CN110129280A - Wide fragmentation pattern vibrio alginolyticus bacteriophage and combinations thereof, kit and application - Google Patents

Wide fragmentation pattern vibrio alginolyticus bacteriophage and combinations thereof, kit and application Download PDF

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CN110129280A
CN110129280A CN201910398914.0A CN201910398914A CN110129280A CN 110129280 A CN110129280 A CN 110129280A CN 201910398914 A CN201910398914 A CN 201910398914A CN 110129280 A CN110129280 A CN 110129280A
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vibrio alginolyticus
vibrio
bacteriophage
vap7
phage
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CN110129280B (en
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乔欢
陈海
徐旭凌
黄杰
费文斌
胡怿林
何四龙
丛郁
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Phagelux Nanjing Biotechnology Co ltd
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Phagelux Nanjing Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • 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
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2795/00032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • 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

Abstract

The invention belongs to the research and development technology fields of vibrio alginolyticus bacteriophage, and in particular to vibrio alginolyticus bacteriophage of wide fragmentation pattern and combinations thereof and kit.The vibrio alginolyticus bacteriophage is vibrio alginolyticus bacteriophage VAP7(Vibrio alginolyticus phage VAP7), deposit number is CCTCC NO:M 2018767.Also disclose the vibrio alginolyticus bacteriophage or vibrio alginolyticus bacteriophage composition or the application of reagent or kit in killing and/or prevention vibrios microorganism belonging to genus containing vibrio alginolyticus bacteriophage or vibrio alginolyticus bacteriophage composition.The present invention has host range wider, still has high toxicity to host strain under low concentration；And it can be preferably proliferated on non-pathogenic bacteria host；Large-scale industrial production can be achieved.The present invention can provide excellent strain resource for the application of phagotherapy.

Description

Wide fragmentation pattern vibrio alginolyticus bacteriophage and combinations thereof, kit and application
Technical field
The invention belongs to the research and development technology fields of vibrio alginolyticus bacteriophage, and in particular to the vibrio alginolyticus phagocytosis of wide fragmentation pattern Body and combinations thereof, kit and application.
Background technique
Vibrio alginolyticus is a kind of halophagia Gram-negative brevibacterium, and no gemma, pod membrane are under the jurisdiction of vibrionaceae, vibrio, It is a kind of common Marine Pathogenic Bacteria, quantity occupies first of seawater class vibrios.People can be caused wound infection, food poisoning, in The diseases such as otitis.Meanwhile it is also a kind of conditioned pathogen of the ocean waters cultivated animals such as fish, shrimp, shellfish, when environmental degradation opportunity The decline of body immunity function, vibrio alginolyticus sickly look are easily broken out.
Bacteriophage is a viroid for special cracking bacterium, and main chemical compositions are made of protein and nucleic acid, deposit extensively It is in soil, empty gas and water and organism, there is stronger specificity.Since it has powerful sterilizing ability, bacteriophage can It is used as a kind of bacterial-infection resisting preparation, many scholars at home and abroad's concern has been constantly subjected to since early 20th century.
About vibrio alginolyticus bacteriophage, Fu Hanqing et al. (" microbiology notification " 25 date of June in 2018) is isolated One plant of vibrio alginolyticus bacteriophage Ф V170, belongs to kind of an inner width by 7 plants in bacteriophage Ф V170 15 plants of vibrio alginolyticus of cleavable Spectrum；Optimal multiplicity of infection is 0.01；One step growth curve shows that incubation period is 10min, burst size 101.3；To 65 DEG C or more Temperature is sensitive.The bacteriophage has the potentiality of vibrio alginolyticus disease during treatment sea-farming.
Patent of invention CN103898015B discloses a kind of vibrio alginolyticus bacteriophage and its answering in sea cucumber disease prevention With bacteriophage has inhibiting effect to vibrio alginolyticus, generates in 12h without bacteriophage resistant strain.
Patent of invention CN107904213A discloses a kind of vibrio alginolyticus bacteriophage and the Bactericidal composite comprising the bacteriophage Object, bacteriophage can quickly inhibit the growth of vibrios in a short time, and inhibiting effect is held time length.In addition, the bacteriophage Safely, effectively, and high specificity, production cost is low, and single phagocytosis is compensated in the control process of pathogenic bacteria for bactericidal composition The deficiency of autogenic therapy.
Currently, in terms of the research of vibrio alginolyticus bacteriophage is concentrated mainly on bactericidal, for more cause of diseases The research of vibrio alginolyticus bacteriophage and its crack characteristic is rarely reported.To sum up, the vibrio alginolyticus for being badly in need of developing wide fragmentation pattern is novel Bacteriophage.
Summary of the invention
The purpose of the present invention is to provide vibrio alginolyticus bacteriophage of a kind of wide fragmentation pattern and combinations thereof, kit and answer With having the characteristics that wide fragmentation pattern.
Concrete scheme is as follows: a kind of vibrio alginolyticus bacteriophage of width fragmentation pattern, the vibrio alginolyticus bacteriophage is molten algae arc Bacterium bacteriophage VAP7 (Vibrio alginolyticus phage VAP7), deposit number are CCTCC NO:M 2018767.
Further, the vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage VAP7) is strong Property bacteriophage, have one in symmetrical head and longer tail portion, head is icosahedral symmetry, and major diameter L is 60~70nm, horizontal Diameter W is 60~70nm, L/W ≈ 1；Tail portion includes contractile flesh sheath and tailfiber, and flesh sheath length is 30~40nm, wide 5~10nm, Tailfiber has 3~6；And pH3~pH11, at 4 DEG C~65 DEG C under the conditions of there is patience, cultivate 8 under the conditions of MOI=0.001 ~10h, potency are 1.8 × 1010pfu/mL。
Further, the vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage VAP7) has Nucleotide sequence shown in SEQ ID No.1.
A kind of composition of the vibrio alginolyticus bacteriophage containing wide fragmentation pattern, the composition contain the vibrio alginolyticus and bite At least one of thallus VAP7 (Vibrio alginolyticus phage VAP7) and other Vibriophages.
Further, the composition contains vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage VAP7), vibrio alginolyticus bacteriophage VAP9 (Vibrio alginolyticus phage VAP9) and vibrio alginolyticus bacteriophage Two or more in VAP21 (Vibrio alginolyticus phage VAP21).
Further, the composition includes vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage It VAP7), further include one of each bacteriophage mutants or a variety of；The mutant at least 90% and corresponding Vibriophage Native sequences it is identical.
A kind of width vibrio alginolyticus bacteriophage of the fragmentation pattern or reagent or kit of vibrio alginolyticus bacteriophage composition.
A kind of vibrio alginolyticus bacteriophage of width fragmentation pattern or vibrio alginolyticus bacteriophage composition or contain vibrio alginolyticus Answering in vibrios microorganism belonging to genus is being killed and/or prevented to the reagent or kit of bacteriophage or vibrio alginolyticus bacteriophage composition With:
The vibrios microorganism belonging to genus includes vibrio alginolyticus V.alginolyticus, Vibrio anguillarum V.anguillarum, comma bacillus V.cholerae, non-Nonther conserved quantity non-O1V.cholerae, Fermi operator V.fischeri, vibrio fluvialis V.fluvialis, Vibrio harveyi V.harveyi, sick Vibrio damsela V.ordalii, vibrio parahaemolytious V.parahaemolyticus, Vibrio salmonicida V.salmonicida, vibrio mimicus V.mimicus, Vibrio splindidus V.splendidus, Vibrio aureus V.azureus, Vibrio gazogenes V.gazogenes, Vibrio vulnificus V.vulnificus, bamboo Pod vibrio piscium V.trachuri, damselfish vibrios V.damsela, floating vibrios V.natriegen, clam worm vibrios V.nereis, fish Enteron aisle vibrios V.ichthyoenteri, shark vibrio V.carchariae, Vibrio campbellii V.campbellii and kill prawn One of vibrios V.penaeicida or a variety of.
Vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage VAP7) of the present invention is in bacteriophage The influence etc. of potency, the optimal multiplicity of infection (MOI) for cracking vibrio alginolyticus, pH stability, temperature to bacteriophage survival stability Aspect have following physiological property and the utility model has the advantages that
(1) potency (referring to table 1) with higher, wherein vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus Phage VAP7) infection vibrio alginolyticus best MOI be respectively 1:1000；And it is strong with high affinity and cracking ability Bacteriophage can crack rapidly vibrio alginolyticus (table 2)；Vibrio alginolyticus bacteriophage VAP7 (the Vibrio alginolyticus Phage VAP7) the impacted minimum of growth activity when pH is 8；It is relatively preferable to the thermal stability of temperature；It is suitable for 4 DEG C low It is gentle to put.
(2) vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage VAP7) virulence genes or not Well-founded because；There is wider host range for examination bacteriophage, to the discriminations of 180 plants of vibrio alginolyticus up to 95%.Three plants of cracking Rate can achieve 96.7%.
(3) vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage VAP7) is to non-pathogenic bacteria Cracking: only can recognize 10 plants of vibrio parahemolyticus bacterial strains in 3 plants, 5 plants floating vibrios in 1 plant and 3 plants of Vibrio aureus In 1 plant.Further, vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage of the present invention Or combinations thereof VAP7), object can not be identified exists including 7 plants of staphylococcus aureuses, 2 Hygrophilous monads and 6 plants of Escherichia coli Interior non-pathogenic bacteria.
(4) vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage VAP7) and nonhost cause a disease Property bacterium interaction, can not identify 62 plants for examination nonhost pathogenic bacteria in any one plant.
(5) vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage VAP7) has following advantage: It has high toxicity for stringent virulent phage and to host strain；With wider host range；In non-pathogenic bacteria place Your Majesty can preferably be proliferated；It can carry out large scale fermentation culture；Its culture solution can stable survival at room temperature, at 4 DEG C can be reserved for 12 months.The present invention does not carry out any genetic modification to for examination bacteriophage.Therefore, vibrio alginolyticus bacteriophage VAP7 of the invention (Vibrio alginolyticus phage VAP7) can provide excellent strain resource for exploitation phagotherapy, and have Good application and development prospect.The more vibrio alginolyticus of cocktail thereof cleavable of heretofore described vibrio alginolyticus bacteriophage, Cleavage rate has stronger cracking performance up to 96% or more.
(6) vibrio alginolyticus bacteriophage VAP7, VAP9 and VAP21 of the present invention or combinations thereof object can be by art technologies Personnel's record according to the present invention and common sense in the field are prepared into the medicine for being applied to disease caused by treating or preventing vibrio alginolyticus Agent or reagent.
(7) product form of vibrio alginolyticus bacteriophage VAP7 of the present invention or combinations thereof object includes but is not limited to carrier The forms such as carrying, concentration sprinkling or medicament immersion are applied to the ranges such as controlled host's body surface, life water body；As embodiment party One of case, it includes but is not limited to property of water-bearing carrier that the carrier, which carries form,；Concentration sprinkling form includes but is not limited to shrimp seedling spray It spills；It includes but is not limited to purificant etc. that medicament, which impregnates form,.
(8) vibrio alginolyticus bacteriophage VAP7 of the present invention is prepared to be applied to the fast of vibrio alginolyticus as effective component Fast detection reagent or kit.Its include but is not limited in the form of test paper, kit etc. to the vibrio alginolyticus in target sample into Row detection, or the target pathogenic bacteria in clinical sample are screened, it can effectively ensure the sensitivity detected.
(9) vibrio alginolyticus bacteriophage VAP7 of the present invention or combinations thereof object, which is produced, is applied to ring frequently as effective component The various products of border disinfection, for example including but be not limited to impregnate by liquid, sprinkling, with property of water-bearing carrier in combination use etc. in the form of pair Water distribution system, aquaculture facility or other environmental surfaces carry out disinfection decontamination, and the growth and activity of target bacteria can be effectively controlled. The liquid impregnates, sprinkling form includes but is not limited to disinfectant etc.；The property of water-bearing carrier includes but is not limited to that phosphate is slow Fliud flushing, seawater etc..
Figure of description
Fig. 1 is the vibrio alginolyticus bacteriophage VAP7 morphosis schematic diagram of Electronic Speculum identification.
Specific embodiment
Following embodiment does not limit effective range of the invention for the present invention is further explained in any manner.
In following instance, involved bacterial strain code name is the naming method number of our company.
Vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage VAP7), deposit number CCTCC NO:M 2018767, depositary institution are China typical culture collection center, and the preservation time is on November 09th, 2018, preservation Address is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University；
Vibrio alginolyticus bacteriophage VAP9 (Vibrio alginolyticus phageVAP9), deposit number are CCTCC NO:M 2018766, depositary institution is China typical culture collection center, and the preservation time is on November 09th, 2018, and preservation address is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University；
Vibrio alginolyticus bacteriophage VAP21 (Vibrio alginolyticus phageVAP21), deposit number are CCTCC NO:M 2018768, depositary institution is China typical culture collection center, and the preservation time is on November 09th, 2018, and preservation address is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University.
In following instance,
The formula of the TSB fluid nutrient medium are as follows: tryptone 17g, soybean papain digestion object 3g, sodium chloride 5g, phosphorus Acid dihydride potassium 2.5g, glucose 2.5g, distilled water 1000ml, pH 7.0.
The formula of TSB (2%NaCl) culture solution are as follows: tryptone 17g, soybean papain digestion object 3g, chlorine Change sodium 20g, potassium dihydrogen phosphate 2.5g, glucose 2.5g, distilled water 1000ml, pH 7.0.
The formula of the TSA solid medium are as follows: tryptone 15g, soybean papain digestion object 5g, sodium chloride 5g, agar 15g, distilled water 1000ml, pH 7.0.
Semi-solid agar culture medium prescription are as follows: tryptone 17g, soybean papain digestion object 3g, sodium chloride 5g, phosphorus Acid dihydride potassium 2.5g, glucose 2.5g, agar 7g, distilled water 1000ml, pH 7.0.
SM formula of liquid are as follows: sodium chloride 8.5g, magnesium sulfate 2g, 1mol/L Tris-HCl 50ml, gelatin 0.25g, distilled water 1000ml。
Vibrio alginolyticus bacteriophage VAP7 (Vibrio alginolyticus phage VAP7), vibrio alginolyticus bacteriophage VAP9 (Vibrio alginolyticus phage phage VAP9) and vibrio alginolyticus bacteriophage VAP21 (Vibrio Alginolyticus phage VAP21) composition (1:1:1) be according to embodiment 8 method prepare.
1 vibrio alginolyticus bacteriophage VAP7 of embodiment is isolated and purified
20ml supernatant is taken after acquiring Fujian marine product sample and Hainan Seawater Samples each 50ml, 5000rpm centrifugation 10min respectively 2 times of TSB fluid nutrient mediums of itself and 20ml and 2ml, are in the vibrio alginolyticus VAHN1 bacterium solution of logarithmic phase by liquid and degerming (108Cfu/ml) uniformly mixing, 150rpm shakes training overnight at 30 DEG C, is enriched with bacteriophage.Sample pregnant solution 5000rpm is centrifuged 10min, the miillpore filter degerming for taking supernatant to cross 0.22 μm obtain the filtrate containing bacteriophage.Take 50 μ l of filtrate and its host molten 300 μ l of algae vibrios bacterium solution is uniformly mixed, and standing 15min combines it sufficiently with the receptor of bacterium surface.Above-mentioned mixed liquor is added Enter 4ml to be cooled in 50 DEG C of semi-solid agar culture medium, be laid on immediately after mixing on the TSA plate solidified, it is solidifying to agar Gu 6-8h is cultivated in 30 DEG C of inversions after, observes plaque growth situation.On the double-layer plate for forming plaque, with sterile pipette tips The big and bright plaque of picking, 0.22 μm excessively of miillpore filter degerming obtains bacteriophage filter after oscillation desorption in 1ml SM liquid Liquid is inoculated in 5ml TSB fluid nutrient medium, and 0.1ml vibrio alginolyticus bacterium solution is added and mixes, 150rpm is stayed overnight at 30 DEG C Training is shaken, 5000rpm centrifugation 10min is filtered after taking supernatant with bacteria filters, observes plaque morphology using double-layer agar technique.It repeats After operation 3-5 times, the consistent plaque of shapes and sizes can be obtained.It is bitten from isolated vibrio alginolyticus in the marine product sample of Fujian Thallus VAP7.Vibrio alginolyticus bacteriophage VAP7 generates single round plaque on vibrio alginolyticus lawn, and center is bright, side Edge has haloing, diameter 5-6mm；Vibrio alginolyticus bacteriophage VAP7 transmission electron microscope photo is referring to Fig. 1.
The deposit number of vibrio alginolyticus bacteriophage VAP7 is CCTCC NO:M 2018767.By nucleotide sequencing, molten algae Vibriophage VAP7 has the nucleotide sequence of SEQ ID NO:1.
The measurement of 2 vibrio alginolyticus bacteriophage VAP7 potency of embodiment
Dilution is done with SM liquid, 10 times of gradients of stoste of vibrio alginolyticus bacteriophage VAP7 (being made by embodiment 1) are diluted step by step To l08Times.L0 is taken respectively5、l06、l07And l08The bacteriophage culture solution l000 μ l of dilution and its host strain vibrio alginolyticus VAHN1 300 μ l of bacterium solution is uniformly mixed, and standing 15min combines it sufficiently with the receptor of bacterium surface.It is cold that 4ml is added in above-mentioned mixed liquor But it into 50 DEG C of semi-solid agar culture medium, is laid on immediately after mixing on the solid agar plate solidified, to agar solidification 6-8h is cultivated in 30 DEG C of inversions afterwards.Each dilution need to do three Duplicate Samples, and when counting takes three Duplicate Samples of this dilution Average.Wherein, phage titer (PFU/ml)=average plaque number × extension rate
As can be drawn from Table 1, vibrio alginolyticus bacteriophage VAP7 has 10 after cultivating 12h10The potency of PFU/ml or more.
Table 1 cultivates the potency of vibrio alginolyticus bacteriophage VAP7 after 12h
Measurement of the 3 vibrio alginolyticus bacteriophage VAP7 of embodiment to vibrio alginolyticus optimal multiplicity of infection (MOI)
Picking vibrio alginolyticus single bacterium colony is inoculated into the test tube for filling 3mlTSB (2%NaCl) culture solution, in 30 DEG C of shaking tables 150rpm shaken cultivation 12h, obtains host bacteria suspension.Bacteria suspension is transferred to l0ml TSB (2%NaCl) with 1:100 ratio Culture solution, 30 DEG C of 150rpm shaken cultivations to logarithm early period.The pure culture liquid of bacteriophage VAP7 is added according to infection multiplicity ratio (being made by embodiment 1) and host strain (MOI=bacteriophage quantity/bacterial number), TSB fluid nutrient medium, which is added, keeps each pipe total Volume is identical.150rpm shakes training overnight in 30 DEG C of shaking tables.5000g is centrifuged l0min and collects supernatant after culture, measures Phage titer.Each point makees double and answers pipe culture to be averaged, and is that best infection is multiple to generate the MOI of highest phage titer Number.Experiment is repeated 3 times.
The results are shown in Table 2, and vibrio alginolyticus bacteriophage VAP7 potency reaches highest (1.8x1010When PFU/ml), MOI For 1:1000.
The potency of vibrio alginolyticus bacteriophage VAP7 under the different infection multiplicities of table 2
The pH stability test of 4 vibrio alginolyticus bacteriophage VAP7 of embodiment
Above-mentioned EP pipe is placed in 25 DEG C of constant temperature after taking sterile EP tube to be separately added into the 900 μ l of TSB culture medium of different pH (1-14) In water-bath, l00 μ l thallus pure culture liquid is added after equalized temperature, stands 1h at room temperature.By sample after to the reaction time Double-layer agar technique is used to measure phage titer after doing appropriate dilution.Above-mentioned mistake is repeated respectively at 4h, 8h, for 24 hours and after 96h later Journey repeats experiment 3 times.
The results are shown in Table 3, its potency is without obvious after vibrio alginolyticus bacteriophage VAP7 is to handle 96h within the scope of 6-8 in pH Variation.
Table 3 reacts vibrio alginolyticus bacteriophage VAP7pH value stability after different time
A. pH stability (the initial potency: 5x10 of vibrio alginolyticus bacteriophage VAP79PFU/ml)
The heat stabilization test of 5 vibrio alginolyticus bacteriophage VAP7 of embodiment
Respectively take l00 μ l bacteriophage pure culture liquid loaded in sterile EP tube, respectively at acted in 55 DEG C, 65 DEG C, 75 DEG C of water-baths 2h, 24h,48h.Sample cell is taken out after action time and is immediately placed on cooling in ice bath, and double-layer plate is used after suitably diluting Method measures phage titer.Experiment is repeated 3 times.
The results are shown in Table 4, and vibrio alginolyticus bacteriophage VAP7 is easier to survive at 55 DEG C in experimental group.Vibrio alginolyticus phagocytosis Body VAP7 has good thermal stability, still there is higher potency after 65 DEG C of water-bath 2h.
Potency of the 4 vibrio alginolyticus bacteriophage VAP7 of table under different temperatures
The survival stability test of 6 vibrio alginolyticus bacteriophage VAP7 of embodiment
5m vibrio alginolyticus bacteriophage VAP7 pure culture liquid is taken to be sub-packed in sterile test tube, respectively at 4 DEG C, 25 DEG C, 30 DEG C of decentralizations It sets, phage titer is periodically measured using double-layer agar technique after suitably diluting.
The results are shown in Table 5, and 50 weeks potency of vibrio alginolyticus bacteriophage VAP7 storage still have 10 under the conditions of 4 DEG C9PFU/ml； VAP7, which stores the potency order of magnitude in 12 weeks, at 25 DEG C does not reduce (109PFU/ml)；Bacteriophage VAP7 can be tieed up in 12 weeks at 30 DEG C Hold 108PFU/ml and the above potency；Bacteriophage has compared with fine melt ability host strain under each storage temperature.Bacteriophage is suitable It is stored at 4 DEG C.
Survival stability of the 5 vibrio alginolyticus bacteriophage VAP7 of table under different storage temperatures
7 vibrio alginolyticus bacteriophage VAP7 virulence gene of embodiment or the detection test of bad gene delection
65 kinds of identified virulence genes (table 6) from lysogenic phage in pathogenetic bacteria body are chosen, by measuring secondary haemolysis Property Vibriophage VAP7 full-length genome simultaneously carries out bioinformatic analysis to it, to determine if containing above-mentioned virulence base Cause.
The results show that not containing following virulence gene for examination bacteriophage.For examination bacteriophage invariably well-founded because.
The main known toxicity gene of lysogenic phage in 6 pathogenetic bacteria body of table
8 vibrio alginolyticus bacteriophage VAP7 of embodiment is to vibrio alginolyticus cracking range test and vibrio alginolyticus bacteriophage VAP7, VAP9 And the preparation and cracking range test of VAP21 composition
The fragmentation pattern of bacteriophage is measured using drop method.Taking potency respectively is 1x108PFU/ml vibrio alginolyticus bacteriophage 3 plants of bacteriophages are uniformly mixed in SM liquid in equal volume, vibrio alginolyticus bacteriophage are made by the stoste of VAP7, VAP9 and VAP21 The cocktail thereof of the 1:1:1 of VAP7, VAP9 and VAP21.
The single colonie of 180 plants of vibrio alginolyticus of picking is inoculated in respectively in the test tube for filling 3ml TSB (2%NaCl), 150rpm cultivates 8h, and each strain bacterial solution is made.It takes 300 μ l bacteria suspensions to mix respectively with semisolid culturemedium and is laid on plain agar On plate, take the cocktail thereof drop of 10 μ l bacteriophage culture solutions and VAP7, VAP9 and VAP21 in the difference of plate respectively On position, it not can contact between when sample-adding each bacteriophage culture solution, in order to avoid influence test result.30 DEG C of culture 6- after natural air drying 8h observes result.Test is in triplicate.
The results are shown in Table 7, and vibrio alginolyticus bacteriophage VAP7 has wider host range.171 plants of VAP7 cleavable molten Algae vibrios, cleavage rate 95%.And the cocktail thereof liquid of VAP7, VAP9 and VAP21 (ratio 1:1:1) can recognize 174 plants Vibrio alginolyticus, cleavage rate 96.7%.Illustrate that vibrio alginolyticus bacteriophage VAP7 has wider host range, while bacteriophage chicken tail The limitation of host range when wine combination can make up bacteriophage single application, has greatly application latent in terms of Phage therapy Power.
The fragmentation pattern measurement result of table 7 vibrio alginolyticus bacteriophage VAP7, VAP9 and VAP21
Note: ++: cracking, plaque are very bright completely；+: cracking, plaque are bright completely；: it is uncracked
Breaking test of the 9 vibrio alginolyticus bacteriophage VAP7 of embodiment to non-pathogenic bacteria
33 plant non-pathogenic bacteria single colonies of the picking including vibrio parahemolyticus, Aeromonas hydrophila etc. are inoculated in respectively In the test tube for filling 3ml TSB, 150rpm cultivates 8h, and each strain bacterial solution is made.300 μ l bacteria suspensions are taken to train respectively with semisolid Base mixing is supported to be laid on plain agar plate.5 μ l bacteriophage culture solutions are taken respectively and (ratio is containing VAP7, VAP9 and VAP21 Cocktail thereof culture drop 1:1:1) is not can contact between when sample-adding each bacteriophage culture solution on the different location of plate, In order to avoid influencing test result.30 DEG C of culture 6-8h after natural air drying observe result.Test is in triplicate.
The results are shown in Table 8, in our current research, vibrio alginolyticus bacteriophage VAP7 10 plants of vibrio parahemolyticus bacterial strains of cleavable In 3 plants；Its cocktail thereof can recognize 5 plants in vibrio parahemolyticus.VAP7 can recognize 1 in 5 plants of floating vibrios simultaneously 1 plant in strain and 3 plants of Vibrio aureus.All bacteriophages can not identify staphylococcus aureus, Escherichia coli and thermophilic aqueous vapor Monad.
The cross reaction of 8 vibrio alginolyticus bacteriophage of table and cocktail and 33 plants of non-pathogenic bacterias
Note: ++: cracking, plaque are very bright completely；+: cracking, plaque are bright completely；: it is uncracked.
10 vibrio alginolyticus bacteriophage VAP7 of embodiment and the mutual of nonhost pathogenic bacteria are tested 62 plants of non-places of picking Main property pathogenic bacteria single colonie is inoculated in respectively in the test tube for filling 3ml TSB, and 150rpm cultivates 8h, and each strain bacterium is made Bacterium solution.It takes 300 μ l bacteria suspensions to mix with semisolid culturemedium respectively to be laid on plain agar plate.10 μ l bacteriophages are taken to train respectively Nutrient solution and containing vibrio alginolyticus bacteriophage VAP7, VAP9 and VAP21 (ratio 1:1:1) cocktail thereof culture drop in On the different location of plate, it not can contact between when sample-adding each bacteriophage culture solution, in order to avoid influence test result.After natural air drying 37 DEG C of culture 6-8h observe result.Test is in triplicate.
Bacteriophage is interacted with nonhost pathogenic bacteria and is reacted, the results show that VAP7 and cocktail thereof without 62 plants of method identification is for trying nonhost pathogenic bacteria (table 9).
The cross reaction of 9 vibrio alginolyticus bacteriophage of table and cocktail thereof and 62 plants of pathogenic bacterias
Note :-: it is uncracked.
It is prepared by the fermentation of 11 vibrio alginolyticus bacteriophage VAP7 of embodiment
Picking vibrio alginolyticus VAHN1 single colonie, is inoculated into the test tube for filling 3mlTSB (2%NaCl) culture solution, 30 DEG C of shaking tables Middle 150rpm shaken cultivation 12h, obtains host bacteria suspension.Bacteria suspension is transferred to 500ml TSB (2% with 1:100 ratio NaCl) culture solution, 30 DEG C of 150rpm shaken cultivations to logarithm early period simultaneously measure its bacteria suspension concentration.Vibrio alginolyticus bacteriophage VAP7 The system of fermentation preparation is 8L, and fermentation medium is TSB culture medium.The initial pH value of fermentation medium is 7.It is connect using flame Kind method inoculation, the bacteriophage (10 of 50ml is respectively connected to its corresponding optimal multiplicity of infection ratio into fermentation medium6PFU/ ) and logarithmic phase host bacterium solution (10 ml9CFU/ml).It is passed through filtrated air in fermentation process, and 3 ‰ defoaming agents are added, fermentation system The standby time is 12h.Every 2h takes the mixed liquor of 20ml bacteriophage and host strain in sterile chamber from sample tap since fermentation, 5000rpm is centrifuged 10min, and the miillpore filter degerming for taking supernatant to cross 0.22 μm obtains the filtrate containing bacteriophage and measures its effect Valence, method is referring to embodiment 2.To after fermentation, whole mixed liquors of bacteriophage and host strain be taken out from sample tap and are accessed In sterile chamber, 5000rpm is centrifuged 10min, takes supernatant to filter through vacuum pump to sterilizing filter device, is bitten Thallus fermentation liquid simultaneously saves at 4 DEG C.
As shown in Table 10, vibrio alginolyticus bacteriophage VAP7 in ferment 12h when potency up to highest, be 3.2x1010PFU/ml。 12h after fermentation phage titer by initial 104PFU/ml is increased to 1010PFU/ml or more improves 6 quantity Grade.Therefore, it is practicable for carrying out the large-scale industry preparation of bacteriophage using fermentation method.
The fermentation dynamic of 10 vibrio alginolyticus bacteriophage VAP7 of table
This specific embodiment is only explanation of the invention, is not limitation of the present invention, and those skilled in the art exist It can according to need the modification that not creative contribution is made to the present embodiment after reading this specification, but as long as in the present invention Scope of the claims in protection all by invention method.
Sequence table
<110>Fei Jileke (Nanjing) Biotechnology Co., Ltd
<120>wide fragmentation pattern vibrio alginolyticus bacteriophage and combinations thereof, kit and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 61885
<212> DNA
<213> Phage VAP7
<400> 1
ggcacgcaat cctttaggtt ttgtgcctgg tattgtttgg gtttgatgac gcccacaagg 60
acaagtgata gtcactgtca ttcctgtttt gcgatcacct ttatattcag atttggctgt 120
accgccacat ggacattgca ttactgttct ccttcaaaag agacataatc gccataaatg 180
tcgccttgtt tatcaacaac aacattcttt aatgtcagta atccaaacaa atgagactga 240
atctcattag cgacctcgtc agtcacgaca cacttgatct tgtcactagt atgcatagag 300
ctgaaaataa aaatgacctt gccatcatca tcgattggct cgtgtgtgag cacaatgact 360
ttgtatggac gaaggtcatc aggtttcttc ataaactttt tgtatataaa accaggtatc 420
ataatgaaat ctccgttcaa tacttgaatc tgaaacatca tatagtggtt ttaaattatg 480
aaacgttaac caggtcagaa aacaacctta tcaatggatc gcagtaataa cacgtccatc 540
actggttctc cgatttcttt gtagtttttg attttgtcaa caacatcatt catatcgaat 600
ttaacctgga gctgacgctt atagttgaat tcttcaacat taacgttttt tgatgccacg 660
agtgttggta cgccgtttaa taagatctta aacattgtaa gacctgggaa aggcgcaacc 720
ataatttccg catcataagt agttttagct gtttctttct tgaaaaagtt tacaaaagat 780
gcaaggctca taatactatc ctccagaatt ttgcccatca taccgtattt agaatcccat 840
gtaaagaaaa acccctcaca tggaggggtt ggagctaata attggcagag gccttgtttg 900
ttgtgatagt tctagagttt gatgactttc atctctggga acattttttg aagctcatcg 960
ataagctcaa cagaatcagg ctttggttct gctgcaacgt tgaagtcaac gaatacattg 1020
atatgccaag ctgtcatcag atttggttca acttgcatag gagcagtcat taggatgtta 1080
cccataccat tttgaacaac attgtatacc aggtcatctt tgaatttacg cactgtgatg 1140
tgttccaggt aaccgtttac caaagcatcc gccaagtaaa gcgttaggaa ctcgcttgac 1200
atttgacacg tagcgccatc aagttcattc atacccggga tagggccagt aaccatgtaa 1260
atcttgtcag cgtttttaga aattgcacag tcaatgatac ctagttgttt gcctttcaaa 1320
cccttgacag cttctgcgaa acgattagac ttcgcttgtt cgctttcaac ataaccacct 1380
tcgataagtt gaatcacttc tgctagcgac ttgtcattta caataaagtt gttttgacct 1440
agtacgcttg caaccatttc gattttctta tcaagtgaaa tttcagtttt cataatatat 1500
tccttgtaaa gggtttttag tgttgtttcg tttgatgtgt ccattatacc taacataaac 1560
ttttgaagtt ttacttaagt ttccaggctt cgtgtttacc acctttatgc cattccgcaa 1620
tctgcattgg gatcacaact tcccattggt ctgggtgcac ttcagcaagc tctgtacgga 1680
tatggtctga acggtatact ttaacagcat gttcagcata tttcagacta gcaaattgtt 1740
tgatgatatc ccaagttaga cgcagacgag tcttttcacg cagtgttgag tcatttttta 1800
gacgttgtaa ctctgcgaat aacattaaac ggaatcgagg aggcaaatag tggacattca 1860
gcccgaatag gtacatcacg cctttctcac caaatgaaac accatcaccc agcttcatgt 1920
taaagaaaaa cactaaaggc cagccatcgt acaccggtaa gatattcttc caacgtggat 1980
cgtagttata gaggaacatg cgaccaatga gccctttatc cgatggtttg gtacgacgac 2040
tctccttaag gatttgacgg tgtacaactt ccgctcgtag gtgggtatcc tttgtgatac 2100
gactcataaa ccatttgttc gattcacggg tatttcgtcg agcattcaac cctttagttt 2160
cgcgatattt cgaaatgtat cgcttaacga taggtggata gttttgctcg cgttgtaaca 2220
ataattgttc tttagccatt atgcttacct taaatacttt caaaatattt aggaaatgag 2280
ttatgcgaga gattagaaat tttattgctc atctgaagca acacggtatt tcacgaaata 2340
accgattccg tgtaacaatc ccactcccac cattagttga ccaggcactt aacccacctg 2400
gtgaccaaga tgcgatatca gatgctatca aaaatggctt ccgtgtcgta tcaattctga 2460
atggtgatgg cattgaagcg cgacgcggtt tacagatcat gtgttcgtca accgagttac 2520
caggtgtcaa cattgatact attgatgtca aacagggtgg tccatcgttc aagattgctt 2580
cgggcacttc taagtctgat atccagttta actttctgat cagtgcagac tgctttgaga 2640
agaacacact tgacctgtgg cgcaaagtca tttttgatag tgatacccgc aaagtggcat 2700
actatgacga ttataaggtg gatattcaaa ttgaggttct ggatgtcaaa gaccgtgttg 2760
tctacagtat caacctggaa gacgcttatc caatcctgtt gagtccaatc accctcaata 2820
agcgtgccgt agatgagatg atgcagtatc gttgtacttt tacctatgct aaggtcagcg 2880
agtttacaca gtcccaaggc atcttatctg gttcggttat tgaagaagta ttggatgatg 2940
tacagaatgg agattggcag tcggcattag ccacttcaag agaacagatc ctaaaagtcg 3000
cacaagggaa ttacactggt gttgcattgg aaatgcatgg gatgatttca gagactacca 3060
aacagattgt tgggatgaat gccactgacg caaaaggagt aatgtccaac tttattccat 3120
ctatccaaac taaccctgga atcagttctg tggacaaagg aagtctcact aagttggtta 3180
aagacttatt ctaataaaaa gccccagaat caatcctggg gctttttgat acgcttaata 3240
acctatagct tagctttgtt ctgcttcttt aagcattttt tcgaactcag ttagctggtc 3300
atcgccatca tcttcttgtt tggtttctgc tgcaccttgc tgagcaccag catcagtctc 3360
ttgacctttg tcttgaccga ttggagtatc ttcttcttgc tcaacaccag taagaccttc 3420
cattttacca ggtgctgcgc caactgaaga atcaactgcc atttgagctt caagtggtgc 3480
atcttcaggg atacccataa ccattttgaa gcgtttttca agttcttcat aggtcttgaa 3540
gttcttgcga tcgtagaagt cttgtagcga ataagtctgt ttccagattt cttcaatctt 3600
ggcttcatca ccaccgaaca gtgcgcttgg tttagcccag gttacacgag catagtcagg 3660
gctgatacaa tcacggccgt tgaacttacg cttttcacca gttaggttga attccaggtc 3720
cgcaccttcc caaggacaga aaacgtcttc aatctttgga tctgttggga acttaggagc 3780
agctgcagca tcaataattt tcttgatacc gcgaccaaat ttaaacaaga aaactttacc 3840
gttgttttca ggtttttgtg cgtcgttaat taccaggatg ttagcatagt aatccgtgtt 3900
tggtagacgc ttcatcaaga ttttcttcag accttcgtcg ttagtttctt tctgttgctt 3960
ccacagcggc tgatcatgct tacgtactgg gcaatcttca tcaaacgtcg ccaagctttt 4020
ctcgatgtat tttgaaccag tcgagccttc aaagaagtgg ctgataatac atgcacaagg 4080
agtcaacaca gcaccttcag gaatttcacc tttttcacgg cgttccatat ctaccattgg 4140
aaccggcagg aaacgaatag tattaacaga aacaaattcg ccaggtttac tttcttccca 4200
agtatgtttc caaatacgtt catccgcttt gaaaccacct tggccttgtt gtgccagacg 4260
ctctttaaga gctttagtgt tttgaccttt gtttgctttt agacgtgcaa gtagtgaaga 4320
catagtagtt accttatatt ttgtccatac tgaggactgg ttttggttta agtgtttgtt 4380
cttgtgaact cgtttatttt acacaacaaa ttaaattgaa gtttgaatat ttagccattc 4440
aattggcttg gcttatcgac aagttctaca taagccgttt caccttcaac aataacttca 4500
agcttctgga tgcggttaaa cggtgcaacc acagtcagat gatgagtcat tgaatcggat 4560
tccttaatca taccaggtgg ttgcttttga acatttgcta cacccggtat tttttctttg 4620
accgcatttg aaatccaagc actaatcatt ttaaaatcct cttagttttt gaacgttgtt 4680
tgattaggtt taaacggata ccctcgcctt ccagatcatt caataattgg tcgttgacat 4740
attgctttgc atcttcaacg gggatatcat attcttcgca agccgccaat aaagactgga 4800
ataaagtatc ttcgttgata ttcgcttgac gcttaacata taagctgaat tcgtcagcgt 4860
tgacaatagt gttagtttgc tttttcatac atcaataacc gatttcggcc ctgctttttt 4920
gtgaatgtct ttcattaggt ttttgaaccc ttctggcgca gccgtatggt ctttgacacc 4980
agttacagtg ccaactttcc caatcactaa cattaccttt gattcgccac atgttaagca 5040
aggttcttcc gtaggcatta atcgattacg gatactcagt gttgcagaag tgacttggtt 5100
gcagccatta catttgtaat tatacttcgg cattacaaca tacctcgcaa catgataagg 5160
aagaatgtga atgttgacca cattactgta taaacaccag ccttctcacc aggtttaact 5220
tgtaagatta acgatttgat aaccgcttct ggtacgtatg tactggtttc cattgcacgt 5280
gcctggaata acacactgtc atgcacacga gcatttaacc agatcatgaa gtgtgacatg 5340
gatttgaaga agcttaaagc cgcaacaaag ataagtacaa cttcaattgc tatttgataa 5400
gcatcaggat taatcactgt cgacattttc tgtctcctca ggggaataag ggttatatcc 5460
acagttgtca atgttatgga taacacgatt aaccatgaac agttttaaca attctttgta 5520
cttaggatct tcttcccaaa gtgtttcaag aattgtttgg tgggatttaa tcccctgttt 5580
gatgtaaagg tcgtaaatac gtaccataac aggggattta atgtgtccaa tatctttggc 5640
catgatgttt gtcctattcg tttaacacgt aggacatttt atcacttacc catcaattga 5700
agacggattt cggaaaggtc tggatggcag ataaggtttg cgaaagagtc aaccaggtca 5760
tcaaccggtt tttcgtactc tttgtttaga agtaactgtt ggtggagttt gatgccccat 5820
agctcgttga aacaatcaat catttgttct ttcttggcgt tacctttacc ttggaactgc 5880
atcttgactt gagttggcgg aatgatttca taatcaatat tcaggttgta aagctcttgt 5940
ttgagttggc cagtacactc accaatctgg aaaacacgat tcgacttgcc acccatcgca 6000
tagccttcta atgatgcttt cgttacttta tttgattgaa gtacggcacg ggcccaaagt 6060
gcgatctcac ggaagcgtgg ttcatcgcta tcaaattttt cttgagcaag gatcacaacg 6120
ttattgaact caccacattt tgatttggtt ttgccgtagt tgtaataacg taggttttcg 6180
tgagtaagtg gtagacttga atcccagata caaatagctg ggccgttcat tgagtagtcg 6240
atgccagcgt agatatcgcc gtcacctaag ataggttgaa tttctgattg tgcgttagac 6300
ataataaagc cctctagtaa ttataaaatt atttagaggg cttaaattct atggattaca 6360
cttgcaagtc aggcgcttgt tcagcttctt gctctttttg ttcagcaatg ttgccagcca 6420
tctggttcac caggtccaat gccatgttac gaagcatctc atggtgattg ttccaggctt 6480
cttcttcacc ctgatacagt tggccgtcag acgggtcaat tgcaatgatg tccgttgtgc 6540
gagcattaaa ttcgcgagca attaccgccg attcgtacgc gagcatggca gggttaccat 6600
tgatattcgg ggcgatgtag acaccaagtt tttcaccttc ggtttgtacc atgaccataa 6660
cagagttaaa catcatgtaa accattggtt ggtcttcaac gctctccagc attgcaacgt 6720
catcccagat ttggatgagt gatggatcca ccttcatgat ggcaacatag ccgtcgcgga 6780
tttcgatcgc ttttttgtaa tgctctttta gacgtggatc tacttttgaa atatccattg 6840
tgataccttt tggtgattgt gtttaatcgt caaacaatgt aatgtgctta acttgtggtt 6900
tagattttgt cggtttttcg acttttggag aataagtcca gtcagtagac ttaacaacat 6960
tctcagggtt tgtgacttga cggttacgac ggtactcgcc catatcttta atattgatgg 7020
cacctgaacg tcggatcgaa acacgtgttg cgtcgtcaac atcaataaac ttggttttga 7080
taaccggaac aataacatcg tccatgcatt tgaaaattgt gctcggtgaa ataatgagca 7140
tatctttcat attagagata cgtttaatag cacgactatg gtcaatagct ttaatgacaa 7200
gtcccttatt tggatgttct tgtaaagcat ccgggttacc actgtaactc aaaatgatag 7260
aaccttttgg aaaaccaagt gcctcaattt cagcttccat gccaacaatt ttaaggttat 7320
ggcgctcggc atacgctaat acagaatcct tcatttcttg tgtccatgtg accggccaca 7380
tagcacgacc aatatcttcg gtacgtacca agttcatgta cccaattgtg ttctctaaga 7440
tgattttctt gctcatgata atgccctaaa taatgttagt tagaagtcac tataataaat 7500
gggttaaaac ttatgaagaa accattacca acgatgccac tcgcacagcg tagttggcat 7560
tgcgcacaaa atgatactga atatgtttac cgtacatgga cgatggctga acaagaaatc 7620
ctggtccagg tacgtgaaga agaaacggat agaaaagaca gaatcgactc cattattcag 7680
gtcgtacaaa actgtgttga gaagccagaa gactttgatg ctcgtaagac tcctacgttc 7740
atcatagagt cgttgttctt gcgtatctat gcaattagca tgggtgaatc acatcatctc 7800
aatatgcgtt gtacgaacgc gatcgataaa gatggtacac tccatgaatg tggcaacaag 7860
attgaattcg acattcctct tgtccaagcg gcggatgtta aagtttatga caatcataaa 7920
aacattatcc cattgccagg tggttacaac ctgaagatgc gttatccttg tttatcaatg 7980
gctgatgaga aaccacctaa agatgttgat gcatcctgga tcattagccg tttcttggaa 8040
tgtatcttta atgatgatgg tgaagtatgg gaagcctccg actacaccag tgaagaaatg 8100
cgtgcgttta tgcgtagcct gggcggtgat atccagcaac ctattatgga agactttttc 8160
cttagtgctc cacacattta ttggaaaatg gcaccaacta aatgtagtaa gtgtggtttc 8220
gaacacaatt atgagatccg gaacttaaac cacgtttttc gctaatgttc gaaatggact 8280
tatcggacct atatgaatta tcatatgccg caaggatgcg tgggataaca ttaacggagt 8340
tctatcaaat ggctccatgg caagtgttaa ctcaaattga tattcacgac tatctcaaag 8400
aacaagagtt agagcgaaga aaacaacagc agaatgggtg atcgtgaggt cgcccatttt 8460
tcatatttat taaatgtgat tacaataggg tgttccaccc aataaattta atattaataa 8520
ttaatataaa tcattaatct ttaatggtgt cccatgggac acatggcctt cggccatcat 8580
ttttatatat tgatcttaaa taatttaagt tattagatca atttataaag agaattacaa 8640
tgcttgaaga cattaaatgg ttttatggca tcatcgaaaa cgttgatgat cctcaaaaat 8700
cgggcttagt ccaagtccga ggcattggta tacatgaact aaacgaagct atcctgccat 8760
cggaattttt gccatggtgt aaggttatga cacctgcaac aagttccagt gttaaaggtg 8820
taggtgttac tccaacgggt atccaaaaag gctctatggc tttgtgcatt gcgatggacg 8880
aagcatacac tgataaccgc attttgttca catggcaagc gggtgacatg aataccaatg 8940
atatcaccga acttgctcgc ggcctaacca atgaaatcat tcgcaaaaaa cttgatgagc 9000
ggaatgagtc taaacgcact ggctttaagt gggctgaacc tcaaaccaac tacggtaatg 9060
tcgtctatcc ttataaccaa gtaacagtat ccagagcggg tcatgttatt gagcttgacg 9120
ataccccaaa ccttgagcgt atccatatcc tacataagga cggttcattc attgaatata 9180
tgcctggtgg cgacgttgtt gaaaaatcaa tgggtaaccg ttatgagatt tatcgacgca 9240
accttaaccg actgatcaat ggtaaagacc gtaagaatgc tgctgatgtg gccgtacatc 9300
attactctaa aacgtatcat ttcaattcgg ctgaacaggc aacattcaca tcagatactg 9360
atatgctatt tgttgcacca tcgactattg aattccatac ggctttatta cagatcattg 9420
gtagtgccaa aatttcacaa accttatatg ttgacgaaat taaagctgct cgtatttcat 9480
gtgacagctt ggatgttgca tcaccaatca atggtactgc gaagtttgct caagctgctg 9540
ctcaggcagg ccgtttaggt ggtggagctc cagctatggg ttcgtcagtg caaccattga 9600
atatccgagc acagttgaaa aactcaactg gtactgaggg tgataacaac gacgctccgg 9660
caggtagtaa caaagcagac gaaaaagtat cccaggagcc cgcatctaaa cagaaatcac 9720
aaactagtgc aaacactagt ggttcaggtt cacaaggctc tggggatact acaggagacc 9780
aacaacaaac aactcctgga acacaagcag aaacgattga tcaggttgtc ggtaaagaga 9840
acattgatga atggtatcgt tcgcagatac ccaacattcc aaagtacaaa tataaccata 9900
ttacgattca tacgactgca acaccactta gtcaattcca tgatgtgcat acaattcgtc 9960
aaatgcataa agcacaaggg tggagtgata ttggttacca cgtattgatt actcgtacgg 10020
gtctgatcca aatcggtcga ccatggaacg tgatgggtgc tcatgttaaa ggacataacc 10080
cgaacaactt tgccgtcgta ttggttggtg gcgttaagct ttctaacggt cgttgggttg 10140
gtgataacaa cttcacacaa gcacagtgga agtcattaca aattgtgatt actgatgttg 10200
cccgtctgta tgatgtgcca gttactaaca tactaggtca ccgagattgg agtccggatg 10260
caaatagtga tggtgttctt gagaaacatg agtggattaa agaatgtccg tgttttgatg 10320
tacgctcatg ggtagcatca tggtgggata cgcgaacagt ataccgacca acaatgccat 10380
caggtaaaac tgaaactatc gatacgaccg aaccgtctaa taagccggtt gtcacggcac 10440
ccgatgaaaa tacagaacat agctcaacaa ctgcgactaa gtttgccgaa gtattgccgg 10500
cgacagaaat tagcaatgtt gtaaaagacc ttagtgaaag acgtgttcaa gatattgcga 10560
ccaatattgc caggggtatt ttatcttcag atgtttcaat ggttgctgcc aacgttgctg 10620
ctttagctgc tgcggatgtg cgtaatgata ccggtccaat ggtggacaaa gctgttacgg 10680
ctttcgagaa atctgaagca gtacaaaaga aagttgaaga agacctaacg tacttcaaaa 10740
accaatatta tggtccaagt agctttacac cagccaaacg tccagacggc accgatccgc 10800
atcctgggga tttgtacttc aatacaatcc aagacaaagt tatggtgtat gttactcgta 10860
ccgatttcat tggttggaca cctattgatg gtggtggtaa aacatgggca acccaaattc 10920
agttcgataa ctcaggtacc gatttaggat cacagaacgt tggttcagca atcaaagagt 10980
tacataaact gtttaaattg tatgaacctg tcgaaggtcc ttcacaaacc ggtgacggac 11040
tcaagcgccg tttcaactct ccgataagcc gtaaagtaga tgataaattg atttgggttt 11100
tcgttgatgg acgtcgaatg aaaccaaccc gagaatatca aatcgaaact gacggcaaaa 11160
ttttattgac tgatgctcca gcaaatggtc tcaaaatcga gtcattgtac tttaacccgt 11220
tgcaaggtgt actaccgtta acaggtggaa cacttgtggg tcgtttaaat ggtacaactt 11280
tgcatatgac gggtaaatgt tacatcaaag agttggaagt cgaaaacgtt acactgattg 11340
agaactcgtt acattcgtca gaccgaacat taaagcgtaa catcagacgt attaagaaac 11400
cattgtctaa gattcaacgt attgacggtg ttagctttaa atggaaagag agtgacaacg 11460
acggacatgg cacaatcgct cagcaattgc aacaagttat gccatcagca gttaaacgcc 11520
ataaaggtaa gttacgagtc gattaccatg ctttgatagc attatgcatt gagtcaatta 11580
aagcccttat ttaccgagtc gaagcgcttg agcggcaagg taaacgtatt aaagagctgg 11640
aaaaacgaat cgaacagatg gagaaattga tcaatgaaaa ccaaagataa cccacgttta 11700
tcattcaaga ctgacgtagt taaaaatgct ttggattctt cacctcgggt ctcttttagg 11760
gacctggagg ttacccgtct tagttggatg cctaaaggtg gtaaaaccct cagcatgaaa 11820
gatatgttga gacgtcaaag ctacagatat tattcggacc gcacaacaaa atttgattcc 11880
aacaaacaaa atttgcgaca aatggctgag tacctatttc ccactcaaac gttggcagcc 11940
aaagggataa ctgtaacctt tacaaatatg acgggcggtg tagttaaagt agacttaacg 12000
aaatcaaccg actttgaagt tgttatggag ctgacgtatt tacgcggtat cgccaagggt 12060
aaaattatac aagtttactt taaaccacaa gacgagatca ttgagtctac tgaaaacctg 12120
tataaagtag aaatgaacgg tagtgtattg aatggattcg cggtccacac tatatatggt 12180
tctatcgaca tttcggcggt cgtaagagag aagtttgatc aacctacagg tggttctgat 12240
aacacaaatg acgaccattg ggaagtcaaa cctacgacac ctgttatagg taaaaaatat 12300
gatttagtta ctgctaccgg attacaatct caccttggct actccagtga acatatccat 12360
tttgctttgg ctgacggtga tcttcaaggc aaagtcactt tggttgcctc tgcggataca 12420
cgcatcggga atgtttatat tcctgttaat gaacgtggtg tacgggtaag atttgccgtt 12480
tatgacctgg gtcataagga tgataaatat tcaaaagcaa aacttgtggc attatcacct 12540
atactaacaa gtacgactgt tgataaaaaa tatctcagtc ataaattgcg ttatatgcta 12600
atcgaaaatc atgagtatcg ttttggttta cagatgtttt cggatcgtgg tccattacaa 12660
ccaccaaacg ccaggtttgt cacagcacag ttaaatacag gtagtgaccg aatacctgat 12720
gatgagtatg atttgaatat ttctgaagcc caacgactca attacagttt agcttattat 12780
gctgaaatag aaacaaagaa aagtggagtt actacatgat aggtgaatta gaacgtgttg 12840
aggaaccagg ttatcattat cgcatcgatg ttggtggtag caaagacgtg tttttattgc 12900
gtttgcacac cgacgtgtgt gagctgtcgt ttattaatgc ccaagcgttg cctttcaact 12960
accaaaagat aacacttatt ttagaacaac aacgtggggc acataaagtc acatggcctg 13020
caaatgtaag ttggccgatg aagcatatcc caattctatc caaccagaaa ggtgagattg 13080
acacgattga gttagtcagt tatgacggcg gaaacacctg gtttgggtat tttactggag 13140
caggatttaa gtacaccaca tcttcatagg aaataccatg catcatttta aacatacacc 13200
aatcaataat gccctacaac tggcattatt atttgataac tttttagacc gtcatgtggg 13260
taataccgac gataacaatg acggtcttta tgtcatcgac caggacggtg tgcttaataa 13320
caacttgcac aaagaagatc gtggctcctc gaatggtgca ccagatggta cagcaacaac 13380
tgaaggccaa gcccttatga tcttgggtta cctatacatg tatgctgcga cagagaatca 13440
gtattggttg gataaatcga cttattattt tgagtcatat attcgcaatt attatttgcg 13500
taaaccaata cctgatactc cacaagtatt ccgttcccat atctacatca atggtcgtgc 13560
accatttatc acagccggac ctaatacttg gcgtaaaggt tcccgtacgg gtactcttga 13620
gtatgatatg gatttcaaga atggtcgcgg cattgttgca tcaggttatc cggaccatgg 13680
cgacgaaatc atatacatac catttgcttt tgacggttac ttaaaatggc gttcaatcgt 13740
tgctgacgta ttgcaacatg gtagcacgtc gaaagaaggt aaaacgtttg atgttgcctg 13800
gtatattgat taccgtggtt ataaggttga cccagaaggt gaaatcattt cacaagtcaa 13860
cactgaacca aaaggcacag tccaacttaa agacaccaca gtcaacggta gataccgcat 13920
taactatgct tcaaacaacg gtagtacaat tgaccgacac gaaccatttg agaactggcc 13980
tatctggatc ccagtacgtg atggcgaaga aggtaatgct gctgatgccg aagaatggtt 14040
tacggactgt gcaggtctca tgtactattt caccaaagat gaacgataca aacgtttgta 14100
tgagtgctgt attgaaacgt tgatcgaaat ctctggcgtt gaccaaaata tgagattctt 14160
ccgtaaagat aagtcagcag gcacaccgtt tactgatggt atcagttatg aatactcata 14220
ctcgcctggt aatgtgagca ttgtatatac tcgtgataat gatggtaaca tcaaaattac 14280
aaaagctgcc gaaccaaacc ctaaagagtt tacggatatc tcattagagc agattgcctc 14340
atggttccat gtagacaata attcgaaagt tattgttgat tacggccata ctgacgcaga 14400
agcgatcaca tctatccgta ttacgattat ggataagcca caaacggaaa aggggataat 14460
gtatgagtat gcattcaaac aagatacgag taccgttaag cgtcatgaga ttccaataca 14520
acacttcgtt gaaaaggaaa acttcataac tggtcgtaag tactacaaac tggatggtac 14580
atcatttgtt ccttatggtg atgcaaaatt tgaaaaacgt actggtgaaa atatacgtgt 14640
tgatgccgat tatcgtgtag accgttttgg gcgtgttatc tttggtaata ccagttattc 14700
cgggattgta tgtggtttct ggtcgattgc atcatcaacg aatggcacat tgccaatggg 14760
ttcaattacc tatcgtaatg tagcccgttc ggatccagct ttagggtcta attgtgatat 14820
tgcgattgtg gatgctgatg gtgtgaaatg gtctatgcaa ctaccgtatc aaccacagtt 14880
tcaaacgttc taccttgatt ggaaaggtcc atggaaacgc actgatggta aacctggtgt 14940
cattaaacca ggcaatttga aacagattga gattcagaag catggtagtg aagatacgag 15000
cgcaactatc gatctgtatg cttttaatgg tgtgccaagt aagttcgacg gtaaaggtgt 15060
tctcaaagag tacaacatga ccattactgg catcaaggcg aacacaatct acactggcga 15120
ctcatttgtg gaatcaggta ccgatgcggc taagttgaat gcaccttata cacctggggt 15180
aatcccattt tcaaccaact tctcggttga gacacaacag gtcgactcat ggcgtggtat 15240
accttacctt ggataccaat atccaatcat ttggggtcgt gacaagaagt atgagtcgcg 15300
tatgaataat atgaccaagt ttatgagaga ctctcaaaca gattaccatc gccgtcgggg 15360
tctcaaggga ccagtaagtg ctgcttatgt ttggccacgt tgggataatg gttcgtatgg 15420
tgagttcaac acatttgttg atgtgttctg gggtagcaaa caaccatggg cagggtatta 15480
ttctcgttgt atgtttggga tgtgtcgttt acgatatcat ttagttcgta ctggccgtac 15540
acctaatgat gacgtggtcc aatttaccga ggattggatt gattatttgg ctgacttcat 15600
gaagaagaat gacaactgga cgccaacagt attccatagt aaccgtgaac catatacgta 15660
tgacggtgac tttactggtc atatgtcagc aagtttcctg gctggggctt gtgagtgtta 15720
ccttgctggt agtcgcaatc caaaccttga gactgtgata gaaggcattt atacacagtt 15780
tgaacgtcag cttattaact tgggtcctgg acaccatatg aatggtggtt acagtgtatg 15840
ggctggaggt aattacttct ttggggtatg gacagaaatc ctgcgtggct tcggtgcata 15900
cattatgtat cataaaaaga aactatacgg tgatagttac acaccgccgt atgagtaaag 15960
gatgaataat gaaagtgcaa tacaaagata ttgatcttgg tatggctaag caccccgtaa 16020
ctggtgatgt agcggtgcgt actgatgtgc gtgccattat gcagtcgatt cgcgagattg 16080
tatttactgc agctggtgaa tgggaagctg aacctgaatt tggtgtaggt atctatagcg 16140
aattgggcga ggaacaacat cctctgattg gtgagaactt gcgtattcga atagaacagg 16200
ctgtagaacg atatgagcct cgtgccgaaa tcgcatcagt ggttattggc agagacggtg 16260
atcatggagt caaaatccaa gtcattttct ttgctttgaa ctctcctgaa ccatttgacc 16320
aagaaatcgt tttagaacgt acccgataga tatgataaaa gcccactgga ttgaccgtgt 16380
gggcttttta ttatctgaga gtatgattat gccaaattgt ccaagaatgt cttcacatcg 16440
aagcacgggc acattttgat aaactcgttt ggtgtaatct tgccgtctcc gttcaggtct 16500
ggactccaat cacgatgacc tttgatatta ccgtctttaa ttcccaaaac acctactaag 16560
tcaatcaata cttcacgcaa gctatcgtat tgctctggtg taaagttatc ttctgcctta 16620
ccattcgaat cgataccccc aaccaggaca atgccaagat tgttacggtt atgtccctta 16680
acatgcgcac cccatcgatt aaacggtcga cctaactcaa tggtgccgtc acgtttgatc 16740
aaaacatgat aaccaatatc attaaaaccg cgtcgcaaat gcattttgcg aatgcttgcc 16800
acgtcatgat cctggtctgg tggtgtagcc gtagcgtgga ccgtaataaa tttccattca 16860
ttgctaagca attgatgtag tttatcttta tcaagaaaca ttgtttttcc tctactgcag 16920
tttcaaatat ttagttcacg ttaatataaa cacctcaacg aaaatactat atattgattg 16980
ttaccactca atattaacta cataccgaca acctaaaaag gaatcacaat gcaagtacaa 17040
aaacgcaatg gccaatttga agacctggac atttcaaaaa tcaccaaagt gatcaactgg 17100
gctgctgaag gcctggaaaa tgtttcggtt tcacaggttg aacttaatgc gaaaatcttg 17160
tttgctgacg gcatgaagac tagcgacatc cacaaaacaa ttattaaagc tgccgctgac 17220
ctgattagcc ctgaaactcc agattatcaa tatctggctg cgcgtcttgc catcttccat 17280
atccgtaaga ttgcgttcgg tcaatacgaa gctcctgacc tttactctca aattgaacgt 17340
atggttaaac ttggtaagta cgacaaacat ctccttgaag actataccaa agaagaaatt 17400
gaacaattga acgaaatcat cgaccatact aaagacctga actatgcgta tgcagcagtt 17460
aaacagcttg aaggtaagta tctagttcaa aaccgtgtaa ccggtgaaat cttcgaaagc 17520
cctcagttca ttttgatgtt ggttccaatg tgtctttttg ccaagaaaaa agatcgcatg 17580
acgaaaatca aagcgttcta tgaatcggtt agcaacttca aaatctcatt gcctaccccg 17640
atcatgagtg gtgtgcgtac tccaacgcgt caattcagtt cttgtgtact tatcgaatct 17700
ggtgatagtc tgaattcaat caacgcttca gctggttcga ttgttcgtta tgtatcacaa 17760
cgtgcaggca ttggtattaa tggtggtcgt attcgtgcta tcggttctga aatccgcggt 17820
ggcgaagcat cacacactgg tgttattccg ttctggaaac acttccaaac agcagttaaa 17880
tgttgttcac aaggtggtgt acgcggtggc gcagcaacgt tattctaccc gatgtggcat 17940
tacgagtatc aatctttggt cgtacttaaa aacaaccgag gtgtcgaaga aaaccgtatc 18000
cgtcatatgg attatggtgt tcagattaac ggcttgcttt acaaacgttt aatcgatggt 18060
aaaaacatct cattgttctc gccttctgat gttcctgggc tgtatgaagc attcttcagt 18120
gaccaagcag agtttgaacg tctatacact gaagcggaag ctaaccctaa gatccgtcgc 18180
aatactattt cagcgatcga agctttctct ggtctaatgc aggaacgtgc gtcaacaggt 18240
cgtatctatg tccaaaacgt tgacaactgt aacatccagg gaccattcga tgaaaaagtg 18300
gctcctgtac gacagtctaa cttatgtctt gagatcgcgt taccaactaa gcctttagag 18360
aatgttgatg atccaaatgg cgaagttgca ctgtgtacat tgtcagcatt taacctaggt 18420
gcaattgaaa gtccggatga gtttggtcca ctagccgaaa tcgttatcga agcacttgat 18480
gcgttattgg attatcaaaa ctacccgtta cctgcagctg agcgttcaac aatgtatcgc 18540
cgtactcttg gtgttggtgt gattggttat gcccactgga ttgccaaaaa tggtttcaaa 18600
tactctgatg gttctgcgaa cgaagctaca cacgaattat tcgaagcaat gcaatatcat 18660
ctgatgcgtg catcggttaa gctggctaaa gaatatggtg cttgtccagg tttccacgag 18720
actaaaatgg ctaaaggtat cttaccagtc gatacttacc gtaaaaacgt tgataagctt 18780
gttaataaca acttgaaatg tgattgggaa ggtttacgtc aagacattct gaaatacggt 18840
atgcgtaact caacagtatc agcgttgatg ccaagtgaaa cgtctagcca gatctcaaac 18900
gaaacaaacg gcattgagcc accacgtgga tttgtatcaa tcaaagcatc aaaagatggt 18960
caaattaaac aggttgtacc ggaactgaaa acattacgtg ataagtacga acttctttgg 19020
gccattaaag acaatacggg ttatctgact cttgtggcga ttatgcagaa gtttgtagac 19080
caagcaatct cggctaatac taactacgat ccgatgaact atcctggcga taaggttcca 19140
atgaagaaac tgctccagga tttgttaaca gcatatcgat acggtgttaa aactttgtat 19200
tatcataaca cccgagacca tgcagaagac aaagaccaaa atgtcgttat cgaaaaagtt 19260
gatgattctt gtggtagtgg tgcttgtaag atttaattgg taatttcaat ttaatacatg 19320
gccctaaaat agtagggcca tttaatttag gagtaagata ataatgagtg ttttcaacac 19380
aacgcacaat gaccaactca aagaaccaat gttcctgggc caagaagtta acgtggctcg 19440
ttatgaccag caaaaatttg aaatttttga aaagttaact gagaaacaat tgggtttctt 19500
ttggcgcccg gaagaagtgg atgtatctca agaccgcatc gactttgcta aattaccaga 19560
aaacgaaaaa cacatcttca tcagtaatct gaaatatcag acactattgg atagtattca 19620
gggtcgtagc cctaacctgg cgttgcttcc gatcacttcg ttaccagagc tcgagaactg 19680
gattgaaacc tggtcattct cagagaccat ccactctcgt tcttatacgc acatcattcg 19740
taatgtattc acggatccga gcattatctt tgatgacatt gtggaaaacg tagaaattcg 19800
taagcgtgca gtggccgtat ccaaatacta cgatgatgtc atccagttaa tcgctcgtcg 19860
tgaagccggt cttgaagtcg acatgtatga gatgaagaaa aagctttact tgtgtctcat 19920
ggcaattaac gttcttgaag ctattcgatt ctatgtttcc tttgcttgtt ctttcgcgtt 19980
tgcagagcgt gaattgatgg aaggcaacgc caagatcatt aaattgattg cgcgtgacga 20040
agcattgcat atgtcaggca cccaacacat gatcaacctt ctacgtaatg gtatggatga 20100
cttctcattc ttacagattg ctgaagaatg taaagacgag tgtattcaaa tcttcaaaga 20160
tgcggtgcaa caagaaaaag actgggctga ttacctattc aaagatggcg gcatgatagg 20220
cttaaacaaa gagattctgt gtcagtatgt tgaataccta acaaacatcc gtatgaatgc 20280
tgtagggctt cctgctatct acgatgtcaa gaccaaccca atcccatgga tcaacgcttg 20340
gctgtcttcc gataacgtcc aggttgcccc acaagaggct gaaatcagtt cttacatggt 20400
aggtcagatt gatagcgaag ttagtacgga tgatttcgca ggctttgaac tttaatattg 20460
aaaagtgtga tctaggtcac acttttttta ttatctgttg tattatgatg tctactcatt 20520
cacaacacta agagtattag acatgtttgc atttgttatc atcgtacaat tggtttttgt 20580
tattgctgga tttgcaagtt gcgaaaagca ataccgttca gactttggag tatttgtggt 20640
tttcgtattc ctggtcgaaa ttttggtttc tatctttaat caaggtcaaa tatcatgagt 20700
aaacgcaaaa tatcatcagc cccatttact gtaacaggtg aatacctagc caattacctt 20760
cgccaaatta tgatcgacaa gggttggcgt tccgcataca aatccgtaat ggaagatatc 20820
gttggaatta acgatgccca aaccattgaa ttcttatcgg gtaaaaagac atttgctggc 20880
gattccaata gtggtattga attcattgat tgggaagata gtgaagaaaa atccctttac 20940
ttagaagaag ttaatgaaaa atacaaaaag atttggtgtt gtgatgaact attgttgact 21000
gttagtgaag tagtcagaga tggtcgttat gtgtcacaac ttgtcaaagg ggatggtgag 21060
tgtcctaacc agggggcttt ctatcgcgaa gttaatccaa atgtgtatgc gaaatatttt 21120
gatgctcgtc attatcaacg tgtaactctg gaaaccgatc ctgatttgcc tgagcgttta 21180
atgcttgtat tttggaaacg ggctgatgct tctttgccgc caccatggat tgaagaatgt 21240
tgtaagagtt gggaagccat ccgacattat gacagttcag aaatccgtat acgtgagctt 21300
gatagttaca aaggtttgaa attccgagaa atggaccgac tacaagctaa tggcatctat 21360
caagctgtag gtgctaatcg tgatgagcgt gtggcactaa tggaacgacg caaagcaata 21420
cttgaccaaa atggtgatga tttcattgaa gttgcaggtc ataaaatacc tagaaagcca 21480
ttgattgctt ggggtttacg ccgcagtgat gccaaacacc ttgccgattg tgattggaaa 21540
gccatctcgc cttctggtga aaaaatgatg aacgattgtg ctgcccacac tgattgggtt 21600
attggcgcag ggtttgatcc agcccatttc tatcgggacc ataaagaaat caatgatgct 21660
gcatttgtct ggtgttatga tttgaacaga aagttatgtt ccgctgactt gttggtattg 21720
acttcttgtc gtggtagtga actacgttcc aaggttcaac aaccacaacc taacgataag 21780
attacgtgtg acatcgtggt cattgaagat gctagtaccc gttggatcaa ggttgctgaa 21840
caggttggta aacgtggtgg tatcataatc tgtgaaaaag gaggccccat gtctcacctg 21900
accatcaaag gacaagatta tggtataacc ctaatgttac acccagaagc ccgtatggtg 21960
taccgtgagg gcattgagct aactgtctat gaaaatggct ttgctagcat taggagcatg 22020
taatgaaacg ttttatctta ttggtaacag cattactagc aagttcttca tatgctacta 22080
cagattgtga aactgtattt ggtcatgacc agctcgtgac catgcagaag gcttacgatt 22140
atggtaaacc ttatgggttg gaatactcat tggccgccat cacctggaag gaatcatcag 22200
ctggtcgtca taacgtgcgg atcgaaactt tcaagaatac taccaaagtg ggttattcac 22260
cattccatat tctattgcgc accgcaatgg ttcatgaggg gtgtgagacc aaacgttgtg 22320
caagtgatat cgcaaataaa ctcatgacca actttgacta tgccgcatct catgcggtac 22380
tagttcttca attttggcag agacggcata ataacaactg gatgaaaaca tggcaggggt 22440
ataatgacgg ctatcaaaat agtcaggatg gccgcaatta cgcccttaat attaagtata 22500
agatcgcata tctcaaacaa tgcgtaaggt tgaaataatg gatcaaactt cacatgcaaa 22560
ggcattggca agttttgtca atgttgcaaa gcaagagctc gatgagttca tgcatcactg 22620
tttggaagaa cagaaaaaag aacccgaaaa ttggcccatt gatagctgga atattagcga 22680
aatgtgggaa caattcgaag cttggcatga ctggaagcat ggagtggaat cgtgttaaat 22740
gaagacgaca ttatatctgc ttttatgggc gatgctgaac cacctgcacc tccactaccc 22800
ttcgacagac cactaccaga aggttggagt cgtgaatacc cgcaaatgga cgatcagcac 22860
atcagtaaca aaattaatcg atttttagaa aagcttgatc attatctagc acatcgtgcc 22920
cgaacgggta ttgaacaaat acaacatatg cctggacatg agcgattgct tgtcattaag 22980
atttatgggg atccaaagtt gtactttgtt ttctttgacg gcatcactgc cgcttggcac 23040
aattaagcct aaatacctaa aacgttaatt tgtaggaaaa agcaatgcct aatttaattg 23100
ataaattgta ccgacctgaa ggtggtcgtg aatctaacat catcgcaacc aataaaggtt 23160
gggttgattt ttcaaaagac cttgataatg acgagttgct tgtggaattc gaccaaaagg 23220
gtggtttcct taaacacctt gccgacaaca acatcacagg tcctggtatt cctggtttga 23280
aaacctttgg ttataaaccg cctggtgata tcactgctaa gggtgacgcg gatactggtg 23340
gttacgaaaa tgattcggtt tacgcactca ctaaagccaa agcagactct acggcagatg 23400
acgtacgttt tgctgcaact gctggtgacc gtgtaacaaa ccttcaagtt ggcttggatc 23460
ctggtgctat tgcagaagtt gcggtttacg aaacatctgg ttcattgcca actaaatttg 23520
ttggtaaagc ccgtgttcgt aacacagatt caaaagtcaa ctggggtaca gctgttgtcg 23580
atattccatt ggaaaatggc aaaacctact gtttggttgt acattcaatc agcggtcagt 23640
tacttgaatt tgccgagcca aatggtattg acaagtatgc cgttgtaaat ggcacattcc 23700
cagcgacttt gcctggtgca gacggcacaa actctgattg gaacgttgct gcatacgcta 23760
tcgttgcgga ttactcaaac gtttaaacaa tctgaaaact aaatcaaggg tgacttcggt 23820
cacccttttt catatctgga acctgtctta taatgattcc acctcaacaa aggaatcatt 23880
atcatgaaga actatcctat tatccgaatt gctgatgtgt tcaaagcatt ggaagcaggt 23940
gagcacactc acttcggtca tggttgtaac tgtcgtaaac aaatgcatcg tgattatggc 24000
ggtggtggtg tggctgctat ggttgccgat tggtgtccag aagcgtttga agtggatcga 24060
atgacatcag gagataaccg tttgggcaca tatacttcag ctgtgaagaa agtggaatca 24120
ggtaatatca ctgtactcaa tatgtactca cagtacgaaa gtggccaagc gccattatta 24180
acactcgcca aaggcatatc caacagcctg gacaaagtta aattgttgat tgacgacaat 24240
actaaatcct tggcaatccc tgcgatcggc tgtggcattg ctggtggtag catttcaatg 24300
cttgtgtcga ctattcaatt ccataacatt ccaaatcttt atctgtatct tcatccagac 24360
gacactatta atatcctgga cttattatat ggcaaagtca tagctaagtt tgaagacgga 24420
tatggtctga tccgtgttat gcgtgagcct caacaaggtc gtcccttata tcgcactatg 24480
gcagttgata agcgtttcca ctcagttgaa gacctattgt catttgcgcg ttcattgtat 24540
ggttcagctg tagtgaacat tccaaattta atcttggatg tagaatattt atccctaaca 24600
acaactcaag agtattaagc acaatgccaa atttattcat taaagctgca gaagcagtac 24660
gcatcaaaat gggacttggt aacaatgtcg ataaacagta tcttgatctg cttgaaaagg 24720
tgttcaaaac tggcgaagac attgaaggcc gcaacggtat ggtacgcaaa acctttggcc 24780
atatgatgga gtttgaccat gaagaaggtt atccgattcc gacttcaaag aaaaccctac 24840
acggtgcagc tgttgcggaa atgactgcgt ttattcgtgg tgaaagttca aacaagaagt 24900
tcatggatct tggttgtaac gtttggaaag gcaactgttt cgctgattac tggatgtcaa 24960
atccaaacaa caagaaacct ggcgatgact taggtcgcat ctatggggta cagtggagac 25020
aatgggatgg tggtattgat cagctcaaag agcttgttga tacgctcatg accaatcctc 25080
atgaccgccg tatgattgtc agctcttgga acccaacaga tatggacaaa atgtcattac 25140
ccccttgtcc tgtgttattc caggtattcg ttcgtgatgg caagtatctt gacttcgcac 25200
tgtatcaacg ttcgtgtgac atgttcttag gcgtgccttt tgatattgtt ggttattcat 25260
atctgcaaac ggtgatcgcc aagttaactg gttataagcc tggcaagttc aaatacttcc 25320
tggcggacac acacatttac cacaaccact ttgaacaagt gaaagagcag ttgggtaacc 25380
gtctgaagac attcccacta ccaaaagccg aactggattg gccttcaacg gacatgaaca 25440
tcacattgga atcgttcagt cctgaagatt tcaaactggt tggttacaaa tcacaacgtt 25500
tcatcaaagc accaatgagt gcatagttca aatctaatat cggggttaaa ataaccccgt 25560
tctttataaa caaaactgtc tttggagtaa aagataatgg aaactacttt caatctgtct 25620
catatggttg cacgcatcat cgagatgcac acaaaatttg gcattcacaa tactgaagaa 25680
catgtattgt cagccgaaga aaccaaattc cgtgttgctg caatgtggga agaatttgaa 25740
gaaggcaaag atgcaacttc aaaagaagaa gaatttgacg cattgattga cctggcagtc 25800
ttcgcacttg gtaccatcga tcgtatgggt atttcagatc agtttgttcc agtgttcaac 25860
actgttccag ttaccaaaac cggccatacg tttgacgatc ttgaaaagat gatccacgtc 25920
gtggcatatg aaatggaaca gcaacctaca agcttagagc aacatgcgat caatattgct 25980
tacttgatgc atgaattgtt tgctgtagtt aaagcaaaag gttacggtga catcttcatg 26040
gctggttaca acctggtaca agatgcaaac tgtgctaaag agcttggtgc aaacactaac 26100
aaagaaggcg gtcgcggtgc attcaagatt gatctgcgta agccagaagg ctggaaagca 26160
cccgaccaca gccaattgat cgaaagcttg gcttaattac tcatcgtggg ctcctatgtg 26220
agcccacatc aaacaaactt aacaaattgg taatatccta tgtctaagac aaaaccgact 26280
ggtatcatct tacttgaagg ccctgacgcg gcgggcaaaa cgactcttgc ggaagcattc 26340
aaaaaagtgg taccaaatac tgaaatcatt catctgactt ggtcaccaaa acttgagact 26400
gtaatgtctg attacaacat tggtctatta gcacatgctg aagaattagc gaaagaccgt 26460
ttggtaatcc tggatcgcca atgtatttcg acttacgtgt atcagtcaac tttccgcccg 26520
gaaaaggcgt cacatcatcc aagtctgtat gcacaacttg cgaaatactg tccaagtatc 26580
agcatcatct tctgtctacc agagtttgac acttggaaac agaacttcct ggacatgtgt 26640
gcatctcgtg aagaaatgta tggtgctgaa aagcttgaac agatgatcga agtgtaccgt 26700
cgttacgacg tcatagctcg tggacctcag acgtctgaag atcagaaact aacctggtat 26760
ccagacaacg tattgatgga ccaaatcatg gatgggggtg tactgaaaca tatgcattct 26820
tatgtgtatg acttcacaaa actgatggac aacgaaggcc tattgtttgc agaaggcttc 26880
ttgcttggtc accaagcaca ccaagaaaaa ttcaagtaaa ctcaacatca aaagtcaagg 26940
tctcagtatt attactggga cctttttatt ttattggaga aatatcatgg caaactatcg 27000
ttatctaatt ggctttggtc aaccaggcta tcattacaca cttgcgggtg gtccatctgc 27060
ttatatcaca tcatcaggcg ttggttacaa caacaaaggt atggacctac gtacagagca 27120
ctatgccgat cactcttatg gagtcgatga gttagtgata ttcttggccc agatgttgaa 27180
gcgtgtatca tcacatggca atcatatcga tctcaaccgt gtatacaatg atcgcatgga 27240
atataaattc taccgcatcc cttccggtgc tattgatctg ttcgattgtg attatcagat 27300
caacattcat gcttatgaga tcagtcaaga gcagttcatt aaggatgtca aagccagtga 27360
tgcatggaat acactgcgtg atgtgtttga taatcacgac caggactacc gtgatgaacc 27420
agaagacatc aatatccgtc tctataccaa tcatattgat gggaagtgga ttcaccatca 27480
tgacgtcctg aatgctcctg atgaaccatg gcaaaagaca actttctctt tacctagtca 27540
tgttacggtt gatcaagtaa actctcacat cacagaaaaa ttggagttga cttatggcat 27600
gtaatccatt aatacaatcg attgttgctc gtgaccttaa caatgttatt ggtcatggtg 27660
acaacgagct agcgtatatc aacaaagacg acatgaagta cttccgcact gtaaccaaaa 27720
atgctgtgtg tattatgggt tcaaagacgt ttgaaagtct tcctggccta ctaaagggtc 27780
gtgaacatat cgtgttgaca tcgcaatcca aagaacaata catgaaaact tcaaaatttg 27840
acaaagaagt attggaattg tcaggggtcg agatcgtcaa taacgttagt caagccttgg 27900
tcgtagccca attgatggcc aagatggaac atcgtcctat ttttgtgatc ggtggcggca 27960
cagtttaccg tcaatttatg gaacatattg atgtgtggca tgtaaccact aacctaacat 28020
caaatccatt caataagcct gtttatttcc atgaatcatt tgactcaaac attaactaca 28080
aactaatcgc tgagacgact catgttgatt gcgatatccc acattcaatc aaaatctatc 28140
aaaaattgta aggaaccact atgttttcag gtgatatgaa cttagctatc gccgaccttg 28200
aaacacttgc gcatactgat gatgcgcaag ttctttcatt tggtctgtgt acagccccag 28260
tcgataagac actaacattc aaacaattgt gtgagacgta ctcaatgttc ctcaagtttg 28320
atgtcaagga acaagctaat aagtacaaac gctcattcga tgagaacgtg atgactgagt 28380
tctggcttaa tgatagccat acgaaccttg aatcacgccg tgtgagtctt taccctagtc 28440
cagaggatgt ttccattggc actatcacag acgccatctg gaagcataca cgagctctgg 28500
gtatggatat caaaacaatg gatatcggtg accgtaacgc atatgaccta tccaaactga 28560
agcatatgtt tgatcagact ggtagtatcc aaccctggtc ataccgtaat ggtttcgaaa 28620
tcacttctgt attgaaggcc tggggagcag accgatatgc tggtatcgat cctaaagata 28680
ttcctggcat ggtttaccac catccagtcc atgatgctgt acttgattgg cttcgcttcc 28740
aataccaagc cattactatt ggtgcattgg ataggcctga aggttatgac ctagactaca 28800
tcctcaataa ctaataaaaa ccctgatgca taacacatca gggtttattt ttattccgaa 28860
tactaaaaag ccccagaatc aatcctgggg ctttcttata tctgactaag atgtttacgt 28920
ggcttcagac tcatttgact ctgtacgtcg tgtatgctcg ctctgataga tacgttcaac 28980
caattcttct tggttagcag tctggctctc gattacttga atcaaactca ttgaaagctt 29040
agccggcaat tcgcctaatg cttgcatgat cagttgtgct tgtgtaggtg ttacagctag 29100
tacgtagcct tcattttggt tatccatgga tatctcctga tgttggatga gtgttaactt 29160
taaagggctt ttcttcaaca aagcgcccag gtttgatgat gacaagctgg ttgtcgcttg 29220
agactgttac aggtacacat aagtcatctt tcaaaagcca tagcttaaca ccacgtttct 29280
cggcttcaat cagaccacaa tgccctgaat tgcgaatatc gcggaagaca accttctcga 29340
tgcctgacat ccataattct ttaacacagt tagcacatgg ttcacgtgta accacggcca 29400
aggcacctat ggctgtctct gtgagacgtt ccagcttctt aagagcgtta ggctcagcat 29460
ggactaaaga tgcgcctggc gcttcacagt cattgctatc gccaggcatt gtaccgttgc 29520
aaccggaagc aattggtgta ttgtatttaa caatgacaca accaacttta tgtttggtag 29580
cgtgtgataa cttggcatga gcttccgcac ataccatatg agcccaaata gtctttggac 29640
agatgtaatc cgatttgatc atgatattct ctctgtgtta tttgttgcga acaaaagtaa 29700
tgatgaaagc aacggtcatg gtgatacaac cgtataggta tgcagtggca atattttcag 29760
caacttttac taagtgtggc catgcgtcat cagcattacc aaacacgaat aaaaagatga 29820
gaccaatcac tgcatagata atctgggata tgatgaatcc acctccccaa atgataccta 29880
aaccctttaa acatttcttc atggtattgt cactcctgaa ttaggaaaat gtttacgatg 29940
aagccccatt atgcgtttgg taaaactttt gaagaatgga ttacccaatc tgttaacgta 30000
ttttgcgatg atgtctccat gacatggctt cgacttagta ggatcatcct ctcgacgaca 30060
gtaacaaccc aggcgcttac caatcagtgt gggcaactgc gccatcagca actcgtcagc 30120
caatagatac ttctcataag cagcgatcgc ttgggaacga gtcatacctt cttcgaaagc 30180
ataattaccc cacatacccc cgcgaccaat gtacacatcg tacttattga agttcttgtg 30240
cacagcttta catttcgcca tgatatttcc tccagataat aaaaaaccct gacaaatgcc 30300
agggtttcat tatattacac ttcaacaatt tggtgctgga tatagatatc gccaagttca 30360
ccttctttac gtgcaatcca gtgagcaatc tcctcacgct cataaggtcc agacaactta 30420
cgctcactag tctggacaat gtgcgcttca agcatgactg ggtctttaat cacgtccgac 30480
attttccaaa tctcctttaa tgttggtgat cagaacttca accgtaccgt ggtcagtgtc 30540
aagcaatcgt gctatcttgt atgtcttctt gtcaatgtag tagacattca ggtcaggatt 30600
gttcttgatc caatcttcca ggatatgatt acgcttacca cgatgcgcca gaacattgct 30660
catgatgaac aacccaccag aggatgcacg attctctgcc atatcaatca gcttcaggtc 30720
atgttcaaac gaccaaccat gcatatagtt cgcattagtg attgtgtatg gcggatccag 30780
gtagtcaatg acatcaggac cggtacgatg catatcaaag aatgtcatga actcatgaga 30840
gctcatatac actgatgtca accagcaatg ggtatcgtac aacgtctgcc agtgatcact 30900
tggattccaa tcgcgattgc ctggaggatt attgaaacca cctgaaccat tccaacgtgg 30960
tgttgacgag aatgcacatc gcacaatacc aatatccaac caaggacaac agtcatgatt 31020
cgcattcacg aagtcacgca tcgcccagaa cgcatcctta ttgcctttgg ttgggccaaa 31080
ctgtgcttgt agcttctcca aatccgccat cacaacatca ggcgacatat atttcaggct 31140
ctcgatcaca cgataacaac atttgtcgta ttcgttatag ttgaccagtg cattgacgtc 31200
ttcaagttca aacatcttcg ccatattaat ggacacaaca cccgaaccac caaacacgtc 31260
attgaagata acgccatcct tcattgcgga ggattgtcgc accaactcca taatgtatgg 31320
caatacttct ttcttatcgc cattgtaacg aagcatgtta tcaataatct ccatcacatt 31380
tccttcctaa ttagaataaa ctgtaagcaa ccattatcac gcagtacgga cttatgaaga 31440
tcacacatcg aatccagcgt atagacggct gccttgtcaa ggtcaacagt cgatccggat 31500
agcgtccagt acataaacga accatgggac aggacacgga caatgaactc atcatccgct 31560
tccaggtctg aataagagac aacaaccatc gccatcagtt atttcttcat ctcaacatgg 31620
atcaggtctt gaccaccaat catgccaaca taattaaatt cagcatcatc attgccgcgt 31680
acacgacgct cattagacgc cacacgccac acactatcac tatggtgaga cacatcctga 31740
tagccggctg ataggagcac agcatattgc gcattgatat catcttcttt gtcggacaca 31800
gtctcagact cggcctcata agcatgagac acaaccgcag tacccgtcac aatgataagc 31860
gtaaagcgac gaacgccagt acccttagca atgttatgga ccaatacttc catcatctca 31920
cccaaagcac gtttaggcaa tgccaagtgt tcagcacgca cagtaatgat atcgccatcc 31980
ttgactctta ataaatccac acccgtagtc tgttccaaca aacgcagaac atgcggattg 32040
ctttcttgat cacccagtgt atagataggc aagttaccat gctcataata agcattagtg 32100
tacccgccag taaagataag cccagcagta tccatatgct tagcaatgca gtgttcaaca 32160
ttacatttac gaacggatac agtacggagt tcaggacctt cttccaacgc caatacataa 32220
gttagctcct ttgtttcttc ttcaaatgtt ctcagctgat ccaagaacgc atcatagtgt 32280
ttagtcatag cctttagtcc tattgttcat atagtaaaag aaaagtggta cagcaaccat 32340
tatgccatac cactttatta tgaacttttg aagaacagac tatcgagcat catgcatctt 32400
acatcgtatc cacgtcttat acccgaatcc cttataggtt gctgtctgtg tatgcgatac 32460
tatttctgca acataaccag caggcagtaa tgcggtattg atatgcgcca accatttatg 32520
cttctcgctc ggtaatgcat cacctaaaca gaagatgagt tcatcagggt ccaggtcatg 32580
ccaatgagat tttagatagc ctttaccctg atatgcatca acgccaggag gtaatggtag 32640
gtcagtacga gagcgtaggt atttgttaag gaggatgagt ctgattttag atttagtctt 32700
ggccattgtg cgattcctaa tcagttaaac tattattatg aaaatattta gcctaatccg 32760
tggaattcaa aaccaggatt tgagtgtttg agagctatat aggagtctat tgcccagggg 32820
aaatattgag aaatgataag aaaggtgtta caaatgccag gaaaggatat gccaaatgat 32880
aagaaatgat aagaaatgcc aggaaagtaa ccgccaggga gcgccagccg acaaaatttc 32940
aaattcgcag gcacaacctt ggcaccggga aagagtttac attcttcaaa agacttttga 33000
agtttcgtct taaataagga accaaaattg cgcctatata gtccgtcatc aaacaaaagc 33060
ctggcacata gcaaatcata ctacacacca ggcaatcgtt caatcaggag ctatacagga 33120
gccagaacag cacgccaaca agagtaatca ctatgaaacc tgcaataaca tccaggatac 33180
tatcataaag cttcttgtga tcgcctgtca actcgccagc caggccagca gacatgaacc 33240
cgtggaagta tccagctata tagacgatag cctccaggag tcctagccaa taatcaacag 33300
cttccttagc cttggaacgg aaccacgtac cgtgcataat gccctacctt acccatacaa 33360
tagatgatga agatgatcag tgcaatgatg aatagattct ccacacacca gatcaggcct 33420
tcaccgaatt tcttcaaaag tttcttcatg acctatgtcc tacgccaaat tatccatgag 33480
cttttgaaca gttgccaatg cagatgatag acgaatgtat tcagggcact cgccaggcac 33540
acgaggggta tggccatcgc cattggtttc ttcttcggca atgtattcca tgccggccat 33600
taacgcatca cgcaactcac ataacgcagc gccagacaca ggagccgtac ggacttcttt 33660
attgaccagt accatcaggt catggatact attgctatcg ataatgcggt ctacgtggta 33720
ttcatcaaga caatcgaata cgtcattgaa gtgtgtgcca ttgtcatgac gaccagatac 33780
gatcacattg ttgttctcga tgacaatgcc gtgaacggtt agtgcctgct tgcggatgtt 33840
aacgttattg cttaggatgc ggaccagttc agccgttagt ttgcactcgc cgttgaagta 33900
aagttttttg atgttcatga tatttctcca aatgatgttt tgagattttg aaaagttcaa 33960
ggaaatgata tcgggttata aacttttgaa gtttttaact ccacagatat ccattgcttc 34020
ttgggtaaag ttgtaatagg tgatgccatc ctttacgata tccacttcca ggacgttgcc 34080
ataatccagc attgcacgac cgctgatcgt caggaacagt gtagaaccca catcctggat 34140
gccctcaaag cacacacgac cacgtttgtt cacataagag cgtaatggct cacccttaat 34200
gcaacaacgc tgtataacgc tgatcatggg ttgtgttagt ttaagcttta gaaccgccat 34260
atcttggcat ctcctctggc taagtgatga tggaatgatt cacgataagg taatgacata 34320
tccaggtgac aatgtgtata gacctggcga gcctgacatg atagatagtc ttgaagacgt 34380
tccatggata gaccggttgg gcgctcaaca tagacactgc ggattcgaca cttattttca 34440
agtacgatat cagtgatcat cacattggtt gtgccaggca ccaggtaaga cttgttctcg 34500
tccgcattat cttcaaggcg atcgagttca tcccataatg ccgggttgtt cttggcatcg 34560
caaatgatga gcacatctga attatcagga ataggaatca ttagttcaac tccaatttat 34620
agacttgtcg gaagtcctgc ttatcttcaa tgaacgcatc tttgaagaat ttcagaccgg 34680
aatgttcaat tgatttgatg ctagccttgt tctcacagtg gacatatgcc attactgtat 34740
caatgccaga tgcgatggcg aatgcgatat gagattccaa tgccttcttc tggatgccct 34800
gaccacgatg tgatggatga acgactgtgc ctgcgaactt ccatgtgttc catcctgctt 34860
ccgcctggtt gcagtcttct acactggcac gagcaaagat agagtaagca atgagcgtac 34920
gcccactgaa cacaccaaca acacgctcgc ggtccaaggc ttcacggata gacacaatat 34980
cacgaggggt gaactggtct ggagttgtgc cacgcatttg aagtttcaac atcttgaccg 35040
catatgaacg attcagtggt ttcagtttca tccgattagt cctcgatgaa gtattcagtg 35100
tctaggcggt tacgagataa gttccaaggt tggatatctt gtgccatgag attacgaagg 35160
ataaccaatg tatggatcgc atcgttaatg gattcaacaa tgatttcgaa tggtgtaaag 35220
cccacaaccc agtcagtaga atttgcatca cgctcttggt aatcgtccca attgcgatcg 35280
ccaaccatga acaggtccat ttcatttctt tcaatgaatt ctgcatcttc cataatggtt 35340
aggtccaatg ggatattgtg atcagcaacc atgccacgga tcattgcagt gcatcgatcg 35400
acagaatgac aattaccagt gaagatgtta tactgctcac gagtcagcgt gataggggtg 35460
ttctgtgctt cttccgcacg gatagcagct tcttgttcac ggcaagcttc aaggccgaat 35520
aggtctacat tagacatgat gattctccat gatatttgat gttttgaagt tcccgacaat 35580
gataaagtgc caggaacttt tgaagttttt atacaggttc ggtaatgaac gaactcaagt 35640
cagcgaagac acgtgcgcct gtgccatcca atacaacagt ggcagtagga tattggccat 35700
cagtacacac atggtccaca taacccgtct ggtcttggta tttgccggaa gtgaatagga 35760
tacgcttacg cttggtgata tcccataggt tcgccaagtg gaatggagtc actgttaaac 35820
gccactcgcc tggatcctca aactcaatat caaagtcggg taagtccaga gtccaatagt 35880
tctctggtga ttgccaaaag ttcccatcaa ccttagggtt ttcttcaaaa gcgatgatgc 35940
caccacgaga accaatcgca aagtagatgg tgtcattctt gatattgcgg cagctatcca 36000
agaacttttg aagttttgga attgagatca tgaacggaat gccgtaaatc ttgacctcaa 36060
tcattgggtg tttatgtact agcattgctt gttctccttc tagtgaacgc catccatttg 36120
atacgaattt tgtgtaatag gatattgaga cacttctcta atggatagta gtctcggttg 36180
atttctacga gattgccaag gtactttctt gggatataac gcggataggc ttcaacaaaa 36240
cctttgaagg tcaaatccac ttcgccatcc tgggtattgt tggcacaatc atatttgata 36300
ttaaacccgc aagtcttatc cgcatataac atgttcacat ccctacgtta aaaggttcag 36360
cacagtcatc caaagccaat gcaatatcgc caaggaatgg taatgcttca gattgtttgc 36420
aacgcataat ccaatcaagg ttgttggcac tcacccatac aatgagtgga tccacactat 36480
cttggaggtg accacaatgg ttctcaaaga gttcttccat aaggccagct tcattactga 36540
catcatgacc tttgagggtt aagctgcgaa gaatcgcagc aatttgaaga ttggtaacaa 36600
agtattccat ttagatcacc gataggtcgc gaagttgttt atccacacca ccaatgaaca 36660
attcagcatc gtcgtcagca accatgttgt atttggcata atcccaagat ttccaattat 36720
caggaatgta gtagatgtta acccattcac caaagatacg ttgctgacct gagtagttta 36780
ggaagattaa cgcattgcca tcgacatgga gattacggaa aaccaccaag ccattgccac 36840
ggtacattag cttaccagtc tctttgtcaa gagcagaaac aaaagtaagt gaagaagtga 36900
tatcgacaac agtattgcca atgttcaaaa gagatttaac catggtctta accccacttt 36960
tcatcaaggt tgaacacgtg accaataatc gcagccacag ggaagaagcc aatgaagaag 37020
gctagggtaa gaagttgcca atgcatttcc ataatgtact ccaaagagtt gtttaagttg 37080
atgaggtaat agtgccaggt tctatgaaca aataacgtga tctagatcgc gcttttataa 37140
gaagtcctat atgtttttct aaatgtcgta aaacccggag gtagtcggac aatcgcagga 37200
ctgaatgtac tatcgcgatg gaagtagggg agaacgattt cgataatgcc gttagcacta 37260
tcaatagaaa gggtatagtc acgcatgtag tgcggacgga tttccaaagc cgaacggatg 37320
cggttatgga tttctttgag cgtttcctgg gatagagtaa agccaggcat tgtctcatga 37380
cggataactg tttggaacat gatgattctc catgatatta aaaagcccag gaacttatac 37440
aagcgcctgg gtggtatttt atcaaacatg aacttttgaa gttttggtat aaacaccata 37500
ttggattagg atattgaata ccgcatctcg ctggtgagtg aacgtctggc catcgtattc 37560
atagccaatc accacacgac ctgatttgtt caagtgtcgg atgatatcat gatcgccttg 37620
ttccaggtgc acagtgacct tatctgggcc atgacgggtg atgtaatact tgaccacttc 37680
atagttcaaa ggatccgcca ggagcttatc aacaatcctg gacacgttca tacgacatgc 37740
ccaagtgctg atgaactctg ccccgtcctt ggtgactctg atgaacggat tacgatgctt 37800
gagtgtcgca tagaccctgg cacgaacctt ttgtttcatc gtttggcggc ccatttacaa 37860
cttctccaac ttttgaagtt tttcattgat tcgggtaatc tgcggatata ccttcttgcc 37920
aatgatgaca ctcacgacgt atagcaatac aaggaacagg atttgctcac tgccaggcaa 37980
tgataatgtc gctttcgtga acaagacact tggccagtag aatggttcat ctgtcatgcc 38040
cgaccagaaa cacgtattca ggataattac acacacgccc gcaatatagg ttaagaacga 38100
cacaaagctt tcgccatcag aattgcgacc gccaagttga ttgataacga tatcattgaa 38160
cttgcgagcg tgtttcttgg caatatggtc tttcatgcca atctgtacca tccagatttt 38220
tgagagcatg ccaatgttca ggtggattac gacgtaaacc ggtgtccaga tgatgaccat 38280
catcatgatg aatgctgctg ttgataacat ttgaagttct ccttatcgct tcttgttaac 38340
tggaacacaa gtgccacgga ttgtgatctt accttgtttg ttgtcgttgg cactttccaa 38400
cgcttgttca cattggctca gactatcgta ataacctggc acttggacgg tatctgcttc 38460
accgccagca gtgatgaaca ctaggtatag gatgaacgct ttcatgacat tagcccctgt 38520
tccaagaatg gggctttctc aagcttggct aggctgttct cccaaccagt ggaatcctgg 38580
acaacgccaa gcgggcacga atcactattg attgtgtccc aatggtcgtt gccataaatc 38640
tgaggcttct catcaaacgc ccagacttcg ccgttcttat ctacggcaaa ccaccagtcc 38700
ttgtcttcat cgaagtaaca tttgacttca tcgacaccaa acgtgctgac tttgacttct 38760
tggtttaaca ctaaaagttt caaactcatg ctttatcctt aaggaccatg cgatccatat 38820
gcgggttggt taataaacgg gttgcgggaa tgccacatgt tttggcaacg tccattaata 38880
cagatgttct ttccgacaac ggtaatactg cttggtaatg gcctggactt ggactcagca 38940
taaagtcaca gtgtagccaa tagttggaca tccaatcttc agaccaggaa tggccatgat 39000
catccttaac agtgactgtg ctcttgtgtg agtcataggt cacaacagta atccattctt 39060
cttccacata atcccataga cggaagttat atggacgaag gccattaggt agcgattgga 39120
gaacatccag gaacgaacgc tctgtatccc aatcaatatc ttcatcattg tgtgcgtatt 39180
tggcacacac tttgatcttg ctagccatta tcagctccta taaacttttg aagttccatt 39240
ggttggcaac gaacttcttc aaacatgaaa tgggttggcc ttcaaccatg atagtgtaca 39300
cacatgattt ggcaatccag gttggaccaa tgacgggcaa atctagtcgc catgctttgt 39360
cagactcatc caggagcatc ccttttggtg gagtagtgcc atcatataac ttcaggtaat 39420
cacgacctct gacgcccgct gagaagtacg tatggagata cttcttgctt tcagttacga 39480
aggcattctt gaacaggtcg caatgctggt tatagtgacg gatacactgg tcccatttct 39540
tcaaaagttc agcttcatca atctcacact tggcatgctt tgcgaagtta cgaaccttat 39600
ggccaagaga acagataggc aatgacttca gcatatcatc atggatcaca cgccaatcgc 39660
caaagcaatc ttgttcttca atggtcactt ccatgcggta attacccgtt ggggtgtcgg 39720
catcccaata tgcaggcgag aacgaataag acattcttct tttcatgata ccacctcaac 39780
atgatgtttt ggcatcattc gccagccatc cacaaccaca tgttggtctt tgatatcggt 39840
gatcataccg cgttggaaat tgccatcagg tgtagtgaaa gtgacaatct tattgaccat 39900
aagttctggg cgaatataat cttggggttc caagtgaagg ccattgcttg ttggagaatt 39960
cgcccaacct tgtgccatgc gctctttctg ttgaggttta tcaccattgg cgatcgctac 40020
tgcacggcca tagtagaagt caaacgaata acacttgtca tgtaatagac aatcttcatt 40080
tggacaccaa acgttattgg agaagtctga attcatttga gtatcgcaac attcacaatt 40140
taacttagac ataacctaat cccactgagt atagatttta agtgtgccaa cacgttgatt 40200
gtaaagcaag gtaatgtaca tgccaccatc aagttcccat tcgatttcca tcagtgtttc 40260
agtgaaatca atgtgcttgg cattgccata tagagccttg tacttctcca ccaggtattg 40320
gaattggaag tcattacaat gcatgatctt aaccacacgg aagcggttag cagttgcacg 40380
atagcggcac gggttgatac ctttaactaa cataactgtg gacatgatta ttccctaccc 40440
ttctgataag tgtacttgac caaatccata tccttcatat tgccatggag gaagttacgg 40500
agatcaacag ttgatgcaaa cgcccaaaca ttttggactt ggtcaaagac cccatagttg 40560
tcaaacaatc tgacttcctt cttggcatgg aaataacaaa tgctatactt gcggaagtag 40620
atcacgccat tgatttctgt tgggatttca acgactgtat gaggaacacg accattcgtc 40680
atgttcttgt ggtagcagta atatccaagt gcacggatat gagccaggat ttcattgtac 40740
tgctctgcaa tcgcatgatt gacacgatta cggttatggc taagggtttt cattacaatg 40800
cctccatgaa catttcaagg gttggcaagt gtgggaattc caatttctga atacatcttc 40860
tccaatctaa ggcttcaggt tttggcatat ccacattgca aagcagtcgg ttgttcatag 40920
tgccatatga agtgaccaac atccaaccaa tagcacctgg aattggttcg ctattccaaa 40980
cgaagacatc accattctga tcaactgtca accagaatgc aatcttgcca ccagaacgcg 41040
ctaggttgcg ctgtacccac aattcattgt gagcattata gaagttccac tcaatgaagt 41100
tcgtgaagcc acgggacaac acagggattt ctacgccatt aatagatgca ttgaatgact 41160
taggtttcag tttgacttct gaaaaggctt tttccaaagc ctttgcaaat tgaatgatag 41220
cttgtttgct gtacatctca gcccaagttt cggcataatc agtcatcgca tagatgtaag 41280
aagtacgctc ttgaatggtg aagtcattgg ttgccaaatc cgcagcaatc ttaacagctg 41340
tagcagagcc attttcgaca atatgtgtca ggattgcttc ttctttgtat ttgaagttct 41400
caacaccagt tagtttgcgg atggtctcaa tagcagtttg gaagttcata acattttcct 41460
tagtggatgt tgtttgtttc gatgggataa tgatatcaaa tgacctgaac caataatgtg 41520
atccaggtca cagtttggat tacttaagca acataacttc tgccatttct ttccaatctg 41580
tcatttttgc ataatccgca agtttcaaaa gtgtacggat agacacttca cgaagttctt 41640
ggacattttc ccacatccaa tcaatcattt ctttagttgc tttatcatca acaccacgtg 41700
aagcaagcag tggagtctct tgagccactt gttggatacg caccatgatt tcagttggag 41760
tgtatacgcc aagtgatagg tagtgtgaac gagatagcag tgcactgaag tgtggagaca 41820
aacgagtgcc acgctcaacc atgccttgga aatcaaggtt agtgatgaag acaacagtac 41880
cttggaaatc aaatacaggt ggaatgtcat tttcttcaag atacttacaa gtagaaccgt 41940
aagaaacctt gcgaacgtcg ccagaatcca aagcagcttt cataatgttc aatgaaactt 42000
catcttggaa gatgttgtca gagtcatcaa gaagaacgat atcatctttt gaactatttt 42060
tgtacagtgt ttcatacaag aatagtggag ttactttacc tttgatatgt tcaaagttac 42120
ggatacgacc ttcttcatgg gctttggtca attctttttc aaatttgtaa gttttgccca 42180
cacctggcgc gcccgaaacg ataagagcat acaaagaatg gtcgataaca ccgtcagcaa 42240
gcatgtcaag aacattgaaa cgtttgcgga tgaacaaatc aagttcttca aggtcaactt 42300
cttttgtttc tggcgttggg ttaacaactg cgatagcacc agattcaatc aattcaatgt 42360
ttgattcttt aacgtgcatg tagaaaggct tgccagtgtc gccatcgata acacgcatag 42420
aaccgtcaag accgtctttt gcgaaaccat ttttgaactc acttggagag taacgacgga 42480
agttttggaa aacttcaaaa gtacctgaaa gttcttgacc gcggtaaaga ccgtttgtga 42540
ttttgactag tgccatggtg ttatcctcat gatgtttggg tgtttcgttt tgatgggata 42600
ataataaaga atcccgacca caaataatgt gatcgggatc gcgttttaag acaaagcacc 42660
aggaaaagtt tttccaacag tatagaaggc gttcccctct ccataatgct tctctagatc 42720
ctggactctc tgtttggcac gaccaggtcc aagtccagtt cgggagacag aacggtcata 42780
gatgtactgg cctcccacaa tataatagat gtagatgtaa tttgtcacag cttctccagg 42840
ttttccaaaa tctgattgat cttgtgtttc gccgtatggt tgctgccacc gccaatacat 42900
tctaatgaac gttggattga ctgcacctgg cgcaagctga ttcgcatatg ggtatttgcg 42960
gctttaacat cagacaacaa ctgcatcatg aagcaattat ccatgaaacc atcttgaaca 43020
ttatcgcgta ccgcaatatt cccaacgtca gtggtcacgc cattatcaga acttgtcatg 43080
acagcatgga gatggccatt aaagatgaac atgccctgga catcatcaaa gaattccata 43140
tccaaacctg tatcaataca ctcgcggatc gcagcaatat gatgtggtgg agtagtgttt 43200
ccagtataaa cctttttcat gatatcacct attcaaagat aatgtggttt agtgtcagga 43260
acgcacccag gcgtgcatgg agattgtcaa actctggagt cgcaacttca tagtcatccg 43320
cactcactgc catgaagtaa tagtcaaact cagcatcata tgcatacagg actttttcgc 43380
cattggcttc gttgaattct gtgatcgcat tttcaacagc gtcgtttggt tcatcaatat 43440
caaagtattg accactgatt cgcaggcttg tgccatccat taagatgagg tgtttaccat 43500
cttcaaaatt agttgccgcc ataattatct tcccaagagt tgttcatgaa ttccaaagct 43560
ttcgctttcc atttttcaca gataaatcca ttgccatttg gacgacctga atataccagc 43620
caggaccaac cttcaccacg gtctttatcg tagatgtcca accattcacc ttcttttgaa 43680
agtttgattt tggaatggac attctcgcgc aggcgttcgc ccgctttttc aacgtgtttg 43740
cacagtagga tcatgccacg cttgacgcca agcttttgca actgttcgtc ggtgctgtga 43800
acttccgcaa agaatgtttg accgatttgg atgtgacctg catttgcatc aaacatgatt 43860
agtaccccga tgtttctatc atgcgacgaa cagcatgctt caggtgttct tcaatacgaa 43920
aagccgtatt cgcacactct ggttcgccct tgtattggtc caggatctta tcaaagatgt 43980
tggtacccag gaagttgcga gcatggatat acacatccat aatcggatcc ataaaagcag 44040
gaacttcgac tttatggagt ttctgtttgt ctctgttatg cagtgcacca aggtcttctt 44100
ctttgacata cattgcagtg tttggaattt ggtaaacaat catgcttcca ccttctcttg 44160
cttaattttg gtgatatccc attcagcatc ccattcaccc gtcatgctat catagaagaa 44220
gtgcacaaca aggtggaaat tgatgttaat gatatcacca tctttcaatc catcccattg 44280
gatggcatcg tccaggtaag tagtgttcag attggtaaag tcatctgtac aactctcatg 44340
gttggcatta gagacttcat cttccaggat ttccatcttg gaatatgggt tagtctccgt 44400
tacatccagg atatggactt tggctgcaac gtcataaact aacattacat ttcaccaaat 44460
gcacggacgg aaacttcata tttctcagct tcatccgaca ggttattgag atgacggatc 44520
gcccattgct tgtaacagtg gatgattttc ttcaaaacac tgtgtggacc atcaacgctt 44580
tggtaaggaa gaacaatttt ctccatacgc atggagttgc caccaaggtg aaggtcaaag 44640
acaacaccat cttcttgggc gtcaacgttg tcaaggtaaa ctgagttgac atcagtttca 44700
gtgaagtcaa acatgcccac agcaacttgg ttgatttcaa gggcgttatt gatgatgcaa 44760
gaccaagtcg cattttgttg aagttgtaat ttgttcatga tgttggttcc tcaatcagga 44820
agtgtaatgg tgtttgttca atgccattat gttcttcaac aaacttttga agtttggaat 44880
atgaagtctc attggcttca gtatagaaga cacactcacc ttgttcattg atcaggttat 44940
gtgtgccatt cacacggtca taggttgccc acagacctat ttggttcgcg tttggcatgg 45000
aatatccagg gaatatgatg cttcaaacat atcgatgcaa acgtcttcgt gtaccaatgc 45060
accattttta tcaatggtga agattcgggt atatcccatt tggtcaacaa atccatcatt 45120
gagacaatgc atattgtctg gaccttgata caaggccata tccgttcttg caaactgtaa 45180
tagatgatca cgatcccatg ggttgagcac accattcaca taacgacgga acaatcgcag 45240
gatggaactg aaaacatcaa caacattaac gccatcagtg acgtaaatgc ggaaagaata 45300
tgaatgcatc ttacgatggg ccaaagtgaa gtcgacataa tcatcatcga tttcaatgat 45360
gttttcaacg gtcacatgac gatagaacgt actgattgta tccaagtaag aatcctttgg 45420
atcaaagcga tgcatatggc tggcaatcgc gccagttagc agaaaactcc attgagcttt 45480
ctgaaaattt tcagacaaca taagtaaggt accccttaga tagtttggat aagtttaagt 45540
gcgatttctt cgttagacat ttgaagttct tcggaagtca actcatggtt taggaactgt 45600
ttaaagttca aagtacgacc atcaccacga cagatattta catcagaaag ctcagtactg 45660
cgacggataa cacaacgagt agttgtgcca acaaaagtga aggtttgagt ttcaaatggt 45720
ttacccaagt atgaaaccgt tttaactttt gaagctttct taatttgcat gacatatccc 45780
ttagtggatg ttgttgtgtc gatggggtaa tgatatcagg ttggcactac caataacgtg 45840
atccaggtca cgttttcaaa caacaggtaa taaaaagccc caggaaattc ccagggcttt 45900
ggtcttaaaa gtaaatccag atcaggttct tctgatgatg ggtccaaaag tctgcatcgt 45960
cataatcgga aagcttattg aacttggcca actggttgcg gaactgttgg cggcgacggc 46020
gattggattc atctttcaat gattgattgc aagagttgtt ccaaccggta tcacgcgaca 46080
gtgttccctt ccagtcttta tggtgtttca attcttcgtc atatgcttct tgaatactca 46140
tcacatgttt caacccagat gcgattgcga atccaggatt gaactttggt tggttgcctt 46200
tggtacgacg gattgttttt gacattttct tttcctctaa cagttaagtt agtggaaaag 46260
catcttcctg tgaattttct tcataatctt atcctaattg cgatagcgct tcattgcgct 46320
cctgtggtgt tttaaagcgt tctttaatta cccaggacat tttacctaca aagagcattg 46380
tactctccag gatttgatac gggagaccgt cggggtcttt tgggtcgtat cgcttcttgt 46440
atttcttttc catggcttaa tcatccagcc aacgaacaaa tgatgtagct tgttcggtgt 46500
aaactttcaa aagttttgga acttcgacga tacgcatctc aatgacaatg cctggctcct 46560
ggtcttcaaa acgtttcgct ggatccgcca tgaataaacc acgtcgtgta tcagttacca 46620
cgtttttccc tggggttaag acttcaagta caggcgtttg atccgcagcg cctttcggta 46680
gttggtagat accagtgatg gaacctgttg gtccaaccca aacgcggtat atataaccat 46740
cttttttgat gtatgcaatg tagtcggtat cttcatccac aagtccaggt gcgatcccat 46800
aacccataga acgaagggta ttgatcaatg caattggtac gttgaaataa ccattatcga 46860
tacattcaag ctggtctccg aactttacgg tgttaactac tgatgcacga tgaaccgatg 46920
acatattctc actgtcaatt ttgtagacaa gtgttgaatc cttgtatttc aacggaacct 46980
tacgataatg actcatgtca ttgttatcca gctgagctgg tggcaggtat tcttcagaag 47040
ataccgcaaa gaggttcaat gattttagaa tgtcacgcat gatgtttgtc tcctaagccc 47100
ggaatatcca tggcttctat ttgggttttg aagttttcaa tagctttctc tgcagcttgg 47160
aaaaacgtta gttgtctact cttggacatt aacttttgaa gttcttgccg ttgggtgtcg 47220
tccaagacga tattccaaat gattgttgca ataccatgtg gtgtcatatc accttggtat 47280
tctctcatta ctttttcaac agtcgcttta tcaagctgca taagaatcct taatttttgg 47340
gtttgttcca agggttacgg gacttaggct tcggatcttt gttcttgatc ttatcggcca 47400
tattcttcaa atctttatcg atagattcga gtgtgtcttt accctcaacg aagtcgataa 47460
tgtaacacaa gatgatggcc atgaaaataa ccaggacaaa aataacccat aacggtgtgg 47520
ccaagaaact gcggatattc ataatgacta ctccaagtct ttgaaattac tatataaatc 47580
cgcaactttt agacgtcgtg gatgttttgg tttatcaaaa agttcgaaca ctttgaacgg 47640
acctttgtct gccaggacag agtatgcata atcatagacg gtctggagtg cagtcatgcc 47700
acattcggta cgagcttcca tctcaactgt ttcttcttca ccgtccttat tggtatactc 47760
aatctcaaac ttgcgaacag gtccataagg gtgtttggca ctttcaatac gggttctaag 47820
cgatttcatt gatctgtacc tcaatactgt taagaattag gatacgacgg tctagcgtgc 47880
gaccacgacg atttttaccc caacgttggt agactgaata tggaccttgg attaggtgac 47940
gttgaacacc tgatatacct ttacactcac ctaatggcat gaagttatca agttcttcat 48000
acatgagtgg tgtggacgat gccatggtat taccgatatc gataatttca cgaccggtct 48060
ccgcaagtat cagctgatcc gtgttcaaca tgattggagt gtagccgtat cggatgtaac 48120
gctgtgccaa taacttgatc agcttacggc caatggttgg cacgtgtcga tcggcattct 48180
cagtgcctgg aatatctaac gaacagatga cactgaacag catacagttg aaatatttgg 48240
cctttagagc gcattcatca agcttaagtc tttccgactc actggtggct tcacgggagc 48300
ccgatttgta ttcacgacgc agttcaatca gtgcccgaac gacattacct aacgtatggc 48360
cgtttttgca gacaacattg ccatattctt gtttgatgat atgcggatag aacatgcgga 48420
actggacgac ggttactttt tcaaataaca tcttctcagc gtagatagag ttggcccgag 48480
caccaaaacg ggttttatcc atgaacgcct tttcgggatt tttaattggg cgaatcggac 48540
cacctaggaa acgacgcagg gttttaatac cgacagaatc ctttaggaaa ttactaccga 48600
taaaattttc aggagagtta gaatcaaaat agatgacttt ggcaagagcc tttttggttt 48660
cttcaaaagt tttcataata tatccctttc ggtttacttc actcgacacc cgatatgaag 48720
ttattttaga acagggatat tattgcttga taaaaacttt tgaagaaaaa ccccgacacc 48780
gaaatgtcag gggatttgtt tatattaatt tgactgcagc aataccagtc aaaccttcag 48840
taccagagta gatatcaatc acttcggcgt tattcacgcc ttttagttta accttgtcat 48900
tagctgccac tgtgatcatt gatgtcaggc taacagtatt aacgtaacta ttaccagcgc 48960
caaccgtaac ttgccgacac cgcttcctgt acctgtactc atgttctacc acttataaac 49020
tgatatgcat tgggtttagc caaacttcat atttgctgtc aaggttctta acagtgtaac 49080
ggaatactcc gccttcaata taggtattag ctacaccttt ggtttgtttg agatcaatag 49140
ggttgatgtc aaatggttcc cagaagttat tattgtctgg gataaattgc cgttcaatat 49200
caagtgtagg actaccagtc agagttgagc gatcgacctg cacaggagca tttggattgg 49260
ttacatctac gattatccat cgcatctgga agtaattagt gtgtgcacta ctcttgtgag 49320
ttttaaccgc acttgcagta ccacgaccac gaatttttaa cataattgtg actccgttaa 49380
tttggacacc acaattattt aagaggattg cagagtgatt gttcagattt aatcgtcagg 49440
aatataaata aaggccacac agattgtatg gcctttggga ttattcaacg tcgagggttt 49500
tcagtagttc gatcgtgtta tcaaggtttt taatcatttt ctcattcatg tcttgcatga 49560
acgcaaagtg actttcttct tgattgacca agaaattacg atgttcttga cataattgca 49620
ttaaatcgtc tttcttaatc gtactatcgc catgagaagc cgctttatat cgcgtcagga 49680
gacccacatt gaacaaaccg tgattgttga tggaattaca taggtttgct gtaacttctc 49740
cacgacggtc ttctgggatg tttccggcac accacacatt attggacaac agtagtttgc 49800
cttcccatgg aatgatgtgg acactgccac ccatgcccaa gtatttctca actaggttca 49860
atgggttgat cgacatcacc atgtaatcac tttcgcgaca caacaggaca ccatcgttca 49920
ccttctggat tttctcatcc cagaatttct tactatcaaa aaagctactt gttggcattg 49980
tctcgctcct ggttaatata acctagccaa gcactcaggc gactctgagc acgttccata 50040
atcttgtcgc gttcactaag gatcacagta ttttgtttga accctggtag gcgacaacga 50100
tattcccatt tcccattgaa gttgtattga acactcgcga tacacagttt acccgcaaat 50160
accttataca tctcgggacc acggatcgca tcaattcgaa caacttcaat ggttgaaatg 50220
acactagctt ctactttcat tttatgcatg ctcctgacgt gcaatgttct ccaagtgtgt 50280
gtcaagaact ttgcgattag tattcagttt ctttaacatg cgggctttct ttgcaagctt 50340
gaaacggtct tcaatcaggt catacatgac atcttgtgca gcaaacgata agatgttcat 50400
tgctttgcca ttaatgtcta tgaacttttc atcaataaca ccatttttat caacatgaca 50460
tgccttgacc atggcaacac gcttcaaacg attaacacgg aacgtcacca accaaaactc 50520
attattgata agtgctgcat tatgtttgga gcaagcctga ccttcgataa ccacaccatt 50580
tttccaggct tgaagttcaa gatgggaaat ggtgcaagtc ttattacgat acaggtcgaa 50640
caatgcacgc tcagaattgt tcaggcggtg ttgttcacca aagaagttga ggtttgcgat 50700
aaaacacttt aatttaaatt tgaaatcttt catgatgttt gttccatttt ctttttgaat 50760
tctttgaatt ctttgcgctt gttgagatgt gttacctgga aaacggcatc tcgtggatcc 50820
ataccttcca ggtcgacact gtgcaacatt aaatatccac aggtgccatc gcaagtgttc 50880
agccatgatg aatcttgttc atgatattct ggcagttgaa catatgccca catttggggt 50940
tcaggtgaga taatgcttga accgtcaact gcaaaccaca ttgaatcagg atatccaaat 51000
tgcatccagg catttaacaa tgcgtcggcc ggtaatggga tgttatcaag tcccatggtt 51060
aggacaattg atactgaatt tactggtttc ataaataacc ttctttggct tgttcttcca 51120
atatttcttg gttaactttt gaagatattt tgccacgtga acgacctttt gaagatttca 51180
caatgacaac ccttgtactt atgtgcggga cgttaaattg ttcacggtca ccattcacat 51240
aatccttgac attctgccta acttcttcca aagaagctcc agacagcttg tatgagctca 51300
tatgacttgc tccaggacca tagcagatcc atcatagtag atatcaatgc ctggagtctc 51360
gcacatcttc tttggcgaag aagcaaatac cttatacaat actggttcac caccaacacg 51420
agaacatgct ctcctggcat acgtcagtgc ataatccgga agtgtcgtga agaacacttt 51480
atccagattc ttggtacgac ctggttcatt aatcccaaac ccatgggatt ccggagggag 51540
aataacatct ccaatctccg catttgaact tgtaccatgg taaaaaatat gactcatttc 51600
tttccttttg ttttcacaaa gttagaatcc gcaggatgtt cactacttcc aaccatttgg 51660
aactgtggca accacaatcg gtaaacgcct gtaatcttat ggcggacata atgaagtcca 51720
gttttaggat tacggtatac gcactgttca tagtacgacg ggatagacat ttacttctcc 51780
aaggtttgga ctgaacgacg acgctgcttt actacacgat tcgcacggac cttacttaaa 51840
cgacggttgc ggcatttcat ttcttcacct catacatctt atgcgggcga taaagttcaa 51900
catttgttaa accattgcat acaaagttga taccatttcc aaagttacgg aattcccaaa 51960
cctgaccttt ttcagatttg attcgcataa cagtcacatc acgcaaatcg acagtgatat 52020
tacaattgcc atggtaacga ctttggaagt tgatgacacc ccattttggg ctatccatta 52080
ggttgttgat cacatgttca aaaccacgac gcgctaacaa acgaccaatg cggaagatag 52140
gacgcttact tttgatcaga tttttctgat tcatgccttc ataaaaggca agtaacaatg 52200
caatattttt catgattatt cacccaactt actgttagca tatttgacaa accctttctg 52260
acgagcttgc aaggacttca gataacgatt gttttccaaa gcttttgtgc gaacttcttc 52320
ccacatcagt ttagttgcag tcttgatgtt gtcatcttgt ttgacaactt tgacatggat 52380
ttcagtcacc aaaccattct ttaaacaatg gttgaattgg ttaggaaccg cgaaattagt 52440
gtaacaatca aattgtttaa ttgttacata gttgctgccc atatctgcaa caagtacaac 52500
aaactctgca tttgggcaat gagttatcaa ttcattcata ggatgttcct tatggatgtt 52560
caagaaagag cccaattaag ggctcatgat ggattataga gatgcaacaa ctttgttaag 52620
agtttcaagg tcaacacgtt ggatagtagt gtacttagca ccttctttga actcaagacc 52680
tagttcgtta agaacgtcag ttgctgattt accttttgaa gggtagaata gtagtccaaa 52740
tgtaccattt ttgattggag tgatttgaag acgaccattg ttaacaacaa gtgagccata 52800
tgtttgacgt actgcaggta ctttaacaac catgtcagag atgttaagag cttcaagagc 52860
ttctacagaa ttgaaaccag agattgcttt aggctttttg atttcaattg ttttaggctt 52920
tttgatttcg ataacttcaa gtttgaaacc tttaaccatt gctttcttgc cgttaagaag 52980
acggttaaaa ccagtagagt cgccagtgta accttgctca actgctttgc ggtagttagc 53040
cgctttagtt gctgcagtgt cgattgcgat agtgatttgg ccgtttgtga taactgtttt 53100
agtgttcata atcaatgttc cttatggatg tttgtttgtg ttgtttgttt cgatgaggtt 53160
atagtaccca attacgagaa tcaaaaacgt gacggagatc acgttttata aaagaaaggg 53220
aaatcttttt agatttccca tataagttat tgatttgcaa ggcgtaacca atcaccaaat 53280
tctttcaaag tcaacggtct gtggaacgtg acataagttt tggtaatttt cagcttggta 53340
tctatcttat tgcagtaagc accctggttg tatgtgatat ctatatttgg gataccaccc 53400
ttgatcttac gcatctgaac attgacggtc atatccgcac gtatgttgac ttcatactta 53460
atgttctgat atcggaaacg aatgcctgta tgcgatcggt tactgaagta ataacattgt 53520
gtcacgttat ggacacggtt aatgacgttg tgtaactcct tggccagaag cttacgggga 53580
ccatgaccgt ccgtggcgtc aaagaagttc ttgacctgat gtgtaacagc catttccaaa 53640
tcaaacacac ctggacgcaa cggtgttgtg cgacgcttgg tcatagactt cttctggggt 53700
tcaggtttag aaccaaataa tccaaagaac caattaatca ttcttttcat gataattacc 53760
tttaatgtct tggatgactt catttaaaat agccaatcca acacagtcat ctttatattg 53820
gatcgctttg ttagaacccc ggcacacgta gtaatgtacg atcggcaacg ggttattctt 53880
caaaaattgt aagtggtatg gatgaccagc aatcttacgc ttcattgcag tgaagataat 53940
gtcatggaac tgttcatgat ccactttacg gttgactgga ggaagtacct tcattttctt 54000
cgcggcgttc ttggcttcac gaccttccat gaatagaagg tcaatgttac ctgtcttaag 54060
atactgccac aagccctcaa tcgtgttgta atgcacctca tggccattga ctacagcaac 54120
accttcgtat cgatagaagt tcgacaggat gcgtcccacc tccaatttgg attccgaata 54180
catgttgata tgactgatgc cttcatactg tggtgtatgc ggaagcaatg atggttccca 54240
accgatacac tcacagatgc catctttaat ttggtgcatc atatgttcaa tgccatcagg 54300
ctcaggtcct gtagtaatca catcgccatc ctggacctgg tcttttaacg gtttgatgcg 54360
agtgtcaatc attgggaaga ttgactgtcg agtaatcttg tagtaaagca tgacctatac 54420
cttatcaata tctgaactga aatatgccaa cgaattctct ggaccatgtt tggacaacac 54480
cacaaggatt cgttcgcttg gtgtcatgta atcccaaatc tttgaatcca ggttgatttc 54540
gtttttctgt gcttgggtta actgctccca ggtcttctca acgataccgt gagcccatgt 54600
accctttgga taggtttgtt catacagctt aacaatatga acgtaccagc ccatggtttt 54660
acgctttgaa cttacagtgt cagccatttc aacatctcca ttgaatgacg ttcaaggttt 54720
gattgcatgc caaccattgt ttgaggggta atatgaataa tccatcgacc attctctttt 54780
gctggacaac aatgccagtt agatgtatgg acttgcaaag tgttgttcag acgcttggca 54840
aaaactttcg catgcatttc atcttcagct gtaatcgaca ccgcaaatgt ttgcagactt 54900
ttagtatcgt tccagtatac aaaaccgtgt gtacggccaa caaaattggc aattgactgg 54960
gataattgga ctgcgacttc accagctaat tgacgacctt tgtcttcgtg gacaaaagcc 55020
actatgctac tctgaccgta ttgatccaac attttcaaag ttaggcattg ttcaggtgaa 55080
ggtagtgctc ttgcaaatga tacttgaatc atgatgtttc tccaataaaa taaaaggacc 55140
aggtaataat acctgatccc aaacttttga agttttaaaa tccacgttgg actttatgca 55200
tgcaacactt cttaaacttc agaccgcttc cacatgcaca tttgtggtta cgttcctggt 55260
tcttaacacg acgtgcagga attcttcttt ctggtacgcg aagcatacga cctagtggtt 55320
tcatatccat ctttattctc ctaaattttt cgccatttag caaattcaaa ttcggctttc 55380
aaaccctgga aggtattgtt ggcaatgtgt cgcaacaatt ccggagttga tacaccattc 55440
ataatatatt cgttgatatc tttaccgtat tgttccagtg agtgtggtaa caagcatacc 55500
ttaacaccgg ctttgatcat tttctcaata cggttaacga tttctcggtt gcgtggttga 55560
ttatctggaa tatagatatc acccttcact ttcaacaaat ccgcatctgc tgatgccata 55620
cagttcggca agaacaggct atcaatcgga ccttcaacga ccagtttggt acggtcaaag 55680
tcgatacgtt ccagaccata acatttggtg gtcttttctt ctttcttaat cgagatatat 55740
cgcaggtcac acttaggatc gaatgaacga ccttgtgcca ttatcatctc gcctcgagca 55800
ttgaagaatg gtatcaccaa acgcggatcc tttggacact tctcagcaga caacatattc 55860
tcagggttca taccgatagc aaaacgctga tagttgttgg taaagaataa tatgtcaaag 55920
aaacgttttg gaacctggcg atttaccagg tagtcgtaac acatatggcc agggccaagc 55980
tccgaaactg gaatacaata agtgtccaag gcgtaatcac tttgaacttc ctgttttttc 56040
ttgaccaatg aacgggtacg ccgagtatct ttgtttgcgg ctttctttgg ttcttctttt 56100
ttcttgagtg ccatgtaaga accagaattg cgtttggttt cctggaagct atcaacaaca 56160
tatgactggt acaaggtagg ctcataagtt ttaagccata accctaaatc ccaccctgat 56220
gcttcaccgc agttatgaca tttgaaccgc atccgatcaa tatcatgatc agcataaaag 56280
tggccacgac gtttattagc tttcttttta ctgtccagac atatcggaca acgaaaacgg 56340
caaatattgt cttgttccca tgtaaatttg tctaacatac ccgacaacat gtttatgtaa 56400
gacttatcaa gttctatgct cataataaaa agcccttaga ataatctaag ggcaatttta 56460
actttattca aacatttgag gttttacaac gttacgtcca acttcaccat aatccttatg 56520
gacaatgatt gctgtcagcg tgcggtttga acgataaccc gcgttattat gccatgcatc 56580
gcgaccagcc aacgtattaa acacttcgac agtacatcca cggtattcct ggatttgttt 56640
atggtgtaag tgtcctgtat accatgaacg atgttctgtt tgaccccaat cgcttggaca 56700
gtcatgtgcc atgatggact caagctcagt gcctttaaca gtatgcgtat gagtcacacc 56760
gatcaggttt ttaccgaaac ggtggtatgt aaatactgaa gggtttacta cgatttcaac 56820
acgaggttcg ttttcatata gacattctaa tgccactgcc agccattgac tactatgttc 56880
atcatggtta ccgattgcat tgatgacttt aactttttcg tgtttctcta gtgctagatc 56940
gatacattga cgcatcaggt taacaccaat acggataatc tttgcccaac gcgaatcaac 57000
atcaagggca ttacccgaat gtgctgtacg gttttgaaca ttgtcagaat ggaagaaatc 57060
acccaggttg acaataagag cagttttggt tgcgggtgtt gattggacca gggattccat 57120
ggcagcaggc aataactgtt ctgcgacctt caagtcataa tccataccag tttcttcgcc 57180
ccatgccatc atgcccaggt ggtaatcacc aatcggataa tggacttccc aatctgttgt 57240
ttcgaccaga ggtgcaggtg ttggcataac tcgtggaaca tcttcacaca tcgcttgaac 57300
aacttcttgc aggatctcga attgttcttc agcatcgcgt tgagtcttca cccattggag 57360
tttcagttca ccgtctttac cgtaaagcgt tgacgtaccc ttgacaccga agccttcagg 57420
aactttctta cgcatatcgt ggtccggact ccaacctttg gatgctgcca cggctttcag 57480
tcgtttaatc gttttggata ctgtttggtg actgattcca agagcacgtg cggctttagt 57540
ttggttgcca tcagctttga taacagcttc aagttgttca gcctggcgtg tagtagtaca 57600
atactctgtt agaccagaca aagcctcttg gacaggtgtg tatttcttat tcttagacat 57660
ctaagatttc ctctttacat taagaacctc acaatattat ctttatttac ggatatgaaa 57720
aagcctatca tttaagatag gcttgtaatt gtattaaagt ttgactgcaa tctcttggtc 57780
catgatgtaa ctggcatcca ggtccatatt gccataaact ggaatctcat catcaggttt 57840
atcatacaga cgacaacgga tggtgccagt attaaggttg agcacaagaa tctcagctgt 57900
ccactttcgc gcgcgttccg cataaaagtc acatgtcgag tacattggat gttcactgag 57960
atcaaccgtt acgcgaggta acggcaatac tccaggtttt ggtttcttag ccatttagtt 58020
agttcctact tttgggaata gattcatcaa atctttcatt gaaacatttg atacgttgat 58080
gtaatggtgt ttagtgttat gcttaacatc atccatattg cgccccagga gtgcacacac 58140
aggttcagct gtcatatcct ttgacatata caccagaaca gagaaaagtt cgctacggct 58200
gttatatgag acctgaacac gtccaacatt agccttacaa gttacataag gtttcttctt 58260
cgcaggtact tttaattcag caccatggat ttccaaatat tccacaagac cattcagtgc 58320
aggcatctca cctttaacaa ttgatggcat tgtcacctta attttggttg gttgtggttc 58380
aacaacttca ataggttttg gattagtgat ttcctcaact tcaataactt taggagttgg 58440
tttaggtgtg actgttggtt tgctattaag agcattgtga gagaatttca aagctttctc 58500
tttgatgaaa ccttttgaag aacacttaca ccaacgtaaa gaacccaaac ggattttctt 58560
atgcatttgt ggtgtcatgt tatactcttt gtcacatgtt ggacaaatgt aaacaaaagg 58620
tcttgtttgt gcacgagtct tttgaccatt cattaacaaa gctttatcaa catccatatt 58680
gtgatatgtc ttaggttcgc aaccaaagtg acgcatcaca ttcttccatt cttttccatg 58740
gggttttgct ctgcgaccgt aaaccttatg aacaacaata tgagcaaatt catgaggaat 58800
ggtatctttg atgaattctt cttcattatc tgcaagaagg gttctgcaaa ggttgatgat 58860
gccgttttcg gtagtaaagc cgttagcgtt accagcaact ttacccattt ctttgatatt 58920
gatttccggc ataccaaaag tttttgaagg tacgatgcgg tttgcaatat caatgtactt 58980
ttgaagaaca tcttggactt gattaacagt aagcatgaca tattccttaa tggatgttgt 59040
ttgtttcgat ggggtaataa taaagaatcc cgaccacaaa taatgtgatc gggatcgcgt 59100
ttttggttat tggattattc ttcttcgtag tcggaacaat aatcccaggt gatgcttggg 59160
ttgatatggt ccaggattat ttttcttgcg ttaacttcca gttttggaca taccccaatt 59220
gactcaccgt tgaagaagac ttcatattgt acaccaatat ctttaaccac atattcagtg 59280
tcgttatgaa tattgatggc aacccgtaca tagtgtgttc caatacggtc tggtgaatat 59340
ccgatcgcac atagccagtt aacaattgat gctgcgaaaa tcgggtcttt tacacgatgt 59400
tcaggaatga taaattgaat atctttggta gattccaaaa tatcttgcac ttcttgcggc 59460
atctttctga attcactttc cgaacattcc acaaataaag tatttccgaa tatatgcatt 59520
ttcttagcca tgagaaaccc ccaaactaaa catcaagaac ataacaacaa agatgaagaa 59580
cttatacatg atgatattgg cgccacttgc ccggtcaatc ttcaaatcaa acatgagcat 59640
tgcaagtaac aataaactga agtcaatcgg taatccatcc gggaaacttg cgatatctac 59700
tatatccaac gatgccaaaa cacctattgt tgaatagaaa cccatacagg tgatcgtaaa 59760
caatgtaatc agtccgatga tatttgtcac acgacctgcg gcaaagtaaa tcgtacacat 59820
tgcaaggatc aacacatgtg cgattgttaa gtggttttca atgatgaagt taaacattac 59880
ggatctcctt ttggatttca tcggcaaatt tttggatatc tggccaggtc ggttcaacat 59940
caccccaatg cttggtacgg gttgctttga tatccaaaga tggaatgttc acctggacgc 60000
gtgtagtcca acagcattct tcattgcaag ggtatgttga aaccttgaag cttggttcaa 60060
atccaagaac ccaattcatg aattcttcaa agccattttc aacttcaaat tctttggaga 60120
ttagaccaaa cttttcgata ccttctttgc taggaaggaa cttgcggaca taatcatcac 60180
ctgtttcaga caatgaaccg atgtaaatac agctggtatc cgttgataca gtcactagac 60240
ctactaaatt aatcttaatt ttcatctttg ccacccttaa ttactgttgg tttccatgct 60300
tggcgtttag gttgtttggt ttcttcttga acggcgtcaa aaaggattgc ttggagcatt 60360
tcattaacac ccgttgagat gtcaaaatgt acccaatctt tttggtcatg acttcctgaa 60420
atagacataa tgccaccatt ggtaatattg ccagggaagc gctgtccatt gaatacgcaa 60480
gagtatagga tatcaccggt tgcagcatca attggaccat acgtgcagtc aatagtacca 60540
aagttcaaag tgattgcctg aagcgaatgt cccgagttga gatcctgaga gtagatacct 60600
ttaggtggtt gtgccacatt tttgaaaggt atgtgattga agcctgttgc aaaggtcaca 60660
taaacgtatt tgtaattctt ggcaacttct tcaataagtg caagatgttg cactgagtag 60720
atattattca tgatattttt ccttatagtt agagatttgg cgttcaatcc aatcttcatt 60780
gagtacatta cgcaccaaag attcttgggc taatacatca cattccaacc aggttactaa 60840
tacagcttct tctttatcga aaatgcattg tttttcgcca atgtcataat attcagaaac 60900
ataaacagca atagcatagt cttgaatacc acattggaag tcatcaaccc ttttcaaatc 60960
ttcatgggag aaccatgcat aaatctgatc aagactattg aatccgaaat acatgatgtc 61020
atagtcacaa tttttgaagt tttcttcatc ggggttcgga tggcgttcat tgtaacgcat 61080
atctccccat agataggtcg caagcgtact tgaagacaca atctcttttg ggttgttatt 61140
ggcatcgcga ccaatgactt taactttttg gtgttgatat ggaccaaggc cattgagcct 61200
ggattcaaca cgaaaaataa ctttgggttc cataacatat tcctatcgga tgttgtttcg 61260
ttagggacat aataaaaaat cccgaccaca aataatgtga tcgggatcgc gtttttggat 61320
ttagacttcg tgagtacctt caacaccacg ttctttgcgt tccgcagtac gcatttcaag 61380
ttctttcaaa ccaatgcgaa gatgatggat agctttagcg ttcgcagcac agttgaactt 61440
tgaagcctgg tagaactcaa gacgttcaat tgccgcaatc agcaagtctt caacaaaaca 61500
accattgtga ccgttttcac caagtggacc tttctggaaa tccacatgca aaccgattgc 61560
tgatgctgaa ccaccaaacg gtaggccatc ttcattcaga ttgttcgtga tatgcatttg 61620
atggtcataa gccaactaga ccagaacaaa ccatctttgt gatgacggag catacaggag 61680
aagcttgaag aagaacaggt ttatcacaaa ctcaagtcaa ttgagttaaa tggtggactg 61740
ccaaaatcca ggacattcgc accagttaaa ccccattctt tagggacaac ttgggaacgg 61800
tccaaagaat gataaaaacg agctaaatct acggcgttct cgttatagta ttttgtcgat 61860
aacattactt attccttttt aattt 61885

Claims (8)

1. a kind of vibrio alginolyticus bacteriophage of width fragmentation pattern, which is characterized in that the vibrio alginolyticus bacteriophage bites for vibrio alginolyticus Thallus VAP7(Vibrio alginolyticus Phage VAP7), deposit number is CCTCC NO:M 2018767.
2. splitting the vibrio alginolyticus bacteriophage of wide fragmentation pattern according to claim 1, which is characterized in that the vibrio alginolyticus phagocytosis Body VAP7(Vibrio alginolyticus Phage VAP7) it is virulent phage, have one in symmetrical head and longer Tail portion, head are icosahedral symmetry, and major diameter L is 60~70nm, and transverse diameter W is 60~70nm, L/W ≈ 1；Tail portion includes shrinkable Flesh sheath and tailfiber, flesh sheath length be 30~40nm, wide 5~10nm, tailfiber have 3~6；Optimal pH be pH6~pH8,4 DEG C~65 DEG C under the conditions of there is patience, 8~10h is cultivated under the conditions of MOI=0.001, potency is 1.8 × 1010 pfu/ mL。
3. the vibrio alginolyticus bacteriophage of wide fragmentation pattern according to claim 1, which is characterized in that the vibrio alginolyticus bacteriophage VAP7(Vibrio alginolyticus Phage VAP7) there is nucleotide sequence shown in SEQ ID No.1.
4. a kind of composition of the vibrio alginolyticus bacteriophage containing any one of claims 1 to 3 wide fragmentation pattern, feature It is, the composition contains the vibrio alginolyticus bacteriophage VAP7(Vibrio alginolyticus Phage VAP7) with And at least one of other Vibriophages.
5. a kind of composition of the vibrio alginolyticus bacteriophage containing width fragmentation pattern described in claim 4, which is characterized in that described group It closes object and contains vibrio alginolyticus bacteriophage VAP7(Vibrio alginolyticus Phage VAP7), vibrio alginolyticus bacteriophage VAP9(Vibrio alginolyticus Phage VAP9) deposit number be CCTCC NO:M 2018766；It is bitten with vibrio alginolyticus Thallus VAP21(Vibrio alginolyticus Phage VAP21) deposit number be CCTCC NO:M 2018768 in two Kind is two or more；
The above bacteriophage is preserved in China typical culture collection center on November 9th, 2018, and classification naming isVibrio alginolyticus phage。
6. the composition of the vibrio alginolyticus bacteriophage of wide fragmentation pattern according to claim 4, which is characterized in that the composition Including vibrio alginolyticus bacteriophage VAP7(Vibrio alginolyticus Phage VAP7), it further include each bacteriophage mutants One of or it is a variety of；Preferably, the mutant at least 90% is identical as the native sequences of corresponding Vibriophage.
7. the examination of a kind of vibrio alginolyticus bacteriophage containing width fragmentation pattern described in claim 5 or vibrio alginolyticus bacteriophage composition Agent or kit.
8. the vibrio alginolyticus bacteriophage or molten algae as claimed in claim 4 of any one of claims 1 to 3 wide fragmentation pattern Vibriophage composition is as claimed in claim 7 containing vibrio alginolyticus bacteriophage or vibrio alginolyticus bacteriophage composition Reagent or kit are being killed and/or are preventing the application in vibrios microorganism belonging to genus:
The vibrios microorganism belonging to genus includes vibrio alginolyticusV.alginolyticus, Vibrio anguillarumV.anguillarum, comma bacillusV.cholerae, non-Nonther conserved quantitynon-O1V.cholerae, Fermi operatorV.fischeri, vibrio fluvialisV.fluvialis, Vibrio harveyiV.harveyi, sick Vibrio damselaV.ordalii, vibrio parahaemolytiousV.parahaemolyticus, Vibrio salmonicidaV.salmonicida, vibrio mimicusV.mimicus, Vibrio splindidusV.splendidus, Vibrio aureusV.azureus, Vibrio gazogenesV.gazogenes, Vibrio vulnificusV.vulnificus, bamboo Pod vibrio pisciumV.trachuri, damselfish vibriosV.damsela, floating vibriosV.natriegen, clam worm vibriosV.nereis, fish Enteron aisle vibriosV.ichthyoenteri, shark vibrioV.carchariae, Vibrio campbelliiV.campbelliiAnd kill prawn arc BacteriumV.penaeicidaOne of or it is a variety of.
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CN110184241A (en) * 2019-05-14 2019-08-30 菲吉乐科(南京)生物科技有限公司 Vibrio alginolyticus bacteriophage resistant to high temperature and combinations thereof, kit and application
CN110184241B (en) * 2019-05-14 2021-01-26 菲吉乐科(南京)生物科技有限公司 High-temperature-resistant vibrio alginolyticus bacteriophage and composition, kit and application thereof
CN110468110A (en) * 2019-09-11 2019-11-19 大连理工大学 A kind of vibrio parahaemolyticus phage and its application in stichopus japonicus disease prevention
CN110468110B (en) * 2019-09-11 2022-08-19 大连理工大学 Vibrio parahaemolyticus bacteriophage and application thereof in disease prevention of stichopus japonicus
CN112852750A (en) * 2020-10-26 2021-05-28 河海大学 Marine-derived vibrio phage, microecological preparation, and preparation method and application thereof
CN112852750B (en) * 2020-10-26 2023-04-21 河海大学 Marine-derived vibrio phage, microecological preparation, and preparation methods and applications thereof
CN112646786A (en) * 2021-01-21 2021-04-13 海南海壹水产种苗有限公司 Rapid preliminary separation method for vibrio kammaticus phage
CN114561363A (en) * 2022-01-27 2022-05-31 青岛百奥安泰生物科技有限公司 Vibrio phage PC-Liy1 with cross-species lysis capability, preparation method and application
CN114703149A (en) * 2022-01-28 2022-07-05 武汉格瑞农生物科技有限公司 Vibrio alginolyticus phage GVA-P21 with high fermentation rate and lasting bacteriostasis and application thereof

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