CN112760296B - 一株副溶血弧菌噬菌体rdp-vp-19010及其应用 - Google Patents
一株副溶血弧菌噬菌体rdp-vp-19010及其应用 Download PDFInfo
- Publication number
- CN112760296B CN112760296B CN202110125461.1A CN202110125461A CN112760296B CN 112760296 B CN112760296 B CN 112760296B CN 202110125461 A CN202110125461 A CN 202110125461A CN 112760296 B CN112760296 B CN 112760296B
- Authority
- CN
- China
- Prior art keywords
- phage
- vibrio parahaemolyticus
- rdp
- host
- vibrio
- 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.)
- Active
Links
- 241000607272 Vibrio parahaemolyticus Species 0.000 title claims abstract description 70
- 241001515965 unidentified phage Species 0.000 title claims description 24
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 230000002265 prevention Effects 0.000 claims abstract description 3
- 208000015181 infectious disease Diseases 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 9
- 230000000844 anti-bacterial effect Effects 0.000 claims description 8
- 239000003242 anti bacterial agent Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000003115 biocidal effect Effects 0.000 claims description 3
- 239000000645 desinfectant Substances 0.000 claims description 3
- 241000701553 Myoviridae Species 0.000 claims description 2
- 239000003674 animal food additive Substances 0.000 claims description 2
- 239000003599 detergent Substances 0.000 claims 1
- 238000009472 formulation Methods 0.000 claims 1
- 230000009089 cytolysis Effects 0.000 abstract description 15
- 238000009360 aquaculture Methods 0.000 abstract description 12
- 244000144974 aquaculture Species 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 10
- 230000001717 pathogenic effect Effects 0.000 abstract description 10
- 238000009776 industrial production Methods 0.000 abstract description 4
- 238000000034 method Methods 0.000 abstract description 4
- 239000010865 sewage Substances 0.000 abstract description 4
- 241000237502 Ostreidae Species 0.000 abstract description 3
- 235000020636 oyster Nutrition 0.000 abstract description 3
- 210000001835 viscera Anatomy 0.000 abstract description 3
- 238000012216 screening Methods 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 description 22
- 230000001580 bacterial effect Effects 0.000 description 12
- 238000005336 cracking Methods 0.000 description 9
- 239000000725 suspension Substances 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 206010059866 Drug resistance Diseases 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 241000238557 Decapoda Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 241000588770 Proteus mirabilis Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000002101 lytic effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000269335 Ambystoma laterale x Ambystoma jeffersonianum Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 241000607598 Vibrio Species 0.000 description 1
- 206010047400 Vibrio infections Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000012459 cleaning agent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000001320 lysogenic effect Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000009364 mariculture Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 230000009278 visceral effect Effects 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
- 206010048282 zoonosis Diseases 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
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- 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
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/381—Microorganisms
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/48—Medical, disinfecting agents, disinfecting, antibacterial, germicidal or antimicrobial compositions
-
- 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
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
- C12N2795/10121—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
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
- C12N2795/10131—Uses of virus other than therapeutic or vaccine, e.g. disinfectant
-
- 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
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10111—Myoviridae
- C12N2795/10132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Pharmacology & Pharmacy (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Animal Husbandry (AREA)
- Birds (AREA)
- Pest Control & Pesticides (AREA)
- Marine Sciences & Fisheries (AREA)
- Insects & Arthropods (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Physiology (AREA)
- Agronomy & Crop Science (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Communicable Diseases (AREA)
Abstract
本发明公开了一株副溶血弧菌噬菌体RDP‑VP‑19010及其在水产养殖中的应用。本发明利用双层平板法从青岛即墨一水产养殖场发病牡蛎内脏筛选得到一株致病性副溶血弧菌ZHBV‑18001，然后以副溶血弧菌ZHBV‑18001为宿主，从养殖场污水中分离出副溶血弧菌噬菌体RDP‑VP‑19010，保藏编号为CGMCC No.19384。本发明提供的副溶血弧菌噬菌体RDP‑VP‑19010对水产养殖环境中致病性副溶血弧菌ZHBV‑18001具有强裂解效用，为工业化生产噬菌体用于水产养殖环境中致病性副溶血弧菌的防治提供了噬菌体来源。
Description
技术领域
本发明涉及生物技术领域，特别是涉及一株副溶血弧菌噬菌体RDP-VP-19010及其应用。
背景技术
近年来，我国的水产养殖业获得迅猛发展。随着海水养殖规模的日益扩大和集约化养殖方式的不断推广，鱼虾疾病的频繁发生对水产品的规模化养殖的健康发展造成了严重的威胁。水产动物的病原菌主要有细菌、病毒，寄生虫等，其中细菌病对水产动物危害程度危害程度较为严重的疾病。
副溶血性弧菌是一种重要的人畜共患病原，不仅引起严重的海水动物弧菌病，而且造成人类的感染，甚至死亡。每年夏秋季节是疾病的爆发期，容易感染鱼类、虾类、贝类以及蟹类等。目前，对于副溶血性弧菌的预防和控制的主要方法是使用抗生素，虽然抗生素具有使用方便，广谱抑菌，见效快等优点，但是，抗生素的滥用也导致了严重的细菌耐药性，在防治副溶血性弧菌所造成的的疾病时效果越来越差，急需开发新型抗副溶血性弧菌的药物。
噬菌体是一种细菌病毒，在自然界中具有较高的丰度，具有宿主专一性强，不会破坏微生态环境。噬菌体侵染宿主菌后，裂解时会释放出新的噬菌体，而少量的噬菌体就能起到防治的效果。副溶血性弧菌噬菌体的作用靶点在原核微生物细胞壁，对动植物细胞上没有相应的作用位点，增加了噬菌体使用的安全性。噬菌体和病原菌可以共同进化，防止细菌耐药性的出现。因此利用细菌的天敌噬菌体来解决细菌具有广阔前景的生物防治途径。
本发明在于相应国家全面禁抗的号令，解决细菌耐药性，抗生素滥用的问题，提供一株治疗禽奇异变形杆菌病的且安全可靠的裂解性奇异变形杆菌噬菌体。
发明内容
本发明的目的在于提供一种副溶血性弧菌噬菌体RDP-VP-19010，旨在解决水产养殖动物因感染副溶血弧菌发病急而治疗不及时，且因致病菌耐药性而导致用药无效等困难。
为实现上述目的，本发明采用如下技术方案：
一株副溶血弧菌噬菌体RDP-VP-19010，其特征在于，透过电镜观察，该噬菌体噬菌斑直径4mm-5mm，圆形透明，无晕圈，其头部为二十面体，长径约61nm，横径约67nm，尾长约112nm，直径约10nm，为有尾病毒目肌尾病毒科，该噬菌体于2020年03月24日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为：北京市朝阳区北辰西路1号院3号，保藏编号CGMCC No.19384。
该噬菌体为烈性噬菌体，其对副溶血弧菌具有强裂解性，裂解率达99％，为工业化、规模化、程序化生产噬菌体及治疗副溶血弧菌引起的水产养殖动物细菌病提供了噬菌体来源。
进一步地，噬菌体RDP-VP-19010在为5.0-9.0范围内，37℃培养1小时，效价仍保持在109pfu/mL以上，在pH＝7.0时，效价达到1010pfu/mL以上，对弱酸弱碱条件有耐受性。
本发明还提供了该副溶血弧菌噬菌体RDP-VP-19010在制备抗副溶血弧菌制剂中的应用。
本发明还提供了该副溶血弧菌噬菌体RDP-VP-19010在制备抗副溶血弧菌ZHBV-18001药物中的应用。
本发明的另一个目的在于提供一种用于防治副溶血弧菌感染的杀菌组合物，包括有效量的副溶血弧菌噬菌体RDP-VP-19010。
本发明还提供了一种用于防治副溶血弧菌感染的杀菌组合物，该杀菌组合物中副溶血弧菌噬菌体RDP-VP-19010的含量≥109PFU/mL。
本发明还提供了一种用于防治副溶血弧菌感染的杀菌组合物，还包括用于抗副溶血弧菌的抗生素。
本发明还提供了一种水产饲料添加剂，其有效成分包括本发明的副溶血性弧菌噬菌体RDP-VP-19010。
本发明还提供了一种清洁剂或消毒剂，其有效成分包括本发明副溶血性弧菌噬菌体RDP-VP-19010。
本发明的有益效果体现在：
(1)本发明从青岛即墨一水产养殖场发病牡蛎内脏组织中筛选得到了一株致病性副溶血弧菌ZHBV-18001，并以该副溶血弧菌ZHBV-18001为宿主，从养殖场污水中分离得到了副溶血弧菌噬菌体RDP-VP-19010，该噬菌体RDP-VP-19010对水产养殖环境中致病性副溶血弧菌ZHBV-18001具有强裂解效用，为工业化生产噬菌体用于水产养殖环境中致病性副溶血弧菌ZHBV-18001的防治提供了噬菌体来源；
(2)本发明提供的副溶血弧菌噬菌体RDP-VP-19010，在pH5.0-9.0范围能维持较好的活性，噬菌体效价没有明显变化，在pH为7.0时，噬菌体效价最高；随着pH值的升高或降低，噬菌体的效价下降明显，能耐受弱酸和弱碱条件；
(3)该副溶血弧菌噬菌体RDP-VP-19010可用于工业生产，可由宿主菌副溶血弧菌ZHBV-18001特异性扩增；该噬菌体还可以作为一种安全的生物消毒剂用于环境消毒和动物体表的消毒，从而治疗水产养殖场污染；该噬菌体还可作为一种药物用于副溶血弧菌引起的疾病的预防和治疗。
附图说明
本发明上述的以及其他的特征、性质和优势将通过下面结合附图和实施例的描述而变的更加明显：
图1为副溶血弧菌噬菌体RDP-VP-19010的电镜图；
图2为本发明副溶血弧菌噬菌体RDP-VP-19010的一步生长曲线图；
图3为本发明副溶血弧菌噬菌体RDP-VP-19010酸碱耐受性实验的示意图；
图4为本发明副溶血弧菌噬菌体RDP-VP-19010对宿主菌ZHBV-18001的裂解曲线图；
图5为本发明副溶血弧菌噬菌体RDP-VP-19010热稳定性实验的示意图；
图6为本发明副溶血弧菌噬菌体RDP-VP-19010的遗传稳定曲线示意图。
图7为本发明副溶血弧菌噬菌体RDP-VP-19010RTD实验的示意图。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
本发明使用从青岛即墨一水产养殖场发病牡蛎内脏中筛选得到副溶血弧菌ZHBV-18001，并通过实验确定了ZHBV-18001的致病性和耐药性，以ZHBV-18001为宿主，从养殖场污水中分离得到副溶血弧菌噬菌体RDP-VP-19010；并通过全基因组序列确定了RDP-VP-19010的基因型。
实施例1致病性副溶血弧菌ZHBV-18001的分离及其鉴定
(1)样品处理：采集发病牡蛎，用清水冲洗，然后把内脏取出后，研磨。
(2)弧菌增值：0.5g样品，置于5mL增值液中，180rpm 37℃培养18h。
(3)将过夜后的菌悬液在TCBS培养基上进行平板划线，放置在37℃的条件下培养12h-18h。
(4)将有副溶血弧菌菌落特征的菌落进行再次平板划线，纯化3次，再制备菌悬液进行保存为后续鉴定做准备。
(5)鉴定：制备好的菌悬液送至华大基因进行鉴定，经分子鉴定为副溶血弧菌，将其中一株命名ZHBV-18001，保存于-80℃冰箱。
实施例2副溶血弧菌噬菌体RDP-VP-19010的分离及鉴定
取发病养殖场不同地点的污水样品，5000r/min离心10min，取上清液过0.22μm的微孔滤膜；将过滤液、3×TSB和混合菌株，100r/min 37℃培养过夜。取上清液，采用双层平板法进行噬菌体分离，抠取透亮的噬菌斑于SM缓冲液中，三次铺板纯化，得到一种噬菌体，并将其命名为RDP-VP-19010；将噬菌体增殖液与60％的甘油按照1：1的比例混合后，于液氮中保存。
实施例3噬菌体RDP-VP-19010的电镜观察
取20μL含噬菌体粗制颗粒的液体滴于铜网上，自然沉淀15min，并用滤纸从侧面吸去多余液体，加一滴2％的磷钨酸(PTA)于铜网上对噬菌体染色10min，然后用滤纸从侧面吸去染色液，待样品干燥后用电子显微镜观察噬菌体形态。
由图1可知，该噬菌体噬菌斑直径4mm-5mm，圆形透明，无晕圈，其头部为二十面体，长径约61nm，横径约67nm，尾长约112nm，直径约10nm；根据国际病毒分类学组织病毒分类第九次报告，可将该噬菌体归类为有尾病毒目肌尾病毒科。
实施例4噬菌体基因组测序
通过对单株噬菌体的富集培养后，在4℃条件下8000r/min离心15分钟，加入10％PEG8000和0.5M NaCl静置过夜，再加入等量氯仿混匀，静置分层后5000r/min离心10min，去处氯仿层和PEG层后，加入限制性核酸内切酶消化处理，并采用梯度密度氯化铯条件下悬浮噬菌体，后期采用TM缓冲液透析3次，每次30min，一部分送华大基因生物测序公司进行全基因组测序。测序结果显示，该噬菌体不具有毒力基因和溶原基因。
实施例5噬菌体生长曲线的绘制
取对数生长期的副溶血弧菌悬液，按照最佳感染复数的比例接种噬菌体RDP-VP-19010，在37℃的条件下，水浴10min，然后常温下12000r/min离心5min，弃上清液，除去未吸附在宿主上的游离噬菌体，如此用2216E培养基洗涤两次。再将沉淀重悬于37℃的2216E肉汤培养基中，迅速置于37℃200r/min的摇床中振荡培养并计时。以时间为横坐标，噬菌体效价对数值为纵坐标，绘制生长曲线，得出噬菌体的潜伏期、裂解期，并计算出平均裂解量。平均裂解量＝暴发末期噬菌体滴度/感染初期宿主菌浓度。
由图2可知，噬菌体感染宿主菌后的30min内效价没有明显变化，表明其潜伏期约30min，感染后30-80min内噬菌体的滴度明显上升，之后趋于稳定，表明噬菌体裂解期约为50min。通过计算，噬菌体裂解量约为163PFU/感染细胞，表明噬菌体具有极强的裂解和复制能力。
实施例6噬菌体pH稳定性的测定
用稀盐酸和稀NaOH溶液调节生理盐水的酸碱度，配成pH值为2、3、4、5、6、7、8、9、10、11、12的缓冲液，再用配置好的缓冲溶液将噬菌体稀释成约1*1010pfu/mL的效价，将稀释液在37℃下，水浴1h，用生理盐水10倍比稀释后铺双平板。37℃倒置培养4-6h后计数。
由图3可知，在不同pH条件下培养1h后，噬菌体RDP-VP-19010在pH5.0-9.0范围能维持较好的活性，噬菌体滴度没有明显变化，效价仍保持在109pfu/mL以上，而且在pH为7.0时，噬菌体效价最高，效价达到1010pfu/mL以上。随着pH值的升高或降低，噬菌体的效价下降明显。表明噬菌体RDP-VP-19010能耐受弱酸和弱碱条件，具有良好的酸碱耐受性。该噬菌体RDP-VP-19010最适生长pH为中性环境。
实施例7最佳感染复数的测定
按照感染复数为10:1、1:1、1:10、1:100、1:1000、1:10000的比例将噬菌体增殖液和宿主加入2216E培养基中，并且确保培养体系的总体积相同。于37℃200rpm振荡培养8h后，常温下12000r/min离心5min，取上清液铺双平板测定其效价。结果如表1所示。
表1噬菌体RDP-VP-19010最佳感染复数结果
| 序号 | 感染复数 | 8h噬菌体效价(pfu/mL) |
| 1 | 10:1 | 6.13×10<sup>9</sup> |
| 2 | 1:1 | 9.62×10<sup>9</sup> |
| 3 | 1:10 | 2.69×10<sup>10</sup> |
| 4 | 1:100 | 7.96×10<sup>10</sup> |
| 5 | 1:1000 | 3.21×10<sup>10</sup> |
| 6 | 1:10000 | 6.61×10<sup>9</sup> |
由表1数据可知，当感染复数为1:100时，培养8h后，增殖液较为清澈，效价最高，说明RDP-VP-19010的最佳感染复数为1:100。
实施例8噬菌体对宿主裂解曲线
在细菌悬液中加入噬菌体，由于噬菌体对宿主的裂解作用，导致OD600的值产生变化，由此得到噬菌体对宿主的裂解曲线。将培养6h的宿主菌悬液以1:100的比例接种到100mL2216E培养基中，37℃200rpm振荡培养2h(OD约1.5，约6.0×108cf/mL)将菌悬液分装在4个灭菌的锥形瓶中，以最佳感染复数，向其中3个加入噬菌体增殖液(经0.22μm膜过滤)，同时以不加噬菌体的作为对照，取其平均值。
由图4可知，副溶血弧菌噬菌体RDP-VP-19010加入到宿主菌ZHBV-18001菌悬液中，作用30分钟后出现一定的裂解效果，约40分钟后，OD600值开始下降，持续作用至150分钟，180分钟后混合菌液开始变得浑浊，OD值逐渐升高。由于前40分钟，噬菌体处于对宿主菌的吸附、侵染阶段，只有少部分噬菌体开始裂解宿主，因此，其裂解宿主的量远远小于宿主的增长量，因此，OD600在前40分钟内，依旧保持一定的上升趋势，伴随着噬菌体数量的指数增加，在40分钟后，其裂解宿主数量大于宿主的增殖速度，所以OD600值开始逐渐降低，混合菌液达最澄清。持续作用2.5小时后，混合菌液出现返浊，原敏感菌被噬菌体裂解，被突变的耐受菌所取代的一种菌***替。
实施例9噬菌体热稳定性试验
将噬菌体原液分装到EP管中，分别在30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃的条件下孵育30min和60min，然后用生理盐水10倍比稀释后，铺双平板测定其效价。
由图5可知，噬菌体在30℃～45℃以内活性保持稳定，效价保持在1010pfu/mL以上，在高温80℃时基本失活。由此可知，噬菌体RDP-VP-19010适合在45℃及以下环境中生存。
实施例10稳定性遗传实验
将RDP-VP-19010连续传代29次，每代培养基中加入等量的RDP-VP-19010和ZHBV-18001，每代培养时间6h，然后铺双平板，测定其效价。
由图6可知，将RDP-VP-19010连续传29代后，测定其效价，其效价稳定在1010pfu/mL以上，说明该噬菌体具有较好的遗传稳定性，适合工业化生产。
实施例11裂解率实验
无菌条件下，分别取1mL样品和1mL宿主菌菌液(1×106CFU/mL)，37℃孵育15分钟，混匀后用生理盐水稀释至10-1-10-3个梯度，每个梯度取100μL涂布于TCBS琼脂平板上，置于37℃，培养24小时，每个梯度重复两次。同时取1mL生理盐水和1mL宿主菌菌液(1×106CFU/mL)作为空白对照，重复以上步骤。选取30-300个菌落数的平板进行计数。该试验重复3次，取其平均值。噬菌体裂解率＝(1-处理组菌落数/对照组菌落数)×100％。
RDP-VP-19010的裂解率达99％，对宿主有较好的裂解效果，适合在水产养殖过程中使用。
将RDP-VP-19010与现已有的副溶血性弧菌噬菌体RDP-VP-19003、VP-HYP2、SX-2、SX-3和VpJYP2进行裂解率的比对，参照上面的裂解率实验方法分别测定裂解率。结果如表2所示。
表2裂解率对比实验
| 噬菌体名称 | 裂解率(％) |
| RDP-VP-19010 | 99 |
| RDP-VP-19003 | 96 |
| VP-HYP2 | 63 |
| SX-2 | 58 |
| SX-3 | 80 |
| VpJYP2 | 74 |
由表2可知，RDP-VP-19010的裂解率明显高于其他5株噬菌体的裂解率，其对宿主菌的裂解率最高，为99％，可有效抑制副溶血性弧菌的生长，表明该噬菌体有替代抗生素治疗副溶血性弧菌引起的疾病的潜力，有广泛的应用前景。
实施例12 RTD实验
在平板上划分区域，将一滴宿主菌(约100μL)滴加到区域中心处，晾干，将不同稀释度的一滴噬菌体液滴加到宿主菌的斑点上，晾干，37℃培养16-24h。根据宿主菌生长状况，凡是不能连片生长的，称为CL浓度(CL浓度是可以完全性裂解环境中宿主菌的浓度，可以用于指导生产实践的稀释方案)。结果如图7所示，RDP-VP-19010在稀释107倍后对宿主菌仍有较好的裂解效果。
应当指出的是，具体实施方式只是本发明比较由代表性的例子，显然本发明的技术方案不限于上述实施例，还可以有很多变形。本领域的普通技术人员，以本发明所明确公开的或根据文件的书面描述毫无异议的得到的，均应认为是本专利所要保护的范围。
序列表
<110> 青岛润达生物科技有限公司
<120> 一株副溶血弧菌噬菌体RDP-VP-19010及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50642
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
aaagataaca aagaaatcca gaaaaaccgt caaaagcgca aggatttaaa gacagctatg 60
cgtaaactgg atgaagctaa caagcaggtt tacaaagacc gtcaacttgc tgagcgcatc 120
aaagctaacg tagctaaaat cactgagtaa tccatcaact agccacagga caataaaatg 180
aagattgaag atattgtaat tggtcaagag taccgtatcg ttcgaaaagg tttggtaaac 240
acaggtcgta ttggtcgtgc tgtaggcaag aatggcttta aagtcaaact actaatgcac 300
cacaacaacg ctgagcttac actgaatgcc atgtctgttg aaccagttga aaaaggcgta 360
aaagctaaga agcaagttga cgttaacgaa aaccgtaagc ctgttgacaa cgagcttgtt 420
aaacctaaaa agaaaccagc taagaagttc gtagaacgtg acgtatttat cgagccagct 480
ccagatgcta tcgttcttat tgaacaaaat ggctctgttg tgtcggtatc aagaagaaac 540
ttagagttct ttgatgaccg tctgctgtca cacggtgtag actatacaaa ctcttacctc 600
ggattcttgc ctcgtggcgc taagttcaaa gttaacaagc ctgacaatat catggagtat 660
cccttttgga ataccgatga aatgcaagat ggcatggaac tcacatttac taattggcgc 720
aatgacttcg gtgctacaat aatcaaaacg gaagaaaact ttgaaattct ggctaaatgg 780
gttgatgtag gtagtctgca caaaatgggc aaggctaagg ttgaagttga tgacgatggt 840
aatgaagtag ttccaccatt ggaagaacaa gctcttaaac gcatggaaaa agatgctaaa 900
gaagctagtg gtcttgcatt cgctctttac tgggattatc gtggtaagct tgagtctggt 960
actcgtaacc atgcagcatg tcactaccac atgcgttgtt tcgttgatgg tgataagcgt 1020
aaagatgcta aggcattgtt tgcagctcag tttattactg accaccacta cggcaacctc 1080
aagaaagacc ttgttgaccg ttacctaact tgggtaatga acgactctcc ttggtctgac 1140
tacatcgtca ctaaagattt ggctgagggt aaagagaagg gtatacgtgt tcgcactgat 1200
attccagcta acgcaatgat gtgtacattc tacgctctgc gttaccctac tgaataccgt 1260
gacgtaatca ctctgtgggg acgttacacg gacgagggct tagacccgac cattgccttt 1320
tacaaagctg aaactcgcgg ctatgaccgt gacacggctt ctcaccacgg cttgttggat 1380
acattctcaa tgacattgca agatgtgatt gattttgcca ataagaaacg tggtaagatt 1440
gtggatgaaa tctatggtgt taaatacttc aactacaaga acgtatctgt acaccacggc 1500
tcatctgtac gccgacttga aaagactgct ctaactgagc ttaaagataa gtttaagcct 1560
aagaaagtca aagaaaaaca ccctgttctg ggcgaaatcg agaaagaaat cgctgcaaca 1620
gagcaaatcg accaattcct aactagcaaa ctggagaaat aagatggaca tggataaact 1680
accgcaaatt atgaaagtta tggtcgtagg ttctggtatg aacctcgact accatcgtat 1740
gctcaacgac atgggtcaca caacagtggc ttgtcgttat gttgagcaaa ttcgtccaat 1800
cttgtacaaa ggtgatattg atgttgtatt atttgtgggt ggggctgacg ttaatcctga 1860
gctttatggt catacgttac atcccacgtc cggtatttct aaagccgaag atgagttgta 1920
ccaacaagct ttctgtcaag ctcaagaatt tgatgaaatc aagtttgtgg gtatttgccg 1980
tggtagtcag ttcttgtgtg ctgctagcgg tggggttctt gttcaacatt gtgataatca 2040
cgctgtgttt aatgggcata gtgttgaaac taagtggggt cacgaattta atgtcacatc 2100
gacgcaccac caaatgcaag acccacggga agtggaacac gaactaatcg cttggactaa 2160
gcgtactacg ttcaaagatt accacattga tggtgagaca gttaccattg aaactcaggc 2220
agaccctgat gagctggata ttgaagttgt ttggtatcca catactaagt cgctagcttt 2280
ccagcctcac cctgaatttg caggagctgg tgagacgtta gcattgttta aacgttgttg 2340
gaatgagtac atgtaatgca gaatgtaaga attgaattgt tgcaaggtcg tcgttttgag 2400
gatgtgccta taggtgatgt gtttctatat aatagctgcc cttacatcaa attgaacgat 2460
gacttaaact ttaactgtaa gaaattcttc tctaaaggta ctggcagtat ttctcgtcag 2520
acttgtgtta tagttcctga agaaattatc attaaataag gcaggtagtt atgaagctgg 2580
atacaagtta gaaatgtaaa tgattactgg ggtggattcc atgtttattt gaagatattt 2640
aattaattac agttttgtgt gtgatttgat gaattctcct taccacccca gtggttatcc 2700
ttattagtat taagtagtaa taataagaat aacttagtta ttagcgatag caaggctatc 2760
aatctgtaaa accattctct tgtatgtaca tactaacaca aaaaagtgtc actgtcaaga 2820
ggtaaatcaa tcaaagttaa gaaaaggtat taaattatgt gtggaattgt aggcgttgta 2880
ggtcttatta cggcaaacga agaaaaagta ttcaagcagt tacttgaagt tgactctcta 2940
cgtggtcgtc attcgactgg cgttattaag ctttcttctg gaggtgtagt agagacacga 3000
aagaaagcga ttgatggtat ggactttgtt aagcttgagg gcaactggat ttcatctggt 3060
gttaaccgta tgcttattgg tcataaccgt tatgcaactc gtggtggtat taacgatgct 3120
aatgctcacc cgttctcaca tggacacatt cacggtgttc ataatggtac actaacaact 3180
caacttggtt tgaaagacca acataaattc cctgttgatt ctgataacct tttctacgat 3240
atggcacatg gtagcctacg tgaaacagct aagaaaatct caggtgcttg gactgttgcg 3300
atgtatgatg aacaagagaa aacgttcaac atgttccgta acactgagcg tccaatgagt 3360
gttgctctct cagaagatgg tacagtaatg tacttcgcta gtgaagctac tatgcttgag 3420
tgggttcttg agcgtaatgg tgttaagtac aaagacattc ttaatctaac tccagccaag 3480
cttatcacaa tcgaaccgac tgccgtagca aacacagacg gtaaacgtat ttctgatttg 3540
atgtctatca ccgacattga gtacaagaaa gttcctgctg ctccggctcg tggagcaaaa 3600
aagttccttt ccgacttcgg gttaaacatt caaaaaaagt atccgttgtc gttaattgga 3660
cgaaaaaaga caaatgcagg gtatgagtat gaagttgaac ttatggttcc tccatacaac 3720
tcatggactg tgtatgctta ccaacctaag actgcggaag ttcttggtaa gttgtacgac 3780
aacgacgaaa ttgcatgttc tgtattaaca attggttaca acagcaatat gccacattgt 3840
tggggtatgg acatcgagcc agctaccgaa gaagctctta aaatccttgc tgatgaagaa 3900
gcggcagcgg cagcggctgt agttgaccaa aacgacgttg atgatttgac agaagatgag 3960
cttagtcaaa tttttgatgc gtatggcatc acccaaatca aaaaggaatg gtatgaccac 4020
aacggtttag ctatgacaga agctgagttt gaaaagactc aagcaggtca gcatggttgt 4080
tgtatgtgtg gcgaccactt ccaagaacca gaagatgtag tgtggaacga tgatggcgac 4140
actgcatttt gtacagattg tgtagaagca tacaacgcta tctacggaac tcaagcacaa 4200
gctaagtaat tcactaacag agagagattt aattatgtca aattcagtat tcggtaaagt 4260
aacactaggc gcagacccag aaatcttcgt agtaaaacct gatggtactc cttggggtgc 4320
attaactact ggctgtaaag gtactaaaga agtgcctgag cctacagagt acggtgcatt 4380
acaagtagat ggtctagccc ttgaatacaa cattcatcca gttgatacct taaatgattg 4440
gatggatttg cacacagcag ctttgacaca cattgctgac acagcagata aagctggtct 4500
tgaaattgac tcaagctctc gtttggactt tacctcatat attgatggta atggtgccac 4560
tgaagatgag ttactatttg gttgtgaccc tgactttaac gcagacacaa tggaagaaaa 4620
caccatgcca gataacgacg gctctatcaa gttccgtact actggtggtc atgttcacat 4680
tggtttagca aactggggtg aactacatgg cggtgatgat aagattgcac acatgatggc 4740
atctaaaatc atctttgtct gtgacgcaat ccttggtcta cgctctgtcc taacagatga 4800
cggcgtagct cgtaaagaat tgtatggtaa agctggtgct taccgtgtta aaccttacgg 4860
tgttgaatac cgtacactgt caaacttctg gctgtttgat gaagaagaaa tgaaattcat 4920
ctttgatgca ctaaacaata tctttgcatc cgatgctgag tttgaacgtg tgtcagagtt 4980
tgcaacatta aatcgttctg atattgttaa agcaatcaac acgaacgata aagagcttgc 5040
tatcaagctg ctagctggtg acgccccatt aactgataaa taaaggagta ccaaatgaag 5100
aacttccatg ataatgcaca cgctaataac cgactatccc gttgctttgt gtgtattaag 5160
attaatggca agtgggtaac taaacgaatc tcagaagcca ccaaacagag tattgtttgt 5220
cgtgatggtg agatttacga cctacatagt gatgatgtta agattacgtt tgataactta 5280
ggttatgtta acactcagca tcaagctatc ttcgttagtc gtactcctaa gcgtatgtgg 5340
aaagctggtc tagtacctga gaatattcag gcagagcaca ttcatccaca gcatctacgc 5400
cacggggata tgattgagtt tgacaagtgt ctagataagc tgatgaataa tgtgtaccca 5460
tcttaccgca tggcttacct gcgagttaag aacggcaagt gtgcagcatc agctttccat 5520
cgtaacttcg ctgtatcatt gagcgacttt acagaacatt tggttcttta ctacaagaac 5580
tacgaaatcg gattcattga taatgattct gaacaaattg tggtgggtga ctcttacacc 5640
catctacgtg aacaaatctt tgaagcaaca ggtatggagt gtatctcatg aagaaaatcg 5700
cagacatctt taaactaaaa cgtcctcgca atgatgactc ttttggtgta gagctggaac 5760
ttgagtataa caaccgtcct gctgcctcag atgaatggaa aatggagcat gatggctctc 5820
tgcgtgtcgc tggtatcgaa tacgtaatga aagagcctat gagcctagct gacagtaaag 5880
tagctttaca gaacctctca gataagttca aagctgttaa gaatcaccct aaagctacta 5940
acttggcatc atcccacatc cacatcaacg tctctcacat gacgttacaa gagtttatcc 6000
agttcgttag tcttgtacta ctattcgaac cacatatcgg taagcatggt ggtcaagacc 6060
gttggatgaa ctacttcgca gtgagttctc aagagagtgg tatcctaatt cgtgagttgt 6120
taaaagtgcg taccgaagaa cagttcgcta aattcatctt acgtgtaaat cagcgtgatt 6180
ggcgctacaa cgggattaac tttgcatctg tggcaaagta tggctcactt gaaattcgct 6240
acttaggggc tatgccagag cctctagatg ttattccttg gcttgagttc tacgatacac 6300
ttcgtgaagc agcaatgaac gacatccaat ggggtatggt gtttgaaaca gtatcggcag 6360
gtgagacatt cttacttgag ggtacattca aaactccatt ccctctaaat caagatgacc 6420
tattacttgg tgctcgtatg gcacaagatt tggtctaccc agtataccat gatgcaaaag 6480
gcgctccttt cgcagaaaca tggaaactta ctaactacta cgcaaacgta taaaggaaat 6540
ttaaacatgg ctatcccaat aaaaggctat gaggggctgt ataccataga caagcatgga 6600
aacatttgga gttacaatgc ttcaagaggt ttacgtaagt ccccaagaca ggttaaaaca 6660
acaaacacaa acggtgcagg ctactgtata gttagcttgt acaaagacgg gaagaagact 6720
tcacactctg tacatagatt agtggcagag cactttatcc ctaaccccaa caactacact 6780
gatgtaaatc atattgatgg ggataagaca aataatgatg tgtcaaacct agagtgggta 6840
acacgtcaag agaatagaat ccactacatt aaatttttaa aggtataata ttatggcgat 6900
taaattacat agctacaaaa acggttctga atctgctaaa ctacttcgtg atgcacttgg 6960
tatcaagatg ttattacctg ccggtaaagg caaatggaaa ggtaaagctg gtgataaact 7020
aatcaactgg ggttgttcta caccacatcc aaacaaaggt ctagctacct acgttaacac 7080
gccagaagca gttcgaagtg cagctaacaa gcttattaca cagcaacgct ttgatgcaac 7140
tcctgcaatg gctaaccata cattgccttg gacaacagaa cgtgttacgg cacatcagtg 7200
gattgagaac ggcgatacag tagtatgtcg tcagaaatta actggtaact ctggcgaggg 7260
tattgtaatc gctgagaaag tggaagacct tgttgatgct cctctataca ctttgtacaa 7320
gaagaagaaa gatgagttcc gtgttcacgt ctttgatggt gaaatcattg atgttcagcg 7380
taaggctcgt aaacacgaag tagaagatga caaagttaac tggaaagtgc gtaacttgga 7440
cggcggtttc atcttcgctc gcaacaacgt tatcgctcct atgccagtgc gcaaagcagc 7500
tattgaagct gttaaagctt tgggtctggt tcatggcgct gttgacattg gtcacggtcg 7560
tcatgggact tacgtttacg aagttaatac cgcttgcggt ctcgctgggt caaccttggt 7620
tacttatgct aaagcatttg ctaagtacct aggcatggac gaagctaatc taaaactaac 7680
taaggaacag ttagacaatt acgttccaat gaacgaagaa aacaaacaac ttgagcaacc 7740
tgagcttgag ccacttccac aagaagtaca ggttggcttg gctcaaggtg aagaaatcgt 7800
agctgttggt gctggcatta atgttggtcg tccaatggct cagatggaag aagaagtagc 7860
ggcatttatt gcagcacaag ccgaagaagc ggaagaagaa aaacctttcc ctgagcaaga 7920
gcttggcaaa aacaagatgc gaatcacagc agacttgaaa gtgaaagatg ttcgtatccg 7980
tttcggcaac attgttgtag atgttaaccg caataacttc gcaaaagttg tacaggcact 8040
gaaataatac ttgacagtga cgcaaataag gtggtagact tgttgaacga aaaagaaaaa 8100
gttctgattc ctaatgagct tcttccacct aagtatcaaa ataaaggcta tcggatgagt 8160
cctgacggtt ctgtatacgg ggtgtatggt aagaagctac gccctaatga ttactcacct 8220
aatggtttta atcatccgat gctttttaat cgcaatttat ggtacattcg agcagccatg 8280
ttcgtcccta accctaataa ttacacaagg gttgtcgaga ttgacaacga agttcactgg 8340
ctgagtgaaa agaaataccg tgaaatggtt gctaaatact taggtcgtga agtagtggag 8400
cgaaagccct tgaatgattg gggtggggta ttgatagaaa tgcttgaaaa gggaacacgc 8460
atagctgatg tagctaaagc tcttggctat tcccgtgcag caatttacaa atacctagag 8520
aagaataact atgaatacaa aaaccgtaga cttaaaaaac gaaagtgaat acgttccatc 8580
agtacgtggt aatcctgctg acgcattgaa tacatacatc gtcacaattt atgataccaa 8640
aactggtaaa tatcacgaag ggctacgaac tcatcattac agtggtgaag cctgtgcttt 8700
ctgggtaatg caaaaccttt cagacggata cgaatttgta tctgcttaca acccagacaa 8760
taatgtaaca atcactaaaa aagacatcct aaaacaagaa gaggtaaaat aattatgaaa 8820
aaagtaatca tgtacgcatc aaacttctca cgctacggta aacttcaagc agttaaatct 8880
actctagctc acctatctcg tgaagcaggc ttcatcttcg ctttcgttaa cgacccagag 8940
cgtggcaaca cagctaagat taagtttggc aacaaacaag tctttgtacg agacttcaat 9000
cctgctcaac taaacaaaca cctagaagaa atgcaagaca tccacgctgt ataaggagat 9060
ttaaatgtca ttaaaccaaa tgttcgatga gttcaactcg ctataccgtg tgaaaggcac 9120
agacatcaac aacgaatcag ccaagggttt tgttaccctt gtgtgtgatg aagtagtaga 9180
gttcgctgag gagttcgaca aagaaactct tgataaagca gcggctgcta aagagggtct 9240
tgatgccatc tatgcaggta tgcaacaact acgaactatg ggtattgact tggaagcagg 9300
gcttaaggct ctgcattcct ctaacatgtc aaaaactgtt aaacgagcag atgctgagaa 9360
agaacttgaa atcgctcgtg aacgttaccc acaagcctac attgatgagg ctcaagccta 9420
ctgtgttatt cgtgatgcag ctacaggcaa agtaattaaa ccaacaactt acactccagc 9480
aactataaac gaggacatct acaatgcgta aacttacaaa agctcacttt aactacctag 9540
cacgtcatat caacctacgt ttccttaact acgagaaaaa gattctcgaa gatattgata 9600
ataaattcaa gggtgttcct aacttctctt ggattatgaa agttcaaggt acatttaatc 9660
cacatcagac tccagaagtg gataaaacaa tccgtgctat tcttgataaa ctgtacgaag 9720
aagttaagta caagaagtat gaacacttca accattcatt caactgcttt atgaagcgtc 9780
tgtgtgtaga acttaagaaa caaaatccta agttcgttga gtctaaattc cgtgaagcat 9840
gtacaaaagg gagcattcag taatatgaca gcattgaaag aacaagtggg aggcgaccac 9900
tacaaacgcc tacaatacca acctcacaca tttgcaatgg acttgtatgc atctggtggt 9960
gtgatgaaag ttgctaagta tcttagccgt gacaaactag accgcacaga ggacttgatg 10020
aaagctcgtc atgtaatgtg cctagatgaa gattgggtta agaacgaatg tgagaataac 10080
gtattcacat tattcgggga aacaggattt aacgctctgt gggaagattg tgttgtgttc 10140
tatgacccag acgaggacga tgagctggta gaacgattct tcgaacagtt cgctggtact 10200
ggtaacgact tcattgtttg gaaactaacc aagcaagcat tctaccaata cctaaaaggt 10260
aatctacctg cggctaaagg tcttatgact aaagcaatca acttagaaaa atcatacaag 10320
gcagtggcac taaagtcact gcgtagttca gaggaagttt aatgacaatt gaggtactag 10380
attttcatgg aagcgataac gccattgcta atgctgctcg tgttagcttt gctagccacg 10440
gcgattggaa ttcaattcca gaagcctaca gtgaagaaaa acgagataag cttattttgt 10500
atcttgctaa gcatcgccat cttactcctt ttcgtcatac ttcagttact atccgatgca 10560
aggagcctat cttccttgcc cgtcagctag gtaaacatca agctggctta agctggaatg 10620
aagtatcaag acgttacgta aaaggtgatg ttgaatttca catcccatca gcatggcgac 10680
tagcccctga tggtagtatt aaacaaggta gtgctggagt tcatcctagt tctggtcaat 10740
tctctgctgc ttatagtgat ttcattaata aagcaaaaga tttctacgat agcatgattg 10800
aaaatggcgt tgcccctgag caagcccgta tggttttacc acagtcaatg ctaactgagt 10860
gggtttggac tggtaatctg ctagctatgg ctcacatcta ccgtgaacga attgcatcag 10920
gagcacaact tgaagcacaa tatttcgcaa gaaaactgga cgacgcctta cagccgcttt 10980
tccctgtggc ttgggcagcg cttacaaaac ctaacctact caaataagga aacaagtatg 11040
gcaacccttg acatacgtaa tcaagatgct caaataaatg atatagtatt tgcaaacaaa 11100
gagggcggcg agggtcgcct ctataagctg gagcgttttg tacctgtaca atttcacctc 11160
gtcacatcca atggctttaa agcccatgtg aaaatggagg atgttgacaa ccttattgaa 11220
gccctaaaga cagcgaaaaa gctatgggca taacaaatta aacgagagaa aaatcatgga 11280
aaagagaaag cacttcttcg gtaactttaa aaagattatc gacagcttgg taatagacac 11340
tcacccacac agtgataatg gcggtgttgt agtagttgcg gggggtttcc tccgtgactt 11400
acatctaaga cgacaaccta aagatattga catcttcttt agtggaccac agtcgtcaaa 11460
aaccatcttt aagagtctca aggagaagaa ccctgaattg tttaaagact gcaagcataa 11520
gttctactgt gatgaaaact atggtatgga agaagaagaa gttaaaggta actttgctgt 11580
ctacgaccta gttaaagttg aacttacaaa tggtttaaac cttgacatca ttgcaacaca 11640
acgtgtgtgg gaacaacctt gggagttgtt aggtaatcat tttgactgta acctaaacgc 11700
cctagcatat gacggtaaga ctttgtttgc acctaatggt atgcctaaca aggatggtat 11760
cttcttccct cttggtggtg catacattga caaccaagtt actaaagagc gtatgattcg 11820
cttagtacag aaagttcctt tcggtaattt ccaagccttt agtcagttct tccctaacga 11880
ttgggatgag attctagaag cacgtaagaa agcacaagaa gttgaacgta aagaactggc 11940
aaaagcagcg caagctgaag ccgcagcaat ggcagcggta gtgccaggta ttattcaaga 12000
tgaagcagca caactggagt tttaataatg gataagcagt ataacaaatc tcgttacgaa 12060
cgtaagcgca aatacaaaaa caaacaacaa caacgtcgtc gccgttatgg tgatgactat 12120
gacaaaggct ttggaggtaa ctgtggagaa taatttcact gttcacacta aacgtacact 12180
agaagcgctc ccattggtgg agcgtttgta catccaagag aaacacaaaa ttcattttaa 12240
gtataagcgt ggtgatattg tttaccattg tttcatgttt taaaggagaa tagatggcaa 12300
aaattattgg cgatacggct tgtcctaagt gtagagaaaa tggtagagac aaaactggca 12360
accacctaat gattttcgat aatggaaacg gctactgcaa tcgctgtggt tttgtccatc 12420
gaaaagataa gatgggtgag gttaagccag ttcctcgacc tattaaatct cctgaagaaa 12480
tccaagctga gttggctgag gttaagacct acccaattca agctcttggt agtcggaaga 12540
ttagtaaaga gactgctgac aagttaggta tccgagtagc gttgagcgag gaagatggta 12600
aaacccagac tcaacattac tatccgtata ctcgtgagag taaactaagt ggctataacg 12660
ttcgtaacct aggtatgaaa tcattctacg gtgttggtga acgtaagggt gttgacccat 12720
tcaactggtt taatgctaga cgtgtagcca cacgtaaaaa gattattatc tgtgaggatg 12780
agctatctac agcatctgtt gttgaggtgt ttgatagatt caccaaggat aagcaatggc 12840
ttcctgctgt tatcggcttg gcaacatccc cacaaggtat tctgcaagac ttgggtcgta 12900
cctatgttaa agatttcatc atgtcatttg atgagattgt ctttgcattg gataatgatg 12960
aggcaggtga ggagggtctt aacttagcac ttcaactatt ccctgatgct aagtttgtag 13020
atttacctct taaagaccct aacgacatga agatgtctga tatggatgta gagttataca 13080
accttatcaa ctataatgca cgtcgtaaac aaccatctgg ttctttgact ttacacgacc 13140
tcaaagagcg tatgcttgct cctgttgaga aaggttactc attaccttgg gagaagctaa 13200
ataaacacac taacgggctt ctgatgggtc agatttggac tatcggttct ggtgtaggta 13260
ttggtaaaac attcttggct aactctatta cagcttggtt atggaaagaa caccgtatca 13320
agtctggtca gattatgttg gagcaacctg ctgaggaatc tggtcgtgct attgtaggtg 13380
cgatgacagg tcagcatttc aatgacccta agattccaat gaacagagag agattggctg 13440
atgctatcga cgacgttggt aacaatattt atttctatga tgctgtgcgt aatgctgatt 13500
gggacgaaat caaaccggtc attagacact gggtggtggt ggaaggcgtc aaagttatac 13560
agcttgacaa tcttactaca ctcactgctc atcttgatag ttctactgca aacaccgaag 13620
taaatagaat catggctgag ctggctatga tgtgtaagaa atatgaattt tctgcattcg 13680
tatacagcca cttgaacccg cctaagactg gcgatagcca tgaagatggg ggtgaggtta 13740
aggaagtaca attaacaggc tctcgtggcg ctatgcgtta ctctagctat gtgattggct 13800
tcgaacgtaa ccgtaaccca gctcttccac aagaggagca aaacctaagc cgtattcgac 13860
taatgaagtg tagacctatt ggttctactc caaccattgt cggtacatat tataatcctg 13920
caacaggatt gattgaacaa cgagaagaaa tgcacctacc taaaacacct aagcttgtgg 13980
gtgggggcga ttaaacgagg aaacatacac gatggctatt tataaaattc aacgagagaa 14040
aattggtaat atcacacacc tagtacgtgt tcaggttctg ccttttgatg tatactgcaa 14100
agtatgtgag gagtatcatg acggtgttat gggaagtgag gatgatttta ctatccaaac 14160
cgaatgtaat tgtgatattg atgacttctt cacagatgaa gaatacgatg atgtcgtaga 14220
agaacgagac cgttgggaaa tcaaagctga agagcttgaa gaagagcggg acgaagctgt 14280
tgcagacaaa gataagcttg aacaggaata cattcaagag attgaaacac ttcgtgctaa 14340
caacgagcta actctaaaca ctctcgatat tgagcgtgaa gaacaccaag aagccatcga 14400
gttacgtgat agtattattg atgaactgga agctaagctt cgtgaattgg agggttaatg 14460
gaatcatttg tttctggact aggtgaggtc tggtgtactg acatcgaatc aacaggtctc 14520
atcgaggaca tgaagaagca atcaaatcca atgcttcact gttttggagc gattaaccca 14580
gagacgggtg agagacgttt gttctctcgc tctaatggta aactaggctc tgaactacag 14640
gcatttctag atagcggtgt cactctagtt atgcacaacg gaatttgttt cgatggtgaa 14700
gcactcaaat atttgggtta cagtgtggac aagtgccgta ttcttgatac tcttgccctt 14760
agttggtacc ttaatcctag gttaacaagg catggtcttg aatactgggg cgaggtattt 14820
ggagtaccta agccacctgt atatgactgg gttaacgaag acccatctgt atacgaccac 14880
cgagtcatgg aagactgtga gattcagtta agactttggc gtatgcagca acaacaactc 14940
aatgagattt atgatacagc taagagcaaa ggtaagctta ttgattacct catgtttaag 15000
atggaacttc aacgtatcca agctcttaac aaatggaaac tggatattga tggctgtcat 15060
aaacttattg cagaacttga ggcacgtaag gatgaacgat tcgatgcact tcaatgctca 15120
atgccaaaag tacctgttaa aagtaagaaa acaagaccag ctaaaccttt caaaaagaat 15180
ggggatttat cggcgacagg tcttaaatgg aaaactctta ccgaggaaca tgggctacca 15240
ttcgaatatg acggcgttat tgaagtcatt acaggatacg tcgaacccaa tgctggctca 15300
catgtacaaa tcaaagattg gttattctcc ctaggatggg aaccacaaac attcgacttt 15360
aaacgtaata aggaaacagg ggaaacacgg aagattcctc agattagtga tggcaatggc 15420
ggtatctgta agtctgtaca gaagctgatt gagaagtatc ctgacaaggg tatcgacaac 15480
ttagcaggtc taggtattat cagccaccgc ttgagtatcg ttaaaggttt cttaagagag 15540
gtagacgatg acggatacat cgcagcaagg tgtaacgggt tcactaacac gttgcgtctc 15600
aaacatgctg gactggttaa tttaccatcc actcgtgttc catacggtaa ggaaattcgg 15660
ggactactcg gctgtccaga cggataccag aacattggct ctgacttatc gtccttggaa 15720
gaccgttgca aacaccactt ccaatggaaa tacgaccctg agtatgttaa actccagatg 15780
caagaggact atgaccccca cctacaagta tgcttactcg gtgggttgct cacggagaaa 15840
cagattgcag cgcacaaagc aaagcaagaa gaccactcag acacaaggca catcggcaag 15900
ggcggtaact acgcttgtca gtacggtgca ggcatcgaaa cgctgatgcg tcagctaggc 15960
attcctctag aggttgctaa agctgtacac tcaggttatt ggaaactaaa ctggtcaatc 16020
aaaaagattg ctcaatcaac aacggtgaaa catgcaaatg gagaaatgtg gcagtataac 16080
ccagtatcag gtctatggta ttggcttaaa gcagacaagg acagattctc aactctttgt 16140
cagggacttg gtgcatacat ctttgacaag tggtgcgaag ctactggtaa tatcgttaga 16200
gagcgatacg gagcaactat gccgttcgtt gcgcaattcc atgacgaact aatccttatg 16260
gtaaaagagg gtacacttgg tgtatggcat gacattctta aagaagccat caaacgtgta 16320
tcagattcac ttaaactaaa ccgagaacta gactgtgatg ttcaaatcgg cgataactat 16380
tcggaaatcc actaatgtac acagaacctt taaattgtac cgtagttaaa gactattcgg 16440
tagaagactt taaacaaata aaaagtattg aagaatttat tttcagtttt gatggtgtag 16500
agtactacac taaccaagat attgatagtg taggtacact aaaatcttat atagaaaact 16560
atgacttaat acttgtacca tctgggagta ttaacacagc aatttttgta gattatatac 16620
aataactact tgacagtgac gctattttct gttataatag ttgtcctttc acgagaatat 16680
ttaaattaaa gattaagtta tacggagaaa ataacacata tgaatatcgg cattccacaa 16740
atcaacaaac caaacaaagc atttaagaaa atcgtagagc aagctgagcc tgtagaagct 16800
ggcatgtaca aagctgtgtt caaccgattc gtaactctag gtttccaaga gagcgacttt 16860
gaagagggta agattcaatt cattactgtc ttcggctttg agcttgttga agatatgatg 16920
ggtaacaaag tacctaatca ttggaaagtg tttgatgacg gttcacgaga agagggtgct 16980
aaaactctta cacgagaata tccattaaac gaccaactac atcacaagtc taacggcttt 17040
gctattgcta aagctctaga cccacaagtt cctactatcc gtaagggtaa ggaaaacgaa 17100
cacgaataca tcgttgactt caactgggac gctcagttag gtaagacggt gttactacaa 17160
gtcaataaga agaaagctaa ggctggtcac ttctacaacc agattcagtc tgtaatgcca 17220
ctaggtgctc cgcttgatgc tcagctacct acagtattct tcaacctata caaccctgac 17280
atgcaagaag tttgggagaa agaagtgtac ggttgggaga agaaaaagat tacagaacgt 17340
cgtgtaatcc caggtgagcc accagttgta tatggtgatg cccctgcaca agaagaacaa 17400
ccacaagagc agcctaaaga agaagcacct caagctatcg tagatgagga agctgatgcg 17460
gatgacgacc gcccattcta atcctaagac ccaccccttg cgggtgggat tttttgaccc 17520
tgcaaaatga ataaaccaca tacactatta cttgactgtg acatcctacg ttatgagttg 17580
ggtagtatct tatcaaaagg agatacaatc accataggag gtactaaaat tgatgtacca 17640
caatcaagag ccaaagttga ggagcttgtt aaagagaaga ttgattatat tattgagaga 17700
actggatgta cttacgttcg aggctacctc tcacaaggaa agaattttcg atttaaacga 17760
gccactatcc aaccttacaa gggaaatcga aaagactttg ttaaaccata tcactggaaa 17820
acggttggag aggttatccg agattacttg gagaacacaa ttgactgtga gcttttcgaa 17880
gcagatgacc atatggcaaa agaccaagat gtggatgaag ggacaacctg cattggctct 17940
agagacaagg acttacgagg tcgtcatgga tggcactact cgtgggcggt gggtgaaagg 18000
agtcctgaga aaccgcttca atggattgat aagcttgaag ctgatagatg gttctataca 18060
caatgtctta caggagattc caccgacaat attcttggat gtgccgttaa gctacctgac 18120
aagaaaggaa atccaagacg caagggcgtt ggtcctaaag cagcagaaca actattagct 18180
gaagcctcct cagagcaaga gatgtttaac atcgttgctg cacaatacca actacattgt 18240
ggcaacttgt ggaaagaaca cctgcttgag aatggcactt tactatttat gtcctacgaa 18300
cttattccgt gggaagaact agattcaaca aaaaggttat tagaaaacta cgaaagagaa 18360
ttatatgcta gataaatatt tccaagaaga aacacaacgt acagttatta agcgcaatgg 18420
tcgtaaagag cctttctctg aggagaagat taaacgatgg tcaaagtatg cagctaagca 18480
tgacgttaat tggaaagagt tatacggtaa aaccttagat agactgccgc ttgaagttac 18540
tacagaagaa attcatagta caatgattaa ggtctgtcta gacaaagaag aacttaaatg 18600
gtctcgtgta gctgctcgtc ttgagtatgc aacactacgt aagaatatgg cacaggttgg 18660
tatctcagat aaggatagct tcctaactat ctaccacaat atgtgtgaac taggtctatg 18720
ggatgctaag tctttccctg aaccagaccc aatctgggat gaatggtaca aggatttgta 18780
tcctaactac ttcgagttct ggcaaatcaa acaatggaat gacaagtatg cagttaaaat 18840
tgatgataaa gcagttgaaa caccacacat gggagctttg gctatggctt tggctatcca 18900
cggtactact caagaagcgt atgacacagc taaagcggtg gtgtacggtc aactcaatct 18960
cccgactcca gttcttaatg ggtgtcgtaa cggcgacttc gattctatta gctgttgcgt 19020
cattactggc ggcgatactg ttgattctat cggtgttgca gagcatatag cttacaagat 19080
gacggctaag aaagctggta tcggtatcga aatggctaca cggtcattgg gtgaaccagt 19140
taaaggcggt cgtatcaaac acttgggcaa ggttccaatc ttcgcaacgg ttgataaagc 19200
tgttaagatg tttacccaag tatctcgtgg tggctctgct acagtggcta ttgacgtata 19260
tgaccttgag gttgtggatt tactactact taaatctcaa cgtacccctg agaatagacg 19320
actcgacaag ttggactatt ctcttgttta tgatgatgag ttcttagatg cagttatcaa 19380
taacaaagac attactctac gtggtaaggt gtctaatatg actaagaaca tcaatgctcg 19440
taaactattg gctacgttct taactgtacg tcaggaaaca ggtcgtgtat actgcttcaa 19500
taaatcagaa gctaaccgtc acacaccatt cattgacgag attcgattaa gtaacctatg 19560
tcaagagatt tgtctaccaa ctaagccata tgaaaatatg tctgacttgt atggtaatgg 19620
catgtctaaa ggtgagacag cattctgtag cctatcagct attaacgtat ctcgtgttca 19680
tacacatgaa gaatatgaac gcctagctta cttggcagtt aagacagttg ataacctaat 19740
tgaacgtgcc ccaatgatga caaactctat gaagatgtct atcactaaac gtcgttctat 19800
tggtatcggt attactggtc tagctggttg gttacatcgt gagggtcgta cttactctga 19860
tgttaatgca attgaacgcc tagctgagat gcactactac tacctgctta aagcttcaca 19920
aaagcttgta gaggagggtc gtacaccttg tgagggtatc aatgaagatt ggctacctat 19980
tgacaccaag cgcactaaga ctgaaccatt catgaattgg gaagcattgc gaggtaaacc 20040
acgtcgtaac tctgtacttg tagctcatat gcctacagag tctagtgctg tgttctctga 20100
tgcaactaac ggtctatacc cagttcgtac tcgtgttatc aacaagcaat cacgctacgg 20160
taacattcaa tacattgcac caaatgtgat tgagacagct tgggatattg ataaccttac 20220
actagcgaga gcttatgcag cagtacaggc ttacactgac caagctattt ctgctgacta 20280
ttacattaca ccgtcacgat ttgagggcgg caaagtacca atgtctaaac taattaaaga 20340
ctgggttgca caagctcgtc taggtaacaa gacaatgtac tactctaaca ctaacgatta 20400
taatggaggc acattctcat cttcagaaga agagggttgt gaagggggtt gtaaactgta 20460
atgactgtat ttaatgcaaa taacaaaggg tactctgagg ggtacccact tttcctaggc 20520
gaagaactag gtctattcga cacaattaac cgtacatatc ctgagctgga agagctatac 20580
caacagcaaa tggctcaaat ctggaatgag tttgaggtag acttgactca agataagatg 20640
gatatgcaga aagttgaccc taaagttaca gagcttatgg ttaaaactat tagttggcaa 20700
catctagctg actctgtagc ttctaaatct atctctggtt tgttaatgaa atatgttaca 20760
aactctgagt tggagggttt gattaactgt tggagcttgt tcgagactat ccatgctcgt 20820
acatatagcc atatcgtcaa gcagacgttc cagaatcctg ttgaaatgct taaggaaacc 20880
tatgacaacc ttaaagtatt agaccgctct caagcgattg tagaggcctt tgatagacta 20940
gaagcactgc ctaaagattc aacccgtaaa gaggtcatgc aagctatttt aggtgcaata 21000
tttgcactgt ttgcacttga ggctattgca tttatggcaa gcttctcagt tacatttggc 21060
attgcagaga ctggtgtatt ccaaggtatt gcacaagatg ttaagcttat ctgtcgtgat 21120
gaggtaatgc atacacgatt tgattatgct atcttatcta ttctacttaa agaccctgag 21180
tggagtcatg tctacaacag caatattggt gagtacaaac gtatcctaga ctctgttgtt 21240
gagggtgaat tagtatggac agattatcta ttcagtgagg gtcgtcagtg tattggtctt 21300
aatgctaagt tggttaaaca gtacgtattg tatatggcac agcctgtgta tgaagcagca 21360
ggtattacct ttggtttcga agcacctaaa gagaacccac taccatacat ggaaaattac 21420
attgatggtt cacgagttca agtagctcca caagaactac aagttaccag ttacaacatc 21480
ggtgctatct cagatgacac cagtgattta gatgatttag attttgagga tttataatgt 21540
ttttaattgc cgaagaaaag ttttacattg aatttaaaga tggttcaagc atgacaggct 21600
ctagtgctga tggtcacaaa ttaactgagc tggaggatag attaattgtt gaggctggct 21660
ctaagcgtag agtgttcttc aatgatacta tcagctatta tgaaattgaa aagacagacc 21720
tagataagac aggtatggac ttagagattg gtattaaata atgtttgata ttgaagatgg 21780
tgtttgggct aaagtacaag cacattggaa caaatacaag aatgatttgt atgtcagaac 21840
agaactactt aaaaaggtta ttgaattgga tattagggtc aaagacttag aaaagaaatt 21900
aaatgaaacc aaatagacga tggatgcagg gtcagcgaaa ggctgactct aaatttgaac 21960
aggagttaaa agatggggtt cttaaagact ttgatttcca gcacggtaag attccttatg 22020
ctattcccca tacttatcgt cctgattttg ttaaagataa tcttatcatt gaagcaaagg 22080
gaagatttag ggatagcgcc gaagcgagaa aatacttatt tattcgagac gccttggagg 22140
aaaaccaaga actcgtattc cttttctata acccacaaac cccgatgccg tttgccaaga 22200
agcgtaagga cggtactaag cagacacatg ctgagtggag tgaaagacac ggatttagat 22260
ggtttacaaa ggagactgta ggtgaactta ttaaccaagc aaataatggc aaggcagaga 22320
agtaatgcca aagccagagg catagagtgg aaacttactt ttgatgagtg gtgttctctt 22380
tgggaaggta aagctcattt aagaggtagg ggttctgatg atttatgtat gtgtaggtat 22440
ggagatactg gtgcatatga aataggcaat gtctttatag gtactggtct tgacaataga 22500
agagaacagc aaaagcgtag atataaagag tttagagata aacataatat tgaacagttt 22560
attaaagacc gtttaagtgg tcttacttac aaacaattaa gagataagta cggcatttct 22620
gaaggaactg tcggtaattt tataaaggag tttattaatg aatccaacac taacacaacc 22680
actagaccta tcaatcaaag cttataagat ggctcaaatt gaagctcaac gtaatggcac 22740
agactgcggt acatatcctc aatggcgtgc tatgatgatg aagaaagata agtttcgtca 22800
ggaactggta gaggctaata agcctaaaga agaagtgaag cctaagcgta agccacgagc 22860
taagaaagaa aaatctgaat aataaaaagc ccccactcta gttaattcta ggtgggggtt 22920
attttttgtt tattgtttag cctcaagacg actaacacgt tgtaatgtat caatcgtacg 22980
gtcgtctatc ttgtctacct ttttctcgat acgtgttagg atttcactaa atccacccca 23040
ctgtgaagct acggtaaggg ttggaatagt gaaagccgca actaacacta ccgcatattc 23100
cacccatctt gccataaaac acctcctata atgctccctt aaatgccgac atcacaggca 23160
agtcctctaa ttccgcttca ccactaacag ccttattaag caagtttgct tgctggtcgt 23220
agatactaaa cgaaacagga accacaactc gtgaaagctg tcctgtagcc gcatcctgta 23280
gataacccat atggattggc atgagagtat tacggtaagc cttcatgaag atttcctcat 23340
cacttaggtt attacctcga acttcgttct ttaaactctc ctgtaagaag ccaacaaaca 23400
tgatggctcc tgtagtagca ccaaatcgtg caaggtctac agccaattct gcaccactct 23460
tacctcgtgg gtcgattaag tcgtacagct ttggtaggat agtgttagca aacatggttg 23520
gataaccacg aagctgggtg aacatagcac cccatcctgt agaaaaccaa actggtttat 23580
ctagagcctc tgggtttagt gctacgtctc tgttgaaccg tgctaccata gatggtttat 23640
actcatcaat gatattcggg ttcttataca tcgctttagc aatgctctca ctgataccta 23700
aatcatttaa tcgttccaca gcactcatgt aagctcgtga gtttttagga cgactattga 23760
ttgtagttag gtcagtttta agttgtgcat ccattgcatg tacagccata ctgtttacca 23820
agtagttgat agcagtctga cctgtagcat ggaagaatgc attgttaaag ttttgcattg 23880
cagggcttaa gaactcaccc tgaatacggt tgttaagtgc agtagttgca ttcataaaac 23940
tacgaccagt cattaatagc tgacggtttg tttcagattt atctacaata ccaccagtaa 24000
atgtactgat taagtctcgt gatacagaac ctaatgcacc tataagggct ttagtacctg 24060
ccacattgcc tactttaata aacaggttaa aaggttctgt caatgatgac agtgcagtaa 24120
gtggtagagc tactacgtta gagatagaac gagcaacaga agctagtttc ttagctgact 24180
cactctggaa ttgaccatat gtaccttggt aagcatccat agcattgtag aatgtatcta 24240
cttctgctgt agtcatagcc tgagcattac cacgctgaga gtttgcttga gcaataagac 24300
catttaagat ttcattgttc ttgcctaact gctctgcata tgtaatacga tgggctgctg 24360
attgtgcata gtttaaaagc tgctctttaa agtttctaga tacagagtaa tcatttaaaa 24420
tatgctgagg aatatgagcc agttgtcgtg agttttctag ttgaccgtac ttaagaggag 24480
aggaagtctc tactctattt ggagctgtaa tattaccatt ctcatcaata cttacacgag 24540
gcatcttagc agcttgtgta tcctgcttct ctaaagctac tttaaatgac tttagttttt 24600
gaaggttttc ctctggagtt actaaaggtt ttacattacc ctcattatct acagttgttt 24660
tatctacctg ttcattatac caacgagtat attgattttg caacccatca atatcaaatg 24720
tttcagggtc cataatagta ggcaggtagt tgttaatata acctgtatct aagttggcac 24780
cttgcttact attagcacgg gcatgaatgt cacccaatac agaatctaac tctctaagtt 24840
cagccgtatc tcgacgtccc tctgcgtaat ccttacgtag tttgtcagca tctttaactg 24900
aagcaaacaa ggtattaata cggttggtta ggtctgctgc cataacctca cgttcaacac 24960
gcatagtctc aatacgttta ttaagttctt gacctgcctc tgttccctct aagaaacgtc 25020
gtacaggacg taatgcagta cccatagtga agttaagggc tgattgcatg aagccatctg 25080
cacctgcaat atcacctgtc tttactgcat ggtcactagc catctctgct agctcagctt 25140
gtttagcttt atctacagtt gcctgagcca cacgtaaagg agctgataca agcataccac 25200
ctacgatagc ctcatcaata tcctctgtgg aaatctcacc agttacaccg tagttacgta 25260
atacttgttg ggcaccatta gtaccaccat caacagcgaa gttcttagct aagtctgtaa 25320
gtacattctt cttagcttct tctgtcaggt tagttttatc tagtttagcc aacataccag 25380
tcatgccttt agctagtgga atatttaata gtgattcagc accagccgta accattgcac 25440
gacctacatc aatttcctct cctgcttcta cttgttggtt aactgtatca ccaaagtatg 25500
aagctgcatt agcgacaaga cctagcttac caaagaacat tgctgccact tctggagcac 25560
ctgtagctag accctcacca atataatcca tcaattgacc aaggctttta atatctttgt 25620
atgaagctac tttagggtta aacttacgcc actctgcata cttctcttct gagtattgtg 25680
caaggtcact agctgtttca tctagaccaa gcttacctgc tagtacagaa gcagcacgag 25740
attgagaagc cagtgcctta tcgacaccag cctcaaaagc cccaacacgt tctactcttg 25800
gctcaaagcc taaatcaact tcattagtag tagcaccaaa atttacgtta gtatctagtg 25860
ggctgccctc accaaacaat tgtggcgtat tgccaagatt atcactcatt aaaactcctt 25920
aatcaactaa tgtggttttg ccaaagaaca taggttcatc atatgctcct gaagcaacac 25980
cttgtcgaat cgcattctta atcgtagtcg gtgtaacagg ttgtttgatt gacttcaaac 26040
ggataatagc atcttctact acagactgtg caaccttgtt atctgctgca tatgacttag 26100
caatagagcg gtcactggtt gatagttcca atggtttatc ctgactgtct agcaatgaag 26160
cttgctgacg ttgacgatta gttaatgcgt tatactctct cacattttgt tctgctgccc 26220
acttagcatc ttcacgagct tgcttagcgg ctgctgcttt ttgttgttgt cgtgcttgta 26280
caccacctgc tagaccttta agtacagcca cacctacatt gttagtttgt gtagcagctt 26340
gagcacctac ggctagcatg gtaatgaagt ctaccttgtc atttagaaaa gaggcacctt 26400
ccgcaacagc atcgccagca ctaccaagta caccagtaac acctttggca tagccacctg 26460
ttgcttcgtc gaatgcatcc caagaattac gtagatactc cagaggtaca ccaagcaagt 26520
cagccgtacg ttgccacgca ttcttacctt cctccgtatt tgggtcagac ttttcaagaa 26580
tcatagctgt cttctgagca tctgtatatt cttcttgtgc ctgtttaaca gcttgctgtt 26640
gttcttcctt agtcaactta ggagttggtg cagctaatac agtacctaca ccctcagtgg 26700
atgtatcagg agctgtatca ccgaagaaac ctttagcaaa cttaatgcca tctttaattg 26760
cagggacgat agcttcccca ccctctttaa ctaatttctt agttgtctcc aaactaaagt 26820
cctcaatacc tgctgcatta agtgcttcct tagtttgttc tttagtttct gtaataaggg 26880
tagaagcttt actagcagcc tcagttactg gctgactacg cttcaaatac tctttctctt 26940
gctctgcggc tcgtgcagca gcagcatcgt aagtactacg aataccctca tctgcttgtt 27000
gtaggagacc agtaatggct ttatcagcag cttcattagt ctttacaggc ttacgttctg 27060
ctgtctgagc tagtaatgcc caagggttag acttgcttgc ctcctgtggg atgaattgtg 27120
ccattaaatt aaacctccat atgttttctc tggtgtaggt gcttcattag tttgatatga 27180
acgggcacct ctcaatggac tctgtaattg catttgtggt ggttgacctt gtaggttctg 27240
tagtaagttc ataccttggt taatgttatt aaggtaatca ccaccaccac ttagaaagcc 27300
tgtagaagct cctgagctgg ctgctggagt taatgaagta gtgctagcag cacctgctcc 27360
aagacctcct gaagttgctg tagcgcctcc tgcgggtgca gacatagaac ccatagaagc 27420
aaatgcccct gagcctagac cacctgaacc aacagcagca ccggaaccaa atccaccacc 27480
tgctgcggca ccaccaccgg caccacctcc agctccagcg gctagggcac cgccacccaa 27540
aaccgccccg aagatactgc ccacaatggc atcagagttc tttacaattt tattgtcttt 27600
atttaaacct aaagagcctt gcatctcagt cattgcatct tggtggttac caaatgcttc 27660
atcagcatca ccactaaata catccttgac aaagttgaaa tcagcttcca tctgggtgat 27720
gggcatcatt gcaatctctc ctaggtcgaa attatcgaaa tcgaagatgt tactgaagaa 27780
gctcattaca atagacctcc actacctgct gctgtacctg tggttgtagc tgtttctgta 27840
ccacccatgt tgcctgaaat aacatcttta taagcaagca aggattgcat gtagctgtta 27900
gctcggctag cttgatattc agacagacct tgatagccac tcattaagtt acctagacct 27960
tgagcacctg ccatacgacg ctgaatgtta ttctgttcag cctcataagc gagctgtgcg 28020
ttcgtctgag cgatttgagc atttaggtca cccattgcta agccttctgc aattgcactg 28080
cgtgagcctg cccctgcgcc tgcctgatta gatgtttgct gaatacccgg aagtacatta 28140
cgttggaagt tagtagtagc tgcctgagca gaagcatcca tagcattgtt gagagcatta 28200
cggtcaacat tactattcat catttccatg tacatgtttt ggctagcacc taggatgtct 28260
tccatctgtc cggtataatc tgaacctaac tgaaagccac cttggttaag ccatgcatca 28320
gcacgattaa gtagttcctc gtagttacca gattggacgg gcttccatac tgaggtacta 28380
tcttgggttt gggattgttt ttggtcaccg cccattataa ctccttataa atagttgtgc 28440
agtaatggtc gtagtcttta tgaattttac gccatgcatc tctgccttta ataatgataa 28500
tatcaatgcc agataatttg gcttcatatt caatctggtt tagtgcttct ttgaagtcac 28560
catcaaattc tcctgctaat gtagctgtat ccagttctaa gattaactgt cgttcatagt 28620
tacgtcttgt tttacctaca attagagcgt tattataacc ccataaaatg taagtaccat 28680
tttgaagtaa ctggacatat tcatctttat ctgtttctag agctggagaa ttatcatata 28740
atctgtctac taactggtaa gctagttcat ctgtaataag ttctttagga atgtatgtaa 28800
gcataagtaa taattattaa taaggacgct taaggaaggt aagtaataag gaaaggaaat 28860
aaataactaa taattaatta tcagtcctta tataaggata ccatcaaaaa gcgtcattgt 28920
caagtattaa atcaaataaa tttgaaatta gttgataata ctgatacctg agttgacttt 28980
actaacctct acgttcactg tactgttagg aaccttgaat gttacaaagt tagatacagg 29040
gaatgactga ccagttgcat gtaggaactg tgctttaagg tcaattgtta agttacctgt 29100
tgcagttgta gcaggaatga atacttccca agctagtgtg ttagttagtt taaaccatgc 29160
ttggttagcg ttctgtgcta caaactcaaa tgcagatgca gcattacctg tagttgaata 29220
ccctgcccca gattcagtaa cagatgttac acggtagtgg taattaaggg tagagcctgt 29280
actgttagca gcagtccata caataccaaa cctatcaacg ttagtacgtg atgtagcaat 29340
caacagcgat gggatacatg taacaacagc cacacggtca tagttctgac gtggaatagt 29400
tacacgagcc ataatagccg taggagtagc tgcaccatta tcaacaaacc attcagacca 29460
atcaggtgta gagctagtaa gtgaactatc tacagcacca ccaacagcac cagtaatgcc 29520
ttggtaagta acctcaccac caaagtaacc attgttagca tataagtcac cagtaggtgt 29580
tactcggaag ttagccgagg ttgatgttgt attacccacc caaatgcggc tgttagagtc 29640
gctagcgtct agaacaacta catcttgacc tgatgtacca ccaatagtga ttcgtgtccc 29700
tgagttaatc tcagtgcctg taatcgtacc tgctgcaatc tggttagctg taatagtatt 29760
agatgcaatc ttactagcag ttactgcacc tgcttgaatc ttattagcag tgatagcatt 29820
ggcagcaatc tgagcagcag tgatagtgtt agctaccaat ttattagctg cgattgtacc 29880
gttgatgatt gcatcaccat ctacaaacag ttctacaggt tgccatgtac ttgatgaacc 29940
agtaccacct gtatagtagc gttgttctga gaactggttt acagagttaa ataatgtaac 30000
agtatcccaa taacgaacta caccatcaga gttagggtca ttgacaatag cattatatgc 30060
ctctaaattg ctccatgagc ttcctgagat agccttagat acattaaggg taccacgggt 30120
accatctgct ccatcctgac catcttgacc cggtggacct tctggaccga taggaccaac 30180
ttcaccttgt tcaccatcct taccagtaac tttgataggg aatgaataag ggtactgagg 30240
caagttacca tcctcttgta ccagaatcca cgtcatccaa actggtagtg ttgggttgtt 30300
aggtgttgtt tctgaccaac ctgttggtgg gtaagtatta cccggaactt gtggttgtgt 30360
agcagcctct ttaaaaaact gcttagtaat cttaccatca atgccatctt gtccatcttc 30420
acctgaaatc ttaacaggga aagaccaagg atatacaggg tcagtgccat cctcacgaac 30480
cttgataaaa gtcatccata caggtagagt aggattctct ggtggagtct tagaccaagg 30540
agatggggct ggataaacat tactaggtac tgatggagtt acagaagctt ctctaaacca 30600
ctgctcaaag atacgaccat ctaaacccgg agcaccatcc tgtccgtctg caccatcttg 30660
tccatcctca acacctgcac taaatggagt tggttgagac catgtatgag tgtctgtatt 30720
ctcatcgtaa gctgctgaca tagtaaccca aatccattca gacttgtcat tcttagcagg 30780
agccacttca gaccaaccgc ttggtggagc agatgatggt gatggagttg atggaggtgt 30840
accatcagca gtaagacggt aagcaaatac tgcaccagta atacttgatg ggtcatcaat 30900
gattttccac catctctcgc gtctgtaaag ccacacaccg gctgaagtga taatcttctg 30960
acctttgtat tgacctacat tataaacgtc gtctagaggc tctgtgaagt acatcatcat 31020
accctcacgc caacgtactg gcatcttacc ttgaggtggg aatgccccac cctccaagat 31080
accattaaca ataccaccta acgccaagag ttcatcattg atatatgttg gcattgactt 31140
ctcttctgta ggtgttggac cagcttggta gtgacctgag cttggacctg aaccaatcat 31200
atctgctcta cttgacatta tctagctcct gttgctttat attctaacca atagccacgc 31260
aattctgcac gactcttttc tgggatttcg aacttaacat gaatgaactt acctgttgct 31320
gtacaaccta ggttagctcg tttgttaggg tcaaacctac caacatatct ccaagctgga 31380
gactgtgtag ttgtctcact accagctacc cacacatcaa cttcaccacc ccgaacgaga 31440
ggccaaatct tattgagtgt tttcgtagtt gtatagtctt ccatgtcaat ccatttcttc 31500
tctacactag ctttaaagtt agcaccatcc caagtaggct catcaatagt tgtatataca 31560
gcacccttac catctgaggt aacaccagcg aacgctaagc cccactgtga gctgttatac 31620
ttaaagtatg aactaatcca tgtatcagtg gcttcttccc atgaagcgct ttctgatggc 31680
gctgtccatg taatatcacc ctctgtaata gataggatag tataagtaga agtcaatgaa 31740
tctggcatat ctcgtttaga ccagtgacgt tcttcccagt tatatacgta tgctacgtta 31800
caacgaccat taacatttgt tgatgcttct ggaatacata cccatgcctc tttctcttga 31860
taacgtacag ctacgaatac cttgtcgata tggtctgggt caatagagtc aaagattctg 31920
ttacgtacaa taccgtctgc tacagattct ttatgtacac cattatgcat atagatgtct 31980
gattgaccaa ccacaaaatg gtagccatca atctcagcca cacaatctgg accaagtaca 32040
cctacgtcgt taaacaattc cttaaaggcg aatactaaga taccgccaat gtatgacatc 32100
tgccatactg ctcgttcacg gtaaatgatg aacgactctc gaagagattt acctccaaga 32160
atcggtccac cattaccgcc tagaaagttc caaccggact ctgtagttgg gtctgcatct 32220
gcccaagaaa cacttaagtc ttttgtgttg atagcactag accaccatac agcagagcgg 32280
tgacgtccct cgttccaagg actgaactta gcttggtatt cttcttctgg cgtttgtggg 32340
tcagccggag gtgtgaagtt atctaccttg aagtccattg ccaccatgtg attgccgtag 32400
gttcgcataa cctgagcctt aacatgtccg gggaatgttt ccagttcttc aaatttactt 32460
actccatccc acacgtaagg cttacctttc atgaagtatg gaacaccatt gattgtatca 32520
ccaaaccaac ggaactcagg ttcatctggg atacctgctg ttggtgtaac atcttggtgt 32580
aagtctttag tgacgatacg gtagatttca ccagtaccat cttgtttggt tgaggcataa 32640
gcccaccagt agttctcatc agtatcatca cgcaatggga ggataatttc aggatatacc 32700
gaaccatcat taagaacacc agtctgacct aagccttgtt cataaccaga taccttctta 32760
gtcttaccat gcttaaacga gacttcatta gcctcgtccc acatagtaga atctaattgt 32820
gatggctcta gtgaaaagtt gacacctttt ggtttctcaa ctttataaac ttggtatgcc 32880
atttatcctc cttatgctgc ttgaccaacg tatacaatga atgctagtac aacgtactct 32940
ggtcggttat caaatgttgg tacgccttcg ttatcaactt ctgctgtcag tccgtgactg 33000
tgtggttgac caccaccaac ttcttcagtc tcagcaccag aaccaccttt aacatctgga 33060
ccagtaccac caccaccaga acctgttgca ttataatcta gtgggtgttt gtgagctgga 33120
atctctgcta gaacaagagt atgttcttct gaaaccaagt gtgttgatag gtcagggttg 33180
ttctcaccac cagttgaacg gttaggttga gctgtatctt taaggtcgtt ataacccatg 33240
ataaacttat cacgtaagtc tacagtctgg aaaccattct gaatagtacc atcagatagc 33300
acccaaccaa ctggaatgtc ctcaataaga ccttggaaca tagtagtctc accaatgaaa 33360
cgaccttgtg taccatcagt cattttgttt agttcagcta ctgtagcagt aacctcacca 33420
tcaatatttg gaaatgattc ctgcaccatt cgctttacag ctttaatctg gtcagcaccc 33480
tcatagcgag ggtctgtagg tgcagggatg ttaatgttca ttccagcaat gtagttacca 33540
ttatcaattg ccattattta cctccttgtg gaatcatacg acctagagcc gtaaagaaac 33600
cagcagcagc catgataaga ccaatctgcc attgctgtag gtatgcctca aagtaaggca 33660
ttacactgat tacaacgccc gctagggacg tcgcaatact cagttgaatg gaatatttct 33720
ttagccaatt catgttattc tcctaaacct aggtgtgctt caatagctgc taggcggtct 33780
ttaagttgtt tgttctcttc agatagttcc tgaatagcct taaatgcaat agcatggaag 33840
tcattaccct tgcgagagat gttcttcata ccgtcctcac cctctatgat aaggtgtggt 33900
agcactgtct ctacttcttg tgcaatgaaa ccatattcaa actgcttaac tacttcatct 33960
tcttcagttt cgtcattacg ctccagtaca tctttttggt atgtatatac aggcaagctg 34020
ttaacaacac tcaatgcatc ttccaccgga ttaacttcag acttgaggcg ttcatcagaa 34080
gcaacactat ctacgtatgc tttacgagca gccatgttag aggcagtagg gttagtaccc 34140
ggtaagtata ccggaactct agcgataacg tttgtggagt taaacagaat ccactggttt 34200
cctgtatcag ataagttctg agaagcaccc atagttagtg cagttccagt tctaccgaag 34260
tataaagggc tacctgtatt agagttaaaa cggatgttta cgttagcatc accgtctgaa 34320
cgatacaggt ctagtttagt agcacctgta gattctactc tcaattcttc tgtattgaag 34380
atagctttgg tacggtcacg gtaaatactt aatgcacgtc ttactacgtt attgtcacga 34440
atacttacgt aatactcgtt gtttactgaa tccgcagcat aacccatctc cgcaccataa 34500
gagccattag actcaaaaca acggaaagct gtgtttacgt ttggctggtc ggcatacaaa 34560
tccatgtaca tagggcttgt accgtagtaa gccatacgca tttggttagg ttgcaagtaa 34620
gtaaagttta catttccgcc ttcagggtag aaacgcatgg tattaccacc ctcaccaaga 34680
gcctgaatct tagcaccatc atcaccaatc tggaaagcat tatatgcaaa attagagttc 34740
gtacctgtaa gtttaagagc tggactaatt gaatgacccc aagagccacc tgctgcaccc 34800
gatgcttggt atttgtcaga aatacttgta ccacttaaga tgattgtatc accagtacct 34860
gcatttagct gtacgttacc atcagaacgt agtacaatat taccattacc attgtcgtta 34920
aaccaagtca tgttctcatt agcagaagaa ccaaaaccaa taaaaccacg acgtgtggcg 34980
tcatcatgag tccattggaa atatgaactt gaaccggtac ctgatgatac accatcttgg 35040
aatgtatatg cagcaccacc tgagtgttca tgtataatta ggccattttc ttgcactacc 35100
aaacggcgag acgggtcaga taaacctcta ggtcgaatct ctacattgtc accattacca 35160
gacaggaatg tgcgaccatc tgcataagcg aacacaccat gttgtgcgcc tgcacgggat 35220
agaccaaaat tattagtacc ctcatactct actagacctc ctggttggaa gctaaagtag 35280
tctgtatcag aagctacatc cataccattt ggacgtactt gaacacctgc cacagagcca 35340
gagccaccaa tacgagtaga acctccatta aacattacaa aaccttgttc tccaccaaca 35400
gtctctcgga ttgtggcagt gatgatatta gttgtaaatg tcttagcacc actaatggtt 35460
tggtctccag tagtcttaac tacttcacca tctaatgtat caatttggtt ttgaagtcca 35520
gacacgagag tatcggtttg tgtagtagta tatgcaccaa cgtcttgagc agtaggttta 35580
tttgctgtgg tgtaaaaata aacccaatca ttaaacacgc cactataacg agtacgatat 35640
gcaacattac cattattacc tgacacacca taaggccaag caacttgggt aatattgcct 35700
gtagagccgt atgtatgtac ttgcgggtaa tggtaccctg agccaccagt tggtttgtta 35760
gcagcagtag taccagataa aacattataa aaacctgcat cctcaagggt atcccaatct 35820
acacctgtac tacctccagt aactttagct gctgccttaa aggtattacc catagggata 35880
ccaacattag caaaagtagg tcggttatca atctgagatt gtaatgatgg tacaatagct 35940
gctgcatcat cagcactttg ttgtgcatct gtagcactct gtgcagcgtt agcttctgaa 36000
gtagctgcat tgctagcact tgttgctgca ttagactcgc tagtggctgc attacttgct 36060
gaagtagctg ctgcattttc acttgccaat gcattagcct cacttgtagc agcagcagat 36120
tcactgttag ctgcatttgt agcagatact tgagcactat cgcttgcagc tacagcagtg 36180
ttagccgctt gtgttgctgt ctgttcaggc acatcccatg tagcaccatt ccagaaacgt 36240
aggtcatgtg ttgtagagtt ccagtatgta gcacctactt gtagtgggtt gccatcatta 36300
tcagttgtag ggtcaactgg cttagcacca aggtattggt cgttaaagtt atcaaaagca 36360
gcttcagcat cagcagcaaa accagcagcg ataacagcag agttagctgc gctagtctca 36420
gagctagctg cattagattc tgattgagca gccgcagcag cgctgttagc agcattggta 36480
gcttgtacta ctgtatcatt gtaaacttgt acagtatcgt tatagacttg gtctgtttgg 36540
tcattgataa cctgagtctg gtcacgaata acttcagcct catctcgata ctgcttagaa 36600
tcttggctat attcgtaaga cttggctgcg gcttcttctg ccttagcaac caaatcctca 36660
atgtaagctg gacgaacagc aacgccatag aagcccttta ggttatctcg ttgagatacg 36720
ttagggtcat tctgaaattg tgtttggttt gtaaagaaac cgttttctgt agccatttat 36780
ttaactccct agtggaactt cccacaaacc accttggtca tcagctcgaa tactccatgc 36840
ttgaatctca gcaatagatg tttccatacg tgagttccag aaagctgctg tctcataatc 36900
acgtacaaac tcataagcat gagataaagc accatacaag taaacttcag gagctacctg 36960
taatagccag tttgtgtcgt catcaaattg tagtgtagtt ggtagagcaa agtagtgtac 37020
ttcaacttta ccgtacttat caaatgtacc agtaaagcca cctacaatat cttcactata 37080
agctttgaga gggaacactt tgtaaagaga accttcacga gcaaacttct gtggaacacc 37140
cccaatgcca ctggtaaagt atttatctac cgcatcttta aattcgtata ctgaaccctt 37200
cttcaaagag tgtctacgta ggactacttc ttcgtcagga ttactttctt taggttctgt 37260
caccagatag tctatagaga taagctctac taaatctgtt ggaatctgta cagaaccatc 37320
tgcatataag tcaacaatct gggttcgttc catcgtagga actcgtagct gtcgagcagc 37380
tttcaatgta actaatcgga taaagtcttt agcacgagga cgaatatcct cacggtcaat 37440
ccaatcaatt acagcatctt ctagttcttt aaaagtgtca tacatgtttt acaccatttt 37500
gcctttccaa acacgcaggt tagcccactc aggactctgt agtttcttcc agaacttagc 37560
tgtcatatca gggtcaacac ccacaagagt catatcaata ccttcttgtt gccactggaa 37620
gataacattc ataggaatac gtgcagctag tttcatatcc ttaccgcctt gactctcttt 37680
ctgaagttcc ttgttagtgt cgataatgtg gtcaactttc tgtacgtgtt taagttctaa 37740
tgaaccatct gcttgttctt tatattctgt acggtaatct tttttcattg tcatttctat 37800
actctccaaa taaaaatagc ccctcccata aagagagagg cttcatcgtt agttattaag 37860
ctactgggac agaagcgaat gggttacctt tcgcaaagtc gataatcata ccagagctta 37920
gttcgttgtt acaacgtaga ccccactcaa cgttaagtgc cttacgcatg tggtcaccag 37980
tcttaggcaa gtcgtaggtg ttgaagctac gtaggaagtc gatagaccac tgtgaagtgt 38040
ctagcgctag tgccatacca ccttgtgtca agtcaccgcc gatgtaacga gaaggcatga 38100
tacgtagagt accaaagtcc gagatgtaca catcaacaac tgactgaaca gtcttgtcct 38160
tagctgattg ctgtagctga ccagtcgctc gacctacgaa agtattcgag attttggtct 38220
tagttttagg gtcaacaatc agtgtgtttg gtgaacgacc tgactccata tagatgccag 38280
tcaataggtc ttgtaggtga gtctcagtaa gctctgctgt agaagcagat gctggctctt 38340
gccatacacc acgaccatta gcaacacctg tcactggagc tggagtatta ccagtcagtg 38400
tagaagtgtt ggtgtcatct acttggataa cgttcttaga aatccagcta gatacagatg 38460
gagtcttacg agctactgca acgttaccac cgttatcagt gtactggtgg tgaccacctg 38520
caagctgcat ctccatatcc atcttcagtt tcttagaaac cttagccatt tggtaagcaa 38580
gctctgagcc acgaccagct ttcttgattg cttcatcagt accagtgata ccgaaaccac 38640
gctcgttaat ctgcatgatt gagcttagtc ggtcagtaga ctgttgctct tgttgagtaa 38700
agtcatgacc ttctggtacg atggtatctg tcgcatgacc tttaccaatt tcatctacct 38760
gccaatccca tacgatgtta tgtgctgaac cacgaccaat cgagttcatt actggggttt 38820
caaacgggtc aacgttgtag attacgtccg ctaggtcttc acgtttaccg actgatactg 38880
tactgttaaa atctgccata attaattatt cctcttatag aaacgctgca aaagcgtcaa 38940
ctgaatcttt gatattacct gttttacgaa gacgtgccat tgactcctga tatttcttct 39000
gagatttatc ttgagtctta cctgtagcac tctttcctga tttgactact ttaggaactt 39060
tacgcttcaa tttcttctct actactttag tctgagaagc tttgtagtcg tcatattcct 39120
tagccttttg gagtagcttg ataacaatag catgtttaag aaggtcttta tcagaatctt 39180
caaaacctag cttttgtgtt tgttcttgta gtccctgcac caacttgtcg tgctcaggtc 39240
cacgccaccc ctcaacaatc tctgctgctc gtttttcctg agctgctaga tagtctgcct 39300
ctttcaagcc tttctctgag gttgattgct caatagcttg tttacgggct tccatcttgg 39360
attgtaacgc tgtacgtgcc tcattcaatt cgtactgaag aattaggaat tgatttgggt 39420
cttcctgttt aagtttattc cagtcaacct cattatactt ctctagttct ttagattctt 39480
cttgaatgag ttggtcaaga gcggttacat atttagtacg ttcagcttct agctcagcat 39540
aatgctcttg tagacgctta cgttcttctg caacttcttg agttttacgt gtatagtctt 39600
gctgacgttg ataaccttga accagttcat caccagttac ttcgatttcc tctccatcaa 39660
ctttgaccaa gaagacttcg ctatcggtag tttcatcttc agattcgtcg gcttcctctt 39720
catcggattc ttcttcggat tcttcatcct catcttcctc ctcagagacc tcttcctcct 39780
ctgattcgtc ctcttctagc tcctcttcaa tttcagtttc atcctcttcg gattccactt 39840
cttgttcgac ttcttcctct tcaatgccta gcatttcgtt agccatcgac tggaatacgt 39900
cgtgctgaga tgccgctgac aaatcgagtc cttcatcaag gttgttcggt ccgtgcccaa 39960
taacaggcgt agcgtttgta ctcataaatc ttactctcct ttatattcgc taagtttgat 40020
ttccaattgg gtgatgatag catcaaccgc cttagcttct cgtttgtatt gaagggagac 40080
ttcatcagcc tccgcaacca ttgagcggat taatgcatcc gcccttaaat catccattgc 40140
ttcttgaaag accttactgt ccagtattgc ctgaatctgt ttggcctttg tttcgtcctt 40200
tgtagagctt tccatctccg attgagacag ctttctctgt ttccttagta aggacgactt 40260
cagcgatgtt aacttgttta gcccactcaa acttttctcg ctccatgtct tgaatgtctt 40320
cttgaagtgc ttgctcacgt tcttgcaacg caatctcacg ttctcgaagt tcaagttctt 40380
taagcttaat tcgactttcc acatcattct tcttcgcttc ctgttgtacc ttaagctggt 40440
caacttgcga atcagactgc ttcttcatag cgtctgaatt agccttaatc tcttcaggtg 40500
taggtttagc tgcttcctct gcacgagctt tcttagtagc taaagcttct tcggtagttg 40560
ggtctaacca gaactcctct acatctgtat aaccagcgtt ttcagtaatc ttagttaata 40620
gattgtaaat cttctgttca ttggttaaga tacctagacc accattaccc acaatgcttt 40680
gtgtcaattc aaacatacgc atcagatgca taagttgttg gtctttatcc ttagtaccgt 40740
tacctacaag aactttcata tccacttggt cacgccaagt agctgggtca attgttacgt 40800
acttaccgtt aagacggaag ctcatctgct catcttggtg tttcattgca agttgcttaa 40860
tagcaataaa caaatcacga acacctgtct cagcaaagat acgagctact aatttcaatc 40920
gtttctcggc ggtagacata actcggttaa cttgaccaga gccagtatga gagtttagaa 40980
ccttgtcatc aagaccttgt gtacgaggag aaacaccagt acggtttgtc ttaatctcat 41040
cgtagtaagc aaccatatcg taagtacctt taggtaactc aggctgtact agaggtcgta 41100
atgcaccttg catcttctca cgaacgacac cacctagctt gttgttttgc atatcagcta 41160
agttaacctg accttcaacg gcagcataac gaccattgtt aatcaagtac atgttgtcta 41220
gttggttacg aagaagtgct gtcttaactt cttgaacatc atgaaccatg tcataaatag 41280
attgaccgta gaactgatgt tgaatagggt taggagttaa tgaagaaaaa ggtacaaagt 41340
cccagtcatc atcactcaga atcgtattac cacacatgat gattcgacgt agctcattaa 41400
aaccatcgcc atccacatcc atacgaacat acgcttcaat aatccaatac ttgtcgttca 41460
ttgggtcatt accgtaatcg tcataatcgt gtgtctgagc accgttatac atctcacgga 41520
ttagtcgtag ttcagaatca gatgggtcat cttcatcgta gttatttaat tcttcaattt 41580
gttcggctgt ataacccatt tcatataggt cagccttagt aaccttacgt tgatgaccaa 41640
caaatgtagc gtctttaaca cacttagcat ttcggtcaat caaaaactct tctggcggaa 41700
caggttctac tttgatgtaa cgtttagttt cttctacaga aatcttaata tcaaacgtac 41760
catcttcatt ctcggcaatc tctagtagct cagcaccatc ttttgcaaga actgctgtaa 41820
caccatcatt agataagcct gtgtagtatt ctgctgtttg gttagtttct tccgtccagt 41880
aatgcttaac aataccacgc ttaaacatta gagcatcttt aaaccattga tgcataatca 41940
tgaaaccttc gttcaggtta aagaatgtgt agttagcgta gttactcata tgttctgcac 42000
catctacatg ggcaggagta cgagcttcat attgcacagc attgtaatct gttgcaaaga 42060
tttccattaa atctggcatc atccagtcaa tggcatctga gacatctcgt gagattactt 42120
gagaacgacc ttcaacttca ttgcccatct tctcgccgta gtagtagcgt tgagcttgtt 42180
tccagttctc ttgtagtttc tcgtcttgat aatcacgagc atctcgaata tatttacgaa 42240
ggtaatgaac tttcttatcc ttctccatta ccttcaattc ttcttgtttt ggcattaaac 42300
tatacctcta ttatcctgta caaagtattc tgtgtatggg tctccaccag aacctacaac 42360
tgcgtatttc tctcgtgaca tgattgcata tcgtgaagca gagattgcat catccttaat 42420
aggaacaatc ttaccatctt gacggtggta ctcacggaac tcttgaagcc agttacgaca 42480
ggttgagaac actttcaagc gtccttgttc catcatctga tacatctcca agataccgac 42540
ttcaacacta ttactacctt tacctgttga tttctcaccc aaggcaatag ggttcttaaa 42600
gtgctctgct gtcatgttca caccttgatt tcggtattgt tgagctagtt ggatacctga 42660
acctttatca tgttgatagc catcgtgagg ccaaacaact ggaatccaac gaggtcgaga 42720
gttaattgct gctgcatgaa caactgctgt ttgtttgttc tgataatgtt catcgtaaat 42780
gtaaacagta tctgactctc tatcccatgc tacccagata gcagcagtag ggtggtcata 42840
accaaagtct aaaccacaaa tacgtggata atggtctggt aaaacaaaag gttctaccac 42900
aatttggtct tcagggaatg ggaatacctg accagaaccg aataccggaa tacctcttgc 42960
acgcatctct cgttcgtgtg gagagtaaac tgcaagtagc tgttgcttgg tgtgctcatc 43020
aatgtgtgga gcatcatccc aagaagcttg aatcaggaac tgacccggtt taagttcgta 43080
cataaatgca cgtaccgttt ctgtcatacc attctctggt gtaaatgtca ttgacacaat 43140
accttttgta gtagctgtac gtgtaataca ctgtgtaaag atattgtgtg gtggttcctc 43200
atctagccaa ataaagtcta gtgctgaacc catgaatttg tgttcgccca tttcatagga 43260
cttaaagccc aatcgagaga cgccacccga cacatgtctg atagtggcgc tctggagagc 43320
atttgggaca cctggtaatc gtgtggtgtc aataatatct tcagcaggaa ttgctcctgt 43380
acctagtgca gtagggtcat ctggttgtcc tagcaactct agctgtagaa tgtcacgggt 43440
tgttacgtta gatacgccac atgcccatgc attaattggt ctatcaaagc gatgtccttc 43500
ccaatcctct gggtatttac cagttaggtg atatgccatt tcagcagcac ctgtaaaggt 43560
tttaccgata cggttagcac acatcgctag tttctgtctg ttgtctttgg aagcgtacat 43620
aaactttttc tgccaatcat atggttcaaa gaacttaagt ttattctttc gtttccaatc 43680
ttcttcagca gcaattagtt tctttaaacg ttctaaatca tccatatact ctcccaagtt 43740
ttaattaagc aaaagtttcc aatgaaatct tgctatccga agcaaccgat actttaaaat 43800
taacagcagg agtttcggga actaggatga tgtcgtctgt gattaccgga ctaggattcc 43860
aaatagcacc tcccggtttt tcattacctg attctaaacc ctgtagaatg aagtaattac 43920
ctaatgcacg ctgagcagca atatcttctg gtaggtatcc gataaagaca agcaagtctc 43980
caatattatc gttagttacc tgtaagaagt tacgatttgg tgaagcccaa acacgagcat 44040
attcaccatc accaatgtta ccacctttag tgacaacacc ctgtactcta tttaagttaa 44100
agcccattat gtatctgctc ctttatcttg tttctggttc tgatgtgtca taggagctga 44160
catccaaaac gcccgtttct tattacgctg tacgtaattc tgattatttc ccaatcgctg 44220
gggtcttact cgctgtacca tttgttctcc atgaacccat gttgtcagta gagccaattg 44280
ccggagcgga taatactcgc ttcatcggga atgcacactc gtcacatggt cgtgtatgaa 44340
cgtcatcttc tacaatcttt attgtggaga atccacagtt atcgcataag aagtctcttg 44400
attttctcat aaagataatc tccctgcctc agacgcttgg tgtctaccac cttctggtct 44460
gtagcttaat agttcgtcac ccttcatatc tcggtagata attgttccat cttttagctg 44520
ttctacctca gagatagggt tgtctggatt tgctaagtta taagatagtg ctcttcgttt 44580
agccaatccc ttaacagact gtccaccaac gtttgctgtg tctaacagtt gtcttgatac 44640
ttcgtcatac tcgccttcca taagagcttt gtttagctta ggaaagtaac tattattaag 44700
tacctgttca cccatattat atgatgcatc tatcaacatt gcctgaagtt caggtggtgc 44760
ttcgttgaag cccttacgca taccccactg acgattaagc agtgctaggt actcccgtgc 44820
tacctcttca tcaggcatat catctggtgc attaactgct tggcgagcta ccgaagtaac 44880
accaagatta ccagttggaa tgtcagtaac attatcacca ctaccctcta atacggctag 44940
atggttaaat actacctctg ttgggtcaaa ctgttgagtt tcagcagcct gttctacttg 45000
ttcgatgcct ctctgtttct gattagcttt agcattttct aaagggtcaa actcagaaag 45060
acgcattcta acatttggta agttaacttt tggtaggctt acctcttgta aagtaacctc 45120
tgggattaga gcctgacggt tataaatatc agtaagtaag ccatctgcca ttagttgtac 45180
ctcgccatca attcttgaat ttctctttgt cgttcctctg gagtcaattc atcagcctta 45240
gtatcctcaa taaccagcgt atctttctga atacctgaat tcttaagaat catctcaaga 45300
gctttaagac gtgtattcca aggaacagtc tcatctgtag ctgcggtaag aagtagtttc 45360
tgtgctaacg ctgaagtctg ccccattcta cgcttaacca ccgccatcac atggtcgttg 45420
tttttatcta aatacttttt agcgttttga gcagcagttt tctcactaaa accagcagca 45480
atgtaggctt gaatatggtc accaccattg tctgcatagg cttctgagaa cttttctaag 45540
cgttccttct caccgatacc acgtttaatt tcggttacgt tgctcattaa ttgctgtctc 45600
catgtcttta gcagagcctt taagttcttc gttttcacgt tctaaaaact ctactcgttg 45660
ttctaagtaa agtacgtatt cttgtactaa tttgcttttg ttgtacatat tgctctccta 45720
tatttggccg agggtatggg attcgaaccc atgcatcctt tcggacgtcg cattagcagt 45780
gcgatacatt aaaccactct gtcaaccctc tataactggc ggaagaacag ggattcgaac 45840
cctgacgtca catcactgta accttcggtt ttcaagaccg ctgccgctac ccagaatctt 45900
tcggcttatc cttcctttaa tttggtactt ctggttggaa tcgaaccaac gtctttcggt 45960
tatcagccga atgtaagagc ccctctacca cagaagtatt taatttggtc tgtgtggcag 46020
gatttgaacc tacgacccta agcttccaaa gctaacactc taccagactg agctacacac 46080
agatattttc cttattattc cttttgtcct cctaactact gtacgtttat acagtaattg 46140
ttgaaacatg tcctatatta tacactaata ttccgtcact gtcaagtata gaaatactaa 46200
atttatcatt ttcttccaaa taatttcaaa ataaagccac caaacccact aaatagtgaa 46260
gaaaggtagt ctaacgggga tggaatgaac ataccaataa tcacccactg ccaccactct 46320
aagctgttgt tgataactgt attacctgaa ctggtaacag cattcttaac atcatccatt 46380
gtatttgtaa ctgattcaat tggaactggt ggaactgtag tagaacaact ggtaagaaat 46440
agaactggaa ctatagtata tagtagtata gtcttactat aggttaatct catacttatt 46500
ccttatttgc tttagattta tcttaatctc tttatttgct ttaggtaaga gtaataatta 46560
gtaagtaata atgattaata attaataatc ttaataacta ttatctagtc cttatataag 46620
gataacataa aaaagtgtcc ctgtcaagta tagttttaca ttaattagtt attttatacc 46680
agttatagta aaatactaca gttttcctcc cccaaaattt taggcatagg ctttacagta 46740
caagggttca atggcatagt gtaaccttca ggggtagttt aagtaaaagt ccccctccgg 46800
tatatggatt caacgtatgc cctatcgcaa gaaacaaaaa tggggtatgg gggtgatata 46860
tgggggagaa attgcatagg tatatataca tactggataa aactacaggt gtatattttt 46920
acataggtgt gagtgtgcat aggggaaggt ttattcaggg tatttgtggc atagtaattg 46980
catagtatta atctataact gtctattcat acagtattca accagttgtt aatctaacgg 47040
tagcacataa cgggtatcag ttaacggtat ggggtagggt attaatcgct atacataatc 47100
tatagcatag ctaggctaag aaatatcatc tataagttat tgattaataa ggataaataa 47160
aaataattat aataaataca aataaaagct tgcaatcatt aggctgttag tattagtatt 47220
tagtcaacgg caagggaaac caaaccgata gctagaaaag gtaattaata tcttgacagc 47280
taaaagataa ggcattaagc tttatcacat gttctttaat aattagtatt tactttgatt 47340
ggattgctaa tcaatgtgct catataaccc atagcagtta tgggaactat cactgataag 47400
tcatcaataa acctttatta aggcggcaag aaaattaaaa taaatactaa attgttattg 47460
acaactaaaa cctagttaga ttaaactagc tagcaagtca gggataacgt agtgatacgg 47520
cgtctaagta gcaagtcata agcatacgaa attatgacac tgacaaaggt gcaataaata 47580
acgataaaaa gttattgaca attagaaacg aatcattata atggttcaca agttaggcaa 47640
gtgttggagg ataagccaat gcagtaatta acagttagac gggcgattgt gcctagactg 47700
gaattcaaag cctcgcatta atcaatacac tgatgcggtt ggtggagggc aaaggtaaaa 47760
caatacaggt tagacgaccg tgctactgta tagtgcaaat tacgtctggg atgctgaggc 47820
tatgtcccat gaaagaagat agccgagcgg ttagtctgcg tctccggtag tgtcgagagg 47880
aatagactgg gtaaaaccat attaaaccta gtagataaag tggctaggat atagatggca 47940
gctttacccc tcattacttg ctatgagtat aattattcat gtggagtatt tataccaatg 48000
gcacgtaaac aaaatcattt gaagaaagtt caaaagcgtt ttaaaaatgc tacacgtaac 48060
aaacagttta ttagaactga gagttatgaa tcacaaaaag gttttggtaa agttgtgcgt 48120
gataaagact cgcatgaata caaaaccatt caatccacca aaggtaaaca aaaagtttac 48180
agaacatcta aaccgttaat ttgaggacta tattatgact aatacagttg caattcttga 48240
accaaaaaca ctaaccgtta aacaaatcga cgatatgatt gtaaatatca agtcccgtgg 48300
taagaaactg gattcagata ttcaagtttg cggtttggct attcttaaac atgtaatgtt 48360
gcatggcgat attactaaag ccaatgctct atacaatgca ctacctaaag gctcacgtcg 48420
cactgcacta gcagcgtggt ttgttgagtt tggtatgctc aagtacagcg agaaaaagga 48480
taaaaaagag aatccacttg cttacaacaa gaaagctacc actaacctac aaggtgcaat 48540
gaataaacct tggtgggaat gtaaaggtac taatgaagat ggcggtcgtc cgtttgactt 48600
agaatctcaa cttaaatcac tacttactcg catggaaaaa gcccgtgaag atggtaagga 48660
agtaaaaact ggtaatgtta atgttgatga gctgaaaaag cttattgcgc aggttaaata 48720
aaaatgaaag ctactcagaa agttgttcgc cagtcacagt tcccgcaagg cggctggcgc 48780
aagcgtgacg ctactctata ttcatcgttt tatagctcta aacaagcccg taacagcgtt 48840
aaacagcgca agcaaggcgc tagcaaggct ttagcatcaa aagctcttag cgaggctttg 48900
agagcgttgt gaacattcga gtagtaaaac cgtgtggtgg cgaaattgtc atcacacgta 48960
acccaattat gaatacttgc ggtcagatga ctgtatctta tcagcgtaaa tggcacatcg 49020
taactaatgg agttatcaca ttatgaatct tgacagctta gcggcaatgg ctgcaaatca 49080
cacccttgtc gatatgatta ctaaaaaggg ctattttgat atttgcactt tagataaagc 49140
attgagaact atgggtattc aagcacaatg tggtaaaagt tacagtatct tgagttctct 49200
acactgtatg gattacaaag atatgccaaa cgaattgcgt cgtagtatcc caatgctagt 49260
gcaaaatgta gtcggtattg atgcagaata ctatgaagct gaacctgaac ctaaaccaga 49320
gcctaaacct aaacgcatgg ctctatcatt cttcaagtaa ggaaatacta tgattaaatt 49380
gagcgattta gtatctgtta acaatccaaa acttaaaaca tttaaccaag atggttttgt 49440
agttggtgta agctttggtg gcggtaatct aataaaagtg aagatgttaa acggtggaac 49500
acacgtattt aaacgtgaaa atcttactag aactaaggtt tggcgcatct ctcactacat 49560
gtaggggtaa atatgatagg tttacttggt ctatttctgc tcgctgtagt tattgttgtg 49620
gcaatcatgg tcttgtttga gaaaaacaag ttcaaatctg ctgtaattac agctctcgtt 49680
ggtctgacag cttatttcgg gctgtcagcc tatatcatgg cacctatgtt tgaggcatta 49740
atgaaatgaa tacaaacatg acactgtatg agttgaactc tcaattcatc ataaaacacg 49800
ctattaagtc tttgaagata gcatatctac agttaaatgc tctaaatgtg ttaaatattg 49860
agccatctgt cgaaaaacaa ggttacttca atgtaacatt gggctgtaat accactgaaa 49920
agacttatat catcactgtt tccatgaaaa acagcttaga acaactgtta aacagcttaa 49980
atctggcatt ggaaacactc acaaaccgtt aagagggtaa aacaatgcgc aaattcttct 50040
atctattcat aatcgtctac gttatcttgt ctattttctt cacttacaag gctgtagctg 50100
ctgaaaatgg cgagtttaag gtaatgtttg caggtttttc gcatcatatt ggtgcgcctg 50160
attatgcaga ccataacgaa aagcacaatt tggtaggtat atcgttcgat aacactgaga 50220
ttttctttat gaagaactcg tttcacaacg attctgtagg gattgcgcat aattttactt 50280
attcttacag ccaatatttg gaatttggtt tccgtgttgg tgctgcaagt ggctacgata 50340
ttgatggaat atacaatatt ggcggtattg caccgtttgc acagccaaat gtgacggtaa 50400
aatatggtaa tttaggggtt gagttgggat atttaccaca aatcagccat tcaacgccgt 50460
atggagcggc aacattaacg tttaattgga gtttatagca tgaaaattca ataccttatc 50520
aagtttttcg acattcttaa ctggttttta gctcgtacta tcgagaaaga taacaaagaa 50580
atccagaaaa accgtcaaaa gcgcaaggat ttaaagacag ctatgcgtaa actggatgaa 50640
gc 50642
Claims (7)
1.一株副溶血弧菌噬菌体RDP-VP-19010，其特征在于，透过电镜观察，该噬菌体噬菌斑直径4mm-5mm，圆形透明，无晕圈，其头部为二十面体，长径约61nm，横径约67nm，尾长约112nm，直径约10nm，为有尾病毒目肌尾病毒科，保藏编号CGMCC No.19384。
2.如权利要求1所述的副溶血弧菌噬菌体RDP-VP-19010在制备抗副溶血弧菌制剂中的应用。
3.一种用于防治副溶血弧菌感染的杀菌组合物，其特征在于，包括有效量的如权利要求1所述的副溶血弧菌噬菌体RDP-VP-19010。
4.如权利要求3所述的用于防治副溶血弧菌感染的杀菌组合物，其特征在于，所述杀菌组合物中副溶血弧菌噬菌体RDP-VP-19010的含量≥109PFU/mL。
5.如权利要求3所述的用于防治副溶血弧菌感染的杀菌组合物，其特征在于，还包括用于抗副溶血弧菌的抗生素。
6.一种水产饲料添加剂，其特征在于，包括如权利要求1所述的副溶血性弧菌噬菌体RDP-VP-19010。
7.一种清洁剂或消毒剂，其特征在于，包括如权利要求1所述的副溶血性弧菌噬菌体RDP-VP-19010。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110125461.1A CN112760296B (zh) | 2021-01-29 | 2021-01-29 | 一株副溶血弧菌噬菌体rdp-vp-19010及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110125461.1A CN112760296B (zh) | 2021-01-29 | 2021-01-29 | 一株副溶血弧菌噬菌体rdp-vp-19010及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112760296A CN112760296A (zh) | 2021-05-07 |
| CN112760296B true CN112760296B (zh) | 2022-06-17 |
Family
ID=75703680
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110125461.1A Active CN112760296B (zh) | 2021-01-29 | 2021-01-29 | 一株副溶血弧菌噬菌体rdp-vp-19010及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112760296B (zh) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20180072055A (ko) * | 2016-12-21 | 2018-06-29 | 주식회사 인트론바이오테크놀로지 | 신규한 비브리오 파라헤몰리티쿠스 박테리오파지 Ｖｉｂ-ＰＡＰ-4 및 이의 비브리오 파라헤몰리티쿠스 균 증식 억제 용도 |
| CN113583974A (zh) * | 2021-08-05 | 2021-11-02 | 瑞科盟(青岛)生物工程有限公司 | 一株裂解性副溶血弧菌噬菌体rdp-vp-21007及其应用 |
| CN114395537A (zh) * | 2022-01-17 | 2022-04-26 | 青岛润达生物科技有限公司 | 一种用于清理水产养殖现场底泥的噬菌体制剂及方法 |
| CN114958778B (zh) * | 2022-01-28 | 2023-10-27 | 武汉格瑞农生物科技有限公司 | 一株副溶血弧菌裂解性噬菌体gvp-p20及其在中华绒螯蟹中副溶血弧菌的防治应用 |
| CN115197919B (zh) * | 2022-07-26 | 2024-05-17 | 中国科学院深圳先进技术研究院 | 弧菌噬菌体组合物及其制备方法和应用 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317792B (zh) * | 2019-06-03 | 2021-05-25 | 台州学院 | 副溶血弧菌噬菌体vp-hyp2及其应用 |
| CN112195158B (zh) * | 2020-10-09 | 2021-11-26 | 瑞科盟(青岛)生物工程有限公司 | 一株裂解性副溶血性弧菌噬菌体rdp-vp-19003及其应用 |
-
2021
- 2021-01-29 CN CN202110125461.1A patent/CN112760296B/zh active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112760296A (zh) | 2021-05-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112195158B (zh) | 一株裂解性副溶血性弧菌噬菌体rdp-vp-19003及其应用 | |
| CN112760296B (zh) | 一株副溶血弧菌噬菌体rdp-vp-19010及其应用 | |
| CN112831474B (zh) | 一株宽裂解谱沙门氏菌噬菌体rdp-nsa-19050及其应用 | |
| CN110317792B (zh) | 副溶血弧菌噬菌体vp-hyp2及其应用 | |
| CN112063594B (zh) | 一株耐高温沙门氏菌噬菌体rdp-sa-18056及其微胶囊的制备工艺 | |
| CN111100844B (zh) | 一株沙门氏菌噬菌体rdp-sa-17118的分离及应用 | |
| CN113430173B (zh) | 一株宽裂解谱、耐高温沙门氏菌噬菌体及其应用 | |
| CN112662635B (zh) | 一株耐高温型大肠杆菌噬菌体rdp-ec-20031及其应用 | |
| CN112662634B (zh) | 一株耐药性肺炎克雷伯菌噬菌体rdp-kp-20004及其应用 | |
| CN108699533B (zh) | 副溶血弧菌噬菌体Vib-PAP-1及其用于抑制副溶血弧菌增殖的用途 | |
| CN111549003A (zh) | 副溶血弧菌噬菌体、蛭弧菌及其应用 | |
| CN112708600A (zh) | 一株裂解性肺炎克雷伯菌噬菌体rdp-kp-20005及其应用 | |
| KR101915486B1 (ko) | 신규한 비브리오 파라헤몰리티쿠스 박테리오파지 Vib-PAP-7 및 이의 비브리오 파라헤몰리티쿠스 균 증식 억제 용도 | |
| CN113166730B (zh) | 新型猪链球菌噬菌体str-sup-1及其在抑制猪链球菌细菌增殖方面的用途 | |
| CN114015662B (zh) | 具有强裂解作用的产气荚膜梭菌噬菌体及其应用 | |
| CN117586966B (zh) | 一株耐酸碱产气荚膜梭菌噬菌体rdp-cp-22005及其应用 | |
| CN113528463A (zh) | 一株裂解性、高效价副溶血弧菌噬菌体rdp-vp-21010及其应用 | |
| CN112368373A (zh) | 新型猪链球菌噬菌体str-sup-3及其在抑制猪链球菌细菌增殖方面的用途 | |
| CN113322242B (zh) | 一株多重耐药性大肠杆菌噬菌体及其应用 | |
| CN113249338B (zh) | 一株耐酸型大肠杆菌噬菌体及其包埋工艺 | |
| CN112522214B (zh) | 一株高裂解性铜绿假单胞菌噬菌体rdp-pa-20001及其应用 | |
| CN110214181A (zh) | 新型副溶血弧菌噬菌体vib-pap-4及其用于抑制副溶血弧菌细菌增殖的用途 | |
| CN112646785B (zh) | 一株耐高温烈性变形杆菌噬菌体rdp-sa-20018及其应用 | |
| CN114574450B (zh) | 一种宽裂解谱耐紫外的奇异变形杆菌噬菌体和其组合物、试剂盒及其应用 | |
| KR20180102040A (ko) | 신규한 비브리오 파라헤몰리티쿠스 박테리오파지 Ｖｉｂ-ＰＡＰ-4 및 이의 비브리오 파라헤몰리티쿠스 균 증식 억제 용도 |
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 | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A Phage RDP-VP-19010 of Vibrio parahaemolyticus and Its Application Effective date of registration: 20221212 Granted publication date: 20220617 Pledgee: Qingdao Jinling Shengtai Financing Guarantee Co.,Ltd. Pledgor: QINGDAO RUNDA BIOTECHNOLOGY Co.,Ltd. Registration number: Y2022370010165 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Granted publication date: 20220617 Pledgee: Qingdao Jinling Shengtai Financing Guarantee Co.,Ltd. Pledgor: QINGDAO RUNDA BIOTECHNOLOGY Co.,Ltd. Registration number: Y2022370010165 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A strain of Vibrio parahaemolyticus bacteriophage RDP-VP-19010 and its application Granted publication date: 20220617 Pledgee: China Construction Bank Co.,Ltd. Jimo Branch Pledgor: QINGDAO RUNDA BIOTECHNOLOGY Co.,Ltd. Registration number: Y2024370010065 |