CN110616197B - 副溶血性弧菌噬菌体vB_VpaP_MGD2、其用途和新型生物杀菌制剂 - Google Patents
副溶血性弧菌噬菌体vB_VpaP_MGD2、其用途和新型生物杀菌制剂 Download PDFInfo
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Abstract
本发明公开了副溶血性弧菌噬菌体vB_VpaP_MGD2、其用途和新型生物杀菌制剂，噬菌体vB_VpaP_MGD2的保藏号为CCTCC M 2019489，其DNA序列如SEQ ID NO:1所示，具有裂解副溶血性弧菌的能力，可用于制备治疗或防控副溶血性弧菌感染疾病的药物，也可用于开发抑制致病性耐药副溶血性弧菌的药物；本发明的副溶血性弧菌噬菌体vB_VpaP_MGD2裂菌谱广，特异性强，且不易使细菌产生抗性，也不会对宿主产生不良影响，有效地解决目前抗生素治疗副溶血性弧菌面临的耐药性问题。
Description
技术领域
本发明属于微生物领域，具体涉及副溶血性弧菌噬菌体vB_VpaP_MGD2、其用途和新型 生物杀菌制剂。
背景技术
副溶血性弧菌(Vibrio parahaemolyticus)是一种革兰氏阴性的嗜盐食源性致病菌，隶属 于弧菌属弧菌科，在沿海地区和海洋动物中广泛分布，尤其在海虾、贝类和海产鱼中该菌具 有很高的检出率。目前研究发现并非所有副溶血性弧菌分离株均有致病性，含有耐热直接溶血素(thermostable direct hemolysin,TDH)和(或)耐热直接溶血素相关溶血素(TDHrelated hemolysin，TRH)的副溶血性弧菌具有致病能力。食用感染致病性副溶血性弧菌生的或未煮 熟的水产品可能会引起急性肠胃炎，常伴有腹泻、头痛、呕吐和恶心等症状。抗生素的长期使用导致多重耐药的食源性致病菌广泛出现，副溶血性弧菌耐药问题也逐渐被重视起来。总 结近年来国内外报道的耐药事件可知，副溶血性弧菌耐药谱和耐药率呈现持续增加的态势。 由于新抗生素的研发周期不断变长，甚至面临着无药可用的窘境，因此寻找新的能杀灭多重 耐药副溶血性弧菌的替代疗法迫在眉睫。
烈性噬菌体能够感染裂解细菌，在上个世纪初就被应用于杀灭病原菌。随后由于廉价有 效的抗生素的发现并大规模应用而逐渐被忽视，只有少数东欧国家(如格鲁吉亚，俄国等)一 直坚持采用噬菌体疗法。近年来由于耐药菌的不断出现，噬菌体再次受到人们重视，尤其近 十几年大量针对耐药菌的新的噬菌体被发现并得到广泛研究。
发明内容
本发明的目的在于有效地解决目前抗生素治疗副溶血性弧菌面临的耐药性问题，在抗生 素过度使用引起副溶血性弧菌耐药性菌株产生的基础上，以致病性副溶血性弧菌为宿主，从 蛤蜊中分离得到1株新的副溶血性弧菌烈性噬菌体，通过形态观察确定该噬菌体的分类地位，并通过全基因组测序确定该噬菌体的基因型。
此外，通过平板点滴法，根据噬菌斑的有无，采用30株单一或多重耐药性副溶血性弧菌 菌株测定噬菌体宿主范围；通过噬菌体对其宿主菌及多重耐药副溶血性弧菌的侵染特性研究， 进一步评估噬菌体在不同感染复数(MOI)下的抑菌能力。
本发明的上述目的通过以下技术方案实现：
副溶血性弧菌噬菌体vB_VpaP_MGD2，保藏于中国典型培养物保藏中心，保藏号为CCTCC M 2019489。
进一步，所述副溶血性弧菌噬菌体vB_VpaP_MGD2来源于蛤蜊。
进一步，所述副溶血性弧菌噬菌体vB_VpaP_MGD2为多价烈性噬菌体，其DNA序列如SEQ ID NO:1所示。
更进一步，所述副溶血性弧菌噬菌体vB_VpaP_MGD2可裂解单一耐药或多重耐药的副 溶血性弧菌。
进一步，所述副溶血性弧菌噬菌体vB_VpaP_MGD2的耐受性温度<60℃，pH为3至10时可稳定存在。
进一步，所述副溶血性弧菌噬菌体vB_VpaP_MGD2可由副溶血性弧菌特异性扩增。
本发明的副溶血性弧菌噬菌体vB_VpaP_MGD2在制备治疗或防控副溶血性弧菌感染疾 病的药物上的用途。
本发明的副溶血性弧菌噬菌体vB_VpaP_MGD2在制备抑制致病性耐药副溶血性弧菌的 药物上的用途。
本发明的另一方面，一种新型生物杀菌制剂，包含所述副溶血性弧菌噬菌体 vB_VpaP_MGD2。
进一步，所述新型生物杀菌制剂以有效剂量的上述副溶血性弧菌噬菌体vB_VpaP_MGD2 为活性成分，还包括药学上可接受的载体或辅料。
本发明的另一方面，所述新型生物杀菌制剂用于抑制或杀灭单一耐药或多重耐药的副溶 血性弧菌。
与现有技术相比，本发明的有益效果在于：
1.本发明基于双层琼脂平板法从水产品中分离和筛选出副溶血性弧菌噬菌体，并研究噬 菌体的生物学特征，获得裂菌谱广、能裂解耐药的致病性副溶血性弧菌噬菌体 vB_VpaP_MGD2，并验证噬菌体的体外裂菌能力，制备新型生物杀菌制剂，可以有效防控副溶血性弧菌感染，解决目前抗生素治疗副溶血性弧菌面临的耐药性问题。
2.本发明的副溶血性弧菌噬菌体vB_VpaP_MGD2可裂解30株单一或多重耐药副溶血性 弧菌菌株中的27株，裂解率为90％，说明该噬菌体为多价烈性噬菌体且具有广谱性，不仅对 单一耐药的副溶血性弧菌有裂解作用，对多重耐药的副溶血性弧菌也具有明显裂解作用。
附图说明
图1为副溶血性弧菌噬菌体vB_VpaP_MGD2的噬菌斑形态。
图2为副溶血性弧菌噬菌体vB_VpaP_MGD2的电镜照片。
图3：A为副溶血性弧菌噬菌体vB_VpaP_MGD2的温度敏感性检测结果；B：副溶血性弧菌噬菌体vB_VpaP_MGD2的pH敏感性检测结果。
图4为副溶血性弧菌噬菌体vB_VpaP_MGD2的一步生长曲线。
图5为以副溶血性弧菌SH03(A)和SH06(B)为指示菌，测定副溶血性弧菌噬菌体vB_VpaP_MGD2的液体裂菌实验的示意图。
图6为副溶血性弧菌噬菌体vB_VpaP_MGD2全基因圈图。
副溶血性弧菌噬菌体vB_VpaP_MGD2(Vibrio parahaemolyticus phage vB_VpaP_MGD2)， 该培养物已于2019年6月25日保藏在位于湖北省武汉市武昌区八一路299号武汉大学校内 的中国典型培养物保藏中心，保藏号为CCTCC M 2019489。
具体实施方式
以下通过具体实施例对本发明做进一步详细说明，应理解以下实施例仅用于说明本发明 而非用于限定本发明的范围。
实施例1：副溶血性弧菌噬菌体的分离
本发明样品采自新鲜蛤蜊，将蛤蜊去壳，称取25g蛤蜊放入无菌均质袋中，每个均质袋 中加入35mL TSB(3％NaCl)，用拍打式均质器拍打4min。加入1mL对数期副溶血性弧菌混合培养液。加入350μL 1M CaCl2增强噬菌体的吸附率，37℃过夜培养。富集后，将液 体富集液转移至50mL无菌离心管中，10,000rpm离心10min，取上清液过0.22μm的微孔滤膜，得到噬菌体原液。
在6孔板中加入1mL TSA(3％NaCl)，待凝固后，取100μL对数期副溶血性弧菌悬 液与1mL融化的50％TSA(3％NaCl)混合后加入6孔板中，取10μL噬菌体悬浮液滴入培 养基表面中间，风干30min，置于37℃恒温培养箱中，观察噬菌斑出现情况。
从6孔板中挑取1个噬菌斑，放置于1mL SM缓冲液中，4℃静置过夜，10倍梯度稀释样品。将对数期副溶血性弧菌悬液100μL(OD600＝0.6)分别与原液及各稀释度的噬菌体悬浮液50μL混合，加入约50℃的50％TSA(3％NaCl)半固体培养基5mL，混合均匀倒入TSA (3％NaCl)固体培养基上，待上层TSA(3％NaCl)半固体琼脂凝固后置于37℃恒温培养 箱中，观察噬菌斑出现情况。双层平板法重复三次，即得到纯的噬菌体，如图1所示。
实施例2：副溶血性弧菌噬菌体vB_VpaP_MGD2增殖与纯化
从第四次纯化后的平板挑取1个噬菌斑，放置于1mL SM缓冲液中，4℃静置过夜。取20μL噬菌体液，加入含400μL 1M CaCl2和200μL对数期副溶血性弧菌SH03的40mL TSB (3％NaCl)中，37℃摇床过夜培养。10,000rpm离心15min除去宿主细胞碎片，随后使用 0.22μm过滤器进行过滤，收集噬菌体液，4℃条件下静置保存。
噬菌体增殖液进行氯化铯密度梯度离心，24,000rpm，4℃离心4h，结束后吸出形成的 条带，为纯化后的噬菌体液。取2mL氯化铯密度梯度离心后的噬菌体溶液于AmiconUltra 50k过滤管，25℃，6,730rpm离心20min，倒出滤液。加入2mL 10％SM缓冲液，25℃，6,730 rpm离心20min，倒出滤液，再用10％SM缓冲液清洗3次后，加入500μL 10％SM缓冲液，用200μL移液枪小心冲洗滤膜，转移所有液体至1.5mL灭菌离心管中，4℃保存备用，即为 纯化后的噬菌体vB_VpaP_MGD2。
实施例3：副溶血性弧菌噬菌体vB_VpaP_MGD2的形态学观察
取纯化后的噬菌体vB_VpaP_MGD2 20μL滴于铜网上，10min后用滤纸吸去多余样品。 滴加20μL 2％磷钨酸染色液在铜网上，2min后用滤纸吸去染液，烘干。采用Tecnai 12microscope透射电子显微镜下进行观察拍照。
如图2所示，该噬菌体头部直径约为56nm，呈六边形，推测是正二十面体结构。尾部直径约为15nm。根据国际病毒分类委员会(ICTV)最新病毒分类系统的标准，该噬菌体属 于有尾噬菌体目(Caudovirales)，短尾噬菌体科(Podoviridae)。
实施例4：温度和pH对副溶血性弧菌噬菌体vB_VpaP_MGD2裂解能力的影响
取400μL噬菌体增殖液置于30-80℃水浴中，作用1h来评估噬菌体的热稳定性，并通 过双层平板法确定效价；将噬菌体增殖液100μL与pH为3-12的SM缓冲液各900μL混合，在37℃水浴中作用1h后，双层平板法确定噬菌体效价。
结果如图3A所示，副溶血性弧菌噬菌体vB_VpaP_MGD2在30℃、40℃、50℃条件下作用1h，能稳定存在；在60℃条件下作用1h后，噬菌体几乎失活；70℃、80℃处理后完 全失活，该噬菌体具有良好温度耐受性(<60℃)。
结果如图3B所示，副溶血性弧菌噬菌体vB_VpaP_MGD2在pH 3至10时稳定，在pH 11下处理1h后几乎失活，在pH 2、12下处理1h后完全失活。
实施例5：副溶血性弧菌噬菌体vB_VpaP_MGD2的一步生长曲线测定
取-80℃甘油管中保存的副溶血性弧菌SH03划线接种于TCBS平板上，37℃培养，挑取 单菌落接种到TSB(3％NaCl)中，37℃摇床过夜培养直至指数中期(OD600＝0.6，约1.0×108CFU mL-1)。将7mL菌液6,730rpm离心5min，将沉淀重悬于7mL SM缓冲液中，并用0.7 mL1.0×108 PFU mL-1噬菌体原液(MOI＝0.1)感染。将混合物在37℃下温育10min。10,400 rpm离心2min后，将沉淀物悬浮在1mL SM缓冲液中并与10mL TSB(3％NaCl)混合。 混合物在37℃下振荡温育，每5min通过双层平板法测定噬菌体效价。
如图4中所示，副溶血性弧菌噬菌体vB_VpaP_MGD2潜伏期为15min，裂解期为40min， 裂解量约为244，表明该噬菌体为裂性噬菌体。非常快速的裂解生长，并伴随相对高的裂解 量表明噬菌体可以非常快速破坏宿主细胞，这有益于噬菌体疗法用于保护食物或各种材料免 于细菌定殖。而副溶血性弧菌噬菌体vB_VpaP_MGD2由于其生长速度快，潜伏期短，裂解量大，潜在应用前景大。
实施例6：副溶血性弧菌噬菌体vB_VpaP_MGD2宿主谱分析
取50μL过夜培养的30株副溶血性弧菌菌株悬液与2mL融化的50％TSA(3％NaCl)混合加入6孔板中，凝固后在培养基表面中间滴加10μL噬菌体液，待液体变干后，37℃培 养过夜，依据噬菌斑的有无来判断噬菌体的裂解范围。
如表1所示，采用点滴法测定噬菌体对30株单一或多重耐药副溶血性弧菌的裂解情况， 副溶血性弧菌噬菌体vB_VpaP_MGD2对27株致病性副溶血性弧菌有裂解作用，包括环境分 离菌株和临床分离菌株，表明该噬菌体均能裂解致病性副溶血性弧菌，且能裂解单一或多重 耐药副溶血性弧菌。
表1：噬菌体vB_VpaP_MGD2的宿主谱分析
注：KAN：卡那霉素；STR：链霉素；SN：庆大霉素；AMP：氨苄西林；AK：阿米卡星
实施例7：副溶血性弧菌噬菌体vB_VpaP_MGD2的体外裂菌能力测定
取-80℃甘油管中保存的副溶血性弧菌SH03、SH06接种到5mL TSB(3％NaCl)液体培养基中，37℃摇床过夜培养。将过夜培养物(约109CFU mL-1)稀释100倍，在100孔板 中加入100μL菌液，再以MOI＝1,10,100加入100μL噬菌体，置于37℃培养中，每隔1h测 量OD600值监测细菌生长情况。
如图5所示，在8h内，当MOI为100时，副溶血性弧菌噬菌体vB_VpaP_MGD2能有 效快速彻底的杀灭宿主副溶血性弧菌SH03菌株，而当MOI为1和10时，从4h开始，OD 值轻微上升。对于副溶血性弧菌SH06菌株，在8h内，当MOI为100时，噬菌体能较好地 抑制细菌生长，而当MOI为1和10时，噬菌体对其裂解效果不明显。
实施例8：副溶血性弧菌噬菌体vB_VpaP_MGD2基因组分析
取氯化铯密度梯度离心后的噬菌体液1mL(108PFU mL-1)加入40μL DNase I放入37℃ 水浴1h。采用NORGEN Phage DNA Isolation Kit提取得到副溶血性弧菌噬菌体 vB_VpaP_MGD2的全基因组DNA。基因组DNA样品采用Illumina Miseq进行全基因组测序 分析。
副溶血性弧菌噬菌体vB_VpaP_MGD2的全基因组测序结果如SEQ ID NO:1所示：副溶 血性弧菌噬菌体vB_VpaP_MGD2是双链DNA噬箘体，其全基因组由44783bp碱基组成，总 G+C含量为43.78％，预测含有48个开放阅读框(ORF)，占基因组的91.11％(40800bp/44783bp)，其中17个ORF(35.4％)是已知功能的蛋白编码基因，而其他27个ORF(56.3％) 为编码假设蛋白质，无编码tRNA基因和毒素基因。在NCBI上通过比对，发现副溶血性弧 菌噬菌体vB_VpaP_MGD2与溶藻弧菌噬菌体Vp670的同源性为91.25％。副溶血性弧菌噬菌 体vB_VpaP_MGD2基因组分为四个基本模块，每个模块由几个功能相关的基因组成(表2)。 这些模块包括复制和调节模块(orf12，orf25，orf29，orf35，orf38，orf42和orf44)，裂解模块(orf3和orf11)，包装模块(orf9，orf10和orf19)，以及结构模块(orf13，orf16，orf17，orf18和orf20)。
表2：噬菌体vB_VpaP_MGD2预测的蛋白编码基因
序列表
<110> 上海海洋大学
<120> 裂副溶血性弧菌噬菌体vB_VpaP_MGD2、其用途和新型生物杀菌制剂
<141> 2019-07-02
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 44783
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
tcacccacag cctcatagag cggttgtctc acagcgtgag acagatagcc cgccttaccg 60
ctctcgccct atatggagcg acacagagcg tcctatccct agactgttat aacataacat 120
tacgcctcag cagctctagt gttataacat aacaattcaa agagctaacc aagaacctac 180
ccaagagcgc accagagcgg cgctagccga tagccaagat aagcaaggca ttcgcccaga 240
gataaggcca agtccgctct gagacgcttt ggtagccccc tatgggggat gtgcgattgc 300
gggagcggga ggagactgct cagccgtaca ctcaaaaata tttaagctat tgagtactta 360
ctaacaatct ccaagcttgc tccaagttat ccatcatacc gactacggaa gctctcatgt 420
ataaggatat tattagggac atccatacca ataatgaagg tatccataat ctcattagag 480
gagtacccat ttaggttaga tgcatcggct tgatacacaa caacaaaggt aagctctggt 540
cggctcctag ctacagagta cagctccaag atattatctc gaatcatact aagagggata 600
cttctcttgc cagagcggtg gtaggttatg ccagtgcact tctcagtgaa ccccgcttta 660
aggttcttag taggcagtgc atatgtatta ccttgtaacc ctcttccaat acccagctta 720
gcaccaaact tcatagctgc tttagctgct cccataccat gccgaccttc tgggttggaa 780
ccaaatacaa atatattatt accagacatc tctgtgatgc ctctagggtc tttcagtttc 840
cagtaacgca tttacataat ctccgtgtca acagcgaaga ctttacaagc ccacttactt 900
aggtacttgt gcattcgctt gattaaatct agatggaact tagatgcatc tttgtcaggt 960
acatccaaga ccaaggttac tttgatacgc ttcatggcct tcctccatta tcatagaaag 1020
tatgattact gcaagtcaat gtaggaatca tcttatctgt ccagtagtta ctgatatcag 1080
tacgagcgta gtgtaattca gtggtagtat agtaactacc attaagtgct tgtctagcaa 1140
cggctctatc ctgctctgtg ataactgaga ggtcagggtg caatgcaata gtccacgaga 1200
actgattagg ttgcaacact acctctctca ctgtatcagg atagaatgga gaagctactc 1260
tacgaagagt aacctctgcc accattactt gacatttata aggttcacca cgagcttcat 1320
gatgaacgtt tagtgctaac caagtctccg cactttcaag aagtcgctga ttagacccca 1380
aggtgttctt acgcaccaga agcccagagc tactataccc actacccaaa tcggtacgtt 1440
gatatcttgg ttcactatct ttcctacttc agttgcttga gttactgcct cagcagaaga 1500
gacagcttga gccacctctc ctgttgcttc tggtagttga gcgcatgcag ttaagataag 1560
gaggaatagc atgtatatcc atctcttcat tatccacctc ttgatgttaa cataggagaa 1620
atattgatac gacctaaacc tgctcttgca tagttagcag ggtaggaaag aacaatcttg 1680
cccttactct cgtagtcttt ggaagtacca ccaccaactg catcgaactc aacctgataa 1740
tcagttgaca aacttgaagg tgtacgggac ttgtaaattt cctgtatatc agtaagcatc 1800
ttaacacctg actgataatc aacggatact ccaaagatat agcgttcatt cagccgatac 1860
tgagtgttag ttgcatgtgg agagatagag gaatctagct catacatggt agctccaata 1920
aaaggaagtt caatgtactc tcttccgcct gattccttaa tctcaaacca gtcatctaat 1980
cctgatgagt ggaagttctg tagtgtattc tcgtgcaagt catataactg acgaccagag 2040
cctttcacat cccttagtct taccttcgtg ttacgagacg tattagtaac aagaatatct 2100
acgtcaccac ctgtgcagtt gtcgaaatca cagtctgtaa tgcggaagtc aatatcagca 2160
gtaccttcta gggatactgg ctgacctttc aagttacgaa gaactgtatc agtaaccttc 2220
aatatacctc tagtgcccat tcggatacct ttagaggatg cggcagttgc ccaaagagga 2280
tgaggttgtt caccatcgat ggtgcagtta gaaaccataa ggttatgacc agagcctgca 2340
ttgaggtcaa tagcatattc tttagggttt acgaatgaac atccatcaat tatcatatct 2400
gcttctggct gagggaagtc accaccagac cactctgtaa taatgtaccc cttatgtgtc 2460
ttgaactgct cattcaagtt gaaggtgcag ttgcttagtg taacattctt aaccttacca 2520
ttagccacac gaatacttga gtaagatggg aagttgaaca cacagttagt aatagtagag 2580
ctatgaagga tttgacgctc cttatcaccg cctagcgaca attgattcgt acctaagaag 2640
aagatacaat tactaacagt atgcgcaccc cctgataagg aaagcaaacc tgagccacca 2700
tcaaaggtgc agtctgtaat gtgcccatca gtaccagtgg caaacatacc agctacaggg 2760
aaggagatga acacgttatc aacgctccac ccagatactt ctacaaagat aggttgccag 2820
ccgccctcaa tccagatatc ggagacacga ggtgggcgac cagaagggtc ttctgttcca 2880
tcggacttcc atcccattac gatgccacca gtagaaccca tgtacaagtg tgatgcaccg 2940
ccattaccaa atagcgtctg tcctttacgt acatatacct taccatcaat cttgtatcta 3000
ccatctggga tatagatgct acgacggaag atggtttcat tatattcatc agcagcacct 3060
gtggctagat tgaaagctgc tgtactatcc actgtacccg ttgggtctgc cccgaagtca 3120
agaacggata atctctcgtc cagcttgtct ttaatagagc gctggattgc catatgtatc 3180
cgagacttga aagcaagcca acgagcacgt ggcttggtta atttagttag tgccaagttg 3240
cacctccttt cttgtttagt tatattcata aagccctctc aaagaaagag cttgagtata 3300
taacttggtt gcgagcctcc ggcttggagg tacacgccac attcgttagt gttacttcat 3360
tactactttg tacgacactt ccgctgcaaa ggtagggtta ggagagatgt acaatttacc 3420
agcattacca tcaacggtta ctggtgtaat atcatcccac acagatagga agccagagaa 3480
ctcaaagcca cggacttcac ctaatctatc gtctaaccat acgaacatgt acttatcctc 3540
agaagcagat gagcctccac catcagcagg tcgagtatca tcaatccata cattgttagc 3600
atcagcgacc tctcggtcat ttgtaatgtt ggccttgatg gttgcctcgt ctttagtccc 3660
acctttaggg aagaaacctg agattagatg atacttgacg gtagggtttg taccgcctcc 3720
tgtgtccgtg aaccagtctg acaagtccac cgtgtgctct gagccagaga tatctgtaaa 3780
cttaatagtc tttgtatctt tatcaggtgt cacgctagtg aacatagaag gagcaagagc 3840
cttagcaaga ccagttcgtg atacgttgta tgattgcttc tctactccat tactgaagaa 3900
cttgaactgc acattattag ggtcagtcca tcccatgcgc atactgtcga acttaggctc 3960
aacagcagac tcagcactac tagctttctg ctctgcctga gttgccagtt gcttagccga 4020
tgcagcttct tgtcctgcct gctcagcagc agaagatgca gaatcagctt tgctctctac 4080
cgccttatgc tcttgttcag taacagcagc gtgcaacact gccttagtac ctttgagtat 4140
ataggacata ccgtcaccta aagtgatacc actgatactc gattctggtt cgttaggtgc 4200
atcaactgtg atgttactag agccacctgt accagatgcg tcccaagggt cgtgaggttc 4260
tacaccagta gcaacataaa tcttgctacc aatcatcaga gccataccag cacgcttgcc 4320
tgattgcgag ttgatgttaa ctggatgctc tgggttattc aagtctgctg ttgtaagtcc 4380
tttagcttca ggtgctaact gcttcattgc agggataata acagagcttg gtttatcgaa 4440
tctagacatt agtcttcctc catttcccac aagaagccat atccatgaat gacatctgaa 4500
ccaactaact cactgatagc tcgtacacga atgctactac ctgacttaac agcaaatact 4560
agtctaacgt actgcttatc tcgccagtct ctaacaaaag aagtataagt gtatgtactc 4620
tcagaagagc ttgcaacttc tacgtgccgt agagagtaag gctgtgcctc ccaagtaata 4680
ccgtcatcgc ttatctgtga ccatatgtaa agctgagatg tacctaaccc atcgttcact 4740
ttatcaatgt caatggtgaa agtacctgag cagttctcaa gtgtccttcc tgatacgtta 4800
cgaaccttat ctgactgaac atcgaaccct actatcttac cttgagttgc taatcctctg 4860
attgcagttg gtcgtaccag accgctagtt gcatcggctt gcattgtctc agtgctacca 4920
gcgccgaacg tagctctttc ctgtcttggc atacgaatcc agacatcagt tggctccgag 4980
cctgtggcta catagatgta atcaccaatc ataactgaca tgccttctcg cttaccagag 5040
cggttcttgt cgtttacaat atgatgaggg ttctgcaaat cccactcagt tgtgtacttg 5100
gctggctctg tgagctgtgt taaaacgggg cagaactctg ctacccctgt atccatacga 5160
gacatctaac ctccttatgc tggtgtgata tccgtagctc cttttgttac tgcaatccat 5220
ttagaagttg gagcatcacc tgccgcttgt agaaggacag gagcgccagc tttcacgatt 5280
gtgataagag cacctagacg cttaccagat tggtctgcgt tgttaacgat atggtctttg 5340
tctgataggt gtgcttcatc cactactggc atgttatgaa gggttacgcc ttctttctgt 5400
actgctgtga atgttgcacc gtcacctgtt agattaccta ctgctgccat gttgtatatc 5460
tcctattgat taatatatag tatagagaat agaagaagaa aggagggact cttaggagtc 5520
cctctagctg agtgctctag acgctctaat agctctataa cgctcttttc ctcttatacg 5580
ctctcctata gggcaactta attctaagtg cctgatttat aaagaaattt agctactgct 5640
aatttaccat ttacggcgtc cagctcggcg tttattaccg aatctgttgc ctattctttg 5700
tacagcccta gctgtaccca gagcggcgtc tccgcgcatt ccgagtgtaa ggttagtcat 5760
gtattgcttc atcgctgtcg ggtcgctcat aacgtcatac cagcgtttct gctgcttgat 5820
ttgacgctgc atcacgactc taagttggtc gtagtccaga ttctcaacaa tctgacgtac 5880
tgcacctgcc agagcatcaa tgcggtcatc atgtcgtaga cagtctttgt cacgcgtgat 5940
atttgacatc tggtggaaca gagagaagtt cattcgctgt tccgcactat atgcttggat 6000
tagctccata tccttctgta cgacctctgg gcgaacgatt aggcggtgtg tggacatcac 6060
tggctcaaga acgtcgataa ttctaagctc tttctgccca gactcttgaa ccgcctcaag 6120
cgttacaggg taatgtttct cgaaataagg cttgatagca gcaacgtgcg caccattacc 6180
atagttattc tctatgaata cggtcttgca ctctgcattc ttcgctgcca ttacaagata 6240
cattaggctt tcttccgaat agccaccctt agttgcacca atatcataga tatagatagt 6300
ggttcccatg agcttgatga tagcgtaagc cgtttcgtct ccgttcttac cgccaccagc 6360
agggtcaatg tacatgatgg tacgctcaaa gtctcccatc tcatagtcaa cctgcatagg 6420
gtagtagaaa ttatctgtct tacgagtacc gaacttaggc gctccttccc agatgcactg 6480
tggtgagtta caccagatag gcataacctg agcacgttct ctgttgaaag gtgcaactat 6540
caagtcgcta agacgtaatg ggtaacggtc tgcgtcagtc aaaccagtgt taagcatgaa 6600
ctgtagcatg aacttagcct tgccttgtgc aagctccttc tcgttaagaa ggtcattatc 6660
atacatctca ggacaagtag gttgtcccca tgtaccatca agaccacctc cagtttgcaa 6720
cgtagagtct ttctggatgt ccttaagaat aataggagca agcttgtctc catagtattc 6780
catttggttc tgattagggt aacgacctgt ccagatacga accttatagc cacgactagg 6840
tagccagtta tagattgatt ctactgactg tggcgtacca agatagatga tgtcaccaaa 6900
ttggttgatt gattcaaact caagagtctg ctccataagc cattcacgac caccaactgt 6960
acgtgagttc tgcaatgact cgatatcatc ggcaatgata atatctgcac gagcaccttg 7020
agcacctgac tcgatagagt aacaggctac tgaaggtgac ttatccgagc cacgtaaaca 7080
ccagtgaata tcaaaggact cgatagagcc tctatcacct gcgtacttat ctgggcgcat 7140
gaagtctaag aagtcaagtg ccgagaagat tttaataatc caaccagcga tttctttcgc 7200
acgtttagag ttctgcgaga aaactacgat acggtagtgc ggattgtgta tgagcatgaa 7260
taccgcatag atagcagtaa gcgtggtctt tgcctgacca cgctgtgcca taatcatacg 7320
atacttatgc cctactagta ggaacttcaa gatgtctgct tgcatcctgt taaggtctgg 7380
attccctaca atagttgtgt taataacaac ctgtgcaaag aaacacaaac cttctacagt 7440
gtttggaaac atttcttgaa gctcatacaa catcttccag cgttctacct gttgctctgc 7500
tgtaagattt ctagccatta ttcgtctccg tcgatgaatg gaataatgtt atcatctgta 7560
ccagtagcac gagcaccttt gctctgctgt gcttccttga tttgacgaag gcgcttagat 7620
agctctgtct cttcgttcat ctcaggggct gcacaagtaa tgctgttttg gtctgctgcc 7680
cacttacctg ctgctgcaag tgccttacca tcaccgatta catcttcgat gttagcacct 7740
tgcttaacaa gtttgagcat ctcttctagc cgctttgagt gcaaagtagt gactagctta 7800
tgaactagac ctacctcgtc ctctgtagca gcattcttat taggtctgcg gtctgctgcc 7860
atttaaatcc cctttctcca tttataaaga atgtagccaa tctgtataac taagtatatc 7920
agagtagcta ttgatacgat atcaggcaaa gagatgccgt agaacttcat gcctacatac 7980
gccgcaggtg gcgctgctgg cagagtctca cggacaactt ctactgcctt tacaatcttt 8040
tcactcacac agcctcctta cccagaaaca atcattgcat agagttcatc accttcttcg 8100
agagactccg ctaggtggat aatagagcca acaacttcgt aagactttga tggaatctga 8160
acaataccat taattgtcaa gatgatacta gagaacgatg cgttagtgtt cacgcttgtc 8220
tcaccgccac tagctgtata ccaccaatta gtagagccat caaatgtgac tggtttagta 8280
ctgaagggtg ttgataaaat agcgaaaacc tcatcaccat cttcaagagg ttctgctagt 8340
acaatcttag aaccaagaat gttaaacgac gagccgatgg actgtacaac accgtttata 8400
gctagcaccg cgtatgatag ttctctatta gtctctaact gaagctctcc acccttagct 8460
atatacctat agttaactac ataggaaggt gcagagggta atgaagattc aagtgataca 8520
agtcttgtat catgctcttt atattgagcc atattaactg agtctttatc atctacacct 8580
ctacctaagt tcacatgctt atgtccaccc atatcgacat tttgcttcat gtagaaacct 8640
ttcggtgtga acccatcaat aagctcttgt gtcaggtaga gctgctgtaa gaaggagttg 8700
ttaacttgcc tatgtccaaa gttattgcca cgagagaaat ctgcgtaagg cttttctata 8760
ggcatctcac gacgaataag aacatcagag ccagcaggag gtgctggtgt aatgctaacg 8820
atgtgaggtg attgtgattg tattacattg ggtacttcct tatcatctac ataagtatga 8880
atatcttcta cacgaaagta ccccatattc tcgccagcca ctgttaaagg gaacagagtc 8940
ttaactccat cccctacgtg gcgaacgaac gagaggttac tgtacgccat tgttttctcc 9000
tatttgttta cctatagggc aaccttattc agttgcaaca ccgaatgtca gattccataa 9060
ggttgaatta taccaaggaa gcactctctg aatagtcttg atagacttct cttcctccct 9120
atttgtcttg tcctccgttg cgagtgacat agcagccgat gttgccttag ctacatcctt 9180
agccatttct actgactgga tacctgagaa gttaccagca cctctcatct cctctggtaa 9240
gaagtttaga gagttcatca tctgtaatgg aagcataggt gcagctagga taccaacacg 9300
gttcataaca ccccagaaca gcccttcatc ttctaggtgc ttgcggatgt attcatcacg 9360
ctcacggcct gagtactgat tggcttgaat gtatgtgttt gctacaagag cgaagtagcc 9420
aaggatagct tgaccagcac ccatagatag caacaaagca cgttcattct tgatgccacg 9480
aagaagcatc ttctcatacg aaccaatagt gaagctcaat agtgtagtca ttactttacc 9540
aagttccttg ttcatataag caggttgctc accgatacct agacgtagga acgagctacc 9600
tatcgtattg cgaacagcag tagataatga gttatatgta tcagcatcta gctgacgaac 9660
tgcattaaag atgtccatat ctttgttatc tcttaggtgc ttcatcacag catcaacctg 9720
ttcactggac ataccgccaa cacggataag tgagttacga acgttgtctg taacctctcc 9780
ttttattgcc atatctctca agttattgac aatagcccta gctgtcatct cttcaccacc 9840
ttgctggaac atcttgaatc cgttcactgt catcgttata tcaagagcat tacctagcat 9900
gttgtcaata cgctttagct tgttctttgt catatcatcg aagtctgcac cattgtagaa 9960
cttgcgtccg aacaggtatt cttgatgacc tgtagcggag aaggtacgtg caaagtccat 10020
catgaactta tcttttgtta cactacgtgt tctgtaatcg aaccatcgtg atgaaggtat 10080
ctgcttaagt gtgttaacaa tgccgttcct tagcattgca actgagtact ccggtattgt 10140
cataagacct gtactacgta gacggactac agaagtagcc ttacgtgcca tacgagtaac 10200
gtccttcata ccatcgtggt cttccagtgg ctctttgtat aaaagacgta atgcctcatc 10260
tagcttatca gaccatctgc cgttctctaa atcagatagt acctcttgag ccttagcttt 10320
tgccttacca tccagacgag ctacatctag acgaatgtcg ttaatagctg catcacgagc 10380
tgctgcaaca gctctctgga attgatgacg agagtggaaa ccgttgctag caaggccggc 10440
gtttgcagca gagtcagata cgtacttcat gtttctgtca atgcttgtgt caatcaagtc 10500
aaccatccat acatcaccag agcgagcttt taggttagga cgtaatgaga acattgcacg 10560
aggtgagatt tgtcccatct cttctttgtc gaacatagct tgcttgatat catcaataac 10620
atctttgtca acattgttag ctaacagctc ttcctcaagc tttctgaact cagcatcaga 10680
cataaagcta tcaaatgtct tgtgggactt gccaccatat gacatagcac gagctacttg 10740
gttcttagct agtcgaagtg cgttctcacg agacaactta ataccacccg tttggtatgc 10800
ggatgcaata cagtcataga taaagtcagc atggttctca ttagctatga tatttgtagg 10860
attaaacaca actgagtgat actcatgtcg atgcttaatc ttatcgaaac caacaacgtt 10920
gtaatccttg ttattcttaa gaccttgctc gtataaacga gaacgagcct tggcaccaag 10980
cataatagca tcatcacctt ctactggctt attacttagc agtgtaccag tagcttgctt 11040
cataacagtc tgtctgtcga actccatctg tagctcatta agtctaccaa tgtcgaactt 11100
accaacacca cgttctttac ggaaagcatc aaatgcattc tgataatcaa gatagtcagg 11160
aactgctcta ttgaacaaag tctctgcaag ctcttcagca gattgatggc cttcaatggt 11220
gcgttgtggg tcttttacta agaacttacc aagaccacga gttgcatcat ctggtgctgt 11280
gtcaatctcc gatgacaagg agcgtaatgg cttgcctatg gctgaagtct caaaacgagt 11340
attacgagga atcttagcag caaaagcctc tgcatcacta agagcattct gaatctcttc 11400
aacatcagtt ggggtaagta attcatcgta ggtctggatg tcatcaaact cttgtcttgc 11460
tctcattgca ccgacacttt gagctggtgg ctcttcctta cctgtctcat cgactactgg 11520
cttacgacca gccttgatgt ccttaccaag agacttggaa ggttctacat caaattctcc 11580
acgttcggct ttaagctcct tgtatcgctg cattaaggac tcaggaacct tgccttgttg 11640
tagcgcatca atctttgagt tagtatccac agccattctg tttgcttcga tggcattatt 11700
aagctcagct tctcttcttg cgattgcacc ttctacttgc tgaatctcag cagctcttgc 11760
tgatggtctt aggttaggac gtgccttaat cttctcgatt acctcaagac gacccttacg 11820
gaactcacga aactcatcac gaagcttttt aatcttcttc tgtgatagaa catcaacacg 11880
ctcacctgcc tctttacgta gttcatcaat aatatctctc tctgacaaat aacgcttacg 11940
agctacaggg tctggttgct gagaggctag cgcctcatct gccatgttat agcctttcac 12000
attctcttga gctttcagct cagctctttg gacactctct acagcattag ttctatccag 12060
tgcattacga gctgccttat aacctagctt agtagcatca atagctcctg agaaggctac 12120
gccgccgata gctgacatat acaaatcttc ttccgttctt tgtgtatcac ccatctttag 12180
aaggtattca gatagaacac cctcagagcc accacgtagc atagaccacc caatcgaacc 12240
tattcgtcct accttactgg tgagtgcagc aggtgcgtca acgaacatca ggggaaccat 12300
agcagggtcg aagatagcag atgctgtttc aatagcgtat ccagtaactc caccaccctc 12360
aagaacctgc ttgacttctc tgtctgcttt gattctgctc tgtctgtgtg caaggttctc 12420
aggcgaaaca gaaccagcta ggaactctat ctcctgctcc gtgtaacctt ccttgttgta 12480
cttcataagt tcagccctat ctactttgta gtcctgctga actccaccct ctttctctgc 12540
ttcatagtgt cgagacagag actcacctac ccaatttcga tacttagctg cctcccacat 12600
ttcagataga gagggctgtt cgccctctgg ctctggtggt tggtaatctt cgctcagtat 12660
agagagtggg atatgagatg gtgtataatc aagaaactct cttctttctg gcattatcta 12720
ttacctcttt gtttgctcca atattcatag aacagcgcca tgtgtgctgc gtcttcttct 12780
cgttgacgct cttcttcgat acgctgttcc cgctctttct ctgctgttgt agctatctct 12840
tcaatacgct cagcttcacc cttctcacca taacgcttaa catcactcat taggaaacga 12900
gcatcgccca cttgctcacc gaacttatcg accattatga gtgtagtacc agtatcgttt 12960
acaacaaact cgatatcttc tggctctact cctaaagggt taatatcttc ttttgatagt 13020
acatttgacg caaagtgctt catgtatcta tcaacaagtt caggtcttac accaatagat 13080
tgtgcaagct caggcttggt cttgttaatc agagtgttgt tgaatgtttg agagtagtta 13140
gctagcttag agcttgcagc agtcttagca ttctgttcaa catccaagcc acctcggtat 13200
aagaaggact gtgcttcttc ccctataacg ttagacaact tttcacgctg ccactccgga 13260
acatccttct tgccgaattg ataccactta gcatttagct catcttcaac tacatctgca 13320
acagcatcac gctgctcagt acgttgttcg ttgcttatgt tgtacggatt tctacggata 13380
tcataagctc tacggaaaga tgaatatggt gagcggttct tcatacccga ctttatgttc 13440
attgccatag ttaagctgtc tttatccgtg aagtacatag ataacgatgt gtcatccatc 13500
atctgaatta tcttaagtgc attagtagca taaggtggta gctcttcttc cgtagtctct 13560
tctgggttta ggttgatgag agcctctaag ttgtctttga gacttggtac aggcattcgc 13620
tgaactctag accagtcaag cttctcttta aggattgcgg tatttgcgtc agcttcgcta 13680
ttaccttgtt ctactaatga cttagccttt tcagcccaag cgccctcaag ttgtttaaca 13740
tagtccttct tctctttcgg tgtgtactta cccagaagac caagaggtat ggcattctca 13800
tcagtgaaag cctttgagga gtcaatagct atattcgcag ttgtcttttg ctttgctatt 13860
gctgttgctc tctgcttctt gagagatgct actctttcct ttgagtaggt gttagggtac 13920
tgcttattaa gtctatcaat cttacgtaat gtagactccc aagatgtgct accacttagg 13980
gcttgtgtct ctatatctgc cattgtatca ccaatgacaa tagcgttctg cttagctaca 14040
aaagagtcat agtccctctg agctttatca agtgcaggta ggttcttaga ccaatctctt 14100
gtctgcaact cagcaagcaa tcggttatca ccatcacctg ctcttgctgc tgcctgatta 14160
acaagagcgg cctttaactc aaactcgttc tgaccaagtg acctcgcacg agctggtatc 14220
tcaacatcaa ctaagtgctt aacatctgca ttagggtttg caagtaggtc attaatacta 14280
atactaacag cttcactcgc cttcttagat tggtactctt tcttaatacg ctcttcagct 14340
tgtacaatag agaactgagc atcttgcact tgaagtgaca cagccttcag agtctgttgg 14400
tcggttccat acttatctaa gataggctgg aacatctgtg tcttcatatc actgatagtc 14460
ttctcatcag catctgggtt ctgattgtac cattccaaga actgattacc agcttccata 14520
gtatcatgct tacctacaat catgttgtag gctaactgac ctgccttagt tgcatcttca 14580
gtgataccct gacgctcttc tgctgtcttc atcaaatcct gagcagctcg gttagcttga 14640
cgtaccttgt cagcctctat ctgcttttga acctcagtgc tagctgctgc gtatccagcc 14700
tctgcgaact taccaagagc acctagtatt tgtgagcgag tgtctacagt cttagtaggt 14760
gttgcaacct ctctagtttg ttgttggaac cctcttcggg ttgttctttt catctcctgc 14820
aatggcatct taacctcctg acttaagtgc tggtgctgct ggttctaatt ctgccttagc 14880
ttgttgataa ccagaatatc cttttaatgc agcagaacca atgtttaaca gtccagccac 14940
agcagatggt ctgcttatag gtcttacatc catactaccc atagcaccgt aacgcatgga 15000
ctcagcttgg tctgctatgt tgtcatactc cgcttgctgg ttctgttgga taatattaaa 15060
gttactaaac ttagtccttt ctaagtcctg taactgactg gcaactgaca ttccaccaac 15120
accagaataa gctgccatta cattaatcct acccttttct tttaggtagt ctttctgcat 15180
tgataatgaa tcctctaacg aagcttgttg ggcttctagc tcttgctcag ataattcatc 15240
atagttagca atcgttgtct caaccaattg ctgattacgg cgctctgccg cttcttcttg 15300
tgcttctgca ttttgattag ctgcatacat agagtaagca gcagatgcaa tgctaacagc 15360
agcagtaact gccgccgctg tagtagctgc tgtagaagaa gcagcagccg caccacctgc 15420
tgctgctact gccatagtac ctccttagat acgtctgcct ctttgtttga acatcccttc 15480
ccattccata tcccgtagga tgaatggtcg gtaatcgtct gtaacaatct caaactgtgc 15540
ctgctgagat tgtaaacgta cagggaatga gaactgacct gccgctagtg gggcgaaacc 15600
gattcggtta ttccagctac ccatacggcg accattgtac tcatacctcc actttctgtt 15660
ctctgagtaa aggtcatata catgtacagt aatattaccg atactttcat agttgagggt 15720
tactttacca agtgtaaacc tatcaagccc catcacacga ccagattggt ctttgatact 15780
aggctgagtc ggtataaact tggaagtgta tttagtgcct acggtaaggt tacacgatgt 15840
tacacctcct agtacatccg ctaagtcatc atatgaccag aacttgttgc cgtccgtatc 15900
aaagataact gaagtacctt tatcagcatc ccaacatcct gtacctctga catactccat 15960
attcttcaaa tctgtaactt ggtatggagt agtccactcc cacctagtac ctgtccagtt 16020
agcagttaca actgctcgtt ggtcaagact tacagggaaa gtaataccgt catcatctgc 16080
gtcattacta ataggcattt gctctaggta tataccgttt cctctatcta ttacgaagta 16140
taccttgtcg cttacaaact tggcaaatag tacatctcct tcaaatatcc acttgtggaa 16200
agcagcctgt actctctgtt cgcctgacca taaccaattg tatgcataca ctatgtggtt 16260
gtttatgtta gtcctaacaa gaagtaagtt aatattagga ctagctatca ggtcaacagg 16320
tataccctca atgtactcag ccacatgctc tgtgattggt ctggctctct tagtatctgt 16380
aagagagtct gtgaacatct cacgaatacc tgcatactta cctgctataa aagagaacat 16440
aactgactca ccagtaactg ctggctgtgc atctatattc attggatacg aagtaacttt 16500
cttaaataca aggctatctt ttgtgatagg ctttgaacca tcaataagga attgaccatt 16560
ctcagcaaag aacactatgt caccctctag tatcgcgtga tgcaataagt tattaacttg 16620
gtctgcgtca gcgaaaacgt caatagggtc atctggtgac tcttcttgag tcgtctccct 16680
gaagaagttg aagtactcgt ttgtcttgcc gaacaccgcc gattcaccag aagtgaacat 16740
catcctgttc tggaacgtac caatactttg tatcgtgtta cccaagaagg atggaaatgg 16800
attagtcttt tcatcaccta ctttacgctc ttcccactca ccatagctca gttcgaaagt 16860
accatcagct aggctgacca attggtgtgg cattgtatta ggattgaact tgtacaatgt 16920
atcttgtgcc gtagactcaa cccaacgaac cttagagcct gattggtctg tcccgttatc 16980
tggtacagct ttcagccaat aagagttcgc ttcaaacccc tccttgttct gtaccttaac 17040
gatgtagttg tcaggtgcat atggtggtag gttagataac tgcctaacct tgtcctgtat 17100
agccaccaag tcatcaccgt tagcactatc aacagtggag atgctaaatg gcttaccatc 17160
aagcctcttt acgaagagtg tgttaccatg caactcagac gaatatggac tatctggtgc 17220
gccaactgct tcatacacaa cttcgataag gttgtctttg tttgagccag tacctagcgt 17280
aatcctatta ccacttatat tgctatagga gacatagcca cctgtagatt tgttgtataa 17340
gctaacaacc tttgatacac tcttagatac atcaatgtaa tatgttgttg atgagctgct 17400
gccagagcct ccagtttgag tctgtgtttt cacacgaaca ctctctgttg ttttctgttt 17460
tccaataagc ttgttgtgta actgctctgc aatatagtta gtacgtacct gctcaatcat 17520
gctagacgtt gcaccatctg gagtgtggac gctagatatt acatcaccat caattatgat 17580
ttgataatct ctaccatatg tcgcatactg gcaatacaca atagccattg atgggtttga 17640
atcagtcttg tctgttaatg tctccgtcac tacagatgta ttagctatga atgtatagtc 17700
agcaatagtc tttagccgta ggagtttctt aggctctgtc accttcaagt aactatctga 17760
aactccttta acagttactg ttctctcatt accatctagg tcgaatacac gaggatactt 17820
attaggctca acgagtatca aataacgctc tgagccacct cggtcgtact gataccaaag 17880
tgaatgaggg tctacattat caagaagttt cttaacgtaa cgagtaccta ctctcttcat 17940
caatccatat aagggggatg gtgttaagtt ttcttgctta gtacattgcc catcaatccg 18000
gtctttgtca gcctgttggc tgactccctg tataggtcgc tcccaactac cagtgattct 18060
tcccataact acctcacatt attatagcca ccagctctgt aaacagtatt acgagcggat 18120
gggttgtcaa acatattaga gcggcgctga cgagcgtcct cagactgtaa tgaaatcata 18180
gaatcctttg cagcttgctc taagttgcgc atcttattag ggtcgccctc tttattgttc 18240
tggaaccaga atgatgctgt gtctgttata gcttgctttg ctgtagtagg tagtgtgtcg 18300
aatggcaagt taacaactaa ttcacaatga agcttaccgt cggtgtctgt aagattacgc 18360
atatcgtagc catacgactg agcatcaaac agcttattac ctcgtaagtt aacaggaagc 18420
ttacgacctc tctgctcctt aactaaacaa actaaggtgt tctgaggtac taccacatat 18480
ccattaacag ggtcagggtc taacttgtgg aactcttctt tattgaacca ccacccttta 18540
cctcggttat gttgaataac ttgagagtat ctatcaagca tcttatccgc atcggaagca 18600
tctatgttcc aatctatctc ttcttctgag ttgataccaa cagaacctat agagtcaaga 18660
actgcattga ttgcatctat cttagtgtcg ataccaaata ctagattgat gtccatataa 18720
ctccttagtc tcacgctgtg agacaaaaat aagcccctac caaagtaggc aggggcaggg 18780
tgttaattat taaccagcag caattactgc tttaacagca gcaacgaatg cagacgcatc 18840
acctgttagg tctgctgact gtgttacttg tgctttacgg gcagcacgtt tgtcaagacc 18900
tgtctcatct gcgctagctt tgataagaag acctagatgc tcccaacggt ctgggattgc 18960
accttctgac atgtagctgt caatgtacca agtcttgtta cctttgttcc accagatttc 19020
accagtaaga gcaatcgagc gaccaacaag cagtgcttcg ctaccgaaga taacagccgc 19080
agctttctct tggtcttcac catcagtgcc tgttaggaca tcgtaacggt agctgttgtt 19140
agcattcgat aggatgtggt gctctgacac ttgaccttca aggtctacgt ggtcgcggtt 19200
cttgttaggg aactggttgg tcggtacaac aggtacgttg taagacttaa gcacgaagcc 19260
agtaactgtc tgaccttgag ctgtgttata acgagcattg cagatacgct ctgcgtcacg 19320
catcgcgttg aactcaggcc aaggcatgaa gattgttagc ttacggacgt caacgttctg 19380
tagtactagg tcttcaatta cccactcgat acctgcgatt agcttgttag ggctgaacag 19440
cgcaccgtct tctttgattt tcttaacaat cgagaagccg tgacctttaa cacgaggttt 19500
ggtacgctta gcttttgtgt tgttgattgc accgaagatt agctgttgaa gcaacatgcg 19560
gtcttcaagg ttcttaagag cagatacttg ctcttcagca agtttacctt taaccatgat 19620
gtcatcttga acatcatcaa gaaggccaac aacgttacga gcaataacag ttgtgtcaat 19680
caatagtgag tttttatcga actccatctc accaccacgg acatctttac caggtgccag 19740
tgcctgtact tgggttacac caaggtattt gttagacacg atgttagtgc ctgtcacttc 19800
ttgtaagtcg aagtacgaca taagacccaa ctgctcatgg taagcgcgct taatctggcc 19860
tgtgaatttc tcaaaagcca gtgacttcgc ttcaccagag gctgatactt tagggtctac 19920
taaattgttt acatcactca tgtagtgaat ctccttctaa ttattacgtt atctataggg 19980
caacttaatt aaagattgcg tttaagacca gcactacgag ctgcgtctac ttttgattgg 20040
tactcaaaat ctgtccagta tttgtcacta tccatgatag tttggaactc atcagatgtt 20100
agatatccct tgtctagtgg agtaccagta ggtgtactgg aagtatcatc accaagtagc 20160
ttgatattct tgtcaccatt ctgcaattta tcagacaact ccatctgtgc tttcacagtc 20220
tgaatgatta gcaactgctg ctcgtggctc tcagattcca tcactgcatt gtacgcagaa 20280
atctgcttgt catcaaagtt attcaagatg aagtcttcca tagcaaccag accttcttca 20340
ccacctacag tagtcatgaa ctcttcagct tgctgcttgc tagtcatctc ttgctgctct 20400
ttgtcagcag cagcagcttt aatagcttgc tcgttcatac ctttgtacat gttcaagtag 20460
ccatcaacca gcgtcttgcc gaacttagac tcaagcttag tacgtgtctc ttcgtctaaa 20520
gaaaagtcac cgtccttctt gaatagttga gacagaagag ctttctcatc aatctctgct 20580
tctttcaaag cattgcttac ctcttctgga acttccaccg aaacagcttc accgttaaag 20640
aagtactcag gttcttcacc agagtcttct ggctcttcct gctcagtctt ctcaggctcc 20700
gtggcagcag actctgcatc cgcatcctcc gcaggtgtag aggcaggtga ctcctcggca 20760
ggttctacag gttccgttgc agtgctctgc tctggctcct gttcttgctg ttcttgcttt 20820
gggtcttctg gctctgtctt accacccgtt aaatcgacaa catccgtaga gttgtaatca 20880
ataatttcta catccattat ttacctcttt gcgtcaggtt gttagctact tgaggcattg 20940
cattagcagc gccttgcatc atcatttctt gttgctgtgc ttgcatacgt tgttcttgtt 21000
gcttctggta ttgctcttct gtcataatcc aaggagtctc aagagacaac tggctagcgg 21060
cagagcgcat gtagtcagcc cagagcatac gctcctgtgc ttgcataggc catgaagcag 21120
gtagattcag catctcagta aactgcatca gcttgtcagc atctgtcatc ttacttagtg 21180
cagagatacc tgtgataaga acagtgttaa caagttcttt aggtaggtcg aaaccaatac 21240
gtcgaagaag caagttgaag taaggacgtt gtagtgtatt agcaagtaaa gagtacgcac 21300
cacctaagtt ctgctccatc tcgatagcat cacggcgaat ctcgtaagcg gtaacacgct 21360
ctgcgttacg ctgtactgct gcacctagca tgaatgctgt gcctagtctt cgctcatact 21420
tatctagtac gtttgcaata ggggtgaagt ctgcatactt ctctaactga agtactccaa 21480
tgtcatctat gttaccaaag ataaactcac cagttggcga ctcaatgagg tggtcaacat 21540
ccgtcacaga cccaggtttg ataaggtact tgatatctgc catgagaatc atacccttac 21600
ctagagcttc tgataggaac tgtaccatgt ggaagtcatt agcatattgc tctaccagag 21660
agcgaccata gtcttctcca tagtttgtct tccaacgtag tacaataaac ggaaagttct 21720
caggtttcac acgataggac ttacctactt tctcacctac aacttcttgc tcgatgtgat 21780
agaagccttg cttaaggact gcacgagtgt acagcttaac ttcttcatca tccttttcct 21840
tgtacttgtt tgaggttcgc atcttagcct taatgatagc ttgcaatgct ggctcaaatg 21900
tatcaatgcc cttgctttct tcgatgatta gttccaatag agtaccagat ttatctcgtc 21960
tattaacaaa cctatccaat gggtagttaa cgagatttcc ttcttctggc atgtacaagc 22020
aagtagtacc acctatcaac aggtgctcca ttgcctcacc taatccgaca cgcccagcta 22080
tatgctcgtg ctccataaga gcagcttgtt ctgcttggac taagccacct tgcgcttcga 22140
ttaggtcttc gtcaccttct cgtaagtgct tcttagcttc ctgtgtcagc tccaacctag 22200
cgaatgtgct atgtggagga aacagtgtca tgatgagttt gttttcgagg ttgtttacac 22260
actgtgcccc gaacgattgc cagcctgttg tgttagtccc agaaccctga ttctcgctgg 22320
ctacttcagg gtaaaggtta ggcaaggtat aagtactgaa ctcacgagca cgagacaaga 22380
agggctcacg ttttaccttg agcttctcat agcgtttctg tattgtattt tcttgcttac 22440
ccttgctatt ctgtttatga cgtatacggt tctttgttag gtcaactgta tcatcttctg 22500
gtaggagttc gatgttcctt gccatagaac ctcctatacg tttagtccag tacctgtagc 22560
tgtacgtgga cggcgtaatc gtttacggcc ttgagataca tcacctgtat caggtgtacc 22620
gagctgaata tcttctggct ctacatctac ctgacgctct ggcttcttag ccggagtctt 22680
ggcttctggg atgtccggtg aactcgccat tacttactcc ttacttgttt acgaaattga 22740
caacccacgt aggagtaccc tagcttctta aagaagcctt cagtctcttc ctcgtggata 22800
ccacttgcaa tactaatctg cacttctact gcaccctgtt catcagccca agcttcccaa 22860
gctttcatta gcttgtatcc aatacttgtc ttacgcttct caggtactac atatagtatc 22920
gtgtcaaaag caagacgggc tttgctccaa ggtaaagtat gacaccagcc ccaaagcagc 22980
cccactggct cgttgtcatc ataagcaagc agtaaacacc catcttctcg ctgtacggtt 23040
atcgctgcat tctgcatagt gtgctcgata tcgacaggga agttgttatg tttagctgcc 23100
tcttctgcat attgatgagc tagtggggca aaggctagta agtcaagcag agtagcctga 23160
cgtactttaa tcatggttcg aatatatcct ctatctgttt cttcagattg cgcttcgtgc 23220
tattggcaac cagcactcca taaggtgtgg taggggactc tgaatcttct gccaacagtc 23280
ccatcaaagt atcgaaagct tcacgcccga tacgcttctt ctgtggtcgc acagagttac 23340
tatctcttgc gaggtcttcc gctacttcca agctacgatt catagcatat ttgatatcgc 23400
tagtagggtc atacctgctc ttggacgtaa tgtgcgagaa tctattcgtc atcgaaatcg 23460
tcctcttcat ctaaggaagc tgctacacat ttctcaacag ctccgattcg tattccatga 23520
ttatgagctt cttcctgcaa gcttacactc acaggcaatc catcttcaat gcgctcatac 23580
aattcatcga agacttcctg tttatctcta ggcatgatta ttatcctatg ttatctatag 23640
ggcaacttaa ttaaaaatcc ggttcagatt tgtcgaatcg caagtcaccg actttcggca 23700
gacggataac accgttcttg ctagaaggct gcaagccata gactcggaat attttaccga 23760
cagggccaca aggcccactt acagggcaaa tcaagtcagc tacacggtac atctcttctg 23820
cgtctttatg agtccaacct ttaccaagca ttgccttaac tgtctttcca cctttgtaac 23880
ggaacagtag gttagcaaca agtcctttgt atttaccagt gccttcttcc caaccaatgc 23940
actctaggtc tacttcgtac cgacgaacaa ctttcatttg atgccagtcc ttagcaccag 24000
ctttccactc aacatcttgt ttgaacacag caccttcatg acctaactta ataagtcggt 24060
cagcgtactg catcaactct tccacgttat tcacaacgtc aatatctagt acgcaatctg 24120
tgtcatacag ctctgtcata agggcagaat atcgctgtgc aaacttacga tgactatgcc 24180
ctgagatgaa ctcacagatt gtaaccatat caaagaaagc gatgtatagg ttgtgtttaa 24240
tctgcaactg cacgtctgtt tcaagaggct tgttacggtt tgggttgagg ataccagata 24300
gtacttcaag tgtgtacgca cagtcagcta ccatttcacc gaagtagaca cctgcatcaa 24360
gacctagctc aacaagacct tcatagtaag ataccagatt ctctgtgttt gcgagttgct 24420
caccagttct accaaagatg ccacacttac catcagcacg aacaaccagc atactgaaca 24480
cgccgtcacg cttgcattga gcgtacattg gaaacgagac tttcttatgc aagatacctt 24540
cttcataatt cttgttcacc tctgcgaagt gctttactag ttgaacaact ttgtttttag 24600
gtcggtagtc tgctggctta ccaagaaatt caaatacgtt cattataatt gctccaagat 24660
ttcagatact gctagagaag gtaatccttc ctgcaatgta atatcaaagc tacggcagaa 24720
gtctggatat agataccgac gactgtcatt gccatatgtg taaccatctc gatgtagtcg 24780
gacaactgtt acattaagcc ctgcatcatg aagtgctctt acttcatcaa tgaatccacc 24840
atcactgaat acaacatcaa agccagtctc atgtgctaac atgcaagctt cagcagctcg 24900
tttaccaaac acatcatctc catacaaagg cttcataatg tcttcagaaa tgtgaatcat 24960
gaactcacga tatgacttac cgccaagatg ctcttgtggt ttctccttct gctctcggtc 25020
attgtactca cgcatgaacg actctttgct cataccagac atggcaagag caatattgaa 25080
catcgtgaac ttgaactctg taacagcaaa tcccttctct tttaataggt ttgcaattgt 25140
gtccttgcca cagaaaggtg gcgcatttaa gataactact ttacgcatgt aaactcctaa 25200
tctcataggg tagcttgttt gcgatgcaat aagtatgtgc taacatcata cctgttgacc 25260
atccgcaatc tgtgtagaaa acagtaagta caccagcttg atgtttccaa gcaaagccag 25320
cttcaatacc tagctcacgc tcttctggta ctgagtcgtc taatacattt ggctgagtgt 25380
acagtaagtg agatgcataa ggtgcctcac ctcgaagcaa tgcatcatgc atacaacgtt 25440
gagcatactc aacgtgagcc tcttctgaat acttagaaga tgctttaaaa ggtgattcaa 25500
taataacagg tttcatcata tactccttca gttgcaataa ggataggctg tctcacagcg 25560
tgagacaacc catagtttat tacagcttac ctaaccaacg accgtcctta ccaagacgca 25620
tcggaaccaa tataggcagt gagtcaacaa ttacagaaca gccaatgata ggcttgtgct 25680
tgaagttctc accataggca aatgccagtg agccaccatc aatcaagcag ccaccataca 25740
tagcccagta caaggcagcg cgagatgctg agtagtcaat agagtactta gcatggaagt 25800
gaccgacaac taggttgcaa cgctcatgag ctgctgcgtc tagtaaatta ccgctagctt 25860
gatgctggaa ttgtacctct tcaccattag gcaaaggtag acggtgtgaa taagcccact 25920
cccaaccttc gccgccaccg ttagggaaca agacttctcg gtatgactta atcatatcag 25980
cagggatacc gtgagtctta gctttacgat gcagcataga gccgtggtta gagtgacaga 26040
ttttcatctc tgggaataca cgctctaatt ggtgcaagaa ctcacgagcc ttaacaagct 26100
ccatccctgc actgtctaag ttagggtcag agtcgtgata gctcaacgca tggtggtctg 26160
tctcatcacc taagttcacc acagtatctg gcttgtactt agctgcaact tcaatcaaga 26220
agtcaagagt atctgggtga tggtaaggtg catgtaagtc agggataact aagatgcttt 26280
cgtgcttagt ctccgacaca ggcttaagag gtttgtcatc taatggtgta ggcttacgca 26340
gcttacggct tagtcgtagc tgacgattcg ctcgtgttag tgtaatgtag aactcaccgt 26400
tcttcttctt ctcttcgtac tgcattgacc agtaacgagc cagctcaggt gatacctcag 26460
taccgtgact gttatcactt aagatagctg acattttctt gaagtcaata gtaccatcgc 26520
ttttagcaga ttctcgaata gcattgatgc gctcttcgtc tgtaaagcgg ttccaaatct 26580
tagcattgct tgccattatt tatccttctt ctttaatgca taacgcttac gtgctttagc 26640
atttcgctta gcacgagcct ctgctggtgt tagatgctct gggtgaatcc actcagtctg 26700
cggaacttga tgcagttcta agtagtcagc cataccgcgt agcatgttaa taatgtcacg 26760
cttgcttgtg caacctcccc atcgaataca gaggttggtt agcttaccct caagaccatt 26820
gattgcacgt ggcaaggcag cgcggataac gccgctcttg tggtcgtggt caactactac 26880
attagtaggt gtcattgaac gtaggtcacg accagtgata gggcacttgc cgttctgtcg 26940
taggataagc tccttcttga tattaggtag gttgtctggg ttgtccttgt gctttgctgc 27000
acgagaacta attgctttac gcagttctat catgccaaaa ctccttatca tttccgtaga 27060
tgacaggaga tttactacgc cagatttcat taggaaaggt ctgcatccaa gcaagacggc 27120
cttgctctag catacggtca tacgctgtaa ggtacgctcc tttccctttc cagaaatccc 27180
aatcaggtgg tggtgcatta taatgcaaca cgtagttatc atagtactct tgcgtaccac 27240
ggaagttagg acaccagtgc ttgccagttc catacacttc cttatacgca cctagtacag 27300
cgtagtacat ctccttctct gtcttacagt tatcaaggag gttataagca aaggtcatgc 27360
ccttaccttt caagcctttg tagttgtctg ctacgtcacc cataataatc tgggcataga 27420
agaactttag tccagtacct tttaaatcgg taatggatgt gctaggtcgc ttgccaacaa 27480
gaacacgttt gaccttctcc tgtcctgcct tagctccacg cttccagaac tcaccagtac 27540
ctacgtactc atagtgattc acttctttat tagagaactt aggattcaac tcacctaatg 27600
gattaacaaa gattagcttc tgagtatcgg gattgtagtg ccaagtaggt gtgattgtac 27660
tgtctttatc aatagagata gtaacagagt cacatagttc cttgtgcatc tcagagccta 27720
gctcaacccc ttgctcttca agtaatcggt atcgtctcca tgcttcaatg ctaagctcat 27780
catctgcttc gttaccgtca gacagcattg ccttaagtaa gaaaatcatc tgtgtcttaa 27840
gctcattaaa gaatggtggt ttatcatcag gtcgctggcc tttatagtca gtggtgtatg 27900
ctaagtctag tcggaagttc ttagcagagt cagtggcata cagcctagca gagtcgcaac 27960
ccgcatctct aatccactta ttcaaagttt gacaaaggct ttcaaatgca gattcaaact 28020
gtggagtatc ttcaatgcat ttgtaatgtc cacctttgac aagcccctta gcagcaagct 28080
cttgcgcagg gtcaataaca aaagctattc ggtaaggaag taggtctgca tctactagcg 28140
cggtacgagt gcccgtctca ggccaaagga tgaagtgctc ttccttctct gctataccac 28200
cgccatagtc aaacccttca tagtatccta tcgtcattat aacctcctaa tcaggattaa 28260
caaaaataag ccccaccgca tctagtgcgg cagggcaagt gacagagagt tattagaact 28320
cttcgtcttc agctacttct tctactggtg cttcagcttc tactgcttct tctgcaacag 28380
gtgcttcttc agcagcttca gcttgctctt tgtacattag gtacgcagct acaacgttag 28440
ccttagctac catcgatttc tcagtagctg tatcaggcgc tgttgcacga gcgaactcta 28500
gaccttctgc gtaatactta agaccgccta gctctgcctg agcttctgcc tctgtcttgt 28560
gagctttcga gcctacaaga ttacctgctt cgtcaacaac aacgaattgg tcaacgaact 28620
taccagcgac tacagtttga agtgatttga ttactagttt agacatgtta atattcctta 28680
gccttacggc agttattaaa gagattcagt tatttatcta tagggcaact taattaagcc 28740
gccctaattt gtctcacgcc gtgagactaa gcgatgtgct taacacgctt tagggtgtga 28800
atagggtagt cgtagttcac gcgcttacct tcttcattga tgaagttaac acaagccata 28860
ccatcacggt acagtaagtg taactcaccg cagccttcac cttcaatgaa gtgagcacca 28920
cggtattgct tagcttgctc ttcggttgtg acatcgttgt catcttcctt agcaagacgc 28980
gcagcaccag ctcgtgctgc gtcttctgga ttcagaatga tttctacttc gctacgtttc 29040
atttagaagt accccgctac tgcgccgatg aatggaatga atacacctac tgcacgaact 29100
acagtctcac tagttgagtt ggtgtccata tctgctacta ggtcatagac gttcatgcca 29160
taacctacac ctgccgcaat aactacggcg ataataagca gcacaagaag tgctgctact 29220
gctttcttag aattcatctt aaaactcctg ctcttcatct agttgctctg ctggctcttg 29280
cggaagctga ccagcgccat tgccttgctg ttgattgcca gattcttctt cgtcatcctc 29340
tgccttgtcc ttagacttag cagtgtaacg ctctgggttc tcatcgtaga ttgattgaat 29400
caactcttgg caagggtgag tacctgctag gaactcttct gtttgcatca ggatacctgc 29460
gaactcacga gtaggatgca agtagtcaag tgcctctttg gtcaattggt cttcacgtag 29520
gaagccaata gcattatcaa gaggtgctag ctctgggtct gcatcaagca tcttacgcat 29580
acggtctgtg attgcagcca ttgacttgaa gttgatgtac ttaggagtac cgtcttcgtt 29640
cttgtcttta ccgcctgtta ggttaacagt ggtcagctca ttaatcatag tgccgaagcc 29700
tttatgacga gccatgccac ccattgcagg gatgaactta gcgtgcaaga atgacttgtc 29760
gcctttctta agagggaaag tcttagtgaa gaacataggc tcaccgtcat caagcttatc 29820
attctcacct agtaggtgga agattgccac agcttgtgga gctgctggtt tcttctgacc 29880
tttgaaagtc tcttggaatg taccaagacg caatagaccg aatagacggg cttcacgctc 29940
accctcttca gggttcttgt atttactaac tacctcggct acctcgccac catagtcaaa 30000
atcagttgca gacatttgct actccttctg tttggatgaa ttaattatct atagggcaac 30060
ttaattaaag cttattaagc tcttcaattg ccttgccagc cgcttcttta gtagcgttag 30120
cacgttcgtg accttcatct agcttatcag cagctcggtc ttccagcatc ttcttaagaa 30180
cttctgctgt ttctagacgc ttgagatata gttcatcttc tacgtcgata cgctctgctg 30240
ctagctccat acgtgtcact tgcttattga ttgcataggt acgtacacgc tctgctagat 30300
tgctgatacg ctctttagct gctagtaata ccttaactac tttatacatc ttatactcct 30360
atttatacta tacgctctat ctatagggca acttaattat atgtagcctg tgagacaaat 30420
tgtacacagg ctgtgagaca agttaatact taggcttatt ccaatcatct aatagagaat 30480
agataattag gaggaaagaa gccagtagaa gtaggttagt gggtgtcatg ccagctatct 30540
cctatcatgt attcacctgc tagtggtact cggaacttca agaactcacc tgctttacgc 30600
atgttatctg ctaggatatg tcccgcacga tgataacgtc ttcgaatagt aaggagtcca 30660
gttccttttc cactgacaat ccttggtgct gaccagattt gtccgtcatc atccacgaac 30720
tgagccttct cttctgtctt ccagtcttct tctttaatct cgtaaagttt ctctttaact 30780
tcatcggaat ttatctccat ctgccattca tcgtgcacat ctgctagcca acaaggatag 30840
ccgttctcat caagtcctat gccttcttcg atgagctgat tctctgccat acatttactg 30900
tatttcatag acaaggaacc tgtcatttga aggagtacgt taagtacagt gtgtattaga 30960
atcttgccac tctgcttacg gatacgtccc caacgaccat caatcgcatg aaggtatccg 31020
taacgctcac ctgctttctc acaagccttg attaagttag caagagaagg tagctctttc 31080
ttgaatcgag caactcgttt ctccatctca cgctcatcaa gaccacacac cttagccaag 31140
ttcttaatac ctgagccgta caagaaggca tagataaatg tcttagccat atcacgaata 31200
ggcaagccag ctaggtgttg gttatgtgta tggatatcgc cattcagaac aatctctgta 31260
tactcagggt cattcatgaa gtgcgcaagc atacgtagct ctaaacctgc accgtcacag 31320
cccagaatca tcttaccttt accagcaatg aacaggtgtc tcagcggatg caagccacga 31380
gaaggaatgt taaccacata cttatgacgc atacggaagg tgttcgtacc aatactaaaa 31440
gcttcagcag gtacacgcca ttcctcatca tagtccgaag ggaactctcc catgttctgg 31500
tagtactgcc acgcttcgca gccatgctct tcattgtaag ccttagctaa taagccttta 31560
cagtgtctac gcccgtcctt ctgcttcggc cactctccgt tagcctcgaa gtactccatg 31620
tctttgacgt taagaatctg agagcgacga gaacgtagta cataccagtc aactatatgc 31680
tctagccact gaggtatctc accatcacga gccttccaag ctttaagaga gtcctcatca 31740
atcttaccag accaaggctt aggtggttca cctgtctcgt tcttgtcatc gttcatccac 31800
ttctcatcat tctctgagaa gtttacaccg agccaaccac gagggtacag cacgttctct 31860
ttgacgtact caaggtttcc taatccaatg tcttcgaaga cgataggagt ataagcacca 31920
gttaccagtg ggtctttggt atcatgagca tttccaacca tctctgggaa gtctttctta 31980
acagcagcag tgtagtcacc cttggctgta gtcaacttcc agatagttgt tcgcttacct 32040
atacgaggct ctgacttaga gatgaagtag ttctgcaagt gactgcgtaa ccacccaaca 32100
tcagcgtcag ggtacgcctt agcgaacgat gtacacctat taagaatgtg actgttcttc 32160
ataggttctg tcttgacacg aggtgggatg tacggttcaa tcttcttgaa cacctcatcc 32220
atctctttac ctatctgaac ccaatcttcc caagcctgtt tcatatcaag acggaagccg 32280
cgctgagcct gacgagcaat accaagagca acctgtgtct ccattcgtaa tgctgagtca 32340
atgcctagcc ctgtacgtgg gttgcgtcca cggcgtagat ggtcagccca atcgttattc 32400
atgagccaga agaacatatc tttaccaatg gcagtatcct ctgcacatcg gtcaaacacc 32460
atgaagtcag taagctttga ccagtcctca ttacatggct tgtatcttcc gatacgaata 32520
ccgtgagcct cgatgctatg agcacctaca ttacccatgc ccatagcgaa tgcttggggt 32580
ggagccttac ggtctgggtt agttagctga gagatgagca ttgtatccat caacttaaga 32640
gggaagtagt cagagtgctc tcgcttctta ccacgacgct ctaagtaatt gaacttccaa 32700
tacttaggcc aaaccttttc gaatgccagt ccgtcatagc caacgcagtt ctgcatgact 32760
aaggactcac actccatcag catatcaaga gcgtcaacta agtaaccatc ttgcgtaccc 32820
tcaccgccta gcatctcacg agcgtttggg tcacgcttct catacgggtc aaagaaaacg 32880
aactcttcat cagtgaatgc atcacggata cagatacagt gcatagagct agggtcatta 32940
taacgaagct ctttaagcaa gccgacaccc tccgcatcgg ctactaagaa gcgacctttg 33000
ccgctcggtt tatcagggtc gcccttcggg tacatagaag ggtaatgttc aagggacata 33060
ccgcctcctt acttacggat tatctctgct tcacatactg gcttagcctc agccactagc 33120
ttaaggatac caacgccaca gcgattctct tttgtgaact ttgatgcagc aatctctgct 33180
agtcgctgag tcttgtgcgg ttctccaatt accttaccac gatggatgag taggaactca 33240
cctacttcat ctagtgggtc agtgttcttg tcggatacaa tagatgtgta accactatct 33300
tttgcattag ctaaagcttt gttatcagca tgtacgttgc gctgcttctg cttaagtgct 33360
gagtgcgaga caacaaatag tttatattta ctattgtatc ctttaccatt gaagacacct 33420
ttcattgttt cgatgtcatc cgtgcagata tgaaagaact ctaggaacca gtcccaagaa 33480
tacttctggt tctttccatt atctacgaag tatacggtaa cgttacctcc aagagttgag 33540
atgattttac cagcccaacc ttgacgactt ccgccgccgt tgtgaacaac tcgcatacca 33600
ttcttaaggc acttgcgcat ttgtttatgg ataggtagaa atttaacagc catgatatta 33660
tcctcatatt atagtagaaa taatcaagag ctgtctcact ctgtgagaca accctttgtt 33720
attaccactc gttagcagct ttgaagtatt cgaacatcgc tttcatcttg cgaactgttg 33780
tcaacttagt aaggtctttg taagagaagt gatagtcagg gaacttacca ccatgcacac 33840
gcacatgaag ctctcccatc aactgactgt gatgccagtc aatcttacag tcgaagaact 33900
taccgcccat ctgagccaag tccttacgaa tcttagtatc cgtaatgaca tcaagcatat 33960
aagaagggac gctcttaata cgtacattga acgtctgtgt cataacttct tcgaacaata 34020
cagcttgagc atgtgtagtt gcaaagttag ctgagtcact tgtgacaatg acttgagcca 34080
ttgcatcacc ttcgcaagcc agctctacca catcatcgtg gctcatagac ataagctctt 34140
ggtagtgttt aagttcttct tgcgtcatct tgtctttgtt gaacatgata ttaatctcca 34200
aatgaattat ctatagggct acttaaaact cttgtgtatc atctacatcg aaaggtacat 34260
cttctggcaa cgtctttggc tctgactctg gtatcgggtc actacgtcta gagcggtcat 34320
cacccatgtc gaagttgtca cgctcccttt gtcctgagtc attggattgc ttacgaccta 34380
cctctggtag ctcatgcaca ccttcgagtt ccttgtactc accagtgcgt atatccttct 34440
cagccactac agtactacct accatgtgac cgatgcctcg gttcttaagg ttacggtaca 34500
gggtaatgca cttgttacgg aaggttgaag caactgtgtt acgctcgatg ccccatacag 34560
cgttagccca gaaagtaatt gaaccagcac cacggaagtc agactcgtac acctcaccac 34620
ccatagtatg tgggatacgc ttacttgcat ccactttaac aaggtgacta aggagcatga 34680
tgttgacagg gttctcatcc ttgaaagtac caagacgttt catggttgca tcaatagctt 34740
gaacaccttt gttaacatta cccttctcat ccttgtgctc gaatgcagtc aggttatcga 34800
cgacgaagta ctgataccct aatgcgaaac actcttccat gacttccatc acagcatcga 34860
catccttaga gccttccaaa tcagcaatga taagacggtc ttgctgctct agtaattcta 34920
gcgcatcatc caagtcttgt tgagtgtagt cacgggcagg gttgtactca taaccttcat 34980
caatctccca ctgctcttgc ggtggtgagt tgaagtcctt gttaactagc atacctgcaa 35040
aggtcttcgt cacttcatca atctggttct caagatagat aacaacaacg tcatgaccta 35100
gcttcattaa gttagagaca tgagccatag ttgtatctgt cttaccaaca ccagtacctg 35160
caccccacac tgacatgtaa tgcagtcgga taccataggt aatcatattg aaacccttaa 35220
gccaatactt ctcacccatt gtggtaatct cacgagcctt ttcctttatg tcggatactc 35280
gcttgagtct tgcgttaggc ttcgactctt ctgcattgaa tacagcagtt acaaactccg 35340
agccacgacc tttcttgagg cagtcattag ggtctttaca acctgctggc attactagac 35400
gcttagactt cccacggaag agctttgatg ctgccttgtt caaggccagt cctgtctcgt 35460
cattgtcgaa cgcccagatt actgtcttga actggttgat atgttccttg ttatcaataa 35520
gttcttgcac acctgcttca cccttattga ctgaccatac atggaacagc ttaagacctt 35580
ctaagtcctt acgacctgcg aagttctcta gtttgttcag ctctttgata agcatctgct 35640
gagcagccat agcgtcacac tgtccaccag tgatgataag catgttcttc atctgaccgg 35700
actctgcgac ctgcttcaat gtgtgcatac cgaacagctc ttgtttaccg aaccgcttac 35760
ccaaatgacc gaagcggaaa tctttaggga tagtacggca ggtcgcaccc atcagctcac 35820
cttcctcata ctgagggtag tagtgacgac atatcttacc cttatcatcg tgccctactc 35880
ttacgttgta gagcttagcg ataaggccat gtatgccacg gtcaactagg tgctgcgtct 35940
tcagaccatc gaaccactgc atctctagtt cccactcagc aagctgagct tcacgctctt 36000
cgtcgttcaa gacttcgaag cggtctttct tacgcatacc agagagagca agctgtcgca 36060
ttagtgggtc tttcaacttg ccagatgctt caagttcttt gaagtcacct accgagtact 36120
ggatatcacc agagataggt aagtctttaa gagcaggggc agagccagcc tgctcgtagt 36180
acgtattgtt atcgctgtgg aactgcttac gataacagaa gccaccacca tctgcgaagc 36240
gcatcaagtg gtcgccagtt ttgtcgtgcc cactttctct acacttagga cacggctcgt 36300
tacggattat atcacccata tctactcctg tctcacactg tgagacaaat tagttacacc 36360
aaccgcgctt catttcacgg atgcgtttac gattacgcga tgagtttgtc tcatgcgcct 36420
tcttgtgatt gtgcttcttt ctaggaagct tctcatctac cttagacatt attcaccttc 36480
gaatgatagt acgtcttctt ctttgaatgt aattactgtg ccacttgctc gtagctctaa 36540
cacgttgtaa gcagagaagg cacgatagcc tttgccattg gttaggttca cactgattaa 36600
gtcatcttta tgagcgatag tagactgacc acctttcaag tctttcttaa caccagtacg 36660
gcaggtcata gtcatgaagt tttcttcgtg tacagaaacc ttgcagtaag ggcatttctg 36720
tagttcttca agacgagcct gagctttctt ggtattaaga taaactttat cgccaacctc 36780
acctgcacct acaacattgc atagctcttc aataatgaag taacgcttag gcgtcttgcg 36840
cttagcaatt acagtgaaaa tcttaccacc gttctgacgg atgatagcct taactagctc 36900
agcgcggttc ttgtctttaa tacgaatagt catgatgttt ctccagttta attaatctat 36960
agggcaactt aataatatcc gtagaactta cggttctctc ttttgtattg aatctgatgt 37020
ttctttatct gattctttac cagcaaatat aaagaagcta tatctaactg ctcctctata 37080
acataacgag ttaagcatac atctttcttt atagtaaaag gtgtatagat atctagtatc 37140
catgcaccat accaaggctt atagccaaca tcaaaatata attgaataac cataataact 37200
ccttgtctca cactgtgaga caactaatgc atcttgttaa gaagagctgt ctcaatctca 37260
gcgtcttcta ctgattctaa gtactcttca tataaaggat ataagtcacc ttcttctggt 37320
aactcgtctt gatatagctc tagccagtca ttgaacgata gcttcttaat cataatcaga 37380
atctcttgtt agtgtgttta tgcatctctc ggttatactt cttcctaatg caattaataa 37440
aacttgagaa gtgtaaagcc gttactggct tacctgtctc tgggcttatc catttagcag 37500
taaagcataa gttacttctt cttataggct tcatgattaa atcctcgtat ttataatcta 37560
tagggctact taattatagc cgctctagta gctcttattc tttcttattc tactaatgct 37620
ctatcctctc tctagccgct ctcctatagg gcaacttaat aatatagaac gagaaaagcc 37680
ctgactcaat taagaatcag ggcagttagt ttaagcaaaa cagtacaagg atttaagaac 37740
ttcttgcaag tcaagctctc ctttctctgg tatagttgct tctatttcag taaggataag 37800
agcttcatta tactctatga agtctttgat tacatcattc tcttcataca tcagtacgaa 37860
agagccagaa agatgcagtc taagtagctc agtattacct gcatgagtac caaaagagtc 37920
atgaataaca gcaatgctag taattccgtt gtcttcgaag gcacaaacag ctagtactaa 37980
gtggcttgca tccatactat gaacaaagtt aggagcggca gagctagcca ttctgcgagg 38040
gtctatcttg tctgtacgta ctgaggtacg gaagaaagtt tcacctagca tctgtgtctt 38100
gatgcgctta tcttctttta cttcataaac agcttgttta acgatgaatc cagtaggagt 38160
catccactca agcggcttgt tatccttagc aatagcagag caaacttcct taatgtactt 38220
catacctgct cttgcagcta ctacaacctc accgattgca gcccaagtaa gcatagaagc 38280
gaatgaaata gcatcacgta gtggcaaatc accatcagaa tcagtgaatg catggatact 38340
acccgctttc atgcctcttg cacgagcctt attgtctgct ttctcttgca aatctttaag 38400
ataatcagca atagagtcac gacaagtgag ctgagagcta ccgtaaggaa gtgtcataac 38460
tggcttctta gtcattgaac gagttacacc aatacgtaac cactcttcac agaacttgta 38520
agccatgata cgagccttgc tttcaaggtc gccagttatc ttagccttct cgaacttaga 38580
tactagtgaa tcaataagct tatcgtagat aggacagtca cgctcatcgt tgttagtgat 38640
agcttccatc cactctacaa caactttaga taccgcaccg tagatatctt gaggcttctc 38700
agatggtaat aagtttactt ccttaccacc tactgaatcc cgtagcatag ccgaataatg 38760
ttggatacca gaacaactcc catccatagc cacagcaatg tgactaggaa agtcctgttc 38820
gttattccct tcatcaatcc aatcaagaag tgctgcgtac tcgtagcacc aagcaaggaa 38880
ctgccaaggc ttatcagctt tcgtccagtc agtgaaagta atcgggtcgg ctgcaatgtc 38940
aaggcacatc tccttaaatt cttctgtctc acagcgtgag acacgttcag cgaaagtctc 39000
tttatcccaa ccccaaacgt ttgcaccgtg taccttgaac cagtacgcgc cagtgctgcc 39060
tagtggcata gagtcagcga atcggataag agccttctgt aagtcaccgc cttgaggcga 39120
tacgaggcta gaatgacagt aaactcgacc acgaaagtca agagtataca cgaaatgtag 39180
gttctcaaag tctttgaact tgtttgcttg gtcaagagta gccacaacct ctcgcacatc 39240
tgacttacgc ttgttctctt tagcgtaaat agatgcaatt tgacgcttcc agctcttgaa 39300
tgctgctttc tggtcgtcat ctagcacttg catcaagtct tcaccacgca aatctgagta 39360
gattgcaggt actgggcaag atggtttgtc gtatttcttg aactctggca taccaagagg 39420
cagttcacgc aagcgaatct cgtttgctat agcgtaaatt gatgggttaa tctgccattt 39480
tacgttctga agagcgttaa gtgcctgata aactttaggc atttgctttc tagtgagacg 39540
tcgaaggtgc ttcttgtctc gtaccttagc aataggtagg ttagaggaca cttctttgct 39600
gtgataacca ccattaaaag gtgatttcca ctccttcgga ggtactacac aaggctcata 39660
ggcaggtgac atgttaccaa ctacttcttt atagccttga atccaagtct caagagcatc 39720
agtagctaca atcttagctg tagttttacc acgaccttga ccgtttactt gcttagcgat 39780
aagaggctta ccatccatta gcatattctt accgaaaatc tcgataagtt tagcccctag 39840
ctgaataaca tcattatcag accaaggtat ccaacggtca atatcaatag cgaactcagc 39900
tttatcaaga agatgctgat atttgtcatt atcaatagaa agaagtctca taatctcaag 39960
cttatcttca cctgcaccga gaagtgcttg gaagtcagct agtaatttta gctctttctc 40020
tgcgtgaacc atgacatcat ggtcgaattt gtaactatta ctgttacgtt tcttcaagct 40080
ctctttgatt gcttgaatgt acttaggtgc tgcattattt agcttagtaa aacgtacttc 40140
gtcttcaata cgacgaccta tattattagc taagttctga gcggttattt catgattaga 40200
aagaccatca aagatagtct tgatagcaat atatgctgct gcttctggcg acaagcactt 40260
aaggtgcatt aagcaagcac taggcttacc gcgtctacca gtgtagtagt caatatatgc 40320
ttgaatggcg tcagccatag ggcgtacaaa ttcacgagtc agacgtctgt accagtctgc 40380
atcagagcca ttgccagatt caagagcgcg ctggttgttc ttctcaaatc gaagaatacc 40440
tgcaccatgc atctcagctt caagctctag ttgaatttgc attaagtctt tgttcataaa 40500
ttactgttcc ttacaattag tgatacgtct agcctgttga ccgacgcacc ataatatatc 40560
tttctcttca ttagagtact caatcttatc agcacctgag caataagtca aaccattgtg 40620
taaccacttc tggtataact caaagtcacg tttaataagt aggcttttaa aagtttgata 40680
aggaatttca gttcgcattg tgattctcct tgtctcacag tgtgaggcta gtttggttaa 40740
agcatatcat aaaggcacct ctaaagatgc cttgagttat gctctatcta ttgcgtttga 40800
taccctgtcg tgcgtcacga gctactagga aggctggctt cttagcgttt gtcgctccct 40860
tctcaatctt aggcttacgg cactcatccc attcaacatt attaatctca tcaatgatta 40920
cttgttgaaa agggaatggc ttattaccca taaactcatt gaagcttact ggcttaaatc 40980
tcaggtcaat acctgcaaga gcgttagccc tagcagcatc ttgagcatga gctacattgg 41040
caggtatgcc aaaagcacct ttcttaatct cgtgatgctc ttgcttgcaa gtcttcacaa 41100
atgatacagg gtctgtaatt actaagtgac caaacttgtc aaccattaca ttcccctcat 41160
gcaagtcaaa ctttgcaata cctttgaaga agtcacgtac tcgaatggca gttctataaa 41220
tagcaagaga ttcgtcatta ccattacagt aaaagatatc tcctagcttg tcgaactgtt 41280
tcataaatga agggtaatca agttctggct tgtctgcctc tctatgataa gctctcttag 41340
ctttaaagat tctaacaagc ttatcataag caactagctt gtccatcaat acaacataac 41400
aagagctgaa acgctcaaga ccatagatag ttggaatacc ttctaagtct tgattatcac 41460
ggcagaatgc agcatacgcc gcgccactgt cttctttctt gaaccctagt ttaatagcat 41520
aattcttgat atcgggatgt gtccatgcct gagagaagaa gccagagcct agcttgacat 41580
agccttgctc tttcgctgcc ttctcaaagt cacgcacttg atagtcgatg taatcgcctt 41640
cacgcacatc acgcaccata gaatccgcta gattgattag tccacgcacg atagggttct 41700
cgaaagttaa cataaaagtt acctcttgtt aggttggtta gtgtatatca tagaagccgc 41760
tctttgaacg acttcgagtt atacacttgt tacttagttt tagcttcttc aatatcggct 41820
agtagttggt ctttggcttt tactagtagc tcaaaagctt cgtgaccgtc accataacca 41880
cacttaatca gctcacggaa tgctttctta acgtgtgtaa ccttcttaga ctctacttga 41940
cctaagccca gtacagcgta aggacacgca ttcttgaact gaggaagcat aggcgcgact 42000
ggtttgcgct ctgacttgct cttaacttct gataaagtct tgttaagctc tgcaatagta 42060
gtgcgtagct cttcaataag cttgtcttta gcatccagct cagctttgtt aactgtctca 42120
tttgcttctt gaagagtatc attctgctgc atttgagcct gtacactgtc gccagtgcct 42180
tccatgtcaa aaggtacgtc gtctgcatta ggaagagagt taagagactc ttctgcctct 42240
tctttcttct cttctgcctg ctctgtcgtc tcttttggtg cttctggctc ttgcttttcc 42300
ggtgcttctg gatacaagat tgcattcaaa gtaccagtat tcaaagagtc attcgcggct 42360
agctctgctg ccttctcaag ttgctcttcg ttagcttctg tagctagtac gtatagtacg 42420
cgcatagcta caccgttgaa tcgcttgtcg ccttcgaaga actcagatac tttcatcagc 42480
ttataagctt gtgacttgcc gatactgaaa ttaagcttac agtattcaac aaattccgct 42540
tgcttcttgc cttcatcttt catgatttca agagcctcac gcagtagctt acctactgct 42600
aaagattctt gctgaatcgt gtcaagtttt gagcaaactt caagcgtgat ttcttgcata 42660
cgtgtaataa cttcactcat gatattatcc tcttaatgat ggttagtgta tatcataaga 42720
gccactactt agaatgactc tgagttatac actacgaaac tagattgatt aaccagatta 42780
gtagaaagac gatagaacta gaaagaaaac ctatcaatag gcatacaaac tctcgcatag 42840
tcatagttaa tttcctctta tttagctaac cagtacttac cttgccagta agtatagcac 42900
tcactagcta ccgcctcttc ctcgctgata gtttggaaag tgccgatttc aactattgag 42960
ccgtcactat tgattaagtg agcactcata tcagccggat tcactgccaa aacagcatct 43020
tggttgtact ctttgatgaa caaatcagct acagcttgaa tgttcttaag agcaacaggc 43080
gcgacgaaag ttagttctgt gcttgctttc tcttcgcctt cttccttcca agagcctaca 43140
caagtttgta ctggcgcatt taccaaggca cctagctttt gagatgcaat gattgagttg 43200
tgcgcgttct ctacttctgt gttagtagct cggaatgcag atgcgaaaat gatagtagct 43260
ttcatgatta tttccttagt tggttgatat atcctcagga gcactcaaag agaatgctca 43320
agagtgatat accatcctaa gagaaccaca tatcgcatat tccatatttt taaagagcgt 43380
cagcctttcg acttggtaaa catcttactt gataggctga ctattgtcaa cctctttctt 43440
aaattattta ttaattaatt tctttatctt gccagccttt acgcttgaac cacttgtaaa 43500
gcttaagcgg tatcttgcaa aactcttgta gcatgacaaa gagaactaag aacaaatcaa 43560
gtattaaacg tagcattcta gtatcctcca gttagttcta tctgtagtga ttcagttatt 43620
gccttaaagg ttaaaccttt aatagtaagt gactggatac aatccatgaa catttcgaaa 43680
gaatcgcagt atattacgta tttcatgata gtacctattg agctaatgag cctatcaagt 43740
gagacgctta cctagttgtt aaagaactag gctagtatta catagtaggc tgactagtgt 43800
caacctattt tatcacattc tgtcatttgt tactaagtca gttgtaaaac ccttaccgtc 43860
ttgatagtaa gtttctaact cttgaactag ttcttgtaag ttatcatcta atgataagta 43920
agagtctaat tgcatgtaac ctgactgaat aagacctgtg tttgagaagt taccgaaaac 43980
aagagcgtct tgtgtctgga aaacttcaaa gtagttacca gattctaatt cgatagtacc 44040
gatgtttgtt gcgttagtaa tatcttgcat agtccagttt ttcatagtag ttaccttatt 44100
aattaatgag cctactatgt aacactaacc tgattgttaa agagcttgtc cgttaggacg 44160
cttattaagt tatcgtatcc ttaatcgctt gtcaacactt aatttcgctt atttcgtctt 44220
aagtgcgtta tagctaatgg ctgaagggct accgagttac aatgttacct attaggcaac 44280
tttatagcta ttgactagca aggatacaag ttattaaaga gcgttccata ttggatgact 44340
gtaatttacc aagagcttac caagttgtca accaagagat tgaaagaaat tactagtacg 44400
ctccaagatg tgaaccaagt cacaaccaag agcgtttggt gagttgctag ctgagtggtc 44460
ataactctct aatgattaat agattattac cttttattgt ttgttctttc tttaaagaca 44520
aactaaaggc tattgctagt cctatctagt acgctccaag agaggaaccc aagagcatac 44580
caagagcaca accaagtaac agctatcaag tactagaacg ctccaagagc aaagaccaag 44640
agcaaccaag catagccaga gcgtacccaa gccagctact aagagcacat tcaagtacaa 44700
acatgaacac caagagcggc gaaggctgaa cactaagagc aaccaagagc attgtgcaat 44760
agcaaagaca tatgccctcc cgc 44783
Claims (2)
1.一种生物杀菌制剂，包含副溶血性弧菌噬菌体（Vibrio parahaemolyticus phage）vB_VpaP_MGD2，以有效剂量的所述副溶血性弧菌噬菌体vB_VpaP_MGD2为活性成分，还包括药学上可接受的载体或辅料；
所述副溶血性弧菌噬菌体vB_VpaP_MGD2保藏于中国典型培养物保藏中心，保藏号为CCTCC M 2019489。
2.权利要求1所述的生物杀菌制剂用于抑制或杀灭单一耐药或多重耐药的副溶血性弧菌。
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