CN117529553A - 具有特异性杀伤产气荚膜梭菌的能力的新型噬菌体及包含其的抗菌组合物 - Google Patents

具有特异性杀伤产气荚膜梭菌的能力的新型噬菌体及包含其的抗菌组合物 Download PDF

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CN117529553A
CN117529553A CN202180096220.7A CN202180096220A CN117529553A CN 117529553 A CN117529553 A CN 117529553A CN 202180096220 A CN202180096220 A CN 202180096220A CN 117529553 A CN117529553 A CN 117529553A
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bacteriophage
clostridium perfringens
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田钟寿
金智恩
文准玉
蔡宗杓
金裕珍
李承殷
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Abstract

本公开涉及一种具有特异性杀伤产气荚膜梭菌的能力的新型噬菌体，和包含其的抗菌组合物。该新型噬菌体CJ_CP_20‑25具有特异性杀伤产气荚膜梭菌的作用，表现出优秀的耐酸性和耐热性，因此能广泛的用于预防或治疗产气荚膜梭菌引起的感染性疾病的抗生素、饲料添加剂、饮用水添加剂、饲料、饮用水、消毒剂或清洁剂。

Description

具有特异性杀伤产气荚膜梭菌的能力的新型噬菌体及包含其 的抗菌组合物
技术领域
本发明涉及具有特异性杀伤产气荚膜梭菌(Clostridium perfringen)的能力的新型噬菌体及包含其的抗菌组合物。
技术背景
产气荚膜梭菌已知为革兰氏阳性、大型专性厌氧菌，其没有鞭毛且形成孢子。产气荚膜梭菌是一种能在动物，尤其是鸡、猪等家畜中引起腹泻等疾病的细菌，与沙门氏菌(Salmonella)引起的禽伤寒一样，被认为是畜牧业中最重要和最致命的病原体之一。
目前，由产气荚膜梭菌感染引起的坏死性肠炎是家禽和养猪业中最常见的疾病之一，其是一种引起鸡或猪小肠下部严重坏死性病变和带血腹泻为主要症状的疾病。这种坏死性肠炎根据病情的严重程度，会使受感染的动物出现脱水和间歇性腹泻症状，使动物的身体逐渐衰弱，并导致生长障碍，并因此是一种已成为畜牧业中的一个重大问题的疾病。此外，产气荚膜梭菌是引起坏死性肠炎的病因，易通过动物粪便传播，因此易于在处于共同饲养空间内的动物间通过土壤或被污染的饲料由经口感染等途径传播，并且由于发病率高，在幼畜中是尤为严重的问题。
在畜牧业等领域，为了预防和治疗牲畜细菌感染引起的疾病，已经使用了各种抗生素，但由于出现了诸如抗生素的滥用或耐药性以及抗生素在动物体内残留的可能性等问题，目前世界各地对抗生素的施用存在一些限制。
因此，噬菌体，其通过感染特定细菌来抑制细菌生长的细菌特异性病毒，最近作为抗生素的新替代品被研究，因为噬菌体表现出比抗生素更强的宿主特异性，但噬菌体的数量仍然绝对不足。因此，为了预防和治疗由产气荚膜梭菌引起的感染性疾病，这是包括猪和家禽在内的畜牧业的一个重要问题，不断地要求开发新的具有广泛的溶菌谱和优异的耐酸性和耐热性并从而具有高度的工业实用性的噬菌体。
引用列表
专利文献
专利文献1US 6942858 B1
公开
技术问题
本公开的目的是提供具有特异性杀伤产气荚膜梭菌的能力并且以保藏号KCCM12934P保藏的噬菌体CJ_CP_20-25，包含其作为活性成分的用于预防或治疗由产气荚膜梭菌引起的感染性疾病的组合物、抗生素、饲料添加剂、饮用水添加剂、消毒剂或清洁剂，包含所述饲料添加剂的饲料，或包含所述饮用水添加剂的饮用水。
本公开的另一个目的是提供使用噬菌体CJ_CP_20-25或包含噬菌体CJ_CP_25作为活性成分的用于预防或治疗由产气荚膜梭菌引起的感染性疾病的组合物以预防或治疗由产气荚膜梭菌引起的感染性疾病的方法。
本公开的又另一个目的是提供噬菌体CJ_CP_20-25或包含其作为活性成分的用于预防或治疗产气荚膜梭菌引起的感染性疾病的组合物的用途，用于制备用于产气荚膜梭菌引起的感染性疾病的预防剂或治疗剂。
技术方案
本公开中公开的每项说明和实施方案也可以应用于其他说明和实施方案。即，在本公开中公开的各种要素的所有组合都属于本公开的范围。进一步地，本公开的范围不受下面具体说明的限制。进一步地，使用不超过常规的实验手段，本领域技术人员将识别或能够确定与本文所述的本公开的具体方面的许多等同物。进一步地，这些等同物应被解释为属于本发明的范围。
一个方面提供了一种具有特异性杀伤产气荚膜梭菌的能力的噬菌体CJ_CP_20-25，并以编号KCCM12934P保存。
如本文所用，术语“产气荚膜梭菌(CP)”是指一种革兰氏阳性厌氧杆菌，其对应于一种在鸡和猪等动物中引起感染性疾病的致病微生物。
如本文所用，术语“噬菌体”是指感染特定细菌并抑制细菌生长的细菌特异性病毒，并且是指含有单链或双链DNA或RNA作为遗传物质的病毒。
另一方面提供了一种用于预防或治疗产气荚膜梭菌引起的感染性疾病的组合物，其包含噬菌体CJ_CP_20-25作为活性成分。
噬菌体CJ_CP_20-25如上所描述。
噬菌体CJ_CP_20-25具有特异性杀伤产气荚膜梭菌的能力，因此当其在产气荚膜梭菌引起的感染性疾病发作前被施用给个体，可通过杀伤进入个体的产气荚膜梭菌，抑制产气荚膜梭菌感染，由此可能预防产气荚膜梭菌引起的感染性疾病。此外，当噬菌体CJ_CP_20-25被施用给患有产气荚膜梭菌引起的感染性疾病的个体时，噬菌体CJ_CP_20-25可通过杀伤个体中现有的产气荚膜梭菌缓解或消除产气荚膜梭菌引起的症状，由此可能治疗产气荚膜梭菌引起的感染性疾病。
如本文所用，术语“预防”可指任何通过噬菌体或组合物的施用而抑制或延缓个体中疾病发作的作用，术语“治疗”可指任何由于噬菌体或组合物的施用而使得个体中疾病的症状被缓解或消失的作用。
产气荚膜梭菌引起的感染性疾病可以是，例如，坏死性肠炎，但不限于此。
如本文所用，术语“坏死性肠炎”是指产气荚膜梭菌引起的感染性疾病之一，对应于频繁发生在诸如肉鸡等牲畜中并造成相当大损害的细菌性疾病。坏死性肠炎的症状是由产气荚膜梭菌在小肠内过度增殖引起的，其包括消化道黏膜坏死和突发性腹泻。例如，猪的超急性坏死性肠炎可在发病后1至2天内致死，并且急性的坏死性肠炎可引起带血的腹泻然后在2至3天内致死。此外，亚急性坏死性肠炎引起5至7天的持续性腹泻(没有血便)和之后虚弱脱水，慢性坏死性肠炎可能引起间歇性腹泻和生长障碍。
用于预防或治疗产气荚膜梭菌引起的感染性疾病的组合物可包含1×102PFU/mL至1×1012PFU/mL或1×105PFU/g至1×1010PFU/g的噬菌体CJ_CP_20-25。
用于预防或治疗产气荚膜梭菌引起的感染性疾病的组合物可进一步包含药学上可接受的载体，可与载体配制，并可作为食物、药物、饲料添加剂、饮用水添加剂等被提供。
如本文所用，术语“药学上可接受的载体”可指不刺激生物体并且不抑制所施用化合物的生物活性和性质的载体或稀释剂。
所述组合物可含有的载体种类没有特别限制，普遍用于本领域的和药学上可接受的任何载体都可被使用。载体非限制性实例包括生理盐水，无菌水，林格氏液，缓冲盐水，白蛋白注射溶液，右旋糖溶液，麦芽糊精溶液和甘油。这些可单独使用或以其两种或更多种混合体的形式使用，如必要也可能添加其他常规添加剂如抗氧化剂、缓冲液和抑菌剂。此外，可选择性的添加稀释剂、分散剂、粘合剂和润滑剂以形成可注射的制剂，如溶液、悬浮液、乳剂或药片、胶囊、颗粒或片剂。
用于预防或治疗产气荚膜梭菌引起的感染性疾病的组合物可口服或胃肠外给药，还可以使用将该组合物应用或喷洒到患病部位的方法。对于胃肠外给药，可用静脉给药、腹腔内给药、肌肉给药、皮下给药或局部给药。所述组合物的适当应用、喷洒和剂量根据配制方法、给药方式、目标动物和患者的年龄、体重、性别、疾病症状的严重程度和食物、给药时间、给药途径、***率和反应敏感性等因素而变化，并且一般技术熟练的医生或兽医可以容易地确定对所需治疗有效的剂量并开出药方。
用于预防或治疗产气荚膜梭菌引起的感染性疾病的组合物的口服给药制剂可包括，例如，片剂、含片(troche)、锭剂(lozenge)、水性或油性悬浮液、粉末或颗粒、乳剂、硬胶囊或软胶囊、糖浆或酏剂。为了将组合物制成如片剂、胶囊等的药剂，其中可包含粘合剂，诸如乳糖、蔗糖、山梨醇、甘露醇、淀粉、支链淀粉、纤维素或明胶；赋形剂，诸如磷酸二钙；崩解剂，诸如玉米淀粉或甘薯淀粉；和润滑油诸如硬脂酸镁、硬脂酸钙、富马酸硬脂酰钠或聚乙二醇蜡。在胶囊制剂的情况下，除了上述物质外，还可以含有液体载体，如脂肪油。
用于预防或治疗产气荚膜梭菌引起的感染性疾病的组合物的胃肠外给药制剂可包括，注射剂形式诸如皮下注射、静脉注射或肌内注射；栓剂注射形式；或喷洒形式诸如可通过呼吸道吸入的气溶胶。为了将组合物制成可注射制剂，本发明的组合物可与稳定剂或缓冲剂在水中混合制成溶液或悬浮液，且可在安瓿瓶或小瓶配制用于单位给药。在用于喷雾的制剂如气溶胶的情况下，推进剂或类似物可与添加剂调和，从而水分散的浓缩液或湿粉末可分散。
用于预防或治疗产气荚膜梭菌引起的感染性疾病的组合物可包含防腐剂、稳定剂、润湿剂或乳化剂、冷冻保护剂、赋形剂等等。
防腐剂、稳定剂或赋形剂所述可以以足够减少组合物变质的有效量包含在组合物中。
如本文所用，术语“变质(deterioration)”可包括所述组合物中所含噬菌体CJ_CP_20-25的细菌数量减少，噬菌体CJ_CP_20-25活力降低，或其组合。
例如，由于组合物包含有效量的足够减少组合物的变质的防腐剂、稳定剂或赋形剂，组合物中的噬菌体CJ_CP_20-25可更长时间地存活或保持其活性，不同于存在于自然中的噬菌体。
作为本领域普遍使用的防腐剂、稳定剂或赋形剂，只要其能减少组合物变质，可被使用而不受限制。例如，稳定剂可以是选自由以下组成的组中的任意一种或多种：海藻酸盐、海藻酸钠、酪蛋白、酪蛋白钠、纤维素、甲基纤维素、羧甲基纤维素、羧甲基纤维素钠、果胶、明胶、***胶、黄原胶、葡聚糖、环糊精和淀粉。
当所述组合物为冻干状态时，其中可包含有效量的足够减少组合物变质的冷冻保护剂。例如，与噬菌体在自然界中存在而在冻干组合物中噬菌体表现出活性降低或不能存活不同，因为组合物包含有效量的足以在冻干状态下减少组合物变质的冷冻保护剂，噬菌体甚至可以在冻干组合物中保持其活性或存活。
作为本领域普遍使用的冷冻保护剂，只要其能减少冻干状态下组合物的变质，可被使用而不受限制。例如冷冻保护剂可以是选自由以下组成的组中的任意一种或多种：甘油、乙二醇、丙二醇、二甲基亚砜(DMSO)、葡萄糖、海藻糖、麦芽糊精、糊精、脱脂牛奶和淀粉。
另一方面提供了一种抗生素，其包含噬菌体CJ_CP_20-25作为活性成分。
噬菌体CJ_CP_20-25如上所描述。
如本文所用，术语“抗生素”是指能够杀伤细菌或抑制细菌生长的试剂，且可以是防腐剂，杀菌剂和抗菌剂的通用术语。
相比传统的抗生素，包含噬菌体CJ_CP_20-25作为活性成分的抗生素对产气荚膜梭菌表现出显著的高特异性，因此能只杀伤特异性的病原体而不杀死有益细菌，且所述抗生素不引起耐药性，因此相比传统的抗生素，可表现出延长产品寿命的效果。
抗生素可通过本领域普遍使用的制造方法制备。
抗生素可包含有效量的足够减少抗生素降解的添加剂，添加剂可以是防腐剂、稳定剂、赋形剂、冷冻保护剂，等等。因为抗生素包含添加剂，其中的噬菌体CJ_CP_20-25可更长时间地存活或保持其活性，不同于存在于自然中的噬菌体。
作为本领域普遍使用的防腐剂、稳定剂、赋形剂或冷冻保护剂，只要其能够减少抗生素的变质，可被使用而不受限制。
另一方面提供了一种饲料添加剂，其包含噬菌体CJ_CP_20-25作为活性成分。再另一方面提供了一种饲料，其包含所述饲料添加剂。再另一方面提供了一种饮用水添加剂，其包含噬菌体CJ_CP_20-25作为活性成分。再另一方面提供了饮用水，其包含所述饮用水添加剂。
噬菌体CJ_CP_20-25如上所描述。
所述饲料添加剂或饮用水添加剂可以以包含噬菌体CJ_CP_20-25的组合物的形式制备，并被混合入饲料或饮用水中，或以这样的方式使用，其在制造饲料或饮用水时直接添加噬菌体CJ_CP_20-25。
包含在饲料添加剂或饮用水添加剂中的噬菌体CJ_CP_20-25可以是流体状态或干燥固体状态，具体地可以是干粉状态。噬菌体CJ_CP_20-25的干燥方法可以是通风干燥、自然干燥、喷雾干燥或冷冻干燥，但不限于此。噬菌体CJ_CP_20-25可以基于饲料添加剂或饮用水添加剂的重量，以按重量0.05％至10％，尤其是按重量0.1％至2％的干粉的形式混合。
如有必要，包含噬菌体CJ_CP_20-25作为活性成分的饲料添加剂或饮用水添加剂可进一步包含其他添加剂。可使用的添加剂的非限制性实例包括但不限于：为防止饲料或饮用水变质而添加的粘合剂、乳化剂、防腐剂等等；和为提高饲料或饮用水效用而添加的氨基酸、维生素、酶、益生菌、调味剂、非蛋白氮化合物(NPN)、硅酸盐剂、缓冲剂、着色剂、萃取剂或低聚糖，而这些可以单独添加，或其中的两种或多种可以共同添加。
作为本领域普遍使用的包含在饲料或饮用水中除了噬菌体CJ_CP_20-25外的原始材料，可被使用而不受限制。所述饲料的非限制性实例包括植物饲料诸如谷物、根茎类水果、食品加工副产品、藻类、纤维、医药副产品、油和脂肪、淀粉、葫芦或谷物副产品；和动物饲料诸如蛋白质、无机材料、油和脂肪、矿物质、单细胞蛋白质、浮游动物或食物。这些可以单独使用或以其中两种或多种的混合物使用。
基于按重量的100份食物，所述饲料添加剂可以以按重量0.05至10份，例如，按肿瘤0.1至2份而被包含。基于按重量的100份饮用水，所述饮用水添加剂可以以按重量0.0001至0.01份，例如，在按重量0.001至0.005份而被包含。
所述饲料添加剂、饲料、饮用水添加剂或饮用水可通过本领域常用的制造方法制备。
所述饲料添加剂、饲料、饮用水添加剂或饮用水可包含有效量的足够减少其变质的添加剂，而所述添加剂可以是防腐剂、稳定剂、赋形剂或冷冻保护剂。由于饲料添加剂、饲料、饮用水添加剂或饮用水中含有添加剂，饲料添加剂、饲料、饮用水添加剂或饮用水中的噬菌体CJ_CP_20-25可以更长时间地存活或保持活性，不同于存在于自然中的噬菌体。作为本领域普遍使用的防腐剂、稳定剂、赋形剂或冷冻保护剂，只要能减少饲料添加剂、饲料、饮用水添加剂或饮用水的变质，可以被使用而不受限制。
另一方面提供了消毒剂，其包含噬菌体CJ_CP_20-25作为活性成分。再另一方面提供了一种清洁剂，其包含噬菌体CJ_CP_20-25作为活性成分。
噬菌体CJ_CP_20-25如上所描述。
消毒剂或清洁剂的配方没有特别限定，且消毒剂或清洁剂可通过制备成本领域普遍了解的配方来使用，所述消毒剂或清洗剂可通过本领域常用的制造方法制备。
所述消毒液可以喷洒以去除产气荚膜梭菌，也可以喷洒在动物活动区域、屠宰场、死亡区域、烹饪场所、烹饪设备等等，但不限于此。
所述清洁剂可用于清洁暴露于或可能暴露于产气荚膜梭菌的动物的皮肤表面或身体部位，但不限于此。
消毒剂或清洁剂可包含有效量的足够减少其变质的添加剂，且该添加剂可以是防腐剂、稳定剂、赋形剂或冷冻保护剂。由于消毒剂或清洁剂中含有添加剂，消毒剂或清洗剂中的噬菌体CJ_CP_20-25更长时间地存活或保持活性，不同于自然中存在的噬菌体。作为本领域常用的防腐剂、稳定剂、赋形剂或冷冻保护剂，只要其能减少消毒剂或清洗剂的变质，可以使用而不受限制。
另一方面提供了预防或治疗产气荚膜梭菌引起的感染性疾病的方法，其包含向除人以外的动物施用噬菌体CJ_CP_20-25或包含其作为活性成分的组合物以预防或治疗产气荚膜梭菌引起的感染性疾病。
噬菌体CJ_CP_20-25、包含其作为活性成分的用于预防或治疗由产气荚膜梭状芽孢杆菌引起的感染性疾病的组合物、以及由产气荚膜梭状芽孢杆菌引起的感染性疾病如上所描述。具体地，产气荚膜梭菌引起的感染性疾病可以是，例如，坏死性肠炎。
所述预防或治疗方法具体地包括向感染了产气荚膜梭菌或处于感染产气荚膜梭菌风险下的人之外的动物施用药学上有效量的噬菌体CJ_CP_20-25或包含其作为活性成分的组合物。噬菌体CJ_CP_20-25或包含其作为活性成分的组合物可以药物制剂的形式被施用给动物，或以饲料添加剂或饮用水添加剂的形式与动物饲料或饮用水混合被施用和饲喂。
噬菌体CJ_CP_20-25或包含其作为活性成分的组合物可通过多种口服或胃肠外的途径施用，只要其能够到达目标组织，可具体通过以下传统的方式被施用：口服、直肠、外用、静脉、腹腔、肌肉、动脉、透皮、鼻内、吸入等途径。
噬菌体CJ_CP_20-25或包含其作为活性成分的组合物的合适的总日用剂量可由治疗医师在健全的医学判断范围内确定，且这对于本领域一般技术人员来说是显而易见的。
噬菌体CJ_CP_20-25或包含其作为活性成分的组合物的具体的药学有效量可考虑到要达到的反应的种类和程度以及个体的年龄、体重、一般健康状况、性别或饮食以及噬菌体CJ_CP_20-25或包含其作为活性成分的组合物的给药时间和给药途径、组合物的分泌速率、治疗周期等而确定，并且可根据许多因素而变化，其包括同时或在相同时间点使用的药物或组合物的其他组分，以及制药领域众所周知的类似因素。
所述人之外的动物没有特殊的限制，只要是可能感染产气荚膜梭菌的动物。例如，所述动物可以是鸟类和哺乳动物，具体的可以包括鸡、鸭、猪等，但不限于此。
另一方面提供了噬菌体CJ_CP_20-25或包含其作为活性成分的组合物用于预防或治疗由产气荚膜梭菌引起的感染性疾病的用途，其用于制备由产气荚膜梭菌引起的感染性疾病的预防性或治疗剂。
噬菌体CJ_CP_20-25、包含其作为活性成分的用于预防或治疗由产气荚膜梭状芽孢杆菌引起的感染性疾病的组合物、以及由产气荚膜梭状芽孢杆菌引起的感染性疾病如上所描述。具体而言，由产气荚膜梭菌引起的感染性疾病可以是，例如，坏死性肠炎。
有益效果
新型噬菌体CJ_CP_20-25具有特异性杀伤产气荚膜梭菌的效果，表现出优秀的耐酸性和耐热性，因此可广泛用于抗生素、饲料添加剂、饮用水添加剂、饲料、饮用水、消毒剂或清洁剂中，用于预防或治疗产气荚膜梭菌引起的感染性疾病。
附图简述
图1是新型噬菌体CJ_CP_20-25的电子显微镜照片；
图2是说明了新型噬菌体CJ_CP_20-25的pH稳定性的证明结果的图示；
图3是说明了新型噬菌体CJ_CP_20-25在60℃下的稳定性的证明结果的图示。
发明详述
在下文中，将参考实施例详细描述本发明。然而这些例子用于一个或多个实施例的说明性目的，且本发明的范围不限于这些实施例。
实施例1.分离具有杀伤产气荚膜梭菌(CP)的能力的噬菌体
实施例1-1.预处理粪便样品溶液的制备
猪、家禽、牛的粪便样品收集自韩国首尔、京畿道、忠清道、庆尚道的畜牧农场，每份样品取20g，用80mL的PBS进行稀释，并在10000rpm下离心15分钟。用0.2μm过滤器过滤上清液之后，向滤液中加入10％(w/v)的氯化钠水溶液，将混合物置于4℃下储存12小时。其后，向其加入10％(w/v)的聚乙二醇8000(Sigma-Aldrich,Cat.No.P2139)，混匀后置于4℃下储存12小时，然后通过在15000rpm条件下离心15分钟去除上清液以进行浓缩。将沉淀溶解于10mL的SM缓冲液(5.8g/L氯化钠,2g/L MgSO4·7H2O,0.05M Tris-Cl(pH 7.5))中，用0.2μm过滤器过滤溶液，将滤液储存于4℃。
实施例1-2.噬菌体浓缩物的制备
将分离自从韩国首尔、京畿道、忠清道、庆尚道的畜牧农场收集的粪便样品的CP菌株和动植物检疫局(Animal and Plant Quarantine Agency)分发的CP菌株接种至BHI(脑心灌注)培养基，置于42℃厌氧环境中静置培养18小时。然后将1mL的CP菌株培养溶液和1mL从实施例1-1获取的预处理的粪便样品溶液接种至50mL的BHI培养基中，混匀后置于42℃静置培养18小时。将混合培养物溶液在6000rpm下离心20分钟，用0.2μm过滤器过滤上清液，向其中加入10％(w/v)的聚乙二醇8000，将混合物置于4℃条件下12小时。然后在15000rpm条件下离心1小时除去上清液以进行浓缩，将沉淀溶解于1mL的SM缓冲液中，用0.2μm过滤器过滤溶液，将滤液储存于4℃。
实施例1-3.噬菌体的筛选和分离
将50μL实施例1-2中制备的噬菌体浓缩物、5mL 0.7％(w/v)的琼脂(BDDIFCO,Cat.No.44164)和振荡培养的与实施例1-2相同的CP菌株的50μL培养溶液混合，使其在600nm的吸光度(O.D.)为2，使用直径为150mm的BHI平板培养基进行双层琼脂斑块试验。用200μL移液器吸头在软琼脂上形成的噬菌斑上打孔，放入0.5mL SM缓冲液中进行洗脱。对含有洗脱的噬菌体的溶液重复进行双层琼脂斑块试验，直到形成同一类型的单个噬菌斑以分离得到含有纯的噬菌体的溶液。用0.2μm过滤器过滤获取的含有噬菌体的溶液，向其中加入10％(w/v)的聚乙二醇8000，并将混合物储存4℃下12小时。然后通过在15000rpm下离心1小时除去上清液以进行浓缩，将沉淀溶解于1mL的SM缓冲液中，用0.2μm过滤器过滤溶液，将滤液置于4℃。
实施例2.分离噬菌体的全基因组测序(WGS)分析
用CsCl梯度法和噬菌体DNA分离试剂盒(Norgen Biotek-Corp.Kit,Cat.No.46800)从实施例1-3中纯化分离的1mL噬菌体浓缩物中提取DNA。全基因组序列分析由Macrogen Inc.进行，使用De novo组装软件(SPAdes 3.13.0)组合基因，并使用GeneMark.hmm和NCBIORFfinder进行开放阅读框(ORF)分析。用BLASTP(E值<0.1)和PSI-BLAST(E值<0.005)程序标注每个ORF的功能。
结果，已证实分离的噬菌体具有SEQ ID NO：1的核苷酸序列，具有51670bp、73个ORF和34.10％的G+C含量，并且这与先前报道的梭菌噬菌体CP3(MF001357.1)具有97％的序列同一性，但已证实不存在所有片段与先前报道的梭菌噬菌体CP3(MF001357.1)的片段具有100％一致性的噬菌体，并且因此发现该噬菌体是新分离的噬菌体。因此，新型噬菌体被命名为噬菌体CJ_CP_20-25，并于2021年1月18日保存在布达佩斯条约下的国际保藏机构“韩国微生物培养中心”，编号为KCCM12934P。
实施例3.噬菌体CJ_CP_20-25的形态分析
为了获得高纯度的噬菌体溶液，采用CsCl梯度法。具体而言，制备每种密度为1.7、1.5、1.45、1.3的溶解于SM缓冲液中的CsCl溶液，在15ml的超离心管(Beckman Coulter,Cat.No.Z00901SCA)中按2ml滴入CsCl溶液，以形成高密度层向低密度层的分层，向顶部滴入实施例1-3中获得的噬菌体CJ_CP_20-25浓缩液2ml。在4℃，25000rpm下离心2小时，然后用注射器(Satorius,Cat.No.17822-K)只收集管中形成的白色噬菌体层。将1μL收集到的噬菌体溶液滴在碳包覆的铜网格上，用2％醋酸铀酰染色15秒，在电子显微镜(TEM,JEOL JEM-101,Tokyo,Japan)下观察其形状。
结果，如图1所示，观察到噬菌体CJ_CP_20-25在形态学上具有二十面体头部和长度约为100nm的可收缩尾部，因此被发现属于肌病毒科，有尾噬菌体目。
实施例4.噬菌体CJ_CP_20-25的pH稳定性评价
为了检测噬菌体CJ_CP_20-25在宽pH范围内是否稳定，制备了每种pH为4、7、7.5或10(pH 4：0.2M醋酸钠溶液；pH 7和7.5：0.2M磷酸钠溶液；以及pH 10：0.2M Tris-HCl溶液)的溶液。取对应各个pH的每种溶液450μL与50μL的2×1010PFU/mL的噬菌体溶液混合并在4℃条件下静置2小时，之后进行双层琼脂斑块试验以评估滴度的增加或减少。
结果，如图2所示，噬菌体CJ_CP_20-25被发现是一种表现出优秀耐酸性的噬菌体，因其在pH 7至10的条件下稳定没有丧失活性，且其活性在pH 4下比pH 7.5组仅降低了约1.3logs。
实施例5.噬菌体CJ_CP_20-25的热稳定性评价
为了检测噬菌体CJ_CP_20-25在高温下是否稳定，将500μL的2×108PFU/mL的噬菌体溶液于60℃静置0、3、6或24小时，进行双层琼脂斑块试验以评估滴度的增加或减少。
结果，如图3所示，相比对照组在60℃暴露0小时，在60℃暴露3小时的噬菌体CJ_CP_20-25的活性降低了约1.2logs，当暴露6小时时活性相比于对照组降低约1.6logs，甚至当暴露了24小时时，活性相比于对照组降低降低了约5.4logs但仍表现出活性。因此，噬菌体CJ_CP_20-25被发现是一种表现出优秀耐热性的噬菌体。
实施例6.噬菌体CJ_CP_20-25的溶菌谱评价
为了检测噬菌体CJ_CP_20-25的溶菌谱，在BHI液体培养基中分别培养了分离自从韩国首尔、京畿道、忠清道、庆尚道的畜牧农场收集的粪便样品的和动植物检疫局(Animaland Plant Quarantine Agency)分发的共45种CP菌株。这之后将50μL每种菌株的培养溶液接种至5mL的0.7％软琼脂，接着将其倒入皮氏培养皿中，盖上盖子，并静置5分钟。其后，将10μL的获取自实施例1-3的噬菌体CJ_CP_20-25浓缩物点在软琼脂上，在42℃下静置培养18小时，培养终止后，根据软琼脂上形成的噬菌斑的存在或缺失情况评估噬菌体CJ_CP_20-25的溶菌谱。
[表1]
(++：形成清晰的噬菌斑；+：形成混乱的噬菌斑)
结果，如表1所示，噬菌体CJ_CP_20-25被证实能够杀伤广泛的CP菌株，因其在所有45种测试的CP菌株中都形成了噬菌斑。
根据以上描述，本公开所属领域的普通技术人员将能够理解，本公开可以以其他特定形式实施而不改变其技术精神或本质特征。在这点上，应该理解以上描述的实施方案在各个方面都是说明性而非限制性的。本公开的范围由所附的权利要求而不是此前的描述加以限定，因此，所有源自接下来的权利要求及其等同形式的意思和范围的变更或修改应该解释为包含在本公开的范围内。
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<110> CJ第一制糖株式会社
<120> 具有特异性杀伤产气荚膜梭菌的能力的新型噬菌体及包含其的抗菌组合物
<130> JL-65312-PCT
<150> KR 10-2021-0025978
<151> 2021-02-25
<160> 1
<170> PatentIn version 3.2
<210> 1
<211> 51670
<212> DNA
<213> 噬菌体 CJ_CP_20-25
<400> 1
ctttttcttc tagtgtactg tttaccatat cgggttacag tgtactctgt actctcatca 60
tctagcttaa gaattttaat tgtcttaggg tcttcaattt cttttatctc ttttacgaga 120
gctgagtctt taggatcttc tacgatctct aaaaaagaat acccaagtcc ttctctccaa 180
tcaactatct cttcaaagac attcttggtt ggtttatcaa aagacagggt atcaattaat 240
tcttccagct gtttatattc agcttccatt tctggagtag gttcatctac cttctcaata 300
tacctaatac ctattccgaa tccagcaata ttatttcggt aggccttttt gcattggggt 360
attattgtag actcccttat aatcacttct aatagttggg ggtcatattc aggggatatc 420
tcatcaactt caattccaag tacactggtt tgttgattag atttatccac tggatccaaa 480
gacttttgga tagggtctcc acatatactt ctggcggata tctttttcaa agttacttta 540
cctcctttct taccttatat atcaaattat aaaatccaga tggcctcata tttcagggag 600
gattacatca actccacata tagtgtcaaa tacggccatt cacatgattt aaaaataact 660
ataatttgat acgtttatta taacataatt cacaaaaatt tgtcaacgtt ttatttttac 720
attattttcc agaagatttt ccgcatatat tccccatata aaattatata attattaatg 780
tatactatat tactatatta atttatgaaa taaattatat agtatatagt atacttatat 840
tacatatata taaaggtggt aactctgata tgccttctat atcacattat tactaatata 900
gagcctagtc aatttcaaaa ctgatatctc acatatcaga ataaaaatta ttggctttaa 960
taggcatatc agttacttag atttttctta atttagatcc aacaggaagg caagctaaga 1020
atagagagtc tgctaagtct ggagaatgta gtcctctttt cttcatttca tccttactct 1080
ccagctttat ttgacctttg gagttgaata ctttaaactt tctggtagtc aattctccca 1140
caagatctgt attatctggt agtcttaact catcatcctc cagtaattcc ttaactacag 1200
aggctatata tgtggttgta tcataataaa aggcataagc ctgggacccc ctctttaatg 1260
gcataccaaa tttacatggt actaccttca accagtccag tttttcttca aattttatct 1320
cctttaatct atcagttact cccccaccta caccagtgtc gtctattttc acaactactg 1380
ctagtttagg gaactcacca tgtagggatt tagcctttct tagcacttct cctactgttt 1440
tcatagtgtc cttaccttgg taatagatag gttcttccac atatccatca attttggtca 1500
taatagaggt cttatctgaa ccaaatctag ctacgtctac acccatatct atagtaactg 1560
gttctgtagg tctggttatt tcactattaa tacctctata tactgtggat aattgtataa 1620
atatgtcatc ttcttctagt gggaagtccc cttttaccag acatctgtag acattggact 1680
cttctccata ctttttcctt aacattttat ggttttcttt atcagttcta tcaacttttg 1740
tagcgtcaat atgaatacat ttaaaatttt ccttaagact gtggtgagaa tcaaagaaga 1800
tacctgtatt tttaacaggg ttaccaatca aaattatctt attttcctca tcggatatag 1860
taccaagtaa tgcttctagt atctcatcat ctattccagg agcctcatca catataatta 1920
gcataaactc actatgaaga ccaagcatat tctcttttct attactagtt ttacacatag 1980
caaaccaagt cataggacta caattcctag tcatagtagt tttatttact gtaaagtaat 2040
cttccacaat agttccttgt agccattttg atatttcagg ccataataca gatgttaact 2100
gtgaaaatgt aggagctgta gcaattacct tacattcagg cctaaaggtc atataccaaa 2160
ttataagaca agcacaggcc atggtcttac ctaccccctg tcctgacctt acagaggttc 2220
tcttattgtc tcttacactc aataaaatat ctttctgatg tggatctaag tgtactccta 2280
gtatatcttc cgagaatcca caaggatcgt catagtatag atctactaat tcttcaaagg 2340
atatttcttc cagagcttcc ttgttaatgt ggaacttatc aactaaagca tctagctcta 2400
atttctcaat tatgctatct aaatcaaaca attatataca cctccttagt aaagtgcttt 2460
atttagtcct gtaatacctt tttacttagt ggtattagat tttcctttac ctgcattctt 2520
tttctttcta tcagccattt tggctctaag aacctcaaga tcttttccct taccagcatt 2580
attctgattt tcctgtattt gtaaaagtaa tttaaccttt tcaagttcaa ctttcatcac 2640
atcagtactt aggccgaagt atacctcaag tctttttagt gcctgaagtc tatcctccat 2700
tttaactgat acgccatctt tactttgttt aatctcattg attaaagtac catctacagc 2760
cttactagac cttaatagaa ctagatcatc cttgaattgg cacacatcag ttatatctgc 2820
aaaggctatt ttcatgtact gccttattac atccatacca tcgcatagta attcttcatc 2880
tctatcggct ctgatagcct ttagagcttc ctgtattttc ttcttcttta atagtttaga 2940
cccatgtgca tgagaattat tcccaggata acctgctcta gcagctgctg tagttgcatt 3000
aaagcacttc atgtaatgaa atacaaacaa ctcttctttc tcatcaagat catacttatc 3060
agccagggtc ttaatagtct tttttgtttt gctactcaat gccacaatat caccttcttc 3120
ttctaaaaat gttatgccct gctctttaaa ctctcttaga cactgcctaa caaaggtctg 3180
tgatacctta gcaaggtttc ttagttgttt aggatgtgta aagggatcag ccttcagggc 3240
attatatatc aattttttgg ctttcttcaa atgttcagga gtacagtact ctggttcagt 3300
gtatgactta tagctcataa tacaatatca cccccttaaa atccagactt tactacgtga 3360
aaaatggttg gcacactaac cttaatgcta gtaataccaa ccaaatggca aaattaacta 3420
tggatttata ccactattat accacatctg gattaattat gtcaacgttt tataatttta 3480
tttcacctgt caatattttt tcatacatat ccacatctat tcttccttcc tgtagcaggt 3540
tatcagctgc aatttgtatc tcttctgggg aatatactgg attaccttta gtatcaagtt 3600
ctataccagt atatgagtct accttacctc tgttcatacc atatccaact gatttatgaa 3660
cttctagttt ttcattatgg tcttcttgcc agtgttttaa ttcttcctga tattccttat 3720
tctcctcttt agtttgttta cattcaatag gtctattatc tttcatcttt tgctttagag 3780
ctgctgtatt gggtttacag ttcttagatc ctccacctga acctctattc ttatcttcct 3840
cataagcttt attaatctca atcccaatta ttagatccat cttctctacc aagtaaagac 3900
attcttttac tattggattg ttaggttgat ttttagaaat gatgtcttga cactctttct 3960
ttaattgatg tggattatta cattggctag ggcatttttc tcctggccca tatggaattg 4020
tatatgctaa gcagttacct gctggatccc ttctataaca tcttctttta catactattt 4080
tactcatggt taatacctcc taatatattt tctttctctt ctaataattt ttcaagttcc 4140
tcaagatttt cttctttatc actattacca gctttagcta tatttagaat agcttctagt 4200
tgttcctcac tcaaaaactc ctttaatttc tcatcctcta atatattaga tacctcttta 4260
ctcatattgt tctacctcca catcattaca aattaattta gctattccct cacttaatct 4320
gtatttctca agatatacca ctttcttaat tcctgcattg attattaaat tagcacagct 4380
ctcacatggg gataaggtta catacatagt agttccttca gttgagatcc catgtttagc 4440
acagtaactt attaagccag cttctgcatg gatcacaata tcacatccag gacctaaaca 4500
agttttacaa tgttctactc ctgaaggtac tccattatat gaagtagcaa ctatcctgtt 4560
atctctaact aatacacaac caactgcttt tcttaggcaa gtagatcttc ctgtcataag 4620
tttagcaact tccatgtaaa gtgtttctct gcttattctg ttgttattat tttccatagt 4680
ttatcacctc aattttatta tatactattc cctatcatat gtcaagagaa actttagaaa 4740
ggtatatcac cataatccag atcaatacat tgggctggct ttggttttac ttttttaggc 4800
tctggagttt tcttgacttt atgatacttt ggagctctgg ctaaatcctt ttttaacttt 4860
ctgactctct tcatatccca ctcatgtatt tcatggtctc taaaatattc attcaatact 4920
cttactgcgg atctctttga aggtgtcttt acagagcagt tattaggagc aagacagcat 4980
cctgtctcct tatctgatat agtataacat actgtattat gaggggctcc tacaggccta 5040
tgaatgaata aggttctgcc catataatca aactcacata atacttccac atatctttta 5100
tactgttgtc cctggtcatt cttacataat acataagtat attctttcat gttaattctc 5160
ctttctatgt taaaagggag atcatctctc cccttaatat ctttttattc ttctgttgtt 5220
ctcatcaacc tcaatcactt cataccttct aaccttatca gatatacctt cctcctgtaa 5280
tactttagga atatatccta tggcctgtct tttgttatca acttcaaaat atctggtcaa 5340
gatccttttt gatgttacaa tcactaatct tactttcata ttatctacct ccatgtggcc 5400
tgaactacag ctccaggtct atgcttagct tcaatcttag cttcttcctc agtatcaaaa 5460
tagattatat cttccttgtg ttcatcctta acagataccc agtgtactcc taaatatttg 5520
ttatgtgcta atacactaac atcagggttc caatccacat caactgtaac ttgtaaaacg 5580
atatatctcc ttttcatatc agttctcctt tttgtattta tttgttgatt ttattataag 5640
agagcctcgg aagtttgtca aagattttct cgaaggaatt cgtataatat cttccgagct 5700
gagcataata tagactataa aacaaaatca aggagttgta ataaatgaaa gataataatt 5760
taataccatt aagagatatt gccaaagagg ttggaataaa tgaaaggaaa gccagaaagc 5820
tattaagatc acatgagaat aaagcaaatc acaattatta ttggatgttt acaccaagag 5880
agaaaagaca tattgtatca gttctaaaga gactgcaata ggttttctaa aaataaggga 5940
gggattaacc ctccttttta taattttttg agttctggaa aaatccttaa ttagttcaag 6000
tttcgtaaag ttctcttgat cttttccatt ctcttcttaa cttggatagc ttcttcttcg 6060
caaggagcat ggccccctag gaattggtgc catcttcttt caagttctct gtattcagat 6120
tgaagttcca tgaactcaag tgaatgattg tccatactat cacctcctca acactattat 6180
aagtgatact gtcatattct gtcaaataaa aaagagagaa tgttattctc cccatcttaa 6240
tatctttata gttgctccat caactccctt gccatccaca tcatggacat acttatcaag 6300
tctcattgtt gagtcctgta taattctttt gtcttctctg tcaaaaacct taatgacatc 6360
agggttaccc ctgtcaatga tgtaagcaat ctcttgagtt ccatctgcat atgttacttg 6420
aagtaatgaa tgtagaggaa taaagtctgg agctgcccaa tcaccaatct taagatcccc 6480
tcctatacta ttcttggtct catatcctgc cacatctgta ggctcatctg atgagtaata 6540
agtaacttca actttgaact ccattccttt aaatgaattt atcatctcaa ttagattttg 6600
tttatcagtt ctcagtttat cattctgata ttttaatcct tctacattgt ccatcagtat 6660
ttcagtctgc tcagttaatt gtttattcga ctcaatatat tcttggacag catttcccca 6720
atgattgttt aggctctgta tggttaacag tagccctcct atcacaatta ttgttagtaa 6780
tgacataatt cttttaatca ttctctttta actcctttat ttttaatttg aatttctcaa 6840
ttccagattt atacttaccc acaatatcaa aggtatcggt tagtattgca taggtttccc 6900
catcagaatt aaatctgaag cttcctctct tgatgtctcc tagcttatta aaggtcttac 6960
cctccttgtc cagatatcct ttatagtcca tatagcacag acaagcagta acaaactcct 7020
gcttattcct gaaattatgc ttgataccca taccatctac ccatttactt aatacaaata 7080
gactttcatt agtcttcttt actttactat tctggattag atccttccac acataatcat 7140
tagcctcttt aaatgactta aatctcttgc ctttcagtat cttactactc tggatcttgg 7200
ctttataatc ctcaaatata cttggccaga agtcttttag atataaatac aaggaaatat 7260
catctaattt agcattcgga tcgttaatct ggattacagg atgaccatcc attgctctga 7320
tattattcct tagttgggta ttctctctct tcattttaga tacttcaata tataatttaa 7380
cgaatgcgtc agacaattca tgattggtta attccctttt taggggctta ccatctttac 7440
tttctttagt agttacaata ttaaagtagt cttgtaacct atcccagtta ttcttcatta 7500
gatctacctc ctatgcttac attatacata tattgtactg atatgtcaaa gtccaggcat 7560
taactgatat ctgatatgcc tatattttta ccccatatca gtctgatatc ccacatatca 7620
gttgaaacta caaaatcaca aaggcattgg tatcactggc gtagagggat atcagttatg 7680
ccccctttat atatacatag tattatatat actatataat aatatattat tatatatagt 7740
atatatatat tacatggtga ttacatcaat ggcaggctat tgccttgcct ccattatcat 7800
tacggcaggc atatccatgc ttccgagctt ttgccctata tagaaaaacc cagacggtca 7860
gcgatcgagc tcttgatttt gttttgtcga gggagagaac agtaagagag aacccccgaa 7920
cgttgacagt tgtacttact tctcttataa tgacagtaaa ggagggataa ccatgaaaac 7980
attatttact gatacaaaac tagtattaat tgaaactact aacttcttca ttatgttaca 8040
agaagaaggt aagtatgtac tatctaaaac aatcgataag gagatgggag aacaaataca 8100
cagagtatct aagttagctg cccttgtata tactaatgag gagtatacta actctaatga 8160
accaagatct atatacttag atgacaattt cttatacgaa gtgcttggta atgggaagta 8220
tattataaga tacagtatat aagagcccac aatcaattct aaggcctttt agatatgtgg 8280
taatgtgatt ttacttataa tgtggtagag acaccttaca ggagctgcta gaggccttta 8340
aaggaggtat atcatgtatt tatgtaatgg taaaatagtc aagaataaaa atgacttaaa 8400
agataagttt ggtggtaaat cagtagtatt catcgatggg gatgtagcta cagtaatgga 8460
ctttagtgac agaaagccaa agataacaaa atatcttatt agggaggtat cacatgataa 8520
acacatcatt attgaagaga ctggaaggga tagaggaaaa gcataagcaa aatactgatg 8580
agataagaaa gaacctaaac tatgccagat atttgattga gaaacagtat tgtcatttaa 8640
caaaagaaca gaagataaaa ttgggacagt tcttaggatt gactgagaag aagttaaaag 8700
aaatacaaga gataaataat cagttaataa atttgagagt ggggctaagt tatgaagtcg 8760
tatgtaataa ccagaaaaaa tagtggggta tctttagcag atacagacat gggacaagta 8820
ttagctaaga tactatgtaa gcatggggga acacttacca ttgaggaaat tgaaccagaa 8880
catattggtc aaactttttc tattgtagaa ggtaaaagaa tatggtataa tagaaaagga 8940
ggtgataata ttggggagaa gaagtgctat gttaattctc aagtatgact gcgaagaaac 9000
tccagaaagg attgaattac gaaagaagtg gttacagatt tgtcttcatt caataatata 9060
ttatagatat gacaacaata tctggacaga ccaacaatgg gatgagactg ccagagaggt 9120
agttaaactt aagaatgaga atccaggttt agtaaacagt atgccattta ggaatgacct 9180
taaagtcttt gatggatcca ctggctttaa cctttcatgt atgcaagata ttaagatgtt 9240
aaggtgggct caaaacttac ttgactttca tgttaaacga ggacttgatg gaaagagtaa 9300
gaaaaagaag aagaagagga ggaaaaagaa atgaacctac ttgagcacta tataaaaaag 9360
gtacacagtg taagagagta ccatgaattt gataatgaac cttgggctaa aggtaaacaa 9420
tatgtagaag tagacatgga ctatatttgt tattctaata caccacaaag agtgaagatg 9480
gtatttactg ttgatgagtg ggagaagata aaatatagag gctactacat gggataggag 9540
gaatagttat gataagagaa atgggcatga gaatgctaat accagaaata catgatataa 9600
aagataggga tatacaaaat tttgtaagaa aggccctaaa tgagatagtt gatgaaaagt 9660
tctttataat acctgcaagt agtactggga aatatcatcc aacttatagt gcaggggtag 9720
gaggtttgtt aagacatact aaagctgctt gttatatagg taaagcatta tgcgaagctg 9780
agatgatatg ttcaagggac agagatatga taatagctgc tttaatatta catgacatta 9840
ataagccaga taaagaacat ccatatatgg ttagagagac actatcaccc ctagaggaag 9900
agtttccaat aatatacaaa gtggtagttg aactaattga gtctcatcat ggtcaatggg 9960
gtagtgttcc tattaatagt tattcccaga gaatagtaca cttggctgat tatatcagtt 10020
cacagaaagg tttaatattc tcatatgacc aaggaactga ttattctgac ctgttatggg 10080
aggagaagta aatggtgcta ggggtaataa ccctagtatc aatattttta ctaggagtgt 10140
ttataggttt tgaggtgaag aaatggaact aggttttgcg atactagcta taatagtatc 10200
aatattttta gaaactaaat atataaagga gtagattatt atgtcaagag caagagctgc 10260
aaggagaaga gccgaaagag agaacaaaaa tcttaatgtt cttaaagctg tggacatcat 10320
gttagcctta tcatgctata cattaaagaa ggaaggttat ggtaagacta gaatgactag 10380
gtttgtggaa gggatgagta aacacacaga ggagattgaa aagggaacac ttaactatga 10440
aaccataatg ggagagatta aggatttagt accaccaggt atatttgatt taggggagga 10500
gtcaaaggag tgatagtatt gggatattta ttatgggcat ttagaatatt aggcatactt 10560
gtaatagtat ggtgtataat ggaggcgatc agttgtgagc aataagttat attttaggta 10620
tagtgctatg aactctggaa agactactca gttaatccag gtagcacata actatgaaga 10680
aaggggaatg aagccattag tagttaagcc cggaattgat actaaaggtg gaccatgtat 10740
aatcagtaga ataggggtag caagaaaggt ggatgttctt ctacctccta gagttaaatt 10800
atcaaaaata ctttctgact ttgagtctaa ttattgggca gtagatgtta tattaattga 10860
tgaggtacaa ttcttatcca gagaacaggt tgatgatctg ttaaatttat cattacatta 10920
tcctattata tgttatggat tgagaactga ctttaaaagg gaaggatttg aaggaagtac 10980
cagattatta caagtggccc ataatattga agaattaaag aacatttgtc aatgtggtaa 11040
aaaagccaca ttctcaatat taaaatacca gggtaaatat actgaccagg gtaaccagat 11100
acaaattgat aaccagcccg aaagtgttga atatgaagcc gtttgtaatg aatgttataa 11160
taaattaatt ttttctaaat aactcgttga caggctatat aggatatgat attatagtat 11220
tgtaatcaaa ttacaaacac caaccaaaac tttataatta gttaatttca aactatacta 11280
aatttgttat ttaaaaatta aaaatatgga ggaatgatta ttatggcaaa aggaaaagca 11340
aaatggttag aggaagcaaa caataaggag ctaattgtaa tcgtacaatc tgctttaaat 11400
tctggagatg agaaagaagt taaaaaggct ttaaaagcta ttgtggaatt agatgaaaga 11460
actcctgaca cagatggaca aataactgta gaggaagcat tagaatcatt agaaatggct 11520
gtaggaactg taaaggctgc ataccaagca gaaatagatg ctgtggaaga tgaaggtgta 11580
gtagatgctg acttcgaaga agttgatgaa gacgaagcag aagaagaaga tgaaaatgat 11640
ttagaatcat tatctaaaaa ggaattagct gctatggcta aagacttagg tatcaaagga 11700
gctaagaaaa aagataaaga tgagttaatc gctttaatca aagaagctca aggtgaagaa 11760
gaagacgaag aggaagaaga agaggatgaa actcctgact actctgaaat gactaagaaa 11820
gacttaattg ctcttgctaa agaaagagga ataaaagtta ataagaaaat gaaaccagct 11880
gagataattg aattattaga agctgatgat gaagaataat tctcatcaaa taataagggg 11940
agcaaacttg ctcctcaaat tttttactta ttttagaaac agatgttaga gggggatgta 12000
taaatggcaa gaggtggaaa ttcaacttat actaaagcag tatattgtta caacaataat 12060
agggagtata gatccttcag atatgcagct aaacagttag gagtatcggc tgttagtata 12120
atgagagtag ttaacgggac taaaccacat atagggggat tagtattcgc tgagatagag 12180
gaaggcacta gaaaattaaa gccaagagga gaagtagttc catacttgaa agaattaggt 12240
tgggataata tagacttata atagggggta taatatatgg acataaagtt cttgaaggat 12300
atattctcta aacaatgtga aaatagagat tacattatcc tagcagctag aaaaggtaaa 12360
gaatggaaag atgtaccaat caaatataat aagaataaca ttgacaaaaa actaaaggac 12420
tttgaacaac agtataaggg ttatgattta tattggagcc caatgcccta tagtaatcct 12480
caaagaagga tagttaactt catagagact aaatacttaa tacaagatat agatgagcat 12540
actgacccat tggggattaa acctaaacca agttatctgt gggaaagttc tcctggaaaa 12600
tatcaggggc tatgggaaat ggataggtat atagaggcta atcaatatga cgaaataaat 12660
ccagcattag ctaaacatat aggctgtgac tcctgctttg atgttactca tgtatataga 12720
ataccaggaa ctattaacta taaatacaag aataaaccaa aagtaaaaag acctatacac 12780
actaaggaga tatataagcc taaggtgatc gctaaggctg ttaaggctgt aagtaaatct 12840
aatgatagtg ttaaggtcaa tataggagga tctgaggcct cacaatctga aagaaagata 12900
tatgctaaat ataatatacc aaagaaggtt agagacttac tggctttaga tgacattact 12960
tctttagata gaagttctac tatatggtat attgagaata aattacatga aataggacta 13020
gagcctaatg agattatatt attagttaag ggctcagctt ttaataaata taagggaaga 13080
aaagatgaag aaacaaggtt aagaaaagaa ttggataaaa tcataggagg agaaatagag 13140
gctgatattg aaaaggctga aagtactaaa ctgagaatag atagttatca ggatgttatg 13200
ggtaataatg gtgccttccc aggttggtta gtacaaggtt tctggggtag aagatctcat 13260
ggaattgtgg ctggacaacc aaaggtattt aaatccacat ttacacagga cttagctata 13320
tcagttgcta gtggaagacc cttcctaggt caatatcctg ttctagaacc aggcccagta 13380
attgtagttc aaaatgagaa tgctgactgg attatgaggg atagaactga aaagataatt 13440
agccacagag gagtagttgg taatgtggat ataaagggta agagaagact taaagttagg 13500
tttgctccag atcttcctat cacttttatt aatcaacagg gatttatgtt agatgaagaa 13560
tcccatagaa aacagataga agaattaatt gatgagataa aacctgtact agtaatattc 13620
gacccattat atcttatgtt taggggagat cttaataatg cagctgatct taatcctgta 13680
ctacaatggt gtcttaaact taagaatgag aagcatacag gagttatgtt aatacaccac 13740
tataataaag gtggaaatgc cactcaaacc agaggtggtc aaaagatggc tggttcattc 13800
atattacatg gttgggtaga atcagcatta tatttaaaaa gacctgatga cttagaaggt 13860
gacgatgagg agattgaagt agatatagat aaccttgata aacaaagcca tttaccaagt 13920
aaaatcatta tggatagaga gttccgtctt gcaggacaat tccctcaaat tgaattgaat 13980
ttatcaatgg gagaatttgg agatccatat tatcatgtag aagtagcaat acccagaaaa 14040
gaagtaattg tgaaacctga ggacaaagcc aaagttatag aggctgtaaa atcaggagct 14100
cataccaagg aggagatagt gtcaatatca ggattaaatt cacagaaggt taatccagtt 14160
ctagatactt taaaagatac tattgtatat tctccggata aaggttataa tataagtaag 14220
aaactaaata ttgggaggaa aaaggcatga taataaagtg taagaggcca gcaggtcata 14280
atcatataaa ggcctttgta gtagtaatta ttgctagagg taagaagaat aaaatcactt 14340
atcattcagg attgaaatat ttctatacct ataaatcagc taaattattt atagacaaag 14400
ttatggaaga atgtccctgg gctgaatttt attgggtgga tgtccatgaa gcccataaga 14460
tccctgaagg cgaagagata cctaagaaga aaatgtggtg tccatattgt caaactattc 14520
aagaatttaa gagaagccaa ggaggttaca agaattgccc tatctgtggt ataagtgacc 14580
aagactttta tgtaaaatgt atgaatataa gatataggga ggctaaggct aatgcgaaga 14640
agaaacttga aggaagtaat actaatccta gggatatcat tgtcaatggg gataatgcta 14700
actcaggggg caaaactaat agaaagtcat caccaaagaa aaagtcaaga aaagttagta 14760
gaaaagcttc ataatgcttt agactataat gttattgaga cttctccatc tggactagat 14820
ggggaggatc ttattatcaa actaaacctt aatagtccta aagaccttac ccctaacaac 14880
ttatctaagt tagctaatga ggttatggtc aataacttag gagctcagaa ttatactata 14940
cttgtttatg atagtaatag accagaaaat cttctggctg ttatagatat acctaaaaat 15000
tctaaaataa cctttgacat aataaattaa ttatggtaat ataaaaatgt agaaggagga 15060
gaatttaatg gtcaataaaa agtattacat cttgaaagac cttaatgatg acaaacccca 15120
actatggaag ggaagtttaa ccactgctac taagatagca ggagcattag gagttagtgt 15180
gataaaacta aataggtatt tagtaagaaa gggatttgcc agaaggtaca atgagcatac 15240
acttgagata atagacccaa tccttgctaa agaacttggt tattatgtaa ctccaagtaa 15300
gtcccttata aatcctttat ttgaatataa tgataaagga gtacaatata tactaaactt 15360
aatagtagaa gatctaaaga atggaggaga gctttaatga ttatcatttt aggaatggat 15420
aactcaggta agaccacaac agctaagaac ctaactaaac acagaggagg agaatatatc 15480
caatcaatgg gacctggttc ttatgaagaa caaagagaat gggtattaac tcaaattgtg 15540
agaaaaggtg aaagggaagc tatacatgat agatttactt gctttgaaga aatggtgtat 15600
ggacctatca tcagagatag tagtaatttc aaccttgaca gtaaggagct aaagatctta 15660
aaacatctat gtaagccaac tattgtatat gctagaccac ctagggaagt gatcttcaat 15720
tttggtgata gagaacagat gccaggagta attgaaagag ctgaacatct actggctaga 15780
tatgatgaac tgatatggaa gttattctgt gatggatgga atgtactggt atatgattat 15840
actacttcca atgtggagaa gctatcccag ttaatagatg actcaagtgc taaagaagct 15900
ataaaccaat ttataaatat atcaagataa gaggagagat tgttatgaat ataaaccatg 15960
cagtagaaga aaaaatagaa ggagatagat tacaagctat ctttaataga caaaaggagt 16020
taatggagaa atatcatcat atagaagcta gatcaggatt atgccaaact gaagactgtc 16080
cagttaactt aaatgataaa agaggtcaag ctagacttaa agattttgct tggagaatga 16140
ctgaagaagt aggagaagca ttggatgctt ataatcatga agaccattac caagaagaat 16200
taattgatgg attacacttc ttaactgaat ttacaattct agctggtaag gattataata 16260
ctatagataa taatgctata ttagttgata gcctagagaa cttatatcat aaggctacta 16320
catcaccaga atttcctgaa atgttagtag aagaagctgt aactgacctg gtaagagaaa 16380
tgggtatgtg ctgtaactgc cttaagaata aaccttggaa acaaacttca atgctaacag 16440
atgtaaatgc ttttaatcaa agattattca atgtatgggt atgttacatt aaattattag 16500
ctgtatctgg cttagaggta gatgacatag taaatatata ccttaagaaa tcacaggtaa 16560
ataaattccg ccaaagatct aattactaaa ataaatactt aggagaaggt aatatgctaa 16620
taagagacta taatgatttt gatgacttat tcttaaacct taatagggaa atgattacca 16680
atccagaaga aactatgatg tatactcaga acatacaggg ctttcaggag gacttagttc 16740
tctcctgtaa gtcccataaa tgtactttaa atctagggga ctttggatat aaagaaggta 16800
agtggggaca cctattaaga tcatacattg attatccaca attaattgaa tttagagaaa 16860
agcttaccaa gataagtggt atgagctata cttattattt caacaggaaa aaagctacta 16920
atggttcttg cttgatagct gcagtagtaa ctagaccaaa aagaaaagga ccttggaaac 16980
acttaaaaat tatgtataga gtatgtgaat tacaaaagaa attcgcagct gatctagtat 17040
taataaacag gttcattgaa gaactacctc aagaagtatg tgagatagat aatattactt 17100
ttcatatgtc tcaagcttat ttgtcaggaa tgtttataaa tggatatttc aattatttta 17160
aggtaccaag aaagagtatt gctaatagca aacatccttg gcataaatcc ttgaatagta 17220
actataacag gttctttaaa tcagaggacc agatacactc ttataaagct ctccagaaga 17280
tgcagttatt acattttggt ctggagaaat ttccaaagat agatatcaat aaattatcta 17340
ttgacaagta ctttaataag tagtataatt aacttaacaa aattaaataa atggaggaat 17400
taaactatga gaatttatat taatgctcaa gaagcatttg aagaagtaaa aagagattta 17460
tgtgaaatgg ggattggggt aagacccaaa actatgcaag ataaagttat agaaggtaat 17520
ccagattatt tcacaaagga gttacaaaac tatagttaca caattctaga aagtaaacct 17580
gaagaagttc caggtgtatc tcaaccttgg gcagatgctg aatttagaga aaggatctac 17640
gatccacaag gtgtcatcaa tcaatactct cttgaagaaa gagaagaatt atttggtata 17700
catccacatc atactaggga ggctttcata aatcctggta aagcttatca attaagacct 17760
gaggtatgga atgaatatct aagagatgga aaatttgggt attcttacaa tgaaagaatt 17820
tggcaataca gacaaattga ggatatcatc aatagaatta aagaagatcc aggctcaaga 17880
caattatggt tatctctgtg gaatccagct attgatccat tcaatatagg aggagtaact 17940
agagtaccat gttcattagg atataacttc caagtaagag aaggtaagct aaatatccat 18000
tatgttatga gaagttctga ttttgctact cactttgcta atgatgttta tcttgctatg 18060
aaactattac attgggtagc tgaacaaact ggctatgaac caggaagttt ctctcataca 18120
attttctctc ttcatgttta caacaaagat attaaggggg tattctaata aatgagttac 18180
atgagaaaag aaagatataa acattctgat cttaataata aggttaacaa gagggaaacc 18240
ctaaagcaat ttattaggaa tactgaaaag gaatttgatt tagaaccagc taaccttggg 18300
gacatgacta ggaatgaact taataactat ataaattata tggatgaact gtggagtaag 18360
taatatgtat aattatagtg atgataacag acctaagaac aataacaatg gctgtcttgg 18420
ttgtttaatt ataatcttag cagctatagg attatgggta ataatttttg atattgccaa 18480
tataatatat catatgatat tctaaattta aagggggcta tcctaatgaa aaatataatt 18540
tgtcctatgt gtaaaaactc agtaacatta aaaagaaaat atggagctat gttttggatc 18600
atgattttct taactggagg cctatgggta gtaaccatac ccttcaaaaa acataaggtc 18660
tgtccagtat gtaactcaat cataaaataa agataagggc ctttaggggt cctttctatt 18720
taggaggtaa tattttaaat gagatgtaac aactgtaacc tatatactca ctcagctccc 18780
tcatgtatag aaggaacttg tgtaggaaag aaaaagaagc caagaataat ggtcataaat 18840
agtttagcta atgataggga tgaggccaat agaatagcta ctcctgataa atcattactt 18900
gataaaatgg aaggtctaga cttttattat accaatgcta ttaagtgtag aactcctaag 18960
ggtactaaaa tcaaagtatc agagattaag aaatgccaag aacatttact taaggaaatt 19020
gaaaaatata aacccgaata tgttatgatc ttaggatctc aagcattaaa gatgctaagt 19080
aatgaaggta taacttcaat atgtggagta cctaagaagc atgagaaata tggctttaag 19140
tttattgcca gctattcacc cggtgtagtt gcatatgacc caactaaagc acaatttgta 19200
gatcaggcct ttaataactt taaagccatg gtaaaaggta aagagcatga attacctgat 19260
cttaacataa aacttattac tagtatgaaa gaactaaacc aggctttcaa atatttaagg 19320
gatgagggtt ataatagagt atcatatgat atagaaacta gaggcttaga taggtttaat 19380
aataacatta ctctatttgg ttttggtaat acccaagtac agtatatact gcctttagaa 19440
gtcaaatata gcccattaag gggtaagccc atagcacaaa gaagattagc taaatccttg 19500
attaaaagac ttaattctga gatgaaggaa agaatagccc agaatggtaa gtttgatgat 19560
aatttcttaa aggagaaata tggtattaag ccaatcataa cctttgatac tttactagcc 19620
tcacattgtt tagatgagaa tactcctaat ggtcttaagg agaatgcttt attacattgt 19680
aatgctaaag actgggatat aaataagaaa ttaaagactg gaaatgtgga gactaaatca 19740
gactttgagg attatgttag gtatctgggg tatgatatat actatacatt tgctctatat 19800
aaaatattta ataaaagact taaaagggat gagagcttat ataaactatt ccaccactta 19860
tacatcccag ctagtaaagc ttatgaggat gttcagttca agggtatata tgtaaatcag 19920
gagaaattca aagaggttga aaaatactta agatctgaac tagataagat tgagactgga 19980
cttaagaagt atactaatgg ccaggatatt aactggagct cacctaaaca ggttggagaa 20040
tttttatatg atatacttgg tcttcctgtg attgaggtta ctgactctgg agcaccagct 20100
actggagaaa gtgtattatt aagactaaga gataaacatc cagcagtaga actactatta 20160
cagcataggg gagttcatat acaaatttcc cactttatag atggttggct aaataggatg 20220
cacaatcata gattatatcc aaactttaaa cttcatggaa ctgtaacagg aagaacctca 20280
agtaataatc ctaatctaca gcaagttcct agagataaga aaatcaggag tttattagga 20340
ccatctcctg gaagagtatt cattgaagcc gatctatccc aggccgaact tagaatagct 20400
gctatgatgg ccgatgagga taatatgaag tttatttacc agactggtgg agatatacat 20460
gactccacat ataatattat atctggggaa gatatcaatg atgagaaaga tccagcagtt 20520
aagaaggaga aaaggaaaaa ggctaaggct gtaaactttg gtttcttata tggtatgcaa 20580
tggaaaaaat tcaaggatta tggaagagac aactatggtc ttaaattaac agatgaggaa 20640
gccaaaacat atagaaggaa cttctttaat aaatacccta aactattaac atggcatgat 20700
aaacaaagaa aaattgttaa agccaatggt gaagtaagat ctctaatagg aagaattaga 20760
agattacccg atatatattc atctgataga tctaaagctg ctgaagccga aagacaatgt 20820
attaactcac cagttcaagg ttttggttca gatatcactt tattaggcct atgtgagatt 20880
acaggctatg ctaaatatgt taatcctgaa tatgtattag ataagtctaa gtttgatgta 20940
ttaggctcag tacatgactc aatattattt gaagtagata aagactatgt ggaagaatta 21000
gcttggaaag taaaatcaat agttgaaaat aataaagtat taaagaaagt atttaaattt 21060
accccaacag tcccaataat catggatata tcagttggtt attcttgggg aggatgtgtt 21120
gaactggatt ttaaaggtga ttggaaaagc cagataagaa aagtgttgac agatgaataa 21180
ttatctgata atataaatat tgtaatgaaa aggaggtata aggatgttaa aaataagtaa 21240
ttccagaatt aacaaattct tgtcttgtcc ttatgcccat tatgttaaat actatgaagg 21300
tctggtacct aaaagaagtg gagctgcctt acaaaggggc tctgctatcc accaggctat 21360
agaagactac cataatggga agagttggaa aaaatctgtt gataaatttt ccaaagagtt 21420
ttacaaaaat acatttaaag aagagatcct tgaatttgga gatattccaa aaatggttta 21480
ttctttatgt gataactatt tccactatta tgatgaaaaa gaagataatg taacctatgt 21540
ggaaaatgaa catcacttcg aattaaaact atgtaagggt gtaactctag aaggctatat 21600
tgatagtgtc ttagatgtgg atggaaagat atgggctaag gaaactaaaa cctacaaaaa 21660
gatgcctgat agaaatttcc tgatcttcaa tagacaatct gctatatata cctgggctct 21720
tctacatgaa tacccaaaag taagtggtac tatatgggat ataatattag ctcagcaacc 21780
aggtagacca gaattaactc aaaaaggggt attatctcaa aagaggatta aatccacacc 21840
cctagagtta gaaagaggaa taagagaatt aggattagat cctaaagatt atgagtctta 21900
tattaattct gctagatggg aagacttctt tgtaagacac ccaataatat tatcaaagaa 21960
tatacttaac agtgtaatga ttgatactat tgagatagct aaacttatta gagatgaggg 22020
ccacaaaaga aaggaaaaga accttggaaa aggttgctct ttctgtgaat ataaatcttt 22080
atgccaagct gaacttttaa atcttgataa agaatttatt attaaggctg actacaaaca 22140
aagggaggaa agtgacaatg gcaaaaaagc aaaaatcaaa atcaaatagt tttgaggaca 22200
gattagtgga tctatatgat atagatgagc ccacaatact aacactttat ggaagatctg 22260
gttcaggtaa aactactatc tcaggaacac tacccaaacc aatattcttt attgatgtaa 22320
aggacaaagg tactctatca gctagaaaca agcttagagt taaaagagga gatatacaag 22380
tatttagtct aaagagtttt gatgacatat acgaggctta tgattaccta tcagaaaaca 22440
ctgataaatt taaaacagta gttatagacc atttaactgc tttacaagaa ttaggtaatg 22500
aaaaggtcaa agctgaagaa ggtaaggacc agatgagcca aagaatgttt ggaaatgtgg 22560
ctaattatat gaaagaggtt ataaaccttt ataaagaatt aaatgaggaa ggtatactac 22620
catgctttat agtacaagat aggttagaat ctggtgatgg tgaaggagaa gaccaattaa 22680
tgcctgaggt aggaccagga ttaatgccat ctgtatctaa atatttatgt gctgtatcaa 22740
gagtaatagg tcatacttat ttatatgaac actcagaaaa agagggtatg aaggttaaga 22800
aagaaatcca gtatagacta agactaggac ctaaccctta ttatattact aaatttacta 22860
gacctcaagg atctgaatgt ccagcttatc tggtacatga tttaaaatca ccaacaacta 22920
tctgggagga tattgaaact attctggctg gtgaatggaa taataagcca gctaaatctg 22980
gtaaaaaaac cagtaagaaa tctggtaaaa agaaaaaata aaattttcta aaaatacctt 23040
tgacaaccaa ttaaaaatct gataatataa acttgtaaac aaagaggtac aatatcttga 23100
caaatattta tctcataatg atattttaaa atttaaggag gacaataaca tggctaaaaa 23160
gactactaag agaggtaaca aaaacaaagg aggattaaag attgatcttt caaacgttga 23220
aacttcagtt actatcccag aaggaaatta cattgtggaa gtagaagatg tagaggttaa 23280
ggtttctgaa aatagtggaa gcaattattt atcatttact tttgtaatag cagaaggaaa 23340
gatgaaagga caaaagttat accacatttg ctcacttcaa ccacaagctt tatttaactt 23400
aaaaggtgtg ttagttgctt taggatttga tatccctgat gaggagttcg aattagatac 23460
agaagctcta gttggtttac aatgtggggt agaagtatca catgaaatat atgagggtaa 23520
gaagaaatca agaataactg attttataaa ccttgacgaa gctgactctg atgatgacga 23580
agatgaggat gatgactcag atgatgatga agaagatgat gaagacgatg agtctgaagt 23640
tgatcttgaa gaattagaca aggatgagct aaaagaatta gctaaggctt taaaaatccc 23700
agctaagaaa atcaagaagg ctaaaactga agaagatcta attgatctaa ttgaagaaga 23760
agctgacgaa gaagaaatag ctgaacaata taatgaccta ttcggagact ctgatgaaga 23820
tgacgaagaa gatgaggatg aagaagagga agatgaagaa gaggaagatg aagaagaaaa 23880
tgactatgag tcaatgactt taaaagaact taaagctgaa gctaaggaca gaggcttaaa 23940
ggttaaaaaa ggaatgtcta aggatgacat catagaaatg ctagaagaag atgatgaaga 24000
ataaaattta attattaaaa tatgagggcc tttaaggtcc tcctttattt acccttggag 24060
gctactatta tgcttgaaag agatgtggtt aaatccataa tgaatatgct taaaaaagaa 24120
tacccaggtt tttggtttaa aactcatggg ggaccatttc aaatagctgg cttacctgat 24180
atactaggtt gccacaaagg taagtttatt ggtattgaag ttaaacttcc tggaaaagaa 24240
aagaacctaa ctcaaaaaca aaaagacatt ataaataaaa taaatctagc aggaggaata 24300
gcttttatgg ctacctcagc tgaatataca aggaggagat tacatgaaaa atttagaaag 24360
acaccaacaa ttcctaggag aactaggaga tctgtatgaa ttaaagaata atctatatgg 24420
ggataatttc cacaagactt atcttgaata tggaaaccct gttctatgta taagacttga 24480
agataagcta ggaagagcta aaagtttatt actaggagat caagatgact tcccttcata 24540
tgctgctcaa aaagaatctg tggttgatac tttactagac ctagctaact atgccattat 24600
ggctgctatg gaactaacta gtgatgataa ctcagaaata catgatctta gtaaagaaga 24660
atatgatgaa gaagacctag atgataatga cgatgatgac ttagatgatg atgacaatgt 24720
ttacgacgaa gaagaattag attttgatag tatgaataaa gaatccttaa aacaatatct 24780
aaaagataat ggggttaaat tccatagtaa ggcctcaaga gatgaacttg taaaactggc 24840
taaggaggta taaggaggag gctttaatgc ctcttctttt ttagtatgaa gagaaaatta 24900
tttaaacatc agaaagaagc attacaacta tttttaaata aagaaaagtt tgccctattt 24960
atggacatgg gtacgggtaa gaccttagtc cctattgtgg cccttgagaa acttgaaggt 25020
ctagatactg tactaatatt ctcacctaaa tctattgtat ttaactggga gtctgagatc 25080
cataaattta ctaaacttaa ggaatatagg atatttaaat tacagggtaa taagaccaag 25140
gttatggaaa cctatagagc tataaaatcc tactcaggat taaagattat tattgccaat 25200
tttgagaagg ctaggttgat ggataaatac cttatgaact taaagccaca gtttattgtt 25260
gttgacgaat cccataaggt aaagaataga aatgcccaga tatctaaggc tctatataaa 25320
attgctacta aatgtaaata taggttgata atgtcaggga ctcccactcc taatggttat 25380
gaagatttat ttatgcaata taaaattatg aactcaaata ttctaggagt taattggaaa 25440
cagtttgaag atgactttat aatcaaagga ggttatatga actatgaaat tgtggggtat 25500
aagaatgagg aaatattaaa gaaccttatg caccagaatt gctatatagt aagaatagaa 25560
gattgtattg atttaccaga acaacttcct gatctgtatt taacctgtga attaaatagt 25620
aaagccagaa aggcctataa tgaccttaga aaggaaatga tagcacaact agatatagta 25680
caggaaaaca ttcctagaaa gcaattaaag gccttattaa ggtctaatgg tataccctat 25740
gagagtaatg agccctatga ggatttattc ttaagggcta atatgtttat caaccaatta 25800
acagctgatc ttactattac ccaatatttg aggctacagc agatctcagg aggatttatc 25860
acaaacaatg taggaaataa tattaatatt gataaaggca aattaggctt gttacaggac 25920
tatctagaag gatataaaaa gcctgttgtt gtaatatgta attttcttga ggaaataaaa 25980
cttatacatg atacttttaa aaagacccac agagtagaat gcttaacagg atctactaag 26040
aatagagctg agatcaataa ggattttcag gagggcaaga ttgatatatt aatactacaa 26100
ataagctcag gtagtgttgg tctaaacctc ttcagggcct caaggttaat attctatagt 26160
tggaactata aatatgatga ttatgtgcaa gctattgcca gaatcaaaag gaatggtcaa 26220
aaggagcctt ggcaaataat acacttaata acggagaata ctattgatga gaaaatatta 26280
aaatcaatac agcttaaaag ggatagagca gaaaaactgt tgactattga taagtaatgt 26340
ggtaatatag aattgaggtg ataagtaatg agaaaagaac aacctagaat aagtaatcat 26400
actgaaatag ctaaggcttt aattgaggct ggtaaaacta ttaaagctct aaccaagatt 26460
aaagtcaagg aggaaaagaa aaaacaaaaa agacttgaaa ggttaagact cctaaatgag 26520
gaaaagaaga aggagagaga attaatgaaa gaatttaatc ccgatgagcc taagaagcca 26580
agaaagaaaa aggacaaacc tataaagaag gttgatctga ataaagttga ggctaaggga 26640
acttacccta caccttctgg aggtaatgca cctacgggaa cagttgactt aaaaatttta 26700
tgtgagtcat taggattaga tccaagtaaa gccagggcca agctaagaaa gcaaggtgta 26760
aacaaaccct ataaatggtc aggatctgaa cttgaggata ttaagaaaat gttaaaataa 26820
gggggaggtt taaaaacctc ttctttttcg ttgacaatat ccacagtata tggtaatata 26880
aatattgtaa tcaaacaaaa aaggagagaa ctgaatatga aagatcttga aaaacttaca 26940
agggaagaat tgattgaact tgtaaaggag ttatctattg attggacagg agcctataat 27000
agaaactata agattgacct tgatgagtat gagattgtat atattgatct taatgatcta 27060
aaagagttca atgataaatt aggtcatcta gaaggagatg tgtatataca atacacggta 27120
aatcttatta aggcccattt acatcctgag aaggatatat tgattagagg gggtggtgat 27180
gagtttgtag ttatcagtga tatagcttat aatctgtgca ataccttgaa tatataccct 27240
gagctatcat gtggctatgg taaaggtaaa actattgaag aagctatagt acaggccgac 27300
tcaatgatgt ataagaataa aaaatctaag aagttaagat actcaaagga ggattagtta 27360
ttatgaggtt attatggtta aaagaatatt tggatatatg caatatcttg ggtattgagc 27420
ccacaatgga atcttgtgct aggtggggaa agaataaaaa actatcaggg ttttcattaa 27480
ccccagtggg agcaactaaa tagttgcttc tttttaaagc gatccgatac ctagcaactt 27540
tcatgccaaa aagaaattcg ggattttctt tgacatagac tccagagtct ggtataataa 27600
tcttgtaaac aaaattaatt caaaggagtt gttaattatg aataacaaaa aatttcaaga 27660
aactttagaa caattaaagg aggaactaga aatacaaggg gtagaattta atgatgagaa 27720
tgacttaata gctgctgttg aatacatcat agaatatgat taccacccag aacaatatgt 27780
ggaagatact ttatgtaact accctgaaat gttcaaggat ctataaaact aaggaggtgt 27840
aaaaacctcc ttttttattt ttctaaaaat ctggatttaa tcgttgacag gatcgggact 27900
atctagtata ataatattgt aatcaaaatt aattgaagga gatgttaact atggaactaa 27960
caagatcaga aaaacttaat gtaataaaag gaattgctca aaaggctatg gataataaaa 28020
tctatccaca atttagaaaa gaactagata aacaggtaaa atcctttgga ctaagcaaca 28080
aagttaggga aagtgaatta ggagactacc tttcttgctg tatggttgcc tatgaagagg 28140
ttatactaaa aatgaaaaaa gccaattaat atgggtggcc aaaaggccat ctattttttt 28200
cgaaaaaatc tccagaatct cgttgacata gtctgggccc tctggtataa taataatatt 28260
gtaatcaata ttaattaatt cttaggagga atgaaaaatg actaaattag aaatgttaaa 28320
tgagatagaa aaaataaata aggtggtttt tgaaaagaac ttccaaaaac aattcttgga 28380
taaggtgata gaactactac aacaaatggg agtaactaaa tattatgtat gtaaacatca 28440
taaaaaagct gagattgaaa aacatcttga ggcttgcatc tgtgcaatat atgatatagg 28500
aagaagggat gatctaggat ggtaaacccc atcctttttc ttcgaagttt ttcgttgaca 28560
gatccagagt tctctcttat aattaaatca taaattaact attggaggta aacaaaatga 28620
gagatatgat agaaaatcaa ttagctgaat atgaggtaaa ggagttatac aatgaggcca 28680
caatgatctt aggaaaagca tttggtgtaa gtgctgttga tctttatata gaatctgatg 28740
tggatgctaa ctactatggt ctatgccata gatcaggtaa taaatttact gggattacaa 28800
ttaacctttt ccctttcagg tttgcacagg aaggagaaaa agcttatgat aaaatcaaaa 28860
aatttggctg ggatattaag gactgtgttc ttgaaactat atgccatgaa ttggctcact 28920
taacttattg gtcacatagt cctttacata aaaaattaac tagtctttat tacaacaaag 28980
tgcaacaggc tagaaaaggt gaaaactcag ctgtagcaca aactagaagt gatatataca 29040
ctttaaaaga tttatgtgaa ttaatagaac tagatccaag aaaggctaga agtatattaa 29100
gaaagaataa tattgagaaa cctggtaaac agtgggagtg ggagcaaggt atgcctaaag 29160
atatatatga tctattaatc aacttaaagg ttaggggaag aaggtaaact tcttctcttt 29220
tttcgttgac atatctcgag agtgctggta taattatatt aacaaataac ttagggggta 29280
aatcacatga tagatatata ttatagtatt caagtagaag ctgaaaactt ctttggttgt 29340
caaaaagaag atggaagctg ggtagcaaca ggagcattta gtatacctgt taatagcata 29400
acttttgaag aggctgatca attatccaat gaggccatag atgggtggtt aaaggagtat 29460
gaggttacta aggtaataag taggagatca catacacagg ttaaattaga ttggtataat 29520
agttatatca aggatgatcc attaaagggt gatataatta atatggtgta ccctgatctt 29580
tcagataaag ctaaaatcaa atatggcttt ggtaagatat gggctagcac tatgtcaaag 29640
gttgctaaga agggtttact taaaacttgc cctagatgtg gaggcactgg ccattactca 29700
agaacctcag atggtaacac tacttgcttt aaatgtaagg gctttaaata tgtgatacca 29760
actaagatct caaagaaatt ttataaatct gtggagaaaa cctttgacat ctctggcaat 29820
aactcttata ataaacttgt aaacaaaaac aaagaggaga tgttaaatat gaacttaact 29880
gcaatggaaa caaagatatt aaatgctatg agaaagaatg aatttgatga tggactagat 29940
gttgactgtg tatgggtatt ctcagtaata gaaaactcag gtatagaagg aactaaggct 30000
agaggtgtga tctcatcact tgttaaaaaa ggtttagtat ttgctgatgg agaggtaata 30060
ggttacacag aagaaggaag aaaggtcttt gataatgctg atggagaaga atgtaactgg 30120
ggaggtccaa aattattaaa agaaatccct gacaaaccag aggaaatctc ttataatgaa 30180
aatgtagata aaacaaataa gaaagaagag gtaaatgata tgaaagattt aaatactatg 30240
aaagctgttg aaattaaaga attagcaaag gaattaaaag ttaaaaactg gtggactatg 30300
aaaaaggctg atctaatagc tgctatccaa caaatccaac aaccacaaga agaaaaagtt 30360
gaagaggtac aacaaccagc tgaacctgaa gaaaaggctg aacaaactaa aattactctt 30420
gatctaacag atgttaagga acctgaagaa gaaaaggttg aaactaagaa aaaaccaaaa 30480
actaaagaag gtgtatttac actaaagatg atacttgaag aattaaatat gaatggtaaa 30540
aaggcaagaa ggatcttaag aaacaaagag gttgtaaaac caggaaagca atgggaatgg 30600
gataatgaag aagaatttaa aaaggttaag gacctacttt ctaaataaag taggcccaac 30660
ctctatcccc tattgattta caggaggatc tgattttctc ttatccatta actgcttcca 30720
tacttgatta ccaaaggaag ctacagctgc acatattaaa ccctgtataa agccattcaa 30780
aaaggtcttg gctgtaaaac ctaattctaa ttgaattgcc atatataaga ttgataatga 30840
tatactgaat atcaaaagta ttaatggtat agaccaatct ggtatataag gcttagcctt 30900
gataaacaga cctaaacagt aacatgatac aatgattatt aaaagctgtg gatcaatgta 30960
attgaatatt aagtttacta aattttcttc catgataaat accccctaaa ttatctctac 31020
atattttgaa cttacataac cttgcttctt accgtggcta gtattatatt ctatatgata 31080
ccaaccattc ttacttccca atatagtaac tctctcatta ctaaatactg agcctattat 31140
attaccattc acatcatctc ttacattaag agaagtgtta actttaacta ttccttgttt 31200
aacatcctca tagaatttct ggctttgctc accctcgtag cgaacgtatc catcagatgg 31260
agtaccatcc tttctataaa atgttattct taaccagcca ttagttctat acattgggct 31320
agccttttga ccaggctgta gttcttcatt atgatctttg atgttaacgt cttcagggtt 31380
ccaccacaca taagttactc cattgtcatt ttgatatttc atgtggtagt caaaacttaa 31440
tctgctgtaa tgttttatgt caatataagc atggtacttc ttattctttc caggatatac 31500
aacttctatg aaatttctag aaggatatat tccgagaaca aataaattat ctaggctatc 31560
aataaatcta ccagggactg gcttacaatc ctcatctaga acttctattg aaccttcccc 31620
cactaaagta gcattagttc ttgtctcatt agactcatag aatcctttgg ggttattgat 31680
tactggtgag cttgagctct ctcctgaatt tactctttct aggaatgtat tccagtaacc 31740
attggctatc attcttctag gacaattttt tctactggca tcatagtgtc ttactacttt 31800
acaattagaa tagtagccat tctttaggta tctaactagc tctacagcat tatcaactgc 31860
cttattgtag tcagagtcag gattaacaca tatttctata gatattgtat ttctattatt 31920
gataccgtat ctattatgac catctccaca attccaagtg gcgtgtttgt gttctacagc 31980
ttgatatacc cctgtatcat cgacataata atggacactg gctttaccta tatttccatt 32040
tgcttgagcc ttacagtggg tttcggctcc agcaccccaa gaggtattat cagtttcgtg 32100
gataattata tacccaggat tatttgcatt tagagtaaca ttcgtagtac ttaatctttt 32160
attgattttc ataataaaca tctcctttat atattatagt gttttattgg gcctgagagg 32220
ctcgctacag cattttaacc acctccttaa tatatttcct tatataaaac ctaacaggcc 32280
ttaaatggcc tgctaggctt ataagttaac ctttgttact aaataggtga tcttttatag 32340
tatctacatc ctctttgata tcctcaacta cattgaattt atcaactagc ttatctatca 32400
cttgatttaa tttattctcc ctttctctag cttcttgtct agtgtcatat aatagccata 32460
caaatagact acaaaagata ccttggctaa gaacttgagt tagtatttct ttttccatag 32520
attacaccct ttccttaaag tcttcaccac atattctttt atagtcctct ggtgtaattt 32580
ctccttccgg attagaagag cattttaccg cctgatgtaa tttgtcctta cctattgctc 32640
caattctata agccatttcc cagaaattca taatccctac ctccttattt attcatcatt 32700
ttaatcttta tatcagctag ttcttgtgtt aatactttca ttatggcctt agtttgcatt 32760
agctctaatt tagtatcagc aagagcctgg gcttgataac caattaattt attaatatcc 32820
agaggattag gatctatagc ctcataggaa gtaaataggt ccttatcttt gatactgtat 32880
agatctgatc tctttttact aatatcaaat ttggttgggc caccctttat gtggtcaaat 32940
aaacttacag tcatttgtat ggctgaaaga ttgtctagat caattacctc agtgttatcc 33000
acaaaggcta aatgcccttg tgaattatga ggtatatgga gacatataat atcttctata 33060
ttatttaata tttcactatc ctctctatag tatagaggtt caggcctatt aattaggtct 33120
aatatcttag taacttcttc atggtcttta tctataaatg attttacatc ttctgagtct 33180
tctgcctgat ttatcagcat ttctttttca tcatcataga atataaggtc tatgatttta 33240
ttagtttcca cagaataatt aaatcctact attctcatgt tttatcaact cctttactca 33300
gtggctaaat aggttagtat tatttgagtt ggcttaccag ctaccatttg ataacatcca 33360
ccattcaaat atgagttatt ccaatagtca atagtacaat aacctgaggt attattagaa 33420
gcgtcattat agttatgtga tactagagat ataggtaata cccaattcca agccttagga 33480
aaatgtatag tataacttac acctccacct gagttagcat aacccgtctt aactatttga 33540
cggactaatc ttagtcctcc cccaagatga catactctaa cgttaccatt gtcattatcc 33600
caataatctc ctttagcaat agatctatta tttacaacga agtcagtaac attctcagta 33660
tatagctttt tgttctcttg tttgaaatac aatcttccat cttcagctcc accataccag 33720
tcatttattc ttaggtattt tctagcccat aggttttcag ctgatagagc cttatcaacc 33780
tctagagatc cgtctctagt agttatcctc atatcaaaat ctttcttact accctttaag 33840
tggaaatcta atataggacc tagttccata actccatcat gtgatataaa agggatacct 33900
gcatagtcat tactagctcc agctaagtcc cattctttta gtttattact gttagcagct 33960
gtagcggttt tacctaaata agcactatca tggttatggt ctgaggctgc agctcctata 34020
tcggatggct taggtttatt gaggttacta taagctatac accattcgga ccatatgcct 34080
ttaaaactac aaaacctagt atatgttttg ccataactat ctgtaaatag ttgagttacc 34140
tcacccccct tgggtaatac caatagagtt ccataaatac catatgtgta aactacaggg 34200
gagtttggta aggtatctac actagatact gagtatatcc cctcttcctt acaaagatta 34260
aaatctacta tatcatcttt tctaatagtt aaataaactc cctcatgggt atggttagtt 34320
ggggatgccc ccacatctgc tgcagttggt ttattatctt catgatagac cttcttattg 34380
ttcttattaa caatatccag agagtctatc tttacaggcc ttccagcata ccctatatgg 34440
actttattat ctgcatctat atagatcaca tagtcatcac tgccttcttt agtcctacag 34500
ctaaatccat aattgtgggc cccaataaat ttctgacccg gtctcatgat tagatggcct 34560
gataattctc cacccgtagt aggtaaggca ccaatatctg ctggagtagg cttattttgt 34620
ttactatata tttcagccca attcgtccat ccggtaccct caatgtttgt agctgttcta 34680
gtaaaaactc ttccaccatc tgcaaagaac atttgagaag cccagtgtcc tgtattccag 34740
gataaaacaa tcaatctccc atctgctagg attggggcat ttctatcgac agaatttcta 34800
actgtagaat aaatacctcc ctctgtaatg gtgtttagat ctacaccctc tagtcttttg 34860
gctgttttat cccaggcccc aatatctgcg ggtgttggct tagctccgga gttataatct 34920
tttctccaag ggaaccaatc cgtattattt actttatacc tccaatactt attaccatta 34980
gtatcataat actcttgcca tatccagtta ttagtattat tatgcttagt ctctgtacta 35040
acatatacta tcagtatacc atattttgta ccagttagtg gggcattagg taactcacct 35100
gagatcaggt attgcccctc agttaaagct gtattaaaat cagttatggc gtccttagga 35160
atgggataag cctttaggtc agcaggggta ggcttattag cctcatcata aaccttatac 35220
cacttactcc aagaattatt tcctgatgtg tagtttctaa catatctagt tattttattg 35280
gttgggtata ggttaataaa ctcctgttgg aatcccctct cacctgtatt tctaaccata 35340
aaaaccctat tactcatatg ggctaaggtt ccctcggggg tatttttaaa gtcaccagtt 35400
cctgtatcaa agtagaagaa ggcattcgct tgtttaaagg aattacaatc tctatctaag 35460
ggagcactaa ccttaataag tgcttgatct tgattagcct ttcggtctac ttgattttgt 35520
aaactggtgt ctttctcaaa taataggtct aatcttttta gcacatcctc aggggctaga 35580
ttataatcag gtactacatt acccctagct aatatgaggt ttagtacctt taatgaggta 35640
cttgaggttg cactctgtac cacaaattgt attttggtat cagtagctcc agatggaata 35700
ctaaatttat gagtttttaa cttgtaccca ttactctgtg gtgtactacc ttgttttaca 35760
atactcctat attgttgacc tccattagtg taagccagtt taatttcata gctagtaaaa 35820
ctaccttcaa tatcttctag aaaggctagt atataatcac tatttggttc tataggcaga 35880
gttttaatct caagagtttg tggacctgtt gacataccag accatttaaa acctcgtctc 35940
tgggttggag tatctgtaat gggtgtgata ttaccaggac cagtataaga ctcagagttg 36000
taccactcaa gcctattaat agtaccaacc cttaaagcat ctagcctatc ctcattagac 36060
ttagacttct tttctacctc agtcattttc tgtttatgtt cggtagcaaa gttctcaatg 36120
ttctcattgt ccttgttaaa atcattcatt tttggcttat ctgttaactc ccacttattt 36180
aaacctaggg taggagtttt actagtactt ggcattttgc atacctcctt tattcagtta 36240
tagcatattc ttcccatttt tcccaggtaa gatctagact gtcccacttg ctccacatat 36300
aatcatatct atcgaacatc tcccaagtaa tataaagtaa tataatttga tatattaggt 36360
gagcaggcat tcttcccctg aatactcttt ctagagcctt taggttaggt ggtacccctg 36420
gtaggtcagt aaagctaatc ttgatcttac tctcttcagc tatgaactct acctttacct 36480
ttccacctgt ataggcctta gtaagatttt tgatagatgt actattaaac tttttccaag 36540
cagatagaac tactgtaata atagctcttt tctcttggtc agttggtggg taatcaaagt 36600
atttatataa ctcaagatca gtaaaaaatc tttctagacc ttccttagga gtggtcttgg 36660
gatccacata gtcttgtaat tcctgtccct tagtatataa cttctgaagt tcagtagcca 36720
taatggtgtt tactaagtta tcagcctctt ttatattatc ataaaattca ggaacataca 36780
atagtaattc tttttgaacc tgttctagac tatacataag ttaccaccac ctcagtaatg 36840
gttggtattt gattagcagg aatagttaaa gattgactac ttccattaag tgtaatggtt 36900
gtataatctt ctactccctc aatatttaaa agcatagccc ctattctatt tatagatagg 36960
ctagtaattt taaatacttg actatttaga aattcttcta gttctttctt gaagtttaat 37020
tttatttgtt cttctgtaac tgaactcaat ttcttaactt gagctcttac agtgatatta 37080
aaggtttggg ctgactcaac agtaaaaaca catcctatag gagctttacc ctcacctaaa 37140
cctttcttgc ctggatccat gtattcttga actttattaa ctagctcagg attagcaggc 37200
ttattttcaa ctgtggttat aataccttta gcagtattta ttccatccca acagggaaga 37260
accacagctt tacccactcc agctacactt ttacaccaat cagttaactg ttgaacattc 37320
ccatctccag atggttgtat agtggcttta gtagctcttt ctcttaactc atcatctgtc 37380
tcctgatctc taccaataac aagtatatct cccatagtag cacttctaag ttctgctata 37440
gcattgatag gaattaaatg tgtacctgtt gatactttat tgggtcctga acctaaggtc 37500
tcacattcaa taatattagg ctctatcact tgccagaaat aatctcctcc ttggaatctt 37560
gaaccaacat cgggttcata gccaacaaat atagctgacc ttctacactt agttggaatt 37620
attctggata tccctacatt ggcagctctc tcatctaaga acttacctgt agcagtactt 37680
agaaatactg agtctaatat ttgattaaca ttaacagcat aaaactcagc tagcttaaca 37740
gcaaaaacca tagccatatc ataggtgaag cttccagttc tagtgtcaat atcatcaggg 37800
gataggccta tcatttcttc taataattct ttatgtgttc gggagaataa atcttcagaa 37860
aatcccatta tatcacccct tccagttttg tatctccata tatggtattt atccatactt 37920
caatatatac attcttacct tgcacattat actttagatc agttacttta gtcactctat 37980
catcttctaa gagacattca gggatagccc tattaatctc catttcaaga agttcaggag 38040
ttactagaga gtcattaatc aaagctctaa tatcacaacc ataagaagta tcttgataaa 38100
tcatataggc atttttagga gttattaatc ttttatataa agattgtttt aaggcctcaa 38160
gaccatccac attatcataa atcctattaa ggtttagatc cagtttatag gtcttggtat 38220
tctccattgt tttctcatca actataagag tatctttaaa gccattaggt agcattatat 38280
caacctccta agatccctaa cataaaatat ctttgtccat catcataggg cataagataa 38340
acagtatcgc cctcactaat agactcaata tgttttggaa catctataat atttccagat 38400
agtactaatt ttctattgtg aagacactca cattttagag ggctagtact aagtacctta 38460
gctctataaa tatttagagg ggtggacata ttactatata cttgttttac acagtctaat 38520
aaattattag ccattaaata tcaccctcct cagtacattt aagttttaaa ttcatggtgt 38580
agtcagtatc aaatttatgg gtatcagact caatgtagaa agccttgtta atttttaaat 38640
gtggtatact aaccacaatg gctttgccac taatacaatc aggtacacct ataacagtta 38700
tagagatatc agttttaggt ctcttacctg tttctagctt tattctagcc ttctcattta 38760
attgggcttg gtttagttca tcactgatag tatcgtataa ttgaaataca ccatatttct 38820
tttggctgct atcatcttta gcagaggcaa ataaagtaat ctcgggatca tctttaccct 38880
taccctgttt atatttacat actagcttta cctggttgac aatactatca aagtttctac 38940
ttcttgaaaa ggttccaata tttttaccat attcgaattt ccacaattca ttattggttc 39000
ttcggtttat aaaatcaaac ttacctgtaa tagaattata gtaaatataa aactgttctc 39060
cagtttgttc ataaacctct tttattacct tcttcataat atcataggga gtttgtttct 39120
cacatactaa actagagaaa gtatatccag ttttagctac tactccaggg gcaaccccaa 39180
aggctttaca taatgactca aatatagctt cgccagtttg gttttcgaat acaaagctat 39240
ctttattatt agcccaataa atcatagggt cataagcttt aaccttaagt aaatgctttt 39300
tctcattatc tgagatattc ataataatac cattaaagat attcttaccc tcatggacta 39360
gtactacctg atctccatca taaattggat gtatatcttg agcattatat tggaatgata 39420
gggtacgagg ggcagatgaa gaagaccctg cccaactctt accactggag gtatttgtaa 39480
tatcaaagga tttatctcct tgtaccacaa ttaattgcat attataacct ccttatataa 39540
tcaccagaac tccaaccttc tttatatcct gaaccattat ctaatttata ctttacataa 39600
tgccatccat tttgagatct taatactgta aagtgttcat taggccatat cttaccctta 39660
gtgggattat tgatacctgg tccagttctt acccatagcc atgagtaaac attagtagta 39720
gtagctgccc aaggttgagt acctccagaa gaactacctc ctgtattacc actataggct 39780
agtttaggtt ttggaattgt aaaactaact gatttaacct caggcttctt atactcagtt 39840
agttttattt ttacttgaag agtaccaata tctccacctt tctcagtata tgagatatta 39900
tcaatagtaa ctaataagtt aatatcagta ctagtaatag taaatcttac tggatcttta 39960
cccatttcaa taagtttagc ataacttact ctagggtctg caatatcagt atactcacat 40020
ccatcgtagt atgagcttgg taactcaaaa ttaaaagaaa aagattttag cttagggtct 40080
cctatgaaag agagttcccc taagccttca atggaaactg tactattatc tctactatca 40140
gagatattta tatcagtggg atttactggt aacctaaacc tagtatctcc ctgtattaac 40200
cagaattgat atttactatg caaggtccag ttcacctccc tcgaacattt cctgatttaa 40260
catttccaca ataaatctgt atactctcat taattcactt tctgagattg ttccatcttc 40320
agcctgtact gtgaaattaa atgagttgtt aatgttcata actttagatc catcatcact 40380
tcctgctcca gattgtctta gtaaatcctg agttttatct gcaggtaata cttgagagcc 40440
tctaggcatt tttgagaatg taggacccat aactaattca ggaccagctt caccaaccat 40500
ggcagcttta tctgtaaaga agtcagtacc ttcagcaagc attggtatct ggggaatatt 40560
aataccctta cccccaataa taggtaccca gtcaggtatc ttaatagtat taagagcacc 40620
aattaagtta ttaatcatac caataatact attaatagga gcttttgcta gggataacat 40680
accttggaaa atacccccaa agatgttctt aacaccttcc caagctttag accaattacc 40740
tgtgaatact ccagccacaa aagctattaa accactcaac atctgcatta atccctgtag 40800
tattcctgat atagtttgaa cggccccagt aacaactgag gagataacag gccatactgt 40860
ttggaaagat gcggctaaaa ggttaagtag tggagttagc atatccacca acccttggaa 40920
tatcatacca aaactctcaa tagctgattg aatatatgga actacattct cagcaaagct 40980
taagaacata ggtagtagtg tattattcca ccaatctgct agtccttgta atattggcat 41040
taggaagtta cctatagctt ccacaacttg tgatatagct ggggctatgt aggtgttaaa 41100
ggcttcagct aatgatacag ctattggcat gaaaacatta gttatcaatt ctccaagagc 41160
tgtaaaagta gcagagaaca tagaacctaa tacttctaat atagggccta acacttccaa 41220
tacagtttgc catacttgag taaagccttg aacataaaga actactaagt ctatggctgg 41280
aattaggtta ttcataaata tagaagctaa agttccaaac atatcaagaa gtacttgacc 41340
tataggagct aatactccca tgataggctc agctaaacca ttaaaggctt cttgtatatt 41400
attattagcc tctgataaat aatctggtag acctattaag atctctccaa aattattata 41460
gatgtcttct ccaagactcc atatcttctc attaatttga tctgccacat cagcaggtaa 41520
gaacttatat aatatatcat tggttagatc tataaagggt tgcatagcat ccccaaactc 41580
ccaaccattc atgatgtaat atatagtctc tcctaaacct tggaaagcag aaacaaccat 41640
tgagaatata ggtcctagtt tttcaaagcc ttcttgtata aatgtgatac catttagaac 41700
agcattagct ataacctcaa cggcaggggc tagggcatct cccacatcta acataactac 41760
agcagctttg gtttttattc tctccatggt tctattaaag cccttatcca ttttttcata 41820
ggcagcttca gtggccccag cactatcctt catctcatta agagcattag taaaggcctc 41880
agtaccttca ccagtcaagg ctagtgcagc tgaaccagct tctacagaac caaagaggtc 41940
attgataccc agattacttt tctgggcatg tttttctaac atctgaaggg cctcttggat 42000
attacctcca cctttaatga agtctttgaa gcccttacca gctatttccc ggaagacttt 42060
atcagtcttg gttcccgatt tagatagctc atctatagca gctcttatct tagtagtagc 42120
tacagatgtt ggaaccccct gagctgttaa tgcagctata gaggcagata cttcttcaaa 42180
agatacccca gcagctgagg ctgaaggtag cacattgaat agggattgtg atagttgctc 42240
aaagttagtc ttaccaagtc ttactgtagt aaacataagg tctgaggccc tttgaacatc 42300
catattctta gctccatatg agttaataac agtagttagt ccatcaaccg cagtttctat 42360
tgatgtgata ccacctatag aagctttacc agctgtttct aagaaactaa aaacgttatc 42420
ctctggaact gatgaggata tagcttgata aagagcaggt actactttat caggcaatat 42480
ccctatatca gaacttaact tttttacctg gccggacatc ttatcaaagg tctcttgagt 42540
agagttgggt aatagagtca taacctcatt taaaccttgc tcaaaatccc caaaggcttt 42600
aacactgtcc ttagtaaaat cccagacagc cttagctgag aatactccag cagctactcc 42660
accaattttg gcaagagtac ccatgaagga attaccacta tcagccatct ggttaaatgc 42720
accagagatc ttatcttggg cagagaatat ggcagtaaat gccgcttgac ttgccattat 42780
atccctcctt tatgattaag agcctttctt taacttatcc ctagcttttt tatcttcctc 42840
taaagccagt tcacatgagg ctataagaaa ggctttccta ttgggagata gtttatcaaa 42900
ttcccagggt agtatattat gcttctggaa taacacatga gcccaaaatg cactagtgtc 42960
ggcctttatt agttttttgc ttctttgatt agatcatcct tattatcgcc taatccattt 43020
atgtccataa cctcactagt tattctagtg aaatctcctg gtaagcttag tacatccata 43080
agtaaatcta ctggatccac aaacttagta gactctaacc attttggatc tttaaagtca 43140
gggaatacta ttgtttctag tatcatttcc ctagtagctc ttctttggtc tgttattgag 43200
ttagttatac ctttgtttct agtaattact gtatttctct tttggatatc ttccatttgc 43260
ttagtagtga ttggctttaa tatccaaggt ataggttggc ccgctaattc atgaccctca 43320
gggaaactaa atctagctac cccttcaact tcaatttcgg gtgattgttt acccttatca 43380
ttttccatga aaaattccat actaaatttt gacataataa tctacctcca caatattctt 43440
aatcttttat aaatatctta cttctgaagc tgagaatgat atttcttctt gaatttcttc 43500
tccctcagca tctagtccaa ttagtggtaa atctccttct aattgtactc ctagtaattg 43560
tactctgtcc tcaccataat tcttaacata gtcagaatct ggatcatcac atacagcttg 43620
tatatccatc ttagggaata taccagtttt taagtattcc ttaatagctt ctcttatcat 43680
tggagtagct ttgaattgag ttatagttcc agttatatca taacctataa cttttgaggt 43740
taatcctttc tgacccataa ctcttttctt agtcacctca ggagtaaata gagcttctag 43800
ttttattaag ctagttactt gcttacctga tacaaaagct ctaccctctc tagcagacat 43860
tctattaact tcactcatgg gctatacctc cttaattatt cttaacagtt acaaagtatt 43920
tcttgaaagt tcttacaggt tgtgtaagta tagtagcata catagtcttt cctttactag 43980
cttcttcatc taccactaca tcagagtcag ggtccacatt ctttaatgct ccttgagctt 44040
gtagtgtagt taatagactt attagtctat cctgagctag ctttatacca tctttgtggt 44100
tagggaaaac attaggaact aagatctgag atcctagatc tataacagca tccaatacag 44160
ccacaacttt gttatctgta aagctttcat cccattcttc tgtaaaagta tggtgtgtat 44220
ttatatcttc ctcaactact acctttactc tttcagaatt tgagtcagtt tgagttgaga 44280
atataaattg acctgttttt aatccatcaa ctatttgaga gtgttttagt cttggattgg 44340
catctactgc tccaggatat gctttataag taaggtcttc agtatatcca gctgcagctc 44400
tagcaccagc tacccatgca gttgcttgag aagctgttaa cttagtgccc ccttctaata 44460
caacaccatt cttaacattg ataatgtatg ggtaatcaca agtagcaaag tcagaagtaa 44520
ctgccacaat attcttacca acttcatctc tgaagtattt aagttttgat aatagagcag 44580
tgtgtaattc actatcagta aatgggaagg ccatagtttt aaagttgtat aactcacagt 44640
tatctaagaa gttgataaca tcagagttat tcatctgagt agaagtacca gtactaaact 44700
cagtcttagc agtctcatct atagaaccac ttccacttac tgtaaaccat tgagacttaa 44760
ctgttgatat ttcagttact cctgtagcct caaagatttt agatccctca agatataata 44820
atatatcctt tcctccaaga ggattggtct taatagttag agataactta ttaccccttt 44880
ctccagcttt agtagctgta aatgttagag accctactac agctttggct ttagttccac 44940
ctgttaggtt atatactaaa acagttgcag cttcttttaa agcctcttta actagtaata 45000
cagtagggtg agttatgtca tacccaagtt gatataatgc ttctaggcca ccttcattgg 45060
ttaccttgat aacatctcca gcattacccc atttaagttg taatggtaag atcataactc 45120
ctagattatt aagcttaggc tcattaagtc caccacttac gaatctagta tacataccag 45180
gtcttacttt attttgtcct ggaataaaag ttcctcctgc catctatttt acctccttct 45240
ttagccagtc ttttataatc tgactagttt gttttattgt taactcttca tcaggtggaa 45300
gggattgaac tgcagcgttg aagacaaact cctcaacctg gaaaagttgt tgacaatggg 45360
ctcttaattg aggtatggaa aatttttgta ctttcactat tttaccccct ttgttatctc 45420
aaagttttcg aagtaagttg gatctttata aagactaacc acaatatcat agtttatctc 45480
tacctgagaa tcttcttcag tttcaccttc aaatacttcc acattcttta ttttgagatt 45540
tttgttggtc ttactaccat ccctattaag tataggaatt tttctatttc tcaagtatat 45600
agcattagct atattctgag caatttcaag agagtcttta ggttcaaata ccttaactct 45660
taaagtattc tcagttctat aactacaaag agtatcattc tgaggattaa ataccttctt 45720
gaaatataat gagggatact tgaacttttc aggtattcgc tcaaaataga ggatagtaat 45780
tccagttaga tcagatatga atttagcaat actccctata tctgctgtca ttaataacta 45840
ccccctaaaa cttcggctaa ccaatcttct aattttcttt ggaaagcctg ttcaaatatt 45900
ttttcatata tttttactgc attgtcccaa taaggtttac caggtacttt cttttgtctt 45960
agtaccatac cagtcttagc tcctggttga taaataaatt tatctcctga ccatatgcca 46020
ggaacgaatc taaatgattg tccttgtgga ttagtgtagt gaccatcgtt tacccactta 46080
gcatatttta ggttagtacc aacttctaaa gttagtcctc catcactact tacccatata 46140
ccattcttac ctcccttttg aaaggagtta agtaataatc tagtgtctac tacttgtagt 46200
ctaattattt catcttgaac cacatcaagg aagtctatcc ctgcagcttc aaaccatact 46260
gctaattgct ttttgaaatc actattagct gcatctctta gcttctttat aaacctttct 46320
atttgtttag catctatttc tatgcctcca gaatttgaag ccataactat ctaaaatcct 46380
tagcctctat accaacctta atttttagac ctcttatatc cctaggagct ttagctatat 46440
atgtgatacc agtctcaagg tccacaattt tatcattttg tctaacatca gttccaatag 46500
caaaatctac tgagtactcc tgggtaatct taaacctagg ttctttgtac tcaagttttt 46560
ctagagttcc tgacttaaaa tgacagggta cctcagtcat gtctggctca tcaggataac 46620
taaagctagg ggtatgggct agcccatatc cgggatcttc agggtcttct aataggtggt 46680
atatattaca tctatgattt aagagcttgt tatatgaatt tccaatcata ttacagtacc 46740
cttaatctca tagtagttgt tctgatagat ggactatcat cttctaagaa aggtttaatc 46800
agaggcataa tatcaggctc tatgacagcc ttatcagttc ttcttgtata tgagtaatca 46860
tcataggtct cggattgaat atcagccttg ctaatgacca aggattgtag tccataatac 46920
tcagcataca atatatgggc taaccttaat tctttactat taggattttt ctccacaata 46980
gtatcatagt ctttatttga atttttaaaa attcctgaga tcttggcttt agctcttagt 47040
atatccatct caatattttt ggggtctctt ttcttaatct caggattact tgaaaagtca 47100
attatttcat ccggagtaat attcatatga gccaccccta tctgtttctt ttattcttgt 47160
ttgtaaatct tctagcaggt tcttctttac tgtcatctgt agaaggtatt tcttctccag 47220
taaaatcttc tagaggttca tcctcaacag gttcatctat gacaatcaca tcatagttct 47280
gttttaaata atttactagg ttatctttaa cttcttctac atggtcttga gtaattctat 47340
gacccttaat aaccagagtt tgtaatttac cctttgctaa tttgacctta gccataatat 47400
ctctcctttc taatgaaaaa gagcccacat ttatagtggg ccttcatgat taagctttta 47460
ccttgattat cttagcagca gctttaggat cttcaaactt aacatccatc ttaagagtta 47520
aaactacata agtagctctt tctctaggag ctctttctaa ttcaattctg atatctcttg 47580
atatacctat aactatattc ttagggtgag ttaagattat atctgatact tgagtagaag 47640
tatcttcata atcttgtagc atagctatac ctttaacagg tacaccataa gcagatttaa 47700
cagagccatt tataaatgag tcatcaccat tagcagttaa tctctctcct actaagtcta 47760
cccaatcagt ttctaaacca tttgagcagt agaatctcca ctcactgggg tttcttaaat 47820
acttagctgg tacagctttc ttagcttgtt ttaataggtc tttacttaaa gcaccccttt 47880
gagcatctac tacatgagca gttaactgtt ttctaacccc atctaattgt tgtaagaact 47940
catcactacc ctcactagta tcaccattaa caattatttc ttccatatca agagcaactc 48000
tttcagctaa taattgcatt acagtgtctt ctattgattt tccttctata tttgtttcta 48060
tagtatcatc agttaactta accacagcta taaattcttt agcttgtaaa gtaactgttc 48120
cagtagttgg agctgaatat ttagactccc ctggatcttt tccttcagtt gcaggattta 48180
atactctttg accaaatcct attgactcaa tctttttaga gtctcctttc attggtacag 48240
ttctagcctc ttttaatatt gttggctcat caattatcat tttaataaaa gcgttagctt 48300
gttctgtgtt taatttacct ccaccactta atactgtaga gttaaatact gctttattta 48360
ttttagccat aatcttttcc tcctatcaat tattcttggt ttccaccaaa taatccatta 48420
aatactgatt tttgaacttc ttgtccttct cctccatttt taacaatttg tttacttcct 48480
acatatggag ccatggcctt agctactgcc tcagctattt tttcttcctc agttttttct 48540
tcttgtttct ggattaagcc agcctctaac atagcttttt ccacagcctt agcaactata 48600
tcttcagtag tagcttgttg accttcactc tttttaactt caccctcaac aggtttagtt 48660
ccatctaatg cctttttgat agcttcgtct atcatttttt gtacttgagt ttcattcatc 48720
tcttcttccc cctccactga gtctaataat tcattaatag cactttgtgc tgtttttaac 48780
ttttccacat tttttgaaga tattttctta cctgccttat tgactggtaa ctcaaaggat 48840
ttagctactt cctcgggagt tcctaataat attccctcca tagtttcaga aaattcttga 48900
agagcttcct ttatcttatc tacatcactt tcaaaagatc tagtgtaatc ccaactagaa 48960
tatggatata gaatatcttc taaggtacta actgcattcc agaaattatc cctttttgca 49020
gtctctttat atttttcaga gtatgcacct ttctgtatat gctttggagt taaccctaag 49080
gcctttccaa tcttatcaag tatagattga cctatagact tctcaatctc atcaatatct 49140
acatcttgtg tagaatattt accagtacca cccatagacc atccagtaat ttcgcctttc 49200
tcaatcttgc tccatagatc atcatcgaca atctcagctt tagctagcca agttccagct 49260
ttaactactg taccttctat tgtagtatct tcctcagtta cccatgattt tacaatcttg 49320
acaccctcaa gagtgttctc attgtgttgt aaatcactac caaggccatt ttcattaaac 49380
cactcacaag ctttcttgat ctcttcctca gtcataaaat tatcatggta gtcagcagtc 49440
attggctcat atactacacc gataacaaaa tgattatctc cttcagactc agactttaag 49500
attggcttat catatctaaa tctattagga gccccaagtt ctttagccac aatccattgc 49560
catttattag ctgctttatc aactaatgaa aggtaatcaa ttttagcatc tgagatctca 49620
atagctttct taactgattt tactggcatt tactctccct cctttacaaa ttatattctg 49680
ctatcacttg atctctgata gcttcttttt cttctttaga taatcctagt atatccttat 49740
caattacagg cccatgggta caatgacaat tcacagtctc agaagctggt aagttgatat 49800
ccctaggaaa tctagcttca taaattccta cagtgaagta ctcaccttta ttaatgacag 49860
taccatccag atcgacatgg tgttgccttg ggtgaatacc ccttccccct gagtgtttcc 49920
actgtattcc tgttactgcg gggcattgat taaaagcctc atccttagca tatgaatgag 49980
ctcttaacat ttcagtaata gcagtagctc tagctctagt tctactaaag ccataagtct 50040
cagataattt accaactaca tcttctacag actctccatc ttcaagagcc ttatccagaa 50100
tattctgtag tttttcatgg gaagttaatt tcatcatttc ccctagttta gaagaccact 50160
catctataaa ggtagtagta tatggagaaa agactttggt tagggataag tccttatcaa 50220
taccttgtaa ataagtatct cctaaatttt ctatagcttc ttgataacaa ctcttaagag 50280
tttctgctaa agaagtatcg atattatctg tacctatttg agactccaca tattccataa 50340
attcttcaat ggtttcaaag tctccatctt ttataagatg ttttaattct tctgtaagga 50400
gatcagaaat agtgtcttct aattcagaga tcttttctat tgtataatca atctcctcat 50460
agccagcttc tttcaattct ttcttaaggc tatcatcttc attctttaag atcttatcaa 50520
ttttagcgat caactcatca gtacccacta ttacttacct ccttttaatt gatagtttaa 50580
taactgtttc ttagcctctt taagtacagc tatcacctca tcactcccat tagagctttg 50640
agccttctca attatacttg ttaagttatt actcattgaa gaggcaactg agtaagtagg 50700
gatatttgcc cattcttcat caataggagt atagtcctta ttgattgatg aatagtactc 50760
ctcattaatc tgattaggag tcatagctct agaggctata gcaaacattc ctttctgata 50820
atcagagttc tcaatattag gactcttaaa gtaagcttcc acatatttaa aaccatagcc 50880
agatagaata acattattta gtatccatgt taggttttct ctttcaggaa caaatacttg 50940
ctcttcagtt tttcttgtag cctctctggc tgtagccaca ttataatctt gggagtaccc 51000
tacatatata tctggtagta agaaagaaga ttgtatcttt ttcctagtat tatccatgta 51060
gtcttggaat aactcatcct tctgtaatat ttcagatagg ggcttgacct caacagtaac 51120
tggcttagaa tcatcaaggg cagtgtctct ctctgcagct tcagtttcta atactaagaa 51180
accatgctgg gaattttcac cttttacatt attcatgtac tcagttaatt cattccaaga 51240
tttatcatta agagtaccac cttgaattag taacattaat ggagtatgtt taccattagt 51300
aaaatatctc cagtttaata attcggcctt tcttgaacct tgagcggcta acataggacc 51360
ttcccatctt ggtgtaccat agacaccata acctgtttta aaatgaagta tactgttagc 51420
tcgatcttta agaggtatct ctcccttttc ataatacttt ccagtattca tatctagatc 51480
tctaggatct ccccactctt taaagaagat tgattgatta tttacagtct gcttaaatct 51540
tctgaacctt tttcttctag tgtactgttt accatatcgg gttacagtgt actctgtact 51600
ctcatcatct agcttaagaa ttttaattgt cttagggtct tcaatttctt ttatctcttt 51660
tacgagagct 51670

Claims (12)

1.一种噬菌体CJ_CP_20-25，其具有特异性杀伤产气荚膜梭菌(Clostridiumperfringens)(CP)的能力，并以保藏号KCCM12934P被保藏。
2.一种用于预防或治疗由产气荚膜梭菌引起的感染性疾病的组合物，其包含权利要求1的噬菌体CJ_CP_20-25作为活性成分。
3.权利要求2的组合物，其中由产气荚膜梭菌引起的感染性疾病是坏死性肠炎。
4.一种抗生素，其包含权利要求1的噬菌体CJ_CP_20-25作为活性成分。
5.一种饲料添加剂，其包含权利要求1的噬菌体CJ_CP_20-25作为活性成分。
6.一种饲料，其包含权利要求5的饲料添加剂。
7.一种饮用水添加剂，其包含权利要求1的噬菌体CJ_CP_20-25作为活性成分。
8.饮用水，其包含权利要求7的饮用水添加剂。
9.一种消毒剂，其包含权利要求1的噬菌体CJ_CP_20-25作为活性成分。
10.一种清洁剂，其包含权利要求1的噬菌体CJ_CP_20-25作为活性成分。
11.一种预防或治疗由产气荚膜梭菌引起的感染性疾病的方法，其包括将权利要求1的噬菌体CJ_CP_20-25或权利要求2的组合物施用给人之外的动物。
12.权利要求11的方法，其中由产气荚膜梭菌引起的感染性疾病是坏死性肠炎。
CN202180096220.7A 2021-02-25 2021-06-15 具有特异性杀伤产气荚膜梭菌的能力的新型噬菌体及包含其的抗菌组合物 Pending CN117529553A (zh)

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PCT/KR2021/007484 WO2022181894A1 (ko) 2021-02-25 2021-06-15 클로스트리디움 퍼프린젠스에 대한 특이적인 사멸능을 가지는 신규한 박테리오파지 및 이를 포함하는 항균 조성물

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