KR20230022739A - 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 및 이의 용도 - Google Patents
대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 및 이의 용도 Download PDFInfo
- Publication number
- KR20230022739A KR20230022739A KR1020210104879A KR20210104879A KR20230022739A KR 20230022739 A KR20230022739 A KR 20230022739A KR 1020210104879 A KR1020210104879 A KR 1020210104879A KR 20210104879 A KR20210104879 A KR 20210104879A KR 20230022739 A KR20230022739 A KR 20230022739A
- Authority
- KR
- South Korea
- Prior art keywords
- bacteriophage
- shigella
- phage
- coli
- escherichia coli
- Prior art date
Links
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 58
- 241000607768 Shigella Species 0.000 title claims abstract description 57
- 241001515965 unidentified phage Species 0.000 title claims abstract description 51
- 230000002147 killing effect Effects 0.000 title claims abstract description 18
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 27
- 241000607762 Shigella flexneri Species 0.000 claims abstract description 7
- 241001646719 Escherichia coli O157:H7 Species 0.000 claims description 31
- 208000015181 infectious disease Diseases 0.000 claims description 20
- 239000004480 active ingredient Substances 0.000 claims description 17
- 201000010099 disease Diseases 0.000 claims description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims description 11
- 235000013373 food additive Nutrition 0.000 claims description 10
- 239000002778 food additive Substances 0.000 claims description 10
- 239000003674 animal food additive Substances 0.000 claims description 8
- 208000004429 Bacillary Dysentery Diseases 0.000 claims description 5
- 239000004599 antimicrobial Substances 0.000 claims description 5
- 206010017915 Gastroenteritis shigella Diseases 0.000 claims description 4
- 201000005113 shigellosis Diseases 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 abstract description 42
- 230000001580 bacterial effect Effects 0.000 abstract description 6
- 230000001717 pathogenic effect Effects 0.000 abstract description 5
- 241001333951 Escherichia coli O157 Species 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 235000013336 milk Nutrition 0.000 description 9
- 239000008267 milk Substances 0.000 description 9
- 210000004080 milk Anatomy 0.000 description 9
- 241000256844 Apis mellifera Species 0.000 description 8
- 229940088710 antibiotic agent Drugs 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 244000005700 microbiome Species 0.000 description 8
- 244000106835 Bindesalat Species 0.000 description 7
- 235000000318 Bindesalat Nutrition 0.000 description 7
- 206010017533 Fungal infection Diseases 0.000 description 6
- 208000031888 Mycoses Diseases 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000701553 Myoviridae Species 0.000 description 5
- 108700026244 Open Reading Frames Proteins 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000012790 confirmation Methods 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 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
- 239000002245 particle Substances 0.000 description 4
- 230000003449 preventive effect Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000006142 Luria-Bertani Agar Substances 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 108020004566 Transfer RNA Proteins 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000010865 sewage Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 229940124597 therapeutic agent Drugs 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
- DEXFNLNNUZKHNO-UHFFFAOYSA-N 6-[3-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperidin-1-yl]-3-oxopropyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1CCN(CC1)C(CCC1=CC2=C(NC(O2)=O)C=C1)=O DEXFNLNNUZKHNO-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-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
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 206010061126 Escherichia infection Diseases 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- 108010061833 Integrases Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000002378 acidificating effect Effects 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
- 238000004458 analytical method Methods 0.000 description 2
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 208000020612 escherichia coli infection Diseases 0.000 description 2
- -1 etc. Substances 0.000 description 2
- 238000011331 genomic analysis Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 230000002101 lytic effect Effects 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
- 230000000877 morphologic effect Effects 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000002265 prevention Effects 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
- 230000002829 reductive effect Effects 0.000 description 2
- 150000003839 salts Chemical class 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
- 239000000126 substance Substances 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
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-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
- 244000215068 Acacia senegal Species 0.000 description 1
- 235000006491 Acacia senegal Nutrition 0.000 description 1
- 241000588626 Acinetobacter baumannii Species 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229920001817 Agar Polymers 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
- 241000301512 Bacillus cereus ATCC 14579 Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 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
- 241000989055 Cronobacter Species 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- 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 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
- 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 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
- 241000194032 Enterococcus faecalis Species 0.000 description 1
- 241000194031 Enterococcus faecium 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
- 241000205840 Escherichia coli O8 Species 0.000 description 1
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 206010016952 Food poisoning Diseases 0.000 description 1
- 208000019331 Foodborne disease Diseases 0.000 description 1
- 241000255896 Galleria mellonella Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 241000588915 Klebsiella aerogenes Species 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 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
- 241000186805 Listeria innocua Species 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 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
- 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 1
- 229920002230 Pectic acid Polymers 0.000 description 1
- 241001529559 Rhipidura Species 0.000 description 1
- 241000607356 Salmonella enterica subsp. arizonae Species 0.000 description 1
- 241000392514 Salmonella enterica subsp. enterica serovar Dublin Species 0.000 description 1
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 1
- 241000607726 Salmonella enterica subsp. enterica serovar Heidelberg Species 0.000 description 1
- 241000531795 Salmonella enterica subsp. enterica serovar Paratyphi A Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 108010079723 Shiga Toxin Proteins 0.000 description 1
- 241000607766 Shigella boydii Species 0.000 description 1
- 241001518902 Shigella flexneri 2a str. 2457T Species 0.000 description 1
- 206010040550 Shigella infections Diseases 0.000 description 1
- 241000607760 Shigella sonnei Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 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
- 239000004098 Tetracycline Substances 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
- 241000607734 Yersinia <bacteria> Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000008272 agar Substances 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
- 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
- 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
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000011575 calcium 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
- 239000002775 capsule Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 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
- 239000013043 chemical agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000012050 conventional carrier Substances 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 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
- 238000001035 drying Methods 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
- 229940092559 enterobacter aerogenes Drugs 0.000 description 1
- 229940032049 enterococcus faecalis Drugs 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
- 238000000605 extraction Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000012634 fragment 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
- 230000002068 genetic effect Effects 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 208000028774 intestinal disease Diseases 0.000 description 1
- 210000000936 intestine Anatomy 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
- 230000001418 larval effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 235000020191 long-life milk Nutrition 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- WVTHODDBDXSFGC-UHFFFAOYSA-K magnesium sodium chloride sulfate Chemical compound [Na+].[Mg+2].[Cl-].[O-]S([O-])(=O)=O WVTHODDBDXSFGC-UHFFFAOYSA-K 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
- 238000004519 manufacturing process Methods 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
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000007481 next generation sequencing Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 244000052769 pathogen Species 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
- 238000001066 phage therapy Methods 0.000 description 1
- 230000007505 plaque formation Effects 0.000 description 1
- 238000007747 plating Methods 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
- 239000003755 preservative agent Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 235000013772 propylene glycol Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 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
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant 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
- 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
- 238000004627 transmission electron microscopy Methods 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
- 238000010200 validation analysis Methods 0.000 description 1
- 230000001018 virulence Effects 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
- 235000010447 xylitol Nutrition 0.000 description 1
- 239000000811 xylitol Substances 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
- A01N63/40—Viruses, e.g. bacteriophages
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
-
- 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
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Communicable Diseases (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Agronomy & Crop Science (AREA)
- Animal Husbandry (AREA)
- Physiology (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Oncology (AREA)
- Dentistry (AREA)
- Pest Control & Pesticides (AREA)
- General Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
Abstract
본 발명은 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 및 이의 용도에 관한 것으로, 본 발명의 박테리오파지인 SME50 (기탁번호: KCTC 14641BP)은 신규한 파지로서 병원성 대장균 O157과 시겔라 플렉시너리를 동시에 제어할 수 있는 우수한 숙주 세균 사멸 범위를 가지고, 고온에서의 안정성과 큰 burst size가 다른 파지들과 대비하여 가지는 것을 특징으로 하여 항균제 등으로 유용하게 활용될 수 있다.
Description
본 발명은 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 및 이의 용도에 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, 접촉감염증이 시겔라 감염증의 주된 전파 방법이다. 세계적으로 매년 2억명의 발병을 보이고 있으며 그중 65만명이 사망하고 있는 실정이다. 우리나라에서 세균성 이질의 발병은 감소 추세를 보이다 1998년이후 전국적으로 집단 발병하는 등 발병이 현격히 증가하여 사회문제화 되고 있다. 또한 1998년 이후 분리된 이질균의 경우 테트라싸이클린, 스트렙토마이신등 항생제에 대하여 내성을 보이는 등 항균제에 대한 내성이 심각한 수준으로 대두되고 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. 또한, 이러한 감염을 예방하기 위하여, 식품 분야에서는 대장균, 시겔라균 오염을 막기 위하여 화학제제등을 사용하기도 하고, 가축 등 경제동물의 사육에도 대장균, 시겔라균에 의한 감염을 막기 위하여 항생제를 사용하는 경우도 있었다.
한편, 박테리오파지(bacteriophage)는 세균에 특이적으로 침투하여 용균하는 바이러스로 유전물질 (단일가닥 DNA, 이중가닥 DNA, RNA)을 단백질 외피가 싸고 있는 유기체다. 상기 박테리오파지는 자연에 존재하는 세균을 감염시킬 수 있으며, 세균이 존재하는 거의 모든 곳에 함께 존재할 수 있다. 이들을 이용한 파지 치료법 (phage therapy)은 인간뿐만 아니라 식물, 동물에게도 적용할 수 있으며, 음식이나 사료 등에서도 감염성 및 부패성 세균을 제어하기 위해 활용 가능하는 등 그 응용분야가 매우 다양하다. 특히 기존의 화학적 항생제에 비해 숙주 세균에 대한 특이성(specificity)이 높고 개발시간 및 개발비용이 상대적으로 적게 소모되며, 숙주 세균이 존재하는 한 스스로 증폭하여 수를 늘리는 self dosing이 가능하여 잠재적 응용성도 높다.
본 발명의 일 양상은 대장균 또는 시겔라속(Shigella) 균에 특이적 사멸능을 갖는 박테리오파지를 제공하는 것을 목적으로 한다.
본 발명의 다른 일 양상은 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 항균제를 제공하는 것을 목적으로 한다.
본 발명의 다른 일 양상은 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것을 목적으로 한다.
본 발명의 다른 일 양상은 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 사료첨가제를 제공하는 것을 목적으로 한다.
본 발명의 다른 일 양상은 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 식품첨가제를 제공하는 것을 목적으로 한다.
본 발명의 일 양상은 대장균 또는 시겔라속(Shigella) 균에 특이적 사멸능을 갖는 박테리오파지 (기탁번호: KCTC 14641BP) 를 제공한다.
상기 박테리오파지는 마이오비리대(Myoviridae) 과에 속하는 것으로서, 보다 상세하게는병원성 대장균과 이질균에 동시 감염능력이 있어 이들을 사멸시킬 수 있는 신규의 박테리오파지이다.
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, 십진 희석하여 한 뒤 용균반 분석(plaque assay) 하여 박테리오파지를 분리할 수 있다 (실험예 1-1. 참조).
본 발명의 박테리오파지는 형태학적 분류상 Myoviridae 과에 속하며, 본 발명에서 상기 박테리오파지를 'SME50'으로 명명하고, 2021년 07월 22일자로 생명공학연구소 유전자은행(korean collection for type cultures, KCTC)에 기탁번호 KCTC 14641BP 로 기탁하였다 (실험예 2-1. 참조).
본 발명의 박테리오파지는 유해한 유전자를 가지고 있지 않은 virulent phage이면서, pH 4 내지 11, 구체적으로 pH 4 내지 5, pH 4 내지 6, pH 4 내지 7, pH 4 내지 8, pH 4 내지 9, pH 4 내지 10, pH 4 내지 11, pH 5 내지 6, pH 5 내지 7, pH 5 내지 8, pH 5 내지 9, pH 5 내지 10, pH 5 내지 11, pH 6 내지 7, pH 6 내지 8, pH 6 내지 9, pH 6 내지 10, pH 6 내지 11, pH 7 내지 8, pH 7 내지 9, pH 7 내지 10, pH 7 내지 11, pH 8 내지 9, pH 8 내지 10, pH 8 내지 11, pH 9 내지 10, pH 9 내지 11 또는 pH10 내지 11에서 감염성을 유지할 수 있다. 또한, 본 발명의 박테리오파지는 4 내지 70℃, 구체적으로, 4 내지 25℃, 4 내지 37℃, 4 내지 50℃, 4 내지 60℃, 4 내지 70℃, 25 내지 37℃, 25 내지 50℃, 25 내지 60℃, 25 내지 70℃, 37 내지 50℃, 37 내지 60℃, 37 내지 70℃, 50 내지 60℃, 50 내지 70℃ 또는 60 내지 70℃에서도 감염성을 유지할 수 있고, 5분 정도의 암흑기, 20분 정도의 잠복기를 가지며, 500 내지 900 PFU/infected cell, 구체적으로 550 내지 850 PFU/infected cell, 더욱 구체적으로는 580 내지 790 PFU/infected cell, 일 예시로 650 PFU/infected cell의 방출량(burst size)을 가지는 생장 특성이 있다 (실험예 2-2. 및 2-4. 참조).
본 발명의 일 구체예로 상기 박테리오파지는 서열번호 1의 염기서열로 표시되는 유전체를 갖는 것일 수 있다. 상기 염기서열은 유전체 크기가 90,298 bp이고 총 138개의 ORF와 25개의 tRNA가 포함되는 것으로 예측되고, 용원성과 연관된 단백질인 integrase 또는 repressor 등이 발견되지 않아 용균성 파지임을 확인하였으며 항생제 저항성 유전자나 독성에 연관된 유전자 또한 발견되지 않아 세포 독성이 없는 것을 알 수 있다. (실험예 2-5. 참조)
본 발명의 박테리오파지는 대장균 또는 시겔라속 균에 특이적 사멸능을 가지는 것을 특징으로 한다. 상기 대장균 또는 시겔라속 균은 이에 한정되지 않지만, 본 발명의 일 구체예로 상기 대장균은 대장균 O157:H7 이고 상기 시겔라속(Shigella) 균은 시겔라 플렉스너리 (Shigella flexneri)일 수 있다.
본 발명의 다른 일 양상은 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 항균제를 제공한다.
상기 항균제는 미생물의 성장을 억제할 뿐 아니라 미생물을 파괴 또는 무력화하는 제제를 의미한다.
상기 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 있다.
본 발명의 일 구체예로 상기 항균제는 대장균 또는 시겔라속(Shigella) 균을 사멸시키는 것일 수 있다. 구체적으로, 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. 구체적으로, 상기 대장균은 대장균 O157:H7 일 수 있고, 상기 시겔라속(Shigella) 균은 시겔라 플렉스너리 (Shigella flexneri)일 수 있다.
상기 대장균 또는 시겔라속 균은 주로 식품 또는 물을 오염시킬 있다. 식품은 미생물의 탄소원 및 질소원 등 수많은 유기물, 무기물을 함유하고 있어 대장균 또는 시겔라속 균이 번식하기 용이하기 때문이다. 따라서 본 발명의 항균제는 식품 등에 사용될 수 있다.
본 발명의 항균제는 유익한 균은 사멸시키지 않고 병원성 균인 대장균 또는 시겔라속 균만을 선택적으로 죽일 수 있어, 부작용이 없으며 병원성 균의 항생제 내성 내지 저항성을 유도하지 않아 기존의 항생물질에 비하여 효과적으로 이용될 수 있다.
본 발명의 항균제는 상기 박테리오파지를 단독 유효성분으로 포함하거나 다른 공지의 유효성분과 혼합되어 제조될 수 있다. 한편, 상기 항균제는 다른 항균제 (가령, propionic acid와 butyric acid 같은 유기산 처리)와 병용 또는 공지의 항균 방법 (가령 열처리 등) 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 사용될 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.
또한, 본 발명의 다른 일 양상은 상기 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물을 제공한다.
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. 구체적으로, 상기 대장균은 대장균 O157:H7 일 수 있고, 상기 시겔라속(Shigella) 균은 시겔라 플렉스너리 (Shigella flexneri)일 수 있다.
본 발명의 일 구체예로 상기 대장균에 의해 유발되는 질환은 대장균 감염증, 보다 바람직하게는 장출혈성 대장균 감염증으로 이루어진 군에서 선택될 수 있다.
또한, 또한 상기 시겔라속 균에 의해 유발되는 질환은 이에 한정되지 않지만 바람직하게는 세균성 이질일 수 있다.
본 발명의 조성물은 상기 박테리오파지를 단독 유효성분으로 포함하거나 다른 공지의 유효성분과 혼합되어 제조될 수 있다. 한편, 상기 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 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, 근육 내 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, 보 통으로 숙련된 의사는 소망하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 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, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐 피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물 유, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유, 덱스트린, 칼슘카보네이트, 프로필 렌글리콜, 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, 증량 제, 결합제, 붕해제, 항응집제, 윤활제, 습윤제, pH 조절제, 영양제, 비타민, 전해질, 알긴산 및 그의 염, 펙트 산 및 그의 염, 보호성 콜로라이드, 글리세린, 향료, 유화제 또는 방부제 등을 추가로 포함할 수 있다.
한편, 본 발명의 다른 일 구체예로, 상기 박테리오파지 또는 이의 배양물을 포함하는 사료첨가제 또는 식품 첨가제를 제공한다
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 시겔라속균을 특이적으로 사멸시킬 수 있다. 구체적으로, 상기 대장균은 대장균 O157:H7 일 수 있고, 상기 시겔라속(Shigella) 균은 시겔라 플렉스너리 (Shigella flexneri)일 수 있다.
상기 식품첨가제가 사용될 수 있는 식품에는 제한이 없으며, 상기 식품첨가제는 대장균 또는 시겔라속 균에 감염되기 쉬운 식품에 당업계에 공지된 방법에 따라 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 박테리오파지인 SME50 (기탁번호: KCTC 14641BP)은 신규한 파지로서 병원성 대장균 O157과 시겔라 플렉시너리를 동시에 제어할 수 있는 우수한 숙주 세균 사멸 범위를 가지고, 고온에서의 안정성과 큰 burst size가 다른 파지들과 대비하여 가지는 것을 특징으로 하여 항균제 등으로 유용하게 활용될 수 있다.
도 1은 SME50의 투과 전자 현미경을 이용한 형태학적 분석 결과를 나타낸 것이다.
도 2는 SME50의 성장곡선 (one-step growth curve)을 나타낸 그래프이다.
도 3은 시험관 내에서의 SME50의 대장균 O157:H7 사멸능을 나타낸 그래프이다.
도 4는 시험관 내에서의 SME50의 시겔라 플렉시너리 사멸능을 나타낸 그래프이다.
도 5는 SME50의 pH 안정성을 나타낸 그래프이다.
도 6은 SME50의 온도 안정성을 나타낸 그래프이다.
도 7은 SME50의 유전자 지도이다.
도 8은 꿀벌부채명나방 유충 모델을 이용한 SME50의 대장균 O157:H7 감염의 치료 효과를 나타낸 그래프이다.
도 9는 꿀벌부채명나방 유충 모델을 이용한 SME50의 대장균 O157:H7 감염의 예방 효과를 나타낸 그래프이다.
도 10은 4℃에서 보관한 로메인상추에서 SME50 (MOI 104)의 대장균 O157:H7 저해 효과를 나타낸 그래프이다.
도 11은 4℃에서 보관한 로메인상추에서 SME50 (MOI 104)의 시겔라 플렉시너리 저해 효과를 나타낸 그래프이다.
도 12는 우유에서 SME50 (MOI 104)의 대장균 O157:H7 저해 (4℃) 효과를 나타낸 그래프이다.
도 13은 우유에서 SME50 (MOI 104)의 시겔라 플렉시너리 저해 (4℃) 효과를 나타낸 그래프이다.
도 14는 우유에서 SME50 (MOI 104)의 대장균 O157:H7 저해 (25℃) 효과를 나타낸 그래프이다.
도 2는 SME50의 성장곡선 (one-step growth curve)을 나타낸 그래프이다.
도 3은 시험관 내에서의 SME50의 대장균 O157:H7 사멸능을 나타낸 그래프이다.
도 4는 시험관 내에서의 SME50의 시겔라 플렉시너리 사멸능을 나타낸 그래프이다.
도 5는 SME50의 pH 안정성을 나타낸 그래프이다.
도 6은 SME50의 온도 안정성을 나타낸 그래프이다.
도 7은 SME50의 유전자 지도이다.
도 8은 꿀벌부채명나방 유충 모델을 이용한 SME50의 대장균 O157:H7 감염의 치료 효과를 나타낸 그래프이다.
도 9는 꿀벌부채명나방 유충 모델을 이용한 SME50의 대장균 O157:H7 감염의 예방 효과를 나타낸 그래프이다.
도 10은 4℃에서 보관한 로메인상추에서 SME50 (MOI 104)의 대장균 O157:H7 저해 효과를 나타낸 그래프이다.
도 11은 4℃에서 보관한 로메인상추에서 SME50 (MOI 104)의 시겔라 플렉시너리 저해 효과를 나타낸 그래프이다.
도 12는 우유에서 SME50 (MOI 104)의 대장균 O157:H7 저해 (4℃) 효과를 나타낸 그래프이다.
도 13은 우유에서 SME50 (MOI 104)의 시겔라 플렉시너리 저해 (4℃) 효과를 나타낸 그래프이다.
도 14는 우유에서 SME50 (MOI 104)의 대장균 O157:H7 저해 (25℃) 효과를 나타낸 그래프이다.
이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
실험예 1: 실험 방법
실험예 1-1. 균주 및 박테리오파지
이하에서 사용된 균주는 모두 도표 1에 명시되어 있다. 사용된 모든 균주는 Luria-Bertani (LB) 배지를 이용하여 37℃에서 호기 조건으로 배양되었다. 대장균 O157:H7 ATCC 43890 (Escherichia coli O157:H7 ATCC 43890)은 파지 SME50의 동정, 정제, 그리고 증폭에 사용되었다.
의정부 하수처리장의 하수 시료에서 파지를 동정하였다. 동정 과정은, 시료를 동량의 2X LB 액체 배지로 8시간 동안 37℃에서 배양 후, 상등액을 0.45 um 크기의 필터로 거른 후 대장균 O157:H7 ATCC 43890 균주에 spotting assay로 분리하였다. 대장균 균주에서 확인된 파지 plaque는 sodium chloride-magnesium sulfate (SM) buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 10 mM MgSO4)를 이용해 십진 희석하여 다시 대장균 배지에서 키워 plaque를 골라내는 방식을 3번 이상 거쳐 순수 파지 plaque를 분리하였다. 순수 분리된 파지는 대장균 O157:H7 ATCC 43890 균주를 이용해 증폭되었고, CsCl 밀도 구배 초고속원심분리를 이용하여 순수한 파지가 농축된 스탁 (stock)를 확보하였다. 구축된 파지 스탁을 이용하여 투과 전자 현미경 (Transmission electron microscope) 관찰을 진행하였다.
| Bacterial strains | Plaque formation a | Resources b |
| Escherichia coli | ||
| MG 1655 | - | |
| DH5α | - | |
| ATCC 25922 | - | ATCC |
| E. coli O157:H7 | ||
| ATCC 35150 | +++ | ATCC |
| ATCC 43890 | +++ | ATCC |
| ATCC 43888 | +++ | ATCC |
| ATCC 43894 | ++ | ATCC |
| ATCC 43895 | +++ | ATCC |
| ATCC 43889 | +++ | ATCC |
| 86-24 | +++ | |
| E2348169 | - | |
| OE50 | +++ | |
| E. coli O157:NM | ||
| 3204-92 | +++ | |
| H-0482 | +++ | |
| E. coli O8:H30 #736 | - | |
| Shigella flexneri | ||
| 2a strain 2457T | +++ | |
| 6 IB1666 | - | |
| ATCC 29903 | - | ATCC |
| ATCC 12022 | +++ | ATCC |
| KCTC 2993 | - | KCTC |
| S. boydii | ||
| IB 2474 | - | |
| ATCC 8700 | - | ATCC |
| S. dysenteniae IB 2476 | - | |
| S. sonnei | ||
| IB 707 | - | |
| ATCC 29930 | - | ATCC |
| Other gram negative bacteria | ||
| Acinetobacter baumannii CCARM 12229 | - | CCARM |
| Cronobacter. sakazakii | ||
| ATCC 29544 | - | ATCC |
| ATCC 51329 | - | ATCC |
| Enterobacter aerogenes | ||
| CCARM 16001 | - | CCARM |
| CCARM 16003 | - | CCARM |
| NCCP 16285 | - | NCCP |
| Klebsiella pneumoniae | ||
| CCARM 10326 | - | CCARM |
| NCCP 15782 | - | NCCP |
| NCCP 15864 | - | NCCP |
| Salmonella Typhimurium | ||
| LT2ⓒ | - | |
| SL1344 | - | |
| ST9 | - | |
| ATCC 14028 | - | ATCC |
| ATCC 19586 | - | ATCC |
| Ty 2-b | - | |
| DT 104 | - | |
| UK-1 | - | |
| KCTC 1925 | - | KCTC |
| S. Paratyphi | ||
| A IB 211 | - | |
| B IB 231 | - | |
| C IB 216 | - | |
| S. Dublin IB 216 | - | |
| S. Arizonae KCCM 41035 | - | |
| S. Enteritidis | ||
| En2 | - | |
| ATCC 13076 | - | |
| S. Heidelberg | - | |
| Yersinia enterocolica ATCC 55075 | - | |
| Gram positive bacteria | ||
| Bacillus cereus ATCC 14579 | - | ATCC |
| B. subtilis ATCC 23857 | - | ATCC |
| Listeria monocytogenes ATCC 19114 | - | ATCC |
| L. innocua I | - | |
| Staphylococcus aureus | ||
| RN 4220 | - | |
| Newman | - | |
| ATCC 29213 | - | ATCC |
| Enterococcus faecalis | ||
| CCARM 5192 | - | CCARM |
| NCCP 16130 | - | NCCP |
| NCCP 16131 | - | NCCP |
| Enterococcus faecium | ||
| CCARM 5512 | - | CCARM |
| CCARM 5527 | - | CCARM |
| CCARM 5528 | - | CCARM |
a +++, EOP of 1 to 0.5; ++, EOP of 0.5 to 0.2; +, EOP less than 0.2; -, no susceptibility to SME50
bATCC, American Type Culture Collection; CCARM, Culture Collection of Antimicrobial Resistant Microbes; NCCP, National Culture Collection for Pathogens; KCTC, Korean Collection for Type Culture
실험예 1-2. 파지의 숙주 사멸 범위와 안전성 확인
파지의 숙주 사멸 범위를 확인하기 위해 사용된 균주는 모두 LB 한천 배지에 double agar layer 방식으로 준비한 후, 파지의 plaque를 십진희석하여 스팟팅하였다. 하룻밤 동안 배양한 배지에서 파지가 각 균주를 감염하였는지의 여부와 파지의 농도 (titer)를 확인하였다. 기준 균주인 대장균 O157:H7 ATCC 43890을 이용하여 파지의 Efficiency of plating (EOP)를 확인하였다.
파지가 다양한 pH 범위에서 얼마나 안정적인지 알아보기 위해서, 파지 SME50 (최종 농도 106 PFU/ml)를 pH 2~12로 조정한 LB 배지에서 한시간 동안 37℃에서 배양하였다. 파지가 다양한 온도에서 안정적인지 확인하기 위해, 파지를 SM buffer에 106 PFU/ml이 되도록 희석 후 4, 25, 37, 50, 60, 70, 그리고 80℃ 에서 한시간 동안 배양하였다. 각 조건에서 활성을 유지한 파지는 standard plaque assay를 통해 확인되었다.
실험예 1-3. One-step growth curve를 통한 파지의 생활사 확인
파지의 생활사와 증폭 정도를 알아보기 위해 One-step growth curve를 확인하였다. 대장균 O157:H7 ATCC 43890에 파지 SME50을 MOI 0.01로 감염시키고, 10분간 상온에서 파지가 숙주 세균에 부착되도록 한 뒤, 45분간 5분 간격으로 시료를 채취하여 분석하였다. 시간별 시료에 어떠한 처리도 하지 않은 실험군과 시료에 2% 클로로포름을 처리하여 인위적으로 숙주 세균의 세포막을 깨트려준 실험군을 적정 농도로 희석 후 standard plaque assay로 파지 입자의 농도를 확인하여 파지의 생장 곡선을 확인하였다.
실험예 1-4. 파지 SME50의 숙주 세균 사멸능 확인
초기 대수기에 있는 숙주 세균 배양액에 파지 SME50을 MOI 0.1, 1, 그리고 10이 되도록 감염하였다. Microplate reader (Spark®, Tecan) 기계를 이용하여 12시간 동안 30분 단위로 시료의 흡광도 값을 측정하여 SME50이 숙주 세균을 얼마나 우수히 저해하는지 확인하였다.
실험예 1-5. 파지 SME50의 유전체 분석
파지 SME50 스탁을 이용하여 파지의 dsDNA를 추출하여 Illumina Miseq (Illumina, San Diego, Ca)을 이용하여 차세대 염기서열 분석을 진행했다. 분석된 서열조각들은 SPAdes (v. 3.13.0)를 이용하여 하나의 contig로 조립하였으며, 완성된 contig를 GenemarkS, FgenesB (softberry), and Glimmer3 (Besemer, Lomsadze, & Borodovsky, 2001; Delcher, Bratke, Powers, & Salzberg, 2007)와 같은 생물정보학적 툴을 이용하여 ORF (Open reading frame)를 분석했다. 파지가 가지고 있는 tRNA는 tRNA-ScanSE를 이용하여 분석되었고, 유전자의 기능은 NCBI BLASTp와 InterProScan database를 활용하여 분석되었다. GeneScene 소프트웨어를 이용하여 유전체 지도를 가시화하였다. 파지 SME50의 전체 유전체 서열은 NCBI Genbank에 기탁되었다 (accession number MT887289).
실험예 1-6. 꿀벌부채명나방 유충을 이용한 생체 내 감염 모델
250~300 mg의 5주령 꿀벌부채명나방 유충을 선별하여 24시간 동안 37℃에서 먹이를 주지 않은 상태를 유지하였다가 실험에 이용하였다. 배양한 숙주 균주를 PBS로 3번 세척하고 2.5 x 105 CFU/유충이 되도록 맞춘 후, 유충 한 마리 당 5 ul를 뒷다리를 통해 주입하였다. 동량의 파지 용액을 MOI 20으로 조절한 후 유충의 반대편 뒷다리에 균 감염 30분 전후로 주입하여 파지의 균 감염 예방 및 치료 효과를 알아보았다. 균의 대조군으로는 PBS, 파지의 대조군으로는 SM 버퍼를 주입하였다. 모든 실험군은 37℃에서 144시간 동안 24시간 주기로 관찰하였다.
실험예 1-7. 식품에서의 파지 SME50의 항균 활성
실험에 사용된 로메인 상추는 실험 전 2 x 2 cm2로 절단하여 증류수와 70% 에탄올로 세척하여 기타 외부 균의 감염을 배제하였다. 이후 증류수로 한 번 더 세척한 후 40분간 생물안전작업대에서 자연 건조한 후 사용하였다. 대장균 O157:H7 ATCC 43890 혹은 시겔라 플렉시너리 2a strain 2457T를 로메인상추의 표면에 오염시킨 후 40분간 건조하였다. 이후, 파지 SME50을 MOI 104 혹은 103의 농도로 맞추어 처리한 후 4℃에서 보관하며 0, 2, 4, 6, 8, 10, 24, 48, 그리고 72시간에서의 오염된 균의 정도를 LB 한천 배지를 이용하여 확인하였다.
인위적으로 오염된 우유 모델의 경우, 멸균 우유 20 ml에 대장균 O157:H7 ATCC 43890 혹은 시겔라 플렉시너리 2a strain 2457T를 오염시키고, 파지 SME50을 MOI 104 혹은 103으로 감염시켜 파지의 균 저해 효과를 관찰하였다. 4 혹은 25℃에서 보관하며 0, 2, 4, 6, 8, 10, 24, 48, 그리고 72시간에서의 오염된 균의 정도를 LB 한천 배지를 이용하여 확인하였다. 로메인 상추와 우유 모델에서 모두 파지를 처리하지 않은 대조군의 경우 파지 대신 동량의 SM 버퍼를 투여하였다.
실험예 2: 실험 결과
실험예 2-1.
Myoviridae
과에 속하는 polyvalent 파지 SME50
하수 샘플에서 순수 분리한 파지 SME50을 다양한 그람 양성 및 그람 음성 세균에 대한 감염능을 확인해본 결과, 시험한 9개 중 8개의 대장균 O157:H7 균주 (88.9%)와 두 가지의 O157:NM 균주를 감염할 수 있었다 (미도시). 일반적으로 단일 파지는 하나의 종 혹은 속의 세균을 감염하는 매우 좁은 숙주 감염 범위를 가지는데, 본 발명의 파지 SME50은 대장균뿐만 아니라 두 가지의 시겔라 플렉시너리 균주를 동시에 감염할 수 있었으며, 그 EOP 또한 기준 균주인 대장균 O157:H7 ATCC 43890과 유사하게 나타났다. 반면에, 파지 SME50은 플렉시너리가 아닌 다른 시겔라 종과 기타 다른 그람 음성 및 양성균은 감염할 수 없는 특징을 지닌다. 이로써 신규 분리된 파지 SME50은 서로 다른 두 속의 균인 대장균 O157과 시겔라 플렉시너리를 감염할 수 있는 polyvalent한 파지임을 증명하였다. 그리고, 상기 파지 SME50은 2021년 07월 22일자로 생명공학연구소 유전자은행(korean collection for type cultures, KCTC)에 기탁번호 KCTC 14641BP 로 기탁하였다.
본 발명의 파지 SME50는 도 1에서 확인되는 바와 같이 파지 플락 (plaque)의 형상은 투명하며 덜 투명한 halo를 가진다. 또한, 투과전자현미경 관찰 결과 파지 SME50은 Myoviridae 과에 속하는 파지로 수축성 꼬리와 대칭형 머리를 가지는 것으로 확인되었다. 파지 머리의 지름은 66.15 ± 4.57 nm (n=12), 수축하지 않은 꼬리는 90.80 ± 11.03 nm (n=7), 그리고 수축한 상태의 꼬리는 40.64 ± 4.67 nm (n=2)인 것으로 나타났다.
실험예 2-2. 숙주 세균 내에서 우수한 증폭 능력을 지닌 파지 SME50
본 발명의 파지 SME50이 숙주 세균 내에서 높은 수준으로 증폭되는 양상을 one-step growth curve를 통해 알 수 있었다. 구체적으로 도 2에서 확인되는 바와 같이 증폭된 파지 입자들이 단 5분의 암흑기만에 생성되었고, 자연적으로는 20분의 잠복기만에 숙주 세균을 용해하고 분비되었다. 감염한 숙주 세균 당 증폭되어 나오는 양을 일컫는 용균 크기(burst size)는 약 650 PFU/ml로 매우 컸다.
또한, 도 3에서 확인되는 바와 같이 파지의 숙주 세균 사멸능을 다양한 MOI에서 확인한 결과, MOI 0.1, 1, 그리고 10에서 모두 우수히 숙주 세균인 대장균 O157:H7 ATCC 43890을 사멸할 수 있었다. 파지 SME50은 6시간까지 숙주 세균을 잘 저해할 수 있었다.
그리고, 도 4에서 확인되는 바와 같이 대장균과 마찬가지로 시겔라 플렉시너리 2a strain 2457T를 숙주 균주로 시험했을 때도 다양한 MOI의 파지가 우수한 사멸능을 가지는 것을 볼 수 있었다.
실험예 2-3. 파지 SME50의 높은 스트레스 환경에 대한 내성
파지 SME50을 37℃에서 2 에서 12 범위의 pH로 조정된 LB 배지에 한시간 동안 배양하였을 때, 숙주 세균에 대한 감염력은 중성에서 pH 7 까지는 비슷하게 유지되었다. 그러나, pH 4에서는 1-log 감소한 결과를 보였으며, 더 강한 산성인 pH 2와 3에서는 파지 SME50이 활성을 잃어 plaque를 형성하지 못하였다. 산성 환경과는 다르게 염기성 환경에서 SME50은 더 안정적인 양상을 보였다. pH 11까지 매우 안정적으로 숙주 세균을 감염하였고, pH 12에서는 감염력을 잃는 것을 확인하였다 (도 5).
파지 SME50은 넓은 온도 범위에서도 안정적으로 숙주 세균을 감염하는 것으로 나타났다. 구체적으로 도 6에서 확인되는 바와 같이, 숙주 세균에 대한 감염력은 4~60℃ 범위에서 안정적으로 유지되었고, 70℃에 한 시간 동안 노출된 파지는 10% 이상의 파지 입자들이 감염력을 유지하였다. 그러나, 더 높은 온도인 80℃에서는 모든 파지 입자들이 불활성화 되었다. 이처럼 넓은 범위의 pH와 온도 조건에서 숙주 세균에 대한 감염력을 유지하는 것은 다른 Myoviridae 과에 속하는 polyvalent 파지인 SFP10, SH7, 그리고 HY01과는 차별적인 특징이라 할 수 있다 (Hamdi et al., 2017; Lee et al., 2016; Park et al., 2012).
실험예 2-4. 파지 SME50의 유전체 분석
도 7에서 확인된 바와 같이, 파지 SME50은 총 90,298 bp의 뉴클레오타이드 (서열번호 1)를 가지며 38.9%의 염기서열이 구아닌과 사이토신으로 이루어져 있고, 총 138개의 ORF와 25개의 tRNA가 예측되었다. 총 95개 ORF(67%)가 기능이 예측되지 않는 hypothetical 단백질을 암호화하여파지 SME50의 신규성을 뒷받침해준다. 파지 SME50의 유전체에서 용원성과 연관된 단백질인 integrase 또는 repressor 등이 발견되지 않았기에 이 파지는 용균성 파지임을 유전체 수준에서 입증할 수 있었다. 항생제 저항성 유전자나 독성에 연관된 유전자 또한 발견되지 않았다.
실험예 2-5. 꿀벌부채명나방 유충 감염 모델을 통한 파지 SME50의 균 감염 치료 및 예방 효과 확인
숙주 균주 감염에 대한 파지 SME50의 치료 및 예방 효과를 생체내 환경에서 알아보고자 꿀벌부채명나방 (Galleria mellonella)의 유충 감염 모델을 활용하였다.
그 결과, 도 8에서 확인된 바와 같이, 먼저 숙주 세균인 대장균 O157:H7 ATCC 43890을 꿀벌부채명나방 유충의 복각에 주입하였을 때, 80% 이상의 유충이 멜라닌화 되면서 하루 안에 죽었으며, 실험에 사용한 유충의 10%만이 실험이 끝날 때까지 살아남았다. 반면에, 파지 SME50을 MOI 20으로 균 감염 30분 후에 주입하였을 때, 실험이 종료되는 144시간까지 모든 유충이 살아남는 결과를 보였다. 이는 파지 SME50은 대장균 O157:H7 감염에 대한 치료 효과가 있음을 시사한다.
위의 실험에 추가적으로 파지 (MOI 20)를 균 감염 30분 전에 주입하여 균 감염에 대한 예방 효과를 알아보았다. 그 결과 도 9에서 확인되는 바와 같이, 파지를 투여한 그룹의 경우에는 100%의 유충이 실험이 끝나는 시점인 144시간까지 생존하는 결과를 보였다. 이러한 결과는 파지 SME50이 대장균 O157:H7 감염에 대해 치료 효과뿐만 아니라 예방 효과까지 가져올 수 있음을 말해준다. 더불어, 파지 SME50이 꿀벌부채명나방에 독성을 나타내는지 알아보기 위해 병원성균 대신에 PBS를 처리하고 파지를 처리한 경우 실험에 사용된 모든 유충이 144시간까지 생존하는 것을 통해 파지 자체에 독성이 없음을 확인하였다. 꿀벌부채명나방 유충을 이용한 생체 내 감염 실험을 통해 파지의 무독성을 증명하고, 생체내에서의 대장균 O157:H7에 대한 강력한 제어 활성을 확인할 수 있었다. 이러한 결과는 향후 파지 SME50이 생체내 감염에 대항할 안전한 항균제로의 가능성을 시사한다.
실험예 2-6.
식품 모델을 통한 식품 항균제로서의 가능성 검증
SME50의 식품 첨가제로 사용될 가능성을 알아보기 위해 실제 식품 모델에서의 항균 활성을 알아보았다.
로메인상추에 인위적으로 대장균 O157:H7 ATCC 43890을 감염하고 파지 용액을 처리하여 4℃에서 보관한 결과대장균 수를 현저히 감소시킬 수 있었다 (도 10). 로메인상추의 유통기한을 고려하여 파지 처리 후 3일까지 관찰한 결과, 감소한 균은 72시간까지 유지됨을 알 수 있었다.
동일하게 시겔라 플렉시너리 2a strain 2457T 균주를 인위적으로 감염하고 파지 SME50을 처리하여 그 효과를 알아본 결과, 파지 처리 6시간 후 오염된 시겔라 균은 1.16-log, 72시간 후 1.92-log 감소한 것으로 나타났다 (도 11).
대장균 O157:H7이 오염된 우유에 파지 SME50을 MOI 104로 처리한 후 4℃에서 보관하였을 때, 대장균 수는 2시간만에 5.48에서 2.75 log CFU/ml로 감소하였으며, 6시간 째에는 2.36 log로 감소하였다 (도 12). 우유에 인위적으로 시겔라 플렉시너리 균주를 감염한 후 파지를 MOI 104로 처리하고 4℃에서 보관하였을 때, 오염된 시겔라 균주는 파지 처리 10시간 후 5.23 에서 3.26 log CFU/ml로 감소하였고, 72시간 이후에는 2.14 log CFU/ml에 도달하였다 (도 13). 또한, 우유를 냉장 보관하지 않은 경우를 모방하기 위하여 우유에 대장균 O157:H7 ATCC 43890을 인위적으로 감염한 후 파지 SME50을 MOI 104 농도로 처리 후 25℃에서 보관 및 관찰하였다. 그 결과, 파지를 처리한 실험군의 경우 대장균이 더 자라지 않고 2시간 째에 1-log 감소하는 양상을 보였지만, 파지를 처리하지 않은 대조군의 경우 시간이 지남에 따라 균이 계속해서 성장하는 양상을 보였다 (도 14).
상기한 결과들을 통해 본 발명의 파지 SME50은 대표적인 식중독균인 대장균 O157:H7과 시겔라 플렉시너리를 동시에 저해할 수 있고, 이를 이용하여 항균제, 식품첨가제, 사료첨가제, 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물 등으로 활용할 수 있다.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.
기탁기관명 : 한국생명공학연구원
수탁번호 : KCTC14641BP
수탁일자 : 20210722
<110> Industry-Academic Cooperation Foundation, Yonsei University
<120> Bacteriophage having killing activity specific to Escherichia
coli or Shigella
<130> PN210142
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 90298
<212> DNA
<213> Artificial Sequence
<220>
<223> Bacteriophage SME50
<400> 1
atgacctatc aagacttctt agcttctttc aaactagaaa ggtttgttca gattgctggg 60
agcactatga cgggggattt aggtattgtc aagttacttt acggtggtaa ggctgtattt 120
gacccaacag attcttctga gattactatt ggggatattt taaagacttt taaaattaac 180
tcaaatggtc ttaaactcac tattgcagat gcttcaaggt cggcaactgt ttatcatact 240
ttgaataaac caagccctaa tgaacttggt atgagaacta atgaagagaa tgatgcaaga 300
tatgcaaggc ttgctgtcac aaataacttc agcattaggc aagcaatcgt aactgatggt 360
gagcagttaa ctcttaagaa ggcaaccaat gcaggtgctt catatattca aggtagagat 420
gctaataatc agcaaacttg gtatgttgga cagggaaatg caaacagtac taatgtttat 480
ttgtacaatc atgctactgg tgcaatgtta acccttgatg caacagcagc atttaataag 540
tcacttagaa tcactggtca agtccaacct tcagatttct ctaacttaga tgctagatac 600
tttactcaga cagtcgctaa tcagagattt gcacagttag ctggtaataa cacttttaca 660
ggtgctaata catttaggac tatgagtatt atctcaaatg ctgctgctat cacactacaa 720
tgtgcatcag caaatgaatc tctgtatcta cttgcaaaag attatcaagg tggtaactta 780
tggtatgttg gtaaaggtag ctcaggtgat ggtgtcacta tctacaacta taccactaac 840
acctctttgg tcttagatgc tgctggagta tcagcaaata aaaatgtcag aatcactggt 900
caagttcaac cttcagattg gactaacatt gactctagat atattccggc agctacttta 960
agtacgattg caagaactaa tgcacaaaat acttttaatg gtgtgcagaa agttgttacg 1020
gataatgaag ccctaattgt gaaaaactcc actcagaaca gaccattgta tattcgtggt 1080
gtggatactg ctaatgtttc aaggtggtgg gtgggtgttg gtggtgcaga cagcaatgat 1140
gtgacactga ataatagctt ctctggcact cagttagttt taggtaactc ttctgcttat 1200
atcaataaga cactgtctct agctggacag attcaacctt cagattggtc taacttagat 1260
gctagatact atacccaatc taccgcaaac tctaggtata tgcttgctta ctcttctggc 1320
agtggtacag aggtgggtga gagtgatggt attgtttgga atgctaaaac tggtctgtat 1380
aatgtgtata ataaatctgg tggctcaaca cagttagtct accatatgta tcagggttca 1440
agttctacac catctgcaca gtttaagttt atctacagga atggtggtat tttttacagg 1500
acagcacgag atgggtatgg ttttgagcag gcatgggcta aaatctatac agaccaagat 1560
aaaccaaccc cttcagaaat tggtgcatac actaaagctg aaactgacca gaagattgca 1620
gaagcaatta gtgactctac agacctgaat aaaatctatc cagtaggtat tgtaacgtgg 1680
tttaacagta atgttaaccc taatacagca cttcctggtc taacttggac gtacctgaat 1740
aatggtgttg gtagaactat tagaattgca gcagcaaacg gttcagatgt tgctacaact 1800
ggtggttcag attctgtaac actatctgtt ggtaacttac cttcacacac gcatagtttc 1860
tctgctacca cttcatcatt tgattatggt actaagacga caaatacgac tggcaaccat 1920
gctcatgata gaggtaacat ggaaatttct ggtgtgtttg gttggttcag aagtgataac 1980
ggtgcattct acactgctag tggtgcattt gtgatgggtg gtagtcaggc ttcgcatggc 2040
ttcacaggct ctaacttcac ttatggtgcg cctgttgatt tccacgcgtc aagaacttgg 2100
acaggtgtaa ccaacacgac tggtaaccac gcccacagtg taggtattgg cgctcacagc 2160
cacacagtta gtggtaacac aggtggtact ggttctggtt cagcatttag tgttactaac 2220
cagttctata agctaatggc ttgggtaagg actgcttaat cccttgttga ctgattgtta 2280
agatggtgtt aatattcttt atgggtattc tcaccatctt ggctgatgag gtgattaaat 2340
gcctacaatc ctagcaattc ttctaaaaaa tctaggtagc ttcttctgga aactcatttt 2400
atcccttcta agtgaataca tgattgagaa agtgttcttt aggcttgcaa gataccttgc 2460
gagtaagaca gacacaccta ttgatgatga gttcgtagat aacttagaaa aagcttttaa 2520
gggggagaat aaatgaagtg gctagaggaa gcttttaaaa ataatattgg tgcaattgta 2580
gttggtattt ttagtgttat tgggatgtat accaccatgc aagtctcgga aggtaaacaa 2640
gaagtgtcta tcacaacaaa attacagcag ttagataatt attccaaaag taattattca 2700
gctattcgtg atttacagtc tgatatgagg ctgcttcagc tagggatgga gaaccagaaa 2760
gtccagttag agaatgttaa gggtgagaac gcaaaactta ctaagactct tgataaattc 2820
tctgatagtg tgaacaatct ggctcaatca gtatcagccc tccaagctat tactgaaaag 2880
aacacaaaga atactgaaaa gtaaagttta aggctcccgt aaaaaggagc cttttttgtt 2940
ttatttacct actgacaaag gtgcttcaat ctttcctgca tgttggtagt tgttaatacc 3000
accaataaag tcacttgcag tgagatgttt taagtcactt aaagtgttaa gtgggatacc 3060
aatttcaaaa gtaggtgcat gaaactcttc attattcatc agttcataaa cctgcttcat 3120
atgattctga taaatctgag tatctccaaa gacacctatc aagtaccgtg gagtgtatcc 3180
agtcatcttg caaagaactt ccagaataaa accataagac gcaatgttaa agggaagtcc 3240
taaaaaggtg tctacagaac gctgatacca ttgtaagtca acttcaccct cgttagtgat 3300
ataaatctga aacagaacat gacaaggtgc taaagccatt gagtttgctg caatatctgc 3360
tgcattccaa gcattaacaa gcatgtaacg gtctgtgata tcacccttca tctttgttac 3420
taaggtctct aactggtcta caacacaacc attatgacct tcaaagtttc tccactgaac 3480
cccatagatt cttccacctg catcttctaa ccaatcttgt tcagaagaat aattagagct 3540
taaccagcgt ttaaaatcat ctgaccagat agtccagcgt tctccgtcat tttcacccca 3600
agtgcggtaa cggagttcac caagcttatt ctctccattc aggaaccata aagcttcccc 3660
aataacttgc cgtgtaaaca cctgtttaga tgttaagagt gggaaaccag ttcgcatatc 3720
aaaacgaaac tgaggtggag caaatgcaga gataacatct ccagttcgtg tagtacgcag 3780
ttcaccgacg gataaaacat ggttcaggat atttttgtaa cttgaatctg cttgtgacat 3840
atcaaaagtg tccttttact tgtggaacat aaatttcaaa agttgcttta ccatcctctg 3900
atggttttga ctgtaccttt gtaaatacac gaccatcata caacttctcg aagaagtttt 3960
caaaaggtaa atgcacagtg acctcttcag taactttatg gaaaactgta tggaacacta 4020
catcagcata cggtaaagca ttgacaagga cacctgcacc accaattaca aagacatctt 4080
catcaataga gctatcaaga tacttcaaaa atgctccaaa tgattcttta ctggctttag 4140
catacattac atcatctttt ccaaagtcta tgcctaagta tggtacagag tttgttaaga 4200
tgaggtttgc acgttctggt aaaggtttac tacccagagt cttaaaagtc tcattcccca 4260
ttactactaa gttgttttta gtgagtctct taaactcttg catgtcctgt ttatgtcgag 4320
gccacggcat accagttgga gtaccaaact caccattttc accacttgca aagattaatt 4380
taatcatttt ataaatcctt tcttaatgaa ccagttgata acattcttca cagctttatt 4440
gctatggtag attaagtgtg gtgtaggctg gtatgagcca taggcccaga atgatagatg 4500
ctcaccacaa ccaacacatg tagtactttc ttcagctata tgaccattaa caatcacatg 4560
aacttcacct tgcaagtctg tacacccaca atgtggacaa cggataggtg ttccatcaga 4620
ctcatagcag tgttcaattt tcatttattt aacctcaaaa actggttcaa gaaccaatat 4680
aaagtctaca cagtcatctt cttcatccag aataccatca aagttcttag gaacgaagtc 4740
gaagaggctc ataacaaaac agtcagaagt aaccctaata gtgcctccca tattctctac 4800
agtcaacact gtacgacttt cttttacctc tcgcagaaga ttgctcacaa agtcatctaa 4860
gaagtacttc atcagtgact taaaaccttt agggtaatct gtaccagagt atgtttcaat 4920
ctgaggtaca agcttaacat ttatgctagt cttcgttaag aacgtgctag aaaacatact 4980
atcaggtaac ttacaaacag tctttccagt accctcatgt tcaatctcaa caaaaccagc 5040
ttcatcacca catgtaacta ttttaaaaga tgcttcattc tcacactgtg aagccataaa 5100
ctttttaagc atcactttaa ggaaatctgc atgagcatct tgtaagtaga tatcaatcat 5160
agttaatcac ctattatggg aacagacact tgatgtcatc atttttgaaa acgaagcttg 5220
tgtagttgtg accgtcaaca tccagccaaa cagcctgaac atctacacca tcaagattgt 5280
ggtaaagctc tttgatgaag tggtctttca caatctgact gctagttgtt tccagcttaa 5340
tgtttgagat tgctgaacct acagccatat aacccatgac acgtttcttc tcagtatgct 5400
taatcttacc atctttgtaa gccataatga gattggaaag aatctttaca ccataatact 5460
tttcaaggtg agtcggtaca ccataaaaac ctaatgcgaa gttttcagag tggataatgt 5520
tttcacgttt catattaatc tgccagcctt aaattaaaat taacaccaaa tttttcacat 5580
acttccaaca taaactcaag agagacatta ccagtgagat taattatgtt ggatacacga 5640
gcctgagaga taccacaaat tttagcaact tgctcttgag aaagacctct agatttaatc 5700
tctctcttaa aacgatgtgc aacgaaatct ctcatttcgt ctacatccat tggacaaata 5760
taactctcca tttcagcctc tctgtcagcc tcccaatcat cttgtggagc ataagggtca 5820
aaaacctcat tcatctttct tcttccgttt acgtgaagtg tcaagcaatg aatctatgtt 5880
tgtagcccta accttttcac gttcatcgtt aagataatcg tcaagagcct tttcaatctg 5940
tgaatcatct tgacagataa ctgtaaagac tgtaaagcca tctgtatcat caattatctt 6000
accatagccg cttctgggat gtaacttttt aagagccata tcaaatacct gtgcagcttc 6060
tgttttctaa gaaatactca acaccataca tagctttttg catcaaatct tctttaaact 6120
gaatacgatg attgagcata gcctgtacat caatatcatc atggtgcttc tctttaatat 6180
ttgcagattg caggaaatac tctgcaagaa cgcgatattg atagtcagtt accttatcag 6240
tcatctctgt taaagactct tcagagattg gtttaagtaa ctcaccatta gccaaagcaa 6300
cacctttaac aatctgacaa gtgtcttctg gaacacctga taagtccaca tttggaagtt 6360
ctgttgcatt gactggtaaa ttcattgcca taaccattga agcggctaat aaaatctttt 6420
tcatgatgac ctcaaagtta tattaaattt tgtattacgt tgagtataaa aataagctat 6480
aacacctgac ttgtcaacag attttatagc ttatttggca gattttttat aaggctactg 6540
ccatccttcc atactttacc gaacatcacc ctactgaacc cgaccctacc tcacccgacc 6600
caaccgaacc gtaccgagca cagccttatt aaaagaccct gtttaaagac cttttaaaaa 6660
ggtaagtctc attttaatgt agctgagact aactacccgc accgaacctc acctgacctg 6720
accgcacccg acctcaccgc gccatgccat accatacctc acttaaaaac ccttttcaaa 6780
gactcttaag tgatgtaatc gaattttaat gtagctccga ttaactaccc atactttacc 6840
taacccgacc gcacctaacc cgacctgacc ctaccagacc ccacctagct aaaccttatt 6900
aaaagaccct gtttaaagac cttttaaaaa ggagcctccg aagaggctgc ctagtcaact 6960
tagaaaggtt ttctacgttg cttgttaata ttagctttca tagcagctaa gtggttttgc 7020
tgttcaatta gtcgtgaacg ttcttggtgg ctaagtgcca tagtattcac attctcaatc 7080
gcttgagtgg cttgctttaa agcacgacca atcttaccag acattttacg ttttgcaata 7140
gttgcttgat gcttaggttc aaccacacga taaccaacac ccattgaagc aaccagataa 7200
atcttctctt ctttaagaag cttttcaata aacttctcaa gtcggtttag acgtgtaagt 7260
gcataagcct tcatttcatc ttcgtactta acaatgtcac cttgataagt tggcttcaca 7320
atacctaaga aggtgtccat ctcagaatgt gatacagtgt caccgtaact aaactttgct 7380
gctaatgctt ttgtattcat aattaaacgt taccttctga aataacttct acagaaaaac 7440
gaccaaagcg tggacgccaa tcacctacac cacaaaggtt gccagcattt tccagaacca 7500
tcaggagttc ctctcgtgta atttgctctt catcgaacat caaaccaaac tctacagacc 7560
aatcacggaa aattggacgg tagctcataa ctcgtgctgt accaatctta acagatttag 7620
cgtagataaa atctggattc ttagcaagtt cttgtgggtt tgctggacag ttcttgatag 7680
tcattgggaa gactacatct gtcagcataa tggctcggtc aattacctta ccaagtttgt 7740
tgagttttgc accagacttg atacaagcct caatcatctc accgttcata acaaaaccta 7800
actgctcatc atagtaacaa gaagttacca gttgactttc tgctaagagc gcatagtctt 7860
cgtcagtctt tttacgttta ctggacaaag acttgtgata ttttgtcaga ggatttaatg 7920
ggtctgacag cgtatcagcg tggctcagga aaggacgagt accagtgatt ttgatattta 7980
acagtttcat agcatttagc tccacacttt agtttattgt taagttgttt gtaaagaacc 8040
ctcaacagaa gactctttag aaacttggta gatttttaac gtggctacta ccatccaccc 8100
atacctcaca tcaccatact atacctgacc ggacctaacc aaactccaca gcaccataca 8160
tcacatcaca caaccttatt aaaagaccct cttcaaagac cttttaaaaa ggtaagtctc 8220
attttaatgt agctgagact aactacccat actgtaccgc acctgacccg acctgaccag 8280
acctaacatt accgtgcaac accgtaccga acagaacatt gccatacctc gcatcactta 8340
aaaaccctct tagaagactc ttaagtgatg ctggtgagta cccgtgggtc tatgctgctc 8400
agtatactca ccaaattttc tatgtcaaca acttttattc ataatctcgt ataaccagcc 8460
caacagggaa ctgtaaagac cccttacgag tcatcttctg gaactgaaca gttaaaggtt 8520
tcccgataaa ctcttcagga tgttcagcaa gatactgttt cttctcgtgt gtagttttcc 8580
atgagacatc tacaaagacg ttaggaagag tctctacaac gaacttgcca tgaccacgct 8640
tatcagtctt tacaccagtc actttaaact cttcagtgtg catcttcttg tgcttaatca 8700
agaagtatga acggtgacaa cactcataga aggagtcttc agagattgaa cggtacattg 8760
caccttcaaa cttagcttca acccacttat catgagcttc atcaaactct tcccaagaat 8820
ttacacgacg agacttaaca gggataactt tacagccatc tctaaagtca ttcaatggtg 8880
aagtctcaat aatatctcta cgctcaggcc acgtcttagt actgtcacag atatcgtacc 8940
agtaaaattt gagcaagtgt ctgtcaggat tgtcttcatt tttaatcatg gagacgatat 9000
cttctaagtc ccacccatga gcataaatct ctccatcaaa ttcatcaaca tctttgtgga 9060
gccttaacat tgccataatg tcaggtatta gctcagtagg tattttataa acagtattct 9120
cacgagagta tgctgtaaat acactaccaa tgcttaagtg ccttgaaatc ctacaacgga 9180
caccatcaag tttcggttga gcatcagcag ggaacttcag atactttgca tgactaacct 9240
ttgcagcatc atgagcaagc tgtacaccca ccttctcagt attctgtgca gactcttttg 9300
tgtaagcata acctttgcgg tcaacctgct ttttatactt tgcagcaact tcaaagagag 9360
cttgctgttc agcgttacgc tcattctttt taccgatgtt tttaggttct gctgtatact 9420
cttcaaacat catcttgcca ttttctttac cgtaggttgt aataactttg tcacctacag 9480
ctacacatga ccagacgttg aaagagccgt ctttattttg tttgtacagt attgtcattt 9540
tattcctctg agtgatacca agtattgcaa gcattacatg cgtaaagctc taagtcattt 9600
ttatctggtt tatgaaaaca gtttgctccg cagtggcaac gaaacggctt tccaccaact 9660
gtcaggatga aagattgaac ctttccatca ggagttcgta cagcattttt atgcttatct 9720
tctttgttca ttttctctta ccaccaatct ctacgtcaat aacctgaaca tcaccagcaa 9780
catcaaactt gtgaatgatg ctttccacag tgtaagtcca accctcgatg cgaaccaatt 9840
caccttctcg tggaactatt gggttacgtt gagctacggt tgtagtggag tgaacaacat 9900
cataacaata gatatagttt actaggactt ccatcgtgaa aacctcactc aataaccgta 9960
gtgtgcgatt gaccattttt gccatagcta taagatgttg ttgtttgaga atttggacat 10020
ctgacaacaa caatatctct gcttatggca tcaccatgta acttatagac tttgcaatct 10080
tgcatctctg gtgggaggct ataagaagcg tttttgtcct cgtaagaggg attgcagcca 10140
gatagaaaga taacactaag cacaattact gctttccaca taatacttct cccattactg 10200
ttcacgatgt tctacttctc tgtacgttcg tcgatagttt ataataagct gtgctaactc 10260
gttgctttta acatctgaac gtagaagttg ccaatcaaaa cctttgccga ctcttccata 10320
taaactaaag aattttactt tgcgtttcat tttggtatct ccagtactca gatgtcaatg 10380
ctaatacgtt cacctttaag gactcctgta cggtcccttg caatcttacc gtcaacaata 10440
aggatatgcg cagaccttaa agactcacta ttaaaacctt ttacgaactt gctgggcatc 10500
accacaataa gcatctcttg aatcagctct ggagtatagc aattgtcgtg aagttgctga 10560
ataacgtctg aaagtgtcat ttttgatgtc tccaatactc aatatcaata agttcttgtg 10620
caatgctttt cagattacct ttcaagaaaa ccttctcaga acctgaaaga gatgaattgt 10680
caattagcct gttcattctc tcaaacaact tctgcttacg gttctttatg gtttcctttg 10740
aagcaccttt catcatcgct gcatgggttc ttccacggtg gctcatagta aactctcaat 10800
atttacacgc tcacctttta agtagcctgt cttatctttt gcgattttac ctccagcaat 10860
aaagatatct actctacgta gaacttcagg gtttaccttc aaattcctac agctactgca 10920
aagaacaata aacatccctt caatctcttt ttgagaataa cctgcacatt gcaactgaac 10980
ttcaatctct gaaagtgtca tgtgacctcc tatacaagat atagtataag gtactcttcc 11040
atgagcacct tgtcaatact aattagtgta cttcaagcca gcttttgcca accttaccag 11100
tgcctgtaac aagcgtttct ttcttaagac ctaagttttt agaagcttgt ccataatacc 11160
agtcagtaac tgctttcaca tctgccgtca tactttctgg acattcccaa ctcgtctcat 11220
cgtgatatgc gcagagcaaa cgagcaccaa tagctggttg acgaccattc aacttcatca 11280
aaccttcatc aatcatacga cgacaagcaa ggttgattgc ctcattttga atctgagcct 11340
ctgaacccat caaaagatag ttcaacagtt tgtgtggaga tttacaccac aaccaagctc 11400
cagcaacctt tacaaaaccc cctttagaga ttgcagcagt tttaccaaac acctcttcaa 11460
gtgccttttt agttgctttg tagtcagctt ccaagtcatc tagcaacttc ttaattttag 11520
gcaaacgaat aaagtaagtc tgtttagtca gtgcaccttc ctgagtagtt gcagctttaa 11580
tcgtcttagc aaacttctca tcacctgcac cgaacagtag tgcatagata ccattcttag 11640
ctttcttacg acccttgcta atctcatgga gaaggttctc atcctgagta gctcgacaag 11700
tcaagatatc ctgctcatca ttcaagctaa agtaaatact gttcagtgtg tgtgcatcag 11760
tccctgaata gacaacgtaa aggtcgttct cagagtcata gcgaaggtac ttatcaatct 11820
ctgggttgag gtactcatca aagtgtttgc agtaatagcg tccatcttct tgacgaataa 11880
actcaatctc ttctttacct tgcgttaccg ctttggtgaa gtctttgtca cccataaagt 11940
tacaaagaag aactagctgg gctgagttca tgtctacaga gacaacgtta gtaccttcct 12000
cacaaatcca tactgccctc attggttccc catagacagc agcaccagac ggtacgttta 12060
caataccata ctgtgtcata cgtccagttg aagtaccaaa taccattgca ccagcactaa 12120
ggcgaccatc aggacgaatc tggttcaacc aacctttttc atcatccttt gagttctcaa 12180
tagttctgcg tcggtgcatc aaagtatagt atttcgcaat cttctgtcca agctcaccct 12240
caatcgtatc ataagatgat tcagtgagtt ttggtgaagt acggattaag caaggctcaa 12300
gcaaatctgt gtatttcttc acagaccagt tatgctcgat gtactgaaca ccctcgtgtt 12360
caacataact caaaccacac cgttccacca tctcctgcca cttaggatgc tttgtaatca 12420
tctttttgtt atctttgaaa cgacaaactt tgacaggacg accgtcagag tctttcttgt 12480
agttccagtc atctggaatc caaccaactg atttcaagta gtctttaaca actgctacct 12540
gagtcatacg agaaacttcg aactcaattg gtgtatatgg tgcatcaatc aaacctgtgt 12600
agcgacttga ctcaagctca aagtggttaa cgacatggct gttatagtac ttcacagttt 12660
taactgtttt tactggcttc cagtctttgc acttcttacc aatctttgca ttcaactcat 12720
tgcaaattgc acgagcatcc ttcattgcta caaaaccctc cttgtactct tcaccagtta 12780
cagagttggt tggtgtatag caattccgct tttcaatgtt aaaaatcttt gtagttggct 12840
taccaaatgg cttgatttcg tatgtctgca tctcaccgtt acgtacctgt tgacgatact 12900
ttgtcttagg gtacttggta attctcttca atccatctgc atgaccgaat gcttcaacat 12960
actcattcca agcttttgca aactcttctc cagtgacttt acctttggtc ttaatagttg 13020
gaggaagatg tggttcaacc tctgaagcaa gctcgttagt caacttgtca agttccttta 13080
catgaaactc cataagctct ttatcagctt tccaaccatt gatagcctgt tgactcatcc 13140
agaaagatgt ttctttagct cgcatgtagg tttcataagt gtctatgcca cacttcttca 13200
gcttgagata ttcattgtcc agtgcacgtt tagttttggc gttaatacgg atatcttcca 13260
caacacgagt gaagatttct gcattccaca caccccaatg ttcaatctct ggtttacgta 13320
caccaacacg agcaccccat gctgccaagc catgagcacc tttataacct tttggagttg 13380
gtctatccat ccactgaaca cgagactgga taagagaatc ctgaaagaaa ttactccacg 13440
gcttacactt tggattatca aagttccaca aatcaggtgc aatgtgattg aaaacccacc 13500
agtcataccc cagaccatta tggatgcaaa gtcgttttgc ctttaacgca aactcaatac 13560
cttcacgcaa accaccctta atgtacttgg tatacttgtg accgaggata ggttcatctg 13620
taaagaccca tacaggcggc tcttcatcgt cagatttata gtctgcgaaa gccattacgt 13680
gcactttagt gaactcaagg agtaaaccat cagtttctgt atcaccaact aaatgtaagt 13740
ttttaaaatc tacgttttcc atttttactc cctctaaaat cattaacctc atacattact 13800
gcaaaaaatt ataaaaagca agttgacaat gtgcttgaca aggtgttaat ctttgcgaag 13860
tgggttttta cttaaaaggt tacttaacag tttaactata cagatacttt aaaactctta 13920
aaagattatt taatagcttt taaaaagctt taaagtataa cgtatagaat acgttaagat 13980
aaaagattaa aacttaatag ttacttaaaa gaggcttaca tgcagaaaat gtttgttcac 14040
ccagacatag ctgactttgt tagagctttg cagaagttgg aagaacttga cattgctgct 14100
caacgggaat atgctcacca tcacaggaga atggttgata tacaacatga aatagagttg 14160
tctgaaaact atgaagatga cttaaaatgc tctgtgtttg agcacatgaa agatgttgct 14220
acagcgagac gtaaagctaa agacacagtt gctttactag attctcttaa aaagaggttg 14280
caaagtggta cagacctgtg taatctagcg tcactgatta acgatgttga aacttcttgg 14340
gatagacatt actatccacg ttctgaaaag acacttgact tttcttcatc agaaaactta 14400
aaatgttcta gaaagaaact taaccaactg agagagaaat aatatgaata ctactgtaga 14460
ccagatgctt gaagaaaaag gtattgacga agcattctta atcaatgccg tagaaagcat 14520
ggctatgatt cttcgtggaa acaactacac agaacagttt atgcctgcaa gctataactt 14580
gcctaaagac aagtctgata acggtgcttt tgcagaacag gttaatatga tgctgtgtga 14640
taaaacactt gctttagcat ctatcgttgt aggtgttgat gctcttgcag agttcatttt 14700
ctatgagatg cagaaaaatg actatattgc catgacctct gaagaaaaag actcgctact 14760
gctcttaggt tatgatgagc tagttgacct gttaatttcg tcaacaagtg ctgttatggc 14820
tgctcttgag aagcgaatgg aagagaagct gtcacgatat gaaaaacttg attggggtgt 14880
agaagagtta aacccacgaa tcaaagagat tatccagcaa actaaagact ctttagcaga 14940
agctgtgaaa gatgattctg tagctgatgt agaaccacta caggtgaagg ttgctctgat 15000
gattccagct atctctacaa tgatgaatat cgttgcacta atccaacttt ctcagatgtt 15060
gggtgtaacc attgagtttg ttgaggagat gactggtggt gtcagtatgc aagcaatcaa 15120
tgatgttttg gtaaacattg gcgcttcaat cattgaagag aagcttcgca ttaactttgg 15180
tgacgatttt gtgaagcaag tctcagctat ggctgaaaaa gagtattcac agattactag 15240
tgaccaactg cactaagtct ttataaatct tacaagcctc cttagttggg ggctttttag 15300
taattaaggg agacaaaatg agtaatatta tcgcatttac tggaaaggca cgttctggaa 15360
aagatacctc atgctctatt gttaagaata tcctagaaga tgagtatggc tacaatgttg 15420
cagtgatggc ctatgcagac aatcttaaac tttctgcatc aaagatattt gacttgacat 15480
ggaatgacct gtacggcgaa actaaagaga ctccacaggt ttttgattta tcatactctg 15540
aacttatgtt caaagttacg gaagctatgg agttcacctt cagagatgaa cgttaccata 15600
tggactttaa gcttatgtca gagttaaccg gacgattaat tatggaactt aagaaggtcg 15660
ctaaaccgac tctattgaca cgtctgggat ttagcaaaaa gtataaattc tcatcaagac 15720
aaattcaaca gatttggggc actgaagtaa tccgtaaagt tatgggtgac aagttctggt 15780
ctaaagacct tgaaaaaaga atggtaaact tctttgacgt ttgctcactt aggaatcaag 15840
aaggtgttgt tttaatcagt gacttaaggt ttgactctga agctgagtgg ctgagcagat 15900
ttgcacacca aactattgaa gtaaaaagag acaatgtgga caaggtttca tcacatgttt 15960
cagaaaatgg aatttctacc aaatatgcgc gtgacattat ccataataat ggtactcttg 16020
aagaccttga aagcaagcta agagctatcc tgaaaattta aaagagagaa tgaagatgag 16080
agtaaaagat aattttaaag ttattgacca tcgtttagtg gaactctcat ctctttccaa 16140
tgaggtaatg attgaacgtc ttaagagagt tgaatcacga aggaaagaga ttgcagacga 16200
aatccatgaa ctggacaaga ttgaaaatgg attgaaggca gaactacaac gaagaggtgc 16260
taatgtctaa aggtcgtaaa ttgaaagagg ctggtcagtt tattggtcat tgtgcatgtc 16320
cacgttgtgg ttcatcagat gctggttcaa tctaccacca tgacgatgat tcttattcga 16380
tgacttgctt tagctgtaac aaaggtttcc cagagtggga ttttgataaa ggacaaatcg 16440
tgagcactta ttctactggt tcagataata aaaaccgtac tttccgtgga atggatttag 16500
atgatgtaaa agaaaaccta gaagcaatgg acttgaaaga caggaagatt cctgcaaaag 16560
tccttgaacg tttaggtatc aaggttgaca ttgatagtga cggtgaaatt gacgcacatt 16620
tctatccaac ttacaaacgt aatgaagatg gtaagctaga gcacgtcggt tatcgtgtac 16680
gtcaccgtta cccagaagac cacccaaaag aacatctacg cggtaagtta aaagactttt 16740
caggtggtgt tggggacatt aaaggtgaac tggcaatgtt cggttcatgg attgctccag 16800
aaggtggtaa ccgtctattc atttgggaag gtgagatgga atgtgccaca gcaatctaca 16860
tgacttctct ggcgattaaa gataagtctc gtcgtaagaa ttactgtcac gtatctgttc 16920
catcaggtgc aaacattaag tctatcaaag acaactatca gtacattaca tcatttgatg 16980
agatttactt gtgttttgat aacgatgagg caggtgctaa agctaccaaa gaggctgctg 17040
gtatcctacc tattgagaag gttcgtttat tccagtatcc agaaggcgta aaagacctta 17100
acgaatggtg gacaaagttc tataaagaga aagacacagt tctggaagga tttaaacagc 17160
gtatctacaa tgcacctcgt tactgccctg ctggtattaa gaactttgca gatggttttg 17220
aggcaatgaa gaatcgtggt cagattccat tgattccctt cccagaatct ttcggagatt 17280
tgaacaggtt gacttatggt ggttacggtt taggtgagat tacaactctg gcagcaccat 17340
cttcagtagg taaatcagca tatacccgtg aaatgattta cactgcttgg aaggaaaccg 17400
attacaatat tggtgtaatt ccagttgaag atacctatga agagttgatg gagatgctct 17460
gtgcaattca tctaagcaag cagatttctg agattcctta tgacgaacgt gattgggatg 17520
aattgaagga agcacacgca gaactatcta aaggccgtcg tatccatatc gttgaccatc 17580
aaggggcaat tgaccaagat aacctgctgg aatttgttga ctatcttgtt aacagcttag 17640
attgcaagat tatcattctt gaccctatca cgttggctct gtcacgttca gatacggatg 17700
aagaggaagt tttgtctgag ctattgcgtc gttgcaaacg ctaccagtat gcacaggtaa 17760
acgtatgtca cgttcgtaag agtgcaggtg gtcagaaagc taactctgaa ggtggagata 17820
tctctgaaga ggatattaag ggttctggtg catacttcca gatttccatg aacaacattc 17880
tgttaatgcg taacaaggtt gacccagacc ctgttaagaa aaacttgaca aaaatcaagt 17940
taactaagtg tcgtcgtcat ggtaagtcaa caggtattgc tggtcatact tggtacaatc 18000
cagatacagg acgtcttatc aaagcttctg gatgtggtgt tgacattgat ggtgcagcag 18060
aaaatattcg tcaacagttt ggtattggtg aagctgaaga ccattacgat gactctttac 18120
ctcattatga ggatgaagtg tttgaccgtg agactggtga agtctatact gaagaacagc 18180
gtcaaagttc aacaattcca cctgtattaa gcgaggatgc agatgactgc ccgttcgaaa 18240
ctgagtgata gttataaaag agaggggttt atacccttct ttgaagaaaa aagtttaaat 18300
aaatttcagg aagagtgttt gacaaagatt gataggttct atagaatgct cctcatgaaa 18360
tgcgatgggg aggttgctat aaaagaagat tcctaccaaa gcacgattac agtcatcatt 18420
acactgcctc agcataatat tagttggctg tttataatta aagaaaatgt gtttgaatat 18480
caagtatatc ggagaatatc atgaagcact ctaaagcatt tgaaaaagtt tttggggatt 18540
ccttaaaagc cactgctggc aaaccagcaa aatactatga agagaagcgt gtaagaactg 18600
gaaaaactgc acgcaaagca gcttctaaag ataagcacaa cttccagtaa ttaatgtttg 18660
acaatagagt atcaacaaat tagaattggt actcttcata aattgagata gaggtttaaa 18720
taatgtctaa agttgttaaa atgaaagctc cggtagagaa gtacaatggt actgaacgtc 18780
aaactctgcg ttacctgctg aaagatgttt ggttttatta tctgaacact tcaccacgtc 18840
cgggaaaagg taaatccatt gataagaaat tcccaggaaa agactgcaac tatagtgttt 18900
caattctggc agaagatggt aacaagctgt ttaaagagtt tactaagtct aagaaaaacc 18960
cagaaggttg ggataaagtt actactgaag cagttgatgc agatgacttc gaagagaagt 19020
ttggttgcaa accaccattt gaagcagaca cttactacat cttgaaagta agtcgtgcag 19080
cagcttataa agatggtgct gtgtggacag ctaaacaatc attccctgta atgctgattg 19140
aagaagtaaa tggtaagcgt gtagctgtta aacagccgat gaagaaaatc aaagctcaag 19200
catctgataa gcatgaagat gacaagaact atgatgtaat tcatccagat attgcagttg 19260
gtaacggttc ttttggtagt gtgattcttt ctactcactt ctacaccttt gagaacaatg 19320
ttctgacaaa acctattcag gaacagttta tcattgatac tcttgtacct tacactggtg 19380
gtaacggtgc taacggcgaa cctgaactgg atgaagatga actggctatg cttggtcttg 19440
atggtgttga agataacggt gaaatcactg aagaagatgc aacagaccac aaaccttcga 19500
atgcttctga tgatggtgat gatgaagact tgccagaccc agatgatgaa gaagatgaag 19560
acttcgatac agaagactaa tctctaaaag ttactttaaa gccctgtact tagtatgggg 19620
ctttttcata tggagagtca caatggagaa gtatacacta acaaaacttc ccgattcagt 19680
tacacatgtc tttattgact ctgatagtat tgcctataaa ggtgcttgtg tagttgagaa 19740
agcaaaatat aaatacgtca ataaactcac agcagaagaa tctgaaccat ttgataatgc 19800
aaaagatgct gcaagatggt tagcagacca gagaatcctt gtagaagagc ttggcctaac 19860
atttgatgaa gatgaatggg aaagacagac ttggaaagaa gctaagagtg aaaaagaagc 19920
tatcatggct actcagcagg ttcttcagga atggcttaag gttgttggta aagaaagaac 19980
ttgggtaggt tacttaacag agaaaggtgt acataaacat aaagacgtta aaggtcttga 20040
gcaccaatat caaggtaacc gtaaagatgc tgtcacacca acacacttag tcgcttgtcg 20100
tgaatatctt ttatccagac cagagttcaa actcttaaag ggtggattcg aagctgattc 20160
aatcgtaatt gctaaagctg aaaagatggg taagaaagct gctcttatga gtattgacaa 20220
agaccttcga caagctgaag ggacttattg tattgatatg acttatgaaa agtcaccact 20280
aatttttatt gctgataata atgttggtga tatctgggat tgtcctatca aatcaaaacc 20340
aaaagctgca aaaacagttg gtgtaggttt taaatttttg tgttatcagg ctgttgctgg 20400
agataatgca gacaactact ttggccttaa aggtgctgga aaagtagctg tgatgaatgc 20460
tttagatggt aaagagactt ataaagagtg tcttgatgct atctatgaac tctatgctaa 20520
aaaagagtct tatacctatg tatcgtggga tggacagact gttaccaaaa caccattaga 20580
gttaatggaa cagcatttct ggatagcata tcaagagaga aatccaaaag ataatttctc 20640
ttttaagaaa tataattggg agccgtgtat gaatgaagat taaaggattt ggcaattgtc 20700
ctacctatgg tcattgggta tctttatgtg gggaagttga ccccactaaa cattttggct 20760
ttgtctattt agtatactgt aagaaaaccg gacaatatta tatcggaaag aaacagctta 20820
atagcgtgac caaaagaaaa gttgctggca agactcggaa gaaggtagtc actaaggaga 20880
gtgattggat gacttatgag acttcttctg agtatattaa aaaagatatt gagagctttg 20940
gaaaagaatt ttttgacttt tacattatcc aaacctacta cacgaaaggt ggtctagttt 21000
atggtgaagc aaacctccaa cataaattcg atgtaatgac aaaaaggatt gactctaagc 21060
tcagactctt ctacaatgcc aatattgcag caattaagtt tattactaaa gaaacttatg 21120
aagatgctga aaaaagaatc cataaggtaa tgaaagcgaa ttgtgcttaa taactattga 21180
gagagaataa aatgtttaac aaaagtaaag ctgtgagtca tgtagcaaag gttgacagca 21240
agattgaaga acttgagcgc attcttgcaa atgccagaga gtcaataatt aaagaagttg 21300
aagctgttga atctcaaatg cagcacctaa tgcttaaacg tcaagagtta cgtgagcatc 21360
tggaatatat tgaaacacga gagctaaaag ttaacagatt tttaaggagt ccgaatgttt 21420
aactcaagag aatcagtaaa aaactggaat cttcgttgcg gaaacaccca aaagcaacct 21480
tacagtgatg agtattggga atctttgaaa tcccagtctc tgtgtatgct tgaagaagca 21540
aaagagcttg taaaagcaat tgaagagaaa gaccccattg agacactgga tgctcaggca 21600
gatttgcaat atgttcttga cggtttgatt tacctgtcac aacatgacca taacggtgct 21660
atgaaagctg tttgccataa taatgacctg aagtatacag acgactatga agaagcttta 21720
aaacgtcttg cagatattga gaagcgtact ggtcaagagt gtattatcag aatgtcagta 21780
gttgatggta aagagtggta tgcgattgtc cgtgcagccg atgggaagat tatgaagcaa 21840
tcaaatctcc ctaaagtaca gcttggtgaa tacattgtag aacttgaaag ccaagaactt 21900
tttgtggtag tatctgagac atgcgttatc tgcaaaggta ttgtaggtag cttaaaggat 21960
ttgggtgtag atggttttgt agaagttaat ccaattacct ctaaagcaga taaagatttc 22020
tgtaaagaga atggactatg gattgcagat attgtctact atgatggtga gcagttccat 22080
gtaacctcat acccgaaact gaattatgat gctaataacc tgaagtgctg gttaaaaggg 22140
gttggttata atggattcac agaacattaa taaagaaggt gtggctcaga agagccactt 22200
ctcaaactac aatatctcta tgacggtgtt tatgaatgac ccgttacttg agaagtatgg 22260
tgagactcca gatacacttc tggataatga acaagtttta aaagcagtcc tgtacaaata 22320
tgggattgat attgagaaag agtattcctt tgaaatctgc caacacagaa atacttttgg 22380
taaagttgtg atggctccac tcttcatggg tgtagaaaga accgactatg gttggttata 22440
tctaaaaaga aacttggaga aataccgtgt ctaaagcaaa aaagttatct tatgatgaca 22500
ttatctcagg tgctaagtta ggtgttgaca gtatcggaca agacgttaag cacggagaca 22560
cagttatgta ctgtgatgac cgaagaggta gaagtgcaat cttgtttgga agaattgttt 22620
gcaagatgcg aggtaattac gttgttgcag acatggatgt gaacgttaca caaaaacttg 22680
aaacacttat ggatgataat acatcatcat catggttctt cctgaattgt atgcacactt 22740
cttcagtcac aaaagtaagt gataagtttt acgatatgtg gcagaatgag caaattttca 22800
agatttaaac tagggagcct cttcggaggc tcttttcatc tgtaggattc aaaaatgatt 22860
aagacaatta aaaaatcaaa cggtacagta gtaagctttg acccagaaag actgaataag 22920
tgggcatcat gggcagataa gcgtggaatt atctggtcag aagtcactat ggaagctatg 22980
aaacgtgtct atgagggttg cactacaaaa gagatgcacc aagccatgat tgatgtttgt 23040
gttgataagc aaactcaaga gtactcagat atggctggac ggctacttct gggaattatc 23100
tacaaagaag cctttggagg ctttactaag gttcctacgc tggttacctt cgttaaaaat 23160
atggagagag caggactttg ggagaagatg gactattctc aggaagagct tgaatacctg 23220
caaggttaca tcgtgcactc aaaagatatc tcttacggtt atgcagtctt gaaacagttc 23280
agagacaagt atggtatccg tgatattaaa actggaagat tgtttgagtc accacagttt 23340
atgtttatgg gtatggctat gaaagccttt gagaagcaac caaagcaccg taggctgcaa 23400
gatgttatca agctgtacac ttacctatct gacctgaaga ttaacgctcc tacgccttat 23460
ctgaatggtt taagagctac taaatcgggt tatgcgtcat gctgtttgat taaggcaaat 23520
gatactgctg aatcactcgg tattgctgca aaggttgctt atgatatgac tacaaagcaa 23580
gctggtattg gtatgttgat ggagactcgc actattggtg atggtattcg tcaaaatact 23640
attgagcaca tgggtaaact accttattac aagcttgtac gttcatctgt agaggcaaac 23700
aaacagaaga gtcgtggtgg ttcagctaac aacttctaca ctgctctaga cccgcagatt 23760
gaagatttac tacgtttgaa gcaccctaca acggttcctt caaaacgtat taacgagatg 23820
gactactcat ttggtacaaa tgattatttc tggcagtgtg ttcaatatga tacagactgg 23880
ttgctattct cttacaagga tgcaccaaag ctctatgaca tgttctacac agcatctgct 23940
gatgagtttg ctatggcagt tggtcatgca gtgcattcag gtgttaaaca cagacgggtg 24000
aaggctcgtg aaattgctaa actgtttatc caacagcgtt atgcaacagg ccgtgtgtat 24060
ccattcttca caaataatgc aaacacacat acaccattta aagagccttt gaagatgtca 24120
aatctttgta tggaaattgt gttgccagtg tacggctttg agaaagagac agacctttac 24180
agagatgatg ctgtgaaaga ggatggtgag gtagctcttt gcttcctagc tagtttggtt 24240
gcagggagaa tttcagaaga tgaatacgct gacgttgctt attatgctct tgcaatggtt 24300
gactccgtta ttgaccttat ggactatccg tatccgtcga tgcgcaacca tgttcagaag 24360
cgtcgttctg ttggtattgg ccttacaaat gtggctcatt accttgcaaa aaattacgtg 24420
aactactctt caagagcagg taagacgaag cttcatgagc ttgctgaaat gcactcttac 24480
tacttacacg aagcttcttt aagacttgct aaagaacgtg gtgtacctga gtatatgcag 24540
ttcactaagt atcctgaagg ttgggttcct ccaaagacag ctaacaggaa gattgatgag 24600
aagcatgatg caaaactaag atatgattgg gatgacttag cacaacgtat taaagaaaat 24660
ggtggaatcc gtaactctgt attagaagct tacatgccta atgagagttc ttcactggca 24720
actaatacga caaacggctt gtatccaatt cgtgacttta ttttaactaa aaagtctgca 24780
actggtaacg tactgtttat tgttccagat tacgaagagt tgaagtatgt ctatgaaatt 24840
gcttgggata ttgacacctt tgacctgatt gattgctatg caattgttca aaagttcact 24900
ggtcaagcta tctcttcaga tttctatgtt gactacgcaa aagctaagaa ggtatcattg 24960
gctcaagctt tgaagtacat gatttatgcc aactcagtag gtatgaaaac catgtactac 25020
cttaacagtc gcatcggtgt aggtaaatct gcactgcaag acgcttattg cgagggttgt 25080
ggtgtttagt tttaataact atgagggtcg taaaagaccc tctaaaatta actttggaga 25140
aactatgaaa gacctaatag aaaaacatga gaaaccaata tacttgttct acaaaccccg 25200
caaaacatta tactacgtga gcagcacagg catcatgata aagcagaatg atgacttatg 25260
ggtaactgga gcctcttaca tctctacagc agatggtaaa atctacgcaa gaccttacga 25320
gatgttcaat gaagaaaatt gggaagtttt agacagaaaa caagccttag agatgataaa 25380
gaaaggagaa atcacactat gattaaccaa cacccaatct ttttaggtgg tgaaagaaaa 25440
acatttgact cccttaataa acactaccca aaaatctttg agctatacaa acaacaaaaa 25500
gcgcaagact ggtcagaaga tgagttccct tttgaacaat cacgtcttga ttttgagagt 25560
gtgccagcat caatgtcagg tgtaatgctt gagattctta agtggcagtg ggaagcagat 25620
acccaagttg ctaagagctt agcgtttgcc tttgcaccat tcatctctga tgacatttat 25680
gcaactgcaa ttatgaagca gtctgagatt gaaaacctac atgctcttac ttactcggag 25740
attgtaagac aatgtattaa aaatcctgaa acaatcttag atgagattaa ccagaacatt 25800
gctgtacaag accgattaaa aactgtgaat cgtgttcttg aagaattact agatgaaggc 25860
ataaactatc gactaagcta tgtccgtgac tcacttctgg acaaagaccc tttacacttc 25920
cataaagtga ttctaaaagg tctatttgca gtgactgcac ttgaaggtat ttcttttatg 25980
gcatcctttg catgtacttt tgcacttgat gctcaagata aatttcaagg tattgctcaa 26040
gctgtccaga aaattatgct tgacgaaatc cttcacacta aaattgatat tgaagtttta 26100
aaagaaactt taagagatga tgagtggcaa aaagctttcc aacaaattct tccagagatt 26160
aaagtaatct tagatgaagt agttgaaagt gaagagaaat ggtcgtatta tatcttctcc 26220
gaaggacgtg ctgtagttgg gttaaataca aaacttcttc atgagtgggt ttactataat 26280
gctgccccac tgtatgatat gtttggcatt cccagagatt ttgtagctcc taaagaacca 26340
cctttgaagt atatgattaa gaagatggaa attgataaag agcagaatgc taatcaggag 26400
caacagaacg gtgcgtattt attgaatact gttgtagatg atttgaatag tggattttta 26460
gaggttcctt aatgacttat gtaatttact ccaaaactgg atgccctcag tgtgagactg 26520
caaagaattt tgcaaaagct cgtggtattg accatgttgt gagaatgttg gggcaggatt 26580
atgaactgtc agacctgatt gatattgcac agatgccagt tcgtcagatg ccattcatca 26640
tgaaaactga cggtcaaaac ctaaaacctg ttggaacgct acagaatttt atggcagagg 26700
tgaataatgc ttaaacgcct ttgggaaggt ttggttgttg atgcaccagc tattgtgatt 26760
ggtatgcttg ttgttaacct atttactgat tttgaacaag gttcgttgtt cggagccatg 26820
ttactatggg ttatctttga aatattagaa atacagttag gcatcactga aaaactaaga 26880
gaacttgttt cgaagttttc taaaaagatt taaaatgaaa gggtctcttc ggagaccttt 26940
ttagcatgta aaggggcaat aatgaacaaa gtacagatta ttaaaaagaa cggctcactt 27000
gaagaacctg atatcaaaaa agttttagca gcagtaacta agtcagctaa cagagttggg 27060
tataaagaac tcccaccaga tgttacccaa gctcttgagt cagcatttat gaggattctg 27120
gtaaagtcca ctaagcagaa taatttgctc atctcggtaa acgatattca cagtattgtt 27180
gaaggtgctt tggcagaagt caatcacgag atttatgagt cttactcaac atacagaaat 27240
taccgtaaag aggttgctca aaattgggat gagctttacc aaaagactaa agacacactc 27300
ttcttaggtg accgtgaaaa cgctaacttt gacagtagtt taatttctac aaaaggctca 27360
attattcgtg gttacctgac taaagaaatc tttaaacagt accatttaac accagaggaa 27420
cttgaagcca ttgaaaaagg gtttatctac atccatgact taagagacct aatttttggt 27480
gggattaact gttgcctgtt cgacattggt aaagtactga aaggtggctt tgaaatgtct 27540
ggcatcgaat actgtgaacc aaagtctgta ctgtcagcct tacaagttat cggtgatgta 27600
gttctttcag caactgcaca gcaatttggt ggatttactt tagcagagat tgataaggtg 27660
cttgtaccgt atgctaagaa gtctctacgc tatcatgctg agaaagcagc atcttacggt 27720
attcctaaag aacattacca taattatgtc atggagcagc tacagattga attaactcaa 27780
ggtttccagt cactagagat gaagctaaat actgtacctt gcagccgtgg tgatttcgca 27840
ttcacaactt taacatttgg tttacttgac tcagacatgt ctaatgaaga caaccgacta 27900
caatacatga ttgcaaacac cattcttgat gttcgcatga atggacaagg taagagcaag 27960
aagcctgttg tattccctaa actggtttat attcatgacc agaagagaca cgatgagaat 28020
atctgtcaag gtcaactgta cagtaaagct attgagtgtt gctctaaagc aatgtatcca 28080
gatttcttaa gcgtatctgg tcatggtgct gtagcagagg cttttgagcg ttctggcaag 28140
gtcatttcac cgatgggtta acaagctcta tagctcatct aaaacgtgcc taaacaggga 28200
aactctacag gtgtagacaa tcctgtgcta aatgatgtac gatgtaagaa actatataaa 28260
ataggagtat tacatgccaa attatcatgt tactgaggac ggaagagtct ttaggaaaaa 28320
tggtgtagaa cttacgcagt ggaaatccaa cactggttat atgaaagtaa ggttttatgg 28380
aaggaagaat cgtgacatgt atgtccatag gttagttgct gaaaagtatg tacctaaccc 28440
taacaatctt ccaattgtaa aacacaaaga tgataacaaa ctgaacaacc atgcttctaa 28500
cttagaatgg ggaactcatt ctgagaatgt taaagaaggc tatgagaatg gttgctacaa 28560
gttttgtaag cgttcatatg ctgtgaaggc tacacataaa gtgacaaaag aagttattgt 28620
tgctaagtca atacgagagt tatctaatgt acttgggtat aatcgcaaga ctatctcttc 28680
aatattgaag ggtgtaaaag agtataataa ctttgaacat gattttgagt acatttaaat 28740
gccgaacgac taaccgtgat gaatgtagcg gtgtagggtc aagcgactcg aaatggtacg 28800
ctacttagaa ataagtagaa gatatagtct ggacttactg gtgacagtaa gcagcttgaa 28860
taaagcgggg taagcgtagc gaacttacct gaacaacaag tgtagagcgt ttttatcacc 28920
atatcttaat gaggatgggg aagagttcta tgttggtcgt gctaacattg gtgctgtatc 28980
tttgaacttg cctatgattt accagtattc taaagagaat ggtttagatt tctggaaaga 29040
gcttgataag tacctagaga tgattcgcag cttccacaag aaacgctacg aaatgattgc 29100
taatatgcca gcaagttcta accctcttgc attcacacaa ggtggtctgt acaaagggac 29160
taagaaacct actgataagg ttggttggga tattgtgaag tctttcacag cttcttttgg 29220
ggttactgct cttgatgagt tgtcggttct tgctgagggt aaacgactcc atgaagttgg 29280
aagctatagt tttgcatacg atgttctggc atacattaac atgaaaactg aggagtttaa 29340
aaatgaagat ggcttcttat atgccgtcta tggtactcca gcagaatcac tttgcgggac 29400
tcagctaaaa cagttcagag atatgtttgg cgttatcaaa ggtgtttctg ataaggaata 29460
ctttacaaac agtttccata tgaatgttgc agcggatatc tcaccatttg agaagcagga 29520
cttagaagaa ccattcttcc atatctgtag aggcggcaga atccagtatg tgagggtagc 29580
taacccagaa aacttaccag cacttaaaag ttgtattaca agaggcatgt tgaagggttt 29640
ttatcaggga cttaactttg acttagcaat ctgtgaacat tgtggtaaca gaccaaaggc 29700
tgatgttgaa gaatgtgagg tttgccattc acatgacatc tctgtgatta acagggtgtg 29760
tggttatctt ggatggacta agattaaagg tgaatcacga atgaacgatg caaaaattgc 29820
tgagattcgt gacagggttt ctatgtaaaa cttgacaagg tggtgtgagt cttgataggc 29880
ttacatcacc ttttttattg gatgttagat atggcagaga gcatcatggg cctgttcata 29940
ggttctgtct tactggggtt ctttttagga ctctcttatt gtgagttgag agataagtta 30000
aaatgtttaa aatatcaaaa acgttgatat acatacttca cattctaatc ttcttttttg 30060
gagttagtgt tatggtatgg ggtttttcag acccacaatg gagtttcgca tatcatggtc 30120
agatggattt atggtcatgc ttcaaaccat ttttagggtt agctatagca ttcagtgcat 30180
taccaactag ggtgaaatta tgattaagtt gaaccaaaaa cagttagagt gggttaaaga 30240
ctacgcctca gagtgtggct cttgcgaaaa gaatcacgta agatactcaa cattccatac 30300
agtcttcaca ttgtacatca gcgacaatgt tctaagtgat tccgtggaag acggtgtagc 30360
gttacctaat gagttacttg ataaactagc tgtagtcact ggaacttggt ctgaagaaga 30420
cggtcatgaa ttatctgatg atattgtctt ctacactctt gaaaacatca tgaatccaga 30480
gtacattatg ctaatgactt gtgcacaaga ctgtgtgcca ttacaaaact tcattaaaga 30540
acactgtgaa gaatttatta ctaaacaggt tccttgtcag gttgtgtttg aataagtaaa 30600
acaattaaat aatattggtg aggtaactag atgaattaca tggagattcg accattcgac 30660
acagctaatg gtgaaggggt tcgtgtaagc ctcttcgtag ctggctgcaa acatcactgt 30720
gaaggctgct ttaacaggga gtcttggaag tttaatgctg gtaaagagtt tacttatgca 30780
aacctctacg gcatcattaa attaatggat gatgaggcca tcagtgggtt gtcaatactt 30840
ggtggagaac ctcttgatga cagaaacatt caagaagtca ccaacatatg taaacgtatt 30900
aaaactgttt acccagaaaa gtctatctgg ctctggacag gttttcagtt acacgaaaaa 30960
attcatctag atgtgatgaa atatgttgac gtggtgattg atggtaagta tgattcttct 31020
aaaccaacag ttaaaccata tcgtggttct gataaccaaa acctctggag aaaagagtat 31080
ggatggcaag gcgattgtca atggcgagca gagtaaatct cgtcgtaaaa ttggctacgg 31140
aaaaggtgag ataatgatta tctcttttaa tagtgcttta ttaggcactt ataaggttaa 31200
gactgaactc cttgatgagt ggaaaaagta tacaaactca ctttttggta atgttggaat 31260
ggaagcctta gcagagattg agaaggctgg aacattcgaa gttgttggta aaggccactt 31320
tacaatgttc tacaaaacaa atttaaacgt tggtgacagt gttgttggtg tcactgaaga 31380
agaacttaaa acattttgtg aaaagattga gagagatatc aaatgggaat ctggaacagt 31440
tttaaatcaa aagtaaaagc agcattggca tcactaggta tgctcactgg tgttgacgtt 31500
actcatctca tgcagaattt caagatggat gagaggctgg ctaatgagat tatgaaaagc 31560
atctatgtag gtcgtggaaa aggtggtaaa aaacaagcac atcgaccaac tggtgcagca 31620
gcaattaaac gtgcagctaa aaaagctcgt aaccgtaaac gtaacaagaa ggctaaataa 31680
tcatgagcaa agtttataac actcgtaaat tacagatttt cgtactgtgc cagtttatgg 31740
ctaaagagta taactgctac tattgtggta cagggttcat cagtgacaat gacggttcat 31800
atctaccatt caaagaggct gtaaaactct tcaatgaaga gaaaagctct aagaaagata 31860
ttgaaaaggt taagctaacc tacagcaaga aagataagaa gattatctgc ctagataact 31920
ttgtgaaagt tactggagaa gcaaaagagt ttatggatga gagtgaaatc tcattcacaa 31980
gcatcttgaa agtagctcag taaacagata ctataagggg actgtaaagg ttcccttttt 32040
tattttggag ataatatgta tctgtcaaat ctgaaacgtt ctgctgcaat gtcagttcta 32100
agactcagct tcgaggaacg tcaagagttc attgactccc accaatacga cccttctaac 32160
tcaaatcaca tgattctctg gaatcgtgat agtacccgtg aaagagcact attccgatat 32220
tacccacact acactgtaga taacttgtat gaatgttttg ttgtgaagaa caccattgca 32280
gttctcaata aactttgcag atacacaggt agtcaatcct tcacattagg tcaccacaag 32340
cctgttacaa aaggtggtga acatcactgc gcaaactggt ttatccaaac taaagctgat 32400
aaccagaagc aaggagatag ccttctaagc actcctaaga tgacctatga agagcaagag 32460
aaatatatca aaaataatat gccagatgtg cttgacaaca actatacaga tttggcaata 32520
tctctcctgt tgaagttcga gacagtttat agggcaactt acaatggcta aagagaagtg 32580
ggagatttta ccactagtca gtgagggtgg taatggttgt gaaatgtata tgatacgagg 32640
tcatgttcca gaaccaattg cacttgagat ggtaaacaat tttacagatg gctcttacaa 32700
agatttagga gaaccaactg tcaagcaaca atgggttaaa cctgtaccag acagcacagg 32760
taactgtagt gtactttatc acgttgtaga ccctgcaaaa tgcaaatctg caatggcagt 32820
aacaaacgta acttttaatt gagagagaaa actatgaaaa catctatccg tgttacagtt 32880
cattcaccaa ctaaagggac tcatgaagaa gagtttaaca tcatccaatt cccttctggt 32940
gagattggtg gacacttttc accagagttt gttgacttca ctgcttatgc agcatcatct 33000
atcaacaatg tgattatgat tgtaaaaggt tatgataaag atacattgtt tgctgtggca 33060
cttgctaaag aggcggtgga caacttagta cctcataagt ttgctatgaa gactgtcatc 33120
ttttactatt tgccaaatgc acgttacgac cgtcacatgt ttaaaggtga tgcagcagct 33180
ttgaaagtgt ttgcacaaca ggttaatgca atgggtttcg atgcagtctg tgcagttgac 33240
tctcacagtt atgtaccaga taacctgttt aactgcttcc agagtatccc ccaaaaggag 33300
attgcagtcc actatgcgaa tgacccactg gttgattact tagtagcccc agatgcaggt 33360
gctgctaaga agattgcaga gaccgctaaa gaggtggata aaccatacat cacaatgtct 33420
aaagtgcgta accttaagac tggtgagatt accggtatgc gaatcctcga tgatgttgat 33480
ttgacagata aaactgtcat gattctggac gacatctgtg atggaggtcg taccttcata 33540
gaggcagcta aacatctccg tgaagcaggt gcaaaacgtg tggaactcta tgtaacacat 33600
ggtatcttct ctaaagatgt tgaaaacctt cttgacaacg gtattgacca catctacact 33660
acaaactctt taggggaagc taaagaccgt ggattaacac attatagtca agttactgta 33720
gcaaatttgg attaggtatt aaaatggata agggttactt cgaaaaatac ctaagatacg 33780
aaccagagac aggtttacta atttggaaag taaccctttg caataccgct attgctggca 33840
atgttgctgg cacaaggtct aagaagggtt atatccaagt tcagataaat aagaagaggt 33900
actatgcaca taatattgca tggattatgt ctggaggtga gataccaagt ggatatgaga 33960
tagaccacat agaccttgat aaaactaata ataagttgga gaacttaaga cttagcacaa 34020
agtcacaaaa tcagaggaat agaggcatac acaagaataa taaaactggt gtgaagggtg 34080
taagtttttg taaacaaaca ggtctttaca aagcaagagt catgctttac cataaagaat 34140
acttttgtgg aagatttaaa acggtagaag aagcaaaaga agctgtaatt aaaaagcgaa 34200
tagaacttca tggtgagttt gcaagacata actaagagag agataaaata tgagtaattc 34260
actttacgca gtaccagcag gtcttaatgc agatgcttac aaggcatcac atatatatca 34320
gtatccagat gctacacagt atcttatgtt gaacctgaca ccacgtagtg acaaatggtt 34380
taacagcccc ttagcaattg acggtgtagt ggcttttggt attcaacgtt ttgttaaaga 34440
ttacctgata gaccactgga acgccacctt cttcgaacgt gacaaaaaag aagcaattga 34500
tgaaatctta gaagtcatga acggcgttct gggtaaagat gctattggtc gagaacattg 34560
ggaagcactc cacgacttag gttatctacc agttgaagtc tacgctgtag aagaaggtac 34620
agttgtccct atgcgtgtgc caatgattgt cttccagaac actgtttcag gtttccattg 34680
ggtagcgggg tatctggaag atgctttctc tgctgagatt tggaaggctt gtaccattgc 34740
aactattgca ttgcactaca aacgtatctg taagaagtgg tctgacctta cttgtgacaa 34800
cgacttgcat ttgccttatc agtgccatga ttttgctatg cgtggtatgt ccggctttac 34860
tgatgacgca tttaacgctg taggccactt aaccagcttt aaagggactg atagcttccc 34920
tgctgtatat acagctaaac gcatctacgg tcagtcctac ccaatctctg atattggtag 34980
ctctgtacca gccactgaac actctgtaat gtgtgcaaac attgcttggg aaggtggtaa 35040
tgagttgatt gaagaagaaa gacgctttaa aggtgagtta caaaccttcc gtcgattctt 35100
aacagagact tacccaactg gtattgcaag tgttgtttca gatacttata atttctggag 35160
aactgtatca gtcatattgc cagaactccg taaagagatt atggaacgtg atggaaaact 35220
ggtaattcgt cctgactctg gagaccctgt acatattgtc acaggctata aagcaatcca 35280
cttagagtgt gctaaaaagg cttattacga acacctaagc aagctggaag ccagcgacac 35340
aatgttgaat gctgttctga acatgaagct tgaaaacatc aactatggta ttgctgggtg 35400
gctactgtca gaaggctacg aaatggttgt tgacagagaa gactttgaag ttgctgatac 35460
agtgctgttg aaaaatgctt acatggttgg ttctgcaaac gttgtaacac gtcctgtagc 35520
agagattgat ggtgctatta agaccctgta taacatcttt ggagggacta tcaactctaa 35580
aggttttaag gtactggatg agcacattgg ccttatttat ggtgactcta tcacgttgga 35640
acgtgcaaac gaaatcctga aacgtttgta tgaaatgggt tttgcaagct ctaacgtagt 35700
gtttggtgta ggttcttata cttaccagta catgactcgt gacacctttg catttgctgt 35760
caaagcaact cttgcaagta ttggtggtaa agagattatg cttgcaaaag accctaaaac 35820
agatagcggt gttaagaagt ctgcttttgg tggtgtagca cccatgtggg atggtgataa 35880
gctgaaagct gtagatggct atggattcca gagctttgca gatgtgcttg aacatccagc 35940
ttgtgcttta cgtttagtct ttagtgactc tgagcagttc ggttacacaa ctcttggtga 36000
tattcgaaat aatattgaca agcaacttta aaagtatatg ataagaggct cctacgggag 36060
cctttttaat ttctggagaa gattatgaaa atcaaagaaa tgaatatcaa cattgtctta 36120
gaagaacgtt gggagaacat caagaagcct gaaaatggtc ataagttcct caacaaaatc 36180
ttagtggcag ctaaagaaga actgactggc aaggttgcag cagcaatcac aataaaggtg 36240
tgtgtaaaag gtcttccaga caatcaccaa tttgcacttg acgagtttaa agaaagcttc 36300
tacaatccaa acaaacagat gcttgaaagt aactttgcag tatctacaag tatcgttcat 36360
gacagaagct ttatccttta caagaatatg agaggtgagt catgcaagca tattggatag 36420
aaattttact gtcacttggt agtgtagcag tgttcgttta ccttctttgc aagtactatg 36480
cagagcacaa aaaatgtgac ttctgtgacg gagaaggtta tacaagagca ggttgctgcc 36540
ctatgtgtgg cggttctggt aaaatgttta ataagtaatt taacagtaaa ctaagaggaa 36600
agtattatgc gcatggtaaa tgaccacgca gaagtgatta agagttcaac ttctttagag 36660
acgtctcaag cacagattac aatgacacct gaaatgttca gccttttgag ttctggtgta 36720
tataccttta aagaaagggc agttattcgt gaactgtcat gtaacgcagt agatgctcag 36780
aaagaagctg gaaaagagaa catcccgttc catgtgcatt tacctactcg ttttgagcct 36840
tactttgaag ttcgtgattt tggaactgga ttgactcatg ataaagttat gagtttgtat 36900
ctaaattacg gggcttctac gaagaatgac tctaatgact acattggtgc aatgggtatc 36960
ggctcaaaat ccccatttgc aattgctcag tcattcacag tatctagcta tgttgatggt 37020
gttgtcaata agtactctgt ttaccttgag aatggtatcc ctcaagtaac taagctgacc 37080
actaacccaa caaaagaacc taatggttta gctgtacgtg tagcagttgc tgaccaccgt 37140
atttcaaagt tctttgaaga agctggtcat gtgtactcat actttgctgt aaaaccagaa 37200
agtaatatcg agtatgatga tgtattagca gatatgaatg tcattgcccg tgaagaaggt 37260
gtttatgatg ccatgattca caaacaaagc tggcgttcta gtggtaacag gacagagttt 37320
aatgtggtga tgggtaatat cgcctatcca gtgaacatgg aagcactact tggtgatgat 37380
ttcttcaaag tcttgccaga gtttttccgt agaagcgtag accttgtaaa catctacatg 37440
cctattggtt cagttgctat tgcagcttct cgtgaagcat tgcagatgaa cgacacgaca 37500
aaaaatgtta tcatcgaggc tactaaaaag ataactgaag caattacaaa ggatgttatc 37560
aagaaagtta atagtcaacc tacactcatg gatgctgcac aggcttatgc tgagttacgc 37620
ttgaactcac gagagatgtt taatgctgtg tgtccaaagc tagagtgggg aggcgttaag 37680
cttgattctc tggaagaaga attactaaac attcgtcgtg gaattatcta cgcagaagac 37740
ggctcagtta tctatgaacg tgatggtaaa ggtaatatta aggttgatag gaacggaaac 37800
aatatcccta aagtagatta tctttataat ccagttgctt atgttaagtt caactcttta 37860
gaaagtaaga ttcgtgcaac agcactttcc tacactcaag aggcaagtat gttcaatatc 37920
tttggtgcaa tgcgcaaaag ccaaattgaa cagtttttgt ttgttatcaa tgaccgtcgc 37980
aataaaaacg gtactgagaa aactgtagga cgcaaccaga ttctgcgtgg tgcatgtcga 38040
gactatgcca gtgagtcaag tctgtttcac agatacaatg gcattgtctt tgtattctct 38100
actgagaaag agctagacga cttaatcaat ctgcacaaac ttgataaaag cttgttaaag 38160
attgtgaaga tgtctgacaa ggaacatcat tatcagcgta aagaagctgt aagaggtgtt 38220
gtaaaactct ggaaagctgt tccagcagaa ggtgtagctt catacaaaga ggtgtcagaa 38280
gacttagata cgattgaaga acctcagctt tacattaagg cagtcggtga cacagttgat 38340
ggtgaatgtt tttgctcatc tccagaagat gtagctaagt cagttgccaa tgtcattgaa 38400
aagacagtct atgttttccg aaaagcaaat tggaaaaaga taccagaaga ctggattgaa 38460
gtagatgaga agctcttgaa cgacagctta actgatattc actggattaa tcataacagg 38520
tatatgacac gtatctatat gaatggtgtc ctagacctta caagcagttg gattattgcc 38580
agaaacttta cgttcaataa caggaagatt tctcgcggct attgctattc acgagacaca 38640
aacaaaacta tcttcttaga aggtaatgaa gatgctgttg aggcaatgtt cggtaaaatc 38700
caatacgttg ctgcaccatt tgcatacact tacactatta gtgtgttgca agatttgaaa 38760
aggtgtcttg ataatgatac aaagctttac aagaaaatta agaaagctgg tgaccgtatg 38820
gtcattaagg tgacaaacta tcttttaaaa agaaaacaag aaaacttctt gctttctcat 38880
ttagattgga ataaagtgtc acccatcgaa gtgagtaagt tcttaggctt tgatgtgaag 38940
tgtgttccag aagggactac aatttacgat taaagtgttt gacaaggggc tttgaagccc 39000
cttagaatat ccttacaaaa tgatttatta actgacaaga gagagtaata agatgactac 39060
taaaactaaa gcacagattg acgcagaaat ttacaaactg gttaaagaag gtaagctgac 39120
taaaacagct attgcacaga agttcaatac ttcaacccgt tcagttggtc gtgctgtaga 39180
gcgtcatgaa gcaaccttaa aaggtaagaa aaaggtagct tcgacaccag ctaagaagac 39240
tttgaagcaa gttgctaaga cctttaaaaa gaaagctgac aaaccagttg aaaaggttgt 39300
taaagagtct gtacagaaag ctccagtaaa taaactgcat gaagctatgc agaaagatga 39360
caagattgag tacatgatta ctggtgactc tgtaattatg acttacggtt cagaatctga 39420
aattgttgag tctactcatc cgaactatca agagattgta gtccatgttg tgaaaggtga 39480
gttcaagaaa gcctttgaac tgatgaatat tcgtaagtct atcgaaaact tcactcaggg 39540
tgctatcaca atcaaaggtg acaagttatt ctacggtgct gttgagatgc gttctactct 39600
ggttgaccgt atccttcaca tgatgaagac tggtgataaa ggttttgaac gccttgtaat 39660
gttcttcgaa aaactgatgg aaaacccatc taaagattct gtagaacaac tttggggatt 39720
cgtatctcac cttgatgttg aaattgatga agaaggctac atcattggtt ggaagaaagt 39780
ctctactcgt gaaggcaagc tggttgactc tcacacctac aaagtaccta atgatttggg 39840
taacattgta gaaatgccac gttggatggt tgataacaac cgtaacgtaa cttgctctca 39900
gggtcttcat gttggtgctt gggattatgt tcgttgcttc tcaggcgata caatcctgaa 39960
ggttcgtgtc catccacgag atgttgtatc tgttccgact gattacaacg atatgaagat 40020
gcgttcatcc cgttatgaag ttgcagcaat cgttgataat caacgtaaag tgctgaaagc 40080
atgggatggt aagactgaag ctttgcatgt catcgttggc actgctggtg aactcatctc 40140
tcaacgtaaa cgtgaaatct aataagtaat ttcttaaaag gctgcttcgg cggccttttt 40200
tatttgtatt ttgtgcagag tgctgtataa ttgttgtcac gataaactaa acaggagaac 40260
caatgaagaa gttgattcta ggtttgtgct taatgtttac agcacactta tcttatgcag 40320
ttgactgccc agagctatca attagccaaa aagtaaacat gttaaaagct taccagtatg 40380
gtgagaataa tatgggtaaa ggttggggta tcactctagc tgctatagcc ttacaagagt 40440
cagagttagg tctgaaggta gagaataaaa agacccatga ctatggtatc ttccagaatc 40500
acttgaagac tgttgtaaag cgtaacaaaa tcagtcctaa tgtggctaaa aagaaactct 40560
taaaagactt tgactatgct gcaaaggaaa cacacaaaga gcttgagttt tggactaagg 40620
tacatggtca accaaagtca aagaagactt tacaaaaagt tttagcgtca tataatgctg 40680
ggtattcgta caaaatccct aaagctaaga aatactctca agatgtttac aacaatatga 40740
aagtcatcgc tcaatgtgaa tttgcaacaa acatttctaa ggtaaaccat gaaaaaatta 40800
agaaagtctg atgaagtcct gtgccatgct tacgacttgc accctcatga gttaggtctt 40860
gattcctgtg tatggactcc agaacagtgc agagattttg aagatactgc aagagaagtt 40920
gtatgttcac ttgaagagtt ccacacatca gaaccaattg tgagcgttgt ggacaaagag 40980
actggacaat ctataggtgt aagacgtgac agtttagtca tagtcaataa agaccttgta 41040
gaaaaaggta acctaatctt agcagatatt gatggtgttc ttacaaattt taatcacgaa 41100
gattgctcaa cggaattgac tgatgggtca ttctcacagt acactaatct tcttgactct 41160
gtaagagcaa aaccaacgta tgttttcagt atcattgatg caattgctaa tcatgctgcc 41220
attggactct tgacagcaag aggtgaatct cagagaatac ctactgagat gtttttaagg 41280
cataacatag agcatgatta catgcttttt atgcgtggtt ttggaactaa ctctataagt 41340
gcagaaagtt taaaagttag gatgattcag tcttgcattc ttccttactt taatatagta 41400
tgctttatag aagatacaga gaagaatgtt cagaaggtaa acagaatcct tccacacatt 41460
aaaaccatgt tagtaaaaca ttgagagaaa acttatgaac aatatcatta cggtagcact 41520
ggacgttacg gcaaacaaat ctgaagtggt tcgtaacatt atcaaaagca actttgaggg 41580
taaaatcttt cgtgctgtca atgtaaaagc agacggtaac atccgagagt atcgtgctct 41640
tctaaatgtt aagaaacacg tcaaaggtgc tggttcaacg actgcacaca aagaaaacct 41700
gatgactatc tatgatatgg gtatggcttc agagttaggt gctgaaggta tcgttaaaga 41760
aggtgctccg tatcgctctt tcaatctgga aactgctctt atgctttcct ttacaagtgg 41820
ttctaaaaca actacttatc tctttactga tgctgcaacg gtatctgcta tcaaggatag 41880
caccgtcaaa gctggggtag ctgctgctgc aaaagcttct tcgatggctg caaatgtcct 41940
tgctaaagtc ctcggttaag gttaagatac aggctccttc gggagccttt ttcatttcag 42000
gagattgtta aatgacattt aaagagttct gtcaagctac tttcataatt gttttcttag 42060
ttggagcagg tgtctgggga ggatactctt acagagacta ccaagttgct gagactgagc 42120
taaataatga gaaactaata agtgttgcta aagatgctta tcaggaaggt ttagccacac 42180
tcagcaccaa ttacaaaaat gatttgaaag atgtgcttgc taagaataag catacaaaag 42240
aggtactaac atatgaaaaa actaagccag agttttataa tgtttgtgtt actgataatt 42300
atgtcagggt gttcaacgaa caaagtgaac agtacattca aaaactccca agtaagtgag 42360
agtgacaagt acactcaaga agagtcaaga ttcactgtca aaggtaatac tggtagtgat 42420
gtggcagcag cccttgagtt ttatcgtgac ggtttttacc agtgcacaat caaagctaat 42480
aaccttattg atatgatttt attaggaaac aaacagcaat gacagagaat gaagacactt 42540
tctatgtaga agggtactta ctgctaccac gaccaaaaga aacttatatg cgggttgatt 42600
tctcaccaac cattatggat aacgtgatgt gccatatctt tatgcaaggt gtcacagcac 42660
agcttaagca tgttggtaaa gagtgcaaaa taaaggttga cactcatcca gaaatcaaag 42720
agaatcacta cacatggttc ttaccagact ctaaagaaat cttagcagtt cttaaaacga 42780
ggaaatgatt atgcaattcc agccaacaaa tcgtggattt tcttatgcag aatttaaaga 42840
tgctaaagga gagatttgta ctttacaaga aagttcatca tatgaaccat gtatctggtt 42900
aggtgctaaa gaggtctctg ttcagtacct agatggtggc tggaaaacac ttgatatccc 42960
agcattagta ggcaaagatt ttgtaggtaa ccaaagaatg aaactcaacc aagaacaggt 43020
gaaagagtta ttgccatacc ttcagtactt tgctgaaaat ggtgaattac caacagagga 43080
acttacaaat gcagattaat ggaagagact ttgtagctgt ttactatgag aaagataaag 43140
aagttggcgt agcacaagta tcttatggta atggtaagtg gttgtatgga acaatcgcag 43200
tagtcggaac aaggagtgat acaaaaactt ttaaagattg tgttgacctt cttgaagaat 43260
ccatccacaa tcattggtgt ctgatatgga tgactgacaa cgaagtgata gaacgcttca 43320
aaaagattgg tatcaacatt gatagcattg agcatgttga tttgtatgag ctaactgaga 43380
aggtaaacta tgaaagtaat actagccaga gataaaaaga cacgtaagat tgtaagttca 43440
gcagtattca aagggagaca tgaagtcgtc ccgtttgcag gagaagatgt gttaacttac 43500
aagaaggtga tgaaacgtga gttcagtaac catttccgtg acttcttctc tgaaccatta 43560
gaagatttta aaagtcgttg tggtaaaggt atcatcactc aagaggtaat tattcatggc 43620
tgacttctgc aaagactgct ctatcgaaat gttcggacgt gatacaggtg acttaaaagg 43680
tcttatcact gaagacgact ttaaagctgg ctatgcaatg ccagtaatct gtgaaggttg 43740
tggatgtatc tgggtagacc atgaagggca acgtgtaaaa ccttcagaag ataaagaatc 43800
ttgggagaga tgctaaatgg gtattgtaaa aattatcaat ggggatatct tctcagcatt 43860
tgataaaggt aagtttgata tcattggtca cggttgcaac tgtatgaact taatgggtgc 43920
aggtatcgct gacaagattt ctaaacttta cccaaaagca tatgagactg atacagaagt 43980
ttatctgtat gcaggaggta taggtcacaa accctgtgaa aatttactag gcaacttctc 44040
tgtagcacgt ttaaaacaag gacgtatagc taacctttac actcagctta agactggtaa 44100
agatgcccga tacagtgctt tagagtcatc tttgaaacag cttaacagat actgtgaagt 44160
caatcagttg aagaaagttg ggttacctat gattggtgca ggtatcggtg gactagaccc 44220
tcaagccgtc acagttatca ttaatcaggt gatgaagagt gtagatgtgt atctgtatgt 44280
ctacgaagga gagatgtacc acaagttacg ctcaggttgg aagaactact gtgaaccaga 44340
atactttgca ggtgtagtag tgttcactga tgatgcagtt accctcttca gacgaagaaa 44400
aggtaagata catcaaagta accctccggt tgaaaagatg tctctaagta acgctctagt 44460
tacccacctg tcgaagagca atcacagaat tgcagtaaca tttggcagtg atgcagatac 44520
atatatctat gcaagaactg atgaggatgt tgaggaaatt ttctcttcac cggaagtaac 44580
cttcttagac gcaaagaact aagaaactct gtagaatatt cacaataggt taagcctttc 44640
ttatctggta aatttttcag gtaaggaggg ctttttcatt tcaggattgt gcaggtagag 44700
ttctggacag aaatctgtag agaaaatttt gaactccaaa gagaaaattc attttcaaac 44760
tgtgcaggta atctccaaag agctttttca ttttcaaaac gtgcagaccc cgctcaggtc 44820
gggcaagaaa aatcatttta gaattgtgca ggtaggtggg tgcaggtggg gatgtcgcca 44880
tttctcacaa ggctaccaca tctgaaaaga aaagtcaaca gaaaaatttg tagcggattg 44940
aaaaataatt cttgattgta gttctgtttt gtggtagtcg cgtgcgcccg tttcattaca 45000
gtttttgcaa aaatattttt aaaattccta ttgactcttt gaatatggtt atctaatatt 45060
tgcatcagac gggcaatcaa gcccacttcc taaactggag aatcaaaaaa tgctttacag 45120
tcgtgaattg atggttacaa ctaacgttaa cacaaaggta aaacgttttt atatgcaagt 45180
gtgcggcgca tggcgtagaa tatcagaagc tgattattgc aactatgtag aaacggcaat 45240
caattctaat tgctacctta cgaagaatac aaagcgacat ataaaacaga cagcggttta 45300
ctattacgat atataaaaag tactttacag ggggcttaaa agctgatatc ttaagcccca 45360
gataaagagc tttacttaat taacccttaa actaggagat tcaaaaatgg cttatcgtgc 45420
acctaaattc atcaacaaag acaactttcg caacgcactg gagaagtcac ttgatgaaaa 45480
ttttaaaggt aacattatcg ttgttcacgc attcaatttt aagtatgatg taaacgggaa 45540
tagaatcaac cattacacag caaccatgtt agatggtacg ctatcaagtg aaaaagctgt 45600
actgaatgca cttgcaggac gtggtaaaaa cctgataaga tgcgataaga gacgttatca 45660
gggtggcgca tatggttatg atgatgctgt ttatcatctt gaaaatatgg gctattcagt 45720
ggaaaaagct ggagtgtctc aaataatcgg cagtgacggc tatgtgacaa ttttcaaaat 45780
taactaataa agtactttac agggggctta tagttcgata aagtaagccc cagataaaga 45840
gctttactta attaaccctt aaactaggag attcaaaaat ggctattatt aaaaacgttg 45900
taattcctgc aaaaactcgt gatgacgctc gcatcatggc gaaaaagtta ggcgggaaag 45960
tggttgacaa cggaaaacaa actgctgtaa ggtggaacgt gaaggcggat aaacaaatga 46020
atttaaaaaa ttcaccaata aaccttttta catgtgtaaa cactatcggg aaaacgaatg 46080
tatacactaa aaaggcatat atcagacgca tcgccatgac gtcacctatt cgcacaatga 46140
gaagtcatgc aaagctgaaa attaagtaat agagtacttt acagggggct taaaagctga 46200
tatcttaagc cccagataaa gagctttacc actcaatcct taaactagga gattcaaaaa 46260
tgaaaactgt agacgctacc tttgaagttg taaaagaaaa attttccatc atttacgaag 46320
ctgtcaattg ctacgaagca agccataatc ttgataattg gaagtttaca gatgaccatc 46380
aaggcggtgt gactattaaa aaccccaact gtcagcgtaa tggatttaaa tatgcaattc 46440
ctttacatta cagccttaaa cagctttcaa ttgactatgc aaaacaaggc cgggaaaatc 46500
catcgaaaga ggcttacatc agcttacaga aagaactaga aagagattta gaggcttcag 46560
aatataacat cgccgcgaaa gttgtagatg caaacggtag cacggtttta gagagttttt 46620
atattggtta tgctttcgat tggtgctaca gtgacggcga tgatttagat gacagactaa 46680
aagaggaagt gaacaacgca gacgcagaat ctgaagtatt agaacgcttg gaagcattaa 46740
aagattcagt gatgaatatc tttaagaact aaatagacct ctttacacgg ggctaacaag 46800
cggatatgat agccccagat aaagagattt atcaaattaa cccttaaact aggagattca 46860
aaaatgaaac tatcaaataa agtccgtgaa aatctaagca agttgcttaa taaaattgat 46920
ttcattgaaa catcaaggaa tagccatgat gttatggaat ggttacacgg aaaaatcaaa 46980
ggccgtcgtg tagtgatttg cttatggaat agttgcaggg aatataaaaa acctgtatta 47040
caagtcaata tctatgatga tacttttaaa agccctgtaa agtctaaaag cgatttatta 47100
gaatcttatg agataactgt aaatggcaag gtatcacgta aagaggaagc accatgttaa 47160
tgatgggtat cactgcaatc attgccatta tagcacttta caaggcttat agcgcatata 47220
atttggcaag taaggcaatc acgcagcagg tcaataataa ccttgtaatg agctttctgg 47280
agcgtttaag cgatgaacaa ataaaacggt tagaaatgag cttcagattt caatcaaaaa 47340
cttatcaaat cagcgacatt ttcaaagatg attttcagct cgtcgatgat tataactctc 47400
tgataaatgc tttaaatatc agtgatttaa aagattatca tgctgtaata gttcaagaat 47460
tgagcaaacg aaaacaaaat tcttgacact gatttttaga tagattaaat tactcatcaa 47520
cggggaggga ttaaccttcc cactaagtaa agtccttaaa ctaggagatt cgaaaatgat 47580
tcaaataggt gataaagttg tctggaacga tatagacaat gatttatgca caaaaattgt 47640
aattgtctta gagcttgatt atataattgg tgttgcaact gttgcagata atgatataat 47700
ctttgatgtt ttaatcagtg agctatcgaa ataagcctta aactaggaga ttcgaaaatg 47760
tatactacta acaacggtcg cactttgaac gtaaccctgc gccactatgt gaacggtgta 47820
atgcacttcg aggatttaca agccgaacaa catatcttgg attggcaagt agcaggactc 47880
caaaaaactg ctacaggata cgggaaaaag attccaacta gttggaaagt atactacgaa 47940
gggcgtttgc gcagaattta tcaagatgta tacagtaata gcgcatcaag ttacatcata 48000
gtaaaaggta aaaagctgca tttagtataa agtactttac aaggggctta tagttcgata 48060
aagtaagccc cagataaaga gctttaacac tcaatcctaa attggagatt ctaaaatggc 48120
tattaataat cgtgagttat caatcttaaa agcacgtttg accgttaatc ggattaatgt 48180
tattacatca tcagcaccgg atgagacatt gcacaacatt atcgggaaga ttcaaagtgt 48240
tatcttagac gttgaaaacg taaaaaactc actggctgac gttgcagcag gtatcacgct 48300
agacggtgcg caatatgaaa tggctgacat gttaggcaaa tctaaggtaa tgaataaaga 48360
gttagacttg aaaatgttta gatttgctgt taaagtgtgg ctatctgtcg agtatgacgc 48420
aaattttgca atcgctgact ttttcgcaac atggttgcaa cgtaatttaa caaatcatga 48480
tttccgtgat gtctgtgatg caatttatgc ggaactctaa aaaaaaaaaa ctgttgacac 48540
tgatttttag atatattaaa ttacgcatta acggggaggg aatgaacttc cccactaagt 48600
aaagtcctta aactaggaga ttctaaaatg gctattatta acggcttaac cattgaaaca 48660
acacacatca aagacattaa agttggtgat gtagtccttt cccacggcgt agagaaaact 48720
gttactgcaa aggatattaa agaggattct ttcatgggta ggactctctt cggtgattct 48780
tattgcttgg gttatcttgc agttttaaaa gttgtcaaaa ataaccgtta aagtacttta 48840
caaggggctt atagttcgat aaagtaagcc ccagataaag agctttacca ctcaatcaag 48900
tccttaaact aggagattca aaaatgaaat gctatcacgg cactactctg aaaaactttg 48960
taaacctgct gaatggtggc gacaagcctg aaggaatctg gaactgctcc tacatggatg 49020
attgtttcta cgtttaccca atgaacaagg ttatcaaaac ggaatgtctg gaagatgagg 49080
aagattctta tatccgtgaa cgctgcattc aaagcgcctt agagtctgga cttattacgg 49140
cagctttcca aatggaaacg caggaagtga ttgttatcga aatggatatc ccagaggaag 49200
aacttgagga cgattattct tgtgacaaca tgagcgaaat tgccagcttt accggatact 49260
ttgataagaa ttggattatc ggtatacact cgacgaaatt tagtgcgatg tatgcgcctt 49320
tctttgtacc ggatacaact aaccgtaatt taggatatat tgacgatgat ttgttaagta 49380
tggcgaaaat tgtacagaat agcggtgata gttccgtgtt ttgtgaccta cttgataaaa 49440
tcacggcaaa catcacagaa cgcaatttaa aagacttttt aggttaatca gtacaaagac 49500
accaacttcc aacttaaggc ccgtacaggg ccatttagag ggcttcaatc atgcttacaa 49560
ccatttacct catcctttcc atttgtaatg gtcattcatg cgattttaaa gggcttgagg 49620
agtttacagg gagtaaagaa aatgttattc atacctgtca gatagcaagg caagactatc 49680
ccgccagtga tgatatacaa tgttacttta agacagaaga taatgacggc gtttattttg 49740
atagcgtaga cggtcaatat gaaattatca ttgaaaaaga ctagacaagc ctgataaaag 49800
tctgtaaatt gtcaatcaac ggggaggtaa taacctccct actaaacaaa atccttaaac 49860
taggagattc gaaaatggct tatgtaaccg taattaccaa taaagaagat tcatcttggt 49920
caactcaagt aagtgacaag atgtcaccta tgcagtgcct aaaatacttt gagcaatgga 49980
acaaaggtga ggaaacaagc ccctttcaag taatgcagat tatccataca gacaacgaag 50040
ggaataaaac tgttttgaat agtgagtact atgcaaccag attcgaaacg agaagtaaag 50100
caatgaaact cttacgtgaa tccggttatt ctcatattgc cgcgctaatc tgggatgacc 50160
tcctaaaaag tcagcgtatc agttatgtaa aacctgaaaa aatattcatc agttaatcaa 50220
caacttacaa aaaactttta aaaaagtgtt gacaatgccc cttgacattg gtaactttgt 50280
tcgagggggt ttatctaaaa gggattaact taaaagattc tttatagata aacttaaaaa 50340
attgcttgca atcagatatc aaaacaagta aattactaat caacgggagg gataacctcc 50400
ccactaagta aagtccttaa actaggagat tcaaaaatga acaaatttca agctatcaat 50460
tacatccgct caaatagtgt tatgtcaaag cctgttaaag acacctatga atttcgttgc 50520
aatggtgtac actttgcaac catcaccaaa tcagaaaacg gggcttatta cgttcatcgc 50580
cgtaatgttg caaccgttgt agtctctcac tttatgcaag ctgtcgcgga acttctcccg 50640
atgtttttag ggctttactt agatgagtgt aagcacattc ataaccatgt taaaagcctt 50700
ttaaacggtt ataaaatggc ttatgagcgt tctatcaagt ccttaaacaa gttttacaac 50760
gcacctgaaa acaccgtatc aatggcatac gctgtaaagg gtgaggcggt gagtctcttc 50820
gaagtggaag aaatcgagct ttatgaccat cgccccaatg gttacgctaa aactctcggt 50880
gaaggtttaa cgttcaatat caagaactta gaaaaaactt taaaagatat tgaaaaagat 50940
attgacgaga caatcaaaag tctgtaaatt gtcaatcaac ggggagggaa tgaccttccc 51000
cactaagtaa agtcctaaac tggagttatc aagatggaaa acgtcgaact gaccacactt 51060
ttacaaattg caaccgacaa agaggatgta gcattattcc accgtatcga aagtgatttg 51120
ttcaatggtg aaatcattaa agaatcttac gaagtggtcg gattcaaaga tggtaaatca 51180
tttggattct ttgacctcga caaaggcaac gctattacag agtatgatgt tttatcttgt 51240
gagattctga caaaatacaa cgaaaaaaga cttgcaacat acgccaacat gcagtaaatt 51300
actaatcaac ggggagggaa tgaccttccc cactaagtaa agtccttaaa ctaggagatt 51360
ctaaaatggc aactatccgc gcaattgtaa attcttatgg ctgctacact ctggacgtta 51420
aatttgatat aggtgggacc gatgagtgtt tcgaactggt gaaaacagtc attaaacaag 51480
ttatcccaga ctataaccct actgatgact tctggaaaga tgcagtaaaa gaggcattga 51540
acgatgaata tcattaccat tattggggtg atttgattgt ctccctgtac tacaataaac 51600
aaggttgcaa ctaatcggcg caacggggca aatagcccca gataccttta tagcccctta 51660
aatggggctt ttttacgtct tatcaaatcc tatacaatcc ttttcctttc ctttccagag 51720
tcttactaga ccttaaaagc aataactcta tagcttactc tataaccttt tatcaaatca 51780
acttaaatat aagcccataa cccgaaaggg aagggcaacc gtttaaacgc tctataaagc 51840
tctataacag ccttatcaac ctaacttaag ggattgcatt aactcaccct aaaaagccct 51900
ttataaagct tcctagaggc ttaaaagaat cttataaggg tgtaagcttt atcatctcgt 51960
tagttccttg tgaatatcta gaaagggctt gttagtgtct taaaagtatg ttataggggc 52020
tataaagtag attaaaagtt acttcaaagt tacgtaaatg ctagccccct taacattttc 52080
ttaacaattt cttaacaaat cctacatagt tatgctttta aggtacttgt tagcaacttg 52140
ttagcccctt gttaaatctc tgttaatacc tcgtaaaagc cttgtaagag tctttggcat 52200
agcctttaaa gcctctgtaa agccctataa agagctttct acatagttgg ggtgtaatta 52260
ctaggaagag actaaaaaga gcttataagg agctataaag ggcttttaag agggctattt 52320
aaagaatcac taagagagaa agggaatgat aagaaaagag atagtgacca tgttaacagc 52380
ttgttaaaaa ggttaaacac ttgttaggaa cttgttaatt atcttaacaa aataactctt 52440
taaaattaac ttgttagctc ttgttagtca tctgttaagc ctctgtaaag actatgtatc 52500
aactgttaag aaactgttaa gctcttgtta gttctgttaa gaatctgtta agacctctga 52560
agagatgtta agaggatgta agaactgtta agagattgtt aatgatattt taaaattctg 52620
caaaggggat gttacaggga tgttaagggc taccaagcag atatacatcc tagacagata 52680
tttttcagtt ccttcaaaga ccctaaaaaa tgacttgata gtcctatttt ataaaaattt 52740
taaaattctg aataattacc tagacagtct tctaaaaata caggcataaa caggtttata 52800
agataccttg ttagtaccgt gttagacctt gtgagataag gtctgtaaag atattcctgt 52860
taagtttgtt aaaacagtct atatagaggc ttagtaatac ctagtgaaca cctatagagg 52920
gtaacatagc tgacatagag attaagaggt gtataggggc ttctaagagg gtgtagagag 52980
atggttaagg tgattgtata ggttgttaag aaggattgtt agagggctat ttagaagggc 53040
tatacagagg ggctattaag accactagat agatactaag atactatata gtactatata 53100
actatatata tagagagtaa gcttaagaag gttgttaaga aggttagata ttttattcac 53160
atatgatggt gacccagagg ttgggacaac cgataaaatt ttataaaaat taaaaagccc 53220
ccaattaagg aggctcttaa gatattttaa accagtgttt ccagaactgt cttcagtgat 53280
gacttggttg tctgataaaa atgagtaaat ccattgttgt tatcgaatac ctgcatcatg 53340
atatcatctc ccttgattgg gtacactcgg atatcatagt ggaacccgtt aaagacagca 53400
gaaccattac agaatgaact aaagtcatcc actgagatgt actcttcgtg acctgtgatt 53460
tttatacaga cacctttgct cggtctctta gggttaatat cttccatcac agacatattc 53520
tgtccaccaa gagataccat agtgaccact ttacggttct tctgtttaaa gtacatctta 53580
actttctgta aagtatcttc atagaacgga taagacttct ctacaaagat tccttcaagt 53640
ggctctttac catttctaac ttctctgata tgacgtactg agaagataag ttgctctagt 53700
atgtcaaggc tttctttgga gagtgcttcc cgaacatcag cccagtcaag gactaggtag 53760
ttgccttcaa cttctttcaa gttatctttt tccatagcta tcacaccatt attgtaaaac 53820
ttggtaatcc ggtactgcaa acttgttgtt gttgttctta atgaggtctt cttctgttac 53880
taagaatgta agaccacaca agtgtgtata tctgctatca tactcaaata ttttaagtaa 53940
gtatttacca tcatggtaaa caaattctac tacacctttt gcaacaatct ttccagtctt 54000
gaaatcttta agagggtata cattaccaac tttaggaact gttttcatct caggttcttt 54060
tagcttctca ccaacttcta catcacctaa caagtgagaa ctctcaaacg ctacatattg 54120
catataagct tctgagcatt cccaaataaa acaatgtaca agaaccttag cagcatgtct 54180
tgaacagttc aactcatcac agatttcttt gtacatttca tctttagttg aattggtaag 54240
cattgctttt cgaactgttt ctttaatttt cataagctct ccgaataacc ctattaagca 54300
tctctaaagt cagctttcca gactctaaat gtggcataac aagctttaaa gctttttcaa 54360
catcatgcac aacatctatt ataccttctt cataggcttc tacaaggata tcttttacat 54420
tttgtgattt acctttttgt ggtatcacta gacgcatatc aaactcattt gcttgaggat 54480
ggttcttacc ttgccaataa ccatctttgt taaagctgat gtagtcaccg tggtaaaagt 54540
gtacaccatt agctttaaac ttctccagca caaggcattg aattgggtat tcagtattcc 54600
actccctgac tcctataacc ttcactgttg cgatgtactc ctgtgttttt gcatctatga 54660
tgtcaagttc ttgaccagct atgatttgca tattcgctac cttctaagtt cctacaaagg 54720
ggctaccaag taacccctca agttattact cagacttctc agtcttctta tcagatttct 54780
tactagtctt cttctcagct ttctgttcag gttccttctc agaagttacc ttgataacca 54840
tgtcaagaag ctctgatagg tcactcttca gcaaagtaac aggctgctca ttgtagaagt 54900
ttttgtcaag ataatctttg aactcttcaa tagaaggaaa tccaaaaatt ggtacaggtg 54960
tcttaatcat tacttagttt ctccataggc atcttttaag attgccttgt taatcatttc 55020
agcgattgca aaacgaacac atacggctat aattgtaggt tgcattcttt acttacctct 55080
tcaagtttaa cctcagagtc cttgagtcgt ttcttatgaa ctctgaatct cattagtgcg 55140
atgatgatac caactaaagg gacagcatac aagtaccaaa catcatatac ctttgcagta 55200
atctgtgtaa tggttaagtc ttgagctatg gaactgattg ctgatactgc cagtaatgtt 55260
gctaggaaaa atgccagtac cattaccgta cttaaacaac ctctggcatg gtgttcatgc 55320
ttaatacact tattcttttc gtcaacgacc ctgttgtagt catttgtaaa gtctctgtag 55380
aaagctttca agtattcttt attgtagcct tcaaagcaac ctgtaccgta ccaataatca 55440
ccaccaaaat gtgctatgat accagtttct ccgtttggtt tagttactct caaagatgtg 55500
tgatgacgag tatcttgata gtctattttg tatctttctt cgtaaatcat tttccaaccc 55560
ctctcaatcg tttgttccac tcaaggtcaa caagtctttg taggacttta cctctaggtg 55620
ttacaccttt taaatctgca tcgtagcaat ggtaaaagtc accttcagcg tgtttaccat 55680
agcatttcct gatattctga cttctacgat actgctctag tagggatagt ctgtagtaat 55740
cttcaactgt agttagtttt ttcattcttt caccacgaat ccttttaaga aactaaaaag 55800
ggaactacct ctcgatagct cccatcatag ttaacattgt atactcggtc aagagtattt 55860
tttaggcagt aatgcactac gcttgttact ctcttcactc aatagtgcga taaattcatc 55920
gcctttccct ttccacggac tgaatgtagg gatgtgctct ctgatagcct caatcactgt 55980
tttcagggca ttgttcttta accaccaatc agacactgcc gaagctacaa tgtagtaagc 56040
accatcatct gtgaaaattg gctcagagat acatggtgaa atcatagtgg taatgataga 56100
gccattattc atgtagtatg ttcttttttc aaccttgtca agaagtttat gaaggtctgc 56160
aaagagactt accacctctt tatggtcaat atctttttca agatttgctc tatattttaa 56220
tagtgcagca tcaacctctt tatcattgaa tttgaagtat tggacttctt cattcacaca 56280
tacactgata ttatttgtgt ttatcgtcaa tttgcacagt ttgactggag agttgatgtt 56340
ggtctggaaa agtgctacac agccttctac attcccgaaa tgtttgacta gtgtaacttc 56400
tggttgaatg tgctcatgtt cttcggacat tacagtttcc tcattaaata ctttttaagt 56460
tgtgtattag gtttctcaaa aacttctata gaagatatct tgacagactc accacgcata 56520
ccagtgtgtt tgaaaatata gtattctaac atacctatgt attttgctgg taagtcatac 56580
ttgacgtaca ctgttacaga atcttcaaat tcaacaacct ttgtaccatc tttgtagtta 56640
aactctttag gatggttgta attgtcgatg tcgtacatta aaaagagtct tttacgctgc 56700
atatctagct tatttgtcaa agactctttg atatctgaaa agtttttata gactgtgaca 56760
tggagcttac tcatcgttaa actctctgac gaagaaatag ttgactttgt tattacctac 56820
cataaggtca aagcaggtgt tctcaaaagg tttgttgtag gcatcatatg aagtgtacgt 56880
atcagctact tgtttagtgt tgagcttttt acttgatgga tttaagaggc caccatacag 56940
tttcttgata gcctcttcta caacagacca gatgatttcg tcagtaattt cttcgtacag 57000
gtcaagttca cgctctagtg agaagtcaca aatagggaaa ataacctgca ctttactcat 57060
taacggataa cctgtgtgac ttgaattgct tcagttacgg acaattcacc tactttctct 57120
tctttgataa aagtgaaggt gagttcaaca ggttcttcct cttcttgctc aactggaaca 57180
ccattaagct catcaaagat tgattgggtt gtccagtctt caccaccatc tgggtagtgg 57240
actgcgtaaa gaggaaactc taaattatac caagcaaaaa cagggcaacc atctgcatca 57300
tctcggcatc tgtggagata tgtgtttgta ccttcaaaat gcaaatggag attaccattc 57360
ttctcaaaga gaagagcatc tcgcaggtct agttcctcat tttcgctgta acggctgaca 57420
tcaactttaa cgtggatatg actgttatta ttcataaaaa cctctctctg cttgttcgat 57480
tttttctctt aaaatttcgc aatgttgtaa atggtagtta gcttcttgtt catgtgttgc 57540
taactgcctt tccagaacag ttttatagta acctgcaagt tgctcttttg tcaatgggtg 57600
ctctgaaata atatcaagcc cactttcacc aagagggttg aattgtccat gctcattgac 57660
attgtaagtc tcaaacttga acaggccatt gttcacagat tcaataggtg acaattcaac 57720
aagctctaca gtaaatcgtg tatacacctt gtcgcctctt gaaaccacat tgtttccgac 57780
aatgcgtaca agcgaaccat tacgtgtgta cttaaaagtt ccaaagtcac tagcccaaag 57840
cttcatagtt ttctccttag aaaataaaaa aggctcccga aggagcctat gaagatatta 57900
ctttgggtat acactgtcaa gataaatgtc agcttccatt ctacgtctgt ttttcagacc 57960
atttgaagtg accttcttac ctttgactgt aaccttgttc caccactgca tagcttctgc 58020
acaacctacc ttgttaccag cattgtggcg cttgataaat gtagactcct gcatagctgt 58080
gataccgatg ttgtatgttt cacttacaag tgcatcgaac tcattctgag aagttggagc 58140
cttgatagct ttattcactg ctgcaacaaa cttctcaaca tctgcgagaa gatactgttc 58200
agcttgttca gccgtgattt tcatacccat cttaacaggt tttccgtcaa tacggattgt 58260
accatacccg attgttggga ttccggcaga gtcttcgtaa gcctctaact tcagaccttc 58320
aaagaactta atagcttcta aaccttttct tgagagttgc attatgcctc ccctgctggg 58380
ttaactgtaa ctgttgccgc attagatgtt acagagccac ctgcaccagt cactacacaa 58440
gtgtagcttc ctgcatcagc agcagttgca ccttctttgg tgtaagttgc agtcgttgcg 58500
tctgggatga cttcctcacc tttcttccac tggtaacctg ttgcaccagt tgccacaaca 58560
ttcagcgtta aggtctcacc ttcagtgatt tcttgattgg ctggttgttg agtgatgact 58620
ggaagagcat taacggtaac agttgcagca ttagatgtta ctgttgtacc acctgcacca 58680
gtaactacac aggtatatga accagcatcg gcagggtctg ctttagcttt cgtgtaagtt 58740
gcaccagttg cactagggat gttcacatta tctttcttcc attgataacc tgttgcacct 58800
gtggcctcaa tactcagcgt caaactgcca ccttcattga ctacttggtt agttggctgc 58860
ttggtaataa ctggcaaagc atttaccgtg acagttgcag ggttagaagt aacagaacct 58920
ccagcaccag ttacaacgca agtgtaagaa cctgcatctg ctggaagcgc accagatttg 58980
ctatagttag catttgtagc tgatgggatg tcagagccat ctttcttcca ttggtagcct 59040
gttgcacctg ttgcagtaac tgataagtca atgtttccac cctcattaat ggtctggcta 59100
gaaggttgct gtgtaatcac aggcagtgcg ttcacagtaa ctgttgctgc atctgaggtg 59160
acacttgttc caccatctcc agatacagca caagtgtatg aaccagcgtc agaaggtgct 59220
acagattgtt ttgtataggt tgcagatgtg gcactggaga tattctcacc atccttcttc 59280
cattgatacc cagtggcatt agttgcgaga acacttaggg taagtgctcc accttcattg 59340
actgtttggt ttgtcggttg ctgagtaatt actggtggct taatggcatc cttcaactta 59400
gcattcaaca ttgagaacgg tttaactctt gccaaccatt cacaatacac tgtgtcaacg 59460
tctttaccat tgataattgc atattgtaaa tccatgaaga agtcagaagt tctcatttga 59520
gcaccgacgc tgtaaagcaa atcatcacta aaaggaactt tgtagtctgg tttgtagtca 59580
aacttcttaa cctctgcgat atcagcttca ggccaatatg agctataggt gagtggtaaa 59640
atagcttctt gatatgttct aaaatcatat ctcttaccag cctttacatt tgcgatgaag 59700
cccttcacaa attctttgaa gtctgggtaa gtctttgctt taatcatttc tttttagcct 59760
ctaaacgtgc gtttaatcta aaatagcctt tatgttgtgg agacacatca ccaacaacga 59820
taccttcaac tttataacct ttaccgatta catactggat atccataaga aatgtaccaa 59880
gtcctacaat gtggaacatt ggatataata acttatctga atctgggaga ttaatatctg 59940
ggttgtaggt aggttgttta ctagtaggtg taatatcttc ttcaggccat ttacttagtc 60000
tccatgtcaa aggtgttggg ataacatcat taagattgta cctcccacca ttctccacgg 60060
aatggatata ggattgtagg aagcaataaa agtcttttgc actcttagct ttcattaaat 60120
atccttataa atagataaag gggcattagc ccctgtttat acttaagctt tggtcttaag 60180
taggttctta agttttttga gattcatgct ctcgtcaaac ttaaaaccaa atccattgac 60240
ataatcaatg aactcggatt tcttactaag tgatagcgcg taactcatat caaagttatt 60300
acttgtttct gcgtctggac tagagttcac atcaaatgaa atttctgtag gctcttgctt 60360
gctacttctc atattatcaa ttaataggca tgtatagttc gctggataga agtttgcact 60420
gccatcaaca tacccatact tattgttgtg aataaggttt gaaaatacct taagaaatgt 60480
ttctgagtcg tggcatacaa gtttgaattg gctgcattct ccaagagcca ctaaagaata 60540
agggctttca ctatcaatgt cgtagtttgg atttgatgtt gtaaccgtca cacgcatcaa 60600
gttattagca atagtcttct gcttgaaaga atttgggaca tagtgaagac ctctatcaca 60660
ctgattaact aactctttaa gtaggtcgtt tgcatatggt gaattaattg atacagagcc 60720
atcactatta tagttaaaac ttactgttaa catttttcag ttctcctgcg atttatgatt 60780
ttgctgcaat agagaaacga agtaaactca ctggaagaaa ccattaagtg ttgttcacta 60840
gattgagcca ttgccctgaa atacattcca gtcaaagcat cgtagtgact attctttcca 60900
attgaggtta tatagctatg actctttgca gttttatata aatctataat atctgcgatt 60960
tctaattggg aactacacat agataatcct cttgtcttct gtaattttga aagaacaggt 61020
acagaccaat ttcagaggat gaatagtaga gtaactctac cctttttgat tccttacagt 61080
gattggtatt tttacctttc ttcgggttgt ttgattttct cattaagtct cctttgtaga 61140
acaaggcaga cctacagctt taagtaagat ggtacagtaa tccatacaac aagtcaagaa 61200
ctattgcaag ttaaaaaagt tgttgacaaa gtacttgaca aggtgtaggc ttataaatac 61260
ttagaagata acttaaaagg ttcctctaaa aaggctattt aaacagatat ctttctggat 61320
atctatcttt tatcttaaaa gccttttaag aggtcttata agaggcatac caatgaaaaa 61380
cagaacaaac aaaggccagt ttaaaaaagg ccagtctggt aacccatctg gaagaccgaa 61440
aggttctcgt aacaaaagct cacttgtaaa agctcaactg accattgata attctgctga 61500
gtttgctgca aagttgtttg aagcgattgt tacaagggac gctgctaagc ttgcagagtt 61560
cgggttaaac acagatgacg taaccatcaa gagcatgatt gaggtcggta aaactatctt 61620
tactcactca gcaggtgaga tgaaagcact tgcagcagac actaagaaga cttctgataa 61680
tggtggtcag tcacagacag ataacaaacc aacgttctct gctgtagcga ctctcaaaaa 61740
ataattttaa aaggtgttga cagactctac aaagttgctc taaagtctgt cacatcaacc 61800
aacaatgaga gaagagagta aaatatgagc gaattattta aacatgcgca ccttcatgca 61860
ggtcgaactg aaaatggtgc tgtaaaccat acttcatcaa tgtctgctct ggtagacttc 61920
tacaaagccg ctggctcaag ccgtagcaat gtagaaatct taccagacct gttctacaaa 61980
gctttgcgtg aggatgttga tgttgcagtt cgtattttac tgcatatgcg agatgtacga 62040
gaaggtatgg gtgagcgtaa agctttccga actgttttac ttcaagcgat tgaagataag 62100
gttttagaac ctacacaggt tcttcgcatt atggataaga ttgcagaact tggtcgtttt 62160
gatgacttca aaatctttgt aggtactcgt tttgagacag atgccttcaa acatttagaa 62220
gcagcattac tagaccctgc aacagcaggt ttagcggcta agtggttacc acgagtaaaa 62280
ccacgccaca aacagtttgt aaaacgtttc tgtaagtttg caaacttaag tgagaaggag 62340
tatcgcacac tgttatctgc actgtccgat acggttgagc aaaaaatctc tgctaatgag 62400
tttggtaaga ttgactacag caagattcct tcactcgctg ctgcacgtta ccaaaagctc 62460
tttaaccgta aagatggtga acgttacaaa gcttatatcg agtcactctc aaagggtgag 62520
gctaagatta acgctggtgc cgtttaccca tacgatgtga ttaaatctat caagtatggt 62580
aatgcaggtg ttgctaatga gcagtggaaa gcactaccaa actggatggc agagggtgaa 62640
aacatcttgt gtatgactga tgtttcaagc tcaatgtctt gggtgaatct tggttcaatc 62700
actgcccttg atattggcgt atcacttgcc ttgtatgtag cagaacgcaa tacaggttgc 62760
tttaagaatg agttaatggt ttattcaaca aaccctcact tcatcgaact gagtggtgat 62820
ttgagaaacc gtcaccgtca ggtaatgcgt catgttgaat atggttcaac taacttacaa 62880
gcagcttttg accgcattct tgaaataggt aagagaaaca acttgactca gaaagatatg 62940
ccaagtaagc ttattatctt ctctgatatg gagtttaatc aggttgatgg tgcaaatggt 63000
cgtacaaact ttgaagcaat tcagagtaag tacaaaaaag ctggatacga aatgccacaa 63060
ctagtattct ggtacttagc aaaccgtaat ggtacttgcg aagtatctgt taaagataat 63120
ggtgtagcaa tggtatcagg gttctctcca gccactttaa aagctctgct tggtggtgag 63180
aagtttgacc caatcagcgt aatgctcaaa gcagtaatga ttgaccgtta catctggtaa 63240
aaagtttcaa aaagggtatt gacaatgtgt ttgataccct ttaatatgtt ctacatagaa 63300
acgaaatgag agcttttcta agatactgaa aaatatttta aaaaagttct tgacaatcac 63360
taaaaaataa tgttaaagtg gttacataga gtttgaaaag tttatctctg tttagctcag 63420
cttggtagag cgttccgttt ggggcggtaa ggccggaggt tcaagtcctc caacagagac 63480
caaattaatg ttccagtaga caaaacggta aagtcaccac tctttcaaag tggatatttg 63540
agggttcaaa tcccttctgg aacgccagtt ttgacagaag accaattgca gcaaacttaa 63600
tcttattcat ctgacacgaa atcggacaaa gaagatattt ggtctggtca ttaagaattg 63660
cgggtataga gaaagggcgt ctcacatgtc tcattagcat ggtatcggca ggttcgactc 63720
ctgcacccgc ctccaatttt gcagaagacc gtattcagca acaaaatact ttttgcggat 63780
aaaagaaaaa acacggtctg gcaatgtatt aaggttagga agcacataag gtatgtgcgg 63840
tcgcctgtta agcgaatggc acagggttcg aatccctgac taaccgcaaa atttaaatgt 63900
gtcgttatcc cgtagatggt agcggtgggg actgtaaatc ccttgtcatt gagactcggt 63960
aggttcgact cctacacggc acaccaacac ttagcgagat acttagttat gagagatgaa 64020
gtatacaaat cacaacttgg tatgagttta ggaactgcac aaaacagact agtaaaagat 64080
ttgctatttg attttatatg taaaacaaat caaaatagat gttttcagtg cggaaaggaa 64140
atgactaggg atgatttctc tatcgagcat aagattccgt ggagacatag tgaaaatgca 64200
aaagagcttt tcttttcact agataacatt tcttatagtc atcttaaatg caacatgaaa 64260
gcagcaagac cagcacagat aagtgaaaga aaacctaaaa caattaaaca cggaactgcg 64320
tatagctatt acaaaggctg tagatgcgat gcgtgtaggc tggctaagaa tgctgagagt 64380
agaagaaact atagtccaga aagacgtagg aagaagtata taagaaatgg tacataggtt 64440
ttggggtatt agtttagacg ggtaaaatac tggattgtct ctcctgagcc gcgagttcga 64500
ttctcgcata cctcgccaat tttgagaggg ctatttagcc cctcccttaa agagttctta 64560
cgagtattct ttaaaggagc agaagaccaa attcagcaag ttacttaaaa tttcaagcca 64620
atttaatttt gaaaaattta aaacttggtc tggctcaaca aatttacaga agaccgttta 64680
cagcaaaact taaacaatct atttctccgg taaagaaaag gcgaaggttc gattccttca 64740
ctcggcagat gtcgagttgg tgtaatggta gcacttaaga tgataaacaa cggtctggta 64800
attgtctttg tagataagtt attaagctta cctgtgagta tgcgaccaca aagggtgaaa 64860
gggtaaattc tagatttaaa cccaacaagg cacatcgcta cagaccttaa gcaagtctgg 64920
gtagcttatc tacaaagata atttaatgga agtgtagcag aatggtgatg cggcagactt 64980
ttaatctgac aggcgatggg ttcgaatccc tccacttcta ccaaaatggt tcagtcgcag 65040
ataaggtaat gcaggggttt cataagccct atgaatgtgg gttcgattcc catctgaacc 65100
tccaatgctg tgaaagcaca tatggatgtg cattcggctg ataaccgaaa ggaagaaggt 65160
tcgaatcctt ctcacagtac caatttcaat aaagttgttg acattgagat gtgaccactt 65220
tatagtaact aaaatacttc ataatatgta taattcaaac accaatacat gagcgttaga 65280
cgtaacacaa tgtggggaaa tttgttgtac atattctgaa tagttttcgt tgcgtagcgt 65340
ctattttgca aatttaaaat aaatgcaaac gacaatgttt ttctggcagt agcttgatag 65400
gctaaacacc agtgaggtct tccaatccct catcaaagaa tttggcgtac tcgcccacgg 65460
tatgattaat aaggtgggca acattctaaa gggtatccct tatggggctt ggcttaaatg 65520
caatgagtac cctttaaaat gttaattacg gggcgtatct cagcggtctt ctaaaccgcc 65580
gttaaccgag taagtggagt atgcgggttc gagtcctgcc gcctcgacca attttggatg 65640
aaatcatgaa gacttgtatt aggtgcaaac ttagcaagga tgaaagtgat ttctacatga 65700
aagatggtag gagaactcat tcatactgta aaaagtgtgt taatgagcaa accatagaaa 65760
gacaaagagc acttaaactt tcagctattg agtacaaagg tggtgtttgt caggattgtg 65820
gtaaaaggta tcatccagca gtgtatgact tccatcattt gaatccttca gagaaagatt 65880
ttaacattgc tcatagaaga agtcttaagt tctctcagga gcttaagaac gaacttgaca 65940
aatgtgtttt gctatgttca aattgccata gggtaagaca tgcaaagtat tagttagcag 66000
attgcttaga cgaccttatc gggtctccgt gtaaggaggt ggtctgtatc tcatgttttc 66060
cagaacatgt aaataatctg agaaggggct ttacagtgtt gcgaaaatac ggtaagcttc 66120
gaattatagt gtgggacgat gttaagactc taaggcatga gcaacggcct ccaaaaccgt 66180
ttcaaagggg ttcaactcct ccgtcccatg ccaaagtctt attaggggta ggtagcggct 66240
aatggcagcc aaacagtctt gaaaactgtt gccactgtag agatacggtg agggttcgac 66300
tcctttactt accgcctaat aagattttac accgtccatg cttgctcgca ttttcggttt 66360
atccctcgtg tattgtcgta cacactgata aagcacctag taggtgtcac ggagaagaga 66420
taatatcaag ctctccaaag gttctagtca ccggattaaa caagactatg caaagaccta 66480
aataggtctt tttagagggc ttctaaggtt attacagctt gttgtggaac cttgttgctt 66540
aggttcttag aagttctcta aaaagattta atgggagatt gacggtaatt ggtaaaccta 66600
ccatccttag aagttggtgt ttgagggttc gaatcccttg tctcctacca aattaatgca 66660
ggtgtagcaa aatggttatg cggctgactc ttaatcagta agacgatggg ttcaattccc 66720
tccacctgta ccaaattggg gtcatagttt atatggttaa aattcgagtt ttgcaaactt 66780
gggaactgag ttcaattctc agtgactcca ccaattagtg catccatagt ttaaacggga 66840
aaattacagt cttccaaact gaggttgagg gttcgattcc ctctggatgc tccaaattaa 66900
tgctcctatc gtataattgg ctattacggt tgccttgtaa gcaacttatc agggttcgaa 66960
tccttgtggg agcaccaatt cagaggtcaa gtgaaagacc gctttgagtc aactgaagac 67020
cgtaacaaat tccacggagt tgagttagcg gcacaacttc agacctcttt catactcgct 67080
tagtttatat ggtaaaacac cacccttaca agatgaagaa aaaggttcaa gtcctttagt 67140
gagtaccatg ttccagtatc ccaattggca gaggatgcaa gctcaaacct tgtactagtg 67200
acggttcgaa tccgtcttgg aacaccaatt ttgcaggatt agttcaaatg gatagagcaa 67260
cagtctacga agctgttaat aggggttcga atcccttatc ctgcgccaat ttaaagaggc 67320
tatttagatg agatatttga agtatgcttc atggattttc ttagctttgt tagaaccatt 67380
agcagcaatc ttagcagtta tcttagcacc tttcgtagtt ccattttaca gtgagaagaa 67440
aggacactta cctttcggat ttagatggat ggaaacttat gacaacccga ttgatggtga 67500
tgaaggtcat gtcaaacgat gggctaagat tagaaagatt ggtaagctcg gtgtctatat 67560
gcagagagtc ggatggctct ggagaaacaa agcttataac ttctcctacc atgtgttagg 67620
aagagatgta aaagatgtta ctaagtggaa aggtaatatc aacgtaagtt ctgaccctga 67680
agataatcag acaggttatc tcttaatgtg gaacagcaat gcttggggat tattcgcttt 67740
tgtaccatca attaaaatct ttggtaaaca attctactgg agaatttatg ttggatggaa 67800
acttaaaagt gttgtcccag aagaaagagc attctcaagg gaaagagtta tgttggcatt 67860
ctttattcat ccactaagaa agtaaagatt taaaggggat tagtttacaa ggttaaaacc 67920
tcggtctttg aaatcgaaga agttggttca attccaacac cccccgccaa tgctccatta 67980
ctccaattgg cagagaggcc agacttaaaa tctgtgttat gtatcggttc gaatccgata 68040
tggagtacca aatttagcgg tatagcataa ctggcaatgc agcagtctct gaagctgtct 68100
tattaaggtt caaatcctta tgccgctgcc acttctaagg attcttacga gagtccttaa 68160
atgtggcctt atcataaatg gtaatgaccc atgctgtgaa catggtctat acgggttcaa 68220
atcccgtagg tcaccccaat ttatagtcca agtagcttat atggttaaag cgcgtgtctg 68280
aaaaacatga gaagagggtt caaatcccac tggactacca atttcaaagg tgcttaatga 68340
aagagatgac agaacaaggt aaggagattt ttaatctctt aaaaactggt agagggtttt 68400
ctaatcccct tattactggt gcagcagttc tcggtggaac cgtagctgcc tctacatcac 68460
ttgtaagctc tattagctct gtaacagacc ctacaataaa ggataagctt attgctgctg 68520
gacttacaac agttctgctt aatagcttta caacgagtct gacaagcact acatcaacta 68580
ctaagacact gacagattac ggtcaaaagt ccattgatga gttttcatca cgtatgcagg 68640
tagcaaaagg gtattccaat gttatgggtg cagcaggaga gcaagttggt tgtacaccat 68700
ttagtggtat tatgggtgtt gctacagagt acggtcaaaa agctattgac acaattaaca 68760
gtacactaga cggtgttaat gatgtattaa gtgacttaca agatgctatt gacaaaggtc 68820
ttgatactgt ttctgattta gctaaccaag ctgttagcaa gattaatgaa ggtatctcga 68880
agattacagc ttatgcagat gaagttgtgc agatgattga agaagaagct gcccttattg 68940
cagagtacct taaaacaaat atcaatgggt tcttagcagg tatcttacca gactggtttg 69000
atgatgcttg taaaactggt gtaattgata ctattgcaac accagaaatg aagaacgcat 69060
tacagaaata atggaagatt aaccctaaaa ggtaagggag cagtttgcta aactgccagt 69120
agctgagaaa tcggtgtacc agttcaagtc tggtatcttc ctccaatttg aatccgtgac 69180
agaaatggat atgtgcctgt ctgcaaaaca ggtttataag ggttcgagtc ccttcggatt 69240
ctccaagtta ttttactctc tctcaattaa aataataata atgccctgct ataagtattc 69300
ttcctttcgc taccgaagag tatttttaga cagggctttt ttacagagtt atactttata 69360
agaagactta tacagtatgt ctctgacgta cattgtggtt tctccttgac ggtctctgta 69420
ccgtcttttt taaggggaaa catggttaac tatttggaga aaacataatg agcgagaccg 69480
tttataaaga actttatgaa gctaacaaaa agctagaatt tatgcagaac actatcatgg 69540
caattgcaga cagattatct gtagcaacag gtattgatat caaagaagcc tctatggatg 69600
cacttcttga tgctgttgat gcaaagttcg aagttaagaa agaagagact gctacagact 69660
ctgaataatt ctatttgcgg aggtgcttaa tggacttaaa tgctgttaag cagaagcgag 69720
tggaagatgt taggaaagtc ctagctggag agttggggct ttctgatgaa gtaaaagaaa 69780
ttatcaaatc attcggtaaa gacccctcta aattccttcc aactcaaatt ctgactttat 69840
taagatacac accagaccaa gttagactta tcttcaaatt gatgactgat aagaactatg 69900
tagcccctca gccgggttct caagaggtct ttttaaatac taatgctgac ttggttctat 69960
atggtggtgc tgctggtgct ggtaaaactg ctgcattgtt aatggactct ttaaggttta 70020
ttgaagaccc taactataat gctgtatatt tccgtcgaaa tacaacacag ttacaaggtg 70080
gtttatggcc tgctgcaaag aaactatttg gtaagtttgg tggggttcct cacgagcaga 70140
agatgactat cacattccct tctggggcaa ctattaagtt cacctaccta gaacttgaaa 70200
agcacgctga aggtcatcag ggtattgaat actcagctat ttactttgac gaaggtacac 70260
acttctctgc ttcacagatt tcatacctac agacccgtct acgttctggt gctgaaggtg 70320
attcatacat gaagatttct atgaacccag atagagacca cttcatttat gattgggtag 70380
agccattctt agatgaagaa ggttacccag accctgagaa gtgtggtcgt attcgttggt 70440
atgttatgaa tgatggtgtg atggtttctg attgggaaag agacaagatt cttgaaatgt 70500
tccctcttga gattcctcag acatacacct tcatctctgg tacgattgat gataacccaa 70560
ttcttgactt cttagaacct aaatatcgtg gtaagttgga aaacaacaca cctgtaaacg 70620
ttgcaagact tcgtttcggt aactggaagg ctcgtgcaga aggttcaaac tattggcaaa 70680
gacaatggtg tgagattgtt gattcactcc cagaagatgt gtttgatgtc agagcatggg 70740
acttagcagc aactttacca tctgagatta accctaatcc agactggaca gcgggtgtta 70800
agatgggtaa atctaaaaaa gacggttgct attatatcat tgatgtagta agatttagag 70860
atagaccctc tggagtcgaa acacaaatta atttgactgc tgaaagtgac ggtaagcgaa 70920
ctggtatttt tatccctcaa gacccaggcg ctgctggtaa atcctatgca acatccctca 70980
tcaggaaact tgccgagaaa ggttatcgcg caagagctaa accaacaaat aaagataaag 71040
ttacccgctt tgcgggattt tctgctgctt ctgaagctgg acttgtaaaa gtcttgagag 71100
gtagctggaa cgaagcttac ttccaagaac ttgaaggttt ttgtggtgat ggcaaaacta 71160
aagatgacca agtggatgct accagtgatg ctttcaacag tcttaatgaa gttaaattat 71220
tcaagccacc atcaatgggt gctcacacag acttagtgag aggaaaccca tacgaggggc 71280
ttagacgttg atagctaggt gagaagaatg gcagaaatta cagaaacaca agaaagctta 71340
ccaccattta gaatgggtga agtaggttct ttgggtctga aggttaagaa tggtagaatc 71400
tatgaagaac ctcgtcaggc actaaggttc cctgaaagta ttaaaacttt ccaattaatg 71460
atgcgtgacc ctgctgtagc agcatctgta aatattatta agatgtttgt cagaaaagtc 71520
aactggagat tcgtacctcc aaagggaaaa gagcaagacc ctaaaatgct tgaaagagca 71580
gacttcttta attctttaat ggatgacatg gagcatgatt gggcagattt tattaactct 71640
gtaatgtcat tctgcactta tgggttctgt gttaacgaaa aggtttataa gaaacgtcag 71700
ggtaaaaaag gaaagtatca gtcaaaattt gatgatggtc taattgggtg ggctaaatta 71760
ccaatcagaa atcaatcaac acttgataag tggtattttg acgaagactt tagaaaagtt 71820
actggtgtta gacaaaatct aagaaatgtt tcacatattg ctggagcaat taatcttgga 71880
gaaagaccac taacaagaaa acttccacga gctaaattca tgctgtttaa gtatgatgat 71940
gagtatggta acccagaagg tcgttcacca ttacttaatg cttatgtacc gtggaagtat 72000
aaagtacaga ttgaagagta tgaagctgtt ggtgtttcaa gagacttagt aggtatgcca 72060
aaaattggtt taccaccaga ttatctggat gaaaatgcag aacctgaaaa gaaagctttc 72120
gtacaatact gcaaaactgt tgttaatgat atgattgcta atgacagagc aggtttaatc 72180
tggcctagat atatcgaccc agaaactaaa gaggatatct ttgagttctc attagtttct 72240
agacagggtg ctaaagcata tgacacaggc tctattattg acagatattc caagcaaatt 72300
atgatggcat ttatgtcaga tgttcttgct atgggtcagt caaaatacgg ttcattctct 72360
cttgcagatt ctaagacaag cttattagca atgtcagtag atattctgct gaagcaaatt 72420
aagaacgtaa ttaaccgtga tttagttgca cagacttatg ctcttaatat gtgggatgat 72480
gaagaacatg tacaaatcac atatgatgat atcgaaactc cagaccttga agcaattggt 72540
tcttatattc agaagactgt tgcagtaggt gctttggaag ttgacaaaga gctatctaat 72600
aaacttagag agcatattgg ccttcctcct gctgatgagt ctcagccagt atctgaaaag 72660
ctttctccaa atagccaaag ccgttcagga gatggttata agaccgctgg agaaggtact 72720
gcaaagacac cttcagcgaa agacccaagc acagcaaata aggcaaataa ataatggctg 72780
aagttatctc tatttcaaat gctacacgag tgcattcgta tagaggtgtc cttatcatta 72840
ctgataagtt atctgtagag gctggctcaa gggtcagcct ttcaggttat gttagtgatg 72900
gtggaatctc tgacgttttc actatttgta ggttacttga tgcaccaatg agtggaaaac 72960
cgtttatttc aggaaattgt agtgaaattg ttaaaattcc atttgacagt tcatgccttt 73020
tgggtgtaaa gttatataac tgcgagaata aacgtatcaa tgttaatagc attgaagccg 73080
ctttcattac cctcgacact gcatttcaat ctccaatgac agttaataaa gaaacaaaca 73140
gacttgaata cattttttca caaaatgatt acaaagtact tgtcaaaggt aaagtatatg 73200
atatgattgt aaatgtggta gatgaatctg gtaaccattc aacagtcctt aaacaaaaag 73260
taaggtttaa ttaatgggaa cattaactat tgatggtaag aataaaatcc tcgccacgct 73320
aaccccaacg actattattt tacacaatgt agacccaacg gcagacccta cagcaaataa 73380
ggttactcag ccagtggcta tttatttttc tgaaccggat aatggcttaa ttgcctcaga 73440
agacacagtt aatatcactg ttccagcttc tgcaacggtc tcacactata gcttgtggga 73500
tgctaacgat aaatgcgtgg caactggtgc tcttagcaaa cctcaattct ttgctgaaga 73560
aggtatctat gttatctctt cagtctctgt agatttaaac aaatagggtg agtaaaaatg 73620
agttcgaata tcttcagact tgctgataga ttattcaacc aacctttact agccactgaa 73680
tcattagctc actcagcagc aacttatgtg aataacagat tgctgggtga tgtccaagca 73740
gcagtaaact ttgataaacc caaaggtgaa gcaagaagtc ttttaaaagt aaaagatgac 73800
attgctatta tccctattat gggtggttta actcatcgta tgacattcat tgatgcaatg 73860
tgtacaggtg gtctaagctc ttatgaaggt ttacgtagag gctttgacga agctttagca 73920
gatgagtcaa ttaagactat tgttctgcat attgattctg gtggtggtga agcttcaggt 73980
tgctttgaat tagcacgtca catcatggct tcaagaggcc aaaagaaaat tattgcttat 74040
gtagatgagt tcgcttgttc cgctgcatat gctcttgcat cttctgctga agaaattatt 74100
gcatcaccag atgcagatgt tggttctatt ggtgtaatta tggttcatca ggaattaact 74160
aaggcatttg aaaagaatgg cgtaacaatt aacgtcatca aagctggtga gtttaaaggt 74220
atgggttcac cattccaagc actttcagaa gaaagcaaag aaagacttca aaagagaatt 74280
aatgatacct acgcaacttt tacaggtttt gtagctgagt ctcgtaatct ctctgaagaa 74340
gctgtaaaga atactgaggc gaatgtttat tctgctcagg aagctcttga acttggttta 74400
attaactcaa tcatgtctca agatgatttc ttaaattact tacaaggttc tgaagaggct 74460
cctgtaagtt taaacgttaa caattcaggt gaagaaatga ctgaacaaga aaagcaagaa 74520
ctagaagctt tgcgtcttca ggttgctcaa atgaaagcta aagaacagga agctgctttg 74580
tcagatttga ctaataagat ttctgcttct gctgaagctt ttggatttga tgcaaaagaa 74640
gctgcaacga ctattttagg tgctggtctt gataaccctc tgagcgttct gtttatgaat 74700
gctatggaag gtgctaacca gaaacttaat gaaactatcg cgtcccatgc ttctgcaatg 74760
gaagagaaag aatcagaaat taccaagctg aaagaaactg ctggtgctgt tcttgaacat 74820
tccaatgcta tggaagaagt gggtaatgac ggcgaagctg atttggttga agaagaaaaa 74880
gaaccagcta agaatgcttc cgaagacacc gctgaacaac gcaaactggc tctccagaat 74940
gctctaaaat ctcttatcaa ataaggaaca caataatggc atatcaaggt tttactaagt 75000
taggtaaaag agaacctctg aatgatatca ttctttggga acaggttacc ccaacaggcc 75060
actctcgtaa agagtacact ccacaggcat ctacagaata tcgtgtaggc gaagttctga 75120
aagcagatgg tactaaagtt acagcagggc aagaagctca ggctgattcg gtatgtatcg 75180
ttaactttta tgcagacctg caactgtctt accacggtca gttgaaagtt gttggtattt 75240
accgcgacgc agaactaaaa gacctgctta ctcttgagtc aggtgttgat gctgccgcag 75300
tcaagacagc tctggctgct aaaggtattg atttcgtacc aactggcctg taataacaat 75360
aataagacat tctggagaat tttacaatgt tgactaattc tgaaaaaagc agatttttcc 75420
ttgctgacct gactggtgaa gtccagtcta tcccaaatac ttatgggtat atttccaact 75480
taggtctgtt ccgttcagca ccaatcaccc aaactacttt ccttatggac ttgactgatt 75540
gggatgttag cttgcttgat gcggtagacc gtgatagccg taaagcagag actagcgcac 75600
ctgagcgtgt tcgtcaaatc agcttcccaa tgatgtattt caaagaagtt gaaagcatca 75660
ctcctgatga aattcagggt gtacgtcagc caggcactgc aaacgaactg actactgaag 75720
ctgtagtacg tgctaagaag ctgatgaaga ttcgtaccaa gttcgatatt actcgtgagt 75780
tcctgtttat gcaagctctg aagggtaaag ttgttgacgc tcgtggtact ctgtacgctg 75840
acctgtacaa gcagttcgac gttgagaaga aaactgttta cttcgacctt gacaacccta 75900
atgctgacat tgacgcttct attgaagaac tgcgtatgca catggaagat gaagcgaaga 75960
ctggcactgt aatcaacggt gaagaaattc acgtagttgt tgaccgtgta ttcttcagca 76020
aactggttaa gcatcctaag attcgtgacg cttatcttgc acagcagact ccgctggctt 76080
ggcaacagat tactggttct ctgagaactg gtggtactga cggcgttcag gctcacatga 76140
acactttcta ctacggtggt gttaagtttg tccagtacaa cggtaagttc aaagacaagc 76200
gtggtaaggt tcacactctg gtgagcattg acagcgtaga agctactgtt ggtgttggtc 76260
atgctttccc taacgtttct atgctgggcg aagcaaacaa catctttgaa gtggcatatg 76320
gcccatgtcc taagatgggt tatgcaaata cacttggtca ggaactgtac gtattcgaat 76380
acgaaaaaga ccgtgacgaa ggtattgact tcgaagctca ctcttacatg ctgccatact 76440
gtactcgtcc tcagttgctg gtagatgttc gcgctgatgc taaaggcgac taatattctt 76500
aaggagggtt atgaatgtgt tatacaggcg acccagccaa taaccctctt gatagagtaa 76560
gaatcctctg cacagacacc aataatgatg aaattcttat tgaacagtct gtgctagagt 76620
ggttctatct agaatctgga aaggatgaaa agaaagcagc catcaaagct cttaaatatt 76680
tactctttca agtagccaag atgggagatg agaaggttgg tggtgtttat ttacgtaact 76740
cttccagatt caaatctctg aaagctgttt atgatgacct tgttaaaagc tctgtttcag 76800
gactacccta tgcaggtggc attaatcagt gtgatattga catgcgtcgt cagaatcctt 76860
gctctgtcaa gaaatacaca gaatatggtg atgctgccag atacgaaggc agagattact 76920
gcaaccgtgt taatggcgta tttattatcg agcgagatga ataatggtta aaagggttat 76980
tcaccccgct agagcaaaat tagtcggggc tatgaagaac ttgcaaacgg ctaatgctca 77040
agttgggtat tttcaagaac aaggtcaaca tagctctggt ttttcttatc ctgctttaat 77100
gtatttacaa gaagttattg gggttccctc agcttctggt aaagtatatc gtaggttgtt 77160
tgaaatcact atgatgctaa acagacagac cttgttagag cagactaaga agaatctata 77220
taagcaactt agcagtctca acacagaccc ttcaaatacc ttagaagcat ttgcaaagaa 77280
tgctcaaaag gcaattaaaa gaggttttgg taattctgct atcctccctc ctaacgcacc 77340
ttccacagtc aagaaaaaag gctttaatgc acctcttgtt gagacaggtg acttaaggga 77400
gaaccttgct tataaaattt ctactaagaa gggtattaaa aaatgaggct cttaaacaga 77460
cacagcttta tagtaaagcg taaagtctct gaagatggtt attataatga tgatggtgat 77520
tgggtagctt cacaagatat tgtagaggtt aactgtaaag gtaatatcca gccatatatc 77580
aaaggttctg taaagaatgg tacacagatt gctttaccgg aaggtattag gcttaccgat 77640
acaagaatcc tgtatactac atataaactt agaacttcag atgatgtaga gtggaatgag 77700
tctgatattg ttataattga tggtcatgag tatgaagtat ttatgactat ggattggtca 77760
cagcaattag cccatacgtc ccattatgaa tatatcatta ttagaaggga taaaatgaat 77820
gcagttagaa acagcagaac ttgaaaaagg tctagttaga accttagtgg atgttattgg 77880
tcacagacta gctcgtgata aaaataatag gccaaatgta attagagctt acccttctga 77940
taactcaaat gacaaaggtt taaaacctga ccagccattt attaccgtat attgtcaaga 78000
tgctgcaaca ccttatggtt gggttcttga taagtttgtt gaggatgatg tagtttgcta 78060
cagaattgct tttcagattc cagtattaat tacggtgaat ggtaaaggtg ctcacagtat 78120
tatgcttgag cttaaacaac gattagagat gagttcagtc agggatttaa ttcttgaaga 78180
gacaggagct acagttttag acactggagc tatcccaaat gactacactt atcttaatac 78240
agatttcgaa aattctgcac ctcttgttgt aactcttgta aaaaattcag tcctgaaaga 78300
tgaacgtgga ggtattattg aacgtgtcat tgttgatggt gaattagttt acgaagaagg 78360
acaagagcca ccagaatata ctatccatct agatgtagac tccaaagggg taaaataaat 78420
gtggaatcca attgttaatg tagatattac attgaacacc gcaggaacta caagagaagg 78480
ttttgggcta ccactatttt tagcttcaac agataacttt gaagaaagag tccgtggtta 78540
cacttcctta actgaagttg ctgaagattt cgatgaaaac tctgctgcat ataaggctgc 78600
taaacaactt tggagtcaga ctcctaaagt aacccagctt tatattggta gacgtgctat 78660
gcagtacact gtatcaattc ctgactctgt tactgaaagt accgattatt ctttaactgt 78720
agctgttaga ggtggagttt ctcaaccatt ccagtacact tcacagtcag aagatactgc 78780
tgagaaggtg ttacagcagc ttaaaacaca gattgaagca gaaccttctg taaaagacct 78840
tgttactgtc aatgtaactg gtactggtgg ttcagcaaca atggtaatca ccaaagctgg 78900
tgacaacgac ttcgtgaagg ttactactac agcacaaaca gtgtcaattg caagcacaac 78960
tgctgacaca gcatcgactg ctctggcagc tattgaagct tattcgactg actggtattt 79020
cattgcagca gaagacagaa ctcaacagtt tgtcttagca atggcttctg agattcaagc 79080
tcgcaagaaa attttcttca cagctaactc tgatgtatca gcactgcaag gtacagaatt 79140
agccagtgca aatgatgttc cagcacaact tgctaagaat aagtacactc gtacagtttg 79200
cttgtggcat cacacagcag cagaagatta tccagaaatg gcatacattg cttatggtgc 79260
tccatatgat gcaggttcaa ttgcttgggg taacgcacag ttaactggtg tagctgcttc 79320
tctacagcca tctaatcaga gacctctgac aagtattcag aagtcagctt tagatgcacg 79380
tcactgtaac tttattgacc ttgatggtgg tgttccagtg gttcgtagag ggattacttc 79440
tggtggggaa tggattgata tcatccgtgg tgttgactgg ttagaatcag acctgaaaac 79500
ttctctgaga gacttgctaa ttaaccagaa gggtggtaag attacttatg atgatactgg 79560
tattacccgt attcgtcaag tcattgaaac ctctctgcaa agagcagtca acagaaactt 79620
cctgtcatct tacacagtta atgttcctaa agcctctcaa gttgctttag cagacaagaa 79680
agctcgtatc ctgaaagatg ttaccttcgc aggcatctta gcaggggcta tcttggatgt 79740
tgacttgaaa ggtacagtgg cttacgaata atagaggtaa attggaatgg ctatgtatca 79800
gcaatattcc cctaaagacg ttgtgtgtag ctggaatggt attgccattg aaggctttgc 79860
cccagactca ttcttacgtc tacagagaac atcaccactt gtgacaccag ttgtaggtgc 79920
aggtggtcaa gttgctctga caagaaatgc agacaagaca ggtactattg agattgagct 79980
aatgcagact tctctctcta atcagatgct ttctgcaatt caagctaaac aagacaatat 80040
ggaacttgaa gaagatatct cttctaactt cgtaatctac gacccatcag gctctgttct 80100
ggcaactggt attaatgctt ggttgcagga actaccacag attgaacttg gtcgtgacca 80160
gaactctaaa acttggattt ttggttgtga gaagctagac tacacttcta caattccagc 80220
gtcaagtgtt taataaatcc tataaggggg agactttaaa aggtcttccc cttttttgtt 80280
tcttttaaaa gtattaagga atcacaatga aaacagaagc tagagtaatt aatggtaaga 80340
aagtaaatat cgttctgctt ggggcaagag atggtattaa gatgtctatg aagttgggta 80400
aaattgttgt tccaactttt gcacagatgc tgtcaagtct gactgataaa gataagaaag 80460
aagctccaat ggttccattt aaagaacttg ttgaagcttg ttttgacaga attgaagaaa 80520
ttaaccttga agaaatggct accctgttat ttcaaggggc aactgttgat gacttcccac 80580
ttaatattga tacatacttc caagcaaact atggtgaatt tattgattac ttagcatttg 80640
cgctggaggc aaacttcgga agttttttcg aagcaagcat tttcaaaagc ctaacttctc 80700
agtaaacatg ggtaacactc tacagacacc actaactgat gctgctgtag aggcaaccta 80760
tgaagaagca gacgagatga aatttgtgct tgctatttat ggtatggaag ggtgtaaaga 80820
aacacttgac caactctttg ctatgacatt ctctgattta ttatcattga gacaatttct 80880
tgaaattcag aggtcgtata aagaggaaat tgcttacaac gaacttagaa gagcaggaaa 80940
aatgtaatgg cacaatatac agttgatagc ttcattgtgg aacttggttt cagtgaaaag 81000
gttgttaagg gcttgcaaag agttgagaag atgtctatgc aagctgctca acgtattgag 81060
agaaatatta ataaagcctt tgatgtgaaa cctaataaaa gttctcagga agcacttaat 81120
agaattgtaa aaaatgctca gtctgcttca ggcagaatca ataaagcact caacagttcc 81180
ttgaaccttg attcacaagg tgtgaagtct cttaagaaac ttgaaactca agcaaaaaag 81240
actgcaaaag gtattaacaa gtccttaaga gatgctatga aggttgacgg taaagttacc 81300
atcaagacag gtagaggaag aggtggtcag aacatccctc ctgttggtgg tggagcaccc 81360
agaggtccga gagtagatgt tgctcagaga caaatggaaa ggatgtttaa taataacttc 81420
tattcagggt taacccgtag actggaaaca atcggtggtc aaggtaacca aatggcagct 81480
tctttcagag gaagcttaca aagtatctat aataaataca agggtactgg taaggttggt 81540
gagtatgaga tggaagttaa aaagctcatc gacgtaacca aacgttgggt tattgcagaa 81600
aatgctagac taaaatcagt taaagaagca gcttggctac aggacagagc taactcatca 81660
ctacgtcaat tagttggtgg atttgtttca gcttatgctt tactggaact atcacaaaag 81720
actattgagg cgggtgtaaa aagacagtct gcacaattag cctctacagc tatctttgga 81780
gcagataccc agcaagccag aatgtttgct gcatcattcg cacaccagat tggtcagaac 81840
tatacagaca ccatgaagca gtactcaaac tttgctgctg gtgctcaacc aacacttggt 81900
tttcagggta ctcaagagtt ctataagaat gctgcaatgt ttgcccgtat caggggtgct 81960
actgatgaag acttgaaagg tatcatggtt gcattccagc agatggcatc aaaaggtaaa 82020
gttcaggctg aagaacttcg tggacagtta ggtgaccgtt tagcaggtgc tgtacaacta 82080
ttcgctgatg ctattggaaa gactccgcaa gaactcgata agatgatgca agacggtaaa 82140
cttcttgctc aagacgttct acctaaagtg tcggaaagaa tggctgaact tgtcaagcaa 82200
gcaggtggta tgaacgccgt atctaagcag actgctacgt caatgggtca agctaaggca 82260
atgtgggata acacacttgt agcactgttt aacaactcta gtgatggtat ttcacagtta 82320
tctaactctg ttgcaatgtt cttacaaggt tctttgggta gtacacaagc tttgggtgtt 82380
gtgattggaa acctattaaa aggtgctagt aatctgcttg actttgttac agacttcatg 82440
tacagaacat ctgcgctata ctactatgca agagcttggt ataaagacct tgacagtagt 82500
cagcaaaaac taatcaaaag tgctggtgaa tttctgggaa cagttgtaac aattggtggt 82560
gcagttgctg tagtatcaaa atcagtcaag ctgctgagtg gtttggttgg tggtggtatc 82620
ttcggtaaaa tcttacaaag acttggtgtt agtgcagcag gtacagcagc agccggagaa 82680
gcagccgcag cagcaggtgg agttacagca acgagaatgg cacttggtac tgttggttct 82740
gcattaatgc tgagaggttc tacagaccca aatgctgcta aaaactacag tgaagttaca 82800
ctaccaaaac catttgaaaa tgctgttgca aatattacaa acccaaaaag gccaatgttc 82860
tttgatgaga atggtcaact tcagtttgca cagtacactc aagacgttga aggtaacaga 82920
aagttaattg acaatggcct atctaattgg gagattatca tggataaact gtcaacatct 82980
attgataatt ttgccaataa gtttaaccag acaccaatga tgatgacacc ttctggttta 83040
cctatgcaga ctaaacaaac cctgaatgtt actttcaatc ttgatggtaa acaaattgct 83100
actaagatgg tggatattac tgacaagaat caagaagaca ttcttctaag ttcaagctat 83160
ccagaggaag aataatgtta tgggattcta atatgcaaat caaatatagt ggcaaagatg 83220
gcatctattt ccacttaaga gacaatgtag atgccttctt aaccttatca gcaactgaaa 83280
atatggaatt tgacagccct atgcaggtga ctacacagaa catgcaatca gggcaaaccg 83340
tcacagataa tgtgcaaaga gcacccagaa caatcactat tagtggtgtc gttgtagttg 83400
gctatgaagg aagcttatta ttaactcgtc agggtcaatt agtagaaaac ttcatcgaca 83460
ctcttgaaag ctggcgtgac cagaagcaaa ttatttcggt catctgtaaa gacggaatta 83520
aaattgatga ttccatcatc acaggcttta aagcctctaa agatgttgga atttcaaatg 83580
gtctaagaat ccagctaact tttcaggaaa ttaacttcaa agctgttgtg ggacaaactg 83640
atatttcggc tgcaactgga aagactgcta ccacgaatga tggtggtgct accagtaaaa 83700
agaatacagg gaatactaca acaagtttag gtaatggtaa attaaactgc cagttattat 83760
ttgacctaga tgctaatggt gtaagggtac tcactagtgc tgaagataat gctcttgcaa 83820
aatgctcaat gtctgcaaaa acaagaaaag gtgttaccac attcagtgac gaggctgaaa 83880
agaatgccgg agcagcatta aataggactg ctggaactgg aaaggcatta caaaaacact 83940
cagtgaatcc gaataagaag gggacttatt aatgtcacaa tatattcctg ttcctgatac 84000
agaatggtct acacaaactg taactctgga tggcactgtc tttgtaattg agctaaagta 84060
taaagagagg cttgacagat ggttcttgac gctatctgat gttgatggta atgtattatt 84120
acatgaaaag aaatgccttg cagaccaatc aatcacagga cgctatgtaa ttccttcatt 84180
agctggagag ctttttgttg aacgaatgta tggtactgat ttacaaccta ccagaaataa 84240
cttcggaaga gaaaaggcat ttgagcttaa ttattacact caagaagata tgagattaat 84300
ggagaactta taatgtctgt aaaagatagc actgctgggg cttcttttag gtgctatcaa 84360
ttggctgtag gtagtgaaac tacagccttt aatgataaac ctacaagcca tgctaaagat 84420
tctatgcaaa tggactattt cgacaacttg caattcactt gtaatgtgtc ttatacgtct 84480
cagaaaaata aagtaacctc tgatgatacc acttttgaaa tctacaatct taataaagag 84540
atgagagcca aattcaaaac cgttggtgca acagttatgc tcagagcagg ttatactact 84600
ggttttaaaa gggactcaaa cggtgacctt attattgaat atgacaacct cccattaatt 84660
tatctaggca ctattgagta tgcttataca tataagcgtg gtgttgatat gattacaaag 84720
gttatctgtt ccaatgataa aatggaaagg accacgatta agacatcaat ttcctataaa 84780
gcagggacaa cacgtaaaag tgtgattaga gatttagtca ataggttagg cttctcactt 84840
attgatgaag acctttcaag tattgatggt tacacttaca agaatggttt tagtgtttgg 84900
gggagtgttg cagaggcgct tacaaaggtt tgtgaagaaa gtagtctacg ttggtataca 84960
tttaataagc aaatccgagt agtacctttt aatgctaagg ctagacaact ttcttgggaa 85020
atttacccat ataatgttat cgactctttg caaggttact atagaagaac cagaaaggtt 85080
ctgaaaaaag agaataagac agttattaaa gttaaaactg gagttcgttg caaaatccat 85140
ttagatggga gaattaagat gggtgataac atcactatca gggaaagtga agactttgaa 85200
ggccagtatc gagtaaaagg tctttctcat aatcttgatt ttactggtgg tgcttggaca 85260
actgaactag atttagagaa ggtggaataa tgaagtcacc agttactaga atgtccgggt 85320
atgtttcaga atgtcttgat gaatttagaa aagaactgta tactggatta ccagctatta 85380
tccagtcttt tgattcaaag actcaaacag ccactgttaa gccactttac tctattaatg 85440
gtttacctat gccagagatt actggtgtgc ctgttcaatt cccaagtggt ggtggagcat 85500
ctttaacatt tcccgtaaaa actaatgaca gatgctggtt agctttctca atgttacctt 85560
tagatgactt cgttgtcaat gacaagaatg ttcagatgga aacaaatatg agaaggacac 85620
acgatatctc agattgtgta gctttcgtag gtatctgtac aagaacacag aattttaaac 85680
cagacccaac agcagtcaga ctccattttg gtgactctgt gttaagaatc actgatgatg 85740
ggaatttcta ctttgaaggt gatgtacaca tttctaaaaa cttgtatgta acagaagaag 85800
tacacggctc agactttatc agtgacacaa ctggtgtgag ctttaatgaa cacacgcacc 85860
attattactg gacagaccct gctggtgagg ctgatactac agaggcacaa taatgaaaac 85920
agactttgca ttaaatctag gtggtgacta tgttgccact ttaggttcag attcagtgta 85980
tgtggctcat ggtgatttaa agattactgg taaccaaatt agaattatcc cagaggatga 86040
taaagctact caggttgctc aaagacttca tattagatgc cttctaaggg ctggtgaagt 86100
cttctttaat acatctgctg ggttcccata tttacaactt gccaaattta aacagaaaac 86160
ttctatcttt gataattaca tgaaagccta ccttgttgaa acaagagatg tgtctaacat 86220
ctataactat tcttcttcaa tggataatgc tcaaagaaaa gtgactgtta attttgatgc 86280
aaccactaca acagatattt taacagacat tacgcaagag gttaatatct aatggcagga 86340
ctaactacaa caggattaca aactctaaga tatcaggaga tttttgataa tatcaaatca 86400
agacttctta gagatatttc accaaacctt gacgtttctg aggatagtca attaggactc 86460
ttcctagctt caattgcaag gtctttagca gatactcatg agattctatc agaagtctat 86520
gacggtggaa caattgacaa ggccgaaggt tttaaccttg atgatattac agctttaaat 86580
gctgtatata gatatgttgc tcaggcaaca agaggtcagg tggagtttac tggaacgact 86640
ggtgctacca ttccttcaac aacaagatta agaagtactg ccggaaacat cttttatcca 86700
gttaacaaca ttacccttac cccgtcatat tgtgttgaag ctatccttga agttaactct 86760
ttacgaactg atgcagatta tgttattatt attgataacg tcattttctc atataaacca 86820
acctcatcag acacaatcac cacacttcta acaaaattag cagaagctat taatggtggt 86880
attgtagcaa aagcagaagt tattaatgat ggagccgcat taagagtcta taaagatgag 86940
ggtgatatca ttgcaagaac aaaccctatg gttgttactg caacaacgtt cctgacattt 87000
acaaaaatta caactatttc agatgtagta gctgaagagg tgggtgctac ccctgcttta 87060
gctggtacac ttattgagat tgagacaact gttgatggtc ttgatagtat ttataacagg 87120
tatgacttaa caactggtag aaacgaagaa actgacacag aactgagaca gagatattta 87180
gaatccttag cagttactgg tgtaggaact ttggatgcaa ttgtagctgc tgttaaaaga 87240
gttcaaggtg tctcggatgc ttcaggtgtt gaaaatgata ctgaagagac aaactcagaa 87300
ggtctcccac caaaatcttt taagattgtt gtagttggtg gtcaaaatga taatgtcgct 87360
caggcaatct gggatacaaa acctgctggt attagggctt atggctcaat ttttggtaca 87420
gcttatgatt taggtggttt ggcacataat atctatttta gcagaccgac accaaaatac 87480
gcatttgtta aggtatctta ctcattatat gatgaagaaa gcttgacaat ccccgaagag 87540
gatatcagag acagtattgt acaaggtatt aatgcctacg gaagaactct taaggttggt 87600
aatgatgtta tccctaacag aatctatgga tatatctatg acgtaattaa aggtattgag 87660
attaatgagg ttaaagtagc cttatcaaac agccaatcag ttccacctag tgacgggcag 87720
tatactacag caagaattac tgttgacggt gaccaatata ctgtttggga aagcagccag 87780
tataccatca ctaaggagta ataatgtttg aaaagattga tagtgtttat tacaaaacac 87840
ttgatgaacg aactgtaaca cagtttaaag ataagtttat ctacacaagc atcttgaaag 87900
caatcactga tgagttacaa acactagagg atgtttgctg gcagatgcac acagagagaa 87960
atatcaggac atcagtaggt caacaacttg ataatattgg ctcactaatt aaagttccta 88020
gacctctagg tgctgatgat gaaacatata gagctatgct ttatatccaa atcttcctaa 88080
gacgctctga cactactcca acattcttgc aaaaagcaat tatgacacta tatggtgcaa 88140
ctttctctca gatttttgag catattactc ctaagactgg tggtatcgtt atccgtatta 88200
acaccagaaa aaacgttctt gatgctgcat acacgctatc aaagatttct gcaacaacaa 88260
ctggttctgc tgttatctta agagatgtta ctttaaaggg gactgcttgg acacctgtag 88320
aggttgctga ttcagcactt gctattgttg atgataaaga gaactggttc gtaactgatg 88380
ctaacagggg tcttgttaca aataacactg gtggctctct ggagtccaac ctagtaggga 88440
gtcttgcaga tgctggtgtt attgatggtt actttaaggt tgacagaaca gcaaactcac 88500
agagtgatga ttacatgact gtcgataggt ctaataatag cggttatttt attgttggta 88560
aagaagctgt tgcaggtggt gcatacggag tcatggctga ggttgcacag attgtcaaag 88620
gcagaaaaga taaatcacaa caagaaggaa attcttaatg gcagttttaa attggtctac 88680
aaatgaagtg gatactgatg gtaatcagtt aaaagtttta ccaccaccgg aaattcaaga 88740
gacaggtctt ttaaagggtg agcctatggg tagacagtgg ttcaactata ttatcaacta 88800
cctgctgaat aaagtaaacg gcactgttgg tgaagttcgc tcatatgcaa ctgagcaacc 88860
agatttagtg gctaatggct ggaatctaat taagtctgag acagggactg catcaacaag 88920
cacaagaaac ctttacacat atgaatatgt aggagcttaa taatggcagt aggtgaaatt 88980
caaattagtg ccttgccaca agcggcctta ccaattgatt tgagtgatat cttccatctt 89040
aagcaaggtg ttgaggataa aagatgtacg cttgaacagt tgttagcacc tcacacatct 89100
ttgaggaata accctcatgg tgtaactaaa tcgcaagttg gtcttgataa tgttattaac 89160
tcactccaat tggtcgcagc taataacttg tcagacattg tagatgttgc tgaggcaaga 89220
gcaaatctac agattatgtc gtcagatgaa gtgaataact taatccagca acatatcgac 89280
gacaaaagta acccacacaa tacaacgaaa gcacaggtag ggttaagtaa tgttcagaac 89340
tggacaacat ccaacctgta caatgaagat gctgataaat atgctaccgc gagagcagtg 89400
aataatctat acaaagctat ccaagcatca tatccagtgg ggactattca tctgtctgtt 89460
aactctgcta acccatccac ttacttgctt tgtggaggta cttgggagtt ggtttctaaa 89520
ggtagggcgc ttgttggtta tgatacagaa gctagacctg ttggttcaac gttcggttct 89580
cagactgtag cattaacaaa taataattta ccagcacaca cccactctat ctatttaaca 89640
ggtggtggtc acacacactc tgcaagcgta tttatctcta gttttgatta tggttcaaaa 89700
agtacatcaa cttttgatta cggtacaaaa acgactaata gtgctggtgc tcacactcac 89760
acgtttagcg gcacgacaag taatgatggt aaccataacc atagagtacc actgagaggt 89820
aatgatagaa gtggggggga tgctattact gcatctgctt atgctggggt taataataat 89880
acgtatactg atttagcagg tgctcacacc catagcttct caggcacaac tgcatcatct 89940
ggtgctcata accatactgt tgcaattggt gctcatagcc acactgttaa tattggttca 90000
cacagtcata cgggcactgt tactgtttct agctctgagc atacacactc agggacatct 90060
ggttctgttg gtggtggtca agctttcagt gttgagcagc catcttttgt cgtatatgta 90120
tggcagagaa cagcttaatt tctttacagg ggctttgtag ccccttttaa gaggaaaatt 90180
tataatggca gattacaaat tgagtcaatt aaactcaatc gacacaattc gttcagaaga 90240
cctattgcac atcagagtta agaaaagacc tgaaatgttg ggcgacgaag accgtaga 90298
Claims (13)
- 대장균 또는 시겔라속(Shigella) 균에 특이적 사멸능을 갖는 박테리오파지 (기탁번호: KCTC 14641BP).
- 제 1항에 있어서, 상기 박테리오파지는 서열번호 1의 염기서열로 표시되는 유전체를 갖는 박테리오파지.
- 제1항에 있어서, 상기 대장균은 대장균 O157:H7 인 박테리오파지.
- 제1항에 있어서, 상기 시겔라속(Shigella) 균은 시겔라 플렉스너리 (Shigella flexneri)인 박테리오파지.
- 제1항의 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 항균제.
- 제5항에 있어서, 상기 항균제는 대장균 또는 시겔라속(Shigella) 균을 사멸시키는 것인 항균제.
- 제6항에 있어서, 상기 대장균은 대장균 O157:H7 인 항균제.
- 제6항에 있어서, 상기 시겔라속(Shigella) 균은 시겔라 플렉스너리 (Shigella flexneri)인 항균제.
- 제1항의 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 대장균 또는 시겔라속 균에 의해 유발되는 질환의 예방 또는 치료용 약학적 조성물.
- 제9항에 있어서, 상기 대장균에 의해 유발되는 질환은 대장균 감염증인 약학적 조성물.
- 제9항에 있어서, 상기 시겔라속 균에 의해 유발되는 질환은 세균성 이질인 약학적 조성물.
- 제1항의 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 사료첨가제.
- 제1항의 박테리오파지 또는 이의 배양물을 유효성분으로 함유하는 식품첨가제.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020210104879A KR20230022739A (ko) | 2021-08-09 | 2021-08-09 | 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 및 이의 용도 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020210104879A KR20230022739A (ko) | 2021-08-09 | 2021-08-09 | 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 및 이의 용도 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20230022739A true KR20230022739A (ko) | 2023-02-16 |
Family
ID=85325749
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020210104879A KR20230022739A (ko) | 2021-08-09 | 2021-08-09 | 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 및 이의 용도 |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR20230022739A (ko) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101830892B1 (ko) | 2015-09-11 | 2018-03-29 | 서울대학교 산학협력단 | 살모넬라, 병원성 대장균 o157 및 이질균에 대해 동시에 숙주감수성을 나타내는 박테리오파지 bsp10 및 이를 함유하는 조성물 |
-
2021
- 2021-08-09 KR KR1020210104879A patent/KR20230022739A/ko not_active Application Discontinuation
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101830892B1 (ko) | 2015-09-11 | 2018-03-29 | 서울대학교 산학협력단 | 살모넬라, 병원성 대장균 o157 및 이질균에 대해 동시에 숙주감수성을 나타내는 박테리오파지 bsp10 및 이를 함유하는 조성물 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101592177B1 (ko) | 대장균에 대한 광범위 항균 활성을 갖는 박테리오파지를 활용한 대장균 감염을 방지 및 처치하는 방법 | |
| KR101761573B1 (ko) | 신규한 장출혈성 대장균 박테리오파지 Ｅｓｃ-ＣＨＰ-1 및 이의 장출혈성 대장균 증식 억제 용도 | |
| CN107109372A (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 | |
| KR101761581B1 (ko) | 신규한 장침입성 대장균 박테리오파지 Ｅｓｃ-ＣＯＰ-4 및 이의 장침입성 대장균 증식 억제 용도 | |
| KR101591789B1 (ko) | 신규 박테리오파지 및 이를 포함하는 조성물 | |
| CN107250352A (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 | |
| CN107109373B (zh) | 新型噬菌体以及包含其的组合物 | |
| CN110846282B (zh) | 耐高温的大肠杆菌噬菌体及其组合物、试剂盒和应用 | |
| KR20200006662A (ko) | 대장균 박테리오파지 Esc-COP-14 및 이의 병원성 대장균 증식 억제 용도 | |
| KR20130028275A (ko) | 살모넬라 티피무륨 감염을 방지 및 처치하는 방법 | |
| KR101587113B1 (ko) | 치주염 유발 장알균(Enterococcus faecalis)을 특이적으로 사멸시키는 신규한 박테리오파지 | |
| KR102125235B1 (ko) | 닭 대장균 예방 및 치료를 위한 신규 박테리오파지 및 이를 이용한 항균조성물 | |
| KR101859974B1 (ko) | 신규한 에로모나스 하이드로필라 박테리오파지 Aer-HYP-2 및 이의 에로모나스 하이드로필라 균 증식 억제 용도 | |
| KR20230022739A (ko) | 대장균 또는 시겔라속 균에 특이적 사멸능을 갖는 박테리오파지 및 이의 용도 | |
| KR20230005365A (ko) | 병원성 대장균의 증식을 억제하기 위한 조성물 및 방법 | |
| KR102193495B1 (ko) | 신규한 아시네토박터 바우마니 균 특이 박테리오파지 ab63 및 이를 포함하는 항균 조성물 | |
| KR102003795B1 (ko) | 신규한 시가톡신 생성 대장균 특이 박테리오파지 ecoh1 및 이를 포함하는 항균 조성물 | |
| CN113583974A (zh) | 一株裂解性副溶血弧菌噬菌体rdp-vp-21007及其应用 | |
| KR102722687B1 (ko) | 살모넬라 사멸능을 갖는 박테리오파지 및 이의 용도 | |
| KR101992013B1 (ko) | 병원성 장내세균에 대해 사멸능을 갖는 신규 박테리오파지 및 이의 용도 | |
| KR20180106345A (ko) | 박테리오파아지 s８ 및 이의 용도 | |
| KR102592424B1 (ko) | 병원성 대장균, 살모넬라 또는 시겔라에 대해 사멸능을 갖는 박테리오파지 및 이를 함유하는 항균 조성물 | |
| KR20230127664A (ko) | 살모넬라 사멸능을 갖는 박테리오파지 및 이의 용도 | |
| KR101993124B1 (ko) | 신규한 살모넬라균 특이 박테리오파지 sg1 및 이를 포함하는 항균 조성물 | |
| KR101101377B1 (ko) | 이질균에 특이적 사멸능을 갖는 박테리오파지 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| E902 | Notification of reason for refusal | ||
| E902 | Notification of reason for refusal |