CN109207435A - The separation identification of one plant of escherichia coli H21 bacteriophage ST31 - Google Patents
The separation identification of one plant of escherichia coli H21 bacteriophage ST31 Download PDFInfo
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- CN109207435A CN109207435A CN201710536101.4A CN201710536101A CN109207435A CN 109207435 A CN109207435 A CN 109207435A CN 201710536101 A CN201710536101 A CN 201710536101A CN 109207435 A CN109207435 A CN 109207435A
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- bacteriophage
- escherichia coli
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- phagocytosis
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- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/04—Disinfection
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- 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
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- 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
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10031—Uses of virus other than therapeutic or vaccine, e.g. disinfectant
Abstract
The present invention relates to the bacteriophage ST31 for removing pathogenic Escherichia coli, belong to bioengineering field.One plant of novel strong coliphage ST0 (gene order SEQ ID NO.1, GenBank Access no.KY962008) is provided, has icosahedral head and short-tail, head diameter about 60nm, tail portion is about 15nm.Its DNA is cyclic annular duplex structure, and gene group leader 170,496bp, prediction has one section of tRNA between 10,055bp and 10,1331bp, and prediction has 45 open reading frame, wherein there is 16 to have similar protein function.The bacteriophage can be used as a kind of biological preventions, the removal for the fields such as sewage disposal system and medical treatment pathogen.
Description
Technical field
The present invention relates to the prevention and control for Escherichia coli H21, belong to bioengineering field.Particularly, the present invention relates to right
From one plant of novel bacteriophage ST31 (gene order SEQ ID NO.1, GenBank Access no. in sewage disposal system
KY962008 separation and genome Basic Characteristics), the especially forecast analysis of its functional gene and albumen.
Background technique
Extensive use with antibiotic in the fields such as medical treatment and agricultural, endurance strain are paid attention to already.In environment
Antibiotic not easily biological-degradable, be easy in water environment and soil environment, or even in atmospheric environment, storage and accumulation.Recently,
Antibiotic has been acknowledged as emerging environmental contaminants, because of the potential adverse effect of antibiotic, to the ecosystem and human health.
Drug tolerant bacteria also increases medical difficulty, has the death of 23000 people related with drug tolerant bacteria every year in the U.S..Drug resistance is thin
The propagation of bacterium and drug resistant gene in the environment is a great public health problem.Obviously, the use of strict control antibiotic
Alternative medicine is extremely urgent with developing.
Compared with antibiotic, Phage therapy has many advantages, such as that specific height, few side effects, low dosage use, especially
Disease caused by being treated using phagotherapy because of drug tolerant bacteria has substitution effect.Phage therapy mainly has single phagocytosis
The mode of body treatment, a variety of bacteriophage co-therapies and bacteriophage and Antibiotic combination treatment.Currently, being raiseeed using bacteriophage
Fowl cultivation, aquaculture and food industry etc. removal pathogen have been cooked more research, and achieve preferable achievement.However
The type and quantity of bacteriophage are the key factors for limiting Phage therapy and further developing, therefore separate and identify phagocytosis
Body is come to enrich bacteriophage kind library be very necessary.
Summary of the invention
It is an object of the invention to: a kind of selectable basic material is provided for the prevention and control of Phage therapy and pathogen,
Bacteriophage kind library is enriched with this, and further develops a kind of novel biological control means.
Another object of the present invention is to: by the Morphological Identification and genome sequencing to bacteriophage ST31, further
Go deep into data mining duty gene and albumen, for Phage therapy and biological control one or more pairs of pathogens is provided, there is cracking to make
Enzyme etc. expands host range with this, is conducive to the popularity and validity of bacteriophage application.
1. the novel virulent phage ST31 separated from sewage disposal system in the present invention, host is Escherichia coli
H21, the bacteriophage can generate bright, sharp-edged plaque on double-layer plate, and phagocytosis spot diameter is at 5-6 millimeters；Transmission
Electronic Speculum shows that bacteriophage ST31 has icosahedral head and short-tail, and head diameter about 60nm, tail portion is about 15nm, tail diameter
About 20nm.
2. the full-length genome size of the bacteriophage ST31 according to claim 1 is 39,693bp, for annular double-strand
Structure, G+C% content are 49.98%, and prediction has 269 open reading frame, and has one between 10,055bp to 10,133bp
The region section tRNA, the accession number of GenBank database are KY962008.
It is industrialized production 3. the bacteriophage according to claim 1 has fine melt effectiveness to Escherichia coli H21
Bacteriophage and prevention and treatment for Escherichia coli in sewage discharge and reusing sewage provide bacteriophage source.
Main advantages of the present invention are: the bacteriophage ST31 according to the present invention is in the experiment for cracking Escherichia coli H21
In show cracking effectiveness rapidly and efficiently, can be used in sewage disposal system the prevention and treatment of the explosive situation of Escherichia coli and
The removal of drug resistance Escherichia coli.
Detailed description of the invention
The double-layer plate plaque morphology of Fig. 1 bacteriophage ST31
Fig. 2 bacteriophage ST31 transmission electron microscope form
Fig. 3 bacteriophage ST31 genome loop graph
Fig. 4 bacteriophage ST31 genome system chadogram
Specific embodiment
Embodiment below is intended only as illustrating purpose, and will be those skilled in the art according to its moditied processing
It can remember, and including in spirit and scope and in the range of accessory claim.Herein cited is all
Publications, patents and patent applications are to merge herein by the way that reference is whole for all purposes.Unless otherwise indicated, according to following
Method separation handles and analyzes coliphage of the invention.That is done all within the spirits and principles of the present invention appoints
What modifications, equivalent substitutions and improvements etc., should all be included in the protection scope of the present invention.
Present invention test is with bacteriophage host strain Escherichia coli H21 (ST130) by heart trouble in Chinese disease prevention and prevention and control
Opportunistic pathogen room provides, and by saving for this room.
Present invention test LB culture solution, culture medium and PEG8000 (being purchased from Amresco company)；DNase, RNaseA
(Sigma company)；Proteinase K (Amresco company)；0.22 μm of miillpore filter (Shanghai City new Asia purifies device factory)；Chloroform is put down
It weighs saturated phenol (Beijing Ding Guo Bioisystech Co., Ltd).
Embodiment 1, bacteriophage isolate and purify
It is to be collected in municipal sewage plant of Beijing aeration tank in March, 2017 to make that the present invention, which is tested with sewage sample,
For the water sample for separating bacteriophage.
Sewage sample 500mL is taken, solid CaCl2 to final concentration 1mmol/L is added, 3000g is centrifuged 10min, big to remove
Impurity, then with 0.22 μm of filtering with microporous membrane degerming.Take 300ml filtrate, 2 × LB of 300mL culture solution and host bacteria suspension
15mL is added together in the conical flask that high pressure sterilization is crossed, and 37 DEG C, after 120rpm shaken cultivation 10h, 3000g is centrifuged at 4 DEG C
10min takes supernatant 100mL, and 2 × LB of 100mL culture solution and corresponding host bacteria suspension 5mL is added, sets 37 DEG C of shaking table 120rpm
After shaken cultivation 12h, 4 DEG C, 5000g is centrifuged 10min, and supernatant is filtered through (0.22 μm) of miillpore filter again, and filtrate is quasi- contain
There is the stoste of corresponding host strain bacteriophage.Using whether having bacteriophage in double-layer agar technique identification filtrate, above-mentioned stoste is taken to pass through
0.1mL is added host's bacterium solution 0.1mL and mixes after appropriate dilution, is added 4mL is cooled to 47 DEG C 0.7% after 37 DEG C of standing 15min
LB semisolid culturemedium pours on LB solid medium immediately after mixing, after its solidification after, 37 DEG C of culture 6-8h, observe whether there is or not
Plaque is grown.If there is plaque, show accordingly to bite containing corresponding bacteriophage conversely, being then shown to be and isolating in filtrate
Thallus needs resampling to separate.
Plaque of the bacteriophage separated for the first time on double-layer plate, form, size are often inconsistent, need further pure
Change.Picking is transparent on the double-layer plate for have plaque clearly bites single bacterial plaque, and leaching is placed in the sterile tube containing 1mL SM liquid
In, it is placed at room temperature for after 1h 4 DEG C overnight, next day takes the above-mentioned solution of 0.1mL after suitably diluting, and does double-deck put down with host bacteria suspension
Plate.It repeats this step 5-6 times, until the size and form of each plaque are almost the same (Fig. 1) in double-layer plate.
The morphologic observation of embodiment 2, bacteriophage
It takes 20 μ L drop of phage suspensions on copper mesh, after its natural sedimentation 10min, is blotted with dry filter paper from side,
It hangs after about 1min on copper mesh plus 1 1% uranium acetate of drop, dyes 2min, it then carefully will from side using dry filter paper
Extra stain blots, and is protected from light after nature hangs 30min, is observed with transmission electron microscope (JEM-1400).
Transmission electron microscope results show (Fig. 2) that the head of bacteriophage ST31 is in icosahedron, head diameter 60nm, short-tail, length
About 15nm is spent, is reported according to international viral taxonomy tissue (ICTV) virus taxis the 8th time, it can be tentatively by this plant of bacteriophage
ST31 is classified as Podoviridae.
Embodiment 3, phage genome analyze and identify
Bacteriophage genome sequencing and analysis: using 2500 sequencing technologies of Illumina HiSeq to the DNA of sample into
Row sequencing, constructs the library 400bp.V.1.3.6 splice software followed by abyss to splice optimization, obtain optimal
Assembling result.Forecast analysis is carried out using open reading frame of the biosoftware PHASTER to genome, and uses NCBI
Blastp complete functional gene preliminary annotation, using tRNAscan-SE (Http:// lowelab.ucsc.edu// tRNAscan-SE/) on-line prediction tRNA, the drafting of full-length genome loop graph, base are completed using 6 software of DNAMAN version
5.05 software component systematic evolution tree of MEGA is utilized in full-length genome data.Bacteriophage ST31 genome sequence has been filed on the U.S.
National Biotechnology Information Center (NCBI) gene database (GenBank), typing KY962008.
Whole genome analysis shows, the genome of bacteriophage ST31 is cyclic annular duplex structure, overall length 39,693bp, 10,
Prediction has one section of tRNA between 055 bp and 10,133bp, and open reading frame (Open Reading Frame, ORF) prediction has 45
A (as shown in table 1 and Fig. 3) wherein there is 16 to have similar protein function, and has carried out functional gene annotation.Through homology ratio
To and genome system phylogenetic analysis (Fig. 4) display, the affiliation of bacteriophage ST31 and bacteriophage bacteriophage PE3-1 are most
Closely, but marking is less than 70, and confidence level is lower, therefore more illustrates that bacteriophage ST31 is novel bacteriophage.
The annotation of 1 bacteriophage ST31 open reading frame of table
Claims (3)
1. one plant of novel virulent phage ST31 separated from sewage disposal system, host is Escherichia coli H21, the phagocytosis
Body can generate bright, sharp-edged plaque on double-layer plate, and phagocytosis spot diameter is at 5-6 millimeters；Transmission electron microscope, which is shown, to be bitten
Thallus ST31 has icosahedral head and short-tail, and head diameter about 60nm, tail portion is about 15nm.
It is annular duplex structure 2. the full-length genome size of the bacteriophage ST31 according to claim 1 is 39,693bp,
G+C% content is 49.98%, and 45 open reading frame have one section of tRNA, the bacteriophage between 10,055bp to 10,133bp
It is KY962008 in the accession number of GenBank database.
It is industrialized production phagocytosis 3. the bacteriophage according to claim 1 has fine melt effectiveness to Escherichia coli H21
Body and prevention and treatment for Escherichia coli in sewage discharge and reusing sewage provide bacteriophage source.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710536101.4A CN109207435A (en) | 2017-07-04 | 2017-07-04 | The separation identification of one plant of escherichia coli H21 bacteriophage ST31 |
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| CN201710536101.4A CN109207435A (en) | 2017-07-04 | 2017-07-04 | The separation identification of one plant of escherichia coli H21 bacteriophage ST31 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994456A (en) * | 2011-09-15 | 2013-03-27 | 蔡志明 | Artificially-synthesized S13 phage and applications thereof |
| CN105324481A (en) * | 2013-02-27 | 2016-02-10 | Cj第一制糖株式会社 | Novel bacteriophage and antibacterial composition comprising the same |
-
2017
- 2017-07-04 CN CN201710536101.4A patent/CN109207435A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994456A (en) * | 2011-09-15 | 2013-03-27 | 蔡志明 | Artificially-synthesized S13 phage and applications thereof |
| CN105324481A (en) * | 2013-02-27 | 2016-02-10 | Cj第一制糖株式会社 | Novel bacteriophage and antibacterial composition comprising the same |
Non-Patent Citations (3)
| Title |
|---|
| KY962008.1: "Escherichia phage ST31, complete genome", 《GENBANK》 * |
| 于龙等: "两株大肠杆菌烈性噬菌体的分离与生物学特征研究", 《军事医学科学院院刊》 * |
| 新浪: "美国科学家14天造奇迹，合成噬菌体", 《新浪新闻》 * |
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