Bacteriophage protein Gp37 and its application in preparing preparations for inhibiting Vibrio cholerae colonization and toxin secretion

Abstract

The present invention relates to the field of biotechnology and discloses a bacteriophage protein Gp37 and its use in preparing a preparation for inhibiting Vibrio cholerae colonization and toxin secretion. The present invention screens a library of phage genes specifically targeting Vibrio cholerae to identify proteins capable of inhibiting Vibrio cholerae colonization and toxin secretion during interaction with the host. The discovery and identification of a novel regulatory protein, Gp37, that inhibits Vibrio cholerae colonization or virulence not only supplements functional information about 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 Gp37 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 small intestinal mucosa. It is classified as a Category A infectious disease in my country and is also an internationally quarantined infectious disease. It has caused seven global pandemics, causing significant damage to people's lives and property. Because cholera epidemics are closely linked to sanitation, sewage treatment systems, and socioeconomic conditions, Vibrio cholerae remains a key pathogen under surveillance in economically underdeveloped countries and regions.

[0003] Vibrio cholerae ( Vibrio cholerae ) enters the body through the human mouth. Some Vibrio cholerae can survive the acidic environment of the stomach and successfully enter the host intestine and colonize on the surface of the epithelial tissue of the small intestine. After bacterial colonization, they begin to multiply and start the expression of a series of virulence factors, the two most important of which are ctxAB Genes encoding cholera toxin CT and tcpA The TCP pili encoded by the gene, the expression of these virulence factors is regulated by a cascade of multiple proteins: in the core regulatory pathway, the virulence genes are directly activated by ToxT, toxT The gene is regulated by ToxR and TcpP, tcpP The transcriptional expression of V. cholerae is positively regulated by both AphA and AphB. The regulatory network governing the virulence and colonization phenotypes of V. cholerae is complex, with numerous regulatory proteins involved. Proteins regulating these two virulence genes are still being discovered and added. In particular, given the emergence of drug resistance in V. cholerae, the discovery of new regulatory proteins that inhibit colonization or virulence is 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 virulence 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. Summary of the Invention

[0006] The purpose of the present invention is to provide a bacteriophage protein Gp37 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 Gp37, 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 Gp37.

[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 Gp37 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 Gp37 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 Gp37 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 Gp37 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 bacteriophage protein Gp37 inhibits Vibrio cholerae colonization and toxin secretion (including non-disease diagnosis and treatment purposes), wherein the gene encoding bacteriophage protein Gp37 is introduced into Vibrio cholerae via a plasmid to obtain recombinant Vibrio cholerae expressing the bacteriophage protein Gp37, 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 Gp37 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-gp37 plasmid constructed in a preferred embodiment of the present invention.

[0027] Figure 4 This is the construction and PCR verification of the gp37 gene overexpression strain in the preferred embodiment of the present invention.

[0028] Figure 5 The preferred embodiment of the present invention is the strain N-gp37 ctxA (left) and tcpA (Right) Analysis of gene transcription levels.

[0029] Figure 6 The effect of Gp37 protein on other regulatory proteins in the virulence regulation pathway in the preferred embodiment of the present invention. A: Gp37 protein inhibits the virulence regulation pathway of Vibrio cholerae. toxR Gene transcription; B: Gp37 protein inhibits the virulence pathway of Vibrio cholerae toxT C: Gp37 protein inhibits the virulence pathway of Vibrio cholerae tcpP Gene transcription.

[0030] Figure 7 This is an analysis of the competitive colonization ability of suckling mice in a preferred embodiment of the present invention.

[0031] Figure 8 In a preferred embodiment of the present invention, the amount of CT toxin secreted is determined by ELISA.

[0032] Figure 9 In the preferred embodiment of the present invention, the phage protein Gp37 and tcpP Binding to gene promoters.

[0033] Figure 10 In the preferred embodiment of the present invention, the phage protein Gp37 and toxR Binding to gene promoters.

[0034] Figure 11 This is a diagram of the Vibrio cholerae virulence regulation network in which Gp37 may be involved in a preferred embodiment of the present invention.

[0035] Figure 12 The technical route flow chart of the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0036] The present invention aims to obtain a bacteriophage protein capable of inhibiting the colonization and virulence of Vibrio cholerae.

[0037] The present invention adopts the following technical solutions:

[0038] 1. Analyze the effects of phage proteins on colonization and virulence-related gene transcription of Vibrio cholerae strains, and preliminarily determine the biological phenotype that causes strain changes;

[0039] 2. Determine whether phage proteins cause changes in gene expression levels in the virulence regulatory pathway of Vibrio cholerae and further clarify its regulatory pathway;

[0040] 3. Verify through animal model experiments or measuring toxin excretion and phenotypic changes of strains caused by phage proteins;

[0041] 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-gp37. 2) Transforming the plasmid into Vibrio cholerae N16961, screening it with a solid LB medium containing tetracycline (Tc, 2 μg / mL), and naming it the N-gp37 strain. 3) Streaking the strain N-gp37 on a solid medium overnight, picking single clones and inoculating them into a liquid LB medium containing tetracycline (Tc, 2 μg / mL) and IPTG (0.05 mM), and shaking and culturing them at 37°C, 200 rpm / min for 8 hours. By detecting the phenotypic experiments such as the virulence and colonization of the strain, 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 ctxABThe 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-gp37 strain in suckling mice was reduced by 98-99%. tcpP, toxR, toxT The promoter of the luminescence reporter gene in the plasmid pBBR1MCS4-LuxCDABE luxCDABE We used the transcriptional fusion reporter plasmid of the V. cholerae strain (GenBank: OK165504.1) to measure the luminescence value reaction virulence regulatory gene promoter activity and other experiments to analyze the transcription of genes related to colonization and virulence in the virulence regulatory pathway. We found that the phage protein Gp37 can effectively inhibit the transcription of related genes in the virulence regulatory pathway of Vibrio cholerae, ultimately inhibiting the colonization of the strain in suckling mice. At the same time, the CT toxin secretion of the strain was greatly reduced, providing important protein candidates and research reference plans for the subsequent control of Vibrio cholerae pathogenicity and the discovery and application of inhibitory virulence peptides.

[0042] 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.

[0043] 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).

[0044] 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).

[0045] 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).

[0046] Escherichia coli SM10λpir was purchased from Shanghai ELISA Biotechnology Co., Ltd.

[0047] The pBBR1MCS4-LuxCDABE plasmid was purchased from Baosai Plasmid Strain Resources Co., Ltd.

[0048] Example 1

[0049] 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:

[0050] (1) Annotation of the bacteriophage VP1 genome and identification of unknown proteins

[0051] 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.

[0052] The phage Gp37 protein sequence is shown in Figure 1 The phage VP1 genome annotation and function prediction can be found in Figure 2 .

[0053] (2) Construction of prokaryotic expression vector

[0054] 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:GATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCAttatccatctaactgttttccttcttccaaaatttca / R:GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCAatgaccggacgtagaaaacctga) were designed and amplified using bacteriophage VP1 as a template. The amplified product was recovered to obtain the gene gp37 encoding a protein of unknown function. To construct a prokaryotic expression vector, we inserted the complete gp37 gene fragment (SEQ ID NO: 2) into the multiple cloning site of the pSRKtc expression plasmid, constructed the overexpression recombinant plasmid pSRKtc-gp37, 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.

[0055] The plasmid map of pSRKtc-gp37 is shown in Figure 3 .

[0056] (3) Construction of overexpression strains

[0057] The recombinant plasmid pSRKtc-gp37 with correct sequencing was transformed into conjugative Escherichia coli SM10 λpir, spread on LB plates containing chloramphenicol, cultured at 37°C overnight, and single clones were picked for PCR verification ( Figure 4 ), ensure the existence of the unknown protein gp37, and at the same time, conjugate with Vibrio cholerae N16961, and transfer the recombinant plasmid pSRKtc-gp37 into N16961 to construct an overexpression strain, named N-gp37.

[0058] (4) Phenotypic identification of overexpression strains

[0059] 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) containing empty plasmid pSRKtc and N-gp37, and the reactive strains were screened by chloramphenicol, streptomycin and tetracycline resistance. ctxA 、 tcpA Luminescent detection of transcription status of strains N-control, N-gp37 (pBBR- ctxA -lux), N-gp37 (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 Gp37 on the virulence genes of the strain ctxA , colonization genes tcpA The impact of expression.

[0060] N-gp37 strains ctxA and tcpA Gene transcription level analysis Figure 5 .

[0061] From the above experimental results, it can be seen that the expression of bacteriophage protein Gp37 in Vibrio cholerae can cause the virulence gene of Vibrio cholerae ctxA and colonization genes tcpA The transcription level was significantly reduced.

[0062] (5) Transcriptional analysis of virulence regulation-related genes

[0063] 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 control and N-gp37 respectively to obtain a fluorescent reporter gene strain containing the target fragment. It 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 420 / OD 600 ). Compare the differences in transcriptional expression of genes related to virulence regulatory pathways.

[0064] The effects of Gp37 on other regulatory proteins in the virulence regulation pathway are shown in Figure 6 .

[0065] From the above experimental results, it can be seen that the phage protein Gp37 can significantly inhibit the virulence regulation pathway of Vibrio cholerae related to virulence and colonization phenotype. toxR 、 toxT 、 tcpP Gene transcription.

[0066] (6) Colonization experiment in suckling mice

[0067] Six 5-7 day old CD-1 suckling mice were taken and the control strain (control) and the experimental strain (N-gp37) 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 to N-gp37 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.

[0068] Competitive colonization analysis of suckling mice Figure 7 .

[0069] From the above experimental results, it can be seen that the phage protein Gp37 inhibits the colonization of Vibrio cholerae in suckling mice, which is reduced by about 98% compared with the control bacteria.

[0070] (7) CT toxin secretion detection

[0071] 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-gp37) 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. Except for the blank wells, 100ul of detection antibody labeled with horseradish peroxidase (HRP) was added to each 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 wells. The accuracy of the results was determined based on the R value. The results of each sample well were calculated based on the OD value. 450 / OD 600 Sure.

[0072] The results of ELISA determination of CT toxin excretion are shown in Figure 8 .

[0073] From the above experimental results, it can be seen that under in vitro culture conditions, phage protein Gp37 can reduce the excretion of Vibrio cholerae CT toxin.

[0074] (8) Analysis of Gp37 and tcpP 、 toxR Promoter binding

[0075] Electron migration assay (EMSA) was used to analyze the effect of Gp37 on virulence regulatory genes. tcpP 、 toxRPromoter 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 scanned.

[0076] EMSA analysis of Gp37 and tcpP 、 toxR The promoter interaction results are shown in Figure 9-10 .

[0077] From the above experimental results, it can be seen that Gp37 can be respectively tcpP 、 toxR Directly binds to the promoter.

[0078] (9) Functional identification model diagram

[0079] Combining the above experimental results and literature review, we used Biorender (https: / / www.biorender.com / ) to draw a functional identification model diagram of the bacteriophage protein gp37 to further clarify the pathogenic mechanism of Vibrio cholerae and the complex regulatory network of its regulatory proteins ( Figure 11 ).

[0080] The above technical route process is shown in Figure 12 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 or improvements may be made based on the present invention. Therefore, such modifications or improvements, which do not depart from the spirit of the present invention, are within the scope of protection claimed by the present invention.

Claims

1. Bacteriophage protein Gp37, 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 respectively.

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.

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 , and their reference sequence numbers in GenBank are DQ773718.1, DQ774024.1, and DQ773728.1, respectively.

8. A method for identifying bacteriophage protein Gp37 that inhibits Vibrio cholerae colonization and toxin secretion, characterized in that: The gene encoding the bacteriophage protein Gp37 is introduced into Vibrio cholerae via a plasmid to obtain a recombinant Vibrio cholerae expressing the bacteriophage protein Gp37, and then the virulence and colonization phenotype of the recombinant Vibrio cholerae are detected by in vivo and in vitro experiments; The bacteriophage protein Gp37 is the same as that described in claim 1; The method is for non-disease diagnosis and treatment purposes.

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