A polypeptide for blocking PTS pathway and its application

Abstract

The present invention provides a polypeptide that blocks the PTS pathway and its use. In vitro binding experiments demonstrate that phage-displayed peptide 1C6 binds to HPr in a dose-dependent manner. 1C6 blocks the PTS pathway, specifically the EI-HPr interaction and phosphotransferase. This suggests that peptide 1C6 has potential antibacterial properties.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a polypeptide for blocking a PTS pathway and an application thereof. Background Art

[0002] The phosphoenolpyruvate:carbohydratephosphotransferase system (PTS), also known as the phosphotransferase system, is a carbohydrate transport and phosphorylation system present in all bacteria and archaea but not yet found in plants or animals. This PTS, which is found only in prokaryotes, is a potential antimicrobial target. The PTS typically consists of three proteins or protein complexes: enzyme I (EI) and the histidine-containing heat-stable protein HPr, which are located in the cytoplasm, and protease II (EII), which is membrane-bound. These proteins are phosphorylated at histidine or cysteine ​​residues. During various sugar utilization processes, the phosphorylation state of carbohydrates is altered by PTS components and transported to the cytoplasm. In pathogenic bacteria, the PTS links carbohydrate metabolism with functions such as chemotaxis and virulence expression.

[0003] Enzyme I includes EI and homologous component EI Ntr , are located intracellularly. EI participates in almost all PTS-sugar transport and has no PTS-sugar specificity. As the first component of the phosphate transfer chain, it directly accepts the phosphate group from PEP and transfers the phosphate group to the next component HPr. Ntr It is a nitrogen source - a key component of PTS.

[0004] HPr, sometimes also referred to as PtsH, is a small intracellular phosphate carrier protein. Like EI, HPr lacks PTS-sugar specificity and is involved in the transport of most PTS-sugars. The histidine-15 position of HPr is phosphorylated upon receiving a phosphate group from EI, transferring the phosphate group to the downstream specific PTS-sugar EIIAB. Summary of the Invention

[0005] To overcome the deficiencies of the prior art, the present invention provides a polypeptide for blocking the PTS pathway and its application.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A first aspect of the present invention provides a polypeptide, wherein the amino acid sequence of the polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0008] Furthermore, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

[0009] The second aspect of the present invention provides a polynucleotide encoding the polypeptide according to the first aspect of the present invention.

[0010] The third aspect of the present invention provides a vector comprising the polynucleotide according to the second aspect of the present invention.

[0011] Furthermore, the vector further comprises a transcription promoter and / or enhancer.

[0012] Furthermore, the vector also includes an operably linked nucleic acid molecule.

[0013] Furthermore, the operably linked nucleic acid molecule includes a tag.

[0014] Furthermore, the tags include epitope tags for positioning and tags for purification.

[0015] The fourth aspect of the present invention provides a host cell, which comprises the vector described in the third aspect of the present invention.

[0016] Furthermore, the host cells include prokaryotic cells and eukaryotic cells.

[0017] Furthermore, the prokaryotic cell includes Escherichia coli.

[0018] Furthermore, the eukaryotic cells include protist cells, animal cells or fungal cells.

[0019] Furthermore, the animal cells include mammalian cells, bird cells, and insect cells.

[0020] The fifth aspect of the present invention provides a polypeptide derivative, which includes a modified product of the polypeptide described in the first aspect of the present invention or a detection agent linked to the polypeptide.

[0021] Furthermore, the modification includes amino modification, methylation modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, phosphorylation modification, sulfation modification, esterification modification, glycosylation modification, and cyclization modification.

[0022] Furthermore, the detection reagent includes fluorescent dyes, radioactive labels, metal ions, and enzymes.

[0023] The sixth aspect of the present invention provides a pharmaceutical composition, which comprises the polypeptide described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the polypeptide derivative described in the fifth aspect of the present invention.

[0024] Furthermore, the pharmaceutical composition also includes pharmaceutical excipients.

[0025] Furthermore, the pharmaceutical excipients include one or more of diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants.

[0026] Furthermore, the dosage form of the pharmaceutical composition includes granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions or solutions.

[0027] The seventh aspect of the present invention provides the use of the polypeptide described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the polypeptide derivative described in the fifth aspect of the present invention in inhibiting bacteria or in preparing drugs for inhibiting bacteria.

[0028] The eighth aspect of the present invention provides a method for inhibiting or killing bacteria, which comprises administering the polypeptide described in the first aspect of the present invention, the polypeptide derivative described in the fifth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention.

[0029] Furthermore, the bacteria are inhibited or killed by blocking the PTS pathway.

[0030] The ninth aspect of the present invention provides a method for preparing the polypeptide described in the first aspect of the present invention, wherein the method comprises culturing the host cell described in the fourth aspect of the present invention.

[0031] Furthermore, the method further comprises purifying the polypeptide.

[0032] Advantages and beneficial effects of the present invention:

[0033] This application demonstrates, through in vitro binding experiments, that phage-displayed peptide 1C6 binds to HPr in a dose-dependent manner. 1C6 can block the PTS pathway, namely, EI-HPr interaction and phosphotransferase (interfering with EI phosphorylation of HPr), suggesting that peptide 1C6 has potential antibacterial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the binding activity diagram of peptide 1C6 and HPr;

[0035] Figure 2 This is a diagram showing that peptide 1C6 blocks the phosphorylation of HPr protein by EI. DETAILED DESCRIPTION

[0036] The following provides definitions of some terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0037] The present invention provides a polypeptide for blocking the PTS pathway, wherein the amino acid sequence of the polypeptide has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0038] In one embodiment, identity / similarity refers to the identity / similarity between two or more nucleic acid sequences, or two or more amino acid sequences, and is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences. When aligned using standard methods, homologs or orthologs of nucleic acid or amino acid sequences have relatively high levels of sequence identity / similarity. The primers disclosed herein are not limited to the exact sequences shown, and as will be appreciated by those skilled in the art, the sequences may be altered, if desired, without significantly affecting the ability of the primers to function.

[0039] The present invention provides a polynucleotide encoding the above polypeptide.

[0040] In one embodiment, polynucleotide (polynucleotide) or nucleic acid molecule (Nucleic acidmolecule) or polynucleic acid (polynucleic acid) refer to the polymeric compound including covalently linked nucleotides, which can be composed of natural subunits (for example, purine or pyrimidine bases) or non-natural subunits (for example, morpholine ring). Purine bases include adenine, guanine, hypoxanthine and xanthine, and pyrimidine bases include uracil, thymine and cytosine. Polynucleotides include polyribonucleic acid (RNA), which includes mRNA, microRNA, siRNA, viral genome RNA and synthetic RNA, and polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA and synthetic DNA, any one of which can be single-stranded or double-stranded. If single-stranded, polynucleotides can be coding strands or non-coding (antisense) strands. The polynucleotides encoding amino acid sequences include all nucleotide sequences encoding identical amino acid sequences. Some versions of nucleotide sequences can also include introns, and their degree makes introns be removed by co-transcription or post-transcription mechanism. In other words, due to redundancy or degeneracy of the genetic code, or by splicing, different nucleotide sequences can encode the same amino acid sequence.

[0041] In one embodiment, the polynucleotide / nucleic acid molecule can be synthesized, for example, by standard chemical synthesis methods and / or recombinant methods, or produced semi-synthetically, for example, by combining chemical synthesis and recombinant methods. The coding sequence can be linked to transcriptional regulatory elements and / or to other amino acid coding sequences using established methods, such as restriction enzyme digestion, ligation, and molecular cloning.

[0042] In one embodiment, the application also relates to variants of the above-mentioned polynucleotides. The variant of the polynucleotide can be a naturally occurring variant, such as a naturally occurring allelic variant, or it can be a non-naturally occurring variant. By mutagenesis techniques, including those mutagenesis techniques that are applied to polynucleotides, cells or organisms, these non-naturally occurring variants of the polynucleotides can be prepared.

[0043] In this regard, variants are those that differ from the polynucleotides described above by nucleotide substitutions, deletions, or additions. The substitutions, deletions, or additions may involve one or more nucleotides. The variants may be altered in the coding region or in the non-coding region, or both. Alterations in the coding region may result in conservative or non-conservative amino acid substitutions, deletions, or additions.

[0044] In one embodiment, the deletion refers to the deletion of one or more amino acids or nucleotides in the amino acid sequence or nucleotide sequence.

[0045] Insertions or additions refer to changes in the amino acid sequence or nucleotide sequence that result in the addition of one or more amino acids or nucleotides compared to the naturally occurring molecule. Substitutions refer to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides.

[0046] The present invention provides a vector comprising the above-mentioned polynucleotide.

[0047] In one embodiment, the vector also includes a transcriptional promoter and an optionally present enhancer, a translation signal, and a transcriptional and translational termination signal. Expression vectors for stable transformation typically have a selectable marker that allows selection and maintenance of transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cell. The vector can also include additional nucleotide sequences that are operably linked to the connected nucleic acid molecule, such as an epitope tag for localization, such as a 6-his tag or a myc tag, or a tag for purification, such as a GST fusion; and sequences for directing protein secretion and / or membrane association.

[0048] The present application is not particularly limited to vectors, and may be vectors capable of replicating and / or expressing polynucleotides in eukaryotic or prokaryotic cells, including mammalian cells (e.g., human, monkey, rabbit, rat, hamster, or mouse cells), yeast cells, insect cells, and bacterial cells (e.g., Escherichia coli). Preferably, the vector includes at least one selectable marker operably linked to a suitable promoter so that the polynucleotide can be expressed in the host cell. For example, the vector may include a polynucleotide introduced into a phage, plasmid, cosmid, minichromosome, virus, or retroviral vector, or other vectors conventionally used, for example, in genetic engineering.

[0049] As an optional mode of the present application, the vector is a virus. The viral vector is used to introduce a non-endogenous nucleic acid sequence encoding a target-specific polypeptide. The viral vector can be a retroviral vector or a lentiviral vector. The viral vector can also include a nucleic acid sequence encoding a transduction marker. Suitable viral vectors include vectors based on RNA viruses, such as vectors derived from retroviruses, such as vectors derived from Moloney murine leukemia virus (MLV), and include more complex vectors derived from retroviruses, such as vectors derived from lentiviruses. HIV-1 derived vectors belong to this class.

[0050] Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses, including adenoviruses, herpes viruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, and cytomegalovirus), and pox viruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, but are not limited to, norwalk viruses, togaviruses, flaviviruses, reoviruses, papillomaviruses, hepatitis viruses, and hepaciviruses. Examples of retroviruses include avian leukosarcoma, mammalian C, B, and D viruses, HTLV-BLV groups, lentiviruses, or foamy viruses.

[0051] As an optional method of the present application, the vector is an expression vector. The expression vector according to the present application can guide the replication and expression of the polynucleotide of the present application in a host.

[0052] Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems)

[0053] pTriEx-Hygro (Novagen) and pCINeo (Promega). Non-limiting examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and a variety of mammalian and avian cells is the multipurpose expression vector pCS2+.

[0054] The present invention provides a host cell, comprising the above-mentioned vector.

[0055] In one embodiment, the host cell is a cell for receiving, maintaining, replicating and amplifying the vector. The host cell can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. In one embodiment, the host cell is a genetic package that can induce expression of variant polypeptides on its surface. In another embodiment, the host cell is infected with the genetic package.

[0056] In one embodiment, host cells are cells used to receive, maintain, replicate, and amplify vectors. These include prokaryotic cells and eukaryotic cells. Eukaryotic cells include, but are not limited to, protist cells and animal cells, including mammalian cells, avian cells, and insect cells. Mammalian cells include, but are not limited to, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293 cells, 293T cells, and 293F cells.

[0057] Methods for transformation into host cells include any method for introducing nucleic acids into organisms, cells, tissues or organs, and can be performed using standard techniques selected according to the type of host cell as known in the art. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, and transformation mediated by PEG, dextran sulfate, lipofectamine, or desiccation / inhibition.

[0058] The present invention provides a polypeptide derivative, which comprises a modified product of the above polypeptide or a detection agent connected to the polypeptide.

[0059] In one embodiment, the detection reagent can be any substance with detectable physical or chemical properties. This type of detectable reagent is well developed in the field of immunoassays, and generally speaking, most of any labeling useful in this type of method can be applied to the provided method. Therefore, the labeling can be any composition detectable by spectroscopy, photochemistry, biochemistry, immunochemistry, electricity, optics or chemical methods. Detectable reagents include but are not limited to fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radioactive labels (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), in particular, radiolabeled (e.g., 157 Gd, 55 Mn, 162 Dy, 52 Cr and 56 Fe), metal ions (e.g. 111 In, 97 Such as 67 Ga, 68 Ga, 72 As、 89 Zr and 201Tl), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), electron transfer agents (e.g., including metal binding proteins and compounds), luminescent and chemiluminescent labels (e.g., luciferin and 2,3-dihydrophthalazines, e.g., luminol), magnetic beads (e.g., DYNABEADS TM ), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex).

[0060] The present invention provides a pharmaceutical composition, which comprises the above-mentioned polypeptide, the above-mentioned polynucleotide, the above-mentioned vector, the above-mentioned host cell or the above-mentioned polypeptide derivative.

[0061] In one embodiment, a pharmaceutical excipient is used to refer to a material that is compatible with a recipient, preferably a mammal, more preferably a human, and suitable for delivering an active agent to a target site without disrupting the activity of the agent. Toxicity or side effects associated with the pharmaceutical excipient, if any, are preferably commensurate with a reasonable risk / benefit ratio for the intended use of the active agent.

[0062] Pharmaceutical excipients include, but are not limited to, diluents, binders, surfactants, humectants, adsorbent carriers, lubricants, fillers, and disintegrants. These pharmaceutical excipients are used, as needed, to enhance the stability of the formulation, improve the activity or bioavailability of the formulation, or produce an acceptable taste or odor when orally administered. The formulations used in such drugs may be in the form of the original compound itself or, optionally, in the form of a pharmaceutically acceptable salt. The formulated drug may be administered by any appropriate method known to those skilled in the art, as needed.

[0063] Among them, diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, and water.

[0064] Binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, and hydroxypropyl methylcellulose.

[0065] Surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, and cetyl alcohol.

[0066] Humectants include, but are not limited to, glycerin.

[0067] Adsorptive supports include, but are not limited to, bentonite, silica gel, kaolin, and bentonite.

[0068] Lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl sulfate.

[0069] Fillers include, but are not limited to, mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugars, coupling sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, and sodium bicarbonate.

[0070] Disintegrants include, but are not limited to, cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, and soybean polysaccharides.

[0071] The present invention provides a method for inhibiting or killing bacteria, which comprises administering the above polypeptide, the above polypeptide derivative or the above pharmaceutical composition.

[0072] In one embodiment, bacteria are inhibited or killed by blocking the PTS pathway, ie, EI-HPr interaction and phosphate transfer (interfering with phosphorylation of HPr by EI).

[0073] The present invention provides a method for preparing the above polypeptide, which comprises culturing the above host cell.

[0074] In one embodiment, cells are cultivated in a nutrient medium suitable for producing the polypeptide using methods well known in the art. For example, the shake flask culture performed under the conditions of expressing and / or separating the polypeptide can be carried out in a suitable medium and by allowing expression and / or separation, and small-scale or large-scale fermentation (comprising continuous, in batches, fed-batch or solid-state fermentation) in a laboratory or industrial fermentor. Cells are cultivated in a suitable nutrient medium using methods known in the art, the nutrient medium comprising a carbon source and a nitrogen source and an inorganic salt. Suitable substratum can be obtained from commercial suppliers or can be prepared according to disclosed composition (for example, in the catalogue of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can directly be recovered from the substratum. If the polypeptide is not secreted into the substratum, it can be recovered from cell lysate (lysate).

[0075] The polypeptides can be detected using methods known in the art that are specific for the polypeptides. These detection methods may include the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, enzyme assays can be used to determine the activity of a polypeptide as described herein.

[0076] The resulting polypeptide can be recovered using methods known in the art. For example, the polypeptide can be recovered from the nutrient medium by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0077] The method further comprises purifying the polypeptide.

[0078] In one embodiment, the polypeptide can be purified to obtain a substantially pure polypeptide by a variety of methods known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic methods (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

[0079] The present invention will be further described below with reference to specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.

[0080] Example

[0081] 1. ELISA detection of the binding activity of phage-displayed peptides to HPr

[0082] HPr protein was diluted to 100 μg / mL in the coating buffer, and 100 μL was added to each well of the ELISA plate. The plate was incubated in a humidified chamber at 4°C overnight. The plate was washed three times with a plate washer and blocked with 5% skim milk-PBST (200 μL per well) at 37°C for 1 hour. The supernatant of the purified 1C6-displaying phage was diluted in the blocking buffer, starting at 1:10 and serially diluted two-fold to 1:640. 100 μL was added to each well of the ELISA plate and incubated at 37°C for 1 hour. The plate was washed three times, and anti-M13-HRP secondary antibody was added and incubated at room temperature for 60 minutes. The plate was washed five times, and color was developed with 100 μL of TMB substrate. The reaction was incubated for 3 minutes and terminated with 100 μL of 2N H2SO4. The plate was read at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0083] The results are as follows Figure 1 As shown, the phage displaying 1C6 prepared in the present application can specifically recognize the HPr protein on the solid phase carrier, and as the dilution of 1C6 phage increases, the binding between 1C6 phage and the target protein has a good concentration dependence. The amino acid sequence of 1C6 is: PCTKQGGGTSHSAILARA (SEQ ID NO: 1).

[0084] 2. Detection of the interference effect of 1C6 on EI phosphorylation of HPr

[0085] Before the assay, EI protein was allowed to return to room temperature to allow for full dimerization. The EI / HPr enzyme reaction was performed in a 100 μL reaction volume in a 5 mM MgCl2, 50 mM HEPES (pH 7.5), 100 mM NaCl buffer containing 160 μM NADH, 5 U LDH, 3 mM PEP, and 80 μM HPr. The reaction solution was preheated to 30°C before the addition of EI (final concentration 300 nM). A blank reaction solution lacked HPr and contained the same volume of DMSO to correct for EI autophosphorylation consumption, NADH autooxidation, and DMSO interference. A control reaction solution contained the same volume of DMSO. The positive control was α-ketoglutarate (final concentration 0.6 mg / mL). For the 1C6 interference assay, 1C6 at a final concentration of 0.6 mg / mL was pre-incubated with EI and HPr for 5 minutes before the addition of PEP. After a 5-minute reaction, the assay samples were inactivated by heating at 95°C for 4 minutes. The sample was diluted to a certain multiple and NAD(P)H-Glo TM The amount of residual NADH in the sample was determined using the Detection System kit (Promega) according to the standard test method, and the interference effect of 1C6 on EI phosphorylation of HPr was determined by the calculation formula: inhibition rate (100%) = (luminescence value of the experimental group - luminescence value of the control group) / (luminescence value of the blank group - luminescence value of the control group) * 100%.

[0086] The results are as follows Figure 2 As shown: 0.6 mg / mL 1C6 can effectively block the phosphorylation of HPr protein by EI, with an inhibition rate of 80±9.12%.

[0087] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. A polynucleotide, characterized in that The polynucleotide encodes the polypeptide of claim 1.

3. A carrier, characterized in that The vector comprises the polynucleotide according to claim 2.

4. The carrier according to claim 3, characterized in that The vector further comprises a transcriptional promoter and / or enhancer.

5. The carrier according to claim 3, characterized in that The vector also includes an operably linked nucleic acid molecule.

6. The carrier according to claim 5, characterized in that The operably linked nucleic acid molecule includes a tag.

7. The carrier according to claim 6, characterized in that The tags include epitope tags for positioning and tags for purification.

8. A host cell, characterized in that The host cell comprises the vector according to any one of claims 3 to 7.

9. The host cell according to claim 8, characterized in that The host cells include prokaryotic cells and eukaryotic cells.

10. The host cell according to claim 9, characterized in that The prokaryotic cells include Escherichia coli.

11. The host cell according to claim 9, characterized in that The eukaryotic cell includes a protist cell, an animal cell or a fungal cell.

12. The host cell according to claim 11, characterized in that The animal cells include mammalian cells, avian cells, and insect cells.

13. A polypeptide derivative, characterized in that: The polypeptide derivative comprises a detection reagent connected to the polypeptide according to claim 1.

14. The polypeptide derivative according to claim 13, characterized in that The detection reagents include fluorescent dyes, radioactive labels, and metal ions.

15. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide according to claim 1, the polynucleotide according to claim 2, the vector according to any one of claims 3 to 7, the host cell according to any one of claims 8 to 12, or the polypeptide derivative according to any one of claims 13 to 14.

16. The pharmaceutical composition according to claim 15, characterized in that The pharmaceutical composition also includes pharmaceutical excipients.

17. The pharmaceutical composition according to claim 16, characterized in that The pharmaceutical excipients include one or more of diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants.

18. The pharmaceutical composition according to claim 16, characterized in that The dosage forms of the pharmaceutical composition include granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, suspensions or solutions.

19. Use of the polypeptide according to claim 1, the polynucleotide according to claim 2, the vector according to any one of claims 3 to 7, the host cell according to any one of claims 8 to 12, or the polypeptide derivative according to any one of claims 13 to 14 in inhibiting bacteria for non-therapeutic purposes.

20. Use of the polypeptide according to claim 1, the polynucleotide according to claim 2, the vector according to any one of claims 3 to 7, the host cell according to any one of claims 8 to 12, or the polypeptide derivative according to any one of claims 13 to 14 in the preparation of a drug for inhibiting bacteria.

21. A method for inhibiting or killing bacteria for non-therapeutic purposes, characterized in that: The method comprises administering the polypeptide of claim 1, the polypeptide derivative of any one of claims 13-14, or the pharmaceutical composition of any one of claims 15-18.

22. The method according to claim 21, characterized in that Inhibit or kill bacteria by blocking the PTS pathway.

23. A method for preparing the polypeptide according to claim 1, characterized in that: The method comprises culturing the host cell according to any one of claims 8 to 12.

24. The method according to claim 23, wherein The method further comprises purifying the polypeptide.

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