Phage protein Gp37 and application thereof in preparation of preparation for inhibiting vibrio cholerae colonization and toxin secretion

Abstract

The invention relates to the technical field of biology, and discloses bacteriophage protein Gp37 and application thereof in preparation of a preparation for inhibiting vibrio cholerae colonization and toxin secretion. According to the invention, a specific bacteriophage gene bank targeting vibrio cholerae is taken as a screening object, a protein capable of inhibiting vibrio cholerae colonization and toxin secretion in an interaction process with a host is searched, and a novel regulatory protein Gp37 for inhibiting vibrio cholerae colonization or toxicity is found and identified, so that functional information of unknown functional proteins of bacteriophages can be supplemented, and the screening efficiency is improved. Main biological functions of a host influenced by the vibrio cholerae can be determined, and a basis is provided for vibrio cholerae prevention and control and virulence control.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to phage protein Gp37 and its application in the preparation of a preparation for inhibiting the colonization and toxin secretion of Vibrio cholerae. Background Art

[0002] Cholera is an acute intestinal infectious disease caused by non-invasive Vibrio cholerae that can colonize the surface of the small intestinal mucosa. It is classified as a Class A infectious disease in China and is also one of the internationally quarantinable infectious diseases. It has caused seven major pandemics in the world, causing huge damage to people's lives and property. Since the prevalence of cholera is closely related to sanitation, sewage treatment systems, and socioeconomic conditions, Vibrio cholerae remains a pathogen that is closely monitored in less developed countries or regions.

[0003] Vibrio cholerae ( Vibrio cholerae ) enters the body through the human mouth. Some Vibrio cholerae can survive by resisting the acidic environment of the stomach, enter the host intestine smoothly, and colonize on the surface of the epithelial tissue of the small intestine. After bacterial colonization, a large number of bacteria start to multiply and initiate the expression of a series of virulence factors. The two most important virulence factors are ctxAB cholera toxin CT encoded by the tcpA gene and toxT TCP pili encoded by the tcpP 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, and the

[0004] Phages can specifically target and lyse pathogenic bacteria. The phage gene bank is the best source for screening regulatory proteins that kill, inhibit growth, or reduce virulence. As a vast amount of phage genomic information is being mined, more than two-thirds of phage gene functions have not been clearly annotated. Screening for proteins that interact with host virulence regulatory proteins from phage proteins with unknown functions is a new research idea and direction. There have been a few research reports in this area. By studying different phage proteins targeting Pseudomonas aeruginosa, it was found that these phage-encoded proteins with unknown functions play important roles during host interaction. For example, phage-encoded Dap1, the novel phage protein Gp21 encoded by the virulent phage vB_Pae_QDWS, and phage protein SrpA have important biological functions in affecting the virulence of Pseudomonas aeruginosa, as well as the metabolism, growth, and division of the host. A polypeptide derived from the Tip protein of Pseudomonas aeruginosa phage can specifically inhibit the assembly of type IV pili (T4P), which is crucial for multiple physiological activities of bacteria, such as adhesion, biofilm formation, and reducing the survival and spread of bacteria in the host. Against drug-resistant strains, this peptide has potential applications in antibacterial therapy and can provide new strategies for clinical treatment.

[0005] Mining new members that inhibit virulence from the phage gene bank specifically targeting Vibrio cholerae can further clarify the pathogenic mechanism of Vibrio cholerae and its complex regulatory network of regulatory proteins, providing a research basis for specifically targeting and inhibiting its virulence and reducing its transmission hazards. Summary of the Invention

[0006] The object of the present invention is to provide a phage protein Gp37 and its application in the preparation of a preparation for inhibiting the colonization and toxin secretion of Vibrio cholerae.

[0007] To achieve the object of the present invention, in the first aspect, the present invention provides a new phage protein Gp37, derived from phage VP1, which is: (a) a protein or polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1; or (b) a protein or polypeptide derived from (a) with one or several amino acids substituted, deleted, or added and having the same function as the sequence shown in SEQ ID NO:1.

[0008] In the second aspect, the present invention provides a nucleic acid molecule encoding the phage protein Gp37.

[0009] In the third aspect, the present invention provides a biological material containing the nucleic acid molecule, and 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.

[0010] Fourth aspect, the present invention provides the application of phage protein Gp37 in the preparation of a preparation for inhibiting the colonization and toxin secretion of Vibrio cholerae.

[0011] Fifth aspect, the present invention provides the application of phage protein Gp37 as a Vibrio cholerae ctxAB gene inhibitor; The ctxAB gene is a gene cluster composed of genes ctxA and ctxB Genes ctxA and ctxB are located within the same operon. The reference sequence numbers of genes ctxA and ctxB in GenBank are DQ774432.1 and DQ774431.1 respectively.

[0012] Sixth aspect, the present invention provides the application of phage protein Gp37 as a Vibrio cholerae tcpA gene inhibitor; The tcpA gene encodes the TcpA protein, and the reference sequence number of this gene in GenBank is DQ773719.1.

[0013] Seventh aspect, the present invention provides phage protein Gp37 as an inhibitor of genes related to colonization and virulence on the Vibrio cholerae virulence regulation pathway, and inhibitors of genes on other branch regulation pathways related to these genes ( Figure 11 ).

[0014] The genes related to 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.

[0015] Eighth aspect, the present invention provides a method for identifying the inhibition of Vibrio cholerae colonization and toxin secretion by phage protein Gp37 (including non-disease diagnosis and treatment purposes). The gene encoding phage protein Gp37 is introduced into Vibrio cholerae through a plasmid to obtain a recombinant Vibrio cholerae expressing the phage protein Gp37, and then in vivo and in vitro experiments are carried out to detect the virulence and colonization phenotypes of the recombinant Vibrio cholerae.

[0016] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects: (1) In view of the serious problem of current pathogenic bacteria drug resistance and the urgent need to reserve new antibiotic replacement therapies, the present invention uses a phage gene library specifically targeting Vibrio cholerae as the screening object to search for proteins that can inhibit the colonization and toxin secretion of Vibrio cholerae during the interaction with the host. This can not only supplement the functional information of unknown phage proteins but also clarify their main biological functions affecting the host, providing a basis for controlling the prevention and control of Vibrio cholerae and its virulence.

[0017] (2) Using the phage gene library as the screening object in the present invention greatly increases the possibility of obtaining positive results. It has the characteristics of specific targeting, its own bactericidal and bacteriostatic effects, and a large number of candidate unknown proteins.

[0018] (3) During the interaction between phage and host, phage proteins have short-term and efficient gene transcription. Their proteins are not only easily expressed in the host but also relatively easy to construct plasmids and express proteins prokaryotically in Escherichia coli in vitro, facilitating phenotypic and functional analysis and identification. Brief Description of the Drawings

[0019] Figure 1 It is the Gp37 protein sequence of the present invention.

[0020] Figure 2 It is the genomic annotation and function prediction of phage VP1 in the preferred embodiment of the present invention.

[0021] Figure 3 It is the plasmid map of pSRKtc-gp37 constructed in the preferred embodiment of the present invention.

[0022] Figure 4 It is the construction and PCR verification of the gp37 gene overexpression strain in the preferred embodiment of the present invention.

[0023] Figure 5 It is for the ctxA (left) and tcpA (right) gene transcription level analysis of the N-gp37 strain in the preferred embodiment of the present invention.

[0024] Figure 6 It is the influence 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 transcription of the toxR gene in the Vibrio cholerae virulence pathway; B: Gp37 protein inhibits the transcription of the toxT gene in the Vibrio cholerae virulence pathway; C: Gp37 protein inhibits the transcription of the tcpP gene in the Vibrio cholerae virulence pathway.

[0025] Figure 7 It is the analysis of the competitive colonization ability of suckling mice in the preferred embodiment of the present invention.

[0026] Figure 8 In a preferred embodiment of the present invention, the ELISA method is used to measure the extracellular secretion amount of CT toxin.

[0027] Figure 9 In a preferred embodiment of the present invention, the binding of phage protein Gp37 to tcpP the gene promoter.

[0028] Figure 10 In a preferred embodiment of the present invention, the binding of phage protein Gp37 to toxR the gene promoter.

[0029] Figure 11 In a preferred embodiment of the present invention, the network diagram of Vibrio cholerae virulence regulation that Gp37 may participate in.

[0030] Figure 12 In a preferred embodiment of the present invention, the flow chart of the technical route. Detailed implementation manners

[0031] The present invention aims to obtain a phage protein that can inhibit the colonization and virulence of Vibrio cholerae.

[0032] The present invention adopts the following technical solutions: 1. Analyze the influence of phage proteins on the transcription of genes related to the colonization and virulence of Vibrio cholerae strains, and preliminarily determine the biological phenotypes that cause changes in the strains; 2. Determine the changes in the expression levels of genes related to the Vibrio cholerae virulence regulation pathway caused by phage proteins, and further clarify its regulatory pathway; 3. Through animal model experiments or measurement of the extracellular secretion amount of toxins, verify the phenotypic changes of the strains caused by phage proteins; Specifically, the present invention provides the discovery and identification of a new phage protein that inhibits the colonization and toxin secretion of Vibrio cholerae, including the following steps: 1) Identify and obtain the prokaryotic expression plasmid pSRKtc-gp37. 2) Transform the plasmid into Vibrio cholerae N16961, and screen it with solid LB medium containing tetracycline (Tc, 2 μg / mL), named strain N-gp37. 3) Streak the strain N-gp37 on solid medium and culture it overnight, and then pick single colonies and inoculate them into liquid LB medium containing tetracycline (Tc, 2 μg / mL) and IPTG (0.05 mM), and culture them at 37 °C with shaking at 200 rpm for 8 h. Through phenotypic experiments such as detecting the virulence and colonization of the strains, it is found that the expression of this protein in Vibrio cholerae can cause a decrease in the transcriptional level of Vibrio cholerae virulence genes ctxAB and can cause ctxAB a decrease in the extracellular secretion amount of the encoded CT toxin protein; at the same time, it can cause a significant decrease in the transcriptional level of the gene tcpA encoding type IV pili, because tcpAThe encoded TcpA is related to strain colonization. We detected that the colonization ability of the N-gp37 strain in suckling mice was reduced by 98 - 99%. By constructing other related genes in the Vibrio cholerae virulence regulation pathway tcpP, toxR, toxT promoter and the transcriptional fusion reporter plasmid of the luciferase reporter gene in the plasmid pBBR1MCS4-LuxCDABE luxCDABE (GenBank: OK165504.1), we measured the luciferase value to reflect the promoter activity of the virulence regulation gene and other experiments, and analyzed the transcription of genes related to colonization and virulence in the virulence regulation pathway. It was found that the phage protein Gp37 could effectively inhibit the transcription of related genes in the Vibrio cholerae virulence regulation pathway, 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 an important protein candidate and research reference plan for subsequent control of Vibrio cholerae pathogenicity and the discovery and application of inhibitory virulence polypeptides.

[0033] The following examples are used to illustrate the present invention, but not 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.

[0034] Vibrio cholerae N16961 used in the following examples was kindly provided by Professor John J Mekalanos of the Department of Microbiology and Molecular Genetics, Harvard Medical School, USA (see the literature Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1556 - 61).

[0035] The phage VP1 was preserved in the Diarrhea Disease Control Laboratory, Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention (see the literature J Virol. 2021 Feb 24;95(6):e02245 - 20).

[0036] The pSRKtc plasmid was kindly provided by Professor Jun Zhu of the Department of Microbiology, University of Pennsylvania, USA (see the literature Infect Immun. 2014 Apr;82(4):1676 - 82).

[0037] Escherichia coli SM10λpir was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

[0038] The pBBR1MCS4-LuxCDABE plasmid was purchased from Baosai Plasmid Strain Resource Company.

[0039] Example 1 This example provides the discovery and identification of a new phage protein that inhibits Vibrio cholerae colonization and toxin secretion, including the following steps: (1) Genome annotation of phage VP1 and determination of unknown proteins The genome of VP1 consists of linear double-stranded DNA with a length of 42,845 bp and a GC content of 45.49%. The online annotation tool RAST (http: / / rast.nmpdr.org / ) was used to annotate the genome, and the CGview tool was used to visualize the genome annotation. A total of 61 putative open reading frames (ORFs) were predicted, of which 16 ORFs were predicted to be proteins with known functions.

[0040] The protein sequence of phage Gp37 is shown in Figure 1 The genome annotation and function prediction of phage VP1 are shown in Figure 2 .

[0041] (2) Construction of prokaryotic expression vector The isolated and identified pSRKtc plasmid was cultured in a liquid agar medium containing 10 μg / ml tetracycline, and the plasmid was extracted and digested with two restriction enzymes to obtain a linearized vector. Homologous recombination primers (F: GATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCAttatccatctaactgttttccttcttccaaaatttca / R: GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCAatgaccggacgtagaaaacctga) were designed, and phage VP1 was used as a template for amplification. The amplified product was recovered to obtain the gene gp37 encoding an unknown function protein. 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. It was spread on an LB plate containing X-Gal (40 μg / ml), IPTG (0.5 mM), and tetracycline (10 μg / ml), and incubated at 37 °C overnight. White monoclonal colonies on the plate were picked for sequencing.

[0042] The plasmid map of pSRKtc-gp37 is shown in Figure 3 .

[0043] (3) Construction of overexpression strain The correctly sequenced recombinant plasmid pSRKtc-gp37 was transformed into the conjugative Escherichia coli SM10 λpir, spread on an LB plate containing chloramphenicol, and incubated at 37 °C overnight. Monoclonal colonies 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 to transfer the recombinant plasmid pSRKtc-gp37 into N16961 to construct an overexpression strain, named N-gp37.

[0044] (4) Phenotypic identification of overexpression strains Transfer the correctly sequenced recombinant plasmids pBBR- ctxA -lux- and pBBR- tcpA- lux into the conjugative Escherichia coli SM10 λpir to obtain the strains SM10 λpir (pBBR- ctxA -lux) and SM10 λpir (pBBR- tcpA- lux), and then conjugate them with the N16961 recipient bacteria (control bacteria control) and N-gp37 containing the empty plasmid pSRKtc respectively. Through the triple resistance screening of chloramphenicol, streptomycin, and tetracycline, obtain the luminescence detection strains N-control, N-gp37(pBBR- ctxA 、 tcpA -lux), N-gp37 (pBBR- ctxA -lux) that can reflect tcpA- lux), and culture them with shaking at 37 °C and 200 rpm / min. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance values of the bacterial liquid at 600 nm and 420 nm respectively, and calculate the unit fluorescence value (OD 420 / OD 600 ), and judge the effect of high expression of Gp37 on the expression of virulence genes ctxA and colonization genes tcpA of the strain.

[0045] The ctxA and tcpA gene transcription level analysis of the N-gp37 strain is shown in Figure 5 .

[0046] It can be seen from the above experimental results that the expression of the phage protein Gp37 in Vibrio cholerae can cause a significant decrease in the transcription levels of the virulence genes ctxA and colonization genes tcpA of Vibrio cholerae.

[0047] (5) Transcription analysis of virulence regulation-related genes Amplify the promoter regions of toxR 、 toxT 、 tcpP by PCR and clone them into the upstream (without promoter region) of the pBBR1MCS4-LuxCDABE plasmid luxCDABE reporter gene to obtain the recombinant plasmids pBBR- tcpP -lux and pBBR- toxR-lux, pBBR- toxT -lux. The recombinant plasmids were respectively transferred into control and N-gp37 to obtain the fluorescent reporter gene strains containing the target fragments. They were respectively diluted and inoculated into 96-well plates and cultured with shaking at 37 °C and 200 rpm / min. The absorbance values of the bacterial solutions at 600 nm and 420 nm were detected by a microplate reader, and the unit fluorescence value (OD 420 / OD 600 ) was calculated. The differences in the transcriptional expression of genes related to the virulence regulation pathway were compared.

[0048] The effects of Gp37 on other regulatory proteins in the virulence regulation pathway are shown in Figure 6 .

[0049] It can be seen from the above experimental results that the phage protein Gp37 can significantly inhibit the toxR , toxT , tcpP genes related to virulence and colonization phenotypes in the virulence regulation pathway of Vibrio cholerae from being transcribed.

[0050] (6)Colonization experiment in suckling mice Six 5- to 7-day-old CD-1 suckling mice were taken. The control strain (control) and the experimental strain (N-gp37) were respectively streaked on LB plates and cultured overnight at 37 °C to form bacterial lawns. The bacterial lawns of each strain were directly picked and diluted in liquid LB to a McFarland turbidity of 2 - 3 (at this time, the bacterial concentration was about 10 8 CFU / ml). They were mixed at a volume ratio of 1:1, and 50 μl of the mixed bacterial solution was inoculated into each suckling mouse. The inoculated suckling mice were placed at 28 °C, and the bacterial solution at the time of inoculation was gradient-diluted and spread on plates containing X-Gal (40 μg / ml), IPTG (1 mM), and tetracycline (2 μg / ml) and cultured overnight at 37 °C, and the blue-white plaque ratio was counted as the input (white:blue) ratio. After about 18 hours, the suckling mice were sacrificed by cervical dislocation, the small intestines were dissected from the suckling mice and placed in 1 ml of PBS respectively, and they were homogenized with a tissue homogenizer to fully release the bacteria colonized in the intestine. Then the small intestine homogenate was gradient-diluted and spread on plates containing X-gal, tetracycline, and streptomycin. It was cultured overnight at 37 °C, the growth of colonies was observed, and the ratio of the control bacteria to N-gp37 was recorded as the output (white:blue) ratio. The output / input was used as the CI value (competition index) to judge whether the colonization ability had changed.

[0051] Analysis of the competitive colonization ability of suckling mice is shown in Figure 7 .

[0052] It can be seen from the above experimental results that the phage protein Gp37 inhibits the colonization of Vibrio cholerae in suckling mice, and compared with the control bacteria, it is reduced by about 98%.

[0053] (7) Detection of CT toxin secretion Use a bacterial cholera toxin (CT) ELISA detection kit (Shanghai Yansheng Industrial Co., Ltd.). Set up standard wells and sample wells (control bacteria, N-gp37) in the strips after equilibrating at room temperature for 20 min. Add 50 μl of standards with different concentrations to the standard wells, add 10 μl of the sample to be tested first and then 40 μl of sample diluent to the sample wells, and do not add anything to the blank wells. Add 100 μl of the detection antibody labeled with horseradish peroxidase (HRP) to each well except the blank well. Seal the reaction wells with a sealing film and incubate at 37 °C for 60 min. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 min, discard the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times. Add 50 μl of substrate A and B to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min. Draw a standard linear regression curve based on the standard wells, determine the result accuracy according to the R value, and determine the results of each sample well according to OD 450 / OD 600 .

[0054] The results of ELISA for determining the extracellular secretion amount of CT toxin are shown in Figure 8 .

[0055] It can be seen from the above experimental results that under in vitro culture conditions, the phage protein Gp37 can reduce the extracellular secretion amount of Vibrio cholerae CT toxin.

[0056] (8) Analyze the binding of Gp37 to tcpP , toxR promoters Use electrophoretic mobility shift assay (EMSA) to analyze the binding of Gp37 to the promoters of virulence regulatory genes tcpP , toxR . Prepare a 6% non-denaturing polyacrylamide gel and perform protein-probe complex reactions, including negative control, sample reaction, and probe cold competition reaction systems, and let stand at room temperature for 30 minutes. Use pre-cooled 0.5×TBE electrophoresis buffer for electrophoresis, pre-electrophorese at 120 V for 20 minutes, then add 2.2 μl of 10×EMSA / Gel-Shift loading buffer, and electrophorese at 120 V for 2 hours. After electrophoresis, transfer the gel to a nylon membrane, transfer at 100 V for 50 minutes, and keep on ice bath throughout the process. Cross-link with ultraviolet light at 70 mJ / cm² for 30 seconds. According to the operation method of the chemiluminescent nucleic acid detection kit (Thermo Fisher Scientific): Heat the nucleic acid detection blocking buffer and 4× washing buffer to 37 °C - 50 °C to dissolve, incubate with the blocking buffer containing horseradish peroxidase conjugate for 15 minutes, and shake. Wash the membrane 3 - 4 times with 1× washing buffer, 5 minutes each time. Finally, develop the color with the HRP-DAB substrate color development kit until the bands are clear, wash 2 times with pure water to terminate the color development, dry the membrane and save it, and scan the figure.

[0057] EMSA analysis of the interaction between Gp37 and tcpP 、 toxR The results of the interaction between the promoter are shown in Figure 9 - Figure 10 。

[0058] From the above experimental results, it can be seen that Gp37 can respectively bind to tcpP 、 toxR the promoter directly.

[0059] (9)Functional identification schematic diagram Combining the above experimental results and literature review, a schematic diagram of the functional identification of the phage protein gp37 was drawn using biorende (https: / / www.biorender.com / ) to further clarify the pathogenic mechanism of Vibrio cholerae and its complex regulatory network of regulatory proteins ( Figure 11 )。

[0060] The above technical route process is shown in Figure 12 。Although the present invention has been described in detail above with general descriptions and specific implementation examples, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. Bacteriophage protein Gp37, characterized in that Its for; (a) a protein or polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; or (b) A protein or polypeptide derived from (a) with equivalent functions, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO:

1.

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

3. A biological material containing the nucleic acid molecule according to claim 2, characterized in that: The biological material is an expression box, a transposon, a plasmid vector, a virus vector, an engineering 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 ctxB Application in gene inhibitors; Said ctxB Genes are ctx and ctxB Gene clusters, genes ctx and ctxB Genes located in the same operon ctx 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; Said 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 for inhibiting 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 in vivo and in vitro experiments are performed to detect the virulence and colonization phenotype of the recombinant Vibrio cholerae; 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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