CN107109372B - 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 - Google Patents

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 Download PDF

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CN107109372B
CN107109372B CN201580071134.5A CN201580071134A CN107109372B CN 107109372 B CN107109372 B CN 107109372B CN 201580071134 A CN201580071134 A CN 201580071134A CN 107109372 B CN107109372 B CN 107109372B
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clostridium perfringens
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bacteriophage
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尹成俊
姜尚铉
全秀娟
孙志洙
白亨琭
朴硕晃
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Abstract

本发明涉及可以特异性地杀死产气荚膜梭菌细胞的肌尾噬菌体科噬菌体Clo‑PEP‑1，其具有由SEQ ID NO：1的核苷酸序列表示的基因组（登记号：KCTC 12664BP），以及使用包含所述噬菌体作为活性成分的组合物来预防和治疗产气荚膜梭菌感染的方法。

Description

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
技术领域
本发明涉及一种从自然界分离的感染和杀死产气荚膜梭菌 (Clostridiumperfringens)细胞的噬菌体，以及使用包含噬菌体作为活性成分的组合物用来预防和治疗产气荚膜梭菌细胞感染的方法。更具体地说，本发明涉及从自然界分离出来的并可以特异性地杀死产气荚膜梭菌细胞的肌尾噬菌体科(Myoviridae)噬菌体Clo-PEP-1，其具有由SEQID NO：1(登记号：KCTC 12664BP)的核苷酸序列表示的基因组以及预防产气荚膜梭菌感染和之后使用包含所述噬菌体的组合物作为活性成分治疗的方法。
背景技术
产气荚膜梭菌是绝对厌氧菌(在氧气存在下几乎不能存活)且是导致严重疾病的致病细菌，包括坏死性肠炎和人类和动物如牛、猪和山羊等的食物中毒。产气荚膜梭菌产生的肠毒素通常是溶血性毒素和坏死毒素，其包括四种主要毒素α、β、ε和ι。根据它们的存在，产气荚膜梭菌分为六种产毒素类型，A至F。A型产气荚膜梭菌是食物中毒的主要致病病原体，C型产气荚膜梭菌是坏死性肠炎的主要致病病原体。
最近，产气荚膜梭菌的感染在家禽业中越来越多。因此，鸡场正在遭受这些病例，因为它们在大量鸡群中流行，且长时间潜伏不表现出症状。特别是在肉鸡中，产气荚膜梭菌的感染往往发生在大范围内，因此被广泛认为是当今的主要病原体。此外，据养猪业报道，产气荚膜梭菌的感染病例也在增加。考虑到这种产气荚膜梭菌对畜牧业的重大损害，迫切需要开发一种预防或治疗产气荚膜梭菌感染的方法。已经使用各种抗生素来预防或治疗产气荚膜梭菌细胞的感染。然而，由于耐抗生素细菌的增加，迫切需要有效的替代方法。
最近，使用噬菌体作为治疗细菌感染的新方法引起了我们的注意。特别地，我们对噬菌体非常感兴趣的原因是因为基于噬菌体的治疗是一种自然友好的方法。噬菌体是感染细菌的极小微生物，简称为噬菌体。一旦噬菌体感染细菌，噬菌体在细菌细胞内增殖。完全增殖后，后代破坏细菌细胞壁逃离宿主，这说明噬菌体具有杀菌能力。噬菌体感染的特征是特异性高，使得特定噬菌体仅感染特异性细菌。也就是说，可以被特定噬菌体感染的细菌是有限的，这表明噬菌体只能杀死特定细菌并且不会伤害其他细菌。
噬菌体首先由英国细菌学家特沃特(Twort)在1915年发现，当时他注意到微球菌菌落被未知的某种东西融化并变得透明。1917年，法国细菌学家d'Herelle发现，痢疾病人粪便的滤液中的痢疾志贺菌(Shigella disentriae)被某物融化，并进一步研究了这种现象。结果是，他独自鉴定了噬菌体，并将其命名为噬菌体，其意思是病菌杀手。从那时起，已经连续鉴定了特异性地作用于志贺菌(Shigella)，伤寒沙门氏菌 (Salmonella Typhi)和霍乱弧菌(Vibrio cholerae)等致病菌的噬菌体。
由于噬菌体杀死细菌的独特能力，已经研究并预期噬菌体是治疗细菌感染的更好的方法。然而，Fleming发现青霉素之后，由于抗生素的普及，只有东欧国家和前苏联的噬菌体研究在继续。2000年以后，常规抗生素的优点由于耐抗生素细菌的增加而褪色。因此，噬菌体再次被视为可以代替常规抗生素的新的抗菌剂。
此外，最近全球政府强化对使用抗生素的监管。对噬菌体感兴趣的越来越多，工业应用也越来越多。
因此，本发明人尝试通过使用从自然界中分离的可以选择性地杀死产气荚膜梭菌的噬菌体而开发可用于预防或治疗产气荚膜梭菌感染的组合物，并进一步建立使用该组合物预防或治疗产气荚膜梭菌感染的方法。结果是，本发明人分离出适合于此目的的噬菌体，并确定了可将本发明的噬菌体与其他噬菌体区分开的基因组的核苷酸序列。然后，我们已经开发了一种包含分离的噬菌体作为活性成分的组合物，并证实该组合物可以有效地用于预防和治疗产气荚膜梭菌感染，完成了本发明。
发明内容
本发明的一个目的是提供一种从自然界分离出来的并可以特异性地杀死产气荚膜梭菌细胞的肌尾噬菌体科噬菌体Clo-PEP-1，其具有由SEQ ID NO：1(登记号：KCTC12664BP)的核苷酸序列表示的基因组。
本发明的另一个目的是提供一种适用于预防产气荚膜梭菌感染的组合物，其包含可以感染和杀死产气荚膜梭菌细胞的噬菌体Clo-PEP-1作为活性成分，以及使用所述组合物预防产气荚膜梭菌感染的方法。
本发明的另一个目的是提供一种适用于治疗产气荚膜梭菌感染的组合物，其包含可以感染和杀死产气荚膜梭菌细胞的噬菌体Clo-PEP-1作为活性成分，以及使用所述组合物治疗产气荚膜梭菌感染的方法。
本发明的另一个目的是提供一种用于使用所述组合物预防和治疗产气荚膜梭菌感染的消毒剂。
本发明的另一个目的是提供一种用于使用所述组合物预防和治疗产气荚膜梭菌感染的饮用水添加剂。
本发明的另一个目的是提供一种通过使用所述组合物预防和治疗产气荚膜梭菌的感染而对养殖业有效的饲料添加剂。
为了实现上述目的，本发明提供了一种从自然界分离出来的并可以特异性地杀死产气荚膜梭菌细胞的肌尾噬菌体科噬菌体Clo-PEP-1，其具有由SEQ ID NO：1(登记号：KCTC12664BP)的核苷酸序列表示的基因组，以及使用包含噬菌体作为活性成分的组合物来预防和治疗产气荚膜梭菌感染的方法。
噬菌体Clo-PEP-1由本发明人分离，然后于2014年8月21日放置于韩国生物科学和生物工艺学研究院韩国典型培养物保藏中心(登记号： KCTC 12664BP)。本发明还提供一种适用于预防或治疗产气荚膜梭菌感染的消毒剂、饮用水添加剂和饲料添加剂，其包含噬菌体Clo-PEP-1作为活性成分。
由于本发明的组合物中包含的噬菌体Clo-PEP-1有效地杀死产气荚膜梭菌细胞，因此被认为对预防或治疗由产气荚膜梭菌引起的各种疾病是有效的。因此，本发明的组合物可用于预防和治疗由产气荚膜梭菌引起的疾病(感染)。
在本说明书中，术语“治疗(treatment)”或“治疗(treat)”表示 (i)抑制由产气荚膜梭菌细胞引起的疾病；和(ii)缓解由产气荚膜梭菌细胞引起的疾病。
在本说明书中，术语“分离(isolation)”或“分离的(isolated)”表示通过使用不同的实验技术分离噬菌体的所有操作，和确保能够区分该噬菌体与其他噬菌体的特征的操作，还包括通过生物工程技术增殖噬菌体使其有用的操作。
包含在本发明组合物中的药学上可接受的载体是通常用于制备药物制剂的药物载体，其实施例为乳糖，葡萄糖，蔗糖，山梨糖醇，甘露醇，淀粉，阿拉伯树胶，磷酸钙，藻酸盐，明胶，硅酸钙，微晶纤维素，聚乙烯吡咯烷酮，纤维素，水，糖浆，甲基纤维素，羟基苯甲酸甲酯，羟基苯甲酸丙酯，滑石，硬脂酸镁和矿物油，但并不限于此。除了上述成分之外，本发明的组合物还可以包括润滑剂，润湿剂，甜味剂，香料，乳化剂，悬浮剂和防腐剂。
在本发明的组合物中，包含噬菌体Clo-PEP-1作为活性成分。此时，噬菌体Clo-PEP-1为以下浓度：1×101pfu/ml～1×1030pfu/ml或1×101pfu/g ～1×1030pfu/g，优选1×104pfu/ml～1×1015pfu/ml或1×104pfu/g～1×1015 pfu/g。
本发明的组合物可以由本领域技术人员通过使用单位剂量形式或在多剂量容器中的药学上可接受的载体和/或赋形剂通过本领域的方法来配制。制剂可以是在油或水溶性介质、提取物、粉末、颗粒、片剂或胶囊中的溶液、悬浮液或乳液的形式。此时，可以另外包含分散剂或稳定剂。
本发明的组合物可以根据使用目的制备为消毒剂、饮用水添加剂或饲料添加剂，但不限于此。
有益效果
使用包含噬菌体Clo-PEP-1作为活性成分的组合物来预防和治疗产气荚膜梭菌感染的方法与基于包括常规抗生素的化学物质的常规方法相比具有对产气荚膜梭菌特异性高的优点。这意味着，本发明的组合物可以特异性地用于预防或治疗产气荚膜梭菌的感染，而不会影响其他有用的寄宿细菌，因此具有较少的副作用。一般来说，当使用如抗生素的化学物质时，一般寄宿细菌也会受到损害，从而削弱动物的免疫力，具有各种副作用。同时，本发明的组合物使用从自然界中分离的噬菌体作为活性成分，因此其是非常自然友好的。
附图说明
参考附图可以最好地理解本发明的优选实施方式的应用，其中：
图1是示出噬菌体Clo-PEP-1的形态的电子显微照片。
图2是说明噬菌体Clo-PEP-1杀死产气荚膜梭菌细胞的能力的照片。盘上的透明区域是通过溶解细菌细胞形成的噬斑。
具体实施方式
本发明的实际和目前优选的实施方式如下列实施例所示。
然而，应当理解的是，本领域技术人员在考虑到本发明公开内容的基础上可以在本发明的精神和范围内进行修改和改进。
实施例1：分离能够杀死产气荚膜梭菌细胞的噬菌体
从自然界中收集样品以筛选具有杀死产气荚膜梭菌细胞能力的噬菌体。用于本文中噬菌体分离的产气荚膜梭菌细胞是本发明人已经分离并鉴定为产气荚膜梭菌的产气荚膜梭菌细胞。
噬菌体的分离过程在下文中详细描述。将收集的样品加入到TSB(胰蛋白酶大豆液体培养基)培养基(酪蛋白的胰消化物，17g/L；大豆的木瓜酶消化物，3g/L；葡萄糖，2.5g/L；氯化钠，5g/L；磷酸氢二钾，2.5g/L)，以1/1000的比例接种产气荚膜梭菌细胞，然后在37℃下厌氧培养12小时。培养完成后，以8000rpm离心20分钟，回收上清液。回收的上清液以1/1000的比例接种产气荚膜梭菌培养物，然后在37℃下厌氧培养12 小时。当样品含有有效噬菌体时，为了提高噬菌体的滴定度，将上述步骤重复5次。重复5次后，培养物溶液以8000rpm离心20分钟，回收上清液。回收的上清液用0.45μm过滤器过滤。将得到的滤液用于斑点测试，以检查是否包括能够杀死产气荚膜梭菌细胞的噬菌体。
斑点测试如下进行：以1/1000的比例在TSB培养基上接种产气荚膜梭菌细胞，37℃厌氧培养过夜。将上述制备的3ml产气荚膜梭菌培养液 (OD600＝2.0)涂在TSA(胰蛋白酶大豆琼脂；酪蛋白的胰消化物，17g/L；大豆的木瓜酶消化物，3g/L；氯化钠5g/L；琼脂，15g/L)板上。将板放在厌氧室中干燥约30分钟。干燥后，将10μl所得滤液直接点在产气荚膜梭菌苔的表面上并干燥约30分钟。干燥后，将板在37℃，无氧条件下培养一天，然后检查在细菌苔表面上透明区的形成。如果在滤液滴下的地方产生透明区域，则可以判断为滤液中包含能够杀灭产气荚膜梭菌细胞的噬菌体。通过上述步骤，可以获得含有能够杀死产气荚膜梭菌细胞的噬菌体的滤液。
之后，从上述确认具有能够杀死产气荚膜梭菌细胞的噬菌体的滤液中分离出噬菌体。常规的斑块测试用于纯噬菌体的分离。具体来说，通过使用灭菌的尖端拾取斑块测试过程中形成的斑块，然后将其加入到产气荚膜梭菌的培养液中，然后在37℃无氧条件下培养12小时。培养完成后，以8000rpm离心20分钟，得到上清液。在回收的上清液中以1/50 的比例接种产气荚膜梭菌培养物，然后在37℃厌氧条件下培养12小时。为了增加噬菌体的滴定度，将上述步骤重复至少5次。然后，以8000rpm 离心20分钟，得到上清液。用获得的上清液进行斑块测试。一般来说，纯噬菌体分离不是一次性完成的，因此通过使用上述形成的斑块重复上述步骤。在重复操作至少5次后，得到含有纯噬菌体的溶液。通常重复分离纯噬菌体的步骤，直到产生的斑块在尺寸和形态上相似。通过电子显微镜观察确认最终的纯噬菌体分离。直到在电子显微镜下确认纯噬菌体分离之前，重复上述步骤。用常规方法进行在电子显微镜下的观察。简言之，将含有纯噬菌体的溶液装载在铜格栅上，随后用2％的乙酸铀酰阴性染色。干燥后，在透射电子显微镜下观察形态。本发明中分离出的噬菌体的电子显微镜照片如图1所示。从形态上观察，上述分离的噬菌体被鉴定为属于肌尾噬菌体科噬菌体。
含有上述确认的纯噬菌体的溶液进行纯化。将产气荚膜梭菌细胞的培养液以噬菌体溶液总体积的1/50的体积加入到含有纯噬菌体的溶液中，然后再培养12小时。培养完成后，以8000rpm离心20分钟，得到上清液。重复该过程5次，得到含有足够数目噬菌体的溶液。将最终离心得到的上清液用0.45μm过滤器过滤，然后用常规聚乙二醇(PEG)沉淀。特别地，将PEG和NaCl加入到100ml滤液中，直到达到10％PEG 8000/0.5M NaCl，在4℃下静置2～3小时。然后，以8000rpm离心30 分钟，得到噬菌体沉淀物。将所得噬菌体沉淀物重新悬浮于5ml缓冲液 (10mM三羟甲基氨基甲烷盐酸盐(Tris-HCl)，10mM MgSO4，0.1％明胶，pH 8.0)中。该溶液被称为噬菌体悬浮液或噬菌体溶液。
结果，收集上述纯化的噬菌体，将其命名为噬菌体Clo-PEP-1，然后于2014年8月21日在韩国生物科学和生物工艺学研究院韩国典型培养物保藏中心保藏(登记号：KCTC12664BP)。
实施例2：噬菌体Clo-PEP-1基因组的分离和序列分析
如下分离噬菌体Clo-PEP-1的基因组。将基因组从实施例1获得的噬菌体悬浮液中分离。首先，为了去除悬浮液中的产气荚膜梭菌细胞的 DNA和RNA，将脱氧核糖核酸酶I和核糖核酸酶A各200U分别加入到 10ml的噬菌体悬浮液中，在37℃下培养30分钟。30分钟后，为了去除脱氧核糖核酸酶I和核糖核酸酶A活性，加入500μl 0.5M乙二胺四乙酸 (EDTA)，培养10分钟。将悬浮液在65℃下进一步培养10分钟，然后加入100μl蛋白酶K(20mg/ml)以破坏噬菌体的外壁，然后在37℃下培养20分钟。之后，加入500μl 10％十二烷基硫酸钠(SDS)溶液，然后在65℃培养1小时。加入10ml比例为25:24:1的苯酚：氯仿：异戊醇混合物，然后充分混合。将混合物以13,000rpm离心15分钟以分离每层。得到上层液体，向上层加入体积为上层体积的1.5倍的异丙醇，然后以 13,000rpm离心10分钟以沉淀噬菌体的基因组。收集沉淀后，向沉淀物中加入70％乙醇，然后以13,000rpm离心10分钟以洗涤沉淀。回收洗涤的沉淀物，真空干燥，然后溶于100μl水中。重复该步骤以获得足够量的噬菌体Clo-PEP-1基因组。
通过在首尔国立大学环境管理国家仪器中心使用Illumina Mi-Seq装置进行下一代测序(NGS)分析上述获得的噬菌体Clo-PEP-1基因组的核苷酸序列。结果表明，噬菌体Clo-PEP-1的最终基因组的大小为 50,401bp，全基因组的核苷酸序列为SEQ ID NO：1。
通过使用网站(http://www.ncbi.nlm.nih.gov/BLAST/)上的BLAST 研究了上述获得的噬菌体Clo-PEP-1的基因组序列与先前报道的噬菌体基因组序列的相似性。从BLAST结果看，很难发现与该噬菌体序列具有50％以上序列同源性的噬菌体序列。基于该结果得出结论，噬菌体 Clo-PEP-1应该是以前未报道的新型噬菌体。
实施例3：噬菌体Clo-PEP-1对产气荚膜梭菌的杀伤能力的研究
研究了分离的噬菌体Clo-PEP-1对产气荚膜梭菌的杀伤能力。为此，通过与实施例1中所述相同的方式通过斑点测试观察透明区域的形成。用于该研究的产气荚膜梭菌共有15个菌株，其已经由本发明人分离并鉴定为产气荚膜梭菌。噬菌体Clo-PEP-1显示了对本实验中使用的12个产气荚膜梭菌细胞的杀伤能力。杀伤能力试验的代表性结果如图2所示。同时，还研究了噬菌体Clo-PEP-1杀死多杀巴斯德菌(Pasteurella multocida)，副猪嗜血杆菌(Haemophilus parasuis)，支气管炎博德特氏菌(Bordetella bronchiseptica)，粪肠球菌(Enterococcus faecalis)，屎肠球菌(Enterococcus faecium)，猪链球菌(Streptococcus suis)，乳房链球菌(Streptococcus uberis)和铜绿假单胞菌(Pseudomonas aeruginosa)的活性。结果是，确定了噬菌体Clo-PEP-1不具有针对这些微生物的杀伤活性。
因此，证实了噬菌体Clo-PEP-1具有杀死产气荚膜梭菌细胞的特异性能力和针对产气荚膜梭菌的广谱抗菌谱，这表明本发明的噬菌体 Clo-PEP-1可用作用于预防和治疗产气荚膜梭菌感染的组合物的活性成分。
实施例4：噬菌体Clo-PEP-1对产气荚膜梭菌感染的预防作用
将100μl噬菌体Clo-PEP-1溶液以1×109pfu/ml加入到含有9ml TSB 的试管中。对于含有9ml的TSB的另一个试管，仅添加相同体积的TSB。然后，将产气荚膜梭菌培养物加入到每个管中以制备OD600为0.5的细菌悬浮液。之后，将试管转移到37℃的厌氧培养箱中，然后培养，期间观察产气荚膜梭菌细胞的生长。如表1所示，在添加有噬菌体Clo-PEP-1 溶液的管中，产气荚膜梭菌细胞的生长受到抑制，而在没有噬菌体Clo-PEP-1溶液的管中，产气荚膜梭菌细胞的生长没有被抑制。
表1
产气荚膜梭菌的生长抑制
Figure GDA0001402545610000101
上述结果表明，噬菌体Clo-PEP-1不仅抑制产气荚膜梭菌细胞的生长，而且可以杀死它们。因此，噬菌体Clo-PEP-1可以用作用于防止产气荚膜梭菌感染的组合物的活性成分。
实施例5：噬菌体Clo-PEP-1对产气荚膜梭菌感染的治疗作用
研究了噬菌体Clo-PEP-1对受产气荚膜梭菌细胞影响的动物的治疗作用。40只2天大小鸡口服1×107cfu产气荚膜梭菌细胞以感染动物。随机分为2组。一个实验组每1g饲料混合1×108pfu的噬菌体Clo-PEP-1。另一对照组以相同的方式喂养，没有噬菌体Clo-PEP-1。在2天的激发之后，测量动物粪便和盲肠内产气荚膜梭菌细胞的数量。在测量中，为了避免其他污染细菌的干预，使用了产气荚膜梭菌细胞的选择性培养基 (TSC琼脂板：OXOID)。结果表明，与没有噬菌体Clo-PEP-1的对照组相比，喂噬菌体Clo-PEP-1的实验组应将粪便中的产气荚膜梭菌细胞减少500倍以上，盲肠含量减少超过300倍。从上述结果可以确认，本发明的噬菌体Clo-PEP-1可以非常有效地治疗由产气荚膜梭菌引起的感染性疾病。
实施例6：饲料添加剂和饲料的制备
使用噬菌体Clo-PEP-1溶液制备含有浓度为1×108pfu/g的噬菌体 Clo-PEP-1的饲料添加剂。其制备方法如下：向噬菌体溶液中加入麦芽糖糊精(40％，w/v)，然后加入海藻糖到终浓度的10％。混合均匀后，将混合物冷冻干燥。最后，将干燥的混合物研磨成细粉末。上述干燥过程可以用真空干燥、温热干燥或在室温下干燥来代替。为了制备对照饲料添加剂进行比较，制备不含噬菌体但仅含有缓冲液(10mM三羟甲基氨基甲烷盐酸盐，10mMMgSO4，0.1％明胶，pH8.0)的饲料添加剂。
将上述两种饲料添加剂分别与1000倍体积的鸡饲料混合，得到两种最终的饲料。
实施例7：饮用水添加剂和消毒剂的制备
饮用水添加剂和消毒剂在使用目的上不同，但组成相同，因此它们以相同的方式制备。使用噬菌体Clo-PEP-1溶液制备含有浓度为1×108 pfu/ml的噬菌体Clo-PEP-1的饮用水添加剂(或消毒剂)。特别地为了制备饮用水添加剂(或消毒剂)，将噬菌体Clo-PEP-1溶液加入到缓冲溶液中达到1×108pfu/ml，混合均匀。为了比较，将上述缓冲溶液本身用作不含噬菌体的饮用水添加剂(或消毒剂)。
制备的两种饮用水添加剂(或消毒剂)以1:1000的比例在水中稀释，然后用作饮用水或消毒剂。
实施例8：对养鸡业的影响
研究了实施例6和实施例7中制备的饲料、饮用水和消毒剂对养鸡业的影响。特别地，研究重点是死亡率。共120只2天大小鸡分为3组，每组40只(A组：饲料试验组，B组：饮用水试验组，C组：消毒剂试验组)。实验持续4周。每组分成两个小组，每小组分别包含20只小鸡。根据噬菌体Clo-PEP-1治疗与否分成两个小组(小组-①：用噬菌体 Clo-PEP-1治疗；和小组-②：未用噬菌体CLO-PEP-1治疗)。将该实验中使用的小鸡分成每个小组，并在分开的彼此间隔足够距离的房间中饲养。每个小组被划分并如表2所示命名。
表2
养鸡实验的小组
Figure GDA0001402545610000121
按照表2所示的常规饲料供给方法提供饲料，提供实施例6中制备的饲料。根据表2中所示的常规供水方法提供饮用水，提供实施例7中制备的饮用水。消毒剂与常规消毒剂一起轮流处理鸡一周三次。也就是说，在喷洒本发明的消毒剂的那一天，没有用常规的消毒剂进行处理。结果示于表3。
表3
养鸡实验死亡率
组别 死亡率(％)
A-① 0
A-② 40
B-① 5
B-② 35
C-① 0
C-② 35
从上述结果可以确认，本发明制备的饲料、饮用水和消毒剂在降低动物死亡率方面是有效的。因此，可以得出结论，本发明的组合物可以有效地用于提高动物养殖的生产率。
本领域技术人员将理解的是，在前面的说明书中公开的概念和具体实施方式可以容易地被用作修改或设计用于实现本发明的相同目的的其他实施方式的基础。本领域技术人员还将理解的是，这些等同的实施方式不脱离如所附权利要求中阐述的本发明的精神和范围。
保藏单位名称：韩国生物科学和生物工艺学研究院
保藏编号：KCTC12664BP
保藏日期：20140821
Figure GDA0001402545610000141
。
<110> 尹特荣生物科技株式会社
<120> 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
<130> DFP17KR3050
<160> 1
<170> KopatentIn 2.0
<210> 1
<211> 50401
<212> DNA
<213> 噬菌体 Clo-PEP-1
<400> 1
attagaatac ccccttaata tctttgttgt aaacatgaag agagaaaatt gtatgagaga 60
aacttcctgg ttcatagcca gtttgttcag ctacccaatg taatagtttc atagcaagat 120
aaacatcatt agcaaagtga gtagcaaagt cagagcttct cataacataa tgaatattta 180
atttaccttc ccttacttgg aagttatatc ctaatgaaca tggtactcta gttactcctc 240
ctatattgaa tggatcaata gctggattcc acagagataa ccataattgt cttgagcctg 300
gatcttcttt aattctattg atgatatctt caatttgtct gtattgccaa attctttcat 360
tgtaagaata tccaaatttc ccatctctta gatactcatt ccatacctca ggtcttaatt 420
gataagcttt acctggattg atgaaagcct ccctagtatg atgtggaggt ataccaaata 480
attcttctct ttcttcaaga gagtattgat tgatgacacc ttgtggatcg tagatccttt 540
ctctaaattc agcatctgcc caaggttgag atacacctgg aacttcttca ggtttacttt 600
ctagaattgt ataactatag ttttgtaact cctttgtgaa ataatctgga ttaccttcta 660
taactttatc ttgcatagtt ttgggtctta cctcaatccc catttcacat aaatctcttt 720
ttacttcttc aaatgcttct tgagcattaa tataaattct catagtttaa ttcctccatt 780
tattgaattt tgttaagtta attatactac ttattaaagt acttgtcaat agataattta 840
ttgatatcta tctttggaaa tttctccaga ccaaaatgta ataactgcat cttctggaga 900
gctttgtaag agtgtatctg gtcctctgat ttaaagaacc tattatagtt actattcaag 960
gatttatgcc aaggatgttt actattagca atactctttc ttggtacctt aaaataattg 1020
aagtatccat ttataaacat tcctgacaaa taagcttgag acatatgaaa agtaatatta 1080
tctatctcac atacttcttg aggtagttct tcaatgaacc tgtttattaa tactagatca 1140
gctgcgaatt tcttttgcaa ttcacatact ctatacataa tttttaagtg tttccaaggt 1200
ccttttcttt ttggtctagt tactactgca gctatcaagc aagaaccatt agtagctttt 1260
ttcctgttga aataataagt atagctcata ccacttatct tggtaagctt ttctctaaat 1320
tcaattaatt gtggataatc aatgtatgat cttaataggt gtccccactt accttcttta 1380
tatccaaagt cccctagatt taaagtacat ttatgggact tacaggagag aactaagtcc 1440
tcctgaaagc cctgtatgtt ctgagtatac atcatagttt cttctggatt ggtaatcatt 1500
tccctattaa gatttaagaa taagtcatca aaatcattat agtctcttat tagcatatta 1560
ccttctccta agtatttatt ttagtaatta gatctttggc ggaatttatt tacctgtgat 1620
ttcttaaggt atatatttac tatgtcatct acctctaagc cagatacagc taataattta 1680
atgtaacata tccatacatt gaatagtctt tgattaaagg catttacatc tgttagcatt 1740
gaagtttgtt tccaaggttt attcttaagg cagttacagc acatacctat ttctctcact 1800
aagtcagtta cagccttttc cacacaaggt ttatcttcat catttactat agcttggtaa 1860
tataagtttg ctaggtgatc ttttcctact ttgttcactg gatctatagt attataatcc 1920
ttaccagcta ggattgtaaa ttcagttaag aagtgtaatc catcaatcaa ttcttcttgg 1980
taatggtctt catggttata agcatctaat gcttctccta cttcttcagt cattctccaa 2040
gcaaaatcct taagtctagc ttgacctctt ttatcattta agttaactgg acagtcttca 2100
gtttggcata atcctgatct agcttctata tgatgatatt tctccatcaa ttccttttgt 2160
ctattaaaga tagcttgtaa tctatctcct tctacttttt cttctactgc atggtttata 2220
ttcataacaa tctctcctct tatcttgata tatttataaa ttggtttata gcttctttag 2280
cacttgagtc atctattaat tgggatagct tctccacatt ggaagtagta taatcatata 2340
ccagtacatt ccatccatca cagaataatt tccatatcag ttcatcatat ctagccagta 2400
gatgttcagc tctttcaatt actcctggca tctgttctct atcaccaaaa ttgaagatca 2460
cttccctagg tggtctagca tatacaatag ttggcttaca tagatgtttt aagatcttta 2520
gctccttact gtcaaggttg aaattactac tatctctgat gataggtcca tacaccattt 2580
cttcaaagca agtaaatcta tcatgtatag cttccctttc accttttctc acaatttgag 2640
ttaataccca ttctctttgt tcttcataag aaccaggtcc cattgattgg atatattctc 2700
ctcctctgtg tttagttagg ttcttagctg ttgtggtctt acctgagtta tccattccta 2760
aaatgataat cattaaagct ctcctccatt ctttagatct tctactatta agtttagtat 2820
atattgtact cctttatcat tatattcaaa taaaggattt ataagggact tacttggagt 2880
tacataataa ccaagttctt tagcaaggat tgggtctatt atttcaagtg tatgctcatt 2940
gtaccttctg gcaaatccct ttcttactaa atacctattt agttttatca cactaactcc 3000
taatgctcct gctatcttag tagcagtggt taaacttccc ttccataatt ggggtttgtc 3060
atcattaagg tctttcaaga tgtaatactt tttcttgacc attaaattct cctccttcta 3120
catttttata ttaccataat taatttatta tgtcaaaggt tattttagaa tttttaggta 3180
tatctataac agccagaaga ttttctggtc tattactatc ataaacaagt atagtataat 3240
tctgagctcc taagttattg accataacct cattagctaa cttagataaa ttgttagggg 3300
taaggtcttt aggactatta aggtttagtt tgataataag atcctcccca tctagtccag 3360
atggagaagt ctcaataaca ttatagtcta aagcattatg aagcttttct actaactttt 3420
cttgactttt tctttggtga tgaccttcta ttagttttgc cccctgagtt agcattatcc 3480
ccattgacaa tgatatccct agaattagta ctacttcctt caagtttctt cttcgcatta 3540
gccttagcct ccctatactt tatattcata cattttacat aaaagtcttg gtcacttata 3600
ccacagatag ggcaattctt gtaacctcct tggcttctct taaattcttg aatagtttga 3660
caatatggac accacatctt ctttttaggt atttcttcgc cttcagggat cttatgagct 3720
tcatggacat ccacccaata aaattcagcc cagggacatt cttccataac tttgtctata 3780
aataatttag ctgatttata ggtatagaaa tatttcaatc ctgaatgata agtgatttta 3840
ttcttcttac ctctagcaat aattactact acaaaggcct ttatatgatt atgacctgct 3900
ggcctcttac actttattat catgcctttt tcctccccat atttagtttc ttacttatat 3960
tataaccttt atccggagaa tatacaatag tatcttttaa agtatctaga actagattaa 4020
ccttctgtga atttaatcct gatattgaca ctatctcctc cttagtatga gctcctgatt 4080
ttacagcctc tataactttg gctttgtcct caggtttcac aattacttct tttccaggta 4140
ttgctacttc tacatgataa tatggatccc caaattctcc cattgataaa tttaattcaa 4200
tttgagggaa ttgtcctgca agacggaact ctctatccat aatgatttta cttggtaaat 4260
ggctttgttt atcaaggtta tctatatcta cctcgatctc ctcatcgtca ccttctaagt 4320
catcaggtct ttttaaatat agtgctgatt ctacccaacc atgtaatatg aatgaaccag 4380
ccatcttttg accaccccta gtttgagtag catttccacc tttattatag tggtgtatta 4440
acataactcc tgtatgcttc tcattcttaa gtttaagaca ccattgtagt acaggattaa 4500
gatcagctgc attattaaga tctccactaa acataagata taatgggtcg aatattacta 4560
gtacgggttt tatctcatca attaaatctt ctatctgttt tctatgggat tcttcatcta 4620
acataaatcc ctgttgatta ataaaagtga taggaagatc tggagcaaac ctaactttaa 4680
gtcttctctt accttttata tccacattac caactactcc tctgtggcta attatctttt 4740
cagttctatc tctcataatc cagtcagcat tctcattttg aactacaatt actgggcctg 4800
gttctagaac aggatattga cctaggaatg gtcttccact agcaactgat atagctaagt 4860
cctgtgtaaa tgtggattta aatacctttg gttgtccagc cacaattcca tgagatcttc 4920
taccccagaa accttgtact aaccaaccag ggaaggctcc attattaccc ataacatcct 4980
gataactatc tattctcagt ttagtacttt cagctttttc aatatcagcc tctatttctc 5040
ctcctataat tttatccaat tcttttctta accttgtttc ttcatctttt cttcccttat 5100
atttattaaa agctgagccc ttaactaata atataatctc attaggctct agtcctattt 5160
catgtaattt attctcaata taccatatag tagaacttct atctaaagaa gtaatatcat 5220
ctaaagccag taagtctcta accttctttg gtatattata tttagcatat atctttcttt 5280
cagattgtga ggcctcagat cctcctatat tgaccttaac actatcatta gatttactta 5340
cagccttaac agccttagcg atcaccttag gcttatatat ctccttagtg tgtataggtc 5400
tttttacttt tggtttattc ttgtatttgt agttaatagt tcctggtatt ctatatacat 5460
gagtaacatc aaagcaggag tcacagccta tatgtttagc taatgctgga tttatttcgt 5520
catactgatt agcctctata tacctatcca tttcccatag cccctgatat tttccaggag 5580
aactttccca cagataactt ggtttaggtt taattcccaa tgggtcagta tgctcatcta 5640
tatcttgtat taagtattta gtctctatga agttaactat ccttctttga ggattactat 5700
aaggcattgg gctccaatat aaatcataac ccttatactg ttgttcaaag tcctttagtt 5760
ttttgtcaat gttattctta ttatatttaa ttggtacatc tttccattct ttaccttttc 5820
tagctgctag gataatgtaa tctccatttt cacattgttt agagaatata tccttcaaga 5880
actttatgtc catatattat accccctatt ataagtctat attatcccaa cctaattctt 5940
tcaagtatgg aactacttct cctcttggct ttaattttct agtgccttcc tctatctcag 6000
cgaatactaa tccccctata tgtggtttag tcccgttaac tactctcatt atactaacag 6060
ccgatactcc taattgttta gctgcatatc tgaaggatct atactcccta ttattgttgt 6120
aacaatatac tgctttagta taagttgaat ttccacctct tgccatttat acatccccct 6180
ctaacatctg tttctaaaat aagtaaaaaa tttgaggagc aagtttgctc cccttataat 6240
ttgatgagaa ttattcttca tcatcagctt ctaataattc aattatctca gctggtttca 6300
ttttcttatt aacttttatt cctctttctt tagcaagagc aattaagtct ttcttagtca 6360
tttcagagta gtcaggagtt tcattcttca ccttgagctt ctttgattaa agcgattaac 6420
tcatctttat cttttttctt agctcctttg atacctaagt ctttagccat agcagctaat 6480
tcctttttag ataatgattc taaatcattt tcgtcttctt ctgcttcgtc ttcatcaact 6540
tcttcgaagt cagcatctac tacatcttca tcttccacag cttctatttc tgcttggtat 6600
gcagccttta cagttcctac agccatttct aatgattcta atgcttcctc tacagttatt 6660
tgtccatctg tgtcaggagt tctttcatct aattccacaa tagcttttaa agccttttta 6720
acttctttct catccccaga atttaaagca gattgtacga ttacaattag ctccttattg 6780
tttgcttcct ctaaccattt tgcttttcct tttgccataa taatcattcc tccatatttt 6840
taatttttaa tttttaaata acaaatttag tatagtttga aattaactaa ttataaagtt 6900
ttggttggtg tttgtaattt gattacaata ctataatatc atatcctata tagcctgtca 6960
acgagttatt tagaaaaaat taatttatta taacactcat tacaaacggc ttcatattca 7020
acactttcgg gctggttatc aatttgtatc tggttaccct ggtcagtata tttaccttga 7080
tattttaata ttgagaatgt ggctttttta ccacattgac aaatgttctt caattcttca 7140
atgttatggg ccacttgtaa taatctggta cttccttcaa atccttccct tttaaagtca 7200
gttctcaatc cataacatat gataggataa tgtaatgata aatttaacag atcatcaacc 7260
tgttctctgg ataagaattg tacctcatca attaatataa catctactgc ccagtaatta 7320
gcctcaaagt cagaaagtat ttttgataat ttaactctag gaggtagaag aacatccacc 7380
ttccttgcta cccctattct actgattata catggtccac ctttagtatc aattcctggc 7440
ttaaccacta atggcttcat tcccctttct tcatagttat gtgctacctg gattaactga 7500
gtagtctttc cagagttcat agcactatac ctaaaatata atttattgct cacaactgat 7560
cgcctccaat atacaccata ctattacaag tatacctaat atcctaaatg cccataataa 7620
atatcccaag actatcactc ctttgactcc tcccctaaat caaatatacc cggtggtact 7680
aaatccttaa tctctcccat tatggtttca tagttaagtg ttcccttttc aatttcctct 7740
gtgtgtttac tcatctcttc cacaaaccta gtcattctag tcttaccata accttccttc 7800
tttagtgtat aacatgataa ggctaacatg atatccacag ctttaagaac attaagattt 7860
ttgttctctc tttcagctct tcttcttgca gctcttgctc ttgacataac aatctactcc 7920
tttatatatt tagtttctaa aaagattgat actactatag ctaatattgc aaatcctagt 7980
tccatttctt cacctcaaaa cctataaaca ctcctagtaa aaatattgat actagggtta 8040
ttacccctag caccatttac ttctcctccc ataacaagtc agagtaatca gttccttggt 8100
catatgagaa tattaatcct ttttgtgaac tgatataatc agctaagtgt actatcctct 8160
tttgataggt attaattggg tactctcccc attgaccatg atgagactca attagctcaa 8220
ctaccccttt gtatattatt ggaaactctt cctttaatgg ggatagtgtc tctctaacta 8280
gatatggatg ctccttagca ggcttattaa tatcatgtag tattaaagca gctagtatta 8340
tatctctgtc ccttgaacat atcatctcag cttcgcataa tgctttacct atataacaag 8400
cagctttagt atgtcttagc aaacctccta cccctgcact ataagttgga tgatatttcc 8460
cagtactact tgcaggtatt ataaagaact tttcatcaac tagctcattt aaggcctttc 8520
ttacaaaatt ttgtatatct ctatctttta tatcatgaat ttctggtatt agcattctca 8580
tgcccatttc tcttatcata actattcctc ctatcccatg tagtagcctc tatattttat 8640
cttctcccac tcatcaacag taaatactaa tttagttctt tgtggtatgt tagagtaaca 8700
aagataatca aggtctactt ctacatattg tttaccttta gcccaaggtt cattatcaaa 8760
ttcatggtac tctcttacac tgtgtacctt ttttatatag tgctcaagta ggttcatttc 8820
tttctccccc ttttcttctt ctttttctta ctctttccat caagtcctcg tttaacatga 8880
aagtcaagta agttttgagc ccaccttagc atcttaatat cttgcataca tgaaagatta 8940
aagccagtgg atccatcaaa gactttaagg tcattcctaa atggcatact gtttactaaa 9000
cctggattct cattcttaag tttaactacc tctctggcag tctcatccca ttgttggtct 9060
gtccagatat tgttatcata cctataatat attattgaat gaagacaaat ctgtaaccac 9120
ttctttcgta attcaattct ttctggagtt tcttcgcagt catacttgag aattaacata 9180
gcacttcttc tccccaatat tatcacctcc ttttctatta taccatattc ttttaccttc 9240
tacaatagaa aaagtttgac caatatgttc gggttcaatt tcctcaatgg taagtgttcc 9300
cccatgctta catagtatct tagctaatac ttgtcccatg tctgtatctg ctaaagatac 9360
cccactattt tttctggtta ttacatacga cttcataact tagccccact ctcaaattta 9420
ttaactgatt atttatctct tgtatttctt ttaacttctt ctcagtcaat cctaagaact 9480
gtcccaattt tatcttctgc tcctttgtta aatgacaata ctgtttctca atcaaatatc 9540
tggcatagtt taggttcttt ctgatctcat cagtattttg cttatgcttt tcctctatcc 9600
cttccagtct cttcaataat gatgtgttta tcatgtgata cctccctaat aagatatttt 9660
gttatctttg gctttctgtc actaaagtcc attactgtgg ctacatcccc atcaatgaat 9720
actactgatt taccaccaaa cttatctttt aagtcatttt tattcttgac tattttacca 9780
ttacataaat acatgatata cctcctttaa aggcctctag cagctcctgt aaggtgtctc 9840
taccacatta taagtaaaat cacattacca catatctaaa aggccttaga attgattgtg 9900
ggctcttata tactgtatct tatgatagct ctattgccaa taacctcata taaatgagta 9960
ttatccttat ataagtatgg atttatcctc atatatagga atacagtact taggtacttg 10020
agatactaat tctccgcaat tcttatctat tgctctagat agaataaatc tgtcctcttc 10080
cattattatg aagaagtcct tgttttcaat gataatttct ttttcattag ataatataac 10140
tttcatcatg gttatccctc ctttactgtc attataagag aagtaagtac aactgtcaac 10200
gttcgggact actgttctct ttccaactgg agaagaactc tctctcaact actgttctct 10260
cttactctct ttccaaccaa acaaaatcaa gagctcgatc gctgaccgtc tgggtttttc 10320
tatatagagc aaaagctcgg aagcatggat atgcctgccg taatgataat ggaggcaagg 10380
caatagcctg ccattgatgt aatcaccatg taatatatat atactatata taataatata 10440
ttattatata gtatatataa tactatgtat atataaaggg ggcataactg atatccctct 10500
acgccagtga taccaatgct tttgtgattt tgtagtttca actgatatgt gggatatcag 10560
actgatatgg ggtaaaaata taggcatatc agatatcagt taatgcctgg actttgacat 10620
atcagtacaa tatatgtata atgtaagcat aggaggtgga tctaatgaag aataactggg 10680
ataggttaca agactacttt aatattgtaa ctactaaaga aagtaaagat ggtaagcccc 10740
taaaaaggga attaaccaat catgaattgt ctgacgcatt cgttaaatta tatattgaag 10800
tatctaaaat gaagagagag aatacccaat taaggaataa tatcagagca atggatggtc 10860
atcctgtaat ccagattaac gatccgaatg ctaaattaga tgatatttcc ttgtatttat 10920
atctaaaaga cttctggcca agtatatttg aggactataa agccaagatc cagagtagta 10980
agatactaaa aggcaagaga tttaagtcat ttaaagaggc taatgattat gtgtggaagg 11040
atctaatcca gaatagtaaa gtaaagaaga ccaatgaaag tctatttgta ttaagtaaat 11100
gggtagatgg tatgggtatc aagcataatt tcaggaataa gcaagagttt gttactgctt 11160
gtctgtgcta tatggactat aaaggatatc tggacaagga gggtaagacc tttaataagc 11220
taggtgatat taagagagga agcttcagat ttaattctga tgaggaaacc tatgcaatac 11280
taactgatac ttttgatact gtgggtaaat ataaatctgg aattgagaaa ttcaaattaa 11340
aaataaagga gttaaaagag aatgattaaa agaattatgt cattactaac aataattgtg 11400
ataggagggc tactgttaac catacagagc ctaaaaaagt aaaaccaaag ccagcccaat 11460
gtattgatct ggattatggt gatatacctt tctaaagttt ctcttgacat atgataggga 11520
atattatata ataaaattga ggtgataaac tatggaaaat aataacaaca gaataagcag 11580
agaaacactt tacatggaag ttgctaaact tatgacagga agatctactt gcctaagaaa 11640
agcagttggt tgtgtattag ttagagataa caggatagtt gctacttcct ataatggagt 11700
accttcagga gtagaacatt gtaaaacttg tttaggacct ggatgtgata ttgtgatcca 11760
tgcagaagct ggcttaataa gttactgtgc taaacatggg atctcaactg aaggaactac 11820
tatgtatgta accttatccc catgtgagag ctgtgctaac ttaataatca atgcaggaat 11880
taagaaagtg gtatatcttg agaaatacag attaagtggg ggaatagcta aattaatttg 11940
taatgatgtg gaggtagaac aatatgagta aaggaaaatt ctttagtgat agtcaattaa 12000
aggccataag agctatggcc tctgcaggta ttgaggaaat taaaaatccc tatgaaagta 12060
acattgatga gtttgataga ttatcagaag aaaaatatat cagagaaaaa ttattgtggg 12120
aagaaagccc aaggattaag ccagttccag ttaaaggatt ggaagaagtt aaagaattta 12180
tagatggact agatactgaa tgtgaagtat ttcctgtact aggaactaat aatatagttg 12240
tgatagaacc tttagtagga gtagatctag tacatgataa agtaagagaa gcatttcaag 12300
gatgtaacat ttccagagta tcaagagcaa tatttattac atttaaggag gataataaca 12360
atgagtaaat taatatgtaa aagaagatgt tatagaaggg atccagcagg taactgctta 12420
gcatatacaa ttccatatgg gccaggagaa aaatgcccta gccaatgtaa taatccacat 12480
caattaaaga aggagtgtca agacatcatt tctaaaaatc aacctaacaa tccaatagta 12540
aaagaatgtc tttacttgat agagaagatg gatctaataa ttggggttga gattaataaa 12600
gcttatgagg aagataagaa tagaggttca ggtggaggat ctaagaactg taaacctaat 12660
acagcagctc taaagcaaaa gatgaaagat aatagaccta ttgaatgtaa acaaactaaa 12720
gaggagaata aggaatatca ggaagaatta aaacactggc aagaagacca taatgaaaaa 12780
ctagaagttc ataaatcagt tggatatggt atgaacaggg gtaaggtaga ctcatatact 12840
ggtatagaac ttgatactaa aggtaatcca gtatattccc cagaagaaat acaaattgca 12900
gctgataacc tgctacagga aggaagaata gatgtggata tgtatgaaaa aatattgaca 12960
ggtgaaataa aattataaaa cgttgacata attaatccag atgtggtata atagtggtat 13020
aaatccatag ttaattttgc catttggttg gtattactag cattaaggtt agtgtgccaa 13080
ccatttttca cgtagtaaag tctggatttt aagggggtga tattgtatta tgagctataa 13140
gtcatacact gaaccagagt actgtactcc tgaacattcg aagaaagcca aaaaattgat 13200
atataatgcc ctgaaggctg atccctttac acatcctaaa caattaagaa accttgctaa 13260
ggtatcacag acctttgtta ggcagtgtct aagagagttt aaagagcagg gcataacatt 13320
tttagaagaa gaaggtgata ttgtggcatt gagtagcaaa acaaaaaaga ctattaagac 13380
cttggctgag aagtatgatc ttgatgagaa agaagagttg tttgtatttc attacatgaa 13440
gtgctttaat gcaactacag cagctgctag ggcaggctat cctgtgaata attctcatgc 13500
acatgggtct aaactattaa agaagaagaa aatacaggaa gctctaaagg ctatcagagc 13560
cgatagagat gaagaattac tatgcgatgg tatggatgta ataaggcagt acatgaaaat 13620
agcctttgca gatataactg atgtgtgcca attcaaggat gacctagttc tattaaggtc 13680
tagtaaggct gtagatggta ctctaatcaa tgagattaaa caaagtaaag acggcgtatc 13740
agttaaaatg gaagatagac ttcaggcact aaaaagactt gaggtatact tcggcttaag 13800
tactgatgtg atgaaagttg aacttgaaaa agttaaatta cttttacaaa tacaggaaaa 13860
tcagaataat gctggtaagg gaaaagatct tgaggttctt agagctaaaa tggctgatag 13920
aaagaaaaag aatgctaagg gaaggaagag tgaataatta tgaatgaaac tgaatttacc 13980
atgattaata gccttcctct ggaggttaag gttagaaaaa ctatgttaag agtacaagaa 14040
tgggtagacc attttggtga agatggagta tatatctcct gtagtggggg taaagatagt 14100
ttagtattag tttatatcat taggaaactt atgggactaa agaatatacc tattgtttat 14160
gtggatagtg gcttagagta ccctgatgta agagaaaacc tattaagaga atatcctgat 14220
gtagtgatta ttaggcctga aatgaagcct aaggatatta taaagaagta tggctatcct 14280
gttatatcca aagctaatgc gtctatatta tacaaactga acaactataa actaaagcca 14340
gaatacagag acaaattatt gaatgggaat gaaaaaggta aggtaggtac actgcctaag 14400
aaatatcata gcctgattgg atgtgaagtt caaataggaa aaggttgctg taatgttata 14460
aagaaaagaa ctttccatag atatgagaaa gaaacaggaa gagtaccatt cacaggagaa 14520
ttagctgtag aaagtaatct tagaaagaag gcttatctag cagaaggatg taatgccttt 14580
caaaggaaat taggacctaa atctatgcca ttaggttttt ggaataccaa tgacgtacta 14640
actatgtgta agaagtacaa tatcaaaata cctaaagtat acggagatat aattgaggac 14700
tacacagatg agaatggaca agttatttat aaaacaactg ctctaagtag aacaggatgt 14760
acttggtgtg gctttggtgc tgaatttgat gataggttta taaggctaaa ggaagtccag 14820
cctagaatgt atgattatat tattaatggt ggagatttta atgacctagg ctattgggag 14880
cctaatgaag aaggtttagg tttaggatat gtactagata agctaggaat aaaatattag 14940
gggggaaggc ctatgtttga tttagataga atgattgaaa aattagagct agatgcttta 15000
gttgataagt tccacatcaa caaggaagcc ctagaagaaa tatcctttga agaattagta 15060
gatctatatt atgacgatcc ttgtggattc tcagaagata tattaggagt acaattagat 15120
ccacatcaaa aggatatttt aataagtgta agagataata agagaacctc tgtaaggtca 15180
ggacaggggg taggtaagac tatggcctgt gcttgcctta taatttggta tatgaccttt 15240
aggcctgaat gtaaggtaat tgctacagct cctacatttt cacagttaac atctgtatta 15300
tggcctgaga tatcaaaatg gctacaggga actattgtgg aggattattt tacagtaaat 15360
aaaactacta tgactaggaa ttgtagtcct atgacttggt ttgctatgtg taaaactagt 15420
aatagaaaag agaatatgct tggtcttcat agtgagttca tgctaattat atgtgatgag 15480
gctcctggaa tagatgatga gatcctagaa gcattacttg gtactatatc agatgaggaa 15540
aataagataa ttttgattgg taaccctgtt aaaaatacag gtatcttctt tgattctcac 15600
cacagtctta aggaaaattt taaatgtatt catattgacg ctacaaaagt tgatagaact 15660
gataaagaaa accacaaaat gttaaggaaa aagtatgggg aagagtccaa tgtctacaga 15720
tgtctggtaa aaggggactt ccccttagaa gaagatgaca tatttataca attatccaca 15780
gtatatagag gtatcaacag tgaaataacc agacctacag aaccagttac catagatatg 15840
ggtgtagacg tagctagatt tggttcagat aagacctcaa tcatgaccaa aatcgatgga 15900
tatgtggaag aacctatcta ttatcaaggt aaggacacca tgaaaacagt gggagaagtg 15960
ctaaggaagg ctaaatccct acatggtgag ttccccaaac tagctgtagt tgtgaaaata 16020
gacgacactg gtgtaggtgg gggagtaact gatagattaa aagagataaa acttgaagaa 16080
aaactggact ggttgaaggt agtaccatgt aagtttggta tgccactaaa gagagggtcc 16140
caggcttatg ccttttatta tgatacaacc acatatatag cctctgtagt taaggaatta 16200
ctggaggatg atgagttaag actaccagat aatacagatc ttgtgggaga attgactacc 16260
agaaagttta aagtattcaa ctccaaaggt caaataaagc tggagagtaa ggatgaaatg 16320
aagaaaagag gactacattc tccagactta gcagactctc tattcttagc ttgccttcct 16380
gttggatcta aattaagaaa aatctagtaa ctgatatgcc tattaaagcc aataattttt 16440
attctgatat gtgagatatc agttttgaaa ttgactaggc tctatattag taataatgtg 16500
atatagaagg catatcagaa ttaccacctt tatatatatg taatataagt atactatata 16560
ctatataatt tatttcataa attaatatag taatatagta tacattaata attatataat 16620
attatatggg gaatatatgc ggaaaatctt ccggaaaata atataaaaat aaaacgttga 16680
caaatttttg tgagttatgt tataataaac gtatcaaatt atagttattt ttaaatcatg 16740
tgaatggccg tatttgacac tatatgtgga gttgatgtag tcctccctga aatatgaggc 16800
catctggatt ttataatttg atatataatg taagaaagga ggtaaagtaa ctttgaaaaa 16860
gatatccgcc agaagtatat gtggagaccc tatccaaaag tctttggatc cagtggataa 16920
atccaatcaa caaaccagtg tacttggaat tgaagttgat gagatatccc ctgactatga 16980
cccccaacta ttagaagtga ttataaggga gtctacaata ataccccaat gcaaaaagac 17040
ctaccgaaat aatattgctg gattcggaat aggtattagg tatattgaga aggtagatga 17100
acctactcca gaaatggaag ctgaatataa acagctggaa gaattaattg ataccctgtc 17160
ttttgataaa ccaaccaaga atgtctttga agagatagtt gattggagag aaggacttgg 17220
gtattctttt ttagagatcg tagaagatcc taaagaccca gctctcgtaa aagagataaa 17280
agaaattgaa gaccctaaga caattaaaat tcttaagcta gatgatgaga gtaccgagta 17340
cactgtaacc agatatggta aacagtacac tagaagaaaa aggttcagaa gatttaagca 17400
gactgtaaat aatcaatcaa tcttctttaa ggaatgggga gatcctagag atctagatat 17460
gaatactgga aagtattatg aaaagggaga gatacccctt aaagatcgag ctaacagtat 17520
acttcatttt aaaacaggtt atggtgttta tggtacacca agatgggaag gtcctatgtt 17580
agccgctcag ggttcaagaa aggccgaatt attaaactgg agatatttta ctaatggtaa 17640
acatactcca ttaatgttac taattcaagg tggtactctt aatgataaat cttggaatga 17700
attaactgag tacatgaata gtgtaaaagg tgaaaattcc cagcatggtt tcttagtatt 17760
agaaactgaa gctgcagaga aagacactgc ccttgatgac tctaagccag ttactgttga 17820
ggtcaagccc ctatctgaaa tattacagaa ggatgagtta ttccaagact acatggataa 17880
tactaggaaa aagatacaat cttctttctt actaccagat atatatgtag ggtactccca 17940
agattataat gtggctacag ccagagaggc tacaagaaaa actgaagagc aagtatttgt 18000
tcctgaaaga gaaaacctaa catggatact aaataatgtt attctatctg gctatggttt 18060
taaatatgtg gaagcttact ttaagagtcc taatattgag aactctgatt atcagaaagg 18120
aatgtttgct atagcctcta gagctatgac tcctaatcag attaatgagg agtattattc 18180
atcaatcaat aaggactata ctcctattga tgaagaatgg gcaaatatcc ctacttactc 18240
agttgcctct tctatgagta ataacttaac aagtataatt gagaaggctc aaaactccaa 18300
tgggagtgat gaggtgatag ctgtacttaa agaggctaag aaacagttat taaactatca 18360
attaaaagga ggtaagcaat agtgggtact gatgagttga tcgctaaaat tgataagatc 18420
ttaaagaatg aagatgatag ccttaagaaa gaattgaaag aagctggcta tgaggagatt 18480
gattatacaa tagaaaagat ctctgaatta gaagacacta tttctgatct ccttacagaa 18540
gaattaaaac atcttataaa agatggagac tttgaaacca ttgaagaatt tatggaatat 18600
gtggagtctc aaataggtac agataatatc gatacttctt tagcagaaac tcttaagagt 18660
tgttatcaag aagctataga aaatttagga gatacttatt tacaaggtat tgataaggac 18720
ttatccctaa ccaaagtctt ttctccatat actactacct ttatagatga gtggtcttct 18780
aaacttgggg aaatgatgaa attaacttcc catgaaaaac tacagaatat tctggataag 18840
gctcttgaag atggagagtc tgtagaagat gtagttggta aattatctga gacttatggc 18900
tttagtagaa ctagagctag agctactgct attactgaaa tgttaagagc ccactcatat 18960
gctaaggatg aggcttttaa tcaatgcccc gcagtaacag gaatacagtg gaaacactca 19020
gggggaaggg gtattcaccc aaggcaacac catgtcgatc tggatggtac tgtcattaat 19080
aaaggtgagt acttcactgt aggaatttat gaagctagat ttcctagaga tatcaactta 19140
cccgcttctg agactgtgaa ttgtcattgt actcatgggc ctgtaattga taaggatata 19200
ctaggattat ctaaagaaga aaaagaagct atcagagatc aagtgatagc agaatataat 19260
ttgtaaagga gggagagtaa atgccagtaa aatcagttaa gaaagctatt gagatctcag 19320
atgctaaaat tgattacctt tcattagttg ataaagcagc taataaatgg caatggattg 19380
tggctaaaga acttggagct cctaatagat ttagatatga taagccaatc ttaaagtctg 19440
agtctgaagg agataatcat tttgttattg gtgtagtata tgagccaatg actgctgact 19500
accatgataa ctttatgact gaggaagaga tcaaaaaagc ttgtgagtgg tttaatgaaa 19560
atggccttgg tagtgattta caacacaatg agaacactct tgagggtgtc aagattgtaa 19620
aatcatgggt aactgaggaa gataccacaa tagaaggtac agtagttaaa gctggaactt 19680
ggttagctaa agctgagatt atcgatgatg atctatggag caagattgag aaaggcgaaa 19740
ttactggatg gtctatgggt ggtactggta aatattctac acaagatgta gatattgatg 19800
agattgagaa gtctataggt caatctatac ttgataaaat tggaaaggcc ctagggttaa 19860
ctccaaagca tatacagaaa ggtgcatact ctgaaaaata taaagagact gcaaaaagaa 19920
ataatttctg gaatgcagtt agtaccttag aagatattct atatccatat tctagttggg 19980
attacactag atcttttgaa agtgatgtag ataagataaa ggaggctctt caagaatttt 20040
ctgaaactat ggagggaata ttattaggaa ctccagaaga agtagctaaa tcttttgagt 20100
taccagtcaa taaggcaggt aagaaaatat cttcaaaaaa tgtggaaaag ttaaaaacag 20160
cacaaagtgc tattaatgaa ttattagact cagtggaggg ggaagaagag atgaatgaaa 20220
ctcaagtaca aaaaatgata gacgaagcta tcaaaaaggc attagatgga actaaacctg 20280
ttgagggtga agttaataag aatgaaggtc aacaagctac tactgaagat atagttgcta 20340
aggctgtgga aaaagctatg ttagaggctg gcttaatcca gaaacaagaa gaaaaaactg 20400
aggaagaaaa aatagctgag gcagtagcta aagccatggc tccatatgta ggaagtaaac 20460
aaattgctaa aaacggagaa gaaggacaag aagttcaaaa atcagtattt aatggattat 20520
ttggtggaaa ccaagaataa taacaattag gagggaaagt tatgtcaaaa gtaaataaag 20580
ctgtatttaa cgctgccgta ttaaatggtg gaggtaaatt aaatccagaa caagctaacg 20640
cttttattaa aatgataatt gatgagccaa caatattaaa agaggctaga actgtaccaa 20700
tgaaaggaga ctctaaaaag attgagtcaa taggatttgg tcaaagagta ttaaatcctg 20760
cagctgaagg aacagatcca ggacaatcta aatattcagc tccaactact ggaacagtta 20820
ctttacaagc taaagagttt atagctgtgg ttaagttaac tgatgatact atagaaacaa 20880
atatagaagg aaaatcaata gaagacactg taatgcaatt attagctgag agagttgctc 20940
ttgacatgga agaaataatt gtaaatggtg atactggaca tagtagcgat actttcttac 21000
aacaattaga tggggttaga aaacaattaa ctgctcatgt agtagatgct aacaaagctg 21060
ctttaagtaa agacctatta aaacaagcta agaaagctgt accagctaag tatttaagaa 21120
acccaggtga atggagattc tactgctcaa atggtttaga aactgattgg gtagacttag 21180
taggagaaag attaactgct aatggggatg actcatttat aaatggtact gttaaatctg 21240
cttatggagt acctgttaaa ggtatagcta tgctacaaga ttatgacgac tcttctactc 21300
aagtatcaga cataatctta actcacccta agaatatagt tataggtatc tcaagagata 21360
tcagaataga gttggaaaga gctcctagag aaagatctac ttatgtagtt ttaactctta 21420
agatggatgt taagtttgaa gatcctaaag ctgctgctaa gatagttaaa gtaaaagctt 21480
aatcatgaag gcccactgta aatgtgggct ctttttcatt agaaaggaga gatattatgg 21540
ctaaggtcaa attagcaaag ggtaaattac aaactctggt tattaagggt catagaatta 21600
cccaagacca tgtagaagaa gttaaagata gtctagtaaa ttatttaaaa cagaactatg 21660
atgtgattgt catagatgaa cctgttgagg atgaacctct agaagatttt actgaagaag 21720
aagtaccttc tacagatgac aataaagaag aacctgctag aagatttaca aacaagaata 21780
aaaatagaaa cagatagggg tggctcatat gaatattact ccggatgaaa taattgactt 21840
ttcaagtaat cctgagatta agaaaagaga ccccaaaaat attgagatgg atatattaag 21900
agctaaagct aagatctcag gaatttttaa aaattcaaat aaagactatg atactattgt 21960
ggagaaaaat cccaatagta aagaattaag attagctcat atactgtatg ctgagtatta 22020
tggactacaa tccttggtca ttagcaaggc tgatattcaa tccgagacct atgatgacta 22080
ctcatataca agaagaactg ataaggctgt tatagagcct gatattatgc ctctgattaa 22140
acctttctta gaagatgata gtccatctat cagaacaact actatgagat taagggtact 22200
gtaatatgat tagaaattca tataacaagc tcttaaatca tagatgtaat atataccacc 22260
tattggaaga ccctgaagat cctggatatg gcctagccca tacccctagc tttagttatc 22320
ctgatgagcc agacatgatt gaggtaccct gtcattttaa atcaggaact ctagaaaaac 22380
ttgagtacaa agaacccagg tttaagatta cccaggagta ctcagtagat tttgctattg 22440
gaactgatgt tagacaaaat gataaaattg tggaccttga gactggtatc acatatatag 22500
ctaaagctcc tagggatata agaggtctaa aaattaaggt tggtatagag gctaaggatt 22560
ttagatagtt atggcttcaa attctggagg catagaaata gatgctaaac aaatagaaag 22620
gtttataaag aagctaagag atgccgctaa tagtgatttc aaaaagcaat tagcagtatg 22680
gtttgaggct gcagggatag acttccttga tgtggttcaa gatgaaataa ttagactaca 22740
agtagtagac actagattat tacttaactc ctttcaaaaa gggggtaaga atggtatatg 22800
ggtaagtagt gatggaggac taaccttaga agttggtact aacctaaaat atgctaagtg 22860
ggtaaacgat ggtcactata ctaatccaca aggacaatca tttagattcg ttcctggcat 22920
atggtcagga gataaattta tttatcaacc aggagctaag actggtatgg tactaagaca 22980
aaagaaagta cctggtaaac cttattggga caatgcagta aaaatatatg aaaaagtatt 23040
tgaacaggct ttccaaagaa aactagaaga ttggttagcc gaagttttag ggggtagtta 23100
ttaatgacag cagatatagg gagtattgct aaattcatat ctgatctaac tggaattact 23160
accctctatt ttgagcgaat acctgaaaag ttcaagtatc cctcattata tttcaagaag 23220
gtatttaatc ctcagaatga tactctttgt agttatagaa ctgagaatac tttaagagtt 23280
aaggtatttg aacctaagga ctctcttgaa attgctcaga atatagctaa tgctatatac 23340
ttgagaaata gaaaaattcc catacttaat agggatggta gtaagaccaa caaaaatctc 23400
aaaataaaga atgtggaagt atttgaaggt gaaactgaag aagattctca ggtagagata 23460
aactatgata ttgtggttag tctttataaa gatccaactt acttcgaaaa ctttgagata 23520
acaaaggggg taaaatagtg aaagtacaaa aattttccat acctcaatta agagcccatt 23580
gtcaacaact tttccaggtt gaggagtttg tcttcaacgc tgcagttcaa tcccttccac 23640
ctgatgaaga gttaacaata aaacaaacta gtcagattat aaaagactgg ctaaagaagg 23700
aggtaaaata gatggcagga ggaactttta ttccaggaca aaataaagta agacctggta 23760
tgtatactag atttgtaagt ggtggactta atgagcctaa gcttaataat ctaggagtta 23820
tgatcttacc attacaactt aagtggggta atgctggaga tgttatcaag gtaaccaatg 23880
aaggtggcct agaagcatta tatcaacttg ggtatgacat aactcaccct actgtattac 23940
tggttaaaga ggctttaaaa gaagctgcaa ctgttttagt atataaccta acaggtggaa 24000
ctaaagccaa agctgtagta gggtctctaa catttacagc tactaaagct ggagaaaggg 24060
gtaataaatt atctctaact attaagacca atcctcttgg aggaaaggat atattattat 24120
atcttgaggg gtctaaaatc tttgaggcta caggagtaac tgaaatatca acagttaagt 24180
ctcaatggtt tacagtaagt ggaagtggct ctatagatga gactgctaag actgagttta 24240
gtactggtac ttctactcag atgaataact ctgatgttat taacttctta gataactgtg 24300
agttatacaa ctttaaaact atggccttcc catttactga tagtgaatta cacactgctc 24360
tattatcaaa acttaaatac ttcagagatg aagttggtaa gaatattgtg gcagttactt 24420
ccgactttgc tacttgtgat tacccatata tcatcaatgt taagaatggg gttgtattag 24480
agggaggcac taatttaaca gcttctcaag caactgcatg ggtagctggt gctagagctg 24540
cagctggata tactgaagac cttacttata aagcatatcc tggagcagta gatgctaatc 24600
caagactaaa acactctcaa atagttgatg gattaaaaac aggtcaattt atattctcaa 24660
ctcaaactga ctcaaattct gaaagagtaa aggtagtagt tgaggaagat ataaatacac 24720
accatacttt tacggaagag tgggatgaaa gctttacaga taataaagtt gtggctgtat 24780
tagatgctgt tatagatcta ggatctcaga tcttagttcc taatgttttc cctaaccaca 24840
aagatggtat aaagctagct caggatagac taataagttt attaactaca ctacaagctc 24900
aaggagcctt aaagaatgtg gatcctgact ctgatgtagt ggtagatgaa gaagctagta 24960
aaggaaagac tatgtatgct actatactta cacaacctgt aagaactttc aagaaatact 25020
ttgtaactgt taagaataat taaggaggta tagcccatga gtgaagttaa tagaatgtct 25080
gctagagagg gtagagcttt tgtatcaggt aagcaagtaa ctagcttaat aaaactagaa 25140
gctctattta ctcctgaggt gactaagaaa agggttatgg gtcagaaagg attaacctca 25200
aaagttatag gttatgatat aactggaact ataactcaat tcaaagctac tccaatgata 25260
agagaagcta ttaaagaata cttaaaaact ggtatattcc ctaagatgga tatacaagct 25320
gtatgtgatg atccagattc tgactatgtt aagaattatg gtgaggacag agtacaatta 25380
ctaggagtac aattagaagg agatttacca ctaattggac tagatgctga gggggaagaa 25440
attcaagaag aaatatcatt ctcagcttca gaagtaagat atttataaaa gattaagaat 25500
attgtggagg tagattatta tgtcaaaatt tagtatggaa tttttcatgg aaaatgataa 25560
gggtaaacaa tcacccgaaa ttgaagttga aggggtagct agatttagtt tccctgaggg 25620
tcatgaatta gcgggccaac ctataccttg gatattaaag ccaatcacta ctaagcaaat 25680
ggaggatatc caaaagagaa atacagtaat tactagaaac aagggtataa ctaactcaat 25740
aacagaccaa agaagagcta ctagggaaat gatactagaa acaatagtat tccctgactt 25800
taaagatcca aaatggttag agtctactaa gtttgtggat ccagtagatt tacttatgga 25860
tgtactaagc ttaccaggag atttcactag aataactagt gaggttatgg atataaatgg 25920
attaggcgat aataaggatg atctaatcaa agaagcaaaa aactaataaa ggccgacact 25980
agtgcatttt gggctcatgt gttattccag aagcataata tactaccctg ggaatttgat 26040
aaactatctc ccaataggaa agcctttctt atagcttcat gtgaactggc tttagaagaa 26100
gataaaaaag ctagggataa gttaaagaaa ggctcttaat cataaaggag ggatataatg 26160
gcaagtcaag ctgcatttac tgccatattc tctgcccaag ataagatctc tggtgcattt 26220
aaccagatgg ctgatagtgg taattccttc atgggtactc ttgccaaaat tggtggagta 26280
gctgctggag tattctcagc taaggctgtc tgggatttta ctaaggacag tgttaaagcc 26340
tttggggatt ttgagcaagg tttaaatgag gttatgactc tattacccaa ctctactcaa 26400
gagacctttg ataagatgtc cggccaggta aaaaaattaa gttctgatat agggatattg 26460
cctgataaag tagtacctgc tctttatcaa gccatatcct catcagttcc agaggataac 26520
gtttttagtt tcttagaaac agctggtaaa gcttctatag gtggtatcac atcaatagaa 26580
actgcggttg atggactaac tactgttatt aactcatatg gagctaagaa tatggatgtt 26640
caaagggcct cagaccttat gtttactaca gtaagacttg gtaagactaa ctttgagcaa 26700
ctatcacaat ccctattcaa tgtgctacct tcagcctcag ctgctggggt atcttttgaa 26760
gaagtatctg cctctatagc tgcattaaca gctcaggggg ttccaacatc tgtagctact 26820
actaagataa gagctgctat agatgagcta tctaaatcgg gaactaagac tgataaggtc 26880
ttccgggaaa tagctggtaa gggtttcaaa gacttcatta aaggtggagg taatatccaa 26940
gaggcccttc agatgttaga aaaacacgcc cagaaaagta atctgggtat caatgacctc 27000
tttggttctg tagaagctgg ttcagctgca ctagccttga ctggtgaagg taccgaggcc 27060
tttactaatg ctcttaatga gatgaagaat agtgcggggg ccactgaagc tgcctatgaa 27120
aaaatggata agggctttaa tagaaccatg gagagaataa aatccaaagc tgctgtagtt 27180
atgttagatg tgggagatgc cctagcccct gccgttgagg ttatagctaa tgctgtccta 27240
aatggtatca catttataca agaaggcttt gaaaaactag gacctatatt ctcaatggtt 27300
gtttctgctt tccaaggttt aggagagact atatattaca tcatgaatgg ttgggagttt 27360
ggggatgcta tgcaaccctt tatagatcta accaatgata tattatataa gttcttacct 27420
gctgatgtgg cagatcaaat taatgagaag atatggagtc ttggagaaga catatataat 27480
aattttggag agatcttagt aggtctacca gattatttat cagaggctaa caataatata 27540
caagaagcct ttagtggttt agctgagcct atcatgggag tattagctcc tataggtcaa 27600
gtacttcttg atatgtttgg aactttagct tctatattta tgaataacct aattccagcc 27660
atagacttag tagttcttta tgttcaagga tttactcaag tatggcaaac tgtattggaa 27720
gtgttaggtc ctatattaga agtattaggt tctatgttct ctgctacttt tacagctctt 27780
ggagaattga taaccaatgt tttcatgcca attgctactt ctttagctga ggcctttgct 27840
acttatatag ctcctgcaat ttctcaagtt gtggaagcta taggtaactt cctaatgcca 27900
atattacaag gactagcaga ttggtggaat aatacactac tacctatgtt cttaagcttt 27960
gctgagaatg tagttccata tattcaatca gctattgaga gttttggtat gatattccaa 28020
gggttggtgg gtatgctaac tccactactt aaccttttag ccgcatcttt ccaaacagta 28080
tggcctgtta tatcctcagt tgttactggg gccgttcaaa ctatatcagg agtactacag 28140
ggattaatgc agatgttgag tggtttaata gcttttgtgg ctggagtatt caccggtaat 28200
tggtctaaag cttgggaagg tgttaagaac atctttgggg gtattttcca aggtatgtta 28260
tccctagcaa aagctcctat taatagtatt attggtatga ttaataacct aattggtgct 28320
cttaatacta tcaagatacc tgactgggta cctattattg gaggtaaggg tattaatatt 28380
ccccagatac caatgcttgc tgaaggtact gacttcttta cagataaagc tgccatggtt 28440
ggtgaagctg gtcctgaatt agttatgggt cctacattct caaaaatgcc tagaggctct 28500
caagtattac ctgcagataa aactcaggat ttactaagac aatctggagc aggaagtgat 28560
gatggatcta aagttatgaa cattaacaac tcatttaatt tcacagtaca ggctgaagat 28620
ggaacaatct cagaaagtga attaatgaga gtatacagat ttattgtgga aatgttaaat 28680
caggaaatgt tcgagggagg tgaactggac cttgcatagt aaatatcaat tctggttaat 28740
acagggagat actaggttta ggttaccagt aaatcccact gatataaata tctctgatag 28800
tagagataat agtacagttt ccattgaagg cttaggggaa ctctctttca taggagaccc 28860
taagctaaaa tctttttctt ttaattttga gttaccaagc tcctactacg atggatgtga 28920
gtatactgat attgcagacc ctagagtaag ttatgctaaa cttattgaaa tgggtaaaga 28980
tccagtaaga tttactatta ctagtactga tattaactta ttagttacta ttgataatat 29040
ctcatatact gagaaaggtg gagatattgg tactcttcaa gtaaaaataa aactaactga 29100
gtataagaag cctgaggtta aatcagttag tttcacaatt ccaaaaccta aactagccta 29160
tagtggtaat acaggaggta gttcttctgg aggtactcaa ccttgggcag ctactactac 29220
taatgtttac tcatggctat gggtaagaac tggacctggt atcaataatc ctactaaggg 29280
taagatatgg cctaatgaac actttacagt attaaggtct caaaatggat ggcattatgt 29340
aaagtataaa ttagataatg gttcaggata taaagaaggt tggagttctg gtgattatat 29400
aaggaggtta taatatgcaa ttaattgtgg tacaaggaga taaatccttt gatattacaa 29460
atacctccag tggtaagagt tgggcagggt cttcttcatc tgcccctcgt accctatcat 29520
tccaatataa tgctcaagat atacatccaa tttatgatgg agatcaggta gtactagtcc 29580
atgagggtaa gaatatcttt aatggtatta ttatgaatat ctcagataat gagaaaaagc 29640
atttacttaa ggttaaagct tatgacccta tgatttattg ggctaataat aaagatagct 29700
ttgtattcga aaaccaaact ggcgaagcta tatttgagtc attatgtaaa gcctttgggg 29760
ttgcccctgg agtagtagct aaaactggat atactttctc tagtttagta tgtgagaaac 29820
aaactcccta tgatattatg aagaaggtaa taaaagaggt ttatgaacaa actggagaac 29880
agttttatat ttattataat tctattacag gtaagtttga ttttataaac cgaagaacca 29940
ataatgaatt gtggaaattc gaatatggta aaaatattgg aactttttca agaagtagaa 30000
actttgatag tattgtcaac caggtaaagc tagtatgtaa atataaacag ggtaagggta 30060
aagatgatcc cgagattact ttatttgcct ctgctaaaga tgatagcagc caaaagaaat 30120
atggtgtatt tcaattatac gatactatca gtgatgaact aaaccaagcc caattaaatg 30180
agaaggctag aataaagcta gaaacaggta agagacctaa aactgatatc tctataactg 30240
ttataggtgt acctgactgt attagtggta aagccattgt ggttagtata ccacatttaa 30300
aaattaataa gtctttctac attgagtctg atactcataa atttgatact gactacacca 30360
tgaacttaaa acttaaatgt actgaggagg gtgatattta atggctaata atttattaga 30420
ctgtgtaaaa caagtatata gtaatatgtc cacccctcta aatatttata gggctaaggt 30480
acttagtact agccctctaa aatgtgagtg tcttcacaat agaaaattag tactatctgg 30540
aaatattata gatgttccaa aacatattga gtctattagt gagggcgata ctgtttatct 30600
tatgccttat gatgatggac aaagatattt tatgttaggg atcttaggag gttgatataa 30660
tgctacctaa tggctttaaa gataccctta tagttgatga gaaaacaatg gagaatacca 30720
agacctataa actggatcta aatcttaata ggatttatga taatgtggat ggtcttgagg 30780
ccttaaaaca atctttatat aaaagattaa taactcctaa aaatgcctat atgatttatc 30840
aagatacttc ttatggttgt gatattagag ctttgattaa tgactctcta gtaactcctg 30900
aacttcttga aatggagatt aatagggcta tccctgaatg tctcttagaa gatgatagag 30960
tgactaaagt aactgatcta aagtataatg tgcaaggtaa gaatgtatat attgaagtat 31020
ggataaatac catatatgga gatacaaaac tggaaggggt gatataatgg gattttctga 31080
agatttattc tcccgaacac ataaagaatt actagaagaa atgataggcc tatcccctga 31140
tgatattgac actaggactg gaagtttcac ctatgatatg gctatggttt ttgctgttaa 31200
gctagctgag ttctatgctg ttaatgttaa ccaaatatta gactcagcat ttctaagtac 31260
tgctacaggt aagttcttag atgagagagc tgccaatgta gggatatcca gaataattcc 31320
aactaaatgt agaaggtcag ctaaatttgt tggctatgaa cccgatgttg gttcaagatt 31380
ccaaggggga gattatttct ggcaagtgat agagcccaat attattgaat gtgagaccct 31440
aggttcagga cctaataaag tatcaacagg tacacatcta attcctatca atgccatagc 31500
agagcttagg agtgctacta tgggagatat acttgttatt ggtagagatc aggagacaga 31560
tgatgagtta agagaaagag ctactaaagc cactatacaa ccatcgggag atggaaatgt 31620
tcaacagtta actgattggt gtaaaagtgt agctggagtg ggtaaagctg tggttcttcc 31680
ctgttgggat ggaataaata ctgctaaagg tattataacc acagttgaaa ataagcctgc 31740
taatcctgag ctagttaata aagttcaaga atacatggat ccaggcaaga aaggtttagg 31800
tgagggtaaa gctcctatag gatgtgtttt tactgttgag tcagcccaaa cctttaatat 31860
cactgtaagg gctcaggtta agaaattgag ttcagttaca gaagaacaaa taaaattaaa 31920
cttcaagaaa gaactagaag agtttctaag cagtcaagta tttaaaatta ctagcctatc 31980
tataaataga ataggggcta tgcttttaaa tattgaggga gtagaagatt atacaaccat 32040
tacacttaat ggaagtagtc aatctttaac tattcctgct aaccaaatac caaccattac 32100
tgaggtggtg gtaacttatg tatagtctag aacaggttca aaaagaatta ctattgtatg 32160
ttcctgaatt ttatgataat ataaaagagg ctgataactt agtaaacact attatggcta 32220
ctgaacttca gaagctatat actaagggac aggaattaca agactatgtg gatcccaaga 32280
ccactcccaa ggaaggacta gaaaggtttt tcactgatct tgaattatat aaatactttg 32340
attacccacc aactgaccaa gagaaaagag ctattattac agtagttcta tctgcttgga 32400
aaaagtttaa tagtacatct atcaaaaatc ttactaaggc ctatacaggg ggaaaggtaa 32460
aggtagagtt catagctgaa gagagtaaga tcaaaattag ctttactgac ctacccgggg 32520
taccacctaa tctaaaggcc ttagaaaggg tattcaggga aagaatgcct gctcatttaa 32580
tatatcaaat tatattactt tatattactt gggagatgtt cgatagatat gattatatgt 32640
ggagcaagtg ggacagtcta gaccttacct gggaaaaatg ggaagaatat gctataactg 32700
aataaaggag gtatgcaaaa tgccaagtac tggtaaaact cctactctag gtttaaataa 32760
gtgggagtta acagataagc caaaaatgaa tgactttaat aaggataatg aaaacattga 32820
gaactttgct accgaacata aacagaaaat gactgaggta gaaaagaagt ctaagtctaa 32880
tgaggatagg ctagatgctt tgagggttgg tactattaat agacttgaat ggtataattc 32940
tgagtcttat acgggtcctg gtaatatcac atctattaca gatgctccaa ctaagagacg 33000
aggttttaaa tggtcaggta tgtcaacagg tccacaaatt cttgaaatta aaaccctgcc 33060
tatagaaccg aatagtgatt atgtactagc cttcctagag gatattgaag gtagttttac 33120
tagctatgag attaaactgg cttacactag tggaggccaa caatataaga gcattgtaaa 33180
gcaaggtagc acatcacaga gtaatgggta taaattaaaa actcataaat ttagtattcc 33240
ctctggagct actgatacta aaatacaatt tgtggtacag ggtgcaacct caagcacatc 33300
attaaaggta ctaaacctat tactagctag aggtaatgta gtacctgact ataacctagc 33360
ccctgaggat gtactgaaaa gactagacct attctttgag aaagatatta atttacaaaa 33420
ccaaataaat caaaaagctg ccaaagtgga tgtgatccaa agggtcgatg tacctgtgga 33480
tagggattgt aattccttta aagcaggaaa ctccttctgc tcctttgata tggggaatgg 33540
tgattttaaa aatacccctg agggaacact gccccagggg agtgctaggg tatttatctt 33600
aaggaatata ggtttaggtg atggtaagaa taggtttcaa caggagttta ttaatctata 33660
tccagatagt aacataaccc gatatattag aaattatatc tatgagagta actcatgggg 33720
taaatggtat aaggtttatg atgaagctaa taagccaaca ccacgagaat taggtgtagc 33780
tgcggttaat atgcccatgg ctgatacaga ccttaataac cataaggaac ctggatttta 33840
ttggggttat caaaatatgg tcaatgcccc tgtacccaat ggtgtagcaa ttatggaggt 33900
agttaagtat aataatgatt gtgtattaca aagatttacc tcagttagag cagatagtaa 33960
ccatactact tatattagaa cctgctattc aaataaatgg tctagctgga agaagattta 34020
tgatgagggt aataagccta ctccaatgga cataggggct tgggataaaa cagccaaaag 34080
actagagggt gtagatctaa acaccattac agaggggggt atttattcta cagttagaaa 34140
ttctattgaa aaaaatgccc caactctagc agatgggaga ttgattgttt tatcctggaa 34200
tacaggacac tgggcttctc aaatgttctt tgcagatggt ggaggagttt ttactagaac 34260
agctacaaac atggagggta ctggatggac gaattgggct gaaatatata gtaaacaaaa 34320
caaacctacc ccagctgata taggggcttc tcctagtaac cataatcata ataaggtata 34380
ttaccctata cctaaacagg ctattactga ttttaattcc ggtatagcta taactgaggg 34440
tcaacatcta gtatcttcta gtacagctat acctaattca cctcctttat atgctcaaga 34500
tggtagtagt atatatggta tacttcatgt atacgttagt gttggtgatt cttataatgg 34560
tgtgaataac tggatatggc aagactttca tgatactagg ggtaatcact actggaggta 34620
taaaattaat aatgctgact ggagtccttg gagagctgta tataattctg ttaataaacc 34680
aaccccccag gctatagggg ctgtagctac tgaaggtgat ggagtaataa atgggaaatt 34740
aactgtagac caaatagtca atgttagcac tgtgggagta ctagctagtc ttaggaatga 34800
taatatgaga gctttattat gtggagtagg aggaacagat gtatatctcc aaaataaaaa 34860
gtctaataaa tttttacagt taagagatga tggtgtatta gcttatagtg ggagtttaat 34920
ttatcatcaa aataataaac ccactccctc tgagatagga gcagcctctt caggccataa 34980
tcatgatagt gcttatttga agaaagaaga tacagctgct aatagtaata aacttaagaa 35040
ctgggactta gctgaaggga gtaataactt tgtgggtata ccatttatac cccatgatgg 35100
agtcatggag gtaggtagaa tggttgactt tcatgagtct ggatctgata aggattttaa 35160
tagtagatta gagtctatta atggagcctt aaagtgttgg caagacttct ctgctgaaaa 35220
tctatgggct aggaaatacc taagaataaa tgactggtat ggtggagctg aagatggaag 35280
attgtggttc aaacaagaga acaaaaagtt atatactgag aatattactg acttcgttgt 35340
aaatggtagg tctattgcta aaggagacta ttgggataat gacaatggta acgttagggt 35400
atgtcatctt gggggaggac taagattagt tcgtcaaata gttaagacgg gctatgctaa 35460
ctcaggtgga ggtgtaagtt atactataca tttctctaag gcttggaatt gggtattacc 35520
tatatctcta gtatcacata actataatga cgcttctaat aatacctcag gttattgtac 35580
tattgattat tggaataact catatttgaa tggtggatgt tatcaaatgg tagctggtaa 35640
gccaactcaa ataatactaa cctatttagc tactgggtaa gggagttgat aaaacatgag 35700
aatagtagga tttaattatt ctgtggaaac taataaggtt atagacctta tattctatga 35760
tgatgaaaaa gaaatgctga taaatcaggc agaagactca gaagatgtaa gatcatttat 35820
agataaagac catgaagaag ttactaaaat attagaccta attaatagac ctgaacctct 35880
atactataga gaggatagtg aaatattaaa taatatagaa gatattgtat gtctccatat 35940
accccataat tcacaagggc atttagcctt tgtggataac actgaggtta ttgatctaga 36000
caatcttcct gttatacaaa tgactgtaag tctatttgac cacataaaga gtggcccaac 36060
taaatttgat attagtaaaa agagatcaga tctatacagt atcaaagata aggacctatt 36120
tacttcatat gaggctatag atcctaatcc tctagatatt aataaattaa ttggttatca 36180
agcccaggct cttgctgata ctaaattaga gctaatgcaa actaaagcca taatgaaagt 36240
attaacacaa gaactagctg atataaaaat taaattgatg aataaataag gaggtagaga 36300
ttatgaattt ctgggaaatg gcttatagaa ttggagcaat aggtaaggac aaattacatc 36360
aggcagtaaa atgctcttct aatccagaag gagaaattac accagaggac tataaaagaa 36420
tatgtggtga agactttaat gaaagggtgt aatctatgga aaaagaacta ctaactcaag 36480
ttcttagcca aggtatcttt tgtagtctat ttgtatggct attatatgac actagaaaag 36540
aggctagaga aagagagaat aaattaaatc aagtgataga taagctagtt gataaattca 36600
acgtagttga ggatattaaa gaggatgtag atactataaa agatcaccta tttagtaaca 36660
aaggttaaat tataagccta gcaggccatt taaggcctgt taggttttat ataagaaaat 36720
atattaaggg ggtggttaaa gtgctgtagc gagcctctca ggcccaataa aaccctataa 36780
tatttaaagg agatgttaat tatgaaaatc aataaaagat taagtactac taatgttact 36840
ctagatgcaa ataatccaga gtatataatt atccacgaaa ctgataatac ctcttggggt 36900
gctggagctg aaacccactg taaggcccaa gcaaatggaa atataggtaa agccagtgtc 36960
cattattatg tcgatgatac aggggtatat caagctgtag aacacaaaca tgccacttgg 37020
aattgtggag atggtcataa tagatatggt atcaataata gaaatacaat atctatagaa 37080
atatgtgtta atcctgactc tgactacaat agagcagttg ataatgctgt agagctagtt 37140
agatatctaa agaatggcta ctattctaat tgtaaagtag taagacacta tgatgccagt 37200
agaaaaaatt gtcctagaag aatgatagcc aatggttact ggaatacatt cctagaaaga 37260
gtaaattcag gagagagctc aagctcacca ataatcaata accctaaagg attctatgag 37320
tctaatgaga caagaactaa tgctacttta gtaggggaag gttcaataga agttctagat 37380
gaggattgta agccagtccc tggtagattt attgatagcc tagataattt atttgttctt 37440
ggaatatatc cttccagaaa tttcatagaa gttgtatatc ctggaaagga taagaaatac 37500
catgcttaca ttgacataaa acattatagc agattaagtt ttgactacca catgaaatat 37560
caaaatgata atggaataac ttatgtgtgg tggaaccctg aagacattaa tgtcaaagat 37620
cataatgaag aattacagcc tggtcaaaag gctagcccaa tgtatagaac taagggctgg 37680
ttaagaataa cattttatag aaaggatggt actccatctg atggatacgt tcgttacgag 37740
ggtgagcaaa gccagaaatt ctatgaggat gttaaacaag gaatagttaa ggttaacact 37800
tctcttaatg taagagatga tgtgaatggt aatataatag gctcagtatc taataatgag 37860
agagttacta tattaggaag taagaatggt tggtatcata tagaatataa tactagccat 37920
ggtaagaagc aaggttatgt aagttcaaaa tatgtagaga taatttaggg ggtatttatc 37980
atggaagaaa atttagtaaa tttaatattc aattacattg atccacagct tttaataatc 38040
attgtatcat gttactgttt aggtctgttt atcaaggcta agccttatat accagattgg 38100
tctataccat taatactttt ggtattcagt atatcattgt caatcttata tatggcaatt 38160
cagttagaac taggttttac agctaagacc tttttgaatg gctttataca aggtttaata 38220
tgtgcagctg tagcttcctt tggtaatcaa gtatggaagc agttaatgga taagagaaaa 38280
tcagatcctc ctgtaaatca ataggggata gaggttgggc ctactttatt tagaaagtag 38340
gtccttaacc tttttaaatt cttcttcatt atcccattcc cattgctttc ctggttttac 38400
aacttctttg tttcttaaga ttcttcttgc ctttttacca ttcatattta attcttcaag 38460
tatcatcttt aatgtgaact taccttcctt agtttttggt tcagcttttg gttcatcctt 38520
tttctctggt tccttaacat ctgttagatc aagagtaatt ttagtttgtt cagccttttc 38580
ttcaggttca gctggttgtt gtacctcttc aactttttct tcttgtggtt gttggatttg 38640
ttggatagca gctattagat cagccttttt catagtccac cagtttttaa cttttaattc 38700
ctttgctaat tctttaattt caacagcttt catagtattt aaatctttca tatcatttac 38760
ctcttctttc ttatttgttt tatctacatt ttcattataa gagatttcct ctggtttgtc 38820
agggatttct tttaataatt ttggacctcc ccagttacat tcttctccat cagcattatc 38880
aaagaccttt cttccttctt ctgtgtaacc tattacctct ccatcagcaa atactaaacc 38940
ttttttaaca agtgatgaga tcacacctct agccttagtt ccttctatac ctgagttttc 39000
tattactgag aatacccata cacagtcaac atctagtccg tcatcaaatt cattctttct 39060
catagcattt aatatcttta tttccattgc agttaagttc atatttaaca tctcctcttt 39120
gtttttgttt acaagtttat tataagagtt attgccagag atgtcaaagg ttttctccac 39180
agatttataa aatttctttg agatcttagt tggtatcaca tatttaaagc cgttacattt 39240
aaagcaagta gtgttaccat ctgaggttct tgagtaatgg ccagtgcctc cacatctagg 39300
gcaagtttta agtaaacctt tcttagcaac ctttgacata gtgctagccc atatcttacc 39360
aaagccatat ttgattttag ctttatctga aaggtcaggg tacaccatat taattatatc 39420
accctttaat ggatcatcct tgatataact attataccaa tccaatttaa cttgggtatg 39480
tgatctccta cttattacct tagtaacctc atactccttt aaccatccat ctatggcctc 39540
attggataat ttatcagcct cttcaaaagt tatgctatta acaggtatac taaatgctcc 39600
tgttgctacc cagcttccat cttctttttg gcaaccaaag aagttttcag cttctacttg 39660
aataccataa tatatatcta tcatgtgatt tacctcctaa gttatttgtt aatataatta 39720
taccagcact ctcgagatat gtcaacgaaa aaagaggaga agttttacct tcttccccta 39780
acctttaagt tgattaatag atcatatata tctttaggca taccttgctc ccactcccat 39840
tgtttacctg gtttctcaat gttattcttt cttaatatac ttcttgcctt tcttggatct 39900
agttctatca attcacataa atcttttaaa gtgtatatat cacttctagt ttgtgctaca 39960
gctgagtttt caccttttct agcctgttgc actttgttgt aataaagact agttaatttt 40020
ttatgtaaag gactatgtga ccaataagtt aagtgagcta attcatggca tatagtttca 40080
agaacacagt ccttaatatt ccagccaaat tttttgattt tatcataagc tttttctcct 40140
tcctgtgcaa acctgaaagg gaaaagatta attgtaatcc cagtaaattt attacctgat 40200
ctatggcata gaccataata attggcatcc acatcagatt ctatataaag atcaacagca 40260
cttacaccaa atgcttttcc taagatcatt gtggcctcat tgtataactc ctttacttca 40320
tattcagcta attgattttc tatcatatct ctcattttgt ttacctccaa tagttaattt 40380
atgatttaat tataagagag aactctggat ctgtcaacga aaaacttcga agaaaaaaaa 40440
aggatggggt ttaccatcct agatcatctc ttcttcctat atcatatatt gcacagatgc 40500
aagcctcaag atgtttttca atctcagctt ttttatgatg tttacataca taatatttag 40560
ttactcccat ttgttgtaat agttctatct tcttatctaa gaattgtttt tggaagttct 40620
tttcaaaaac taccttctgt attttttcta tctcatttaa catttctaat ttagtcattt 40680
ttcattcctc ctaagaatta attaatattg attacaatat tattatacca gagggcccag 40740
actatgtcaa cgagattctg gagatttttc gaaaaaaata gatggccttt tggccaccca 40800
tattaattgg cttttttcat ttttaggata acctcatcat aggcaaccat acagcaagaa 40860
aggtagtctc ctaattcact ttccctaact ttgttgctta gtccaaggga ttttacctgt 40920
ttatcaagtt cctttctaaa ttgtggatag attttattat ccatagcctt ttgagcaatt 40980
ccctttatta cattaagttt ttctgatctt gttagttcca tagttaacat ctccttagtt 41040
aattttgatt acaatattat tatactagat agtcccgacc ctgtcaacga ttaaatccag 41100
attttttcga aaaataaaaa ggaggttttt acacctcctt agttttatag atccttgaac 41160
atttcagggt agttacataa agtatcttcc acatattgtt ctgggtggta atcatattct 41220
atgatgtatt caacagcagc tattaagtca ttctcatcat taaattctac cccttgtatt 41280
tctagttcct cctttaattg ttctaaagtt tcttgaaatt ttttgttatt cataattaac 41340
aactcctttg aattaatttt gtttacaaga ttattatacc agactctgga gtccatgtca 41400
aagaaaatcc cgaatttctt ttttggcatg aaagttgcta ggtatcggat cgctttaaaa 41460
agaagcaact atttagttgc tcctatgggg gttaatgaaa accctgataa ttttttattc 41520
tttccccacc tagcacaagc ttccattgtg ggctcaatac ccaagatatt gcatatatcc 41580
aaatattctt ttaaccataa taacctcata ataattaatc ctcctttgag tatcttaact 41640
tcttagattt tttattctta tacatcattg agtcagcctg tactatggct tcttcaatag 41700
ttttaccttt accatagcca catgatagct cagggtatat attcaaggta ttgcatagat 41760
tataagctat atcactgata actacaaact catcaccacc ccacctaatc aatatatcct 41820
tctcaggatg taaatgggcc ttgataagat ttacagtgta ttgtatatac acatctcctt 41880
ctagatggcc taacttatca ttgaactctt tcagatcatt aagatcaata tatacaatct 41940
catactcctc aaggtcaatc ttatagtttc tattataggc tcctgtccaa tcaatagata 42000
actcctttac aagttcaatc aattcttccc ttgtaagttt ttcaagatct ttcatattca 42060
gttctctcct ttttgtttga ttacaatatt tatattacca tatactgtgg atattgtcaa 42120
cgaaaaagaa gaggttttta aacctccccc tattttaaca ttttcttaat atcctcaagt 42180
tcagatcctg accatttata tggtttgttt acaccttgct ttcttagctt ggccctggct 42240
ttacttggat ctaatcctaa tgactcacat aaagttttta agtcaactgt tccagtaggt 42300
gcattacctc cagaaggtgt agggtaagtt cccttagcct caactttatt cagatcaacc 42360
ttcttaagag gtttgtcctt tttctttctt ggcttcttag gctcattagg attaaattct 42420
ttcattaatt ctctctcctt cttcttttcc tcatttagga gtcttagcct ttcaaggcct 42480
ttttgttttt tcttttcctc cttgacttta atcttggtta gggccttaat agttttccca 42540
gcttcaatca aagctttagc tatttcagta tgattactta ttctaggttg ttcttttctc 42600
attatttatc acctcaattc tatattacca cattacttat cagtagtcaa cagtttttct 42660
gctctatccc ttttaagctg tattgatttt aatattttct catcaatagt attctcagtt 42720
attaagtgta ttatttgcca aggttccttt tgaccatttc ttttaattct ggcaatagct 42780
tgcacatagt catcatactt atagttccaa ctatagaata ttaaccttga ggccctgaag 42840
aggtttagac caacactacc tgaacttatt tgtagtatta atatgtcaat cttgccctcc 42900
tgaaaatcct tattgatctc agctctattc ttagtagatc ctgttaagca ttctactctg 42960
tgggtctttt taaaagtatc atgtataagt tttatttctt caagaaaatt acatattacc 43020
acaacaggtt ttttatatcc ttctagatag tcctgtaaca agcctaattt gcctttatca 43080
atgttaatac tatttcctac attgtttgta ataaatcctc ctgagatctg ctgtagcctt 43140
aaatattggg taatagtaag atcagctgtt aattgattga taaacatatt agctcttaag 43200
aataaatcct catatggctc attactctca taaggtatac cattagacct taataaggcc 43260
tttaattgct tcctaggaat gttttcctgt actatatcta gttgtgctat catttccttt 43320
ctaaggtcat tgtaggcctt tctagcttta ctatttagtt cacaggttaa atacagatca 43380
ggaagttgtt ctggtaaatc aatacaatct tctattctta ctatatagca attctggtgc 43440
ataaggttct ttaatatttc ctcattctta taccccacaa tttcatagtt catataacct 43500
cccttgatta taaagtcatc ctcaaactgt ttccagttaa ctcctagaat atttgagttc 43560
ataattttat attgcataaa taaatcttca taaccattag gagtaggagt ccctgtcatt 43620
atcaatctat atttacattt agtagcaatt ttatatagag ccttagatat ctgggcattt 43680
ctattcttta ccttatggga ttcgtcaaca acaataaact gtggctttaa gttcataagg 43740
tatttatcca tcaacctagc cttctcaaaa ttggcaataa taatctttaa tcctgagtat 43800
gattttatag ctctataagt ttccataacc ttggctttat taccttgtaa tttaaatatt 43860
ttatattcct taagtttagt aaatttatgg atctcagact cccagttaaa tacaatagac 43920
ttaggtgaga atattagtac agtatctaaa ccttcaagtt tctcaagggc cacaatagga 43980
actaaggtct tacccgtacc catgtccata aataaggcaa acttttcttt atttaaaaat 44040
agttgtaatg cttctttctg atgtttaaat aattttctct tcatactaaa aaagaagagg 44100
cattaaagcc tcctccttat acctccttag ccagttttac aagttcatct cttgaggcct 44160
tactatggaa tttaacccca ttatctttta ggtattgttt taaggattct ttattcatac 44220
tatcaaagtc taattcttct tcgtcgtaaa tgttatcatc atcctctaag tcctcatcgt 44280
cattatcatc tatgtcttct tcatcatatt cttctttact aagatcatgt atttctgagt 44340
tatcatcact agttaattcc atagcagcca taatagcata gttagctaag tctagtaaag 44400
tatcaaccac agattctttt tgagcagcat atgaagggaa gtcatcttga tctcctagta 44460
ataaactttt agctcttcct agcttatctt caagtcttat acataggaca gggtttccat 44520
attcaagata agtcttgtgg aaattatccc catatagatt attctttaat tcatacagat 44580
ctcctagttc tcctaggaat tgttggtgtc tttctaaatt tttcatgtaa tctcctcctt 44640
gtatattcag ctgaggtagc cataaaagct attcctcctg ctagatttat tttatttata 44700
atgtcttttt gtttttgagt taggttcttt tcttttccag gaagtttaac ttcaatacca 44760
ataaacttac ctttgtggca acctagtata tcaggtaagc cagctatttg aaatggtccc 44820
ccatgagttt taaaccaaaa accggggtat tcttttttaa gcatattcat tatggattta 44880
accacatctc tttcaagcat aatagtagcc tccaagggta aataaaggag gaccttaaag 44940
gccctcatat tttaataatt aaattttatt cttcatcatc ttcttctagc atttctatga 45000
tgtcatcctt agacattcct tttttaacct ttaagcctct gtccttagct tcagctttaa 45060
gttcttttaa agtcattgac tcatagtcat tttcttcttc atcttcctct tcttcatcag 45120
agtctccgaa taggtcatta tattgttcag ctatttcttc ttcgtcagct tcttcctcaa 45180
ttagatcaat tagatcttct tcagttttag ccttcttgat tttcttagct gggattttta 45240
aagccttagc taattctttt agctcatcct tgtctaattc ttcaagatca acttcagact 45300
catcgtcttc atcatcttct tcgtcatctg agtcatcatc ctcatcttcg tcatcatcag 45360
agtcagcttc gtcaaggttt ataaaatcag ttattcttga tttcttctta ccttcatata 45420
tttcatgtga tacttctacc ccacattgta aaccaactag agcttctgta tctaattcga 45480
actcctcatc agggatatca aatcctaaag caactaacac accttttaag ttaaataaag 45540
cttgtggttg aagtgagcaa atgtggtata acttttgtcc tttcatcttt ccttctgcta 45600
ttacaaaagt aaatgataaa taattgcttc cactattttc agaaacctta acctctacat 45660
cttctacttc cacaatgtaa ttcccttctg ggatagtaac tgaagtttca acgtttgaaa 45720
gatcaatctt taatcctcct ttgtttttgt tacctctctt agtagtcttt ttagccatgt 45780
tattgtcctc cttaaatttt aaaatatcat tatgagataa atatttgtca agatattgta 45840
cctctttgtt tacaagttta tattatcaga tttttaattg gttgtcaaag gtatttttag 45900
aaaattttta ttttttcttt ttaccagatt tcttactggt ttttttacca gatttagctg 45960
gcttattatt ccattcacca gccagaatag tttcaatatc ctcccagata gttgttggtg 46020
attttaaatc atgtactaga taagctggac attcagatcc ttgaggtcta gtaaacttag 46080
taatataata ggggttaggt cccagtctta atctatactg gatttctttc ttaaccttca 46140
taccctcttt ttctgagtgt tcatataaat aagtatgacc tattactctt gatacagcac 46200
ataaatattt agatacagat ggcattaatc ctggtcctac ctcaggcatt aattggtctt 46260
ctccttctcc atcaccagat tctaatctat cttgtactat aaagcatggt agtatacctt 46320
cctcatttaa ttctttataa aggtttataa cctctttcat ataattagcc acatttccaa 46380
acattctttg gctcatctgg tctttgcctt cctcagcttt gaccttttca ttacctaatt 46440
cttgtaaagc agttaaatgg tctataacta ctgttttaaa tttatcagtg ttttctgata 46500
ggtaatcata agcctcgtat atgtcatcaa aactctttag actaaatact tgtatatctc 46560
ctcttttaac tctaagcttg tttctagctg atagagtacc tttgtccttt acatcaataa 46620
agaatattgg tttgggtagt gttcctgaga tggtagtttt acctgaacca gatcttccat 46680
aaagtgttaa tattgtgggc tcatctatat catatagatc cactaatctg tcctcaaaac 46740
tatttgattt tgatttttgc ttttttgcca ttgtcacttt cctccctttg tctatagtca 46800
gccttaataa taaattcttt atcaagattt aaaagttcag cttgacataa agacttatat 46860
tcacagaaag agcaaccttt tccaaggttc ttttcctttc ttttgtggcc ctcatctcta 46920
ataagtttag ctatctcaat agtatcatcc attacactgt taagtatatt ctttgataat 46980
attattgggt gtcttacaaa gaagtcttcc catctagcag aattaatata ggactcataa 47040
tctttaggat ctaatcctaa ttcttttatt cccctttcta actctaaagg tgtggattta 47100
atcctctttt gagataatac ccctttttga gttaattcag gcctacctgg ttgctgagct 47160
aatattatat cccatatagt accacttact tttgggtatt catgtagaag agcccaggta 47220
tatatagcag attgtctatt gaagatcagg aaatttctat caggcatctt tttataggtt 47280
ttagtttcct tagcccatat ctttccatcc acatctaaga cactatcaat atagccttct 47340
aggtttacac ccttacatag ttttaattca aagtgatgtt cattttccac ataggttaca 47400
ttatcttctt tttcatcata atagtggaaa tagttatcac ataaagaata aaccattttt 47460
ggaatatctc caaattcaag gatctcttcc ttaaatgtat ttttgtaaaa ctctttggaa 47520
aatttatcaa cagatttttt ccaactcttc ccattatggt agtcttctat agcctggtgg 47580
atagcagagc ccctttgtaa ggcagctcca cttcttttag gtaccagacc ttcatagtac 47640
ttaacataat gggcataagg acaagacaag aatttgttaa ttctggaatt acttattttt 47700
aacatcctta tacctccttt tcattacaat atttatatta tcagataatt attcatctgt 47760
caacactttt cttatctggc ttttccaatc acctttaaaa tccagttcca cacatcctcc 47820
ccaagaataa ccaactgata tatccatgat tattgggact gttggagtaa atttaaatac 47880
tttctttaat accttattat tttcaactat tgattttact ttccaagcta attcttccac 47940
ataatcttta tctacttcaa ataatattga gtcatgtact gagcctaata catcaaactt 48000
agacttatct aatacatatt caggattaac atatttagca tagcctgtaa tctcacatag 48060
gcctaataaa gtgatatctg aaccaaaacc ttgaacaggt gagttaatac attgtctttc 48120
ggcttcagca gctttagatc tatcagatga atatatatct ggtaatcttc taattcttcc 48180
tattggagat cttacttcac cattggcttt aacaattttt ctttgtttat catgccatgt 48240
taatagttta gggtatttat taaagaagtt ccttctatat gttttggctt cctcatctgt 48300
taatttaagg ccatagttgt ctcttccata atccttgaac tttttccatt gcataccata 48360
taagaaacca aagtttacag ccttagcctt tttccttttc tccttcttaa ctgctggatc 48420
tttctcatca ttgatatctt ccccagatat aatattatat gtggagtcat gtatatctcc 48480
accagtctgg taaataaatt tcatattatc ctcatcggcc atcatagcag ctattctaag 48540
ttcggcctgg gatagatcgg cttcaatgaa tactcttcca ggagatggtc ctaataaact 48600
cctgattttc ttatctctag gaacttgctg tagattagga ttattacttg aggttcttcc 48660
tgttacagtt ccatgaagtt taaagtttgg atataatcta tgattgtgca ttctatttaa 48720
ccaaccatct ataaagtgag aaatttgtat atggactccc ctatgctgta atagtagttc 48780
tactgctgga tgtttatctc ttagtcttaa taatacactt tccccagtag caggtgctcc 48840
tgagtcagta acctcaatca caggaagacc aagggtatca tataaaaatt ctccaacctg 48900
tttaggtgag ctccagttaa tatccttacc attagtatac ttcttaagtc cagtctcaat 48960
cttatctagt tccgatctta agtatttttc aacctctttg aatttctcct gatttacata 49020
tatacctttg aactgaacat cctcataagc tttactagct gggatgtata agtggtggaa 49080
taatttatat aagctctcat ctcttttaag tctcttatta aatattttat atagagcaaa 49140
tgtatagtat atatcatatc ccagatatct aacataatcc tcaaagtctg atttagtctc 49200
cacatttcca gtctttaatt tcttatttat atcccagtct ttagcattac aatgtaataa 49260
agcattctcc ttaagaccat taggggtatt ctcatctaaa caatgtgagg ctagtaaagt 49320
atcaaaggtt atgattggct taataccata tttctccttt aagaaattat catcaaactt 49380
accattctgg gctatccttt ccttcatctc agaattaagt cttttaatca aggatttagc 49440
taatcttctt tgtgctatgg gcttacccct taatggacta tatttgactt ctaaaggcag 49500
tatatactgt acttgagtat taccaaaacc aaatagggta atgttattat taaacctatc 49560
taggcctcta gtttctatat catatgatac tctattataa ccttcctctc ttaaatattt 49620
gaaagcctgg tttaactctt tcatattggt aataagtttt atgttaagct ctggtaattc 49680
atgctcttta ccttttacca tggctttaaa attattaaag gcctgatcta caaattgtgc 49740
tttagttggg tcatatgcaa ctacacctgg tgaatagctg gcaataaact taaagccata 49800
tttctcatgc tttttaggta ctccacatat tgaggttata ccttcattac ttagcatctt 49860
taaggcttga gatcctaaga tcataacata ctcaggttta tatttttcaa tttccttaag 49920
taagtgttct tggcatttct taatctctga tactttgatt ttagtaccct taggagttct 49980
acacttaata gcattggtat aataaaagtc tagaccttcc attttatcaa gtaatgattt 50040
atcaggagta gctattctat tggcctcatc cctatcatta gctaaactat ttataaccat 50100
tattcttggc ttctttttct ttcctacaca agttccttct atacatgagg gagctgagtg 50160
agtatatagg ttacagttgt tacatctcat ttaaagtatt acctcctaaa tagaaaggac 50220
ccctaaaggc ccttatcttt actttatgat tgaattacat actggacaga ccttatgttt 50280
tttgaaaggt atggttacta cccataggcc tccagttaag aaaatcatga tccaaaacat 50340
agctccatat tttcttttta atgttactga gtttttacac ataggacaaa ttatattttt 50400
c 50401

Claims (8)

1.一种能够特异性地杀死产气荚膜梭菌的肌尾噬菌体科噬菌体Clo-PEP-1，其基因组由SEQ ID NO：1的核苷酸序列表示，且以登记号KCTC 12664BP保藏。
2.一种用于预防和治疗产气荚膜梭菌感染的组合物，其包含权利要求1所述的噬菌体作为活性成分。
3.一种饲料添加剂，其包含权利要求2所述的组合物。
4.一种用于除人类以外的动物的饮用水添加剂，其包含权利要求2所述的组合物。
5.一种消毒剂，其包含权利要求2所述的组合物。
6.权利要求1所述的噬菌体在预防和治疗产气荚膜梭菌感染的药物制备中的用途。
7.权利要求2所述的组合物在预防和治疗产气荚膜梭菌感染的药物制备中的用途。
8.根据权利要求7所述的用途，其中所述组合物以饲料添加剂、用于除人类以外的动物的饮用水添加剂或消毒剂的形式给药到受试者。
CN201580071134.5A 2014-12-29 2015-12-28 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 Active CN107109372B (zh)

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CN111662880B (zh) * 2020-05-25 2022-06-17 江苏省农业科学院 一株产气荚膜梭菌噬菌体、包括噬菌体的抑菌剂及制备方法和应用
KR102586836B1 (ko) * 2021-02-23 2023-10-10 씨제이제일제당 주식회사 클로스트리디움 퍼프린젠스에 대한 특이적인 사멸능을 가지는 신규한 박테리오파지 및 이를 포함하는 항균 조성물
KR102586837B1 (ko) * 2021-02-23 2023-10-10 씨제이제일제당 주식회사 클로스트리디움 퍼프린젠스에 대한 특이적인 사멸능을 가지는 신규한 박테리오파지 및 이를 포함하는 항균 조성물
KR102586838B1 (ko) * 2021-02-25 2023-10-10 씨제이제일제당 주식회사 클로스트리디움 퍼프린젠스에 대한 특이적인 사멸능을 가지는 신규한 박테리오파지 및 이를 포함하는 항균 조성물
KR102586839B1 (ko) * 2021-02-25 2023-10-10 씨제이제일제당 주식회사 클로스트리디움 퍼프린젠스에 대한 특이적인 사멸능을 가지는 신규한 박테리오파지 및 이를 포함하는 항균 조성물
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US10028984B2 (en) 2018-07-24
RU2662986C1 (ru) 2018-07-31
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CN107109372A (zh) 2017-08-29

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