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the Gp61 protein sequence of the present invention.

[0025] Figure 2 This is the genome annotation and function prediction of bacteriophage VP1 in a preferred embodiment of the present invention.

[0026] Figure 3 This is a map of the pSRKtc-gp61 plasmid constructed in a preferred embodiment of the present invention.

[0027] Figure 4 The preferred embodiment of the present invention is the N-gp61 strain ctxA (left) and tcpA (Right) Analysis of gene transcription levels.

[0028] Figure 5 The effect of Gp61 on other regulatory proteins in the virulence regulation pathway in the preferred embodiment of the present invention A: Gp61 protein inhibits the virulence pathway of Vibrio cholerae toxR Gene transcription; B: Gp61 protein inhibits the virulence pathway of Vibrio cholerae tcpP C: Gp61 protein inhibits the virulence pathway of Vibrio cholerae toxT Gene transcription.

[0029] Figure 6 This is an analysis of the competitive colonization ability of suckling mice in a preferred embodiment of the present invention.

[0030] Figure 7 In a preferred embodiment of the present invention, the amount of CT toxin secreted is determined by ELISA.

[0031] Figure 8 In the preferred embodiment of the present invention, the phage protein Gp61 and toxR Binding to gene promoters.

[0032] Figure 9 In the preferred embodiment of the present invention, the phage protein Gp61 and tcpP Binding to gene promoters.

[0033] Figure 10 A diagram of the Vibrio cholerae virulence regulation network in which Gp61 may be involved in a preferred embodiment of the present invention.

[0034] Figure 11 This is a technical route flow chart of a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0035] The present invention aims to obtain a bacteriophage protein capable of inhibiting the colonization and virulence of Vibrio cholerae.

[0036] The present invention adopts the following technical solutions:

[0037] 1. Analyze the Vibrio cholerae phage genome, annotate it, and make preliminary functional predictions, identify proteins with known and unknown functions, and preliminarily identify candidate proteins;

[0038] 2. Construct a prokaryotic expression plasmid for the phage protein and a Vibrio cholerae overexpression strain, and preliminarily analyze the effects of the phage protein on the colonization and virulence phenotype of Vibrio cholerae;

[0039] 3. Further phenotypic verification will be conducted by analyzing the effects of phage proteins on the transcription of genes involved in the colonization and virulence regulation pathways of Vibrio cholerae strains to clarify their regulatory pathways;

[0040] 4. Through animal model experiments, the colonization ability of the strain in suckling mice and the amount of toxin secretion were analyzed, and the phenotypic changes of the strain caused by phage protein were verified;

[0041] 5. Identify the direct interaction targets between phage proteins and genes related to the regulation of Vibrio cholerae colonization and virulence phenotypes, determine the regulatory genes that they directly target and bind to, and clarify the molecular mechanism by which they exert virulence regulation.

[0042] Specifically, the present invention provides the discovery and identification of a new phage protein that inhibits the colonization and toxin secretion of Vibrio cholerae, comprising the following steps: 1) Identifying and obtaining the prokaryotic expression plasmid pSRKtc-gp61. 2) Transforming the plasmid into the toxin-producing strain N16961 of Vibrio cholerae, screening it with a solid LB medium containing tetracycline (Tc, 2μg / mL), and naming it the N-gp61 strain. 3) The strain N-gp61 was streaked and cultured overnight on a solid LB medium, and single clones were picked and inoculated into a liquid LB medium containing tetracycline (Tc, 2μg / mL) and IPTG (0.05mM), at 37°C, shaking at 200rpm, and culturing for 8 hours. By detecting the virulence and colonization phenotypic experiments of the strains, it was found that the expression of the protein in Vibrio cholerae can cause the virulence gene of Vibrio cholerae. ctxAB Decreased transcription levels can cause ctxAB The amount of CT toxin protein encoded by the gene is reduced; at the same time, it can cause the gene of type IV pili tcpA The transcription level of tcpA The encoded TcpA is related to the colonization of the strain. We detected that the colonization ability of the N-gp61 strain in suckling mice was significantly inhibited. By constructing other related genes in the virulence regulation pathway of Vibrio cholerae tcpP, toxR, toxT We conducted experiments such as measuring the activity of virulence regulatory gene promoters in response to luminescence values ​​and analyzing the transcription of genes related to colonization and virulence in the virulence regulatory pathway. We found that the bacteriophage protein Gp61 can effectively inhibit the transcription of related genes in the virulence regulatory pathway of Vibrio cholerae, and ultimately inhibit the colonization of the strain in suckling mice. At the same time, we detected that the CT toxin secretion of the strain was greatly reduced, and clarified that the Gp61 protein can regulate genes respectively. tcpP 、 toxR The molecular mechanism of promoter binding provides important protein candidates and research reference plans for the subsequent control of Vibrio cholerae pathogenicity and the discovery and application of virulence-inhibiting peptides.

[0043] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0044] Vibrio cholerae N16961 used in the following examples was kindly provided by Professor John J Mekalanos, Department of Microbiology and Molecular Genetics, Harvard Medical School, USA (see Proc Natl Acad Sci USA. 2002 Feb 5;99(3):1556-61).

[0045] Phage VP1 is maintained by the Diarrheal Disease Control Laboratory, Institute of Infectious Disease Prevention and Control, Chinese Center for Disease Control and Prevention (see J Virol. 2021 Feb 24;95(6):e02245-20).

[0046] The pSRKtc plasmid was kindly donated by Professor Jun Zhu of the Department of Microbiology, Pennsylvania State University (see InfectImmun. 2014 Apr;82(4):1676-82).

[0047] Escherichia coli SM10λpir was purchased from Shanghai ELISA Biotechnology Co., Ltd.

[0048] The pBBR1MCS4-LuxCDABE plasmid was purchased from Baosai Plasmid Strain Resources Co., Ltd.

[0049] Example 1

[0050] This example provides a method for discovering and identifying a new phage protein that inhibits Vibrio cholerae colonization and toxin secretion, comprising the following steps:

[0051] (1) Phage genome annotation and identification of unknown proteins

[0052] The VP1 genome consists of linear double-stranded DNA with a length of 42,845 bp and a GC content of 45.49%. The genome was annotated using the online annotation tool RAST (http: / / rast.nmpdr.org / ) and visualized using the CGview tool. 61 putative open reading frames (ORFs) were predicted, of which 16 ORFs were predicted to be proteins with known functions.

[0053] The phage Gp61 protein sequence is shown in Figure 1 The phage VP1 genome annotation and function prediction can be found in Figure 2 .

[0054] (2) Construction of prokaryotic expression vector

[0055] The pSRKtc plasmid was isolated and identified and cultured in liquid agar medium containing 10 μg / mL tetracycline. The plasmid was extracted and digested with double enzymes to obtain a linearized vector. Homologous recombination primers (F: GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCAatgagcagacgaagcagacg, R: GATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCActagttacgtgtccatcggct) were designed and amplified using bacteriophage VP1 as a template. The amplified product was recovered to obtain the gene gp61 encoding a protein of unknown function. To construct a prokaryotic expression vector, we inserted the complete gp61 gene fragment (SEQ ID NO: 2) into the multiple cloning site of the pSRKtc expression plasmid, constructed the overexpression recombinant plasmid pSRKtc-gp61, and transformed it into Escherichia coli Trans1-T1. The plasmid was spread on an LB plate containing X-Gal (40ug / ml), IPTG (0.5mM), and tetracycline (10ug / ml), and incubated at 37°C overnight. The white single colonies on the plate were picked for sequencing.

[0056] The plasmid map of pSRKtc-gp61 is shown in Figure 3 .

[0057] (3) Construction of overexpression strains

[0058] The correctly sequenced recombinant plasmid pSRKtc-gp61 was transformed into conjugative Escherichia coli SM10 λpir, spread on LB plates containing chloramphenicol, and cultured overnight at 37°C. Single clones were picked for PCR verification to ensure the presence of the unknown protein gp61. At the same time, it was conjugated with Vibrio cholerae N16961, and the recombinant plasmid pSRKtc-gp61 was transferred into N16961 to construct an overexpression strain named N-gp61.

[0059] (4) Phenotypic identification of overexpression strains

[0060] The recombinant plasmid pBBR- ctxA -lux-、pBBR- tcpA- lux was transformed into conjugative Escherichia coli SM10 λpir to obtain SM10 λpir (pBBR- ctxA -lux) and SM10 λpir (pBBR- tcpA- lux) strain, and then conjugated with N16961 recipient bacteria (control bacteria, control) containing empty plasmid pSRKtc and N-gp61, and the reactive strains were screened by chloramphenicol, streptomycin and tetracycline resistance. ctxA 、 tcpALuminescence detection control bacteria for transcription, N-gp61 (pBBR- ctxA -lux), N-gp61 (pBBR- tcpA- lux), 37℃, 200rpm / min shaking culture. Use microplate reader to detect the absorbance of bacterial solution at 600nm and 420nm respectively, and calculate the unit fluorescence value (OD 420 / OD 600 ), to determine the effect of high expression of Gp61 on the virulence genes of the strain ctxA , colonization genes tcpA The impact of expression.

[0061] N-gp61 strains ctxA and tcpA Gene transcription level analysis Figure 4 .

[0062] From the above experimental results, it can be seen that the expression of bacteriophage protein Gp61 in Vibrio cholerae can significantly inhibit the virulence genes of Vibrio cholerae. ctxA and colonization genes tcpA Transcription.

[0063] (5) Transcriptional analysis of virulence regulation-related genes

[0064] Amplification by PCR toxR 、 toxT 、 tcpP The promoter region was cloned into the pBBR1MCS4-LuxCDABE plasmid luxCDABE Upstream of the reporter gene (without promoter region), the recombinant plasmid pBBR- tcpP -lux、pBBR- toxR -lux、pBBR- toxT -lux. The recombinant plasmid was transferred into the control bacteria and N-gp61, respectively, to obtain a cold light reporter gene strain containing the target fragment. The strain was diluted and inoculated into a 96-well plate, and cultured at 37°C with shaking at 200 rpm / min. The absorbance of the bacterial solution at 600nm and 420nm was measured using a microplate reader, and the unit fluorescence value (OD) was calculated. 420 / OD 600 ). Compare the differences in transcriptional expression of genes related to virulence regulatory pathways.

[0065] The effects of Gp61 on other regulatory proteins in the virulence regulation pathway are shown in Figure 5 .

[0066] From the above experimental results, it can be seen that the phage protein Gp61 can significantly inhibit the virulence regulation pathway of Vibrio cholerae related to virulence and colonization phenotype. toxR 、 toxT 、 tcpP Gene transcription.

[0067] (6) Colonization experiment in suckling mice

[0068] Six 5-7 day old CD-1 suckling mice were taken and the control strain (control) and the experimental strain (N-gp61) were streaked onto LB plates and cultured overnight at 37°C to form bacterial lawns. The bacterial lawns of each strain were directly picked and placed in liquid LB to dilute to a McFarland turbidity of 2-3 (the bacterial concentration was about 10 8 CFU / ml). Each puppy mouse was inoculated with 50 μl of the mixed bacterial solution at a 1:1 volume ratio. After inoculation, the puppies were placed at 28°C. Serial dilutions of the inoculated bacterial solution were plated onto plates containing X-Gal (40 μg / ml), IPTG (1 mM), and tetracycline (2 μg / ml) and cultured overnight at 37°C. The ratio of blue to white colonies was calculated as the input (white:blue) ratio. Approximately 18 hours later, the puppies were sacrificed by cervical dislocation. The small intestines were dissected and isolated, each plated in 1 ml of PBS, and homogenized using a tissue homogenizer to fully release the intestinal colonizing bacteria. Serial dilutions of the small intestine homogenate were then plated onto plates containing X-Gal, tetracycline, and streptomycin. The plates were cultured overnight at 37°C. Colony growth was observed, and the ratio of control bacteria and N-gp61 was recorded as the output (white:blue) ratio. The output / input ratio was calculated as the CI (competitive index) to assess changes in colonization ability.

[0069] Competitive colonization ability analysis of suckling mice Figure 6 .

[0070] From the above experimental results, it can be seen that the phage protein Gp61 significantly inhibits the colonization of Vibrio cholerae in suckling mice.

[0071] (7) CT toxin secretion detection

[0072] The bacterial cholera toxin (CT) ELISA detection kit (Shanghai Yansheng Industrial Co., Ltd.) was used. After equilibration at room temperature for 20 minutes, standard wells and sample wells (control bacteria, N-gp61) were set up in the strips. 50ul of standard of different concentrations were added to each standard well. 10ul of the sample to be tested was first added to the sample well, followed by 40ul of sample diluent. No addition was made to the blank wells. 100ul of detection antibody labeled with horseradish peroxidase (HRP) was added to each well except the blank well. The reaction wells were sealed with a sealing film and incubated at 37°C for 60 minutes. The liquid was discarded, patted dry on absorbent paper, and each well was filled with washing solution. The plate was allowed to stand for 1 minute, the washing solution was shaken off, and the plate was patted dry on absorbent paper. The washing was repeated 5 times. 50ul of substrate A and B were added to each well. Within 15 minutes, the OD value of each well was measured at a wavelength of 450nm. A linear regression curve of the standard was drawn based on the standard well. The accuracy of the result was determined based on the R value. The results of each sample well were calculated based on the OD value. 450 / OD 600Sure.

[0073] The results of ELISA determination of CT toxin excretion are shown in Figure 7 .

[0074] From the above experimental results, it can be seen that under in vitro culture conditions, phage protein Gp61 can reduce the excretion of Vibrio cholerae CT toxin.

[0075] (8) Analysis of Gp61 and tcpP 、 toxR Promoter binding

[0076] Electron migration assay (EMSA) was used to analyze the effect of Gp61 on virulence regulatory genes. tcpP 、 toxR Promoter binding. A 6% non-denaturing polyacrylamide gel was prepared and the protein-probe complex reaction, including negative controls, sample reactions, and a cold competition reaction for the probe, was run at room temperature for 30 minutes. Electrophoresis was performed using pre-chilled 0.5× TBE electrophoresis buffer at 120V for 20 minutes. Subsequently, 2.2 μl of 10× EMSA / Gel-Shift loading buffer was added and electrophoresis was continued at 120V for 2 hours. After electrophoresis, the gel was transferred to a nylon membrane at 100V for 50 minutes, on ice throughout. UV crosslinking was performed at 70 mJ / cm² for 30 seconds. The following protocol was used for the Chemiluminescent Nucleic Acid Detection Kit (Thermo Fisher Scientific): Nucleic Acid Detection Blocking Buffer and 4× Wash Buffer were heated to 37°C–50°C to dissolve the mixture. The mixture was then incubated with blocking buffer containing horseradish peroxidase conjugate for 15 minutes with shaking. The membrane was washed three to four times with 1× Wash Buffer for 5 minutes each. Finally, the HRP-DAB substrate color development kit was used to develop the color until the bands were clear, and the color development was terminated by washing twice with pure water. The membrane was dried and stored, and the image was scanned.

[0077] EMSA analysis of Gp61 and toxR、tcpP The promoter interaction results are shown in Figure 8-Figure 9 .

[0078] From the above experimental results, it can be seen that Gp61 can be respectively toxR 、 tcpP Directly binds to the promoter.

[0079] (9) Functional identification model diagram

[0080] Combining the above experimental results and literature review,

[0081] Biorende (https: / / www.biorender.com / ) was used to draw a pattern diagram for the identification of bacteriophage protein gp61, further clarifying the pathogenic mechanism of Vibrio cholerae and the complex regulatory network of its regulatory proteins ( Figure 10 ).

[0082] The above technical route process is shown in Figure 11 .

[0083] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Bacteriophage protein Gp61, characterized in that Its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The biological material containing the nucleic acid molecule according to claim 2, characterized in that The biological material is an expression cassette, a transposon, a plasmid vector, a virus vector, an engineered bacterium or a transgenic cell line.

4. Use of the protein according to claim 1 in the preparation of a preparation for inhibiting Vibrio cholerae colonization and toxin secretion.

5. The protein of claim 1 as a ctxAB Application in gene inhibitors; described ctxAB Genes are made up of genes ctxA and ctxB Gene clusters, genes ctxA and ctxB Genes located in the same operon ctxA and ctxB The reference sequence numbers in GenBank are DQ774432.1 and DQ774431.1; The applications are for non-disease diagnosis and treatment purposes.

6. The protein of claim 1 as a tcpA Application in gene inhibitors; described tcpA The reference sequence number of the gene in GenBank is DQ773719.1; The applications are for non-disease diagnosis and treatment purposes.

7. Use of the protein of claim 1 as an inhibitor of genes related to colonization and virulence in the virulence regulatory pathway of Vibrio cholerae and genes in other branch regulatory pathways related to these genes; The genes associated with colonization and virulence include genes tcpP, toxR, toxT , their reference sequence numbers in GenBank are DQ773718.1, DQ774024.1, and DQ773728.1; The applications are for non-disease diagnosis and treatment purposes.

8. A method for identifying bacteriophage protein Gp61 that inhibits Vibrio cholerae colonization and toxin secretion, characterized in that: The gene encoding the bacteriophage protein Gp61 is introduced into Vibrio cholerae via a plasmid to obtain a recombinant Vibrio cholerae expressing the bacteriophage protein Gp61, and then the virulence and colonization phenotype of the recombinant Vibrio cholerae are detected by in vivo and in vitro experiments; The bacteriophage protein Gp61 is the same as that described in claim 1; The method is for non-disease diagnosis and treatment purposes.

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