CN104845943A - Streptococcus phage and application thereof - Google Patents
Streptococcus phage and application thereof Download PDFInfo
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- CN104845943A CN104845943A CN201510268679.7A CN201510268679A CN104845943A CN 104845943 A CN104845943 A CN 104845943A CN 201510268679 A CN201510268679 A CN 201510268679A CN 104845943 A CN104845943 A CN 104845943A
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Abstract
The invention discloses a strongly-lytic streptococcus phage with wide host range. The preservation number of the streptococcus phage is CCTCC M 2014430, and the preservation date is September 19th, 2014. The streptococcus phage provided by the invention has the characteristics of strong lysis, high hydrophily, wide host range and the like, and cow mastitis caused by streptococcus can be prevented and cured by individual or complex use of the purified streptococcus.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of streptococcus phage bacterial strain and application thereof, particularly a strain broad host range, fine melt streptococcus phage and the application in control mammitis of cow thereof.
Background technology
Mammitis of cow is an international difficult problem, is also the disease that adult dairy cattle is the most expensive.Foreign data is reported, the sickness rate of mastitis is about 25-60%, and China's investigation finds, sickness rate 20-70%.In the U.S., the financial loss that mastitis causes every year reaches 2,000,000,000 dollars, and worldwide, annual milk-producing loss counts roughly 3,800,000 tons.Mammitis of cow not only brings massive losses to the economic benefit of milk cow production, and some mastitis pathogenic bacteria easily produces various noxious products, causes milk and milk products to pollute, brings significant damage to human health.Therefore, the key measure becoming Cow product of mastitis how is prevented.
Streptococcus is the important pathogen causing mammitis of cow.The streptococcus pathogenic agent of mammitis of cow is caused to be mainly the streptococcus agalactiae (Streptococcus agalactiae) of Lan Shi B group, C group streptococcus dysgalactiae (Streptococcusdysgalactiae) and streptococcus uberis (Streptococcus uberis), wherein streptococcus agalactiae and streptococcus dysgalactiae are contagion pathogen, and streptococcus uberis is the pathogenic pathogenic agent of environment.Due to the diversity of pathogenic bacterium, the continuous generation of Resistant strain, suis is more and more strong to the resistance of common antibiotics, brings very large difficulty to control.Although microbiotic remains the Main Means controlling mammitis of cow at present, but depend merely on microbiotic and can not fundamentally solve mammitis of cow problem, the invalid mastitis case of antibiotic therapy gets more and more, so, find novel antibacterial, biocide preparation extremely urgent, exploring the methods for the treatment of of seeking new mammitis of cow has become the important topic that the whole world faces.
Rockefeller University of U.S. professor Vincent takes the lead in having carried out the research adopting streptococcus pneumoniae phage and lyase treatment pneumonia thereof, and this has reopened the research of the phagotherapy of bacteriosis.Phage is the virus of a class bacterial infection, widely distributed at occurring in nature, in close relations with bacterium, can not to cause in body flora imbalance or cause target flora to occur resistance.In streptococcus phage, the U.S. has carried out extensive research for streptococcus pneumoniae (Streptococcus pneumoniae) phage, and obtains many strains lytic phage and part lyase.The research that Loeffler etc. deliver on Science magazine shows: the lywallzyme Pal of its S.pneumoniae phage Dp21 obtained can make the CFU of 15 strain S.pneumoniae reduce by 1 × 10 in 30s
4/ mL, wherein 3 strains have strong resistance to mould.At present, the domestic relevant report not yet having streptococcus phage to treat mammitis of cow.In view of the specificity of phage to host bacteria dissolved destruction and the many advantages of Phage therapy, therefore, phage is considered to a kind of effective means for the treatment of mammitis of cow and environment disinfected.
Summary of the invention
The technical problem to be solved in the present invention is, provides a kind of broad host range, fine melt streptococcus phage.
The technical problem that the present invention also will solve is, provides the application of above-mentioned phage in control mammitis of cow.
For solving above technical problem, the present invention adopts following technical scheme:
A kind of streptococcus phage, this Phagus is in Myoviridae, and head is regular polygon, and head diameter is about 78nm, and tail is about as 118nm.By this phage called after vB_StrM_L1, in China typical culture collection center preservation, depositary institution address is: Luo Jia Shan, wuchang, wuhan, postcode is 430072, preserving number: CCTCC M 2014430, Classification And Nomenclature: streptococcus phage (Streptococcus phage), preservation date is on September 19th, 2014.
In March, 2014 this phage collect from the sewage of Xi Gang diary farm, Nanjing, and gathering people is Bao Hongduo, and the telephone number of picker is 025-84391627.
Wherein, this phage has splitting action to streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis and Streptococcus viridans.
The application of above-mentioned streptococcus phage in preparation control mammitis of cow reagent is also within protection scope of the present invention.
A kind of pharmaceutical composition, this pharmaceutical composition comprises above-mentioned streptococcus phage.
Wherein, described pharmaceutical composition is made spray and is sprayed in cowshed, control streptococcic pollution, comprise and described pharmaceutical composition sprayed in the breeding environment of the body surface of milk cow, milk cattle cultivating utensil, milk cow.
Wherein, the pharmaceutical composition described in utilization carries out nipple dipping to the streptococcic milk cow of infection, control mammitis of cow.
Wherein, the pharmaceutical composition described in utilization carries out breast perfusion to the streptococcic milk cow of infection, control mammitis of cow.
Beneficial effect:
(1) phage specificities cracking mammitis of cow suis is adopted can to kill the suis with multi-drug resistant.
(2) streptococcus phage provided by the invention causes the suis pathogenic bacterium of mammitis of cow all to have stronger to kill activity to multiple;
(3) phage provided by the invention is product and the means of a kind of novel control mammitis of cow suis pathogenic bacterium;
(4) through mouse test, streptococcus phage toxic side effect provided by the invention is little, and security is high;
(5) streptococcus phage has good wetting ability, is easily mixed with flushing liquor, dip or injection liquid, kills environment or intramammary suis.
Accompanying drawing explanation
The plaque morphology of Fig. 1 phage.
The transmission electron microscope photo of Fig. 2 phage.
Fig. 3 temperature is on the impact of bacteriophage activity.
Fig. 4 pH value is on the impact of bacteriophage activity.
Fig. 5 phage sterilization effect in the medium.
The sterilization effect of Fig. 6 phage on milk cow hopper.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1: phage is separated and preparation.
It is streptococcus agalactiae reference culture ATCC12403 that the present invention tests with the Host Strains of phage.
Inventive samples picks up from Xi Gang diary farm, Nanjing sewage, gets supernatant and filters through double-layer filter paper, the centrifugal 20min of 10000rpm, then uses 0.22 μm of membrane filtration supernatant.Get 10ml and filter supernatant, add 0.5ml Host Strains overnight culture, then add aseptic CaCl
2after mother liquor mixes to final concentration 1.25mM, add 20ml THB substratum, room temperature effect 30min, then be positioned over 37 DEG C, after being cultured to 6 ~ 8h, get above-mentioned culture with 12000rpm, 4 DEG C, centrifugal 30min, gets supernatant; Get this supernatant 10ml again, add 0.5mL Host Strains overnight culture, add aseptic CaCl
2mother liquor adds 20mLTHB substratum after mixing to final concentration 1.25mM, cultivates centrifugal by above-mentioned cultural method, obtains the supernatant of enrichment; According to above test method again enrichment once, by supernatant 0.22 μm of membrane filtration of enrichment three times, form phage stoste.
Solid THB flat board is divided into 2 regions: draw Host Strains liquid 0.1ml and drip in dull and stereotyped centre, with painting rod, bacterium liquid is spreadable equably; After it dries, get phage stoste 10 μ l and drip in one of them region; After naturally drying, after being placed in 37 DEG C of incubators cultivation 10h, observing and drip phage region with or without plaque test.
If there is plaque test, prove that phage exists.Get phage stoste 100 μ l, carry out a series of 10 times of dilutions, get 10
-2, 10
-4with 10
-6the Host Strains liquid 0.1ml of each 0.1ml of diluent and incubated overnight mixes, after room temperature effect 15min, add about 4ml 0.6%LB substratum, THB culture medium flat plate upper strata is poured into rapidly after mixing, shake up horizontal 10min, treat that it solidifies, be placed in after 12h cultivated by 37 DEG C of incubators and observe, obtain the double-layer plate forming single plaque.
Embodiment 2: Phage amplification and purifying.
On the double-layer plate forming plaque, with the single plaque that the rifle choicest cut-off footpath of pipettor is larger, be inoculated in 3 ~ 5mL THB liquid nutrient medium, add phage Host Strains liquid 0.1mL, mixing, room temperature effect 15min, cultivate 10 ~ 14h, 12000rpm, 4 DEG C of centrifugal 10min for 37 DEG C, get supernatant, add 0.3% chloroform.Picking 4-5 single plaque, becomes equirotal plaque by phage purifying so repeatedly.
Get the Host Strains of 1mL fresh culture, add 0.3mL phage splitting liquid (with single phage culture and Host Strains respectively according to the ratio of 1:1,1:10 and 1:100).37 DEG C of incubation 20min, make phage particle be adsorbed in Host Strains; Add 100mL THB liquid nutrient medium, then add CaCl
2mother liquor is to final concentration 1.25mM, and 37 DEG C of shakes cultivate 12 ~ 16h, 12000rpm, and 4 DEG C of centrifugal 10min, get supernatant, obtain a large amount of phage splitting liquid.
In lysate, add RNase A, DNase I to final concentration is 1 μ g/mL, 37 DEG C of incubation 30min; Add 9.3gPEG 8000,5.8g NaCl, shake up to dissolving, ice bath 1h or 4 DEG C spends the night; 4 DEG C of centrifugal 10000rpm 10min, remove supernatant liquor; Add 2mL SM liquid, abundant washing precipitation, room temperature effect 1h; Add isopyknic chloroform, gentle vibration 30s; 4 DEG C of centrifugal 10min of 5000rpm, to be separated organic phase and aqueous favoring, reclaim the aqueous favoring containing phage particle, obtain the phage of purifying, and double-layer plate detects the phage particle (Fig. 1) of purifying.
Embodiment 3: phage transmission electron microscope detects.
The phage particle getting purifying does electron microscopic observation, add 10 μ l sample drop on copper mesh, treat its precipitation 15min, suck unnecessary liquid with filter paper, phospho-wolframic acid (PTA) with 2% dyes 1-2min, and after dry, transmission electron microscope (Hitachi H-7650) is observed.As shown in Figure 2, Phagus is in Myoviridae, and head is regular polygon, and head diameter is about 78nm, and tail is about as 118nm.
Embodiment 4: temperature and pH are on the impact of phage.
Get 0.1ml 1 × 10
8the phage of pfu/ml purifying acts on 60min respectively in 30 DEG C ~ 90 DEG C water-baths, tires surveying it after sample cooling; Get the peptone water and 1 × 10 of pH3.0 ~ 12.0 respectively
8pfu/ml purified phage balanced mix, surveys it after 37 DEG C of water-bath effect 2h and tires.
As shown in Figure 3, after phage acts on 1h respectively at 30 ~ 70 DEG C, it is active in noticeable change for result; After 80 ~ 90 DEG C of effect 1h, survive without phage.
Result as shown in Figure 4, when pH is 3, can't detect phage; When pH is 5.0 ~ 10.0, it is tired and there was no significant difference of initially tiring; And when pH is 11, can't detect phage.
Embodiment 5: the safety experiment of phage.
6 ~ 8 week age female SPF BALB/c mouse, mean body weight 25 ± 2g, totally 40, purchased from Yangzhou University's comparative medicine center.Mouse is divided into 2 groups at random, often organizes 20; Wherein one group of oral phage vB_StrM_L110
8pfu/0.25mL/ only (embodiment 2 provides); The oral equal-volume PBS of control group.After continuous oral 14d, often de-lethal 5 mouse of neck of group, observe internal organ, digestive tube and mucous membrane changing conditions; Often organize remaining 15 to continue to feed, get its ight soil every day and detect phage number change.
Result shows, and this dosage phage does not affect mouse health and daily behavior, dissects inspection and does not show any abnormalities, and after oral phage terminates 5 days, in stool in mice, can't detect phage.
Embodiment 6: phage host range is analyzed.
Do this experiment with the phage vB_StrM_L1 provided of embodiment 2, tired and be adjusted to 1 × 10
8pfu/mL is for subsequent use.
Test and Selection 3 strain streptococcus agalactiae reference culture and 40 strains are from suffering from the multi-drug resistant strains of streptococcus (comprising streptococcus agalactiae, Streptococcus viridans, streptococcus dysgalactiae and streptococcus uberis) be separated the sick ox of mammitis of cow, the host range of phage is analyzed, concrete operations are as follows: get streptococcic overnight culture 100 μ l, drip in 1.5%THB culture medium flat plate central authorities, with painting rod, they are coated uniformly lawn.Then each flat board is divided into two regions, one of them region, get 10 μ l phages and drip on lawn surface, another region drips 10 μ l physiological saline and compares, and is inverted in 37 DEG C and cultivates 12 ~ 16h, observations after droplet drying.
Result is as shown in table 1, and this phage has splitting action to 26 strains in 43 strain suis, cleavage rate 60.46%.Illustrating that this phage has the characteristic across kind of cracking, is a kind of phage of broad host range.
The host range analysis of table 1 phage
Embodiment 7: phage sterilization effect in the medium.
Get the suis bacterium liquid 1.0mL of incubated overnight, with LB by its OD
600nmvalue is adjusted to 1.0 and (is about 1 × 10
9pfu/mL), (tire is 1 × 10 to add phage vB_StrM_L1
8pfu/mL), finally its OD is adjusted with LB
600nmbe about about 0.5, in 37 DEG C of quiescent culture, simultaneously not add phage as a control group.Cultivate 0,1,2,3,4,5,6,7h detects OD
600nmthe change of value.
The results are shown in Figure 5, compared with bacterial controls group, phage vB_StrM_L1 treatment group, after 1h, suis quantity sharply declines, and during 3-4h, streptococcic quantity is controlled to lower value, and, OD during 5-7h
600nmclose to 0, phage vB_StrM_L1 is described, and sterilization is rapid and thorough in the medium.
Embodiment 8: the suis that phage controls in the environment of diary farm pollutes.
Be 10 by concentration
5the suis of cfu/ml is sprayed at milk cow hopper surface, then with concentration for 10
8pfu/ml phage vB_StrM_L1 implements to spray to hopper, after 2h, starts detection of streptococcus.
Detected result such as Fig. 6 shows, and after 2h, the streptococcic quantity in hopper surface drops to below 1000CFU, and after 5h, the streptococcic quantity in hopper surface drops to below 10CFU, illustrates that this phage effectively can kill the suis in breeding environment.
Embodiment 9: phage Quality Initiative coccus mammitis of cow is tested.
This test is carried out in Xi Gang diary farm, Nanjing, chooses 12 holstein cows lactation period of suffering from recessive mammitis of cow (microbial primarily of hammer) in field and carries out therapeutic test.Be divided into two groups at random, phage experimental group and microbiotic control group, often organize 6 cow heads.(tire eventually is 10 to the phage diluent of the purifying that experimental group ox embodiment 2 provides
8pfu/mL), before milking He after milking, carry out nipple dipping with phage, nipple dipping every day 3 times, each 1min at every turn, continue use 30 days.The ox of control group is treated according to the antibiotic therapy mode that diary farm is conventional.After the treatment phase terminates, application Beijing mastitis reagent (BMT) the latent mammitis sickness rate to tested milk cow is added up.
Result is as shown in table 2, and before and after test, the positive udder region of phage dipping group bovine subclinical mastitis has lacked 11, and morbidity head number decreases 5; The positive udder region of microbiotic control group ox latent mammitis decreases 3, and morbidity head number decreases 2.Experimental result shows, phage has certain prevention effect to mammitis of cow.
The recall rate of table 2 latent mammitis
Embodiment 10: phage controls milk cow feed streptococcus intermedius and pollutes.
It is 10 that the suis of incubated overnight is adjusted concentration
5cfu/ml, to be evenly sprayed at the 100g silage surface of spreading out by 1ml bacterium liquid with miniaturised nebuliser (bottle), then by the phage vB_StrM_L1 of purifying with 1ml 10
8the concentration of pfu/ml is implemented to spray, and starts detection of streptococcus after 2h.
Detected result shows, with 10
8pfu/ml concentration is implemented to spray rear 4h, and in silage, the quantity of Host Strains drops to below 1cfu/g, illustrates that this phage can effectively kill the suis polluted in milk cow feed.
Claims (7)
1. a streptococcus phage, is characterized in that, the deposit number of this streptococcus phage is: CCTCC M2014430, and preservation date is on September 19th, 2014.
2. the application of streptococcus phage according to claim 1 in preparation control mammitis of cow medicine.
3. a pharmaceutical composition, is characterized in that, this pharmaceutical composition comprises streptococcus phage according to claim 1.
4. the application of pharmaceutical composition according to claim 3 in preparation control mammitis of cow medicine.
5. the using method of pharmaceutical composition according to claim 3, is characterized in that, this pharmaceutical composition is made spray and is sprayed in cowshed.
6. the using method of pharmaceutical composition according to claim 3, is characterized in that, pharmaceutical composition is carried out nipple dipping to milk cow.
7. the using method of pharmaceutical composition according to claim 3, is characterized in that, utilizes this pharmaceutical composition to pour into cow breast.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103029A (en) * | 2017-12-12 | 2018-06-01 | 江苏省农业科学院 | The bacteriophage of one plant of cleavable ox source Streptococcusagalactiae and its application |
| CN108272828A (en) * | 2017-12-13 | 2018-07-13 | 中国水产科学研究院珠江水产研究所 | One plant of Streptococcusagalactiae bacteriophage and its application |
| CN113082060A (en) * | 2021-04-30 | 2021-07-09 | 石河子大学 | Preparation and application of phage cocktail preparation for treating dairy cow mastitis |
| CN113207913A (en) * | 2021-04-30 | 2021-08-06 | 石河子大学 | Preparation and application of biological disinfectant for cow mastitis |
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| CN102198265A (en) * | 2011-03-22 | 2011-09-28 | 上海交通大学 | Method for degrading streptococcus suis biofilm by applying phage lyase |
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| CN102198265A (en) * | 2011-03-22 | 2011-09-28 | 上海交通大学 | Method for degrading streptococcus suis biofilm by applying phage lyase |
Non-Patent Citations (2)
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| BAI Q等: "Characterization and genome sequencing of a novel bacteriophage infecting Streptococcus agalactiae with high similarity to a phage from Streptococcus pyogenes", 《ARCH VIROL.》 * |
| 李陇平 等: "金黄色葡萄球菌烈性噬菌体的分离鉴定和最佳保存方法研究", 《中国畜牧兽医》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103029A (en) * | 2017-12-12 | 2018-06-01 | 江苏省农业科学院 | The bacteriophage of one plant of cleavable ox source Streptococcusagalactiae and its application |
| CN108103029B (en) * | 2017-12-12 | 2020-11-10 | 江苏省农业科学院 | Bacteriophage capable of cleaving bovine streptococcus agalactiae and application thereof |
| CN108272828A (en) * | 2017-12-13 | 2018-07-13 | 中国水产科学研究院珠江水产研究所 | One plant of Streptococcusagalactiae bacteriophage and its application |
| CN113082060A (en) * | 2021-04-30 | 2021-07-09 | 石河子大学 | Preparation and application of phage cocktail preparation for treating dairy cow mastitis |
| CN113207913A (en) * | 2021-04-30 | 2021-08-06 | 石河子大学 | Preparation and application of biological disinfectant for cow mastitis |
| CN113207913B (en) * | 2021-04-30 | 2022-06-14 | 石河子大学 | Preparation and application of biological disinfectant for dairy cow mastitis |
| US20220347241A1 (en) * | 2021-04-30 | 2022-11-03 | Shihezi University | Preparation of phage cocktail as therapeutic agent for cow mastitis and use thereof |
| CN113082060B (en) * | 2021-04-30 | 2024-03-08 | 石河子大学 | Preparation and application of phage cocktail preparation for treating dairy cow mastitis |
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