KR20110041670A - 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 - Google Patents
대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 Download PDFInfo
- Publication number
- KR20110041670A KR20110041670A KR1020090098609A KR20090098609A KR20110041670A KR 20110041670 A KR20110041670 A KR 20110041670A KR 1020090098609 A KR1020090098609 A KR 1020090098609A KR 20090098609 A KR20090098609 A KR 20090098609A KR 20110041670 A KR20110041670 A KR 20110041670A
- Authority
- KR
- South Korea
- Prior art keywords
- bacteriophage
- shigella
- coli
- present
- culture
- Prior art date
Links
- 241001515965 unidentified phage Species 0.000 title claims abstract description 103
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 70
- 241000607768 Shigella Species 0.000 title claims abstract description 66
- 230000002147 killing effect Effects 0.000 title description 19
- 201000010099 disease Diseases 0.000 claims abstract description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 17
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 16
- 239000004480 active ingredient Substances 0.000 claims abstract description 14
- 235000013373 food additive Nutrition 0.000 claims abstract description 11
- 239000002778 food additive Substances 0.000 claims abstract description 11
- 239000003674 animal food additive Substances 0.000 claims abstract description 10
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 9
- 241000894006 Bacteria Species 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 14
- 239000004599 antimicrobial Substances 0.000 claims description 13
- 208000015181 infectious disease Diseases 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 230000003115 biocidal effect Effects 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 241000233866 Fungi Species 0.000 claims 2
- 239000003139 biocide Substances 0.000 claims 1
- 241000607760 Shigella sonnei Species 0.000 abstract description 4
- 229940115939 shigella sonnei Drugs 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 230000006907 apoptotic process Effects 0.000 abstract 2
- 244000005700 microbiome Species 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 229940088710 antibiotic agent Drugs 0.000 description 7
- 235000013305 food Nutrition 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 230000002265 prevention Effects 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 241001138501 Salmonella enterica Species 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 239000006150 trypticase soy agar Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 4
- 241000237858 Gastropoda Species 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 208000001848 dysentery Diseases 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 2
- 241000702202 Siphoviridae Species 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 235000010446 mineral oil Nutrition 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000001066 phage therapy Methods 0.000 description 2
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 2
- 229960003415 propylparaben Drugs 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000454 talc Substances 0.000 description 2
- 229910052623 talc Inorganic materials 0.000 description 2
- 235000012222 talc Nutrition 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 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
- 241000220479 Acacia Species 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000588732 Atlantibacter hermannii Species 0.000 description 1
- 208000004429 Bacillary Dysentery Diseases 0.000 description 1
- PTHCMJGKKRQCBF-UHFFFAOYSA-N Cellulose, microcrystalline Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC)C(CO)O1 PTHCMJGKKRQCBF-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 241000588720 Escherichia fergusonii Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 108091092584 GDNA Proteins 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
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 206010065764 Mucosal infection Diseases 0.000 description 1
- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 229920002230 Pectic acid Polymers 0.000 description 1
- 239000004353 Polyethylene glycol 8000 Substances 0.000 description 1
- 241000588733 Pseudescherichia vulneris Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000533331 Salmonella bongori Species 0.000 description 1
- 241000266403 Salmonella enterica subsp. enterica serovar Choleraesuis Species 0.000 description 1
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108010079723 Shiga Toxin Proteins 0.000 description 1
- 241000607764 Shigella dysenteriae Species 0.000 description 1
- 241000607762 Shigella flexneri Species 0.000 description 1
- 206010040550 Shigella infections Diseases 0.000 description 1
- 241000588717 Shimwellia blattae Species 0.000 description 1
- 229920002472 Starch Polymers 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
- 239000004098 Tetracycline Substances 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 235000015278 beef Nutrition 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
- 239000011230 binding agent Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 239000012050 conventional carrier Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 229940037395 electrolytes Drugs 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000000369 enteropathogenic effect Effects 0.000 description 1
- 230000000688 enterotoxigenic effect Effects 0.000 description 1
- 239000000147 enterotoxin Substances 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 208000028774 intestinal disease Diseases 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000000845 maltitol Substances 0.000 description 1
- 235000010449 maltitol Nutrition 0.000 description 1
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 1
- 229940035436 maltitol Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- LCLHHZYHLXDRQG-ZNKJPWOQSA-N pectic acid Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)O[C@H](C(O)=O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](OC2[C@@H]([C@@H](O)[C@@H](O)[C@H](O2)C(O)=O)O)[C@@H](C(O)=O)O1 LCLHHZYHLXDRQG-ZNKJPWOQSA-N 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229940085678 polyethylene glycol 8000 Drugs 0.000 description 1
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 description 1
- 239000010318 polygalacturonic acid Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000013772 propylene glycol Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 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
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- 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
-
- 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
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
-
- 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
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/00031—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
- C12N2795/00032—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)
- Zoology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Polymers & Plastics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Nutrition Science (AREA)
- General Engineering & Computer Science (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
Abstract
본 발명은 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지에 관한 것으로 구체적으로 본 발명은 대장균 또는 시겔라속(Shigella) 균에 특이적 사멸능을 갖는 신규한 박테리오파지, 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 항균제, 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물, 사료첨가제 또는 식품첨가제에 관한 것이다. 본 발명의 박테리오파지는 대장균 또는 시겔라속(Shigella) 균을 특이적으로 사멸시킬 수 있어, 상기 박테리오파지 또는 이의 배양물은 항균제, 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물, 사료첨가제 또는 식품첨가제로 효과적으로 사용될 수 있다.
대장균, 시겔라, 박테리오파지, 항균제
Description
본 발명은 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지에 관한 것으로 구체적으로 본 발명은 기탁번호 KCTC 11428BP를 가지는 대장균 또는 시겔라속(Shigella) 균에 특이적 사멸능을 갖는 박테리오파지, 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 항균제, 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물, 사료첨가제 또는 식품첨가제에 관한 것이다.
대장균(Escherichia coli)는 전체 박테리아의 0.1%정도를 차지하며 인간뿐만 아니라 다른 동물의 장에서 주로 서식한다. 대장균은 대부분이 유해성 박테리아가 아니지만 병원성 대장균도 많이 존재한다. 예를 들어, Enterotoxigenic E.coli (ETEC), enteroinvasive E.coli (EIEC), enteropathogenic E.coli (EPEC), enteroaggregative E.coli (EAEC) 및 enterohemorrhagic E.coli (EHEC)들은 장독소 (enterotoxin), 시가 독소 (shiga toxin), 세포 독소 (cytotoxin) 및 내독소 (endotoxin) 등의 독소를 배출하여 설사나 장내 질병을 일으킨다. 또한 이들 병원성 대장균은 개발도상국의 여행자들에게서 빈번히 발견되며, 살균되지 않은 우유, 요리되지 않은 쇠고기, 상한 물 등을 섭취함으로써 쉽게 감염된다.
또한 시겔라(Shegella)속에 속하는 균은 장내세균과에 속하는 세균으로 운동성이 없고 협막도 없으며 아포도 형성하지 않는 그람음성 세균으로 S.flexneri, S.sonnei, S.doydii, S.dysenteriae의 4종으로 나뉜다. 상기 균들은 세균성 이질을 발생시키는 데, 상기 질환은 감염력이 강해 매개체 내의 증식없이도 환자나 보균자의 손에 묻어있는 세균만으로도 전파가 가능하며, 점촉감염증이 쉬겔라 감염증의 주된 전파 방법이다. 세계적으로 매년 2억명의 발병을 보이고 있으며 그중 65만명이 사망하고 있는 실정이다. 우리나라에서 세균성 이질의 발병은 감소추세를 보이다 1998년이후 전국적으로 집단 발병하는등 발병이 현격히 증가하여 사회문제화 되고 있다. 또한 1998년 이후 분리된 이질균의 경우 테트라싸이클린, 스트렙토마이신등 항생제에 대하여 내성을 보이는 등 항균제에 대한 내성이 심각한 수준으로 대두대고 있다.
상기 대장균 또는 시겔라속 균과 같은 미생물에 의한 감염이 발생하는 경우 그 치료를 위하여 주로 항생제를 사용한다. 하지만 항생제가 남용되는 경우 항생제 내성 미생물들이 발생하여 부작용이 발생할 수 있다. 구체적으로 항생제가 남용되는 경우 미생물들은 항생제에 대한 내성을 획득하게 되었고 내성을 지닌 미생물들은 다른 미생물에게 내성을 전달하여 확산 속도가 빠르게 퍼져나갔다. 따라서 상기 항생제가 아닌 다른 방법으로 대장균 또는 시겔라속 균을 사멸시킬 수 있는 방법이 필요한 실정이다.
한편, 박테리오파지(bacteriophage)는 세균에 특이적으로 침투하여 용균하는 바이러스로 유전물질 (single DNA, double DNA, RNA)을 단백질 외피가 싸고 있는 유기체다. 상기 박테리오파지는 자연에 존재하는 모든 생물체를 감염시킬 수 있으며, 생물이 존재하는 거의 모든 곳에 함께 존재할 수 있다. 뿐만 아니라 그 수와 다양성도 엄청난 것으로 밝혀지고 있고, 실제로 해양이나 토양의 경우 박테리아의 수보다 많이 존재하는 것으로 추정되고 있으며 그 다양성도 어마어마한 것으로 알려지고 있다. 이들을 이용한 파지 치료법 (phage therapy)은 인간뿐만 아니라 식물, 동물, 음식 등 그 응용분야가 매우 다양할 뿐 아니라 잠재적 응용성도 많다고 할 수 있으며 나아가 항생제보다 보다 특이적인 치료가 가능하다.
이에 본 발명자들은 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지에 대하여 연구하던 중, 하수로부터 신규한 박테리오파지를 분리하고 상기 박테리오파지가 대장균 또는 시겔라속 균에 특이적 사멸능이 있음을 밝혀 본 발명을 완성하였다.
따라서 본 발명은 대장균 또는 시겔라속(Shigella) 균에 특이적 사멸능을 갖는 박테리오파지 (기탁번호 KCTC 11428BP)를 제공하는 것을 목적으로 한다.
또한 본 발명은 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 항균제를 제공하는 것을 목적으로 한다.
또한 본 발명은 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것을 목적으로 한다.
또한 본 발명은 상기 박테리오파지 또는 이의 배양물을 포함하는 사료첨가제를 제공하는 것을 목적으로 한다.
또한 본 발명은 상기 박테리오파지 또는 이의 배양물을 포함하는 식품첨가제를 제공하는 것을 목적으로 한다.
상기 과제를 해결하기 위하여, 본 발명은 대장균 또는 시겔라속(Shigella) 균에 특이적 사멸능을 갖는 박테리오파지 (기탁번호 KCTC 11428BP)를 제공한다.
또한 본 발명은 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 항균제를 제공한다.
또한 본 발명은 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물을 제공한다.
또한 본 발명은 상기 박테리오파지 또는 이의 배양물을 포함하는 사료첨가제를 제공한다.
또한 본 발명은 상기 박테리오파지 또는 이의 배양물을 포함하는 식품첨가제를 제공한다.
이하 본 발명을 보다 상세히 설명한다.
본 발명에서 항균제는 미생물의 성장을 억제할 뿐 아니라 미생물을 파괴 또는 무력화하는 제제를 의미한다.
본 발명에서 배양물은 본 발명의 박테리오파지의 배양액, 상기 배양액의 농축액, 상기 배양액 또는 농축액의 건조물을 포함한다. 상기 배양물은 본 발명의 박테리오파지를 당업계에 공지된 박테리오파지 배양용 배지에 본 발명의 박테리오파지를 접종하여 제조될 수 있으며, 상기 배지는 이에 한정되지 않지만 TSA(Tryptic Soy Agar)일 수 있다.
본 발명의 박테리오파지는 대장균 또는 시겔라속 균이 서식하는 곳에서 당업계에 공지된 방법에 따라 분리될 수 있다. 이에 한정되지 않지만 바람직하게는 박테리아는 여과하지만 박테리오파지는 통과시킬 수 있는 구멍크기의 필터로, 대장균 또는 시겔라속 균이 서식하는 곳에서 채취한 시료를 여과시킨 다음, 원심분리하여 상등액을 회수하고 상기 상등액을 이용하여 용균반 분석(plaque assay)을 함으로써 대장균 또는 시겔라속 균을 특이적으로 사멸시킬 수 있는 본 발명의 박테리오파지를 분리할 수 있다 (<실시예 1-1> 참조).
본 발명의 박테리오파지는 형태학적 분류상 시포비리대(Siphoviridae) 및 람다 유사 파지(lamda-like phage)속에 속하며, 본 발명에서 상기 박테리오파지를 'bacteriophage EP23'으로 명명하고, 2008년 11월 19일자로 생명공학연구소 유전자은행(korean collection for type cultures, KCTC)에 기탁번호 KCTC 11428BP로 기탁하였다 (<실시예 1-2> 참조).
본 발명의 박테리오파지는 35분 정도의 암흑기, 45분 정도의 잠복기를 가지며, 10±1 PFU/infected cell의 방출량(burst size)을 가지는 생장 특성이 있다 (<실시예 1-3> 참조).
본 발명의 박테리오파지는 서열번호 1의 염기서열로 표시되는 유전체를 가지며, 상기 염기서열은 gDNA 서열이다. 또한 본 발명의 박테리오파지의 DNA 중합 효소 단백질의 DNA 및 아미노산 서열을 다른 박테리오파지와 비교한 결과, 본 발명의 박테리오파지는 다른 박테리오파지와 동일하지 않으며 신규한 것이다 (<실시예 1-4> 참조).
본 발명의 박테리오파지는 대장균 또는 시겔라속 균에 특이적 사멸능을 가지는 것을 특징으로 한다. 상기 시겔라속 균은 이에 한정되지 않지만 바람직하게는 시겔라소네이(Shigella sonnei ) 균일 수 있다. 구체적으로 본 발명의 박테리오파지는 대장균 또는 시겔라속 균에 대해서 특이적인 사멸효과가 있으며, Escherichia blattae , Escherichia hermannii , Escherichia vulneris , Escherichia fergusonii 및 살모넬라 속 균에 대해서는 사멸효과가 없다 (<실시예 2> 참조).
한편, 본 발명의 항균제는 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 것을 특징으로 한다.
상기 박테리오파지 또는 이의 배양물은 상기 기재한 바와 같이 대장균 또는 시겔라속 균에 특이적 사멸능이 있어, 상기 성분을 함유하는 항균제는 대장균 또는 시겔라속 균을 사멸시킬 수 있는 특성이 있다. 상기 시겔라속 균은 이에 한정되지 않지만 바람직하게는 시겔라소네이(Shigella sonnei ) 균일 수 있다.
상기 대장균 또는 시겔라속 균은 주로 식품 또는 화장품을 오염시킬 있다. 식품은 미생물의 탄소원 및 질소원 등 수 많은 유기물, 무기물을 함유하고 있으며, 화장품은 기름이나 물을 주성분으로 하며, 미생물의 탄소원이 되는 글리세린이나 솔비톨 등과 질소원이 되는 아미노산 유도체, 단백질 등이 배합된 것이 많아, 대장균 또는 시겔라속 균이 번식하기 용이하기 때문이다. 따라서 본 발명의 항균제는 식품이나 화장품에 첨가되어 사용될 수 있다.
본 발명의 항균제는 유익한 균은 사멸시키지 않고 병원성 균인 대장균 또는 시겔라속 균만을 선택적으로 죽일 수 있어, 부작용이 없으며 병원성 균의 내성 내지 저항성을 유도하지 않아 기존의 항생물질에 비하여 효과적으로 이용될 수 있다.
본 발명의 항균제는 통상적인 항균제 제조방법으로 제조될 수 있으며, 유효성분인 본 발명의 박테리오파지 또는 이의 배양물 외에 필요에 따라 담체, 기타 보조제 등을 추가적으로 포함할 수 있는데, 상기 담체는 생리식염수, 증류수, 사료, 박테리아용 배지(예, Tryptic Soy Agar) 등일 수 있으며, 보조제는 pH 교정 등의 목적으로 완충액 등일 수 있으나 반드시 이에 한정되는 것은 아니다. 또한, 본 발명의 항균제는 액상, 반 고체상 또는 고체상으로 제제 형태는 분말제, 과립제, 정제 등의 고체상 제제, 현탁제 또는 주사액제 등의 액상 제제 및 크림, 로션, 젤, 페이스트 등의 반 고체상 제제 등으로 제형화될 수 있다.
한편, 본 발명의 약학적 조성물은 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용인 것을 특징으로 한다.
상기 박테리오파지 또는 이의 배양물은 상기 기재한 바와 같이 대장균 또는 시겔라속 균에 특이적 사멸능이 있어, 상기 성분을 유효성분으로 함유하는 본 발명의 약학적 조성물은 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용으로 효과적으로 사용될 수 있다. 상기 시겔라속 균은 이에 한정되지 않지만 바람직하게는 시겔라소네이(Shigella sonnei ) 균일 수 있다.
상기 대장균에 의해 유발되는 질환은 이에 한정되지 않지만 바람직하게는 대 장균 감염증, 보다 바람직하게는 장침습성 대장균 감염증, 장출혈성 대장균 감염증, 장독소성 대장균 감염증 및 장병원성 대장균 감염증으로 이루어진 군에서 선택될 수 있다.
또한 상기 시겔라속 균에 의해 유발되는 질환은 이에 한정되지 않지만 바람직하게는 세균성 이질일 수 있다.
본 발명의 약학적 조성물은 상기 질환 부위에의 도포 또는 분무하는 방법으로 이용할 수 있으며, 그 밖에 경구 또는 비경구 투여를 통해 투여할 수도 있으며, 비경구 투여의 경우 경피 투여, 정맥내 투여, 복강내 투여, 근육내 투여, 피하 투여 또는 국부 투여를 이용하여 투여할 수 있다.
본 발명의 약학적 조성물의 적합한 도포, 분무 및 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 질병 증상의 정도, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. 일반적으로, 본 발명의 약학적 조성물은 이에 한정되지 않지만 바람직하게는 1x103 내지 1x 1010 pfu/ml의 박테리오파지를 포함할 수 있다.
본 발명의 약학적 조성물은 당업자가 용이하게 실시할 수 있는 방법에 따라, 약리학적으로 허용 가능한 담체, 부형제, 희석제 또는 부성분을 추가로 포함할 수 있다. 상기 약학적으로 허용되는 담체, 부형제 또는 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카 시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐 피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유, 덱스트린, 칼슘카보네이트, 프로필렌글리콜, 리퀴드 파라핀 및 생리식염수로 이루어진 군에서 선택된 1 이상을 들 수 있으나, 이에 한정되는 것은 아니며 통상의 담체, 부형제 또는 희석제 모두 사용 가능하다. 또한, 상기 약학 조성물은 통상의 충진제, 증량제, 결합제, 붕해제, 항응집제, 윤활제, 습윤제, pH 조절제, 영양제, 비타민, 전해질, 알긴산 및 그의 염, 펙트산 및 그의 염, 보호성 콜로라이드, 글리세린, 향료, 유화제 또는 방부제 등을 추가로 포함할 수 있다.
한편, 본 발명의 사료첨가제 또는 식품첨가제는 상기 박테리오파지 또는 이의 배양물을 포함하는 것을 특징으로 한다.
상기 박테리오파지 또는 이의 배양물은 상기 기재한 바와 같이 대장균 또는 시겔라속 균에 특이적 사멸능이 있어, 상기 성분을 유효성분으로 함유하는 본 발명의 사료첨가제 또는 식품첨가제는 대장균 또는 시겔라속 균을 특이적으로 사멸시킬 수 있다. 상기 시겔라속 균은 이에 한정되지 않지만 바람직하게는 시겔라소네이(Shigella sonnei ) 균일 수 있다.
상기 식품첨가제가 사용될 수 있는 식품에는 제한이 없으며, 상기 식품첨가제는 대장균 또는 시겔라속 균에 감염되기 쉬운 식품에 당업계에 공지된 방법에 따 라 첨가제의 형태로 제조된 것을 말한다. 상기 사료첨가제는 이에 한정되지 않지만 가축용 및 어류용 사료에 첨가제의 형태로 제조된 것을 말한다. 상기 사료첨가제 또는 식품첨가제의 첨가량은 당업자라면 용이하게 정할 수 있으며, 대장균 또는 시겔라속 균의 감염 용이성, 감염 정도에 따라 달리 정할 수 있다.
본 발명의 박테리오파지는 대장균 또는 시겔라속(Shigella) 균을 특이적으로 사멸시킬 수 있어, 상기 박테리오파지 또는 이의 배양물은 항균제, 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물, 사료첨가제 또는 식품첨가제로 효과적으로 사용될 수 있다.
이하, 본 발명을 실시예에 의해 상세히 설명한다.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
<
실시예
1>
본 발명의 박테리오파지의 분리 및 동정
<1-1> 본 발명의 박테리오파지의 분리
대장균을 특이적으로 사멸시킬 수 있는 박테리오파지를 분리하기 위하여, 대장균이 존재할 것으로 예상되는 곳에서 시료를 채집한 후 이의 배양액을 대장균에 접종하여 대장균을 사멸시키는 박테리오파지를 분리하였다.
구체적으로 박테리오파지가 존재할 것이라 예상되는 하천수에서 검체를 채집하여 여과 종이로 일차 여과한 후에 상기 회수된 여과액을 0.22 ㎛의 필터를 이용하여 여과한 다음, 여과액을 대장균과 함께 30℃에서 1일 동안 배양하고 다시 8,000 rpm에서 20분간 원심분리하여 상등액을 회수하였다. 상기 회수된 상등액을 0.22 ㎛의 필터를 이용하여 다시 여과한 다음, 그 여과액을 이용해 용균반 분석(plaque assay) (Calson, K. (2004) Appendix. Working with Bacteriophages: Common Techniques and Methodological Approaches. In the Bacteriophages: Biology and Applications (Kutter, E & Sulakvelidze, A eds) pp 437-494, CRC Press, Florida)을 함으로써 대장균을 특이적으로 사멸시킬 수 있는 박테리오파지를 분리하였다.
<1-2> 본 발명의 박테리오파지의 형태학적 분류 및 기탁
상기 <실시예 1-1>에서 수득한 박테리오파지의 형태학적 특성을 살펴보기 위해, 상기 박테리오파지를 10%(w/v) 폴리에틸렌 글리콜 8000에 넣은 후 4℃에서 하룻밤 동안 클로로포름으로 대장균과 파괴된 세포 잔해를 제거하였다. 이 후 염화세슘(CsCl) 밀도 구배법(밀도: 1.15 g/㎖, 1.45 g/㎖, 1.50 g/㎖ 및 1.70 g/㎖)을 이용한 원심분리(20,000 rpm, 2시간, 4℃)를 통하여 박테리오파지를 정제하였다.
상기 정제된 박테리오파지를 구리 그리드(copper grid)에 묻고, 2% 우라닐 아세테이트(uranyl acetate)로 대조 염색(negative staining) 및 건조한 다음, 투 과전자현미경을 이용하여 형태를 관찰하고 그 결과를 도 1에 기재하였다.
상기 도 1에 나타낸 바와 같이, 본 발명의 박테리오파지는 형태학적 분류상 시포비리대(Siphoviridae) 속 및 람다 유사 파지(lamda-like phage)에 속하는 것을 알 수 있고 도 3에 나타낸 바와 같이 계통학적으로 람다 유사 파지에 속하는 것을 알 수 있다.
상기와 같이 분리한 박테리오파지를 'bacteriophage EP23'으로 명명하고 2008년 11월 19일자로 생명공학연구소 유전자은행(Korean Collection for Type Cultures, KCTC)에 기탁번호 KCTC 11428BP로 기탁하였다.
<1-3> 본 발명의 박테리오파지의 생장 특성
본 발명의 박테리오파지의 생장특성을 살펴보기 위하여 생장곡선을 작성하였다. 구체적으로 one-step growth curve test를 실시하였으며, 보다 구체적으로 대장균을 TSB (tyrptic soy broth, Difco)에 접종한 후 30℃에서 exponential time (OD 0.6-0.7)까지 20 ㎖에서 배양한 후 원심 분리하고 세포를 수집한 후, TSB (trypic soy broth, Difco) 0.25 배 (v/v)를 추가로 첨가하였다. MOI (multiplicity of infection) 0.1 수준의 본 발명의 박테리오파지를 접종 한 후 상온에서 5분 동안 흡착시킨 다음, 원심 분리하여 상등액을 버리고 TSB 20 ㎖을 추가 첨가 하였다. 상기 첨가 후, 상온에서 5분 간격으로 샘플링하고 한 셋은 바로 파지 적정(phage titration)을 위해 대장균이 도말된 플레이트 위에 도말하였다. 또한 다른 한 셋은 세포내 파지(intracellular phages)의 해방을 위해 1% 클로로포름을 처리 한 후 파지 적정(phage titration)을 위해 대장균이 도말된 플레이트 위에 도말하였다. 이후, 시간 추이에 따른 적정된 박테리오파지의 수(plaque form unit, PFU)를 측정한 결과를 도 2에 기재하였다.
상기 도 2에 기재한 바와 같이, 본 발명의 박테리오파지는 35분 정도의 암흑기가 있으며, 45분 정도의 잠복기를 가진다. 이들의 방출량(burst size)은 10±1 PFU/infected cell임을 알 수 있다.
<1-4> 본 발명의 박테리오파지의 유전자 서열 분석 및 비교
1L의 TSB에서 30℃에서 배양한 후 본 발명의 박테리오파지를 접종하여 용균 현상이 관찰될 때 폴리에틸린글리콜 침전 및 염화세슘 고속원심분리를 통하여 박테리오파지를 농축하였다(Calson, K. (2004) Appendix. Working with Bacteriophages: Common Techniques and Methodological Approaches. In the Bacteriophages: Biology and Applications (Kutter, E & Sulakvelidze, A eds) pp 437-494, CRC Press, Florida).상기 농축된 박테리오 파지에서 DNA를 분리하여 (Sambrook, J. & Russell, D. W. (2001). Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York.) 박테리오파지의 전체 유전체 서열 (44077개의 DNA 서열)을 밝혀 서열번호 1에 기재하였다.
또한 본 발명의 박테리오파지의 DNA 중합 효소 단백질의 DNA 서열(서열번호 2)을 다른 박테리오파지와 비교한 결과, 도 3에 기재한 바와 같이 본 발명의 박테리오파지는 다른 박테리오파지와 동일하지 않으며 차이가 있음을 알 수 있으며 가 장 가까운 엔테로파지 SSL-2009a와 DNA 서열과 92% 상동성을 가지는 것에 불과하여 신규한 것임을 알 수 있었다.
<
실시예
2>
본 발명의 박테리오파지의 대장균 또는
시겔라속
균주에 대한 특이적 사멸 효과
본 발명의 박테리오파지의 숙주에 대한 감염성을 조사하기 위하여, 대장균 및 시겔라속 균주 등를 숙주로 하고 실험을 수행하였다. 상기 균주를 TSA(tryptic soy agar, Difco) 배지에 배양하고 페이퍼 디스크(paper disc)를 가운데 놓은 후, <실시예 1-2>에서 정제된 박테리오파지 30ul를 디스트 위에 접종하였다. 상기 접종 후, 30℃에서 24시간 동안 배양한 후, 환이 생기면 본 발명의 박테리오파지의 용균(lysis) 활성이 있는 것으로 나타냈으며, 상기 결과를 하기 표 1에 기재하였다.
번호 | 기탁번호 | 균주의 종류 | 사멸여부 |
1 | KCTC2223 | Escherichia coli | + |
2 | KCTC2293 | Escherichia coli | + |
3 | ATCC21278 | Escherichia coli | + |
4 | DSM4481 | Escherichia blattae | - |
5 | DSM4560 | Escherichia hermannii | - |
6 | DSM4564 | Escherichia vulneris | - |
7 | DSM13698 | Escherichia fergusonii | - |
8 | KCTC2518 | Shigella sonnei | + |
9 | DSM5570 | Shigella sonnei | + |
10 | DSM 14848 | Salmonella choleraesuis indica | - |
11 | DSM 14846 | Salmonella choleraesuis choleraesuis | - |
12 | DSM 13772 | Salmonella bongori | - |
13 | DSM 9386 | Salmonella choleraesuis arizonae | - |
14 | DSM 9221 | Salmonella choleraesuis hountenae | - |
15 | DSM 9220 | Salmonella choleraesuis salamae | - |
16 | ATCC 13311 | Salmonella typhimurium | - |
17 | ATCC 13076 | Salmonella enteritidis | - |
18 | DSM 14847 | Salmonella choleraesuis diarizonae | - |
- KCTC: the Korean Collection for Type Cultures
- DSM: the German Collection of Microorganisms and Cell Cultures
- ATCC: the American Type Culture Collection
상기 표 1에 나타낸 바와 같이, 본 발명의 박테리오파지는 대장균 또는 시겔라속 균주에 대한 특이적 사멸 효과가 우수함을 알 수 있다.
도 1은 본 발명의 박테리오파지를 투과전자현미경으로 관찰한 사진이다.
도 2는 본 발명의 박테리오파지의 생장 특성을 나타낸 one-step 생장곡선이다. (점선: 클로로포름을 처리한 경우, 실선: 클로로포름을 처리하지 않은 경우)
도 3은 본 발명의 박테리오파지의 단백질 서열 중 하나인 DNA 중합 효소 단백질의 아미노산 서열을 다른 박테리오파지와 비교하여 계통학적 위치를 나타낸 것이다.
<110> Industry Academic Cooperation Foundation of KyungHee University
<120> Bacteriophage having killing activity specific to Escherichia
coli or Shigella
<130> DPP20094090KR
<160> 2
<170> KopatentIn 1.71
<210> 1
<211> 44077
<212> DNA
<213> Bacteriophage EP23
<400> 1
aggatttagt ctttcaggtc gtcatcggtc gccgaaagcg ttatggggat ctccgatttg 60
atgactaaat tcgacgaccc atatatggcc ccggtgtgcg ggtctgtata gaaaccaatc 120
acgccgcgct cgaccatatc gtcgattgcc ttcgtcgaag tgccaccctt agcgagtccc 180
gcctggcgta caattgccga tttactgaat ccagtatggt cagggttatc tgccatcagt 240
ttttccaggg ccgcataaag cactttctct ttttcgttgc gcggaccggc ggtgttctct 300
tccttaactg cggctttgcc cgccgccttc gcctgttcta tcgcgaacgg tgccagggct 360
accggtacga ggtatagagt ctcgtcgcgc ggggtggacg caattggttt gacgtcccaa 420
ccggcaacca catcgccgaa gtctggcccg gtgctggtgg actggtgggc ttcgaacgct 480
tcggacttgc ggagatccac cttaaccttg cacttgagca ggacgaagcc gcgcggcgct 540
tgcttgatgc cgttacgtgc cttttcgtga tacaggttca attgacctgg ctggttgtcg 600
tccggttgct ccaggaagaa cgcagaatcg acagcaccat gcagcgcgcc ggacccacgg 660
ggagatctgc ggccgtttgc acctttcgcc gggtggtgga tgacgcccgc actgccgcct 720
gtattttccg caatggcctt cagcgctttg accacttcgc ccatatcgct ggcgttgttc 780
tcgtcgaacg gttttgcatc ggcggaccgc agcgcaacag tctggttgag tgagtcgaac 840
gccaccaggc caaccggctc gccgttagcg gtgcggttga tgaagctgat aacttcacgc 900
aagcctgatt tgctggtgat atcccagccg cgtgattgca ggtcaataat gtgcagcttg 960
tccaggtcgt gctcatattt gatctgcaat gcttccttac gctggttgga ggcttcgcca 1020
ccttccgcgt cgaaatagaa gcaatgagac tggacgacct tcgcaccgcc gaacggaatc 1080
cccgcggcaa ccgcgcccat ttggcccagc acgtagaacg atttaccgat gttcgactcg 1140
ccagccatgt accaggtcga tttgaagttg accaggcctt cgatgatcgg gtcgtgctta 1200
ctgaatagtg cgaccggctc gttatccaga tccgcatcgg tgcacacgcc gtattcttcg 1260
ccgtcgccaa ctgcatcaac aatatgatca tataaggatt cgacttcctc gtcgtccaga 1320
cctggcagga tggcggcgat ttgcttacgc gacatgttgt gcggctcgac gttcaggtgg 1380
tccggtatcc cgataagacg tagcgccagg tgctggtgtc tatttagctc agagcaacac 1440
tcgttgcctg tgtgcataca cttgaaacgg atctcgccat ccttgaccat gatagccgtc 1500
gacccttcgc cgctgtgcag atgttcgttc gggcatggca cttcataacc gcgaccggaa 1560
gataactcct caaatcctgc ctggaagcac cagtccagaa tcccgtccgc cactgcgtcg 1620
tcgccctggg taagctcgcg gcgtacttct tccttctcca cgccaagcgt atccgccagg 1680
cgaagcagac gacggacgcg ggcacgtttg ccgtagtggc acttaataac cgattgctgg 1740
tgcggcaggt acatcaggcg gtttgtgtct ttcgtgcaat cgtcgaagtc cacatccgac 1800
agaccaagtt gatgtaatag cgcatactgg acacggccca ggtcttcagc cgggaccggc 1860
ttatcagtga gtaccaggaa gcgagcgcaa cgcgtgccgc ctttaagcgg gtggcggtcg 1920
ccggtggttg tgtggaatac gtgttccaga cccgcgtcaa tgaacgcctt acggcagcgg 1980
cgtaaggtgc gagtgtcggt gcggtcaata tcaaagaaca ggatcgagcg gtccaccgcg 2040
ctggtcttgc ggcgtaagcc gtcactggta tacgctacga tgccatcggc cttacgcttg 2100
cggcgcgcat actccgcttt atcctcggtc ttgttgatat gcaccgatga tatttctttc 2160
gcgtcattga taaattcgaa aaattcatcg gtggtcatct ggaagttttc cgccctggct 2220
ttttcgcctt ttttagcggg tcgtgttaca gcgaatttaa ttaagctcat tgccgttgcc 2280
ttacttcgaa gttttgttat tgcgtaccca ggagcgcacg gtgctctgat taattcctgt 2340
caactcagcg atcatcgccc aggattttcc ctcggcgcgc aaacggataa cctgtttttt 2400
gtattcttcc atttcttcat tgtgcttttg cagcttgctg gctgccgtac cgttgagtaa 2460
ttccagcttc gcgcgcaatt ctttttcggt aagtcgcaga cgttcgattt caaatagcac 2520
cgcccccacg gcatcggtat acgcccagga ccggctagtc tcactgcaga cagtcttcgt 2580
tagcatttcg attgcgtcgt tgatattcat tattctgtct ccggttggtt aatgaccaat 2640
gaaactacac ctaaaaccgc agggtgtcaa ctcctttggg atattgaaac gggcgcgcac 2700
gcgtgtaata atactcccgt ctcattgtct cttattggtt agcgccgtcg cattttgctt 2760
cggcgctttt tttttttaaa ttagttgttg actcccttct gcaaccaatc taatattcac 2820
ctcgtcgaca ggatgcagga cgcaaactgg agaggggcaa cgcaggggcc ggatcgaagt 2880
gtcaggattc ccaaccgtga ttatataacc aggtccacta cccgaacggg agaacgcgta 2940
acctggcact atgttaaggc caccgacgaa gttaatggtc gacacctaaa cttaaccaat 3000
cggagatata ccatgtttga gcaaatcgta aacctgatca ctcgcggcgt agtagcactg 3060
gaaatcatcg ctaagcatta tgaaaccgtt ggcgctgccg ttggtaaaca ggaagacaag 3120
aagccgtcta aggccaaaaa agaagtcgta gaagatgacg acgctgaaga cgaagccccg 3180
gcgaaaacca aaaaagcacc ggcgaaaggt aaggccaaaa aagaagtcgt agaagatgac 3240
gacgacgatg ccgatgccga agacgaaaaa ccggctaagt ctaagaaagc accggcgaaa 3300
ggcaaaggta agaaagatgc cgatccgctg gcggagatgc gcgacgaaat caaacagtat 3360
gccgcgatta tcgccggtgg cgatgacgac gatgcgaacg acgaattcga cgaccttctg 3420
gaatcgttcg agatcaaatc aatcgctaag ctggaagatg acgacgtcga agacttccac 3480
aaagagctaa aagaaatcgt cgaaacttat ttcgaactgg aagattaata tcatggacta 3540
tgaaatgaga atcatgctcg atttcatagc agaaaattgg gcggcattca ccgccttctg 3600
cgaagaaaga ggcgacgacc cggacgaggt ttataaacaa ctcggcggtg aagattaata 3660
ttatccggcc cgcatattag cgggcctctt tttagaggac gagacaatgg cacgattcac 3720
gctaatcgag gcggtggatt cccgcgacca gcgcccgtac tattggctat tcgaacgcat 3780
cggcagcttt caatcccgcg ttgccgtaat agataaacgc cgcaacaccc ccgcgcaaat 3840
taagcgcacc acctttacta accccgattt ttatatttgg gctgactcca atctggaata 3900
tgttcgcttc gccgttgcat ccgaagcaaa aacaattgac cgctgggaga caaaataatg 3960
aaatttttcg tgctggcaac ttgccacgaa ttaaacgacc atttctcttt tcttgttgtt 4020
aacaaggacg cctcgatgtg ctggaccgat gcgcagcatg aagtgcgcag ccgtgtcgaa 4080
aaagcgggct tggcgaatgt gaaatatgta attgtccagt ttacacgggt ggaataatgg 4140
cgattaaacc aaaacgtaaa acgcaatccg gtagtaatac ggaacactca ttacttggcc 4200
catccggtgc taaaaaatgg atggggtgtc ccgccgccct ggtggtggaa aaagatatcc 4260
cgaacgaatc cgggcaagcg gcgattaacg gtacgagtat gcacaccgta tctgaagtgg 4320
tgctaaaccg catcattgcc ggggagaagc tgataaccgc taagacgtac aaagggtgct 4380
acgtagagaa cgaaggtaag gggccggtaa aggcccaccc gaaagcaccg aaaggcggcg 4440
tactggtcaa cgacgacatg gtgaaacagt gcgacgcgta catcgaccac tggcgaccgc 4500
tcctcgaagt ggcggagttt gttcagcttg agatgcgcgc cgatctgacc cgcgtccttc 4560
actcaggctt cgagatcgac ggcaaccggg ttaagacgtt cggtactgcc gatatggtca 4620
tggtcatgaa gaagaccgac ggcacgtaca tgctgattgt tggcgacctg aagacggggc 4680
ggcataaggt cgaggcgaaa gaaaacaaac agcttatgtt gtatgcgctg ggcttgctgc 4740
gcaagctgaa aactatgtac gacattacaa ccgtgcgcct tgttatcttc cagccgtatt 4800
gcggcggggc gtcggagtgg gatatttcgg tagaagccct ggagatattc gctaagtttg 4860
cgtccaagcg tgctatcgcc gccctggaag cgtactcccg cggaaaaaag ggactcaccc 4920
gcgccgactt ccgaccgagc gttgatgcgt gtcagtggtg cagatttgcg gatcagtgta 4980
gcgcccgcgc taaggctgca atcggcacga tgaccccacc aacggcaaca gacgaagacc 5040
tgggcgacga cgtgagtatt gcgacagagg aagaatgcgc caatcacgac cgcaatatgg 5100
cccgcgaagc ccgcaaagcg gcacgacgtg cgaagcgtgg caagaaagaa gcggcccccg 5160
gcgctatgac cgccgcggaa ctgcgcaagg cttatgaagg cctggacgca atgcgccagc 5220
acatcaaggc tatcgagtcc gcggtattca aggctgtgat ggcgggcgac ggcgaatcgc 5280
tgggcctcaa gatggttgca ggtaaggaag ggattcgcaa gtgggcggac gaaagcgaag 5340
tgattgaaat cttcacgaag gcccggatca agcgcgatgt gatgtacaaa gaaacattac 5400
tcagccctac tgatgccgag aaggtattga aggacgagaa gccgaaagta tgggcgaagc 5460
tgtgtgataa aatcacgcgc gcaccagcta aaccggtatt agcacccatc gacgacccgc 5520
gccctgcgtg gtctgtagct actgacgagg acttaagcaa tgaataaagt attcgcaatc 5580
ttattgatgt ttgttatcgg cgcgctgggc tttatcctgc attgtgtgca aggcgtgtta 5640
atggtggcat ggatgattag acatccgacc cgctggctgg atgacggcta tagcgaagct 5700
gccgtcgatt gggtggagaa gaacatcaac cgcccgccgt taagctggat cgagaacgcg 5760
accaactacg ttacccgaaa tctttttaat gaaaatgctt gaccgctcag gcgcaaccaa 5820
gtaaattagc aaccaagccg aacggggtgg cttccaataa ccccgataca tgtgaaaatg 5880
aggaattgga aaatggctaa agtcaacctg aaaaatgtgc gcgtatgttt ccttaaaatt 5940
tgggaacgtg atacccctaa acaggatgga caaaaaccgg cataccgtgc agtgattctt 6000
cttgataaag aagatccgca ggtggacaaa gtggaagcgg cagcgcgcgc agtgctaacc 6060
gacaagctga aatccgagaa gaatgctgat aagtggatgg accgccacta cgcccaggat 6120
tctaaggaat gcgcagttcg cgacggtgac gagcgcgacg aagtaaccga agaattcgaa 6180
ggcatgttgt acatcaacgc caaatcgttc aaacagccga tcatccagac ttccgaaggc 6240
gagaagcaga cggagcaggg tctgaccatc gacggcgacg aaatcgaagg gaaggaaatc 6300
tattccggtt gctattgcaa cgtttcctta gatatttggg cctggaacaa taccaacggc 6360
aaaggcttag gctgtggtct gctgggtatg cgcttccgtg atgacggcga agccttcggc 6420
ggtggcgggt cttcatgttc cgacgaagac ctgggcgacg atgacgaaga tgaaagcccg 6480
cgtaagtcca aaaagtcaaa acgtcgtgat gacgatgacg aagacgacga cgaagacgaa 6540
gcgccacgta aacgtcgccg ccgttaaggg caaagcccac tattcaagcc cgcttaatgc 6600
gggcttttta acaggacaat gaaaaatgcg tattaaagcc agcgaagtaa aagtcggaat 6660
gcaagtatgg tccaagctac tgggggaata tttcacagtt gccgagattc gcaacaacgg 6720
cgaagagatc gccctgtccg acggcatctt ctccatgatc ggcagcgctg acgccgtagt 6780
gaggataaag caatgaaaga cttcgaacgc ctgtttctcg acaccgagac atttagcggg 6840
gtggacctga agaaagttgg ggcgtacgcc tacgcggagc acccaaccac agagattatg 6900
atttgcacct acgccataga cgagggccgc gtgcagacat gggatgccac tgaatccccc 6960
acaatgccgc gtgaactgcg caaggcgctc cgccgggtat cccgcaagaa ggctaaaata 7020
gtgatggcta acggcctcct gtttgaccgc ctggttatcc gcgagaagtg gggtattgac 7080
ctgccggtaa gccagatcga agacactatg attatggcct ttcgccacgc gctgcccggt 7140
agcctcgata tgcagtgcca ggttctgggc gttgacgcag agcacgcgaa ggataaggca 7200
ggcaaggcgc tgattaaccg attctgtaag cctaccccta agacctacaa aatccgccgc 7260
tacactcgcg agactcaccc ggaagaatgg gccaaatttc tgcgctatgc cgcgctggat 7320
attatcgcaa tgcgtgaagt ctactggcgc ataccggact ggggaaactc gcctaaggaa 7380
gatgaaatcc tgctcatcga ccagttgatt aacgaccgcg gcttctacgt cgacgttgac 7440
ctggcgaacg ccgcgatcaa agcggtgcag gcgcataagg aagaattgaa ggaagaagcc 7500
tgggagcgat ttggcggtaa gctgacaggc aatgacttcc tgccaatcct gcgtgatatc 7560
gccccggcgt tcacgatcca caacgcgcag aaatccacgc ttaacgacct gcttgaagac 7620
ccggacttcc ccgacgaagg caaagcgctt atcgaaatgc gcctcggggc gtcatcaacg 7680
gcatcgacta agtacaatcc gctagttaac ggcttatccg cagatggtcg ccgccgtggg 7740
tgtcttcagt atggcggcgc aaagcgcacg ctgcgctggg cgggcaaagg cttccagcct 7800
caaaacctgg cgcgtggcga gtacagcgac gaccacgaag ggaagattaa acgccgcgag 7860
ggtgaaagcg acgtctcgtt ttgggtccgg tcgcacatgc tcaccaacgg catcaactcc 7920
ctgttacgcg ggaccgcaca ctgggcgtat gacatatcga agctaacggc ctcgaccgtt 7980
cgcgggtgca ttattccggc gaagggcaag aagtttgtcg tagcggacta ctccaatgtg 8040
gaagggcgtg gacttgcctg gatcgctggc gagaaaacag cgttaatggt ctttaaggcc 8100
ggtcgcgata tctattgcga gactgccggt aagatgttcg gccttgaccc ggattacatt 8160
aaagccaacc gcaaggactt acgccagatc ggtaaggctt gcttgcaccg ccatacccaa 8220
gtattgacca atggcgggtt caaggatatt atggccgtaa catcaacgga taaagtatgg 8280
aacggcgaaa aatgggtgag tacaaaaggc gcgcacctta tggggtggaa accagtgata 8340
gacgtggacg gggtcttgat gacagaagac cacaaaatct tgacgcattc atggaaggag 8400
gcaaggcaac tcgtttcaga caggtgcacg atggtccgcg ccctggcgag aggtatggac 8460
gcctggttat cctgcgcgaa ttaccaaaac gacagggtca cagacaactg ctggcccaat 8520
gtgattgcgg agcgatgcca ggacgcgtcg ggtatgacaa cgtccgaagg ggtaaaacat 8580
cccaatgcaa cctctgcgca attgaagcga caaagcggta tcgtaaacag tatttttgct 8640
acgatgacgc aatgccagac gatgcgcaca gaacgcggct actcaaccgg ctgtcagcgg 8700
caatatcacg aacgacaaac ccccgcgcca aagggtataa aaactacggc ggtcgtggga 8760
ttgccgtatg tgacgaatgg aagagagata aaaggtcgtt tcttctctac gttcaaacgc 8820
tggacggctg ggataagcca gacttcgaaa tggacaggat cgatacagac ggcgactatg 8880
agcctggaaa ccttcggttt gtcagccgtt cggaaaactg ccgtaaccgg cgcagcgtgc 8940
ccgagcttca gagaaagtac gatgaagccc ttacccgcat tgctgaactg gaacgggaag 9000
ttaacctatt gcgagccggt ttacgacctg attgatgtgg aagacggaaa ccggttcctt 9060
attgcgtcta agtctggttt tctggtggcg cataactgcg agcttggcct gggctatggt 9120
ggcggcgtcg cggcgttctt gcagttcgct aaaaacctgg gcctcgacct ttacactatg 9180
gctgacgtca tgaagggcac attcccggat cacatttggg cggcggcgaa gcggggttat 9240
gagtacgctc gcattaacga ggagaagcgc ccgccgaagc ctggcaagaa agacgagcgc 9300
ccaacttaca tactgccgaa aaacgtgtgg ctgacgtgtg acgcgatcaa acgtatgtgg 9360
cgtgagtctc acccgatgac ggtagccttt tgggccgaac tggaggacgc ggtgctctgc 9420
gcgatccgca atccgggcaa agcgtactgg gctggcgcga acgttcgccc ggatggcagg 9480
aaagcgctca aaattgttcg aacgaaggcg aagcatgacc cgaccttcga cgaggagcgc 9540
gacgacccga acgcggcggg gtggtggctt aaaattgagt tgccgtcagg gcgcatcatg 9600
agctaccctg gcatcgcgct gtctgtcacg accgagatcg acgaagacac cggcaagaag 9660
cgcaccagca cacgcattaa atatcagggc gagaatcaga cgacgcgcca gtggggattc 9720
cagtatacct acggcggcaa actgacggaa aacatcgtac aggcgttgtg ccgcgatatt 9780
ctcgcatggt ccatgcctgg cgttgaagct gcggggtatg agatcgtact ttccgtacac 9840
gatgagctga taaccgaagt gccggatacc gacgactaca caaccgagga gttgtgcgcg 9900
ctgatgtgcg acctgccggt ttgggcgaaa ggcttcccac ttgccgcaga gggcgattgc 9960
atgtatcgct accgcaagta atgacaccag aaggcaagat tcaaagccac ctgatgagac 10020
gggtgaaaga tgttggcgga ttctgccgaa aactggcatg ggaggggcgc gcaggtgcgc 10080
ccgacctgat tattatcatc aacggcaaaa tagtgttcgt cgaagtgaag cgccccggag 10140
gaaaaccgaa gccgcaccag gtacgtgaac atgagcgaat ggcccgccgc ggtgctgacg 10200
ttcgcgttat cgacaacata tccgactgcg atttattggt tgctgaattg gtcgcatagc 10260
ggtatagtgt gccggttagc catatcgagg attttagaga tgattgaaga aaaacgttgt 10320
agtaattgcg gctgtgtaaa gccgctatcc cagttccaca aatacaccgg taagactgcg 10380
cgcagtccag acgggtatcg ggcagagtgc aaatcgtgcc gcaataagaa agagcgcgaa 10440
cggcagcgcc gctaccgtgc agaacaaaaa taattaatac ggcgggcttg tcccgccatt 10500
tttgggagac atgacgatga aagaacatta ccgactcacc aagctgggcg aatgtaaacc 10560
aggcgatcgc gtgtggttta tcgcgggcct aaattaccca ctgccgttga cggtgtgctg 10620
gcaaaaattt aagagcttcc gggctaccgg tgtccacgtc ggacacgcca ccaactacga 10680
cgccacaaag aggaactggg atagtgaaac tgaagtctgg aaaaaggcgg aataaagaaa 10740
tggcacgttt ccgacgtcgc gaatatcaga agttgataac cgcgttcatc ctcaagcata 10800
agcgatgcaa tatatgggcg acaatggggt cgggtaaaac cggcgcgacc atgtgggcgc 10860
tgaataaaat gttccagact ggcatcctgt ccaaccagga tgaccgcgta ctggtgctgg 10920
ccccgctgcg cgttgcgtcc ggtacatggc ccccggaaca ggagaaatgg cgcttcccag 10980
ccttgctggt agtagacggc actgggccag agaaggaccg catcgctgcg cttgagagcg 11040
acgctaacgt ggtttgtatt aactatgatg tagttgagtg gcttgttgag tattgcggcg 11100
acgagtggcc ctttacggtc gtggttgccg atgaatccac taagctgaaa ggctatagaa 11160
gcaagcaggg aagtaagcgc gcccgtgcgc tggcagcgat cgcacataag cacgtcaagc 11220
gctggattaa cctgactggc acacccgcac cgaacgggct gaaagaccta tggggtcaga 11280
cgtggtttgt cgacggcggc gaacgtctcg gcagtagcta taaggcgttc acggaccgct 11340
ggtttattag caagcctgtt aagcctggca gcttcacgat gcagcacgcc ccgcttaaga 11400
actccgagaa agaaatacaa caacgactgt ccgacgtgtc gctaaccgtt gatgcggctg 11460
aatacttcgg gtgtgacaag ccaatctata cgccggttgt tgtggacctg ccgaagaaag 11520
cccgcaagat ttatgaccag tttgaaagcg agctatttgc cgaactggaa agcggaacag 11580
tggaagcggc gaacgccgcg gcgaagacaa ttaagtgcct tcagcttgca tccggcgctg 11640
tgtataaggt ggacgaagac ggcgaacgga ccgacgagtg ggtgaagatt cacgacgcga 11700
agctggacgc gctggaaagt atcgtggagg agttgaacgg cgcgccgttg ctcgttgcgt 11760
atcagtacaa gcatgatctg gcacgtctta agaagaaatt tccgcacggc gtcgcgttgg 11820
gtaagggtaa gcagggcaat aaggatatgg aagcgtggaa caggggcgag atccccatca 11880
tgtttgcaca cccggcatcg gcaggccacg gccttaactt gcaggacggc ggacatcacc 11940
tggcaatctt ctccgatacc tggaactttg agcacttttc gcagatcgtt gaacgtatcg 12000
ggccggttcg ccagatgcaa gccggacacc ctcgaccagt attcgtctac attatccagg 12060
ctcgcgggac actggacgaa ttggtcgccg agcgtcgcga cagtaggcga gatatccagg 12120
acgacttgat ggaatacatg aaagctaaga aaacgaggaa aatttaaaat gacattcgaa 12180
ccaatcgatt tttcggtcgc aatgactaac cagctattca cctacgacag caaaaccgga 12240
gtcatccgtc tgaaatccac cggctgcaag attggcaacc tcaatactac tgatggcggt 12300
actcgtatcg agtgcaacgg caagacgctg tctggcgcac gcgtggcgtg gattatcgag 12360
actggcaata caatacctcc aggctatgcc gtagtgtgcc gtcagcgcac acctggccac 12420
tacggcaacc gcattagtaa cctttacttg gtagacgttg cgcgcgaccc aaatttttat 12480
gcgtaaattg gttgcataag catattggtt gcattatatt gagggtgtcc ggttggacgc 12540
cctttttaat acctggagac aacacgatga gaattttaca agaagaaaaa gattactggg 12600
ccgcgcaatg cctcgaagcc cgcgaacagg ccgaacgggc taacgcgttg gtggacacca 12660
accaggcgct atatgaagga gagaagaaag tgcgccagga gctagaggag cgcttcgaac 12720
tgcttttcaa attgatgggg attatgcagc gcaccggata caccaccagc gttgcactaa 12780
accgcatcac tgccgatggt ctgcgtgagc attgcaaacg ggccgaggag tacgtgcgtc 12840
cgaagggccg ccagtctaag attgaccgga aagacctggt gctggttgcg tggcggggga 12900
acgggtctta ctactccgac tgccgaccac tcggcaccga ggaatcaaag cgcgtgcaac 12960
gcgttctaaa cagtaataag aaaattgccc tgtacgcgat agatccagat catgcagaag 13020
agtgagcaaa aagatagctg gcgcacgccg ccagctttat ttaaaccgct gcgccggaag 13080
taccgattcc gttacgatat ggcggcgagc gatgccaacc accttctacc gcgttacttc 13140
acggcagagc acagcgcgct tgaggtggac tggtcagagt tgaaagggtg gaagtggtgc 13200
aacccgccgt attccgatcc gctgccgtgg gtcaagtgcg cagcgcacgg acgccggacc 13260
gtcatgctac tgaatcagga cacgtcgact aagtgggcga agctggcccg gaagactgca 13320
aacctgataa ttcttctgga ctggcgtatc cgatttattc acgctgtaac tggtgaagtc 13380
gggccgagca ataacaagtg ccagcaattg ataatcttcg acgaagcgcc gcccaccggc 13440
agcgcacaaa ttgaaataat gagcgaggga gaactacgtg aatatcttaa tcgttgatcg 13500
cagtatgcgc gtcgtggacg aaattgttaa acgctaccaa atcaacccga gggagaacaa 13560
agtgcgtcgc gtgtcacggc cagaacacct cctgggcatg gaccttactg gctggcttgt 13620
aatgactgcc cgctgtcatc ttgtgtgtga cacgttttcc gcccgtactg ttcgtgcaaa 13680
tttgattgag gaattgaaat gaaattactg atttgtgatc tggatggcgt tattaatgga 13740
tcaagcgatg cgcgcgccga cctggtgccg gggataaaaa cgaaatccac cttttgggcg 13800
aagtggcaca aagcgcacgt ccgcgaagac ctgaacatgg atatgctgcc gctgctgcgc 13860
atgtataaag agcaaggctt cgagatcgcg tatctgacaa accggcagcg agaatgttgg 13920
gacaccaccg ccgagcaatt ggaggtcttc ccagtaggcc ggttgtttat gcgccacatg 13980
ctggacgaca ccccgccgcc tgagtttaag gcgtgcgccg tgttcaaaat ggtctgctat 14040
gcggacgtgt cggaactggt catcattgag gactgcccga agaacatcgc ggcaattcgt 14100
cgggcgtgtg gtgatttggt cccgaaaatt tacgcaataa acgttgcaaa attcacttgc 14160
aaccaatagg ttggttgcac tatagtaaac gtgtcggggg caatacagcc ccctcgctaa 14220
aagggtaata ctgtgaaagc atacgaatac tgcaaaaacg ccgccaccac tatggaacag 14280
cgcgggaaag aaaacggcta tgacaacgcg aaagaagaac gcagcgctaa acaaatcgct 14340
gccgtgttta acgcgcttac cggtcgcgac ttgacagagc aagaagcctg gaccttccta 14400
atctgcctga agctggtgcg ccagcaccgt aagcaccagg aagataacat cgtggatctg 14460
gtggcgtatg cctccctgct gggcgagtct tacatgaccg tgcatgacga gattcagatc 14520
gatacaacgg cagaacagtt tgaaagcctc aaaggcctgt ccgtgcgcct ggacggtgag 14580
attaatgcgt ataacagcgc aattcgcgtt gcggaggaca cggtagcctc gttcggcaag 14640
actgttaagg ccggcgattt tggcatcgca gatcaggacg cccagcctat ggtcgtgctc 14700
accggcgaag acggaccgaa aatctgcatg acgccggaag acttccgcaa catggaaaaa 14760
acactggtgc gtagcgcact gaaataacac taatcgcggg cttcggcccg cagcaatggg 14820
ggattcacaa tgttcggaga atttgaaata accggtaaac aattccacct ggcggcaatg 14880
ttagcaagca tcgagtttga tcaatggcgt ttaaagtcag acgcgtgcaa acaaagccgt 14940
ctcgtcggtg tgcttggcga gatgtttgcg ggcatgtacc ttgaggggca ggcaggcggg 15000
cgtagttgca taccgcaagg cttgctaatg cgtactggcc tgtttagcgc caacaccatc 15060
gaccgcggcg atattattat ggtcggtaag cgcaaggtta aatccggccc cgactaccgt 15120
gagcacatga tcgatgccgt ttggacctac gaggtgaaag ccaccagcgg catccgtcgc 15180
ggactggttg aggcacgttg cgcgcacgag tacctcacac gtcgagttgc cggtgttatc 15240
ctggttggtg tgtcattcgg gcagcacagc gcgcacggcg taattgagga cattgcggaa 15300
ccctacgtga ttgtgaacga ttggcccttg ttggaagtgg aggggaagga atattacgaa 15360
agcccgctgg tgcggcgcaa aatggactgg gaaaactcaa actaaaaaag gggccgtgtg 15420
gccccctctt ttattttttc aattctgcaa tcgccgcttt taactctgca atctctgctt 15480
tattagcctc gtcacgcgcc cgcaatgctt taattgccgc tagggcgtcc agtagcattg 15540
gtgtctgatc aagatgatag tacccgccga tctctttcgt gtactgctta tcaatctggc 15600
ggatctgctg cgagataacg ccgcgtcggt agctacgttc cggcccatca ctcaggaacg 15660
agaaaatttt aaaatccatt cggtttatgt tatccagcgc accttctaga ttaagatttc 15720
cccggacagt tttcaggcgc tcatccgaca ccgcagaggt agttacctgc ttgtagttac 15780
caaaaacgct actattactg gaacggagat acatcgcccc gcccgcgcta tcctgaaaca 15840
ccaactggca gcggtaattg gcggatgatc ctgatgtttg aagcatcgac atgccgagtg 15900
ttcctgaaat tggattgccg acgtcagcga cggtacccca ataaagcccc ggcgcaaggt 15960
ttgtggcgtt cgtcgccctc ggtccttgcg agggagccgc tacgccccag cctgtaattt 16020
ctggaaggtt tacttcttcc ttgtatgcaa taaatcgcgt ctgcccgctg gcaggcgcgg 16080
tgaagtggac ggcggcctgc ccattggcat ctgagccatt tcgccagtag agcgccatgc 16140
ggttatcact cgaatcatgc tcaaagacaa cgcgatagcc gacctctgtt ggtgacatgc 16200
tggttgtaat aaaccttaag ccgggccagt cgcttttaga caggtcaacc ccctcaaatt 16260
gcgcacgctg agtgcggcct agtgacagat tgtcccgcgc cgtatctttg ttcgctacgt 16320
cgctaaggtt ttgcgactta agcatgaact gtgatgggtt gatttggttt ttaaactgct 16380
ctgcttcgtc gcgataccct tgcgcctggc tgcgggcgtt ctccgcacca gtttttgctg 16440
tattagccgc atcgcgcgct gattctgcgc ctgttttggc ggtgttggcc tgatcacgtg 16500
cggtgttggc ctggttgcgg gcgttctccg cgccagtttt tgctgtatta gccgcatcgc 16560
gcgctgattc tgcctggccc ttcgcagtaa cagcctggtt ttttgccgtc tcggcctgtt 16620
gttgcaggtt gcgcatttct acggggtcga tctgcttcat tagctgcgac agatagcccc 16680
atgacatgcc ctgcataacc tgcccgttag gtaacgtcac tgttatttcc tcttcttctc 16740
cgaagattgc gcgccagttg ttctcatctg cgatcatcaa tcgcaatgct ttttgcgcct 16800
ggacggttac gcccatcgtc gccaatgaca tggtgtcacg tttgatgccg tcccaggcca 16860
gacccgtcgc cgttgggccg tcgaacggca acaccagtgt gagcgcggtt gcgctttcga 16920
tggacgcgat cgccacggtg taaggcactt gcccaaccac cagcgataag aagtcaccag 16980
ccttgtactc ggtaaacgtt gtgccaaccc ctactacagc cttgctattg ttcgttacgg 17040
agagcgtacc cgctgccata gctaaatcct cataaaattg gttgcattct tggttgagcg 17100
gtgtaatact aaaactcaac ccaatacagg agatgataac atgaaaactt taatcaaaaa 17160
tctgataata cccgcggcgt gccttctgat gacggcatgt ggcagcggct taagcacagg 17220
cgttccaaat tccggtgtaa tacctcagcc tattgagcga catgaggacg ggtctttggt 17280
ttacgatacc gaaaagctac catataccgg tcagtggtgc caggagatta gccacgaaca 17340
ccgcaggatt ggttccccat caaactgcgt cattgattat taaaagatag cccctcaagc 17400
gaggggctat ttgcttattg cgaccagcta cctgtcccac gagtcattaa aattacagac 17460
gggtgcaatg tgctgccgta gacgtttgta tactcttctt tcacagtaat tgtcacactt 17520
cccgccgtta cacctttcgc agctaaagga atcgttgtat ggattgcgtt tccgcttgcc 17580
gctcgttcaa tagtccttga gactactttc ccgccaatat tggcggtaat agcaatgcgg 17640
cttgactggc ttgcggtgta cccgaccagt gagtagctta acagcaatac aaagtctttg 17700
agttttgttg aattggatga gtcgttgaaa gtcaccgacc acgaccttga tccattcctg 17760
gctgtgttgg gtattgcttt cgcaccgata acacacatat ttgcaacgtc accgacaaaa 17820
ctggtggctt cgaccgttcc ggtaaatacc ccatcccttg cataaaccgt gccccggaac 17880
tcaccatcat tcgcataaac ggtgccgcgg actgtcacat tgttaagctc cgcattacca 17940
gatttaggca ggttccaccc gcgtgatccc gctacgaagt tatctgaccg cagcgtgtcc 18000
gtgatctgcg cgaagtttat ggacgcgttc ttgatgcggg ctgtgtctat atacacctgt 18060
cccccatcca caataaacgc cgatgacagc gtgccgttgg acgggtgcat gatgtagaac 18120
tggtccgcct tgaatgcgat gcgtgattta accgcgttgc ccgacgtcac gacggaaagc 18180
tgcataccgg catcgtagta agtcccgtta tacgtcacgc ccgccttaag gctgtacgtg 18240
gcgctaccac cggagtggtt aaacgaagac gtcatcttct gctcaatagc ggcagactgc 18300
tgcccaaact gtgctgcgac ctgggtctta tactccgcca gtgcgccagc tacgtccgcc 18360
gttgccgtct gtacctgctg gatagcggct tcctgctccg agaacttagc ggcaacagtc 18420
tgctgatacc gggcgaaggc ctgctcttgc gtactctgcg ccgtctgcaa cgtgtgaatc 18480
gccgcttcag cttcgtcaag gcgcgtgccg atcaacgtct ccagttgcgc gacggcctgg 18540
tgggcgtctg ctgccacgtt atacgccacc ttgatttccg cataaatccc ctggtactca 18600
gcgcgggcgt tgaacatttg ctgcgcgtcg gtgagcatgg cctcgtaaat gccttgcggc 18660
atggtgtcga tcgcttcctg gaagcgttta ccatcctcag aagtcaagaa ctcatcgtca 18720
agccccttca tgtactcgtc aactttgtta cttacctgcc cttcaaacgg ggccgaccag 18780
tccgacaggt tgccgatttt atccgctacg cgggcgcgca actgctgata gataccgaac 18840
tccagagacg tttgtgcata ctccgcccct ggatatggga cgttggtaag aaatttccat 18900
tccgtctcac cggtcttgcg cacttcaagc tcggtgtatg agatatccgt cgcggcgtta 18960
ttatggttcc acttccacaa aatctggtgg aggccagaaa tcgcgttggt gatgatcggc 19020
gcgttaagcc cgccgaggcg accttccacc ggcgtgatgg cggactccac ccacggggat 19080
gaaatctcac ccataccgat cgcgcggacg cgtgcctggt agttgccgga gaacacgtta 19140
gggatgtgcg cgccgttcga tgccgtgcgt ggaaggttca cccagtcgtt accccactgg 19200
atttccatat tctgcaagct ggtgcgccgc cactggattt cgtacacaac ggcatctttc 19260
accggcggga aaacaatatc cacgttggtg attagctggc cttgcgatga gatctgcgac 19320
ggggtgatga ttaccgactc cggccccttc attacgccgt caggaacttg cgtgaagatg 19380
cccggatcaa ggcgagcgcc attatcaacg gcatcgtatt tgctgtcgtt gtattcaatc 19440
ccgcgcaccg agaaagtgct ggtagcgtcg tcatattcca ggtcggtgat gcggaacttc 19500
tgcaactgga tatcgtcgcc atcaacgcaa aacggttgat cgggaatcgg gtcagcgttc 19560
agtgctgttt tcagcgtaac cttgttgccg ttgacgctgg caatctcgcg agtctccaca 19620
ttcccgctat cggtgcgcac caagaattta tcgccagggc ggaacgtgat ttcgctgtcc 19680
gtgttcagca cagtacgcgt ccctggatct gacaacaggc gtccgccgcg tgtctcgaag 19740
ccgccggaaa tattagtatc cgcgatcgca actacgcttc ccgggataaa aaacagccct 19800
tccatgccaa ccttaaagct aacttgctgg tcaagctggt tggagattag cgcccagtgc 19860
ccgcggcgtt gcgcttcgga ctcacgcgta cagccgatcg ccgtaatatc gaggcggtta 19920
atcttatggc ggcgcaccag ttcgacgcgc tggcccgttg cgatggcgtc ttcgtagtgg 19980
ttcttcgggt tgccgtatgc taccgcgaac gtggaatacc gggttttctg gctaccggat 20040
gcgtagtcga atcgaccatc aatcaccgag gcgtttgtga tagtgtgcga taccgcggct 20100
accggctggt cactgacgat atttaacatc tgattgcccc accaggtcat accggcaaag 20160
atagcggcta aatccttaag cacggtccag gcgtcggtct gcgactggat gtacatatca 20220
ttcttaaatc ggatttcctg cccgccgttg ccgtcgctga ccagctgatc gcagcgctgc 20280
gcaacctggt aaagcgcgta tcggtttagc atttgcggtt tgatacgacg accgaggcca 20340
aagcgctcag taatacaaat atcgtaccag tgccacgcag ggttatccgt ccatgcgcgt 20400
ttaaacgtgc catcccagtc ccccgaatag gtgtgattaa tcgggtcgta gttgttcggg 20460
accaacacct tgcgaccttt gcacttgatt gacacgcgcg ggatattctg gaaagcctta 20520
gcgtcgaact ccacgaacaa cagcgcggtt agcgggtatc gcaggcgggc gtcgataatc 20580
tccgtaatgg attgcacccg gatatcattg accactttcg cagaggtgga atcagccgtt 20640
aagcggtgca cctccacgcg ccagccggta ttcacgttag acggtagctc gatgcggtgg 20700
tcgcgctcgt acagcgacat tgtttttcca acggcagcga agcggaactc gcgcggatcg 20760
gcgttatccg tatacacaac gattttatag tcgatgcgcc cgcccaccag gtcgccatta 20820
tcttcgttag tgtacagggc cggtataccg actcgaatgc gcacggcgtc caaactggtg 20880
tcgttaatct ggcgcaccca cggcgtgccg tattccagtt ttacacccac tgtctgctcg 20940
ctttccaccg cggaaaatcc gtgaataacg tcctggtgct gtgtgcctgg gcggaactcc 21000
acgcgcgcgc cggggaagtt ttcggatcgg tcggcgttct ggatcggcgt gccgtccaaa 21060
taaacgcgct gccgtaattc gttgtcggtc gggatgcctt caaactcacc ttcacccagt 21120
gctagcaaga tgcgggcttt agccagtgaa tgcaggctgt caggcgtctc ctgtggggtg 21180
tgcggttttg cgccgccgcc tttatggccc gtgacgtcgt agataatcgt cattcgttaa 21240
aatcctcggt tacaattccg gctgaaataa tagcgccgcc gatctcacgc tcgccccata 21300
atacaccaag tgcggtacct tgcgctgtcg tgtttaccgg tccgccgaac gcatacgacg 21360
ctttgttgtc cacgtcctgc cgtgtctgca atcctttcgg ctgtggcgac agcaattgca 21420
taacgccgcc cagcgccatt gacatcccga acgagaaagt tgctgtgcca atagcggcag 21480
cggtcgcggc tgaagcacca aaagcggcag tagcaagccc gccggtgtag aatgacgcca 21540
ccacgattgc tacgcctgcc agcaactgac ccagtcctct ttttgcgcca gtcataaccg 21600
gcgcgatggt cactacgcgg tcgccgtgga actcgcccaa ctcgtcgtat ccggtcattg 21660
tgtgattgcc cttgcgcacc ttgaacataa tgttttccag gtgggccgac attagaaagt 21720
cctgtaagcc aggtaactta acgcacaggc cggtgatgcc ttcgcctggt gtgaagcagt 21780
ccagcttgtg cactttacca aaacgacgcc ctaagacgcc gtacaattta aacgtggtca 21840
ttgccattct gtaatatccc caggtaagtc tttatgacgc acccatttaa ccacattgtc 21900
gcggaaaaat cctgcccgga aggggacgac gctcgacaag ctgccgtaga tatggtgtag 21960
tagctggttg cctggaagga agatccccgc atggtttggc accggcgcgc taatctgcat 22020
gatgaccatc gcccctggtg tattctcctc gcattccctg aagcctgctc cgtaccagtt 22080
atccatatac aggttttcgc cagtctccca ccagttgtac ggcacgcgga agtctggcag 22140
aatcacgccc tgctttttgt gccagtccat tacgaggcca tagcaatccg taattccgag 22200
aacaaatcgg cggcctacca gcggcgggtc ttccgggtca atctcgataa agtcgccgta 22260
ctcattagta atggtccaca cgacgccgct gccgttgcac ccgttaacgt cgccaagtga 22320
cggggttagc tcgcgctggc ctgggtgtga gtggacaacg cgaacaacgt cgcccatatc 22380
ctcggcctcc atccaggcca tatcactaat ggtaaacgcg tctctacgtg atcgcgctcc 22440
ggtcgggtct tcgaatacgt tcatgcacgg gacaaaaact tgtttgcggc ccaccatgac 22500
aaccaggccg caggcttcct ccggcagaca tgccgcaacg tgcgcggcaa tatcgctttt 22560
aacctttttc gttaacatga ctacccctta attaatcgtg accccggaaa gccgccgaag 22620
tccagcaccg ccgctttagg atccgcaagc ccttgcccaa atcgaagata acagtctgac 22680
aaacagccgc cgcatcggtc catgttcatc gcgatgacct cattaccgcg ttcgtcgtaa 22740
tacttgatac ccggctttgc aaggttccag gtgcatccgt ccccgctggc gtacttccca 22800
ttgagcgccc actcgcacat attcatgatt tggcggcggg ggactacttg cccctccata 22860
tccgcgggcg aggataatgc gaaggtgatt tgtttgcccg gattgcggcc cacctgtgag 22920
tcgatgtaaa actcctggac acgtttttgc gttgggtctg gatccggccc gttgtccagg 22980
aacttcgcga acgtgtcgat gatttcaacc ttcgcgccca gtagctggtc gtgattcagt 23040
gatagcctgg tgatgacccc gccaatatcg acgatagtta gctgcggttc tgctgccgtt 23100
gatgtggaga agttaaggcc ggacaaatcg aagggccaaa attcgaactc ctgcccgccg 23160
aagaagatag attttggctt tagcttgtcc tcgtccccac cggcggcagc aatatcctcg 23220
gcagtgtgtg gaataaagca atagtggaaa tagtgatggc ccgcgccgat cgcaccgtcg 23280
cctggttcca cgtcctgcgc gtcaatgtga atcaggcgaa cgcgacccga tgggtcaaag 23340
cgcgtcgcct cacgatatag tttattcatc tcacgaatcc aatagctgtt ttaagggtgg 23400
ctgataccgt tgccgtgtag tgcccgttgt ccgtgtacga caagctcgaa cgctcgacaa 23460
cgaataaacc tgtttcgcca tccggtgctg tgacgacaaa cggttttacg aagtgaccgc 23520
gaaggaagtt gtaaaccagt ttagggtcgc accatttgtc agtagtcagc ccgacgaatt 23580
gcacggggaa ctgtttcgtt tcggcgttcg ggccgtccgc gatgttctgc ccgtagccat 23640
taccgaattg caattcctcc gaccggaaag ctaactgcac gtcgccaccg ccctgcaatt 23700
gcatgtgcca gtaatagggc cattcgtttg atcgcgggtc gtgtgcttcg attagctcat 23760
acgccgcacc ggatttcgtt ggaccggccc agttatcttt cagtgtaatg accgtattcg 23820
agttgaatgc tgcaattttg ccggtgtagt cgttgccgtc gatgtgggtg aatacgcgta 23880
ccttcccgat ctcgctaagg aacgccgtcc ccacgcccgt aacggtctta ctccccttag 23940
ttaatgtgat tgtaccttgt gacataaaaa agcccccacg atggatgtga gggccatttt 24000
atcatcgttt gtaaagtaca ccacccggtt tcgtcgcttt cacgatctcg gccttcaccg 24060
cctcggtgat ttgcttgccg aacgcttgcg cagtacgggg gtccggcccg ctgacactgg 24120
tatccgtttt accagtcccc gtgtaaacgt tagtcgtcac gaccatacca ccgcctgcgc 24180
ccgacgcggc aacgcccagt ttcccgtccg gcccgcgctt cagtggcatg atggcctctg 24240
gtccagcctc gcccatcaat cccgcaccct tagcgaacgc ctggacgcca taactaaaga 24300
aggtaggctg gcttaccact ttccccgaat aggccgcgag attgcctcct gcgtacgctc 24360
cgccgtccgc attcttagtg aggccagaga agatagcgcc gaacaatccg tttttgccgc 24420
cgtcaccacc gaaagcgcta acgatagact tgaacgcctg cgaggtagcc agcttgatta 24480
actcgctcag gatggattta accatatcct ttgcgttcaa cttgcccgtc tcgaaaaact 24540
cgttcattgt gtcctgcata gcgctgaacg tgaatttcgt gagcgacgcc atctgcgtat 24600
atccgttacc catttcatcc acgctatcct taagcccgcg actgaatcca tcccatagcg 24660
aggcgttagc gccggtctgc gcctcctgca cttcccgcag cttagacacc gccttgtcaa 24720
tatcggtatc gttagcgcct acacgcttca ggcggtcgat tagctgcaat tcttcacgca 24780
ggtttgccgc ttctcgagta gaaagcgcat agcccgccgc ggtggctttt agctcggcat 24840
tctggttctt aacgaaggcg tcgatctgcg cgtgcgcctt aacctgcttc tctttcgcgt 24900
ccagtagttt ggcttcttcg acaatctgct cgcgcacttc cttcacgcgt ccgaattcta 24960
ccatctgctg cttctctgct gccgttagcg ccctttcttt gctcgccgcg attaactgct 25020
cattgtcggc gatcagcttg tttaaggccg cttgagcgcg ttgcgtgcgg gtctgcgtag 25080
acgcgttggc ttcgatctgc ttgccggatt ctcgaagctg ggccagttcg ctccgtgcga 25140
cttccagcat tttcataccg gcatcgactc gtacagcggc ggccttcttc ggggcggggt 25200
ctttataacg ctcgtttacc tgcttcaccg cctcggcaag ctctttctca tcaataacgc 25260
cgcgcttatt cagcaaattg aggcgggcga tctcgtcggc gcgcttcttc gcgttagttg 25320
cgtactcctt ctcgaacgca gcgcggtcgc gggcggtgcg gttggcctcc tcgttcgcct 25380
ttgcggattc gtctgcggcg cgggcgttgt ctcgctcagc acggttacgc agggtgagcg 25440
acgccaccac tttttcctgg gcgaccagtt cgctgtaaag catgttaata cgtgctttct 25500
gcgcttcagt cattacccca ccggcagcgt ttaccgcttt catctcggca tagtaagccg 25560
tggtcatgcg ctgtagtgat tcgcgcgctt tcatctcgga ctcattggag gactgcgcgc 25620
ggcccacatc caggatggcg tcccacattg atttggctgc cgatgtgacg acgttcatag 25680
atcgctcaag cgtacccatg ttcgccttga tctcattggt catgctggtg aacgacgcag 25740
cggctgtttt gttagccaac gcaaccgcgt ctgtctcacg acccgcatca acgagcgact 25800
gtacctgctg gatctgcgct tcgtttacga cgtgatactt atccgccagc gcacgcagtc 25860
cgccaacggg gtctgttgac agcttcgcca ccgccgctac cacatcctcg atcggctggc 25920
tggacacctt cgcaaagtca ttaactacct tagccaatgc gccgaagtca ccacccgctt 25980
tcacacccgc cgccgccagc gcctggattg tgtcacgcgt cttgcgaaat gacccgccca 26040
tgcgctcggc gttctcggac agcacaagga tctggttagc tgtgaggcct gaaatattac 26100
cggacagcgc cagcgtctta ttaagcgccg ccacctggtt ctctgcgcgg gtcgtaattg 26160
ccacccctac gcctaacgtc gccgttaatg ctgaaaggcc gatccctact gggccgagaa 26220
agcccgcaac atatcgcagc gtgttaccca gtccgccaaa cgccccagta agctgcggta 26280
actgctgaag cataacgcgg tgcaccggca tacccatctc aagcgtcacc gcaatatcct 26340
gtaactggaa tgcggcgttc cgcgatgcga atcccaggtt tttggtcgac atgcctaatc 26400
ggctggcgag tcgctcctgt ttcgtcatgc catcggcggc acgctgcgat tcagtcgcta 26460
acttttccat tccgcccgcg gcgtcgccag taaccttttt cgacttgccg agggtgtcgt 26520
ttaatttctt tactttctgt tctgtgttct cggatttctg cgcaaatgtg tccagcacag 26580
tattagccgt tttaatttgc gcgacgtccg ccttgagcgt gatgcccgct gcctgatctg 26640
ccattatgca gtcccccttt tttatgcagt tgccgtcggt cattgtatca gcgaatttaa 26700
aacaaacctt attcagtctc agacgagggg ggccgctgac ccccgtctag tctgaaagac 26760
tgggagttgt cccgcctgtc ccgatcggtc aaaacccgcg gcagcactga ctgggacagg 26820
ggtgctgtcc cgctttgtcc cgcctcattc tgtcccgctt tctgtcccgt ttgttttatg 26880
cagaaaaacc gcatagccat tcacttttga ggggcgggac agatcgggac agcacccctg 26940
tcccgctttt agtttatgca atcgtagtgg gtaaatatgc agtttttaga tgtgcgggac 27000
agaacgggat aaatcgggac gtcccgcctg tcccgatttg ctatgcgggg ttatgccgcg 27060
ccatttcgtc cagcgcaatg cgttccatta gcttaatatc tttaaacgca cgctgcgcgt 27120
tttcgatgcc gtgcatctta aaaagccatt ccagtacgcc gtagtcaatg ccgtatgcac 27180
caccgaatcc ggtgcgccat tgcgtggagc aatctgtgaa gactgcgact accttcgcat 27240
tgtcgggcca tagtgcaacc ggggggcaaa catcttccgg ggtcgccccc cataaactcg 27300
cggctaaatc ttcggagggt ggcggcgggc cataaaagcg tcgcgccgcc tcaattagtt 27360
tttttcacgc aatcccatca gttcgagata gtacgaagtg tggaacacgc cgaacgcgcg 27420
cggatagttg accaccaggc ggcggacgtt ctccgcgtta aattcgtccg gcagattcca 27480
gccttctgct acgtgcatga tggcctcgac cataacgtta aagccgtccg ggtcattgtc 27540
gtcatattcc gataacttgc gctcagtatc ctgcatggtc tgcgaaaact cgtcgatcgg 27600
acggtggcgc acggtaaacg tcattttgcc cggttcgttt tcgcctgggc gcgggatttc 27660
aatcggcagc ttaaaagtcg gcttagggtc aagtgtaaag agtggtgctt tagccattgt 27720
agtattctcc aataaaaaaa gggccagtac ggccccatat taacacccgc tatttaagcg 27780
accaaatcac gaccgcgctt caaccaggtc cttacggtaa ataaccatat cggattgcat 27840
agtgagggtg gactccaccg tctccacgtt gtttacttca gccgacggaa tcttgcggaa 27900
agacacctta gccgggtaaa tacggatttc acctttgccg cttgccgctg ccgggttagt 27960
gaacttaatc acagtggttt tctgtgtgtc gtccaggtct tccagaatcg ggcggatcgg 28020
gtcttcttcg tcgtgggtga aagtgtaggt ctgtaccaac gggtttttcg tggtgttcag 28080
actgatcgcg gtattcacct gcaacggctg gaaggtagtg gtctgctggt caccacccga 28140
cacggcaacg ttagtgataa acgggaagtc gataaaattc gacactttca ccacttcgcc 28200
tggggttccc gccccaaaag caccggcagg atatttggtg acgtccgagg tgtcgaagcg 28260
cgccagcgta acggcgttct cggctacttt acctacgata aacgcgcggt tcagcgcacg 28320
gacccacggg gttttagtga acataaccac gtcaccaact tgcaggccat gagaagccgc 28380
gcaggtgata acgcaacctt tcgtaagatc agagatcgac gcggcggcgt tggtagctgc 28440
tgtaacagcg atgccggtgc caagtacgga cccaatctgg acgctggacc cgttaggaag 28500
ttgataaccc ataatgaacg ctcctgttta gatatacggt tcaatgatac agcacgcgca 28560
gaatagcgcc aaatcaggcg caacaggtgt aggggactgt aaccacgatg cgggcgcgat 28620
caacgtcctc aaccactgaa ctggtataag gcgagtcctg aagctggaag gccgctccgg 28680
aaatccttac ggcctgccat ttatccgacg tcgcgatggc gtccgccagt tcgctcgcat 28740
actgtgtacc cgtgcccgcg ggaaacacta cggcaacctg atatacgcca gcatagatcc 28800
ggcccttctg ctggaagccc acaaactggg tcggggccgg tagcaggtac ggctccaggt 28860
agacaccgct gccgtttgcg tggtcgccgc tcacgttctc ccagttaacc tggatcgggc 28920
gctggctagt gctcaggctt accgataact cttctaccac gctgttaagc gctttgcgga 28980
ttgcggtatt gctcattatt tcaccttcga attaatttct gccgctacga cgcgaacaat 29040
ccccgcgggc gcttgtttac tccagccata ctcaagtcgc tgcgcgtatg gcacgttgtt 29100
agtaaaccag atcgacccgt attcactcga atcgtaatgc gccagcactt cacgcccagc 29160
ggctaatgtt gccgttccgg ccttgtcaac gcggtcaata gcaccgacag ctggtcgatc 29220
aaatgtcacc tgccagttac cccggaagcg ccccccggtg tagttcttcg cggcccagcc 29280
gcgctggcgg aacgtgacgt tgccgttttt agtcttgaat gataccagca cactggcgtg 29340
cttcttctgc ccgcgcttca gtttgccgcc attcttgcga cgctgtgcgg cgttaacctt 29400
gttcgcgtgc tggcgggcca ttgcataagc cttgttaatc ttccaccggc gagggtcgcc 29460
caccggggag atctcaataa ggcgtccgag tatttgcatc ccgtacgccc gcaccgcctt 29520
atcgttgttc tccttcgtct tatccaccca tgtagcaata gtggcggcga atgagtagac 29580
ttcagccacg ttagcccctt agctgtagtt gaaacagcat ggtctggccc gccggttgca 29640
gggggttggg attgataacc ctgtagtctg tattgttcag gctaaccaaa tccccgacct 29700
tcacctgctt gacggcctga cacaaaaact tcacatcccc ggcaacgata cgcgtcccgt 29760
cgatctcgct tggcttgtac tcctcgcgaa gtccgatgac gttaaacgag gtcggaggga 29820
tgaccacttc gtcgccgtct acgcggtcga ctgtaccggg gcgcgtcacc gtagccgtca 29880
tgccaaactt tttaatcaac ggattaactt tccgctttaa gcccgagtaa ttaaagcccg 29940
ccattatcat cctcctgtac cggtggttcc ggttctggcg ctggttccgg ttctggttgg 30000
cgtttagtat ttgcggcgat aatagccgcc agtaatgcag gattcatatc acccccggaa 30060
tacgtcaaaa ttacccgctg cgttgccgtc tgaatcaatc cagtggccca gcaagccatc 30120
ccaccacggg aacgagacac cgctaccaat cgtcgcgggg tcatattcca tcgtgatcgg 30180
gccaacagtt tcgcggatag tctccttacc gctacccaca ggagaaatat caacctcgtc 30240
cgccaccaac atcgccaacc tgtaaacggc ttgctcaacg gcaacgggga ttgatgcgaa 30300
gtctacgatt tgccctgcag gcactactgc caaaacttca gtgagcgtgt ctcgcaccgg 30360
cttaccgtcc gactggtaat taaggcgggg ccatgcgtcg atgccggtct ggtctgcggg 30420
ttcgccgatc cagttgatgc cgttaaggaa gtcattaacg acggtgaggt gtcgcgttgc 30480
atcgcccagc gtgacttcaa cgcctcgcgt tgcggcataa tcaacaaagg tttgcgggtc 30540
gccatacatt gtattcactc ctataaaaaa ggcgggcata agcccgccca tttgggatgt 30600
gcgcctgtta cgacgcggtg gtagcagtca gcgtaaccag tacgcccgcg gtttccttga 30660
tgttacgggt aggcactgcc tgcgctgtct gcgtacggcg gcggcctttg gtatcgctat 30720
cgctgccaac atcctgcacg gttgccggtg cgttgtctac ctggccctgg tccagttccc 30780
agttggcctt atcggtgata tcggacagct tgaacgagcg aacgccttcg accggagtac 30840
gagcggacgc cttcagacga tagcctttaa cagccacgtt gaagtcgaac tcgccctgcc 30900
accagcgctc aatgttctcg ttaccgcctt tttcctgcgc cagcatatct aggccgttgg 30960
tggtaactgc aaccgcgcca gggaccagac ccagcatttt acccgcgccc attgcatcgg 31020
cagcggcatc ggagataatg aaacggcggc ccaggccatc gcccattacc tgcagatcgc 31080
cgatcgcgaa tacctgctcg gcagatggaa gcgcctggta agcgatgaag tttgcccagg 31140
taacgccgtc cataaaccag gacttaatca aagcagcctg atcgccgaac ttagaagccg 31200
ccagcgggaa gtctgccagg gtcgggaagg tacgaccgcc aacgccgtca acgcgcgcag 31260
gttgggtgta gtttgccgct gcgttgcttt caatagcggc tttgcccgca ccgatacccg 31320
ctttgaggta atgcagcata atcgcttgag tagcctgtgc cgcaatctca gccgcaacgc 31380
tattaacgtt ggtttcgatc ttagccatca tggccttagt gattgccacc ggacccactt 31440
tcgcggacag gttaaccgag ttggtcagca tacgcgccag cacttttgcg gtcgccggag 31500
taccgacagg ggcgtaggcg ttacggtcgg taacgaggtt ggcgatcaag cctacggaca 31560
ttttttctac aacgtccttc agaacttcac ccgtgccgag aacaacagca ccgttagcgg 31620
cagcgttgaa cacgttcagg ttatcgggaa tcatttgcgt aaccgcggtg acgagtttac 31680
gctggaacac tgttaaagac atattagaat cctttgagtt gtgagcattc gctcagtgtc 31740
gttaatgtta ccttagattt acgcgctcgc aaaatcgccc gctttcgcgg gcgatctgct 31800
tactcgtcgt cgcccatttt agcgatgata tcccctgcct gtttcgttaa gtcagtgata 31860
ccgaagctgt ggcccatagt gatttttgac ttcacatctt cgacaacctg cgtcggtttg 31920
ctaggagcgc cgcctgctgg cgtgccagcc aggacggatg caaaatcggc gttattacgg 31980
aactcttttt caagctcttc catagtcagc gcggacggtt tgccatcctg caagacccgc 32040
actttcaagc tgccgtcttc gccgtcttcc agcgtcaggc ggtccataac gtgacgttgc 32100
atgattccgg cgttcttacc gaacagcttc gacgccagat ctttggcggc gctgccgatg 32160
gtcaggtcgt ggatctgctt gcggtaggct tcgattttcc ccgatgtgtc cgcttcatgt 32220
tttgcgaact tatctttcca ggatttgtcg attgcttcga ggtcaccatt tttgcgcgcg 32280
gcggcttcct ctttctcttt cgcaaggcgc tcggcttcgg cgcgacgttc ggcttctgct 32340
ttcttctcgc taaccagctt cgcgttgttg tctttcagac ccttgatctc cgcctgtact 32400
tcttcggcgg tcatgaacgt ggatttatac ccatcgccat cggcaacaaa taaagccttc 32460
atgccttctg gcaacgcgtc atattctgct ttagtaagtt tcatgctatc cccctgggat 32520
tgtggcaaag cgggccaccc gcttgacttc acgccttaat attatcagcg ataaaaataa 32580
tcgcaaaatt cgcttgcatt gcagatgttg gttgcattat attggttgca caccaacaac 32640
gaggaaatga taatgattaa agttaactat gccgatctta aagccgctat gatgtgcgcc 32700
gcaactaatg acgtgcgata ctatctgaat ggtgtatttt tcgacgagaa aggcttcatt 32760
gtggcctccg acggacatcg tttgttctta gggtccgcgg ttgttcccga aggtgaaagt 32820
aaaatagtat caattaaagg ccgcttacct gctaaattcg aatactgcaa cattgacgac 32880
acctccgcag cgttctttga tagtaaagac gtattaatag ggaccctgcc cgtagagaac 32940
atagacggac gattcccaga ctggcgtaga gtttcgtctt tcgccagcgc taaagtggag 33000
gctatcgggt ttaatggggc atacctatcc gacgccttca aaattgctaa attgttcgac 33060
cgtaagtttg atagcttaaa actggaattc cggggggtcg acaaggcgac acgtgtgtcg 33120
ttcaaaggtg gggccttcct tgttatcatg ccgatgcgtt tgtagcgaac aaagcggcct 33180
gttggtggcg gcatcaaatt caactaaacc aaacggggcg aaagccccaa caacgaggac 33240
acaaaaatga gactgacgaa agacattaaa aatcagatcc tggctaacat cctgcgcgac 33300
cacgaaatcg caaccgaagc caaatcaatc atgatggact cccgcgatct ggcgtacgca 33360
atcacgctgg actgtatgcc ggacggcatt cgcacattcg ccgcgctacg cgagcatatc 33420
ctgttaatcc tcgatgacca gtcgcagatg tacggcgtca gcgtatacac caacaaggcg 33480
acgtatagtt ataacaccga ccgtccgaag ggtatcggca gcgtttacga cgccactttc 33540
gaaatcaacg caggcggaaa tgttcgcacc ttgtccttga gcggtgacgg tcgccaatac 33600
cgcgtacatc gcgggtactg ggaagagaaa tatatgaata tgctcgcgcc tgatctcggc 33660
gatatcgttc cggggttcga gacggttatg ctcgcgtcag tgtgcgaagt ggacgaggac 33720
gggaaagcgc tgccgatata tcttccacgc ggtcgcgtag actacccggc agatcatgat 33780
ttctgtaaac gcctggaccg caacgacaaa gcccgtcgcg agttacgcgc gaagtatgat 33840
gcgttcaaac tcaccgttac cggtgtgctg gacctgcaca atactgacaa gaagctgatt 33900
aaggcatggc ctgaagttga gcaattcata ccatacccgg acaagccaaa atccacggcg 33960
gtggcgctgg atgtgaaaac tctcaacgag atttgcggta ttccgcgctg acgaaaaggg 34020
gccattacgg ccccttaatt atttgctgca actccaggac ctgcgccctc agcttcgccc 34080
cgcattccat atttttcacg tcaatcatca aatcctcgtc ggggtctgcg gacgggctat 34140
cgaacttgca cggcggtcgc agcgccgact ccgttagcgg catcaacggc acgttgcttg 34200
atccgcacgc gctcagcatc aaaactacaa tcagggcggt tagggcgcgt aacatattta 34260
acaacctctt tcgtgatagt ccggtattct acgtcgcggg cctgttcggc ctgcacggag 34320
cgctcaacaa ctttagtttg ctgccgttgg gcttgggctt gcgcttcttg cgccgcctcc 34380
gccactacgc cagcgcgata ggagtcacct agccagaatc ctaccgccag accgacaccc 34440
agtccaatcg ctgcatattt aatcacgcca taacccccgg tttttaagct gcgcaatagt 34500
cattagttcg cctgaatcgg tgaacatctt cggcactttc acgccgcgca taatctggtc 34560
ggcgcgttgc actccgtata attcttccag gatatggcgc ggctggcgct gcacccatga 34620
gaagaaatca gtctgcgcgt cgacctgctc actcagcaat cgacccgcgt cagccttcag 34680
tgccggacgc ttaccactgg gccaatcttc catgccttta accttccacg tctccgtcga 34740
ccggcaacaa taatgcaact ttcccggacc agcgccatat tgtgacccag ccaccatttt 34800
cccgccgcgc tttcctttgg tgtcgggagt aacatcaacc ggataaaaca ggcgatcgcg 34860
tagctggcac atgggagaag tatgcgtgtc caatgtagat aaccattggc gaccttcgag 34920
aatatcgtca ttagctttta ccattaattc gcgggctgtc gccgcatagt ggttgactgc 34980
ggacttaaca acgcttgaga tagcttgcgc actacgaccg ccgagcgcgc gcctcacgtc 35040
cgctataatt tccatagtcg gcttaccctg gatgaatcca gcgcgcacct gattcccgat 35100
ctgcgtcctg gtccactcag acaatgaatc gggccattgc atcatggtat ttccctggaa 35160
cgggtttttc atcgcagtcg ctgcgatctg cgcaccggtc actacgccaa ccacctcgat 35220
cgcaggtaca gcgaccaccg gctggatcgc tgccgttaat gtgtcggcga gataatcggc 35280
ctccgtgtcg gcaaactcct gaaggctttc agccaacgcc ttaaactcgg tgcgcaattc 35340
tgctttaact gtcttgtcga gttgcgttaa cagcgtggat agtgccttgt tattcatgga 35400
ttttttgcca tcaagtaact cggtcagctt accaagtaac gagggacgga acttctccca 35460
catagccagc accctccgcg cctggtcatt gctcaggcgc tgggtgaata tgtgacggcg 35520
aatcattcga tccgccatat attgattaat tgtcttcgcc attgtccgct accttgtccg 35580
ggtcttcttc ggtagattct accgcatccg gggacatttg aggcaatgga ttctgatttc 35640
gcaattcgtc ttccacctgc tcaacggttt gcgagtcatc aatgacgccc tgggccatca 35700
tccacttgat aaagtccgcc agacggaccg taccggtctg cacaccagcc atcatcgcgg 35760
tcataagctg tgcgtcaacc gtgatttcgg tgtagaactt attcagcgtt acgcgttgat 35820
ctgcggtgtc gccagtgaac agtttgacga tttgcagcgc acggttgaat gccttctcaa 35880
cgttaccggc aatcagtgac aggatactgt tatcagtctg cgcatcgtag gccgcttcag 35940
ttgccgtctt aggtgcggtc cccttctcga ccagggccgc gcccagttta gccatttgca 36000
attcgcgtcg ctcacacagc gcaactgaca ggtttctctc ctcagcctga agcagtttag 36060
cgtccatgtt ctggcccagg ataacgccct tagtcgcgcc tagtgcgatg ccacccttca 36120
ggtttttatc agcccatgat tgggtaagac cggtcgcaat aagtgtaggt tggcccacaa 36180
tgtgcgcaat ctcggcgacg tctgcttcgg cgttgtagtg cttaatgttg atggatgcaa 36240
tatccgccag cggtgcggcg tccggcgtcg cgttgttgtc cacagcacca caccagcaaa 36300
atggcagttg agtaagcgcg ttaccgtcag catccaccag tggaacaagg tcggttcggg 36360
tgaatccccg cggcaagtcc atttgcactt ctgacgtgtc cgcattgtaa aaccagcggc 36420
gggcgtgtgc cttgccgtca atcatgcgaa gctcggtcca gatggtcacc tcgtggtttg 36480
cgaagtcgtc ctggtcttcg gtgctgattt cgtccgtttc cttcagcacg attagcgtgt 36540
cgaccccgtg ggtctgccgc cagttgatga tctgttcagc cgtatacagg cggataagcg 36600
ggcggtgtgc cgccatttca gcggctgtct gcggcactac gttacccagt tcgtcaaaat 36660
gcgcctggcg gtcatagtcc accataaagc ctgcgcggcc cgattgcagc acttccgaca 36720
tagcgccgcg caggagttgc gccaggggca tcccgctgcc gtctacgtct tcgatcaggt 36780
cagccattgc cccggaaagg tccatactca ccggtttggc gaaggcgacg cctaacaagg 36840
cattcagtgt gcgggcggtg gcgttcagga agacagcgcg ttgcttatat gccttatagc 36900
gcgcacgcga tttcgggtct gtggtgtcgc cgctggctgg gtgtggcaga tacaatggtc 36960
cgcacgcctt cacggcgcgc tcccctgcta cacaatcgcg aatcattttc cactctggcg 37020
cgatgcgcgc atagagtggg tgctggttgt caacgttaac ggtcatggtc ggtttcctta 37080
gtagaagtct acagttggaa caactgcgat cggacgaatc acagggaaca agtgcgcaat 37140
agggtagccc gcggcatcgt tcatgtggtc caccccggcg gttttatccg gctcgccttt 37200
cttcaggtcg taaacctgct gttccagaca tttcgctagt gtagggcatt tatccaggtt 37260
gacaaaatac tggcgcacgc ccttcgagtt gcacatcatc gtattcatcg caatcaatcg 37320
gtctttcaca ggcgggttaa cgctgtcata ctccacctcg aagcccgcat cttccagttg 37380
tgcgatatcg gacgacgacg cgttgaccgt cttgcgggat ttgccgctgc tatccgggta 37440
aacgacaaca cgaccggcag cacaatgatc ggggtagcgc tcctctatcg cctcaatcat 37500
agccggggtg tcaaacaggt ccacaaattc atccaccgcg tgaatctcgt cgcggaactc 37560
cttgttctcg gtgactcgct gccgtctgac gtacacaacc gccgccatct tcgtgacgtt 37620
aaagtccata ccgataatca gcgtgtcgtc cggcttcact tcttcggtgc tggcgttctc 37680
ctcgcggttg aacatcttgt ataccgcgcc cgacgtaagg ttaacaaaca ggccgttcag 37740
gtacgcgtcg atcaggttag ctgggtactg actacggagc gtgtcgataa agtctgccgg 37800
taaatggtgg ttgtccgtgg tcttcgcccg gatcaagcgt ttctggttgt ctttctccac 37860
ctcgaaaatt tggtacatcg cccggtagcc ttcaggcgtt gacacgatga cgaactggcg 37920
gacgaagccc gcacgcagac gaccgagcaa tttgtgatat gccgccagcg cgacgtcttg 37980
cttcgtagtg tcgaattcat cggcaacaat ccacgcggcg ttgaccccga tcaggcgggt 38040
atagttctcc attgattcac agataacgcg tgtccatttg cctttcacca gcacattgta 38100
gatcttatcc tgcttattga acttccaccg gaagccagcc tcgtcgaatg ccttctccag 38160
ttccggatac atgattttaa ccaggagtgg aatagtcggt tcggtcacga tgccgtcata 38220
accagggtta agtgtcagaa gctggatgac cttacgcgca gctacccacg actttccgcc 38280
accgaagccg gaacacaggc cgaggatttt agtctttgtg tcgcgcagta gttcaacctg 38340
gtgcggcaag caatcaggct ggtaaaggcg gacagcctgc gcaacgcgtg tcgggcgcgg 38400
aggtctgttt cgctttgcga tcgctcgttc aagggcgcgg tgttctgccg catagttacg 38460
cattattcac cctggccttt gccggaaggt tcgacaacta cgtcgccgtt atcatcttcg 38520
tcgtcatcca gcggtacatc gtcgcggacg ccgtggtttg ctttaagcag gaatgtagcg 38580
aagcccgccg cgatggtcac gccgccagct tccatcagga aggttttctg caactccatt 38640
gcgtccgcca tcgcctcggc aaactcctca tgcgcacgcg cccagcgata caggcaagct 38700
acgcccacgc cgatttcagc ggcaaatcgg ccgaaggttg gcattttgtt gcgggggatt 38760
acttgcgcag cgcctttatc ggagtagttg acctgccagg cgtcggcgtc ggcaaaatag 38820
cggcgaagct gatcgcagta aacggatctg tagtcagtag ggcgtccgca tttgccgcct 38880
ttacggcgct ttccgtactc gatttcaggt ccaagattgg tgtgctcggt ttgctcgcct 38940
ggtttacggc gcggggttac tgctactcgt ccgtccagtt tggcgcgtcg gcccttaact 39000
ttgatttctg ccatgatata agcccttttg ttcatatgcg ccgaccatcg gcaacatagg 39060
agacggccct gccatctaag ggggattata acaggcacaa aaaagcccac caaatcggca 39120
ggctatgcgg tttaaacgtc gcgcccgtag tatcgggttg acatgctttg tacttcgcgt 39180
atagcgccct ctgtgtagcg tatttgcgac agtatctcga ttcgctcatc catcgacata 39240
ccgactgccg ctgccgttcg ttcgaatcgg cgcaatcctt ccagttgttt gcgtagttct 39300
ttcaggcgtg cggcctgcgt gctcatgaat acttgtctga tcatttcaac atttccgggc 39360
taatggttaa gcgcgctact tctccgtagt tggcgctata ggtgatgacg tttgcgctgc 39420
gaccgctcat ccagccgcca cgactggcgt aggcgtcttt cgctgcgagc gtcctgtgct 39480
gttcgacaat catgttgcgg gattcaacaa tcttctggtg gtgcaaatgg cctacgtgcg 39540
cgtagctgta ttcgctcgcc ccgaacgcct gtctgaattt agcgatcata acctgctcga 39600
taacatcgaa tcgcgctttg tgcccgtggt ggaaaaatag cgtagtctta ccgtgctgca 39660
ccattttgta gacgtccggt gacgtgtcca catcgacgcg cggctcgttc tcatacaagg 39720
tattgaacat ctcagcaagc caaatcatac cagattcgtc atgattaccc tggacgatta 39780
gcagttttac tttgtggtgt ttttgcagcg ccaggtctac cacgttgcgc accatgcgaa 39840
tcatatagcg gaccaacttc tggtatcgtg tgtcggcgtc cagtacgtga ccgcttgccg 39900
gtgtgacggc atcaaggcta tcaaagtggg ccatatcccc gaggatattg ataacggcag 39960
ttcctgcgtc cggtgcgcgg gcgaaggctt cgacaaacca gcggtagaac aggtcttcag 40020
caatcgccat atcccagtct tcgccagtct cctcacccca tgccagcatt cccaggtgaa 40080
agtctgacac ggtgtacagg ttgagcaagt cgagtgcctg gactgggact ggcggggctt 40140
ttactggcgc tgccggtgtg atttcgctgt tcataccatc gacaacagcg cgcatagctt 40200
cgagttggcg ctctttgtcc tgctcagtga tgacccacga catgacctct tcgccattgc 40260
cgcgcaccag ggccgatacg cgcttaaccc caaaaccatc ggcaacgcgc ttacggatca 40320
ctgcatcatt gccgtgacca atcccctttt tagacaggcg ggctttgcgc atatagacat 40380
ttcgaagtgc gatgccgtac ttcttcgcaa tctctttaac tgtagtccct tcctgaattt 40440
ctgcgatcaa ttgctcatcg gtaattttag tttgcgggtg catatttaat taattcctga 40500
ttagtggacc gcggacaata taccacggtc cgtatggcgg cgggctatat caattcgcaa 40560
ataaccatgt cgataaaagc cgtggtggtc cagatcgcta atacccaaat atctttacgt 40620
gccatcttga ttactcctct cgttgattgg tcgcagtata gagtctatgt tggttgcaag 40680
tcaagcaata aaaagcccgg ttaattccgg gcttgattgt ttacgctgca ggaccagatt 40740
cctcctgcgg tggtgggctt ggcagcttcg catcgaagac cccagcgtgg ttgatagagt 40800
ccgctatgat agcgcgtgcg cctgggtctt cggttttagc cagtgcggca agcgcgtccg 40860
cattatgatc aagaatgctt tttaacatac tgtgttacct cgtattatgc agcgttacga 40920
ccgattcgtt cggcaacgtc ccactgctta ccatcaaaca acgcctgtct gccgattgca 40980
cggcgtacca gacccttaag cacttcgccg ccttgcttgc ggaactgcgg cagcgtagca 41040
cggaccttcg cccagtcgcc caaccgtacc gcatcatcga aatcattcgc gacgtcatcc 41100
ggtacgatcg gaccaggacc ggcgttgatt accaggtcca ccagcgcgtc gaattgcgcc 41160
tggttaatgc tcggatgtgc gtatttgtta acccagtcct cagcatacgc cacatcgcgc 41220
ttgagcagtg ccagcgcttc ctcgcgggta atgcttcgag gcggattagt gccggtatga 41280
ccataacccc aggtgtacag gccgcgggcc tgctctttct tggtcgcgaa atacggcacc 41340
gggctgaaag cctcccaggc tgccgtaaat cgtagaccgt tatcactgaa tcgcatgttt 41400
taccccattc aaaacagcat tacgcagggt gcgcaatgca aacagccaga ttaacgcggt 41460
ccatgtggta tcagatacgt ctcctgtttg cagcaatccg ataatacaga ccattgccgt 41520
taacagatac agcacccgac cgaaaatgcc atcccggacg gacggcgcat acacgttaaa 41580
cagggacgaa gccccgatga taacaaacaa caggacggag ataatcattt tgcgcccccc 41640
ggaagtcgga acgacgcaat agtctccctg atccggtcgg cgattggcat ccagaagatt 41700
gcggttacga agcctaaacc ggcgacagtg cgctcacccg acaaaccagc ccactcggca 41760
atagccggtg cgccaaatat ggcacacaag aagccaacta caacatacaa aacgaagttt 41820
agtggcccct tcgtagcctc accatgaatg cgtgcgccta cgaagccacc cgccagacat 41880
gccagggcga atagccactc gtttagctta tccataaaaa agtcccagta agttttagct 41940
cactgggact atatcacgct tgcataatta atcgaaaacg gaccacttgc cgacggcttt 42000
ccactcccac ccggatgagc ggacggcttc gaatgttcga ccgcctatca tatcccgata 42060
ccacagccac cacatccgaa gcctggtcgg gtgctccgtt attccacggc gcttgtagat 42120
ctcgaatgtc atccgggtaa tgctggcagc ttccaccgca ggccatacca ggaataccca 42180
tattgcgccc agcactcccg taaccaggca taacatgctg gccataccaa accaataaat 42240
cacatcatga atcataatct gccccatagg tatttaacca gactaataac cgacgccaac 42300
agcaccaggg ccgccagact accgccacat actaccgcga agacgtatag catcgaattt 42360
ccccttccgg tttgccaata tggcgcgccc atgcacgcgc cacgaagtaa tttcgaaata 42420
cccgaccgcc tatttcaaag cggttgtgct gaagcatatc acggatcgtc gcactcattt 42480
cagaatcacc gagcgcgggt cgatggcttt catgctgccg tctcgttcca cctggatgaa 42540
tccaacaccg tattcctgcg catggcgttg tgcgttcgtc acggcatcaa agcgattgac 42600
gatttggtgt acggcgcggc ccatcttcgc ttgcacctgg tgggagtcgt aggttgcgcg 42660
ctcatgaggc tggcggttgc gcaggcagta tttgcgcccc acgcgctcgt agtggattag 42720
cccgaagtac gtcataaccc tctgtatgtt acccactact ccgcccttgc gggtttgcat 42780
acgcaagccg tgctggctgg tgtgctcaac gacccacaat tgcccgtcgc gaatatcacg 42840
cagcacatcg ccgcgctcaa tatcatgcgt gaggatttcg tatttctccg gcagctttgc 42900
cttattcgcc caccagacac gatcgcttgc ggcgtggtga agtcgcacat gggtgtcaag 42960
ggtctgtatt ggcttaagaa tgcggccatc tgattttcgg cggaatagta atttcatttg 43020
ctcagtctcc agtgtttgcc tacccgctcg actgcgggct tccggcgtaa tgtaatacgg 43080
atccccattg gttgcaagga ggtccgcaca tagaaatcat gttgcgcggc gaactggtgc 43140
aagtagcgag tctgcgtcaa cggcttaacg tcgtcgcgat agcaccagta cacagagtcc 43200
cccgcattcg cattcttagc ccatattcgt atgcaatgca gggggatatg ccacgaaagc 43260
tgttttgcgc gtctacgggt cattttggcc tcttatttcg caagctgggc gaatagctca 43320
cgctgggctt taatccgggc aacgtttgtc actgccgtct ctccgatccc gaagcgatac 43380
gccaggcgtt cacgggcaaa tgcaagcgcg tagtcctgga tagcgttcgc caacggctgg 43440
atctccgcca tcgtgtcgaa gcggcgctca agcatacagg cgatggcctg ctgcgtgacg 43500
cgaacaacgg accacgctgc cgatagtctc tctactgctt ccgccccctc cccggcgaac 43560
tccgccgctt cttgtgcctt cgccatagcc attgcgtaat tccagataat cgctttcatt 43620
gttcagccct catgttggtg tgcaaccaat ctagcagggt gctggcatga atgcaaccaa 43680
tattaatcat gcgcatgaaa aagttgatcg ctatcagctg tgactgcaaa ctgcattttt 43740
cacaccacat tctgcaactc gcattggttg cccagttcat cgacgaccgc gatttccaat 43800
gtgctgccgt gtattttgca atcccggatt ttatgcagtc tcagacgagg ggggccgctg 43860
acccccgtct agtctgaaag actgggagtt gtcccgcctg tcccgctcct ctccaggccg 43920
cgtcatccgg ggcgggacgg gggtgctgtc ccgctttgtc ccgccccaag tttgtcccgc 43980
tttttgtccc gcttacttta tgcagtttta ttgcataact attcactata tttatactgc 44040
acggcaacca atgcgaccag ctaaaaacac tcaaaat 44077
<210> 2
<211> 3189
<212> DNA
<213> DNA polymerase of bacteriophage EP23
<400> 2
atgaaagact tcgaacgcct gtttctcgac accgagacat ttagcggggt ggacctgaag 60
aaagttgggg cgtacgccta cgcggagcac ccaaccacag agattatgat ttgcacctac 120
gccatagacg agggccgcgt gcagacatgg gatgccactg aatcccccac aatgccgcgt 180
gaactgcgca aggcgctccg ccgggtatcc cgcaagaagg ctaaaatagt gatggctaac 240
ggcctcctgt ttgaccgcct ggttatccgc gagaagtggg gtattgacct gccggtaagc 300
cagatcgaag acactatgat tatggccttt cgccacgcgc tgcccggtag cctcgatatg 360
cagtgccagg ttctgggcgt tgacgcagag cacgcgaagg ataaggcagg caaggcgctg 420
attaaccgat tctgtaagcc tacccctaag acctacaaaa tccgccgcta cactcgcgag 480
actcacccgg aagaatgggc caaatttctg cgctatgccg cgctggatat tatcgcaatg 540
cgtgaagtct actggcgcat accggactgg ggaaactcgc ctaaggaaga tgaaatcctg 600
ctcatcgacc agttgattaa cgaccgcggc ttctacgtcg acgttgacct ggcgaacgcc 660
gcgatcaaag cggtgcaggc gcataaggaa gaattgaagg aagaagcctg ggagcgattt 720
ggcggtaagc tgacaggcaa tgacttcctg ccaatcctgc gtgatatcgc cccggcgttc 780
acgatccaca acgcgcagaa atccacgctt aacgacctgc ttgaagaccc ggacttcccc 840
gacgaaggca aagcgcttat cgaaatgcgc ctcggggcgt catcaacggc atcgactaag 900
tacaatccgc tagttaacgg cttatccgca gatggtcgcc gccgtgggtg tcttcagtat 960
ggcggcgcaa agcgcacgct gcgctgggcg ggcaaaggct tccagcctca aaacctggcg 1020
cgtggcgagt acagcgacga ccacgaaggg aagattaaac gccgcgaggg tgaaagcgac 1080
gtctcgtttt gggtccggtc gcacatgctc accaacggca tcaactccct gttacgcggg 1140
accgcacact gggcgtatga catatcgaag ctaacggcct cgaccgttcg cgggtgcatt 1200
attccggcga agggcaagaa gtttgtcgta gcggactact ccaatgtgga agggcgtgga 1260
cttgcctgga tcgctggcga gaaaacagcg ttaatggtct ttaaggccgg tcgcgatatc 1320
tattgcgaga ctgccggtaa gatgttcggc cttgacccgg attacattaa agccaaccgc 1380
aaggacttac gccagatcgg taaggcttgc ttgcaccgcc atacccaagt attgaccaat 1440
ggcgggttca aggatattat ggccgtaaca tcaacggata aagtatggaa cggcgaaaaa 1500
tgggtgagta caaaaggcgc gcaccttatg gggtggaaac cagtgataga cgtggacggg 1560
gtcttgatga cagaagacca caaaatcttg acgcattcat ggaaggaggc aaggcaactc 1620
gtttcagaca ggtgcacgat ggtccgcgcc ctggcgagag gtatggacgc ctggttatcc 1680
tgcgcgaatt accaaaacga cagggtcaca gacaactgct ggcccaatgt gattgcggag 1740
cgatgccagg acgcgtcggg tatgacaacg tccgaagggg taaaacatcc caatgcaacc 1800
tctgcgcaat tgaagcgaca aagcggtatc gtaaacagta tttttgctac gatgacgcaa 1860
tgccagacga tgcgcacaga acgcggctac tcaaccggct gtcagcggca atatcacgaa 1920
cgacaaaccc ccgcgccaaa gggtataaaa actacggcgg tcgtgggatt gccgtatgtg 1980
acgaatggaa gagagataaa aggtcgtttc ttctctacgt tcaaacgctg gacggctggg 2040
ataagccaga cttcgaaatg gacaggatcg atacagacgg cgactatgag cctggaaacc 2100
ttcggtttgt cagccgttcg gaaaactgcc gtaaccggcg cagcgtgccc gagcttcaga 2160
gaaagtacga tgaagccctt acccgcattg ctgaactgga acgggaagtt aacctattgc 2220
gagccggttt acgacctgat tgatgtggaa gacggaaacc ggttccttat tgcgtctaag 2280
tctggttttc tggtggcgca taactgcgag cttggcctgg gctatggtgg cggcgtcgcg 2340
gcgttcttgc agttcgctaa aaacctgggc ctcgaccttt acactatggc tgacgtcatg 2400
aagggcacat tcccggatca catttgggcg gcggcgaagc ggggttatga gtacgctcgc 2460
attaacgagg agaagcgccc gccgaagcct ggcaagaaag acgagcgccc aacttacata 2520
ctgccgaaaa acgtgtggct gacgtgtgac gcgatcaaac gtatgtggcg tgagtctcac 2580
ccgatgacgg tagccttttg ggccgaactg gaggacgcgg tgctctgcgc gatccgcaat 2640
ccgggcaaag cgtactgggc tggcgcgaac gttcgcccgg atggcaggaa agcgctcaaa 2700
attgttcgaa cgaaggcgaa gcatgacccg accttcgacg aggagcgcga cgacccgaac 2760
gcggcggggt ggtggcttaa aattgagttg ccgtcagggc gcatcatgag ctaccctggc 2820
atcgcgctgt ctgtcacgac cgagatcgac gaagacaccg gcaagaagcg caccagcaca 2880
cgcattaaat atcagggcga gaatcagacg acgcgccagt ggggattcca gtatacctac 2940
ggcggcaaac tgacggaaaa catcgtacag gcgttgtgcc gcgatattct cgcatggtcc 3000
atgcctggcg ttgaagctgc ggggtatgag atcgtacttt ccgtacacga tgagctgata 3060
accgaagtgc cggataccga cgactacaca accgaggagt tgtgcgcgct gatgtgcgac 3120
ctgccggttt gggcgaaagg cttcccactt gccgcagagg gcgattgcat gtatcgctac 3180
cgcaagtaa 3189
Claims (11)
- 대장균 또는 시겔라속(Shigella) 균에 특이적 사멸능을 갖는 박테리오파지 (기탁번호 KCTC 11428BP).
- 제1항에 있어서, 상기 박테리오파지는 서열번호 1의 염기서열로 표시되는 유전체를 갖는 박테리오 파지.
- 제1항에 있어서, 상기 시겔라속 균은 시겔라소네이(Shigella sonnei ) 균인 박테리오 파지.
- 제1항의 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 항균제.
- 제4항에 있어서, 상기 항균제는 대장균 또는 시겔라속(Shigella) 균을 사멸시키는 것인 항균제.
- 제5항에 있어서, 상기 시겔라속 균은 시겔라소네이(Shigella sonnei ) 균인 항균제.
- 제1항의 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 대장균 또 는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물.
- 제7항에 있어서, 상기 대장균에 의해 유발되는 질환은 대장균 감염증인 약학적 조성물.
- 제7항에 있어서, 상기 시겔라속 균에 의해 유발되는 질환은 세균성 이질인 약학적 조성물.
- 제1항의 박테리오파지 또는 이의 배양물을 포함하는 사료첨가제.
- 제1항의 박테리오파지 또는 이의 배양물을 포함하는 식품첨가제.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020090098609A KR101101376B1 (ko) | 2009-10-16 | 2009-10-16 | 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020090098609A KR101101376B1 (ko) | 2009-10-16 | 2009-10-16 | 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 |
Publications (2)
Publication Number | Publication Date |
---|---|
KR20110041670A true KR20110041670A (ko) | 2011-04-22 |
KR101101376B1 KR101101376B1 (ko) | 2012-01-02 |
Family
ID=44047484
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
KR1020090098609A KR101101376B1 (ko) | 2009-10-16 | 2009-10-16 | 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 |
Country Status (1)
Country | Link |
---|---|
KR (1) | KR101101376B1 (ko) |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101299179B1 (ko) * | 2012-04-18 | 2013-08-22 | 씨제이제일제당 (주) | 신규 박테리오파지 및 이를 포함하는 항균 조성물 |
WO2014133290A1 (en) * | 2013-02-27 | 2014-09-04 | Cj Cheiljedang Corporation | Novel bacteriophage and antibacterial composition comprising the same |
WO2014133289A1 (en) * | 2013-02-27 | 2014-09-04 | Cj Cheiljedang Corporation | Novel bacteriophage and antibacterial composition comprising the same |
KR20150117445A (ko) * | 2014-04-10 | 2015-10-20 | 씨제이제일제당 (주) | 신규 박테리오파지 및 이를 포함하는 조성물 |
WO2016108538A1 (ko) * | 2014-12-29 | 2016-07-07 | 주식회사 인트론바이오테크놀로지 | 신규한 장출혈성 대장균 박테리오파지 Esc-CHP-1 및 이의 장출혈성 대장균 증식 억제 용도 |
WO2016108542A1 (ko) * | 2014-12-30 | 2016-07-07 | 주식회사 인트론바이오테크놀로지 | 신규한 장침입성 대장균 박테리오파지 Esc-COP-4 및 이의 장침입성 대장균 증식 억제 용도 |
WO2016108540A1 (ko) * | 2014-12-30 | 2016-07-07 | 주식회사 인트론바이오테크놀로지 | 신규한 장병원성 대장균 박테리오파지 Esc-CHP-2 및 이의 장병원성 대장균 증식 억제 용도 |
US9950018B2 (en) | 2014-04-15 | 2018-04-24 | Cj Cheiljedang Corporation | Bacteriophage and composition comprising same |
EP3253215A4 (en) * | 2015-01-23 | 2018-07-18 | Intralytix, Inc. | Novel shigella bacteriophages and uses thereof |
WO2018101595A3 (ko) * | 2016-11-30 | 2018-08-09 | 주식회사 인트론바이오테크놀로지 | 대장균 박테리오파지 Esc-COP-9 및 이의 병원성 대장균 증식 억제 용도 |
KR20190018968A (ko) * | 2017-08-16 | 2019-02-26 | 경북대학교 산학협력단 | 병원성 장내세균에 대해 사멸능을 갖는 신규 박테리오파지 및 이의 용도 |
US10265355B2 (en) | 2014-12-30 | 2019-04-23 | Intron Biotechnology, Inc. | Shigatoxin-producing F18 type E. coli bacteriophage esc-COP-1 and use thereof for inhibiting proliferation of shigatoxin-producing F18 type E. coli |
US10286020B2 (en) | 2014-04-15 | 2019-05-14 | Cj Cheiljedang Corporation | Bacteriophage and composition comprising same |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016003307A1 (en) | 2014-07-02 | 2016-01-07 | Limited Liability Company "Bphage" | Bacteriophage strains, compositions and related methods |
US11058131B2 (en) | 2015-04-16 | 2021-07-13 | Kennesaw State University Research And Service Foundation, Inc. | Escherichia coli O157:H7 bacteriophage Φ241 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK1458856T3 (da) | 2001-12-13 | 2012-07-09 | Nestle Sa | Isolerede fager og deres anvendelse i fødevare- eller kæledyrsfoderprodukter |
KR100943041B1 (ko) * | 2007-09-20 | 2010-02-19 | 재단법인서울대학교산학협력재단 | 시포비리대에 속하는 박테리오파지 및 이를 포함하는 세균사멸용 조성물 |
KR100949389B1 (ko) * | 2007-10-08 | 2010-03-25 | 재단법인서울대학교산학협력재단 | 박테리오파지 kctc11120bp를 함유하는 세균증식 억제 또는 사멸용 제제 |
KR101101604B1 (ko) * | 2009-04-22 | 2012-01-02 | 경북대학교 산학협력단 | 병원성 장내세균을 사멸시키는 박테리오파지 |
-
2009
- 2009-10-16 KR KR1020090098609A patent/KR101101376B1/ko not_active IP Right Cessation
Cited By (27)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101299179B1 (ko) * | 2012-04-18 | 2013-08-22 | 씨제이제일제당 (주) | 신규 박테리오파지 및 이를 포함하는 항균 조성물 |
WO2014133290A1 (en) * | 2013-02-27 | 2014-09-04 | Cj Cheiljedang Corporation | Novel bacteriophage and antibacterial composition comprising the same |
WO2014133289A1 (en) * | 2013-02-27 | 2014-09-04 | Cj Cheiljedang Corporation | Novel bacteriophage and antibacterial composition comprising the same |
US9862935B2 (en) | 2013-02-27 | 2018-01-09 | Cj Cheiljedang Corporation | Bacteriophage and antibacterial composition comprising the same |
CN105324481A (zh) * | 2013-02-27 | 2016-02-10 | Cj第一制糖株式会社 | 新颖噬菌体和包括所述新颖噬菌体的抗细菌组成物 |
US9657277B2 (en) | 2013-02-27 | 2017-05-23 | Cj Cheiljedang Corporation | Bacteriophage and antibacterial composition comprising the same |
US11785948B2 (en) | 2014-04-10 | 2023-10-17 | Cj Cheiljedang Corporation | Bacteriophage and composition comprising same |
US10577590B2 (en) | 2014-04-10 | 2020-03-03 | Cj Cheiljedang Corporation | Bacteriophage and composition comprising same |
KR20150117445A (ko) * | 2014-04-10 | 2015-10-20 | 씨제이제일제당 (주) | 신규 박테리오파지 및 이를 포함하는 조성물 |
US10925909B2 (en) | 2014-04-15 | 2021-02-23 | Cj Cheiljedang Corporation | Bacteriophage and composition comprising same |
US9950018B2 (en) | 2014-04-15 | 2018-04-24 | Cj Cheiljedang Corporation | Bacteriophage and composition comprising same |
US10286020B2 (en) | 2014-04-15 | 2019-05-14 | Cj Cheiljedang Corporation | Bacteriophage and composition comprising same |
WO2016108538A1 (ko) * | 2014-12-29 | 2016-07-07 | 주식회사 인트론바이오테크놀로지 | 신규한 장출혈성 대장균 박테리오파지 Esc-CHP-1 및 이의 장출혈성 대장균 증식 억제 용도 |
US10265353B2 (en) | 2014-12-29 | 2019-04-23 | Intron Biotechnology, Inc. | Enterohemorrhagic E. coli bacteriophage Esc-CHP-1 and use thereof for inhibiting proliferation of enterohemorrhagic E. coli |
US10265355B2 (en) | 2014-12-30 | 2019-04-23 | Intron Biotechnology, Inc. | Shigatoxin-producing F18 type E. coli bacteriophage esc-COP-1 and use thereof for inhibiting proliferation of shigatoxin-producing F18 type E. coli |
US10265356B2 (en) | 2014-12-30 | 2019-04-23 | Intron Biotechnology, Inc. | Enteroinvasive E. coli bacteriophage Esc-COP-4 and use thereof for inhibiting proliferation of enteroinvasive E. coli |
US10265354B2 (en) | 2014-12-30 | 2019-04-23 | Intron Biotechnology, Inc. | Enteropathogenic E. coli bacteriophage Esc-CHP-2 and use thereof for inhibiting proliferation of enteropathogenic E. coli |
CN107208067A (zh) * | 2014-12-30 | 2017-09-26 | 尹特荣生物科技株式会社 | 新型肠侵袭性大肠杆菌噬菌体Esc‑COP‑4及其用于抑制肠侵袭性大肠杆菌增殖的用途 |
WO2016108540A1 (ko) * | 2014-12-30 | 2016-07-07 | 주식회사 인트론바이오테크놀로지 | 신규한 장병원성 대장균 박테리오파지 Esc-CHP-2 및 이의 장병원성 대장균 증식 억제 용도 |
WO2016108542A1 (ko) * | 2014-12-30 | 2016-07-07 | 주식회사 인트론바이오테크놀로지 | 신규한 장침입성 대장균 박테리오파지 Esc-COP-4 및 이의 장침입성 대장균 증식 억제 용도 |
CN108471756A (zh) * | 2015-01-23 | 2018-08-31 | 因特拉蒂克斯有限公司 | 新型志贺菌噬菌体及其用途 |
EP3253215A4 (en) * | 2015-01-23 | 2018-07-18 | Intralytix, Inc. | Novel shigella bacteriophages and uses thereof |
US10711252B2 (en) | 2015-01-23 | 2020-07-14 | Intralytix, Inc. | Shigella bacteriophages and uses thereof |
AU2016209252B2 (en) * | 2015-01-23 | 2020-07-16 | Intralytix, Inc. | Novel Shigella bacteriophages and uses thereof |
WO2018101595A3 (ko) * | 2016-11-30 | 2018-08-09 | 주식회사 인트론바이오테크놀로지 | 대장균 박테리오파지 Esc-COP-9 및 이의 병원성 대장균 증식 억제 용도 |
US11213050B2 (en) | 2016-11-30 | 2022-01-04 | Intron Biotechnology, Inc. | Escherichia coli bacteriophage Esc-COP-9 and use for inhibiting proliferation of pathogenic Escherichia coli thereof |
KR20190018968A (ko) * | 2017-08-16 | 2019-02-26 | 경북대학교 산학협력단 | 병원성 장내세균에 대해 사멸능을 갖는 신규 박테리오파지 및 이의 용도 |
Also Published As
Publication number | Publication date |
---|---|
KR101101376B1 (ko) | 2012-01-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR101101376B1 (ko) | 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 | |
KR101070938B1 (ko) | 신규한 박테리오파지 및 이를 포함하는 항균 조성물 | |
CN107250352B (zh) | 新型肠致病性大肠杆菌噬菌体Esc-CHP-2及其用于抑制肠致病性大肠杆菌增殖的用途 | |
KR101151516B1 (ko) | 신규한 박테리오파지 및 이를 포함하는 항균 조성물 | |
CN107109372B (zh) | 新型产气荚膜梭菌噬菌体Clo-PEP-1及其用于抑制产气荚膜梭菌增殖的用途 | |
KR101151532B1 (ko) | 신규한 박테리오파지 및 이를 포함하는 항균 조성물 | |
KR101261873B1 (ko) | 식중독을 유발하는 살모넬라에 특이적 사멸능을 갖는 박테리오파지 | |
US11412760B2 (en) | Escherichia coli bacteriophage Esc-COP-7, and use thereof for suppressing proliferation of pathogenic Escherichia coli | |
KR101591795B1 (ko) | 신규 박테리오파지 및 이를 포함하는 조성물 | |
Ghasemi et al. | Preliminary characterization of Lactococcus garvieae bacteriophage isolated from wastewater as a potential agent for biological control of lactococcosis in aquaculture | |
Suanyuk et al. | Mortality and pathology of hybrid catfish, C larias macrocephalus (G ünther)× C larias gariepinus (B urchell), associated with E dwardsiella ictaluri infection in s outhern T hailand | |
CN111778216A (zh) | 一种地毯草黄单胞菌噬菌体及其组合物、试剂盒和应用 | |
US20190160121A1 (en) | Novel vibrio parahaemolyticus bacteriophage vib-pap-5 and use thereof for suppressing proliferation of vibrio parahaemolyticus bacteria | |
CN108179136B (zh) | 大肠杆菌噬菌体Esc-COP-9及其致病性大肠杆菌增殖抑制用途 | |
KR20100035879A (ko) | 엔테로코쿠스 및 스트렙토코쿠스 특이적 사멸능을 갖는 신규한 리신 단백질 | |
US11679138B2 (en) | Vibrio parahaemolyticus bacteriophage Vib-PAP-7 and use of same for inhibiting vibrio parahaemolyticus bacteria proliferation | |
CN113166730A (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 | |
KR101859973B1 (ko) | 신규한 에로모나스 하이드로필라 박테리오파지 Aer-HYP-1 및 이의 에로모나스 하이드로필라 균 증식 억제 용도 | |
US11679137B2 (en) | Vibrio parahaemolyticus bacteriophage Vib-PAP-4 and use thereof in inhibiting proliferation of Vibrio parahaemoliticus bacteria | |
US11213050B2 (en) | Escherichia coli bacteriophage Esc-COP-9 and use for inhibiting proliferation of pathogenic Escherichia coli thereof | |
CN111742046A (zh) | 新型嗜水气单胞菌噬菌体Aer-HYP-3及其在抑制嗜水气单胞菌细菌生长方面的用途 | |
KR102396828B1 (ko) | 내열성 박테리오파지 ts3, ts6, ts13 및 이를 포함하는 살모넬라 억제용 조성물 | |
KR102423986B1 (ko) | 액티노바실러스 플루로뉴모니애 균에 대하여 용균활성을 갖는 항균물질 및 이의 제조방법 | |
KR102438863B1 (ko) | 액티노바실러스 플루로뉴모니애 균에 대하여 용균활성을 갖는 항균물질 및 이의 제조방법 | |
Sorour et al. | Virulence Range and New Pathological Pictures of Salmonella enteridits and Salmonella typhimurium Isolated from Ducklings in Experimental Infected Chicks |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A201 | Request for examination | ||
E902 | Notification of reason for refusal | ||
E701 | Decision to grant or registration of patent right | ||
GRNT | Written decision to grant | ||
FPAY | Annual fee payment |
Payment date: 20150807 Year of fee payment: 5 |
|
LAPS | Lapse due to unpaid annual fee |