CN111575243A - 坎氏弧菌噬菌体及其应用 - Google Patents
坎氏弧菌噬菌体及其应用 Download PDFInfo
- Publication number
- CN111575243A CN111575243A CN202010318177.1A CN202010318177A CN111575243A CN 111575243 A CN111575243 A CN 111575243A CN 202010318177 A CN202010318177 A CN 202010318177A CN 111575243 A CN111575243 A CN 111575243A
- Authority
- CN
- China
- Prior art keywords
- vibrio
- candelilla
- phage
- bacteriophage
- composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000607598 Vibrio Species 0.000 title claims abstract description 82
- 241001515965 unidentified phage Species 0.000 title claims abstract description 41
- 241000589876 Campylobacter Species 0.000 title claims abstract description 25
- -1 pplication Species 0.000 title abstract description 5
- 241001553290 Euphorbia antisyphilitica Species 0.000 claims abstract description 46
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 239000000203 mixture Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 16
- 230000002147 killing effect Effects 0.000 claims description 8
- 239000003242 anti bacterial agent Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000003674 animal food additive Substances 0.000 claims description 5
- 239000012629 purifying agent Substances 0.000 claims description 5
- 239000002671 adjuvant Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000006071 cream Substances 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims description 2
- 239000002674 ointment Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 238000009360 aquaculture Methods 0.000 abstract description 10
- 244000144974 aquaculture Species 0.000 abstract description 10
- 201000010099 disease Diseases 0.000 abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 8
- 238000005336 cracking Methods 0.000 abstract description 2
- 241000238557 Decapoda Species 0.000 description 19
- 241000894006 Bacteria Species 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 12
- 239000007788 liquid Substances 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 6
- 235000013372 meat Nutrition 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 239000011550 stock solution Substances 0.000 description 6
- 230000009089 cytolysis Effects 0.000 description 5
- 239000000706 filtrate Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 241000607618 Vibrio harveyi Species 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000009545 invasion Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 241000607528 Aeromonas hydrophila Species 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- 241000607594 Vibrio alginolyticus Species 0.000 description 2
- 241000607323 Vibrio campbellii Species 0.000 description 2
- 241000607272 Vibrio parahaemolyticus Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 235000015255 meat loaf Nutrition 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 230000000384 rearing effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000604933 Bdellovibrio Species 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000237858 Gastropoda Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000237891 Haliotidae Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 235000017845 Persicaria vulgaris Nutrition 0.000 description 1
- 244000170788 Persicaria vulgaris Species 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 206010047400 Vibrio infections Diseases 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N aldehydo-N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- UORBWUNQSRMCRM-UHFFFAOYSA-N butylbenzene chloroform Chemical compound ClC(Cl)Cl.C(CCC)C1=CC=CC=C1 UORBWUNQSRMCRM-UHFFFAOYSA-N 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- BKHZIBWEHPHYAI-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol Chemical compound ClC(Cl)Cl.CC(C)CCO BKHZIBWEHPHYAI-UHFFFAOYSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 229940014259 gelatin Drugs 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000001706 oxygenating effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
- A01K61/13—Prevention or treatment of fish diseases
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
- A23B4/22—Microorganisms; Enzymes; Antibiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/04—Disinfection
-
- 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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Wood Science & Technology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
- Environmental Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Physiology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供一种有效裂解坎氏弧菌的坎氏弧菌噬菌体及其应用。该坎氏弧菌噬菌体具有特定的核苷酸序列。本发明实施例所提供的坎氏弧菌噬菌体能够特异性裂解坎氏弧菌,效价较高,从而可以在单独或混合使用后起到对水产养殖坎氏弧菌病害的有效控制。
Description
技术领域
本发明涉及水产养殖技术领域,尤其是涉及一种坎氏弧菌噬菌体及其应用。
背景技术
近年来,我国水产养殖业迅猛发展,收益颇丰,但养殖病害在此发展过程中也逐渐成为制约水产养殖发展的首要问题之一。目前,主要的养殖病害种类中,细菌性病害的危害性最为严重,而这之中又以弧菌属(Vibrio)的病原菌危害最大。弧菌病害流行面广、传播快、发病率和死亡率高,是水产养殖病害研究的重点。坎氏弧菌(Vibrio campbellii)是一种革兰氏阴性、兼性厌氧的常见菌,是水产养殖动物的重要条件致病菌,可以引起养殖对虾暴发红腿病、鲍鱼患脓毒败血症等,还与螺类和养殖幼鱼的发病相关,给水产养殖业造成了巨大的经济损失。
水产养殖业中解决坎氏弧菌病害的常用方法是抗生素药物,但这些抗生素药物在长期的大量使用过程中,带来了诸如环境污染、耐药性产生以及食品安全等问题。因此,养殖人员逐渐将关注重点转移到微生物防治上。微生物防治病菌侵害的方法大体包括拮抗菌防治法、蛭弧菌防治法和噬菌体防治法。以噬菌体防治法为例,噬菌体是侵染细菌的病毒,分为烈性噬菌体和温和噬菌体,温和噬菌体在侵入细菌后不会引起宿主细胞裂解,只是将自己的基因组整合到宿主的基因组上,伴随着宿主基因组同步复制,不能够引起宿主的细胞裂解,一般不会出现有害影响宿主细胞。而烈性噬菌体在短时间内能连续完成吸附、侵入、增殖、装配、裂解这五个阶段从而实现其繁殖的目的。烈性噬菌体在进入菌体后就会改变宿主的性质,使之成为制造噬菌体的工厂,大量产生新的噬菌体,最后导致菌体裂解死亡。而噬菌体防治法也正是基于这一原理来达到防治病菌侵害的目的。因此,有必要提供一种能够有效裂解坎氏弧菌的噬菌体。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此,本发明提出一种有效裂解坎氏弧菌的坎氏弧菌噬菌体及其应用。
第一方面,本发明的一个实施例提供了一种坎氏弧菌噬菌体,该坎氏弧菌噬菌体具有如SEQ ID No.1所示的核苷酸序列。
本发明实施例的坎氏弧菌噬菌体至少具有如下有益效果:
本发明实施例所提供的坎氏弧菌噬菌体能够特异性裂解坎氏弧菌,效价较高,从而可以在单独或混合使用后起到对水产养殖坎氏弧菌病害的有效控制。
第二方面,本发明的一个实施例提供了一种组合物,该组合物包括上述的坎氏弧菌噬菌体。包括该坎氏弧菌噬菌体的组合物在保证噬菌体有效量的前提下,可以有效防治坎氏弧菌。该组合物因而可以是诸如用于杀灭坎氏弧菌、抑制坎氏弧菌生长和相关炎症反应、防治坎氏弧菌所引起的水产动物疾病等作用的组合物,具体像抗菌剂、水体净化剂、添加剂等产品。
根据本发明的一些实施例的组合物,除上述的坎氏弧菌噬菌体外,还包括辅料。辅料的成分根据组合物的具体应用不同而发生相应的改变,可以是诸如缓冲液、表面活性剂、明胶、海藻酸盐、葡萄糖、蔗糖、麦芽糊精等至少一种。
根据本发明的一些实施例的组合物,其剂型选自粉剂、水剂、冻干剂、凝胶剂、霜剂或膏剂。利用辅料将噬菌体等有效成分包装成为不同剂型的产品,从而适应于具体的应用场景,更有效、更针对性地杀灭或抑制坎氏弧菌感染。
第三方面,本发明的一个实施例提供了上述的坎氏弧菌噬菌体在制备抗菌剂、水体净化剂或饲料添加剂中的应用。上述的坎氏弧菌噬菌体可以应用到抗菌剂、水体净化剂、饲料添加剂等产品中,实现对坎氏弧菌的特异性高效杀菌或预防相应的感染的目的。
根据本发明的一些实施例的应用,抗菌剂、水体净化剂、饲料添加剂用于预防和/或杀灭坎氏弧菌。
第四方面,本发明的一个实施例提供了一种杀灭水体中的坎氏弧菌的方法,包括以下步骤:
向水体投入上述的坎氏弧菌噬菌体;或投入上述的组合物。
将上述的坎氏弧菌噬菌体或包含该噬菌体的组合物按照有效浓度投入水体中,可以有效杀灭水体中的坎氏弧菌,从而种苗投进水体前达到有效预防坎氏弧菌感染的目的。
第五方面,本发明的一个实施例提供了一种水产品的处理方法,包括以下步骤:
将非活体的水产品与上述的坎氏弧菌噬菌体,或上述的组合物混合。
将非活体的水产品与上述的坎氏弧菌噬菌体或组合物进行混合后,能够有效杀灭水产品中的坎氏弧菌,起到食品安全保护剂的作用。
附图说明
图1是本发明的实施例4的坎氏弧菌噬菌体对菌株编号为40-2的坎氏弧菌裂解结果。
图2是本发明的实施例6的坎氏弧菌噬菌体对坎氏弧菌的杀灭效果。
图3是本发明的实施例7的15天虾苗实验的对比实验结果。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
实施例1
坎氏弧菌噬菌体的分离、纯化和扩增
本实施例中使用的菌种来源如下:
副溶血性弧菌(Vibrio parahaemolyticus)ATCC17802,购自广东省微生物保藏中心;
溶藻弧菌(Vibrio alginolyticus)ATCC33787,购自广东省微生物保藏中心;
哈氏弧菌(Vibrio harveyi)ATCC33842,购自广东省微生物保藏中心;
嗜水气单胞菌(Aeromonas hydrophila)ATCC35654,购自广东省微生物保藏中心;
其他菌株,均为广东海大畜牧兽医研究院有限公司分离保藏。
1.噬菌体的分离
采自野外的环境中的水样品,利用多层纱布对采集的水样品进行初步过滤,然后将滤液置于4℃,10000g离心15min。取上清液20mL加入事先灭菌的锥形瓶中,并加入20mL双倍浓缩的液体培养基与处在对数期的坎氏弧菌40-2 400μL,37℃过夜培养。次日,培养液经5000rpm离心10min,用0.22μm孔径的一次性滤器对上清液进行过滤,将滤液放于4℃备用,得到噬菌体原液0,收集于无菌离心管中,贴好标签,4℃保存备用。
取200μL噬菌体0原液,加入20mL双倍浓缩的2216E液体培养基与400μL对数期的坎氏弧菌40-2菌液,37℃过夜培养。次日,培养液经5000rpm离心10min,用0.22μm孔径的一次性滤器对上清液进行过滤,将滤液放于4℃备用,得到噬菌体原液Ⅰ,收集于无菌离心管中,贴好标签,4℃保存备用。
取200μL噬菌体I原液,加入5mL的2216E液体培养基与200μL对数期的坎氏弧菌40-2菌液37℃过夜培养。次日,培养液5000rpm离心10min,用0.22μm孔径的一次性滤器对上清液进行过滤,将滤液放于4℃备用,得到噬菌体原液II,收集于无菌离心管中,贴好标签,4℃保存备用。
采用双层平板法,用无菌SM缓冲液对噬菌体原液II进行倍比稀释,得到噬菌体稀释液,取对数期的坎氏弧菌40-2菌液100μL与适当稀释梯度的噬菌体稀释液100μL混匀,然后加入到5mL半固体培养基中,混匀后倒入事先准备的固体琼脂平板上,制成双层平板。凝固后倒置于37℃隔水式电热恒温培养箱中培养。
2.噬菌体的纯化
从双层平板上挑取1个边缘光滑、透明的较大噬菌斑,接种到1mL的无菌SM缓冲液中,4℃过夜。用SM缓冲液将这1mL噬菌体液进行倍比稀释,取适当梯度的噬菌体液100μL与对数生长期的细菌I菌液100μL中,37℃振荡培养6h,4℃,10000r/min离心,上清液用0.22μm滤膜过滤除菌后,用双层琼脂平板法重复培养5次,即可得到纯化的噬菌体。
3.噬菌体扩增
从经纯化后的噬菌体的双层平板上挑取1个边缘光滑、透明的较大噬菌斑,接种到1mL的无菌SM缓冲液中,4℃过夜。取500μL含噬菌体的SM缓冲液与500μL对数期生长的坎氏弧菌40-2于5mL液体培养基中,37℃振荡培养6h。收集培养液于4℃,10000g,15min离心,0.22μm孔径的一次性滤器对上清液进行过滤,滤液即为噬菌体增殖液,放于4℃备用。
实施例2
噬菌体的测序
取实施例1制得的噬菌体100mL,加入终浓度1μg/mL的DNaseI,RNaseA,37℃条件下孵育60min后加入5.84g NaCl(终浓度1mol/L),溶解后置于冰浴中1h。
4℃条件下,11000rpm离心10min,将上清转移至新的离心管中。加入固体PEG8000(终浓度10%),完全溶解后,冰浴1h。4℃条件下,11000rpm离心20min,用少量SM液重悬沉淀。加入等体积的氯仿异戊醇抽提,温和震荡30s,8000rpm离心1min后,吸取上清,重复抽提至澄清。
再次加入DNase I、RNase A至终浓度1μg/mL,37℃反应30~60min。加入EDTA至终浓度20mmol/L,同时每1mLSM液中加入10μL10%SDS。使用病毒DNA/RNA提取试剂盒提取。凝胶电泳验证DNA后用Eppendorf BioPhotometer Plus测定其浓度和纯度。产物送至测序公司测序。
测序结果显示,实施例1纯化得到的坎氏弧菌噬菌体具有如SEQ ID No.1所示的核苷酸序列。以该核苷酸序列可以人工合成实施例1筛选得到的坎氏弧菌噬菌体。
实施例3
噬菌体效价测定
用SM缓冲液对实施例1中的噬菌体增殖液进行连续10倍倍比稀释,得到相应稀释度的噬菌体稀释液。取适当稀释度的噬菌体稀释液100μL与培养至对数期的坎氏弧菌40-2菌液100μL混合,静置3-5min铺双层板,37℃培养箱培养8h后进行噬菌斑计数。结果显示该坎氏弧菌噬菌体的效价较高,能达到1010pfu/mL以上。
实施例4
坎氏弧菌噬菌体裂解谱的确定
将不同的细菌单菌落活化至对数生长期,分别取菌液200μL滴到固体平板中,然后用无菌涂布棒将菌液涂布均匀至吸收,取10μL噬菌体增殖液分别滴到固体平板表面,在超净台内放置30min后置于37℃培养箱中培养8-12h。观察平板表面是否有噬菌体空斑出现。
结果如下表1和图1所示,图1是本发明的实施例4的坎氏弧菌噬菌体对菌株编号为40-2的坎氏弧菌裂解结果,从图1中可以看出,平板表面有大量的噬菌体透明空斑,表明该宿主菌对实施例中的坎氏弧菌噬菌体敏感。
表1.坎氏弧菌噬菌体裂解谱
从表1中可以看出,本发明实施例所提供的坎氏弧菌噬菌体能够特异性裂解坎氏弧菌,对于弧菌属的其它弧菌以及其它属的细菌不具有相应的裂解效果。
实施例5
噬菌体热稳定性实验
取效价约108pfu/mL的噬菌体增殖液1.0mL于无菌EP管中,分别于20℃、30℃、40℃、50℃、60℃、70℃的恒温水浴锅中作用1h,待作用时间结束后取出,进行10倍倍比稀释后用双层平板法进行计数,重复三次取平均值,观察噬菌体效价的变化,结果如下表2所示。
表2.噬菌体热稳定性实验结果
从表2中可以看到,本发明实施例所提供的噬菌体可以耐受20℃至60℃的温度环境。在20℃至60℃作用1h,噬菌体效价变化不大;在70℃作用1h,噬菌体效价降为0;这说明分离到的噬菌体可以耐受60℃高温。
实施例6
噬菌体对虾肉样品的杀菌效果评价
噬菌体效价:107pfu/mL,100μL;
坎氏弧菌计数:107cfu/mL,100μL。
评价方法如下:
(1)从市场买回活虾,用灭菌的手术刀剪在超净工作台中,去除虾壳,将虾肉切成面积为1×1cm的小块(约1g),将肉块过火焰然后放在一次性平皿中备用。
(2)在超净工作台中,将100μL坎氏弧菌菌液均匀涂布在肉块表面。
(3)15min后,实验组滴加100μL噬菌体在肉块表面,对照组滴加100μL PBS在肉块表面。
(4)将制备好的样品置于25℃,于1h、2h、3h、6h、12h时对坎氏弧菌计数。
结果如图2所示,图2是本发明的实施例6的坎氏弧菌噬菌体对坎氏弧菌的杀灭效果,从图中可以看出,实验组样品在短时间内的细菌数目显著减少,说明噬菌体能够杀灭样品中的坎氏弧菌,起到食品安全保护剂的作用。
实施例7
15天虾苗实验
1.虾苗暂养
将虾苗放入装有人工海水的塑料水箱内暂养24h。暂养期间每12h换水一次,持续充氧。
同时取虾苗原水体涂TCBS板检验有无弧菌。
2.坎氏弧菌扩增
挑取菌株编号40-2的单菌落接种于2216E液体培养基中,置于37℃恒温振荡培养箱中培养3-4h(菌落数增殖至109cfu/mL),将培养液4000rpm离心20min后取沉淀,用无菌生理盐水洗涤三次后复溶,4℃冷藏备用。
3.噬菌体施用实验
每组取50条虾,4组,实验各组别在暂养后细菌/噬菌体的添加条件设置如下表3所示。
细菌量108cfu/mL,20mL,加入细菌30min后,再按照MOI加入1.6×109pfu/mL的噬菌体20mL。
不加细菌和/或不加噬菌体时,加入等体积的生理盐水。
表3.实验组别添加条件
噬菌体 | 不加噬菌体 | |
细菌 | 组1 | 组2 |
不加细菌 | 组3 | 组4 |
添加细菌/噬菌体后隔0、12h、24h、36h、48h后,每组挑3条虾,其他虾继续养殖。
4.噬菌体和细菌的检测
各组取出的3条虾加入1ml PBS,用组织破碎机将虾绞碎,离心;
(1)取上清液过滤,梯度稀释,铺双层平板,噬菌斑计数,检测虾肉中的噬菌体含量。
(2)取沉淀,加入1ml PBS,涡旋混匀,梯度稀释,100ul涂TCBS板,菌落计数,检测虾肉中的坎氏弧菌含量。
结果如图3所示,图3是本发明的实施例7的15天虾苗实验的对比实验结果,从图中可以看出,在水体中使用噬菌体后,能够显著降低虾体内的弧菌含量,从而减少虾苗的发病机会。
上面结合附图对本发明实施例作了详细说明,但是本发明不限于上述实施例,在所述技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
SEQUENCE LISTING
<110> 华南农业大学
广东海大畜牧兽医研究院有限公司
<120> 坎氏弧菌噬菌体及其应用
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 21219
<212> DNA
<213> Vibrio phage
<400> 1
tcgcagttcg ctacacgcag gacgcttttt cacgttctgg ttggttgtgg cctgttgatg 60
ctaaaggtga gccgcttaaa gctaccagtt atatggctgt tggtttctat gtggctaaat 120
acgttaacaa aaagtcagat atggaccttg ctgctaaagg tctaggagct aaagaatgga 180
acaactcact aaaaaccaag ctgtcgctac ttcccaagaa gctgttcaga atcagaatga 240
gccgcaactt cgggatgaaa atgctcacaa tgacaaatct gtccacggag tgcttaatcc 300
aacttaccaa gctgggttac gacgcgacgc cgttcaacca gatattgaag cagaacgcaa 360
aaagagagat gagattgagg ctgggaaaag ttactgtagc cgacgttttg gcggcgcaac 420
ctgtgacgac aaatctgctc aaatttatgc gcgcttcgat aaaaatgatt ggcgtatcca 480
acctgcagag ttttatcgct tccatgacgc agaagttaac actttcggat atttctgatg 540
agtcgaaaaa ttatcttgat aaagcaggaa ttactactgc ttgtttacga attaaatcga 600
agtggactgc tggcggaaaa tgagaaaatt cgacctatcc ttgcgcagct cgagaagctc 660
ttactttgcg acctttcgcc atcaactaac gattctgtca aaaactgacg cgttggatga 720
ggagaagtgg cttaatatgc ttggcacgtt cgtcaaggac tggtttagat atgagtcaca 780
ttttgttcat ggtagagatt ctcttgttga cattttaaaa gagcgtggat tactatctga 840
gtccgatgct gttcaaccac taataggtaa gaaatcatga gtcaagttac tgaacaatcc 900
gtacgtttcc agaccgcttt ggcctctatt aagctcattc aggcttctgc cgttttggat 960
ttaaccgaag atgatttcga ttttctgacg agtaacaaag tttggattgc tactgaccgc 1020
tctcgtgctc gtcgctgcgt tgaggcttgc gtttatggta cgctggactt tgtaggatac 1080
cctcgctttc ctgctcctgt tgagtttatt gctgccgtca ttgcttatta tgttcatccc 1140
gtcaacattc aaacggcctg tctcatcatg gaaggcgctg aatttacgga aaacattatt 1200
aatggcgtcg agcgtccggt taaagccgct gaattgttcg cgtttacctt gcgtgtacgc 1260
gcaggaaaca ctgacgttct tactgacgca gaagaaaacg tgcgtcaaaa attacgtgca 1320
gaaggagtga tgtaatgtct aaaggtaaaa aacgttctgg cgctcgccct ggtcgtccgc 1380
agccgttgcg aggtactaaa ggcaagcgta aaggcgctcg tctttggtat gtaggtggtc 1440
aacaatttta attgcagggg cttcggcccc ttacttgagg ataaattatg tctaatattc 1500
aaactggcgc cgagcgtatg ccgcatgacc tttcccatct tggcttcctt gctggtcaga 1560
ttggtcgtct tattaccatt tcaactactc cggttatcgc tggcgactcc ttcgagatgg 1620
acgccgttgg cgctctccgt ctttctccat tgcgtcgtgg ccttgctatt gactctactg 1680
tagacatttt tactttttat gtccctcatc gtcacgttta tggtgaacag tggattaagt 1740
tcatgaagga tggtgttaat gccactcctc tcccgactgt taacactact ggttatattg 1800
accatgccgc ttttcttggc acgattaacc ctgataccaa taaaatccct aagcatttgt 1860
ttcagggtta tttgaatatc tataacaact attttaaagc gccgtggatg cctgaccgta 1920
ccgaggctaa ccctaatgag cttaatcaag atgatgctcg ttatggtttc cgttgctgcc 1980
atctcaaaaa catttggact gctccgcttc ctcctgagac tgagctttct cgccaaatga 2040
cgacttctac cacatctatt gacattatgg gtctgcaagc tgcttatgct aatttgcata 2100
ctgaccaaga acgtgattac ttcatgcagc gttaccatga tgttatttct tcatttggag 2160
gtaaaacctc ttatgacgct gacaaccgtc ctttacttgt catgcgctct aatctctggg 2220
catctggcta tgatgttgat ggaactgacc aaacgtcgtt aggccagttt tctggtcgtg 2280
ttcaacagac ctataaacat tctgtgccgc gtttctttgt tcctgagcat ggcactatgt 2340
ttactcttgc gcttgttcgt tttccgccta ctgcgactaa agagattcag taccttaacg 2400
ctaaaggtgc tttgacttat accgatattg ctggcgaccc tgttttgtat ggcaacttgc 2460
cgccgcgtga aatttctatg aaggatgttt tccgttctgg tgattcgtct aagaagttta 2520
agattgctga gggtcagtgg tatcgttatg cgccttcgta tgtttctcct gcttatcacc 2580
ttcttgaagg cttcccattc attcaggaac cgccttctgg tgatttgcaa gaacgcgtac 2640
ttattcgcca ccatgattat gaccagtgtt tccagtccgt tcagttgttg cagtggaata 2700
gtcaggttaa atttaatgtg accgtttatc gcaatctgcc gaccactcgc gattcaatca 2760
tgacttcgtg ataaaagatt gagtgtgagg ttataacgcc gaagcggtaa aaattttaat 2820
ttttgccgct gaggggttga ccaagcgaag cgcggtaggt tttctgctta ggagtttaat 2880
catgtttcag acttttattt ctcgccataa ttcaaacttt ttttctgata agctggttct 2940
cacttctgtt actccagctt cttcggcacc tgttttacag acacctaaag ctacatcgtc 3000
aacgttatat tttgatagtt tgacggttaa tgctggtaat ggtggttttc ttcattgcat 3060
tcagatggat acatctgtca acgccgctaa tcaggttgtt tctgttggtg ctgatattgc 3120
ttttgatgcc gaccctaaat tttttgcctg tttggttcgc tttgagtctt cttcggttcc 3180
gactaccctc ccgactgcct atgatgttta tcctttggat ggtcgccatg atggtggtta 3240
ttataccgtc aaggactgtg tgactattga cgtccttccc cgtacgccgg gcaataatgt 3300
ttatgttggt ttcatggttt ggtctaactt taccgctact aaatgccgcg gattggtttc 3360
gctgaatcag gttattaaag agattatttg tctccagcca cttaagtgag gtgatttatg 3420
tttggtgcta ttgctggcgg tattgcttct gctcttgctg gtggcgccat gtctaaattg 3480
tttggaggcg gtcaaaaagc cgcctccggt ggcattcaag gtgatgtgct tgctaccgat 3540
aacaatactg taggcatggg tgatgctggt attaaatctg ccattcaagg ctctaatgtt 3600
cctaaccctg atgaggccgt ccctagtttt gtttctggtg ctatggctaa agctggtaaa 3660
ggacttcttg aaggtacgtt gcaggctggc acttctgccg tttctgataa gttgcttgat 3720
ttggttggac ttggtggcaa gtctgccgct gataaaggaa aggatactcg tgattatctt 3780
gctgctgcat ttcctgagct taatgcttgg gagcgtgctg gtgctgatgc ttcctctgct 3840
ggtatggttg acgccggatt tgagaatcaa aaagagctta ctaaaatgca actggacaat 3900
cagaaagaga ttgccgagat gcaaaatgag actcaaaaag agattgctgg cattcagtcg 3960
gcgacttcac gccagaatac gaaagaccag gtatatgcac aaaatgagat gcttgcttat 4020
caacagaagg agtctactgc tcgcgttgcg tctattatgg aaaacaccaa tctttccaag 4080
caacagcagg tttccgagat tatgcgccaa atgcttactc aagctcaaac ggctggtcag 4140
tattttacca atgaccaaat caaagaaatg actcgcaagg ttagtgctga ggttgactta 4200
gttcatcagc aaacgcagaa tcagcggtat ggctcttctc atattggcgc tactgcaaag 4260
gatatttcta atgtcgtcac tgatgctgct tctggtgtgg ttgatatttt tcatggtatt 4320
gataaagctg ttgccgatac ttggaacaat ttctggaaag acggtaaagc tgatggtatt 4380
ggctctaatt tgtctaggaa ataaccgtca ggattgacac cctcccaatt gtatgttttc 4440
atgcctccaa atcttggagg cttttttatg gttcgttctt attacccttc tgaatgtcac 4500
gctgattatt ttgactttga gcgtatcgag gctcttaaac ctgctattga ggcttgtggc 4560
atttctactc tttctcaatc cccaatgctt ggcttccata agcagatgga taaccgcatc 4620
aagctcttgg aagagattct gtcttttcgt atgcagggcg ttgagttcga taatggtgat 4680
atgtatgttg acggccataa ggctgcttct gacgttcgtg atgagtttgt atctgttact 4740
gagaagttaa tggatgaatt ggcacaatgc tacaatgtgc tcccccaact tgatattaat 4800
aacactatag accaccgccc cgaaggggac gaaaaatggt ttttagagaa cgagaagacg 4860
gttacgcagt tttgccgcaa gctggctgct gaacgccctc ttaaggatat tcgcgatgag 4920
tataattacc ccaaaaagaa aggtattaag gatgagtgtt caagattgct ggaggcctcc 4980
actatgaaat cgcgtagagg ctttgctatt cagcgtttga tgaatgcaat gcgacaggct 5040
catgctgatg gttggtttat cgtttttgac actctcacgt tggctgacga ccgattagag 5100
gcgttttatg ataatcccaa tgctttgcgt gactattttc gtgatattgg tcgtatggtt 5160
cttgctgccg agggtcgcaa ggctaatgat tcacacgccg actgctatca gtatttttgt 5220
gtgcctgagt atggtacagc taatggccgt cttcatttcc atgcggtgca ctttatgcgg 5280
acacttccta caggtagcgt tgaccctaat tttggtcgtc gggtacgcaa tcgccgccag 5340
ttaaatagct tgcaaaatac gtggccttat ggttacagta tgcccatcgc agttcgctac 5400
acgcaggacg ctttttcacg ttctggttgg ttgtggcctg ttgatgctaa aggtgagccg 5460
cttaaagcta ccagttatat ggctgttggt ttctatgtgg ctaaatacgt taatcttccg 5520
atctagcact tcagtctcac caaaacgttt gtgaatattt tctaacttaa tcatcgtacg 5580
tacgccttat tcagttttgc ttctgcccag atttgtacgc gagtcagaat caccaccact 5640
ccccagtaaa taagagcaac cgcaaggaaa gcttcaaaga aacggaaact tgatgaggct 5700
tccatctgtg ctttcgccat gatttcggcc acaccaagcg tgaaagcgag agacgttgac 5760
ttgatcatat cgatgaaata gttcatcaat gaaggcagtg cgacacgcgt tgcttgaggt 5820
aaaatcactc ggcgcattgc ttgggatgtc gtcataccga ccgataagct cgcttccatt 5880
tggctgcgat caatgccaat aatggcggca cgaatacttt ccgccatata agcggcaaag 5940
tgcagcgtta aaccgatcac cgctgctgaa aaggcgtcga taccgaccat caatggaaaa 6000
atttgcggta agccgtaata aagcaggaaa agctgaacca acagtggggt gccacggaag 6060
aagctgatgt acagttggct taattgatcc agaactggca gtttaaatac gcgaatattg 6120
gctaaaatga cggataaaat cagcgaaaac accaatcccc atgtcgccat ttccatggtt 6180
gtgccaagat atttcagcaa tatcggcagc agttccaaca tgtaattaaa gtcaaatccc 6240
atacggctta cctaaaggct agagaaaaga aaaggtggaa aacaagttcc acctttacgt 6300
aagtttattt tagtccgaga gtgataccac tttgcggcct aaagtacaaa gagttcttat 6360
ttcgtgatgt cagcgccaaa ccacttcacg gaaattttct ctactgtacc atctgcgcgc 6420
atctcagcca gtgctttgtt tacttcttct tgcagcttac gacctttctc gttgtcaacg 6480
aaaggccatg cgttttgaat ggtctcaaac ggttcgcctg ccaattccag aggaaggcca 6540
gttttcttaa tcagctctaa cgcagaaaga cgatccatta caaacgcgtc tgcacgaccg 6600
agcgccacgt catgttcgat acctgtgtcg tacgttttaa tgttgatctt gccgtctttg 6660
tcgtattgac gcagcagttg ctcaaagtta gagcctaggt tcaccgcaac ggttttgccc 6720
gcaagatcgt caacaccttt gatgctgtcg ttgcctttac gcacggtaat ttgtgcgcca 6780
tctacaacgt aagggtcagc aaataggtat ttagctttac gctcatcagt catggtgatt 6840
tggttggaga tcgtatcgat acgaccagtt tctaaaagac caaataaacc agagaagttg 6900
gctgttacgt attcgatttt gtaatcgtta cgtttaccga tttcatccca catgtcgact 6960
tcaaagcctt gtagcttgtc ttgctttaca aaagtaaacg ggaagtagcg acctgacata 7020
ccaactttga cttcagtagc ggcttgtacg gttgctgccg acagtgcaat ggctgcaacg 7080
gcaaccttaa tccagttttt cataatacat ctccataatt ttatggttgg aaatactact 7140
gttagttagc tcaattaaat aaataaccaa gagcaataac ctagacctgt gagttattag 7200
ataatagaag aagaatgtta aatgtgacga aaatgcgaac tgaacctagc gaaatgatcg 7260
acaaaatcag cattgggaat aaaataatac acagacttat caccaatcgc tcgatcttgt 7320
gaattttgcc caattgtgga tcgttgtgtg ggcaaatttg gggcaaggtg tgttgatctc 7380
atcaagtttg cagaaataat tgtgaataac ttggatctta ttcactggat cgtcgatcaa 7440
tcgctggcga tcttggttat caacaggtaa aattgccagt cttttccgat aattcaatta 7500
ctcagtgggg gcaccgtgtc atcttcgctt tggctgcaat gtttgcagca gcttcaagaa 7560
gagctaccag ctacagaatt cagcatgtgg gttcgtccgc tacaagcgga gctcaatgac 7620
aatactctca ctctattcgc gccaaaccgc tttgtgttgg actgggtgcg tgataagtat 7680
ctgaacaata ttactcgctt actgcaggaa tactgcggtg gcgatattcc taacttacgt 7740
tttgaagtgg ggagccgacc tgtctctgca ccaaaaccag cgccgacacg tacgcctgca 7800
gatgtggctg ccgagtcatc tgcacctgcg caattacagg cgcgtaagcc agtacacaaa 7860
acctgggatg acgacgctca agcgattgcg gctatcaatc accgctcaaa catgaaccca 7920
aaacacaagt tcgataactt tgttgaaggt aagtcgaacc aactgggttt ggctgcggcg 7980
cgtcaggtat cggataatcc tggtgcagca tacaacccgc tgttcctata cggcggcacg 8040
ggcttgggta aaacgcactt gttgcatgcg gtgggcaacg cgattgtgga taacaacccg 8100
aacgcaaaag tggtctacat gcactcagaa cgttttgtgc aagacatggt aaaagcgctg 8160
caaaacaatg cgattgaaga attcaaacgc tactaccgca gcgtggatgc gctccttatc 8220
gatgacatcc aattctttgc taataaagaa cgttcacaag aagaattctt ccataccttc 8280
aacgcactat tggaaggtaa tcagcagatc attctaacgt ctgaccgtta tcctaaagaa 8340
attaatggcg ttgaagatcg tcttaaatct cgctttggtt ggggtcttac tgttgcgatc 8400
gagccgcctg agcttgaaac acgtgtggcg atcttgatga aaaaagctga agatcaccaa 8460
atccaccttg ctgatgaagt ggcattcttc attgccaagc gtctgcgttc gaacgttcgt 8520
gagctagaag gtgcactgaa ccgtgttatc gcgaacgcga actttactgg ccgtccaatt 8580
acgattgatt ttgttcgtga agcgctgcgc gatctgcttg ctctacaaga gaagcttgtg 8640
acaatcgata acattcaaaa gaccgtggcg gaatactaca agattaaagt agcggacttg 8700
ctgtctaagc gtcgttctcg ttctgtagcg cgtcctcgtc agctggctat ggcgttagca 8760
aaagagctaa ccaaccacag tttgcctgaa attggcgatg cgtttggggg ccgcgatcat 8820
acgaccgtac ttcatgcttg tcgcaagatt gagcagctgc gtgaagagag ccacgacatt 8880
aaagaagatt actctaactt gattcgcacc ctgtcttcgt aatcggttag aataacggta 8940
ttctaagcaa atcatttatt gagctgacag cgtaaagagc caagcatgaa atttaccatt 9000
gaacgtagtc atttaatcaa accgctacag caggtttcgg gcgctttagg tggccgacca 9060
accctgccaa tcctgggtaa cctacttctt aaagtagaag agaacgtatt gtcgatgacg 9120
gcaaccgatc ttgaagtaga gttagtaagc aaagtgacgc tagaaggtga ttttgaagcg 9180
ggcagcatta ccgttccttc gcgtaagttt ctcgatatct gccgtggttt accggacgat 9240
gcgatcatca catttgtatt agaaggtgat cgcgtacaag ttcgctctgg tcgcagccgt 9300
ttctcgttgg caacgcttcc tgcgaacgat ttccctaata ttgaagactg gcaaagcgaa 9360
gtggaagtat cactgagcca agccgatcta cgtacgctga tcgacaaaac tcagttctcg 9420
atggcgaatc aagacgttcg ttactacctc aacggcatgt tgtttgaaat tgacggcacc 9480
acgttacgca gcgtggcgac ggacggccac cgtatggcgg tatcgcaaac tcagctaggt 9540
gcagactttg cgcagaagca aatcatcgtg ccacgcaaag gtgtgcaaga attagtaaag 9600
ttgatggatg cgccagagca gcctgtagta cttcagatcg gtagctccaa tgttcgtgct 9660
gaggtgaata actttacttt tacctctaag ttggttgatg gtcgtttccc tgactaccgc 9720
cgtgtattgc ctcagagcac caataagaca ctaatagcaa gttgtgacga gttgcgccaa 9780
gccttctctc gtgctgcgat tctttccaac gaaaaattcc gtggcgtgcg tgtgaatctg 9840
gctgatactg aaatgcgcat aaccgcgaat aacccagagc aagaagaagc ggaagagatg 9900
cttgatgtaa cctttgaagg tgacgccatc gaaatcggct ttaacgtaag ctatgtgctt 9960
gatgttctga acacgttgcg ttgtgaaaat gtacgtgtgt cgatgtcgga tgctaacgct 10020
agcgcattga ttgaaaatgc cgacgatgac agtgccatgt acgtggtgat gccaattcgt 10080
ctataagcca actgaatgcc tctttctcgt ctcatcattc agcagtttcg aaacattaaa 10140
gcctgtgata ttgagttatc agcaggcttt aactttctta tcgggccgaa cggcagcggt 10200
aaaaccagtg tcttagaagc ggtttatctg ctcgggcatg gccgctcatt caagagctct 10260
ctcaccggac gagtgataca aaatgagtgt gacgaactgt ttgttcatgg tcgttttttg 10320
aactcggatc aatttgagct acctatcggc attaataagc agcgtgatgg ctcaacagag 10380
gttaaaatag gcggtcaatc tggacaaaaa ttagcgcagt tggcacaagt tctgcctctg 10440
cagttgatac acccagaagg gtttgattta ctgacagatg gccctaaaca tcgtcgtgcg 10500
tttattgatt ggggcgtgtt tcataccgag cccgcttttt atcaagcatg gggaaggttc 10560
aagcgcctga acaagcagcg caacgcgttg ttgaaaaccg ccaatagtta tcgtgagctc 10620
agttattggg atcaggaaat ggcaggattg gctgaaaaca tcagccaatg gcgagcaagt 10680
tacatcgagc agatgaaaac tgtcgcagaa actatctgtc agacattttt gccagaattt 10740
gagatccaat tgaagtatta tcgtggctgg gataaagaca ctccatacca cgagatacta 10800
gaaaagaatt ttgagcgtga tcaggcattg ggatacacct ttagtggccc aaacaaagct 10860
gatttgcgca ttaaagtgaa cggaacgccc gttgaagatg ttctgtcacg cggtcaatta 10920
aaactcatgg tgtgtgcatt gcgtgtagcg caagggcaac acctgacaga gatggccggt 10980
aaacaatgca tttacctaat tgatgacttc gcgtcggaat tagatagcca acgtcgcaag 11040
cgtcttgctg actgcttaaa agagacgggc gcacaagtat ttgtaagctc tatcactgaa 11100
agccaaatcg ccgatatgtt ggacgataac ggtaaattgt ttcatgtgga acatggcagg 11160
atagagtcaa actagaagag agaaactcat gtctgaaaat tacgattcat cgagtattaa 11220
ggtactgaag ggtctggatg cggtacgtaa gcgtccagga atgtacatcg gcgatacgga 11280
cgacggcacc ggccttcacc acatggtttt cgaggtggtg gataactcaa ttgatgaagc 11340
gttggcaggt cactgtaaag acatcgttgt gacaattcat gaggataact cggtttccgt 11400
tagcgatgat ggtcgtggca ttccaacaga aatccactca gaggaaaatg tctctgcagc 11460
agaagtaatc atgacggttc ttcacgcggg tggtaagttc gacgataact catacaaagt 11520
atcgggtggt cttcacgggg taggtgtttc agtagtaaac gcactatcag agaaagttga 11580
gctaacgatt catcgtggtg gccatatcca tacgcaaacc taccgacatg gtgagcctca 11640
agcgccacta gccgttgtgg gtgatacgga taaaaccggt acacaaattc gtttctggcc 11700
aagtgccgag acgttctcta acactgagtt ccactatgac attctggcga aacgcctgcg 11760
tgaactgtca ttcctgaact ctggtgtgtc gatcaaattg gttgatgagc gtgaagcgga 11820
caaacacgat cacttcatgt atgaaggtgg tattcaagcg ttcgttgatc acctaaacac 11880
caacaaaacg ccaatcatcg aaaaaatctt ccactttaac tctgagcgtg aagacggcat 11940
ttcagttgaa gtggcgatgc aatggaacga tggtttccaa gagaacatct tctgctttac 12000
caacaatatc ccacagcgtg atggtggtac tcaccttgct ggtttccgtg ctgcgctaac 12060
acgtacattg aacagcttta tggataaaga aggtttctcg aagaaagcga aaacagcgac 12120
ttcaggcgac gatgcgcgtg aaggtctaac agcggttgtt tcggtgaaag tgcctgatcc 12180
taagttctca agccaaacaa aagacaaact ggtttcttct gaagtgaaat cagctgttga 12240
gtctgcaatg ggtgaaaaac tgtctgagtt cctgattgag aacccgacag aagcgaagat 12300
ggtttgttcg aaaatcatcg atgcagcacg tgcacgtgaa gctgcgcgta aagctcgtga 12360
aatgacgcgc cgtaaaggtg cactagacct agcaggtctt ccaggtaaac ttgcagactg 12420
tcaggaaaaa gatccggcac tctctgaact atacatagtg gagggtgatt cggcaggcgg 12480
ctccgcaaaa caaggccgta accgtaagaa ccaagcaatc ctaccgctaa aaggtaagat 12540
tcttaacgta gaaaaagcac gtttcgacaa gatgctatct tctcaagaag tagcaacact 12600
gatcaccgca ctaggttgtg gtatcggtcg tgacgagtac aacccggaca agcttcgtta 12660
ccacaacatc atcatcatga ccgatgccga tgtcgatggt tcgcacatcc gtacgctact 12720
gttgaccttc ttctaccgtc aaatgccaga gcttatcgag cgtggctatg tctacatcgc 12780
tcagccacca ctatacaaag tgaaaaaagg caaacaagag tctacaagag cattatgctg 12840
agttagaagc tgaacgtact aaatatgtaa ccgctcttga ccaactcata ctcgcgatgg 12900
cgtagagaaa gacaagcgtg cgcttgaaat cgaacagatg cagcttaaag aagcgaagaa 12960
agacctaact gaagaattcc agattcttga gggcggcctt ctaaaccgtg ttaaagctgt 13020
attgattgaa ggcggttact ctgaagcgaa actagacaca actgatcgta agaagtggct 13080
agaactaacg ctagaagatg acgcactaca aactcagctt gagcaacttg ctgagcagtg 13140
ggatgagcta aaagccgact tcgataagaa gttcgaaact aagcgtcgta agatcactca 13200
aggtgatgat ttagcacctg gtgtactgaa gatcgttaag gtttacctag cggttaaacg 13260
tcgcattcag cctggtgata agatggcggg tcgtcacggt aacaagggtg taatctctaa 13320
gatcaaccct gttgaagaca tgccatacga tgagaaaggt cagcctgtag acatcgtact 13380
taacccactg ggtgtacctt cgcgtatgaa catcggtcag atcctagaag tacacttagg 13440
tctggcagcg aaaggtatcg gtgacaagat caaccagatg gttaaagagc agcaagaact 13500
agcgaaattc cgcgagttcc tacaaaaagt ttacgatcta ggcgacactc gccagaaagt 13560
agacattgct tctctatctg atgatgaagt acgtactctg atcaagaact tgcgtggcgg 13620
tctaccaatc gcgactcctg tatttgatgg cgctccagaa gagtcaatca aagcacttct 13680
tgagctagca gatctaccaa catcaggtca gttaacactg tttgatggtc gcacaggtga 13740
tgcgtttgag cgtcctgtaa ctgttggtta catgtacatg ctgaaactga accaccttgt 13800
tgatgacaag atgcacgctc gttcaactgg ttcgtacagc ctagtaactc agcagccact 13860
aggtggtaaa gctcagttcg gtggtcagcg tttcggtgag atggaagtat gggcactaga 13920
agcatacggt gctgcttaca cgctacaaga aatgctaacg gttaagtcgg atgacgttaa 13980
cggccgtact aagatgtaca agaacatcgt agacggtaac cacagcatgg aacctggcat 14040
gccagaatcg ttcaacgtac tgttgaaaga gatccgctcg ctaggtatca acatcgagct 14100
agaagacgaa gagtaatcct tttctctctt gatgaaagtc gagagaatca aagcgattat 14160
ttggaagaag gtgctcactt tagcaagtga gctcctttta actccttaca ggagctgatt 14220
gtgaaagact tattaaactt tctaaaagca cagcataaga ccgaagaatt tgatgcaatc 14280
aaaatcggtc tatcttcacc agacatgatc cgttcatggt ctttcggtga agttaaaaaa 14340
cctgaaacga tcaactatcg tacgttcaaa cctgaacgcg atggtctgtt ctgtgcgcgt 14400
atctttggtc cagttaaaga ctacgaatgt ctttgtggta agtacaagcg cctgaagcac 14460
cgtggtgtta tctgtgagaa gtgtggtgta gaagttacac aaactaaagt tcgtcgtgac 14520
cgtatgggcc acattgaact ggcttcacca gtagctcaca tctggttcct aaaatcgcta 14580
ccgtctcgta tcggtctact aatggatatc cctctacgtg atatcgaacg cgttctgtac 14640
ttcgaaatgt acgtagtaac agagcctggt atgactgatc tagaaaaagg tcagatgctt 14700
actgaagaag agtatctaga tcgtctagaa gagtggggcg acgagttcac tgctaagatg 14760
ggtgcggaag ctatcaagga cctactaggt tctatggata tgcacgctga agcagaacaa 14820
atgcgtgaag agttagagac aacaaactct gaaactaagc gtaagaaagt aactaagcgt 14880
cttaagctgg tagaagcgtt catcgcatcg ggtaacaacc ctgagtggat gatcctaact 14940
gtacttccag tacttccgcc agatctacgt cctctagtac ctctagatgg cggtcgcttt 15000
gcgacttctg atcttaacga tctataccgt cgtgtgatca accgtaacaa ccgtttgaag 15060
cgtcttctag agctagctgc tccggacatc atcgtacgta acgaaaaacg tatgctgcaa 15120
gagtctgttg atgcgctact agataacggt cgtcgtggtc gtgcgatcac aggttctaac 15180
aagcgtcctc tgaaatctct tgctgatatg atcaagggta aacaaggtcg tttccgtcag 15240
aaccttctag gtaaacgtgt agactactct ggccgttctg taatcacacc aagaaccaac 15300
atctgaccat cttggatcat gacagaggtg tttaactgtc gtttagcaaa gcgaacgtca 15360
accgcaccgt ttgcgcctaa tacgttggaa acttcttgtt caatgtttaa ctgtacagag 15420
tcaccttcgt taatttgtgg cactactttg agtttgatac cgacttcttt acggtcaacc 15480
gtttggaatg ggttgtcgtt gtttgagcca gccgtagagc ccgtaataac cggaacctct 15540
tcacccacaa taaatgatgc ttcaccatta tccattacag tgatacttgg agaagataga 15600
atgtttgagt tggagtcaga agcaacagca ctaattagcg ctgtccagtc acccatgaca 15660
atactcattg ccgcaccatt tactccgcta agtgctgacg ctagcgtaga gtagtcacct 15720
gattcagtaa catcatatgg cgttcgttcg ccattagagt tggtatagta ttcagttcgg 15780
gtttgatctt ttgcttcttc tagcccaacc atcacctgac caatcggggc tcctgagtta 15840
ccgtactgaa ttacagcacc cgtttctaat gaaccccact gaacacctag gttaatacca 15900
tcaccttcag acatttcgac aattaacgct tcaataagaa cctgagcacg acgaatatcg 15960
agttgagaaa tgacgtcttg taatgccatc ataatatctg gtggcgctgt taaaactaac 16020
gagttagttg cagcgtgagc agcaatgacg acctcaccac gcgatgcacc tgatgttttc 16080
tgaccggctt gtttttcagc ttgcaggtta tcggaaacac ctttcaatac atccaccaaa 16140
tcttcagctt tcgcattttt caggtacaca actcggttgt taccttttgt cgccatttca 16200
acatcaagtt gacgaatcaa acgtttcaag cgggctctta cttttggatc accagaaatt 16260
aaaatcgcgt tagtacgttc atcggccaca attttaggtt caaggaactc tggcgtgctt 16320
ttctgattcg cacttttatt cagtgcttca acaatgcgta ccatttcagc agcagatgca 16380
ttacgcagct ctaccaattc aatttcttta tcaccggctt ggtcaacacg cttgatgatc 16440
tctgctaagc gattcactac tgccgcacga cctgtgatca agataatatt cgctggatca 16500
taatgaacca cgttaccggc accggcgtta tcaatcagct gacgaagtag aggagacaac 16560
tctcgaaccg acacattacg caccgcgaca actcgagtga taacactatc gccttgcgcg 16620
ctgccatcac ctaacacagg gattgctgag gtttttgagt cttttgcttt gataacctta 16680
agtaccccat tgtccatctc tacgactgcg taaccgtata cttcaaggac attaaggaaa 16740
aagctgtagt attgctcttc gttaagaacg tcatagctgc ggacatcgat tttaccgcgt 16800
actgaaggat caacaatgat ggtcttttcg aggttacgac ccactatgtt gatgaattct 16860
tgaatatcag tgcctttaaa gctcgcacta aattcgtttg ccattgcacc tggtacgcag 16920
agtaaacttc ctgctagtag ccaagcactt ttgctgaacc aatgtttcac gtatgtctcc 16980
ctgaacttac gccggttgcc taagtcttac aattggatat agatatcgta ctgttggccg 17040
tctcgctcaa cagttaaatt taattcttgc ggatcgctca tcacttgcat cagttgtact 17100
gaagaagacg ggtcacttaa atcgatcccg ttcagttgaa ccgcaatatc gccaggttgt 17160
agtcctacat cattaaataa tgcggcatca cgaccaggtg atactcgata accaacaatg 17220
tctccgtctt ttttgacggg tgaaatggtg atgtatttaa aaacgctttg tggatctttc 17280
gcaatttcct cgcggatttg ctctaatttc tctccaacgt cagacgtcgc tgcttctgct 17340
cgctgagctt gcgccctggc tacctgcggc gattcagata gcttcttata ttctatacct 17400
tccatcatta aggtctcgtc tcgaccttgg ttatcaatga tgacgcgatc caccaatacc 17460
gcttttagtg tcgcacgtgt cccttcgatt tcttcaccca aaccgtacgt agcttgctta 17520
cctcggttag caataacagc aaggctggtg ttggcatcac tacttgcaac cgcaccgacc 17580
agcgtaaggt taaggcgcgt cttaggggcg tctttaacaa taggctgttc tacaaccgcc 17640
gctttttgct cagtgtattt accgaatagg ttggcattct gaagggcatt aaaatctatc 17700
gcgtcgcctt tattgctcgc agcaacgcta ctcgccgatg gacgccattg cggcacttcc 17760
gacgattgag gtatggctaa ccacacaatt ttgccaagta tccatccggt tatcgcaata 17820
aacagacagg taagcaacaa gctaagtttg gcttgaatgg tataatgatt ggacttgatc 17880
tgagccagta gcggtctgct tgataatcct gcacttagct ctaaacgttt cactgaatgg 17940
attcctttat tctaacgtcc gtcatttaag ctataacgat agctatcgac tttaaattgc 18000
taaatttgca ataaaattat tactcaacaa agagcagtga actatatcac aagttgtctt 18060
tcataccttt ttcttatgca gtttttgcca actcccttga aaaaaatacg ccttgactcc 18120
atctgtaggt gaataatcca tcactcattg ccgtattcgg gcagtgatta gatagcgtag 18180
tgttctatga gttccctaaa tgaagccgtt aggctagata aatggctatg ggccgcaaga 18240
ttttacaaaa cccgctccat cgcacgtaac atggtcgatg gcggtaaagt ccactataat 18300
ggccaacgaa gcaagccgag taaaatcgtt gaagtgggcg ctgaagtcag acttcgtcaa 18360
ggcaacgaag aaaaaatcgt gatcatcgaa caaatctcag atcaacgccg aggcgctccc 18420
atcgcacaaa ccttatatcg cgaaaccaat gagagtatcg cgaaacgtga ggaacacgcg 18480
actcaacgta agcttaatgc ccacaaccca agtccggaac gccgcccaga taaaaagcag 18540
cgcagagaca tcattaagtt caaacaccaa taaaagccgg aattaccaac gaggaaagca 18600
ctccatggca aataatgttt taaaccgcta cctattcgaa gatctttcag tacgtggcga 18660
gttggtacaa ttggatgaag cgtaccaacg tgttatctcc agcaaggaat atccggcagc 18720
aatagagaaa ctgctgggtg agctactcgt ttcaactact cttttaactg ccactcttaa 18780
gttcgaaggt tcgatcacca tccagctaca aggcgacggt cctgtgtctc ttgccgttat 18840
caacggtgac cataaccaac gtgttcgtgg cgtagctcgc tgggaaggcg atgttgccga 18900
tgacgcaaac attcacgatc taatgggtaa gggctacctt gtgatcacca tcgaaccgaa 18960
aaacggcgag cgttaccaag gcgtcgttgg attagaaggt gacactttag cagaagtgct 19020
agaaggctac tttgcaaact cagagcagtt aaaaacgcgc ctatggatcc gcactggtga 19080
gcatgagggc aaagcacacg cagcaggtat gttgattcaa gtgatccctg acggcactgg 19140
ctctccagat gatttcgaac atctagagca gctcactgac acagtaaaag atgaagaatt 19200
gtttggttta gaagcgaacg agttattgta tcgactatat aaccaagaca aagtacgtct 19260
gtacgagccg caacctgtcg agttccattg tggctgttca cgtgagcgta gcggtgccgc 19320
aatcatcacc gttgagaaag ctgaaattta tgacattttg gcagaagttg gatcagtttc 19380
tttacattgt gattactgtg gcacaactta tacgttcgac gaatctgaag tcactgaact 19440
gtatgcacaa gcttctacag acaagaaaac cctgcattaa tttacatagc ttatcaagca 19500
cgtaatttac cctacaaaag gccagcttaa tgctggcctt tttgtgatca acccctcgta 19560
ataactagcc tcaagcgtaa taaaacaact gcttaatttt aaacaattaa taatgatttt 19620
gccctagcgc aaaggattgc gtgcacgata gactattccc accaatatgt gacgggttac 19680
ctaaaacctt attgaaatca tgggtaaatt ttgagctatc tccctgtttt ctccccgccc 19740
cgttgctagc atggtgagca gattaaaatt aaaacattct tacaaaatcc ctacaaaatc 19800
cttataagga gcacctatga ccgctatgga acatactaag gctgcaacac ttgatctaac 19860
caagcacgga ctgcataacg taaaagaggt tattcgcaac ccaagctacg aaatgttgtt 19920
tgaggaagaa acgcgtgaag acctaaccgg ttatgagcgc ggcgtggtta ctgaactagg 19980
tgcggttgcc gttgacactg gcatctttac tggtcgctca ccaaaagata aatacatcgt 20040
caaagacgct acgacagaag aacatatgtg gtggacgtct gatcaagtga aaaatgataa 20100
caagccaatc actcctgaag tgtgggacga gctaaaagaa ctggtcacaa accagctttc 20160
taacaaacgc ttgtttgttg ttgacggtta ctgtggtgca aacccagata cacgtttgag 20220
cattcgtgtt atcacagaag tcgcatggca agcgcacttc gttaaaaaca tgtttattcg 20280
cccgacggaa gaagagttag cgacatttga accagacttc gtagtaatga atggcgcaaa 20340
atgcactaat ccaaaatggg aagagcaagg cctgaactca gaaaacttca ccgtatttaa 20400
cctaactgag cgcatgcaac ttatcggtgg tacttggtac ggtggtgaaa tgaagaaagg 20460
tatgttcgcg atgatgaact acttccttcc actaaaagac atcgcatcaa tgcactgtag 20520
cgctaacatg ggcaaagatg gcgatgtagc ggtattcttc ggtctatctg gtacgggtaa 20580
aacgaccctg tcgactgatc ctaaacgtgc gctaattggt gatgatgagc acggctggga 20640
tgatgacggt gtcttcaact tcgaaggtgg ttgttacgca aaaaccatca aactttctaa 20700
agaagcggag ccggacatct acaacgcaat tcgtcgtgat gcactgctag aaaacgtaac 20760
agtacgtggc gatggctcta tcgactttga tgatggttcg aaaacagaaa acacacgtgt 20820
ttcttaccct atctaccata tcgataatat cgttaagcca gtatcaaaag gcggtcacgc 20880
gaataaagtg atcttcctat cagccgatgc atttggcgta ctgcctccag tatctaaatt 20940
gacacccgag caaactaagt accacttcct gtctggcttt accgccaaac tagcgggaac 21000
agagcgcggt attactgagc caacaccaac tttctctgct tgtttcggcg cggcgttcct 21060
aacgcttcac ccaactaaat acgcagaagt actggttaaa cgcatggaag cagcaggcgc 21120
tgaggcttac ctagtgaata ctggttggaa cggcactggc aaacgtatct ctattcaaga 21180
tacacgcgga attattgatg ccattcttga tggttcgat 21219
Claims (8)
1.一种坎氏弧菌噬菌体,其特征在于,具有如SEQ ID No.1所示的核苷酸序列。
2.一种组合物,其特征在于,包括权利要求1所述的坎氏弧菌噬菌体。
3.根据权利要求2所述的组合物,其特征在于,还包括辅料。
4.根据权利要求2所述的组合物,其特征在于,所述组合物的剂型选自粉剂、水剂、冻干剂、凝胶剂、霜剂或膏剂。
5.权利要求1所述的坎氏弧菌噬菌体在制备抗菌剂、水体净化剂或饲料添加剂中的应用。
6.根据权利要求5所述的应用,其特征在于,所述抗菌剂、所述水体净化剂或所述饲料添加剂用于预防和/或杀灭坎氏弧菌。
7.一种杀灭水体中的坎氏弧菌的方法,其特征在于,包括以下步骤:向所述水体中投入权利要求1所述的坎氏弧菌噬菌体,或投入权利要求2至4任一项所述的组合物。
8.一种水产品的处理方法,其特征在于,包括以下步骤:将非活体的所述水产品与权利要求1所述的坎氏弧菌噬菌体混合,或与权利要求2至4任一项所述的组合物混合。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010318177.1A CN111575243B (zh) | 2020-04-21 | 2020-04-21 | 坎氏弧菌噬菌体及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010318177.1A CN111575243B (zh) | 2020-04-21 | 2020-04-21 | 坎氏弧菌噬菌体及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111575243A true CN111575243A (zh) | 2020-08-25 |
CN111575243B CN111575243B (zh) | 2022-06-03 |
Family
ID=72119954
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010318177.1A Active CN111575243B (zh) | 2020-04-21 | 2020-04-21 | 坎氏弧菌噬菌体及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111575243B (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112646786A (zh) * | 2021-01-21 | 2021-04-13 | 海南海壹水产种苗有限公司 | 一株坎式弧菌噬菌体快速初步分离方法 |
CN115725570A (zh) * | 2022-11-02 | 2023-03-03 | 北京金匙医学检验实验室有限公司 | 基于纳米孔测序的血液样本耐药/毒力基因检测方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140105866A1 (en) * | 2012-10-17 | 2014-04-17 | Universidad De Chile | Bacteriophages useful for the prophylaxis and therapy of vibrio anguillarum |
CN110205305A (zh) * | 2019-05-14 | 2019-09-06 | 菲吉乐科(南京)生物科技有限公司 | 耐碱的溶藻弧菌噬菌体及其组合物、试剂盒和应用 |
CN110684741A (zh) * | 2019-08-13 | 2020-01-14 | 广东海大畜牧兽医研究院有限公司 | 一种弧菌噬菌体及其应用 |
-
2020
- 2020-04-21 CN CN202010318177.1A patent/CN111575243B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140105866A1 (en) * | 2012-10-17 | 2014-04-17 | Universidad De Chile | Bacteriophages useful for the prophylaxis and therapy of vibrio anguillarum |
CN110205305A (zh) * | 2019-05-14 | 2019-09-06 | 菲吉乐科(南京)生物科技有限公司 | 耐碱的溶藻弧菌噬菌体及其组合物、试剂盒和应用 |
CN110684741A (zh) * | 2019-08-13 | 2020-01-14 | 广东海大畜牧兽医研究院有限公司 | 一种弧菌噬菌体及其应用 |
Non-Patent Citations (2)
Title |
---|
NICOLA LORENZ 等: "Identification and Initial Characterization of Prophages in Vibrio campbellii", 《PLOS ONE》 * |
张美霞 等: "哈维弧菌对水产动物的致病性及其噬菌体防控研究进展", 《中国抗生素杂志》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112646786A (zh) * | 2021-01-21 | 2021-04-13 | 海南海壹水产种苗有限公司 | 一株坎式弧菌噬菌体快速初步分离方法 |
CN115725570A (zh) * | 2022-11-02 | 2023-03-03 | 北京金匙医学检验实验室有限公司 | 基于纳米孔测序的血液样本耐药/毒力基因检测方法 |
CN115725570B (zh) * | 2022-11-02 | 2023-10-03 | 北京金匙医学检验实验室有限公司 | 基于纳米孔测序的血液样本耐药/毒力基因检测方法 |
Also Published As
Publication number | Publication date |
---|---|
CN111575243B (zh) | 2022-06-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112029732B (zh) | 一种耐高温宽裂解谱的沙门氏菌噬菌体及其组合物 | |
US20210046131A1 (en) | Wide-spectrum salmonella phage and application thereof | |
CN109735471B (zh) | 一株微小杆菌及其作为益生菌在水产上的应用 | |
CN110484513B (zh) | 噬菌体pAhMJG及其在治疗嗜水气单胞菌引发的鱼类疾病中的应用 | |
CN113583971B (zh) | 一株可同时裂解大肠杆菌的沙门氏菌噬菌体及其应用 | |
CN111254121B (zh) | 一种沙门氏菌噬菌体及在防治沙门氏菌感染的疾病的药物中的应用 | |
CN113583972A (zh) | 一株可降低抗生素耐药性的大肠杆菌噬菌体及其应用 | |
CN111575243B (zh) | 坎氏弧菌噬菌体及其应用 | |
CN113430173A (zh) | 一株宽裂解谱、耐高温沙门氏菌噬菌体及其应用 | |
CN113201501A (zh) | 一株具有跨种裂解能力的弧菌噬菌体及其应用 | |
CN112375712A (zh) | 一株乳酸乳球菌及其应用 | |
CN107805629B (zh) | 溶藻弧菌噬菌体及包含该噬菌体的杀菌组合物 | |
Le et al. | Use of bacteriophages to control Vibrio contamination of microalgae used as a food source for oyster larvae during hatchery culture | |
CN113430176B (zh) | 一株稳定高效的烈性沙门氏菌噬菌体rdp-sa-21004及其应用 | |
Yeoh et al. | The Vibrio-predatory filamentous bacteria effectively removed acute hepatopancreatic necrosis disease (AHPND) causative Vibrio parahaemolyticus in vitro | |
CN117070472A (zh) | 一株针对高致病性弧菌及耐药弧菌的副溶血弧菌噬菌体及应用 | |
CN111363723A (zh) | 新型霍乱弧菌噬菌体及其应用 | |
Wang et al. | Distribution of Vibrio parahaemolyticus ATCC17802 in tissues of adult Pacific oysters (Crassostrea gigas) under starvation conditions | |
CN113444695B (zh) | 一株发酵效率高且临床效果好的大肠杆菌噬菌体及其应用 | |
CN112592851B (zh) | 一株对水产病原菌有广谱拮抗作用的嗜酸乳杆菌及其应用 | |
CN110468110B (zh) | 一种副溶血弧菌噬菌体及其在刺参疾病预防中的应用 | |
CN109735506B (zh) | 李斯特菌噬菌体组合物及其应用 | |
CN116098973B (zh) | 一种中药组合物及其应用 | |
CN112553171B (zh) | 一种弧菌噬菌体制剂及其应用 | |
CN114774371B (zh) | 一种弧菌噬菌体及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |