CN103555671A - Vibrio harveyi giant phage VP4B and application thereof - Google Patents
Vibrio harveyi giant phage VP4B and application thereof Download PDFInfo
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- CN103555671A CN103555671A CN201310263942.4A CN201310263942A CN103555671A CN 103555671 A CN103555671 A CN 103555671A CN 201310263942 A CN201310263942 A CN 201310263942A CN 103555671 A CN103555671 A CN 103555671A
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Abstract
The invention belongs to the technical field of biology, and specifically relates to giant phage and an application thereof. The present invention provides a rare giant phage VP4B, wherein double flat plate separation purification, genome extraction, nucleic acid sequence sequencing and other technologies are adopted to carry out determination and analysis on the genome nucleic acid sequence of the giant phage VP4B obtained from the environment so as to obtain the 236,053 bp nucleic acid sequence. The giant phage VP4B provides a significant growth inhibition effect for pathogenic Vibrio harveyi, and can be used for biological prevention and control of vibrio diseases in mariculture.
Description
Technical field:
The invention belongs to biological technical field, specifically giant bacteriophage and application thereof.
Background technology:
Bacterial infection is modal type in catching, and in the past few decades, microbiotic is considered to treat the best method that bacterium infects.But along with abuse of antibiotics phenomenon is day by day serious in recent years, the resistance problem of bacterium is increasingly sharpened, people are selecting the sight of other antiseptic-germicides to transfer on phage.Compare traditional microbiotic, the advantage of phage is that Host Strains is had to height specificity, can continue self-reproduction, and antagonistic drug bacterial strain has infection ability again, and environment is not polluted, non-stimulated.
Along with the development of biotechnology, the status of phage is more and more important.Gene to phage is studied, not only diversity and the evolutionary path for phage provides resource, also for the interaction between the biological characteristics of phage and itself and host provides evidence, simultaneously also for security and the controllability of Phage therapy provides guarantee.
Summary of the invention:
The object of the present invention is to provide a kind of giant bacteriophage VP4B and application thereof.
In order to realize object of the present invention, contriver utilizes the technology such as double-layer plate separation and purification, genome extraction and determining nucleic acid sequence to obtain phage VP4B from environment, it is carried out to mensuration and the analysis of genomic nucleic acid sequence, find its genome sequence total length 246,964bp, GC content is 43.29%, the ORF that contains 210 predictions.Contriver is preserved in (address: Luo Jia Shan Wuhan University preservation center, wuchang, wuhan, Hubei), Chinese Typical Representative culture collection center on May 9th, 2013 by this phage, preserving number CCTCC NO:M2013192, its Classification And Nomenclature is Vibrio harveyi phage VP4B (Vibrio harveyi Phage VP4B).
Giant bacteriophage VP4B of the present invention can be used for phage functional genomics, new virus vector construction, the prevention of carrying out vibrios disease and quick diagnosis, and phage is adsorbed, infects, the research of cracking vibrios mechanism.
According to embodiment below, the giant bacteriophage VP4B in the present invention is lytic phage, can infect Vibrio harveyi, and copy propagation in vibrios body after, cell walls that can cracking Vibrio harveyi, causes vibrios cracking dead.Can effectively control quantity and the rate of propagation of Vibrio harveyi, can be for the control of the Sino-Kazakhstan Vickers vibrios of aquaculture disease.In embodiment, giant bacteriophage VP4B of the present invention can significantly suppress the Vibrio harveyi speed of growth and concentration.
The present invention can come for the vibrios in aquaculture, particularly Vibrio harveyi according to the Bactericidal curves of giant bacteriophage, carries out the Application and Development of disease control and disease control enzyme material.
The present invention also provides the bacterium that comprises giant bacteriophage of the present invention.Preferably, described bacterium is Vibrio harveyi.
The present invention also provides a kind of method that produces giant bacteriophage, and described method is included in the step of cultivating giant bacteriophage of the present invention in bacterium.In one embodiment, described bacterium is Vibrio harveyi, and for example German microbial strains preservation center is with preserving number: the bacterium of DSM-6904 preservation.
Accompanying drawing explanation:
Fig. 1 suppresses Vibrio harveyi growth OD graphic representation for breathing out Vickers giant bacteriophage VP4B.
Embodiment:
1. the collection of phage and concentrated
(1) to 200mL liquid 2216E substratum (1g yeast powder, 5g peptone, 0.01g high ferric phosphate, the old seawater of 1L) in triangular flask, add 0.1mL Vibrio harveyi (purchased from DSMZ, preserving number: DSM-6904) solution, and add 0.1mL Kazakhstan of the present invention Vickers giant bacteriophage VP4B (preserving number: CCTCC NO:M2013192) solution, is placed in 37 ℃ of incubators, shake-flask culture 18 hours in described triangular flask.To adding enzyme inhibitors phenylmethylsulfonyl fluoride PMSF to make its final concentration in the liquid after cultivating, be 1mM, with 15000 * g, carry out the centrifugal of 20min.
(2) get supernatant and add NaCl, making its final concentration is 1M; Add polyoxyethylene glycol PEG6000 (Sigma, 25322-68-3), making its final concentration is 10%, at 4 ℃, places 1-3h.
(3) with 15000 * g centrifugal 1h at 4 ℃.
(4) remove supernatant, resuspended with the SM damping fluid (Amrecso, J614-500ML) of 2mL, with 3000 * g, carry out 15min centrifugal, go precipitation.
(5) by centrifuged supernatant in cesium chloride solution (1.5g/mL), with 220000 * g centrifugal 24h at 4 ℃.
(6) with one-shot injector, collect phage.
2. the extraction of phage genome
(1) in above-mentioned steps 1, in the 1mL phage particle suspension of preparation, add DNase/RNase mixture (Takara, 2210CA/R0675) (final concentration is 2mL/L), mix, in the water-bath of 37 ℃, incubate and bathe 45min.
(2) add 150 μ L TE damping fluids (10mM Tris-HCl, 1mM EDTA, pH=8.0) and 30 μ L10% (w/v) SDS solution, turn upside down, in the water-bath of 70 ℃, hatch 10min.
(3) add 125 μ L acetic acid-potassium acetate damping fluids (pH4.3), in ice-water bath, hatch 30min-1h.
(4) with 14,000 * g centrifugal 15min at 4 ℃.
(5) in centrifuged supernatant, add equal-volume phenol/chloroform/primary isoamyl alcohol (volume ratio is 25:24:1), acutely mix 5min, with 14,000 * g centrifugal 5min at 4 ℃.Repeat this step until without white protein impurity.
(6) centrifuged supernatant is divided into two pipes, with equal-volume Virahol static 1h at-20 ℃.
(7) with 14,000 * g centrifugal 20min at 4 ℃.
(8) discard centrifuged supernatant, by 80% washing with alcohol precipitation.
(9) be placed in super clean bench air-dry.
3. the phage genome DNA that pair step 2 is extracted carries out mensuration, splicing and the analysis of nucleotide sequence:
By Shenzhen, Hua Da gene research and development centre completes order-checking.Adopt Glimmer software from assembling result, to obtain the ORF of gene order, and BLAST carry out functional analysis.Sequential analysis shows, its genome sequence total length 246, and 964bp, GC content is 43.29%, the ORF that contains 210 predictions.34046 Tetrahydrofolate dehydrogenases to 34561 nucleotide sequence coded phages wherein, the 35408th the sweet acid enzyme of the thymus gland to 36316 nucleotide sequence coded phages, its the 86182nd adjusting albumen to the 86889th nucleotide sequence coded phage, the 112547th RNA polymerase to 114532 nucleotide sequence coded phages, the 201168th DNA ligase to the 203135th nucleotide sequence coded phage, the 207525th rnase to the 208049th nucleotide sequence coded phage.
Also carry out sequence alignment with known Vibrio harveyi phage (GenBank:AY283928).Result shows, VP4B and its can form well to be compared, and also has the phenomenon of gene displacement simultaneously, proves that this is a kind of brand-new Vibrio harveyi phage.
4. phage suppresses the OD graphic representation of Vibrio harveyi propagation
(1) to two, 500mL liquid 2216E substratum (1g yeast powder is respectively housed, 5g peptone, 0.01g high ferric phosphate, the old seawater of 1L) in triangular flask A, B, add 0.1mL Vibrio harveyi (purchased from German microbial strains preservation center (DSMZ), preserving number: DSM-6904) solution, in A bottle, add the giant bacteriophage VP4B solution of preparing in 0.1mL step 1, be placed in 37 ℃ of incubators, shake-flask culture.
(2) every one hour, get nutrient solution 1mL in A, B bottle, with 8000 * g centrifugal 5min at 4 ℃, with clean 2216E substratum washing precipitation 3 times, with clean 2216E substratum resuspension.Absorbancy with spectrophotometric determination OD600.
(3) do three parallel sample for every group, try to achieve standard deviation.
Result shows as shown in Figure 1, and giant bacteriophage VP4B of the present invention can significantly suppress the Vibrio harveyi speed of growth and concentration.
Claims (9)
1. a giant bacteriophage, it is preserved in Chinese Typical Representative culture collection center with preserving number M2013192.
2. the giant bacteriophage of claim 1 is for suppressing the purposes of Vibrio harveyi.
3. the purposes of the control of the aquaculture vibrios disease that the giant bacteriophage of claim 1 is used for.
4. the purposes of claim 3, wherein said vibrios is Vibrio harveyi.
5. the bacterium that comprises the giant bacteriophage of claim 1.
6. the bacterium of claim 5, described bacterium is Vibrio harveyi.
7. produce a method for giant bacteriophage, described method is included in the step of cultivating the giant bacteriophage of claim 1 in bacterium.
8. the method for claim 7, described bacterium is Vibrio harveyi.
9. the method for claim 8, wherein said Vibrio harveyi is German microbial strains preservation center with preserving number: the bacterium of DSM-6904 preservation.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192842A (en) * | 2018-01-17 | 2018-06-22 | 厦门昶科生物工程有限公司 | A kind of Vibrio harveyi bacteriophage probiotics and its preparation method and application |
| CN112063595A (en) * | 2020-09-24 | 2020-12-11 | 瑞科盟(青岛)生物工程有限公司 | Lytic vibrio harveyi phage RDP-VP-19012 and application thereof |
| CN112391355A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB _ Vhas-yong3 and application thereof |
| CN112391356A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB _ Vhas-yong2 and application thereof |
| CN112442487A (en) * | 2019-08-14 | 2021-03-05 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB-Vhas-yong1 and application thereof |
-
2013
- 2013-06-28 CN CN201310263942.4A patent/CN103555671A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192842A (en) * | 2018-01-17 | 2018-06-22 | 厦门昶科生物工程有限公司 | A kind of Vibrio harveyi bacteriophage probiotics and its preparation method and application |
| CN112391355A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB _ Vhas-yong3 and application thereof |
| CN112391356A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB _ Vhas-yong2 and application thereof |
| CN112442487A (en) * | 2019-08-14 | 2021-03-05 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB-Vhas-yong1 and application thereof |
| CN112391355B (en) * | 2019-08-14 | 2023-12-01 | 宁波大学 | Vibrio harveyi efficient lytic phage vB_VhaS-yong3 and application thereof |
| CN112063595A (en) * | 2020-09-24 | 2020-12-11 | 瑞科盟(青岛)生物工程有限公司 | Lytic vibrio harveyi phage RDP-VP-19012 and application thereof |
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Application publication date: 20140205 |