Use of bacteriophage protein Gp17 in the preparation of a preparation for inhibiting colonization and toxin secretion by Vibrio cholerae

Abstract

The present application relates to the field of biotechnology, and discloses application of a bacteriophage protein Gp17 in preparation of a preparation for inhibiting colonization and toxin secretion of Vibrio cholerae. The newly identified bacteriophage protein Gp17 not only participates in the structure and function of bacteriophage, but also can significantly inhibit expression of virulence genes of Vibrio cholerae and reduce the colonization ability in the intestinal tract, which indicates that the bacteriophage structural protein may have a new functional mode in regulating pathogenicity of the host, and provides a new technical idea for targeting virulence regulation of Vibrio cholerae. The present application screens a new type of protein from a Vibrio cholerae specific bacteriophage gene library, which not only provides a new tool for elucidating and inhibiting the complex regulation network of virulence and colonization of Vibrio cholerae, but also can discover key target points that can be intervened. The protein screened by the present application for inhibiting colonization and virulence of Vibrio cholerae can be used as a focus for application research and exploration, and provides a new potential strategy for controlling spread of Vibrio cholerae and treating infection of Vibrio cholerae.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to the application of bacteriophage protein Gp17 in the preparation of formulations that inhibit Vibrio cholerae colonization and toxin secretion. Background Technology

[0002] Cholera is caused by a Gram-negative bacterium—Vibrio cholerae (Cholera). Vibrio cholerae Vibrio cholerae, an acute intestinal infectious disease caused by bacteria, poses a serious threat to human health. It can colonize the human intestine and attach to the surface of planktonic organisms, spreading through contaminated water sources and seafood. In recent years, influenced by global warming, the abundance of Vibrio in aquatic environments has been continuously increasing, which may further alter the epidemiological characteristics of Vibrio-related diseases. Furthermore, due to the complex and variable drug resistance patterns of Vibrio cholerae, prevention and control efforts face continuous and severe challenges.

[0003] The pathogenesis of cholera is complex, with its core causative factors being cholera toxin (CT) and toxin-co-regulated fimbriae (TCP). Cholera toxin CT is composed of... ctxAB Encoded in genes, it is secreted into the intestine via the type II secretion system, causing a sharp increase in intracellular cAMP levels in host cells, which in turn leads to water, sodium, and potassium metabolism disorders, resulting in "rice-water"-like diarrhea. The major subunit of the toxin-co-regulated fimbriae TCP is... tcpA The type IV fimbriae encoded by the gene mediate the colonization of Vibrio cholerae in the small intestinal mucosa through bacterial self-aggregation and are essential bacterial structures for its infection in the gut. Studies have shown that Vibrio cholerae coordinates the expression of its virulence and colonization-related genes through a complex, multi-level regulatory network in response to various signals in the host gut environment. This regulatory network is dominated by a complex multi-level signal cascade system. This system integrates gut microenvironment signals (such as osmotic pressure, pH, oxygen, bile, and bicarbonate levels) and precisely controls the expression of virulence genes through the central cascade of ToxR / TcpP-ToxT. The upstream regulators AphA / AphB synergistically induce... tcpP The transcriptional expression of ToxR, in turn, synergistically activates the downstream transcriptional regulator ToxT with TcpP, ultimately driving... ctxAB and tcpA Transcriptional expression of virulence genes is also affected. Furthermore, environmental factors such as anaerobic conditions, quorum sensing (QS), cyclic nucleotide signaling molecules (e.g., cAMP, c-di-GMP), and reactive oxygen species / nitrogen species (ROS / RNS) influence virulence gene expression through various regulatory factors like AphA / AphB and LuxO / HapR, enabling Vibrio cholerae to adapt to different stages of survival and pathogenicity induction from the in vitro environment to host infection. Therefore, a deeper understanding of the expression regulatory network of Vibrio cholerae toxins and fimbriae, and the discovery of novel regulatory proteins, can provide new strategies for cholera prevention and control.

[0004] Bacteriophages, viruses that specifically infect bacteria, are widespread in nature and pose a survival challenge for Vibrio cholerae. The phage genome encodes a large number of functionally diverse phage proteins, which demonstrate significant potential in self-replication, inhibiting host bacterial growth, interfering with virulence expression, and regulating host physiological states. However, the lack of functional information annotation for two-thirds of phage proteins makes in-depth research on phage-Vibrio cholerae interactions worthy of further investigation. Based on their roles in viral particle formation and infection, phage proteins are divided into structure-associated proteins and non-structure-associated proteins. Structural proteins directly participate in the assembly and stability of phage particles; for example, capsid proteins protect viral nucleic acids, while tail filament proteins mediate recognition and adsorption to the host cell surface. Non-structural proteins are mostly expressed post-infection, participating in the regulation of the phage replication cycle, integration process, or modification of host metabolic pathways, thereby assisting phage proliferation. Phage structure-related proteins also participate in other functions. Studies have shown that the capsid protein GpE of phage λ, upon expression, can strongly activate the Avs8 system, increasing the host bacteria's defense against phages; the Aqs1 protein encoded by Pseudomonas aeruginosa phage DMS3 can specifically inhibit the bacterial quorum sensing system, reducing host virulence and biofilm formation; furthermore, antimicrobial peptides derived from phage display technology, such as N10 and NB2, can synergistically disrupt Acinetobacter baumannii biofilms. These findings indicate that phage proteins not only participate in the viral life cycle during phage-host interactions but also function as important regulators of host biological functions. Summary of the Invention

[0005] The purpose of this invention is to provide the application of phage protein Gp17 in the preparation of formulations that inhibit Vibrio cholerae colonization and toxin secretion.

[0006] This invention uses the VP1 phage genome as the research object. Through sequence alignment and protein structure prediction analysis, phage proteins are screened to preliminarily identify candidate proteins with potential regulatory functions. This invention also constructs prokaryotic expression plasmids for the candidate phage proteins and introduces them into Vibrio cholerae to obtain the corresponding expression strain N-Gp37. The effects of phage proteins on the colonization ability and virulence-related phenotypes of Vibrio cholerae are preliminarily evaluated. Further research is conducted using cold light value detection and real-time quantitative PCR (RT-qPCR) analysis to investigate the regulatory role of phage proteins on the transcriptional levels of genes related to virulence and colonization in Vibrio cholerae. Simultaneously, a suckling mouse infection model is used to detect and compare the colonization ability and pathological characteristics of the expressed strain in vivo, thereby evaluating the effect of phage proteins on the colonization ability of Vibrio cholerae in suckling mice.

[0007] To achieve the objectives of this invention, in a first aspect, this invention provides a phage protein Gp17, 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 the sequence shown in SEQ ID NO:1 substituted, deleted or added with one or more amino acids and having the same function.

[0008] In a second aspect, the present invention provides a nucleic acid molecule encoding the protein Gp17.

[0009] The nucleotide sequence of the gene encoding the phage protein Gp17 of this invention is as follows: i) The nucleotide sequence shown in SEQ ID NO:2; ii) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:2 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the solution; iv) Nucleotide sequences that have more than 90% homology with nucleotide sequences i), ii), or iii) and express the same functional protein; or, v) A nucleotide sequence that is completely complementary to the nucleotide sequences of i), ii), iii) or iv).

[0010] Thirdly, the present invention provides biological materials containing the nucleic acid molecules, including but not limited to expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0011] Furthermore, the present invention provides a recombinant expression vector comprising the nucleic acid molecule and operatively linked to an expression regulatory sequence.

[0012] Furthermore, the present invention provides a recombinant host cell comprising the recombinant expression vector or integrating the nucleic acid molecule into its genome.

[0013] Fourthly, the present invention provides the protein Gp17 in the preparation of a preparation of a vaccine that inhibits Vibrio cholerae ( ). Vibrio cholerae Application in formulations of ).

[0014] Furthermore, the formulation is used to inhibit the colonization of Vibrio cholerae in the host gut and / or the secretion of cholera toxin (CT).

[0015] Furthermore, the inhibition is achieved by downregulating the transcriptional levels of virulence-related genes and / or colonization-related genes in Vibrio cholerae; The gene is selected from ctxA , tcpA , tcpP , toxR and toxT At least one of them, whose reference sequence numbers in GenBank are DQ774432.1, DQ773719.1, DQ773718.1, DQ774024.1, and DQ773728.1, respectively.

[0016] Furthermore, the formulation is also used to reduce the environmental tolerance of Vibrio cholerae to bile salts, pH, or salt concentration.

[0017] Fifthly, the present invention provides a pharmaceutical composition comprising at least one of the protein Gp17, a nucleic acid molecule encoding the protein Gp17, or a biological material containing the nucleic acid molecule, and a pharmaceutically acceptable carrier.

[0018] In a sixth aspect, the present invention provides the use of the protein Gp17 as an inhibitor of Vibrio cholerae virulence-related genes and / or colonization-related genes; The gene is selected from ctxA , tcpA , tcpP , toxR and toxT At least one of them, whose reference sequence numbers in GenBank are DQ774432.1, DQ773719.1, DQ773718.1, DQ774024.1, and DQ773728.1, respectively.

[0019] In a seventh aspect, the present invention provides a method for identifying the inhibition of Vibrio cholerae colonization and / or toxin secretion by the phage protein Gp17, wherein the gene encoding the phage protein Gp17 is introduced into Vibrio cholerae via a plasmid to obtain recombinant Vibrio cholerae expressing the phage protein Gp17, and then in vivo and in vitro experiments are performed to detect the virulence and colonization phenotype of the recombinant Vibrio cholerae; the method is not for disease diagnosis and treatment purposes.

[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) The newly identified phage protein Gp17 in this invention is characterized as a phage structure-related protein. Experiments show that this protein not only participates in phage structure and function but also significantly inhibits the expression of Vibrio cholerae virulence genes and reduces the colonization ability of the strain in the intestines of suckling mice. This suggests that phage structural proteins may have a new functional mode in regulating host pathogenicity, providing a new technical approach for targeting the regulation of Vibrio cholerae virulence. This invention screens novel proteins from a Vibrio cholerae-specific phage gene library, providing a new tool for elucidating the complex regulatory network that affects host virulence and colonization, and enabling the discovery of key interventional targets. The proteins regulating colonization and virulence screened in this invention can serve as a focus for applied research, providing a new potential strategy for controlling the spread of Vibrio cholerae and its infection treatment.

[0021] (II) The phage protein Gp17 provided by this invention is a novel protein. Sequence conservation comparison analysis and spatial structure prediction show that the protein is a phage structural protein with a conserved structure. Therefore, studying the biological function and functional conservation of this protein in the process of interaction with the host is of great significance for cross-species prevention and treatment.

[0022] (iii) Using a phage gene library as a screening target for antiviral proteins can greatly increase the likelihood of obtaining positive results. Phages have the characteristic of specific targeting, and because they have bactericidal and bacteriostatic effects, there are many potential candidate unknown proteins.

[0023] (iv) During the interaction between bacteriophage and host, bacteriophage can utilize host resources to achieve rapid and efficient gene transcription and expression of its own proteins. These proteins are not only easily expressed in host cells, but can also be efficiently prepared in prokaryotic expression systems such as Escherichia coli using plasmid systems, thus facilitating phenotypic analysis and functional identification. Attached Figure Description

[0024] Figure 1 This is the amino acid sequence of the phage protein Gp17 of this invention.

[0025] Figure 2 This invention provides a preferred embodiment of Gp17 protein structure prediction and sequence alignment. Based on the Gp17 protein sequence, the spatial structure of Gp17 is predicted using the Alphafold3 online software according to the conservation of spatial structure, and the protein structure model is visualized using PyMOL software.

[0026] Figure 3 The image shows the pSRKtc-gp17 plasmid in a preferred embodiment of the present invention.

[0027] Figure 4 The N-Gp17 strain is a preferred embodiment of the present invention. ctxA (A) andtcpA( B) Gene transcription level analysis.

[0028] Figure 5 In a preferred embodiment of the present invention, this describes the effect of Gp17 on other regulatory proteins in the virulence regulation pathway. A: Gp17 protein inhibits the virulence pathway of Vibrio cholerae. tcpP Gene transcription; B: Gp17 protein inhibits the virulence pathway of Vibrio cholerae. toxR Gene transcription.

[0029] Figure 6 In a preferred embodiment of the present invention, RT-qPCR analysis of Gp17 for... toxR (left) and toxT( The transcriptional level of the right side is affected.

[0030] Figure 7 Analysis of the competitive colonization ability and pathological characteristics of suckling mice in a preferred embodiment of the present invention—Gp17 inhibits the colonization of Vibrio cholerae in suckling mice and alleviates the pathological characteristics of suckling mice. A: Competition index; B: Weight loss; C: Change in small intestinal fluid volume; D: Change in diarrhea volume.

[0031] Figure 8 This is a graph showing the results of ELISA determination of CT toxin excretion in a preferred embodiment of the present invention. A: Linear regression curve of standard; B: Difference analysis of CT secretion.

[0032] Figure 9 The figures show the experimental results of environmental adaptability phenotypes in a preferred embodiment of the present invention. A: Growth curves under different pH conditions; B: Growth curves under different salt concentration conditions; C: Growth curves under different bile salt concentrations.

[0033] Figure 10 This is a flowchart illustrating the technical route of the present invention.

[0034] In the picture, express P <0.01, express P <0.001. Detailed Implementation

[0035] With the increasing severity of antibiotic resistance, the effectiveness of traditional antibacterial methods in controlling bacterial infections is limited. Bacteriophages and their encoded proteins, due to their strong host specificity and unique mechanisms of action, show potential in replacing or supplementing existing antibacterial strategies. The pathogenicity of Vibrio cholerae mainly depends on its colonization ability and toxin secretion regulation within the host. However, current interventions targeting Vibrio cholerae intestinal colonization and toxin secretion regulation primarily focus on antibiotics or immunization strategies, while molecular-level regulation of the virulence regulation pathways themselves remains lacking. Therefore, this invention uses a phage gene library specifically infected with Vibrio cholerae as the research object to screen and identify a novel phage protein, Gp17, that can inhibit Vibrio cholerae intestinal colonization and toxin secretion. This function is discovered and studied for the first time.

[0036] The present invention adopts the following technical solution: This invention provides a novel bacteriophage protein—Gp17—that inhibits the secretion and colonization of Vibrio cholerae toxin, and reveals its function for the first time. The steps include the following: (1) Based on the phage whole genome annotation results, potential functional proteins were systematically screened to identify the phage target protein Gp17.

[0037] (2) The prokaryotic expression plasmid pSRKtc-gp17 was constructed and transformed into the donor strain SM10 λpir. Subsequently, it was introduced into Vibrio cholerae strain N16961 via conjugation transfer. The strain was screened on a solid medium containing tetracycline (2 μg / mL) and streptomycin (100 μg / mL) to obtain a recombinant strain that stably expresses Gp17, which was named N-Gp17 strain.

[0038] (3) Through design ctxA, tcpA, toxR and tcpP Upstream start-up sub-region ， With pBBR1MCS4- luxCDABE Homologous recombination primers for plasmids were used to amplify virulence-related genes of Vibrio cholerae using PCR. ctxA, tcpA, toxR and tcpP The promoter region sequence was obtained. After purification, the amplified product was inserted into the cold light reporter plasmid pBBR1MCS4- via seamless cloning. luxCDABE (abbreviated as pBBR-) lux Upstream of the cold light expression gene (purchased from Hangzhou Baosai Biotechnology Co., Ltd.), recombinant reporter plasmids were constructed, and the correctly sequenced recombinant plasmid pBBR- ctxA - lux pBBR- tcpA-lux pBBR- tcpP- lux pBBR- toxR-luxTransformed into conjugated Escherichia coli SM10 λpir to obtain SM10 λpir (pBBR- ctxA - lux ), SM10 λpir (pBBR- tcpA-lux ), SM10 λpir (pBBR- tcpP-lux ), SM10 λpir (pBBR- toxR- lux The strains were conjugated and transferred into the N-Gp17 strain, and single clones were obtained by screening on solid medium containing streptomycin (100 μg / mL), tetracycline (1 μg / mL) and chloramphenicol (1 μg / mL).

[0039] (4) By detecting the cold light value of the recombinant strain, the transcriptional level of the virulence regulation pathway and colonization-related genes of Vibrio cholerae was analyzed. The results showed that Gp17 could significantly inhibit the transcription of key genes in the virulence and colonization-related regulatory pathway.

[0040] (5) Total RNA was further extracted from N-Gp17 strain, and RT-qPCR was used to analyze genes related to virulence and colonization pathways. ctxA, tcpA, tcpP and toxR The transcriptional level of Gp17 was verified, and the results were consistent with the reporter gene experiment, confirming that Gp17 has a significant inhibitory effect on the expression of related genes.

[0041] (6) In vivo experiments were conducted using a suckling mouse colonization model. The results showed that compared with the wild-type strain, the N-Gp17 strain had a significantly reduced colonization ability in the intestines of suckling mice, and the pathological characteristics of suckling mice were also significantly alleviated.

[0042] (7) The amount of cholera toxin secreted was detected by ELISA kit. The results showed that Gp17 could inhibit the secretion of cholera toxin compared with the control bacteria.

[0043] (8) Phenotypic results show that Gp17 can reduce the environmental adaptability of Vibrio cholerae: decreased bile salt tolerance, decreased salt resistance, and decreased tolerance to acid and alkali.

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the 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.

[0045] The Vibrio cholerae N16961 used in the following examples was kindly provided by Professor John J Mekalanos of the Department of Microbiology and Molecular Genetics at Harvard Medical School (see Proc Natl Acad Sci US A. 2002 Feb 5;99(3):1556-61).

[0046] Phage VP1 was preserved in the Diarrhea Disease Control Laboratory of the Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention (see J Virol. 2021 Feb 24;95(6):e02245-20).

[0047] The pSRKtc plasmid was kindly provided by Professor Jun Zhu of the Department of Microbiology at the University of Pennsylvania (see InfectImmun. 2014 Apr;82(4):1676-82).

[0048] Escherichia coli SM10 λpir was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0049] pBBR1MCS4- luxCDABE The plasmid was purchased from Hangzhou Baosai Biotechnology Co., Ltd.

[0050] Example 1: Discovery and identification of the novel bacteriophage protein Gp17 that inhibits Vibrio cholerae toxin secretion and colonization. 1. Identification of the target protein Based on the VP1 phage genome annotation and sequence alignment and spatial structure prediction of proteins with unknown functions, the target protein phage protein Gp17 was obtained. This protein has a high C-terminus similarity (43.4%) to the tail spike protein Gp9 of Salmonella Typhi P22 phage.

[0051] Predicted protein sequence, gene sequence, and spatial structure of phage Gp17 can be found in [link to phage Gp17 protein sequence]. Figure 1 , Figure 2 .

[0052] 2. Construction of prokaryotic expression vectors Homologous recombination primers (F: GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCAatgagttatgccgcccgc and R: GATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCAttaaataggctggtagacaactgtaccg, SEQ ID NO: 3-4) were designed using the pSRKtc vector and the Gp17 sequence template. The gp17 gene fragment was amplified by PCR using the phage VP1 genome as a template. Simultaneously, the extracted pSRKtc plasmid was double-digested with enzymes to prepare a linearized vector. Subsequently, the pSRKtc linear vector was ligated to the gp17 fragment using a seamless cloning method, transformed into E. coli Trans1-T1, and plated on LB solid plates containing X-Gal (40 ug / ml), IPTG (0.5 mM), and tetracycline (10 ug / ml) for blue-white screening. After incubation at 37°C overnight, white colonies on the plates were picked, plasmids were extracted, and sequenced to obtain successfully recombined plasmids.

[0053] The pSRKtc-gp17 plasmid image can be found in [link / image]. Figure 3 .

[0054] 3. Construction of expression strains The correctly sequenced recombinant plasmid pSRKtc-gp17 was chemically transformed into Escherichia coli SM10 λpir and plated on LB agar plates containing tetracycline (10 ug / ml). After colonies grew, single colonies were picked for PCR verification to ensure that the PCR amplification product size was consistent with gp17. Subsequently, the donor bacterium SM10 λpir-pSRKtc-gp17 was conjugated with the recipient bacterium Vibrio cholerae N16961 and transferred. The conjugate was plated on agar plates containing streptomycin (100 ug / ml) and tetracycline (1 ug / ml), and a single colony was picked and named N-Gp17.

[0055] 4. Virulence and colonization gene transcription level analysis The existing SM10 λpir (pBBR- ctxA - lux ), SM10 λpir (pBBR- tcpA -lux), SM10 λpir (pBBR) -tcpP - lux ), SM10 λpir (pBBR- toxR - luxThe strain was activated on solid medium containing chloramphenicol (15 μg / ml), and then conjugated with control strain N16961-pSRKtc (N16961 carrying the empty pSRKtc vector) and N-Gp17, respectively. Conjugates containing the target fragment were selected using streptomycin (100 μg / ml), tetracycline (1 μg / ml), and chloramphenicol (1 μg / ml) resistant solid plates to obtain fluorescent reporter gene strains for subsequent cryoluminescence detection. The cryoluminescence signal and OD of each strain during growth were measured using a multi-functional microplate reader. 600 Value, and calculate the unit fluorescence value (cold light value / OD). 600 This was used to evaluate the virulence of Gp17 expression against Vibrio cholerae and colonization-related genes. ctxA, tcpA, tcpP and toxR The effect of transcriptional levels.

[0056] N-Gp17 strain ctxA, tcpA, tcpP, toxR See gene transcription level analysis Figure 4 , Figure 5 .

[0057] The experimental results above show that expression of the bacteriophage protein Gp17 in Vibrio cholerae can significantly inhibit the virulence of Vibrio cholerae and colonization genes. ctxA, tcpA, tcpP, toxR Transcription.

[0058] 5. RT-qPCR experiment Control bacteria (N16961-pSRKtc) and N-Gp17 were cultured overnight, and the next day, they were transferred to liquid medium containing IPTG (final concentration 0.5 mM) at a 1:100 volume ratio. The cultures were incubated at 37°C with shaking until the logarithmic growth phase. One ml of each culture was collected, and cells were lysed using TRIzol reagent. After thorough mixing with chloroform, liquid chromatography was performed. The supernatant was collected, precipitated with isopropanol, and washed with 75% ethanol to obtain total RNA. The RNA concentration was determined, and residual genomic DNA was removed by DNase I digestion. An equal amount of RNA was used for reverse transcription to synthesize cDNA. Using the obtained cDNA as a template, SYBR Green real-time quantitative PCR was used to detect virulence regulatory genes. toxR and toxT The expression level of Vibrio cholerae housekeeping genes was selected. recA As an internal control, three technical replicates were performed for each sample. The CT values ​​were compared using a 2-1 method. -ΔΔCT Methods: The fold change in expression of the target gene in N-Gp17 strains relative to the control strain was calculated to assess the impact of Gp17 expression on the ToxR-ToxT virulence regulation pathway.

[0059] RT-qPCR results analysis of Gp17 for toxR and toxTThe effect of transcriptional levels is seen in Figure 6 .

[0060] 6. Analysis of colonization and pathological characteristics in suckling mice Ten 3-5 day old CD-1 suckling mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used. The control strain (N-pSRKtc) and the experimental strain (N-Gp17) were streaked separately onto LB agar plates and incubated overnight at 37°C. Single clones of both the control and experimental strains were then picked and cultured in liquid LB agar until the McFarland turbidity reached 2-3 (at which point the bacterial concentration was approximately 10⁻⁶). 8 The two strains were then mixed at a 1:1 volume ratio and serially diluted 10-fold on plates containing tetracycline (1 ug / ml), X-Gal (40 ug / ml), and IPTG (1 mM). The blue-white ratio (input:blue) was recorded. Each suckling mouse was inoculated with 50 μl of the mixed bacterial solution and placed at 28°C. Approximately 18 hours later, the suckling mice were euthanized by cervical dislocation, and their small intestines were isolated and placed in EP tubes containing 1 ml PBS. The small intestines were homogenized to fully release the colonized bacteria. The small intestine homogenate was then serially diluted and spread on plates containing tetracycline (1 ug / ml), X-Gal (40 ug / ml), and IPTG (1 mM), and incubated overnight at 37°C. The blue-white ratio was recorded as the output (white:blue) ratio. The output / input ratio was used as the CI (competition index) to determine colonization ability.

[0061] Ten CD-1 suckling mice aged 3-5 days (5 mice in the experimental group and 5 mice in the control group) were selected. Each group of mice was numbered and placed in a feeding box with a known weight of bedding (Bedding_Weight 1). The control group (N-pSRKtc) and experimental group (N-Gp17) bacterial strains were cultured overnight. The next day, they were transferred at a 1:100 volume ratio to 3 ml of fresh LB medium (streptomycin Sm 100ug / ml, tetracycline Tc 1ug / ml) and cultured until McFarland turbidity reached 2-3 (logarithmic growth phase). Suckling mice were separated from their mothers 2 hours before inoculation. Each suckling mouse was inoculated with 50 μl of bacterial suspension. Immediately after inoculation, each suckling mouse was weighed (Weight 1) and numbered. Eighteen hours later, the suckling mice were weighed a second time (Weight 1). 2) Then, the suckling mice were euthanized by dislocating their cervical vertebrae, and their complete small intestines were dissected and weighed (SI_weight). The bedding in each feeding box (now containing all the feces and diarrhea fluid excreted by the suckling mice within 18 hours) was weighed again (Bedding_Weight2).

[0062] Diarrhea amount=Bedding_Weight 2-Bedding_Weight 1 Small intestinal fluid accumulation = SI_weight / (Weight 2 - SI_weight) Weight change = Weight 2 - Weight 1 Analysis of competitive colonization ability and pathological characteristics in newborn mice can be found in [the following text is incomplete and requires further context]. Figure 7 .

[0063] 7. CT scan toxin secretion level detection The control strain (N-pSRKtc) and the experimental strain (N-Gp17) were cultured overnight, respectively. The next day, they were transferred to liquid medium (0.5 mM) containing IPTG at a 1:100 volume ratio and cultured at 37°C. The OD of the bacterial culture was measured at 3 and 5 hours. 600 Centrifuge, collect the supernatant, and filter for later use. Use a bacterial cholera toxin (CT) ELISA kit (Shanghai Yansheng Industrial Co., Ltd.). Equilibrate the strips at room temperature for 20 min and set up standard wells and sample wells. The sample wells contain control bacterial samples and experimental bacterial samples. Add 50 μL of different concentrations of standard to each standard well. Add 10 μL of the test sample to each sample well, followed by 40 μL of sample diluent. Do not add any to the blank wells. Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well. Do not add any to the blank wells. Seal the strips with sealing film and incubate at 37°C for 60 min. Discard the liquid and blot dry on absorbent paper. Fill each well with washing buffer, let stand for 1 min, shake off the washing buffer, and pat dry on absorbent paper. Repeat this washing process 5 times. Subsequently, 50 μL each of substrates A and B from the kit were added to each well, and the OD value of each well was measured at 450 nm within 15 min. A linear regression curve of the standard was plotted based on the standard wells, and the accuracy of the results was determined according to the R value. The results of each sample well were calculated based on the OD value. 450 / OD 600 Sure.

[0064] The results of ELISA measurement of CT toxin excretion are shown in the figure. Figure 8 .

[0065] The experimental results above show that the phage protein Gp17 significantly inhibits the colonization of cholerae arcta in suckling mice.

[0066] 8. Environmental adaptability phenotyping experiment The overnight culture of the experimental strain (N-Gp17) was inoculated into 3 mL of fresh LB medium at a volume ratio of 1:100 and cultured with shaking at 37°C and 200 rpm until OD. 600The bacterial culture was diluted 1:1000 by volume and inoculated into 96-well plates containing different concentrations of bile salts (0.1%, 0.3%, 0.5%), pH (6, 7, 8), and salts (0.5%, 2.5%, 5%). The culture was incubated at 37°C and 200 rpm with shaking using an automated growth curve analyzer, and the OD value was measured every hour. 600 The value was continuously monitored for 24-36 hours. Growth curves were plotted based on the obtained data to assess the effect of Gp17 protein expression on the tolerance of Vibrio cholerae to different bile salts, pH, and sodium chloride (NaCl). All experiments were performed in triplicate. Results of environmental adaptation phenotype experiments are shown below. Figure 9 .

[0067] The above technical route and process can be found in [link / document]. Figure 10 .

[0068] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Phage protein Gp17, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the protein Gp17 of claim 1.

3. A biomaterial containing the nucleic acid molecule of claim 2, characterized in that, The biological materials are expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

4. The protein Gp17 described in claim 1 is used in the preparation of a preparation that inhibits Vibrio cholerae (…). Vibrio cholerae Application in formulations of ).

5. The application according to claim 4, characterized in that, The formulation is used to inhibit the colonization of Vibrio cholerae in the host's intestine and / or the secretion of cholera toxin.

6. The application according to claim 4 or 5, characterized in that, The inhibition is achieved by downregulating the transcriptional levels of virulence-related genes and / or colonization-related genes in Vibrio cholerae. The gene is selected from ctxA , tcpA , TCP / IP , toxR and toxT At least one of them, whose reference sequence numbers in GenBank are DQ774432.1, DQ773719.1, DQ773718.1, DQ774024.1, and DQ773728.1, respectively.

7. The application according to claim 4 or 5, characterized in that, The formulation is also used to reduce the environmental tolerance of Vibrio cholerae to bile salts, pH, or salt concentration.

8. A pharmaceutical composition, characterized in that, It comprises at least one of the protein of claim 1, the nucleic acid molecule of claim 2, or the biological material of claim 3, and a pharmaceutically acceptable carrier.

9. The use of the protein Gp17 of claim 1 as an inhibitor of Vibrio cholerae virulence-related genes and / or colonization-related genes; The gene is selected from ctxA , tcpA , TCP / IP , toxR and toxT At least one of them, whose reference sequence numbers in GenBank are DQ774432.1, DQ773719.1, DQ773718.1, DQ774024.1, and DQ773728.1, respectively.

10. A method for identifying the inhibition of Vibrio cholerae colonization and / or toxin secretion by phage protein Gp17, characterized in that, The gene encoding the phage protein Gp17 was introduced into Vibrio cholerae via a plasmid to obtain recombinant Vibrio cholerae expressing the phage protein Gp17. Then, in vivo and in vitro experiments were conducted to detect the virulence, colonization phenotype, and environmental adaptability phenotype of the recombinant Vibrio cholerae. The phage protein Gp17 is the same as that described in claim 1; The method described is not for disease diagnosis and treatment purposes.

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