CN117024531B - Phage targeting protein molecule and application thereof in identifying shiga toxin-producing escherichia coli of O111 antigen serotype - Google Patents
Phage targeting protein molecule and application thereof in identifying shiga toxin-producing escherichia coli of O111 antigen serotype Download PDFInfo
- Publication number
- CN117024531B CN117024531B CN202311306831.7A CN202311306831A CN117024531B CN 117024531 B CN117024531 B CN 117024531B CN 202311306831 A CN202311306831 A CN 202311306831A CN 117024531 B CN117024531 B CN 117024531B
- Authority
- CN
- China
- Prior art keywords
- escherichia coli
- shiga toxin
- producing escherichia
- protein
- antigen serotype
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 40
- 108010079723 Shiga Toxin Proteins 0.000 title claims abstract description 34
- 239000000427 antigen Substances 0.000 title claims abstract description 34
- 102000036639 antigens Human genes 0.000 title claims abstract description 33
- 108091007433 antigens Proteins 0.000 title claims abstract description 33
- 108090000623 proteins and genes Proteins 0.000 title abstract description 35
- 102000004169 proteins and genes Human genes 0.000 title abstract description 34
- 230000008685 targeting Effects 0.000 title abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 108700010839 phage proteins Proteins 0.000 claims description 27
- 244000005700 microbiome Species 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000003550 marker Substances 0.000 claims description 5
- 238000003501 co-culture Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000000872 buffer Substances 0.000 description 19
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 19
- 239000000243 solution Substances 0.000 description 14
- 239000007788 liquid Substances 0.000 description 13
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 12
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 7
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 241000283707 Capra Species 0.000 description 4
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 235000020183 skimmed milk Nutrition 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 241000588626 Acinetobacter baumannii Species 0.000 description 2
- 208000037157 Azotemia Diseases 0.000 description 2
- 241001470651 Clostridium perfringens ATCC 13124 Species 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 241000194031 Enterococcus faecium Species 0.000 description 2
- 208000032759 Hemolytic-Uremic Syndrome Diseases 0.000 description 2
- 241000588747 Klebsiella pneumoniae Species 0.000 description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- 241000607762 Shigella flexneri Species 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052759 nickel Inorganic materials 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000004952 protein activity Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241001515965 unidentified phage Species 0.000 description 2
- 208000009852 uremia Diseases 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- VQTBINYMFPKLQD-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-(3-hydroxy-6-oxoxanthen-9-yl)benzoate Chemical compound C=12C=CC(=O)C=C2OC2=CC(O)=CC=C2C=1C1=CC=CC=C1C(=O)ON1C(=O)CCC1=O VQTBINYMFPKLQD-UHFFFAOYSA-N 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 241000194033 Enterococcus Species 0.000 description 1
- 241000943303 Enterococcus faecalis ATCC 29212 Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 108010017898 Shiga Toxins Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 244000078673 foodborn pathogen Species 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/245—Escherichia (G)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Immunology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Wood Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a phage targeting protein molecule and application thereof in identifying shiga toxin-producing escherichia coli of O111 antigen serotype, and relates to the field of biology, wherein the amino acid sequence of the phage targeting protein molecule is shown in SEQ ID NO: 1. The phage targeting protein molecule can specifically identify the shiga toxin-producing escherichia coli of the O111 antigen serotype, can be used for identifying the shiga toxin-producing escherichia coli of the O111 antigen serotype, has the advantages of simplicity, rapidness, accuracy and the like, and has high application value.
Description
Technical Field
The present invention relates to the field of biology. In particular, the invention relates to phage targeting protein molecules and their use in the identification of shiga toxin-producing escherichia coli of the O111 antigen serotype.
Background
Shiga toxin-producing escherichia coli (STEC), also known as Vero cytotoxin-producing escherichia coli (VTEC), is a food-borne pathogen that can cause human fatal diseases. Patients may develop diarrhea, hemorrhagic enteritis, and hemolytic uremia (Haemolytic uraemic syndrome, HUS). In China, the serotype STEC O111 has high popularity in human beings and animals, and seriously threatens the life health of the human beings.
At present, no study on the identification of STEC O111 by using phage targeting protein molecules is yet carried out in China.
Disclosure of Invention
The present invention aims to solve, at least to some extent, the technical problems existing in the prior art. Therefore, the invention provides a phage protein which can specifically identify the shiga toxin-producing escherichia coli of the O111 antigen serotype, can be used for identifying and enriching the shiga toxin-producing escherichia coli of the O111 antigen serotype, has the advantages of simplicity, rapidness, accuracy and the like, and has high application value.
In one aspect of the invention, the invention provides a phage protein. According to an embodiment of the invention, the amino acid sequence of the phage protein is as shown in SEQ ID NO: 1. Thus, the invention has the sequence as set forth in SEQ ID NO:1 (also referred to herein as "phage targeting protein molecules") can specifically recognize shiga toxin-producing escherichia coli of O111 antigen serotype, rapidly and accurately identify and enrich shiga toxin-producing escherichia coli of O111 antigen serotype, is helpful for pathogenic escherichia coli research, disease diagnosis and treatment, and has important significance for defending and controlling pathogenic escherichia coli transmission. This phage protein is designated herein as "phage protein ppO111", and the nucleic acid molecule encoding phage protein ppO111 has the sequence set forth in SEQ ID NO:2, and a nucleotide sequence shown in the following formula.
RIVNLANAVDDRDAVPLGQLKTMNQSSWQARNESLQFRNEAEAFKNQAEGFKNESSTNAANTYKWRNEAEGFRNEAEQFKNTATQQATAAGNSAAAAHQSEVNSANSATASAKSATLAEQQADRAESEADKLGNMNDFAGAIDSVDPVSHNVTMKGAMMVSDAIQARNPSGYNFVSGDLDVGRDLAVKGRATRNGNTMVTKEDLKKSEFAWNSSLIFPGESDGLNVFRKNGENPLGLKYNGAIEVRPMAGWDTANNLGRSPMITFHWPGKIGEALFMTEYGELAWGDPNGTWNRLANTHQMSGGNSLRGWRVDPANPSVAEVWGYAEQVPPNTQVNINIIPDTGFPYTPVSGGVILTQVSTSTRAQYQEANTVTWWVQRGSVFAISSGSTVRDNFFWFAKIDFQY（SEQ ID NO：1）。
cgtatcgtgaacctagcgaacgctgttgatgaccgtgacgctgttccgttaggtcaacttaagaccatgaaccagagttcttggcaagctcgtaatgagtccttacagttccgcaatgaagcagaggctttcaagaaccaagcggagggctttaagaacgagtccagcactaacgctgctaatacctataagtggcgtaatgaggctgaggggttccgaaatgaggccgaacagttcaagaacactgctacacaacaggctaccgctgctggtaactctgcggctgctgcccatcaatccgaggtgaactctgcgaactcagctactgcttccgctaaatctgctactttggcagaacagcaagcagaccgtgctgagagtgaggcagataaacttggtaacatgaacgattttgctggagcaatcgactctgtggacccagttagtcacaacgtcaccatgaaaggtgccatgatggttagcgatgcaatccaagcgcgtaacccttctggttataattttgtctccggcgacctagacgttggtagggatcttgcagttaaaggtcgagccacccgaaacggtaacactatggtaaccaaagaagacctgaaaaagtctgagtttgcgtggaactccagtttgatattccccggtgaatcagacggccttaatgttttccgaaagaatggtgagaacccattaggtttaaaatataatggtgctatagaggttagaccaatggctggttgggacacagcgaacaatctaggtcgaagtcctatgataacgttccactggcccggtaagattggtgaagcgctattcatgacggagtacggagaattagcatggggagaccctaatgggacgtggaacagattggctaatacgcatcaaatgtcaggcggcaactcactacgtggttggagagttgaccctgcaaacccttccgtggcagaagtgtggggttatgcggagcaggttccgcctaacactcaggtaaacataaacataattccagatactggtttcccttatacaccggtaagtggaggagtgatcttaacgcaggtgtcaacgtctactcgcgcccagtatcaagaggccaacactgttacgtggtgggtacagcgcggtagtgtatttgccatctcctctggctcaacagtccgagacaacttcttctggtttgctaaaattgatttccaatattaa（SEQ ID NO：2）
In a further aspect of the invention, the use of the phage proteins described above for the identification and/or enrichment of shiga toxin-producing escherichia coli of the O111 antigen serotype for non-diagnostic purposes is presented. Thus, the phage protein of the invention can be used for biological research of shiga toxin-producing escherichia coli with different antigen serotypes.
In yet another aspect of the invention, the invention provides a method of identifying shiga toxin-producing escherichia coli of the O111 antigen serotype for non-diagnostic purposes. According to an embodiment of the invention, the method comprises: co-culturing the phage proteins with a microorganism to be tested; determining whether the microorganism to be tested is shiga toxin-producing escherichia coli of an O111 antigen serotype based on whether the phage protein binds to the microorganism to be tested. As described above, the phage proteins of the invention can specifically recognize shiga toxin-producing escherichia coli of the O111 antigen serotype, which is helpful for identifying shiga toxin-producing escherichia coli of the O111 antigen serotype.
According to the embodiment of the invention, when the phage protein is combined with the microorganism to be detected, determining that the microorganism to be detected is shiga toxin-producing escherichia coli of O111 antigen serotype; the phage protein is not combined with the microorganism to be detected, and the microorganism to be detected is determined not to be shiga toxin-producing escherichia coli of O111 antigen serotype; the phage protein is connected with a marker molecule, and whether the phage protein is combined with the microorganism to be detected is determined by detecting the marker molecule.
According to an embodiment of the invention, the marker molecules comprise at least one of the following: GFP protein, mCherry protein, FITC, TRITC, NHS-fluorescein and NHS-rhodamine.
According to the embodiment of the invention, the pH value of the co-culture reaction is 8-9, and the temperature is 4-37 ℃. Therefore, the phage targeting protein molecule has higher bioactivity, and can be more rapidly and specifically combined with the shiga toxin-producing escherichia coli of the O111 antigen serotype.
In a further aspect of the invention, the invention proposes the use of a bacteriophage protein as described hereinbefore for the preparation of a kit. According to an embodiment of the invention, the kit is used for diagnosing diseases and/or symptoms caused by shiga toxin-producing escherichia coli infected with the O111 antigen serotype.
According to an embodiment of the invention, the disease and/or condition is selected from at least one of the following: diarrhea, colitis and uremia.
According to an embodiment of the invention, the kit contains a nucleic acid molecule encoding the bacteriophage protein described above and/or an expression vector carrying the nucleic acid molecule.
In yet another aspect of the invention, the invention provides a method of enriching a shiga toxin-producing escherichia coli of the O111 antigen serotype. According to an embodiment of the invention, the method comprises: co-culturing a sample to be treated of shiga toxin-producing escherichia coli containing an O111 antigen serotype with the phage protein, wherein a magnetic bead probe is connected to the phage protein; the magnetic bead probe is adsorbed by magnetic force so as to enrich the shiga toxin-producing escherichia coli of the O111 antigen serotype from the sample to be treated. As described above, the phage protein of the invention can specifically recognize the shiga toxin-producing escherichia coli of the O111 antigen serotype, and is helpful for enriching the shiga toxin-producing escherichia coli of the O111 antigen serotype.
According to the embodiment of the invention, the pH value of the co-culture reaction is 8-9, and the temperature is 4-37 ℃. Therefore, the phage targeting protein molecule has higher bioactivity, and can be more rapidly and specifically combined with the shiga toxin-producing escherichia coli of the O111 antigen serotype.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows an electrophoretogram according to one embodiment of the invention;
FIG. 2 shows the OD of ppO protein at various pH values according to one embodiment of the invention;
FIG. 3 shows OD values of ppO protein at different temperature conditions according to one embodiment of the invention;
FIG. 4 shows STEC O111 bacterial OD values at different bacterial concentrations according to one embodiment of the present invention;
FIG. 5 shows STEC O111 bacterial OD values at different concentrations of different ppO111 proteins according to one embodiment of the invention;
FIG. 6 shows a schematic diagram of ppO protein-specific assay according to one embodiment of the invention.
Detailed Description
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the following examples, the main test materials are as follows:
pET-28a-sumo vector: purchased from Invitrogen.
pET-28a-sumo-eGFP vector: preserving the laboratory
BL21 (DE 3) competent cells: purchased from Invitrogen.
The formula of the eluent of the washing liquid comprises: tris 20 mmol/L, naCl mmol/L, imidazole 50-500 mmol, glycerol 5%, tween0.05%.
EXAMPLE 1 construction and expression of recombinant proteins
1. According to SEQ ID NO:2 to obtain the target fragment gene ppO111.
2. Construction of recombinant plasmid pET-28a-sumo-eGFP-pO111
(1) The target fragment and pET-28a-sumo-eGFP vector are respectively combined with restriction enzymeSacI andXhoi, enzyme cutting at 37 ℃ for 1.5 hours;
(2) The products after enzyme digestion are connected at 4 ℃ overnight under the action of T4 ligase, and the connection system conditions are as follows:
(3) After thawing competent cells frozen at-80 ℃ on ice, taking 100 mu L of the cells in a sterile 1.5mL EP tube, adding 10 mu L of a connection product, uniformly mixing, and placing the cells on ice for 30min;
(4) Heat-beating in a water bath at 42 ℃ for 90s, and putting back on ice for 2min;
(5) Adding 400 mu L of LB liquid medium, and carrying out shake culture at 37 ℃ and 200rpm for 35min;
(6) 100 mu L of bacterial liquid is coated on a Kan (50 mg/mL) resistance plate, and the bacterial liquid is cultured at 37 ℃ until single colony appears.
The recombinant plasmid pET-28a-sumo-eGFP-pO111 with correct base sequence was introduced into BL21 (DE 3) competent cells according to the conventional method.
3. Expression of ppO111 protein
pET-28a-sumo-eGFP-pO111 of recombinant BL21 (DE 3) was added in a 1:100 ratio to 1000mL LB broth, and kana antibiotic was added at a final concentration of 50. Mu.g/mL, and when cultured with shaking at 37℃at 180rpm to OD 600=0.6, IPTG was added at a final concentration of 0.6mM, and cultured with shaking at 16℃at 140rpm for 14h.
And collecting thalli, crushing the thalli by using an ultrasonic crusher, and setting ultrasonic waves for 3 seconds to stop for 4 seconds, wherein the total time is 20 minutes. 12000rpm (4 ℃) and centrifuging for 3min, collecting supernatant, and storing in a refrigerator at 4 ℃.
4. purification and identification of ppO111 protein
(1) The supernatant was combined with a nickel chromatography gel in a shaker at 4℃for two hours, the mixture was passed through the column, and after washing three times (1 mL each time) with a washing solution (20 mmol of imidazole), the eluates were eluted (50 mmol, 100mmol, 150mmol, 200mmol, 250mmol, 300mmol, 350mmol, 400mmol, 450mmol, 500 mmol) with different imidazole concentrations, and SDS-PAGE was performed separately from the supernatant, the flow through, the eluate.
(2) The expression amount and the size of the protein are determined by configuring the gel.
(3) Proteins were purified using His-tag nickel columns.
(4) Imidazole was dialyzed against PBS (ph=9.0) and the protein of interest was concentrated using a 50-kDa protein concentration column;
5. Western-Blot detection of recombinant proteins
(1) SDS-PAGE gels were prepared as described above.
(2) And (3) transferring the film by using a film transfer instrument, and setting parameter current to 400mA for 20min.
(3) Washing with PBST for 3-5 times each for 5min, adding 5% skimmed milk after washing for 2h, washing with PBST for 3-5 times each for 5min after blocking, adding anti-eGFP antibody, and incubating overnight at 4deg.C.
(4) The next day, PBST is washed 3-5 times for 5min each time, then goat anti-rabbit secondary antibody is added, and the mixture is incubated for 2h at room temperature. The ECL chemiluminescent solution was added immediately and observed after washing 3-5 times with PBST for 5min each.
The results showed that the ppO111 protein was 82.1kDa in size after purification. The detection result of the purity of ppO111 protein is 68.8 mu g/mL. Western-Blot detection ppO protein size was consistent with expected results (FIG. 1).
Example 2 pH stability of the pp O111 protein
（1）180μL 10 5 CFU/mL STEC O111 was added with 20. Mu.L of the coating solution, and incubated overnight in a 4℃refrigerator. The control group was sterile PBS (ph=9.0) buffer.
(2) The next day, the wells were discarded and the water was drained, 200 μl of PBST buffer was added to each well, the liquid was decanted, and repeated 3-5 times. 200. Mu.L of 5% skim milk was added to each well and blocked at 37℃for 2h.
(3) The wells were discarded, 200. Mu.L of PBST buffer was added to each well, the liquid was decanted, and the procedure was repeated 3-5 times.
(4) mu.L of ppO111 protein (340. Mu.g) was taken, dissolved in 900. Mu.L of buffer (ph=2, 3,4,5,6,7,8,9,10, 11) and incubated for 1h at 37℃in an incubator. mu.L of ppO91, ppO, 103, ppO111 protein was added to each well and incubated for 2h at 37 ℃.
(5) The wells were discarded, and 200. Mu.L of PBST buffer was added to each well to pour out the liquid, and the procedure was repeated 3-5 times. mu.L of GFP antibody diluent (1:5000) was added to each well and incubated for 1h at 37 ℃.
(6) One anti-dilution was discarded, 200. Mu.L of PBST buffer was added to each well and the solution was poured off and repeated 3-5 times. mu.L of goat anti-rabbit secondary antibody diluent (1:7000) was added to each well and incubated for 1h at 37℃in an incubator.
(7) The secondary antibody dilutions were discarded, 200 μl of PBST buffer was added to each well and the solution was decanted and repeated 3-5 times. mu.L of a color development solution was added to each well, and incubated in a 37℃incubator for 30min in the absence of light.
(8) The reaction was stopped by adding 50. Mu.L of 2moL/L concentrated sulfuric acid to each well, and OD450 was measured.
The results showed that recombinant protein ppO111 remained highly active at pH8-9 (fig. 2).
Example 3 temperature stability of the ppo111 protein
The sensitivity test of recombinant protein ppO111 to different temperatures was consistent with the pH test method described above. Recombinant protein ppO111 was incubated at 4 ℃, 25 ℃,37 ℃, 42 ℃, 55 ℃, 65 ℃, 75 ℃ and 85 ℃ respectively for 1h. The OD450 was finally determined.
The results showed that protein ppO111 was relatively stable in activity between 4-37 ℃, decreasing protein activity by more than 20% at 42 ℃ and little protein activity at 85 ℃ (figure 3).
Example 4 detection of the lowest concentration of bacteria by the pp theta 111 protein
（1）180μL（0.6×10 8 —0.6×10 1 CFU), i.e. 3.3x10 8 —3.3×10 0 CFU/mL was added with 20. Mu.L of coating solution, and incubated overnight in a 4℃refrigerator. The control group was sterile PBS (ph=9.0) buffer.
(2) The next day, the wells were discarded and the water was drained, 200 μl of PBST buffer was added to each well, the liquid was decanted, and repeated 3-5 times. 200. Mu.L of 5% skim milk was added to each well and blocked at 37℃for 2h.
(3) The wells were discarded, 200. Mu.L of PBST buffer was added to each well, the liquid was decanted, and the procedure was repeated 3-5 times.
(4) ppO111 protein (34. Mu.g) was added to each well and incubated for 2h at 37 ℃.
(5) The wells were discarded, and 200. Mu.L of PBST buffer was added to each well to pour out the liquid, and the procedure was repeated 3-5 times. mu.L of GFP antibody diluent (1:5000) was added to each well and incubated for 1h at 37 ℃.
(6) One anti-dilution was discarded, 200. Mu.L of PBST buffer was added to each well and the solution was poured off and repeated 3-5 times. mu.L of goat anti-rabbit secondary antibody diluent (1:7000) was added to each well and incubated for 1h at 37℃in an incubator.
(7) The secondary antibody dilutions were discarded, 200 μl of PBST buffer was added to each well and the solution was decanted and repeated 3-5 times. mu.L of a color development solution was added to each well, and incubated in a 37℃incubator for 30min in the absence of light.
(8) The reaction was stopped by adding 50. Mu.L of 2moL/L concentrated sulfuric acid to each well, and OD450 was measured. Prism 8.0 software compares FL differences between groupsP＜0.001）。
The results showed that recombinant protein ppO111 showed significant differences from the PBS control group when the detected bacterial concentration was 33 CFU/mL (fig. 4).
Example 5 lowest detection concentration of the ppo111 protein
In agreement with the above pH assay, 100. Mu.L of ppO111 protein (68.8-0.07. Mu.g/mL) was added per well and incubated for 2h at 37 ℃. The OD450 was finally determined. prism 8.0 software compares FL differences between groupsP＜0.001）。
The results showed that ppO protein at a concentration of 0.5 μg/mL still detected STEC O111 bacteria, with a significant difference from the PBS control group (fig. 5).
Example 6 specificity of the pp O111 protein
(1) 180 mu L of 10 are added to each well 5 CFU/mL of STEC O1, O2, O3, O4, O5, O6, O15, O16, O17, O18, O26, O27, O29, O33, O36, O45, O58, O64, O83, O87, O91, O99, O105, O111, O116, O123, O128, O131, O145, O157, O173, O180, E.coli ATCC25922, salmonella ATCC14028, shigella flexneri ATCC12022, pseudomonas aeruginosa ATCC27853, klebsiella pneumoniae ATCC700603, acinetobacter baumannii ATCC BAA-1605, E.faecium ATCC19434, clostridium perfringens ATCC13124, staphylococcus aureus ATTCC27217, E.faecalis ATCC 29212 were added with 20. Mu.L of coating solution per well, and incubated overnight at 4 ℃. The control group was sterile PBS (ph=9.0) buffer.
(2) The next day, the wells were discarded and the water was drained, 200 μl of PBST buffer was added to each well, the liquid was decanted, and repeated 3-5 times. 200. Mu.L of 5% skim milk was added to each well and blocked at 37℃for 2h.
(3) The wells were discarded, 200. Mu.L of PBST buffer was added to each well, the liquid was decanted, and the procedure was repeated 3-5 times.
(4) mu.L of ppO111 protein (34. Mu.g) was added to each well and incubated for 2h at 37 ℃.
(5) The wells were discarded, and 200. Mu.L of PBST buffer was added to each well to pour out the liquid, and the procedure was repeated 3-5 times. mu.L of GFP antibody diluent (1:5000) was added to each well and incubated for 1h at 37 ℃.
(6) One anti-dilution was discarded, 200. Mu.L of PBST buffer was added to each well and the solution was poured off and repeated 3-5 times. mu.L of goat anti-rabbit secondary antibody diluent (1:7000) was added to each well and incubated for 1h at 37℃in an incubator.
(7) The secondary antibody dilutions were discarded, 200 μl of PBST buffer was added to each well and the solution was decanted and repeated 3-5 times. mu.L of a color development solution was added to each well, and incubated in a 37℃incubator for 30min in the absence of light.
(8) The reaction was stopped by adding 50. Mu.L of 2moL/L concentrated sulfuric acid to each well, and OD450 was measured.
As a result, it was revealed that the recombinant protein ppO111 acted only on STEC O111, and did not act on other 32 strains of E.coli (O1, O2, O3, O4, O5, O6, O15, O16, O17, O18, O26, O27, O29, O33, O36, O45, O58, O64, O83, O87, O91, O99, O103, O105, O116, O123, O128, O131, O145, O157, O173, O180) and 10 other bacteria (E.coli ATCC25922, salmonella ATCC14028, shigella flexneri ATCC12022, pseudomonas aeruginosa ATCC27853, klebsiella pneumoniae ATCC700603, acinetobacter baumannii ATCC BAA-1605, enterococcus faecium ATCC19434, clostridium perfringens ATCC13124, staphylococcus aureus ATTCC27217, enterococcus ATCC 29212) with respect to the other 32 different O-antigens (FIG. 6).
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (5)
1. A phage protein, characterized in that the phage protein has an amino acid sequence as set forth in SEQ ID NO: 1.
2. Use of a phage protein according to claim 1 for the non-diagnostic identification of shiga toxin-producing escherichia coli of the O111 antigen serotype.
3. A method for non-diagnostic identification of shiga toxin-producing escherichia coli of the O111 antigen serotype, comprising:
co-culturing the phage protein of claim 1 with a microorganism to be tested;
determining whether the microorganism to be tested is shiga toxin-producing escherichia coli of an O111 antigen serotype based on whether the phage protein binds to the microorganism to be tested.
4. The method for non-diagnostic identification of shiga toxin-producing escherichia coli of O111 antigen serotype according to claim 3, wherein the phage protein is bound to a microorganism to be tested, and the microorganism to be tested is determined to be shiga toxin-producing escherichia coli of O111 antigen serotype;
the phage protein is not combined with the microorganism to be detected, and the microorganism to be detected is determined not to be shiga toxin-producing escherichia coli of O111 antigen serotype;
the phage protein is connected with a marker molecule, and whether the phage protein is combined with the microorganism to be detected is determined by detecting the marker molecule.
5. The method for non-diagnostic identification of shiga toxin-producing escherichia coli of O111 antigen serotype according to claim 3, wherein the co-culture reaction pH is 8-9 and the temperature is 4-37 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311306831.7A CN117024531B (en) | 2023-10-10 | 2023-10-10 | Phage targeting protein molecule and application thereof in identifying shiga toxin-producing escherichia coli of O111 antigen serotype |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311306831.7A CN117024531B (en) | 2023-10-10 | 2023-10-10 | Phage targeting protein molecule and application thereof in identifying shiga toxin-producing escherichia coli of O111 antigen serotype |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117024531A CN117024531A (en) | 2023-11-10 |
| CN117024531B true CN117024531B (en) | 2023-12-12 |
Family
ID=88624940
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311306831.7A Active CN117024531B (en) | 2023-10-10 | 2023-10-10 | Phage targeting protein molecule and application thereof in identifying shiga toxin-producing escherichia coli of O111 antigen serotype |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117024531B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103890192A (en) * | 2011-09-28 | 2014-06-25 | 纳幕尔杜邦公司 | Sequences and their use for detection and characterization of stec bacteria |
| CN109661464A (en) * | 2016-06-30 | 2019-04-19 | 技术创新动力基金（以色列）有限合伙公司 | With the extension bacteriophage variant of host range, preparation method and its in the purposes that nucleic acid is transduceed into interested host |
| CN114015746A (en) * | 2021-12-08 | 2022-02-08 | 山东省农业科学院畜牧兽医研究所 | Application of phage depolymerase ORF38 protein in identification of pasteurella multocida capsule typing |
| CN116003531A (en) * | 2022-12-28 | 2023-04-25 | 山东省农业科学院畜牧兽医研究所 | Application of phage receptor binding protein PO86 in genotyping identification of E.coli O antigen |
| CN116715736A (en) * | 2023-08-07 | 2023-09-08 | 山东省农业科学院畜牧兽医研究所 | Application of phage tail fibrin in identification of O2 antigen serotype avian pathogenic escherichia coli |
-
2023
- 2023-10-10 CN CN202311306831.7A patent/CN117024531B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103890192A (en) * | 2011-09-28 | 2014-06-25 | 纳幕尔杜邦公司 | Sequences and their use for detection and characterization of stec bacteria |
| CN109661464A (en) * | 2016-06-30 | 2019-04-19 | 技术创新动力基金（以色列）有限合伙公司 | With the extension bacteriophage variant of host range, preparation method and its in the purposes that nucleic acid is transduceed into interested host |
| CN114015746A (en) * | 2021-12-08 | 2022-02-08 | 山东省农业科学院畜牧兽医研究所 | Application of phage depolymerase ORF38 protein in identification of pasteurella multocida capsule typing |
| CN116003531A (en) * | 2022-12-28 | 2023-04-25 | 山东省农业科学院畜牧兽医研究所 | Application of phage receptor binding protein PO86 in genotyping identification of E.coli O antigen |
| CN116715736A (en) * | 2023-08-07 | 2023-09-08 | 山东省农业科学院畜牧兽医研究所 | Application of phage tail fibrin in identification of O2 antigen serotype avian pathogenic escherichia coli |
Non-Patent Citations (3)
| Title |
|---|
| A Novel Tail-Associated O91-Specific Polysaccharide Depolymerase from a Podophage Reveals Lytic Efficacy of Shiga Toxin-Producing Escherichia coli;Chen, Yibao等;APPLIED AND ENVIRONMENTAL MICROBIOLOGY;第86卷(第9期);e00145-20 * |
| Biogenesis of a Bacteriophage Long Non-Contractile Tail;Anait Seul等;Journal of Molecular Biology;第433卷;167112 * |
| 产志贺毒素大肠杆菌噬菌体vBEcoMPHB05的生物学特性及全基因组分析;陈义宝等;中国兽医科学;第48卷(第4期);428-437 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117024531A (en) | 2023-11-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rojas et al. | Purification and characterization of a surface protein from Lactobacillus fermentum 104R that binds to porcine small intestinal mucus and gastric mucin | |
| Jonquieres et al. | Interaction between the protein InlB of Listeria monocytogenes and lipoteichoic acid: a novel mechanism of protein association at the surface of Gram‐positive bacteria | |
| JP4249483B2 (en) | Serpins in bifidobacteria | |
| Yang et al. | Magnetic nano-beads based separation combined with propidium monoazide treatment and multiplex PCR assay for simultaneous detection of viable Salmonella Typhimurium, Escherichia coli O157: H7 and Listeria monocytogenes in food products | |
| Neely et al. | Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes | |
| Behari et al. | A homolog of CcpA mediates catabolite control in Listeria monocytogenes but not carbon source regulation of virulence genes | |
| Gleinser et al. | Improved adhesive properties of recombinant bifidobacteria expressing the Bifidobacterium bifidum-specific lipoprotein BopA | |
| JP2000501293A (en) | Compositions and methods for screening antimicrobial agents | |
| US5652102A (en) | Assay for enterohemorrhagic Escherichia coli 0157:H7 by the polymerase chain reaction | |
| US5374551A (en) | Methods for detection, identification and speciation of members of the genus Listeria | |
| CN116715736B (en) | Application of phage tail fibrin in identification of O2 antigen serotype avian pathogenic escherichia coli | |
| CN115960222A (en) | Preparation and application of nano antibody for helicobacter pylori typing detection | |
| Li et al. | Xanthomonas campestris sensor kinase HpaS co‐opts the orphan response regulator VemR to form a branched two‐component system that regulates motility | |
| Heimer et al. | Interaction of Proteus mirabilis urease apoenzyme and accessory proteins identified with yeast two-hybrid technology | |
| CN117024531B (en) | Phage targeting protein molecule and application thereof in identifying shiga toxin-producing escherichia coli of O111 antigen serotype | |
| CN116903710B (en) | Phage targeting protein molecule and application thereof in identifying shiga toxin-producing escherichia coli of O103 antigen serotype | |
| Li et al. | McvR, a single domain response regulator regulates motility and virulence in the plant pathogen Xanthomonas campestris | |
| Miljkovic et al. | AggLr, a novel aggregation factor in Lactococcus raffinolactis BGTRK10-1: its role in surface adhesion | |
| WO2019179174A1 (en) | Colloidal gold test strip and kit for detecting clostridium difficile | |
| CN102707054B (en) | Indirect ELISA (enzyme linked immunosorbent assay) detection method for escherichia coli OmpT (Outer-membrane protease T) antibody | |
| Cheng et al. | Mass spectrometry-based Shiga toxin identification: An optimized approach | |
| CN116023479A (en) | Bovine viral diarrhea virus nano antibody and preparation method and application thereof | |
| Smith et al. | Gene encoding the group B streptococcal protein R4, its presence in clinical reference laboratory isolates & R4 protein pepsin sensitivity | |
| CN100368546C (en) | Food-poisoning fungus concatenated fusion mycinamicin and use thereof | |
| US20230140486A1 (en) | Fusion protein and method of detecting bacteria having pseudaminic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |