CN101798568A - Separated vibrio parahaemolyticus phage and applications in sterilization and microbe-proofing thereof - Google Patents
Separated vibrio parahaemolyticus phage and applications in sterilization and microbe-proofing thereof Download PDFInfo
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Abstract
The invention discloses separated vibrio parahaemolyticus phage and applications in sterilization and microbe-proofing thereof. The separated phage monomer is named as BPH-VP-1 and has broad-spectrum sterilizing property to vibrio parahaemolyticus. The phage BPH-VP-1 is conserved in the China Center for Type Culture Collection (address: China Center for Type Culture Collection, Wuhan University, Mountain Lojia, Wuchang District, Wuhan City), the conservation date is March 24, 2009, and the CCTCC NO is M209058.
Description
Technical field
The present invention relates to a kind of can specificity cracking Vibrio parahaemolyticus (Vibrio Parahaemolyticus) bacterial strain the phage isolate and sterilization and fungi-proofing in application, belong to bioengineering field.
Background technology
(bacteriophage phage) is a kind of bacteriophage to phage, can specific infection and cracking is a kind of or a few bacterium.Phage has strict host specificity, only resides in the susceptible host thalline, and animal and plant cells and other bacteriums are not had infectivity.The security of phage is guaranteed.Phage is divided into two classes, behind the class phage-infect bacterium its genetic material is incorporated in the DNA of bacterium, breeds along with the breeding of bacterium, in case ambient conditions is suitable for the cracking bacterial cell, and causes bacterium death, and this phage is called temperate phage.In case another kind of phage bacterial infection just utilizes bacterium to synthesize progeny phage, cracking bacterial cell and discharging then, this phage becomes virulent phage.On sterilization effect, virulent phage has a wide range of applications, and the infection that especially utilizes its treatment to be caused by " superbacteria " has obtained good effect.
Phage isolate among the present invention is from the isolating virulent phage of occurring in nature, proves that through toxicological experiment it is safe, without any side effects, and Vibrio parahaemolyticus is had the intensive splitting action.This phage is through the highly purified pollution that is expected to be developed to into biological bactericidal agent for preventing and treating Vibrio parahaemolyticus.
Summary of the invention
The objective of the invention is to develop the phage isolate that Vibrio parahaemolyticus is had strong splitting action, this phage isolate can be used alone or as a mixture, can be specifically deactivation Vibrio parahaemolyticus partially or completely, for suitability for industrialized production phage sterilant provides phage strain source.
Another object of the present invention is the water surrounding biological bactericide of a kind of novel safety of exploitation, in order to kill the Vibrio parahaemolyticus in the water body specifically, improves the water body microenvironment.
Another purpose of the present invention provides a kind of biological bactericide that is used for aquaculture, transportation and preservation, in order to the pollution of control pathogenic Vibrio parahaemolyticus in aquaculture, transportation and preservation process.
A further object of the present invention is to provide potential medicine for treating the infection that is caused by Vibrio parahaemolyticus.
The present invention separates from occurring in nature and obtains a kind of phage isolate, and this phage isolate comprises that one or more have the phage of splitting action, the purified phage monomer that gets to Vibrio parahaemolyticus.Prove that phage in this phage isolate is used alone or as a mixture Vibrio parahaemolyticus is had the essence splitting action.Above-mentioned separated phage monomer (vibrio parahaemolyticus phage, latin name Vibrio Parahaemolyticus phage) called after BPH-VP-1 has the sterilizing ability of wide spectrum to Vibrio parahaemolyticus.Phage BPH-VP-1 is preserved in (address: Lopa Nationality an ancient woman's ornament mountain, wuchang, wuhan Wuhan University Chinese in the school typical culture collection center), Chinese typical culture collection center (CCTCC), preservation date is on March 24th, 2009, and deposit number is CCTCC M 209058.
Phage BPH-VP-1 among the present invention gives 10 through the mouse toxicological experiment
10The phage sample of pfu/kg is not seen difference to experiment mice, give 15 days continuously after, the disconnected neck of mouse is put to death, postmortem does not find that its internal organ have difference to change.Identical with other experimental results.This phage is safe.
Phage BPH-VP-1 among the present invention can be used alone or as a mixture, and is 10 in concentration
2-10
3In the Vibrio parahaemolyticus substratum of cfu/ml, 10
6-10
7The phage of pfu/ml reaches more than 99% the sterilization rate of the Vibrio parahaemolyticus of this concentration.Phage among the present invention can be applicable to prevent and treat in the environment, includes but not limited to the pollution of the Vibrio parahaemolyticus in the water body.
Phage BPH-VP-1 among the present invention can be used alone or as a mixture, can be used as sterilant is sprayed in the water body, the control hydrocoles infects the disease that Vibrio parahaemolyticus causes, and can prevent and treat the pollution of Vibrio parahaemolyticus in aquaculture, transportation and preservation process.
Phage BPH-VP-1 among the present invention can be used alone or as a mixture, to the solid food surface sprinkling 10 of being polluted by Vibrio parahaemolyticus
6Pfu/ml-10
7The phage of the pfu/ml order of magnitude, 10
8Pfu/ml-10
11Pfu/ml has better effect, can be in 12 hours the complete inactivation Vibrio parahaemolyticus.Phage among the present invention can be applicable to prevent and treat solid food, includes but not limited to that the Vibrio parahaemolyticus of fishery products pollutes.
Phage BPH-VP-1 among the present invention can be used alone or as a mixture, and can be used as sterilant and is sprayed on the foodstuff production workshop, but specificity, continue to prevent existence and the breeding of Vibrio parahaemolyticus in this environment, the pollution of Vibrio parahaemolyticus in the control food processing process.
Phage BPH-VP-1 among the present invention can be used alone or as a mixture, and can be used as the infectation of bacteria that a kind of potential drug treatment is caused by Vibrio parahaemolyticus.
Phage among the present invention can be united use with other phages, with or the phagocytosis scope of broad.
Phage among the present invention can mix with other antiseptic-germicides uses, when obtaining broad-spectrum antibacterial property, to the Vibrio parahaemolyticus specific killing.Can include but not limited to microbiotic and chemical antiseptic-germicide with the antiseptic-germicide that the phage among the present invention is united use.
Phage among the present invention can be applicable to industrial production, can be by host bacterium Vibrio parahaemolyticus specific amplification, but the application standard viral purification methods is highly purified, can be developed as a kind of biological bactericide and be used to prevent and treat Vibrio parahaemolyticus and pollute.
Description of drawings
The phage BPH-VP-1 that the present invention adopts is a vibrio parahaemolyticus phage, Latin is called Vibrio parahemolyticusphage BVP-40-1, be preserved in (address: Lopa Nationality an ancient woman's ornament mountain, wuchang, wuhan Wuhan University Chinese in the school typical culture collection center), Chinese typical culture collection center (CCTCC), preservation date is on March 24th, 2009, and deposit number is CCTCC M209058.
Fig. 1 is the test result figure of phage stability under the normal temperature;
The test result figure of phage stability when Fig. 2 is 4 ℃;
Fig. 3 is the sterilization effect figure of phage of the present invention in seawater, and the Vibrio parahaemolyticus inoculum size is 1000cfu/ml;
Fig. 4 is the sterilization effect figure of phage of the present invention in seawater, and the Vibrio parahaemolyticus inoculum size is 100cfu/ml;
Fig. 5 is the sterilization effect figure of phage of the present invention to the shellfish sample, and the Vibrio parahaemolyticus inoculum size is 100cfu/ml.
Embodiment
Embodiment 1: screening of phage and purifying
(1) sample collecting and processing
Sample among the present invention picks up from Zhuhai, comprises seawater, the marine fish culture field, Zhuhai City at Zhongshan University's the 5th affiliated hospital's untreated sewage, city, Zhuhai City effluent sewerage, blowdown estuario place.
The sample of gathering is regulated with NaOH, makes pH be about 7.8, through the centrifugal 15min of 4000r/min, gets supernatant liquor with 0.45 μ m membrane filtration.Get the sample 75ml after the filtration sterilization, add 4 times high salt TSB substratum of 25ml (1% sodium-chlor TSB substratum).
(2) in the sample at the phage specific amplification of Vibrio parahaemolyticus
In the sample of handling well, add the good Vibrio parahaemolyticus (OD of amplification in advance
600Be about 0.8, additional proportion is 1: 100), be placed on 30 ℃ of constant temperature shaking tables, 250r/min cultivates 12-24h.
(3) spot measuring specific phage has or not
Vibrio parahaemolyticus is double-deck to detect dull and stereotyped preparation: 1. the high salt TSB of high-temperature sterilization 1.50%agar solid medium, and room temperature is placed to about 40-60 ℃, gets 10-15ml and is poured onto in the culture dish, evenly is laid at the bottom of the ware, and room temperature placement 30min solidifies it; 2. will melt 40 ℃ of-48 ℃ of water bath heat preservations through 0.5% high salt TSB semisolid medium of high-temperature sterilization; 3. get Vibrio parahaemolyticus (10
6-10
8Pfu/ml) 0.2ml-0.8ml adds 0.5% high salt TSB substratum 3.6-5ml of step 2, and mixing is poured in the culture dish that step 1 makes, and room temperature is placed and solidified; 4. the double-deck flat board that detects of Vibrio parahaemolyticus is put 37 ℃ of heat insulating culture 1h.
Get above-mentioned sample 1ml after the Vibrio parahaemolyticus amplification, 8000rpm, centrifugal 15min, supernatant liquor is through 0.22 μ m filtering membrane filtration sterilization.Get 5 μ l points and detect on the flat board in Vibrio parahaemolyticus is double-deck, 37 ℃ of incubated overnight, observation has or not plaque, if the step of carrying out (four) is arranged.(above operation is all carried out under aseptic condition)
(4) amplification of the separation of phage sample and phage sample
The sample of plaque will be presented in the double-deck detection of Vibrio parahaemolyticus flat board in the step (three), through dilution, purifying as follows: the high salt TSB flat board that will prepare, put 37 ℃ of balance 0.5h, make its surface temperature reach room temperature (can omit this step) if place at ambient temperature; Get sample and 4ml 0.5% high salt TSB substratum mixing after 0.4ml logarithmic phase (between the OD 0.4-0.8), the 0.1ml amplification, fall on flat board, vibration is paved it gently, and room temperature is placed about 0.5h, and the surface is thoroughly solidified; Double-layer plate is put 37 ℃ cultivate the 4-18h observation; Select single plaque.With select phage mono-clonal, add in the corresponding bacterial classification of 2ml logarithmic phase, cultivate 2h-6h for 37 ℃, observe.Repeated cloning step 3-5 time obtains phage mono-clonal sample, and with its called after BPH-VP-1.
(5) purifying of phage
Using host bacterium Vibrio parahaemolyticus highly increases phage BPH-VP-1, after treating the substratum clarification, the centrifugal 10min of 8000g takes out impurity, and adding solid polyethylene glycol (PEG8000) to final concentration is 10%w/v, stirring and dissolving, 4 ℃ are spent the night, and 4 ℃ of centrifugal 20min of 8000g abandon precipitation, be resuspended in damping fluid (100mM NaCl, 5mM MgSO
4, 10mM pH7.2Tris-HCl) in, in 37 ℃ of insulation 30min, during vibration for several times.Add chloroform, whirlpool mixing 30min, 3000rpm, centrifugal 5min carefully collects water, and 4 ℃ of dialyzed overnights (molecular weight cut-off 3kd) are removed salt, repeat dialysis once.It is stand-by that the phage dialyzate that obtains is put 4 ℃ of preservations.
Embodiment 2: toxicological experiment
Kunming mice, cleaning level, body weight 22 ± 3g, 2 monthly ages (conformity certification number: SCXK (capital) 2004-0001), provide by consonance medical university laboratory animal research center.Experimental animal is raised in cleaning level receptacle, room temperature 18-22 ℃, relative humidity 40%-70%, 12h sunshine and dim circulation.20 of experiment mices, male and female half and half, adaptability was raised after 3 days, was divided into two groups (phage group, control groups) at random, 10 every group (each 5 of male and female), giving phage batch total amount is 10
10The phage BPH-VP-1 of pfu/kg, control group gives equivalent physiological saline, and successive administration 15d puts to death the disconnected neck of experimental mouse, checks the internal organ situation.
Test-results shows that the phage BPH-VP-1 of this metering is to the not influence of mouse daily behavior.Dissect and check the internal organ no abnormality seen.
Embodiment 3: phage BPH-VP-1 stability is measured
1. phage method of counting (tiring)
Gained phage sample is diluted by 10 multiple proportions example, get the wherein sample 100 μ l of certain Dilution ratio, spread double-deck plate according to the method in the embodiment 1 described step (four), the plate of getting proper ratio calculates the plaque number, and a plaque is represented a phage monomer.
2. the phage sample is adjusted to pH 6.0,6.5,7.0,7.5,8.0 respectively, respectively at 4 ℃ and room temperature (Zhuhai, room temperature is at 25-33 ℃) placement, after sample preparation is finished, at first measure initially tiring of phage, measure tiring of phage at setting-up time then.Test result is seen Fig. 1 and Fig. 2.
The result shows: when room temperature keeps 4 days, and the small size rising of tiring of phage.Under each pH condition, the activity in 14 days all keeps more than 60%, is good with pH8.0 especially.In the time of 4 ℃, the active maintenance effect of phage is better than room temperature.Having good stability of phage is for the preservation of phage with use the condition of providing convenience.
Embodiment 4: detect the sterilization effect of phage sample in seawater
1. phage method of counting: with embodiment 3 described methods.
2. Vibrio parahaemolyticus method of counting (tiring)
Adopt TCBS vibrios selective medium, use the method counting of coating, cultivate 24h, the positive bacterium colony of blue-greenish colour bacterium colony for 37 ℃.
3. method of seawater treatment
Seawater is collected in Tang Jiawan marine site, Zhuhai, with the centrifugal 10min of seawater sample 3000r/min that gathers, takes out insolubles, with the disposable filter filtration of 0.22 μ l, uses sample through checking no bacterial growth promptly to get to test.
4. sterilization effect experiment in the seawater
By the dilution of 10 multiple proportions example, wherein a part is measured by method among the embodiment 2 and is tired with the phage sample, and another part detects sterilization effect as follows:
Get 2ml 1.4 * 10
8The Vibrio parahaemolyticus of pfu/ml logarithmic phase, the centrifugal 10min of 4000r/min abandons supernatant, and the seawater after precipitation is handled with equivalent hangs again, and is standby; Take the seawater sample 98ml after the processing respectively, add 1ml respectively through the Vibrio parahaemolyticus sample of suitably dilution and the phage sample of the suitable dilution of 1ml warp, make the final concentration of Vibrio parahaemolyticus be respectively 100cfu/ml and 1000cfu/ml, the inoculum density of phage sample is respectively 10
6Pfu/ml and 10
7Pfu/ml; Detect the residual quantity of Vibrio parahaemolyticus respectively at 1h, 2h, 3h, 4h, 5h.
Test-results shows: the phage concentration in seawater reaches 10
7-10
8During pfu/ml, the inactivation ratio of Vibrio parahaemolyticus is reached 100%.When phage concentration 10
6During pfu/ml, the inactivation ratio of Vibrio parahaemolyticus in the seawater is reached more than 99%.Test result is seen Fig. 3 and Fig. 4.
Embodiment 5: the transportation pnagus medius is to the influence of Vibrio parahaemolyticus residual quantity in the body of shellfish sample
1. phage method of counting: with embodiment 3 described methods.
2. Vibrio parahaemolyticus method of counting: with embodiment 4 described methods.
3. the sterilization effect evaluation of shellfish sample
Fresh shellfish sample with buying from the market after water is rinsed the surface well, divides into groups at random, is divided into control group successively, contrasts parallel group of parallel group, phage group, phage, and every group adds the 1L seawater, inoculate the 100cfu/ml Vibrio parahaemolyticus respectively.Control group and contrast parallel group and do not do other processing, phage group and phage add 10 for parallel group
7Pfu/ml phage sample.Measure the concentration (measuring method is with reference to GB-T4789.7-2003) of the Vibrio parahaemolyticus in the shellfish sample subsequently respectively at 0.5h, 2h, 4h.
Test-results shows: after using the phage processing, the number of Vibrio parahaemolyticus in the shellfish sample significantly reduces at short notice, show that phage can kill Vibrio parahaemolyticus in actual shellfish sample, can be than killing the Vibrio parahaemolyticus that might invade more completely.
Claims (6)
1. phage BPH-VP-1, deposit number is CCTCC M 209058.
2. the application of the described phage of claim 1 aspect the pollution of control Vibrio parahaemolyticus.
3. the described phage of claim 1 is used for preventing and treating the application of aquaculture, transportation and preservation process Vibrio parahaemolyticus pollution aspect as sterilant.
4. the described phage of claim 1 is used to prevent and treat the application of Vibrio parahaemolyticus pollution aspect as food additive.
5. the described phage of claim 1 is sprayed on the foodstuff production workshop as sterilant, is used for preventing existence and the breeding of this environment Vibrio parahaemolyticus, the application of Vibrio parahaemolyticus pollution aspect in the control food processing process.
6. the described phage of claim 1 is as a kind of application for the treatment of the medicine of Vibrio parahaemolyticus infection.
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