CN111349618A - 一种大肠杆菌噬菌体组合物及其应用 - Google Patents
一种大肠杆菌噬菌体组合物及其应用 Download PDFInfo
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Abstract
本发明公开了一种基于鸡尾酒防控方法建立的以大肠杆菌噬菌体PD06与大肠杆菌噬菌体PD114的复配形成的噬菌体组合物,及其在防治鸡大肠杆菌病的药物中的应用,还公开了以上述噬菌体组合物为活性成分的生物抑菌剂、消毒剂和食品添加剂,不仅可快速、高效、安全杀灭致病性大肠杆菌乃至耐药性大肠杆菌,有效降低发病雏鸡的死亡率,作为水线清洁剂,可有效降解水线中因大肠杆菌富集形成的生物膜,以及净化饮水的特点,所述噬菌体组合物还具有安全、无残留、无副作用的优点。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及一种大肠杆菌噬菌体组合物及其应用。
背景技术
禽大肠杆菌病是由某些致病性埃希大肠杆菌引起的一种禽类常见性疾病,大肠杆菌病的发病时间没有季节性,各个阶段的鸡都有可能感染,尤其是雏鸡;大肠杆菌随粪便排出,粪便污染蛋壳后,可导致鸡胚死亡率升高、弱雏比例增大。本病也可通过被大肠杆菌污染的饮水、饲料、空气、笼具等传染媒介,经消化道、呼吸道、脐带等途径感染,从而引起泻殖腔、输卵管炎症。根据病型不同可引起多种症状,主要包括大肠杆菌败血症、气囊炎、肉芽肿、肝周炎、腹膜炎、输卵管炎、滑膜炎及脐炎等多种疾病。
舍内的地面、空气与水线进行消毒,对防止疫病发生和传播起着至关重要的作用,尤其是规模化养殖场,由于饲养密度大,动物相互接触的机会多,病原微生物传播的速度也快,因此,做好环境消毒至关重要;另外,水作为鸡生长过程中所必需的重要成分,重视饮水系统卫生质量安全是预防鸡群发病的一个重要环节,因此选择一种适宜的消毒剂与水线清洁剂极为重要。
现如今规模化鸡场多选用乳头式自动饮水系统作为饮水设备,而乳头式自动饮水系统管道全封闭,内部清洁较困难,且鸡场为提高饲料转化率、产蛋率和增强鸡的免疫力、抗应激能力等,通常通过饮水系统添加多维等产品,加速了饮水系统污染、细菌超标等,甚至形成生物膜,从而导致鸡群发病,进而降低生产性能,增加成本。
目前,水线的清理主要是使用酸化剂、除垢剂以及消毒剂等,但是,酸化剂的长期使用会造成水线中细菌和真菌的大量增殖;而使用消毒剂的情况下,家禽在使用其他饮水途径的药物或者疫苗时,水线中消毒剂会降低药效或使疫苗失活。因此,目前还没有发现对水线中细菌消毒效果好、无副作用的水线清洁剂。
因此,现有技术有待进一步改进。
发明内容
针对现有水线清洁剂的多种弊端,本发明提供了一种基于鸡尾酒防控方法建立的具有宽宿主谱、强裂解作用的噬菌体组合物及其应用,不仅能解决鸡场饮水系统中大肠杆菌超标问题,还能有效清除由大肠杆菌富集产生的生物膜,降低水线管道和水中大肠杆菌数量,达到有效清洁水线的目的;此外,所述噬菌体组合物是安全有效、无残留的、无毒副作用的绿色抗菌剂产品。
由于噬菌体是一种能特异性感染细菌等微生物的病毒,其可通过诱导细菌裂解来调节细菌菌群,是一种天然的抗菌剂;噬菌体具有很强的特异性,只感染特定种属的病原菌,不干扰正常的菌群。噬菌体作为新型消毒剂,既可以实现带禽消毒,亦可以作为水线清洗剂,清洁管道,净化水源;另外,噬菌体作为一种特异性的细菌病毒,由于其自身能够分泌多糖解聚酶,故可以有效降解敏感细菌的生物膜,甚至可以到达深层细菌表面并对其进行裂解,所以噬菌体消除法对水线清洁以及水线中生物膜的降解有显著效果。
与传统药物相比,噬菌体具有无法比拟的优势:噬菌体的特异性强,只感染特定的宿主菌,杀菌速度快,在杀菌的同时伴随着噬菌体自身数量的指数增殖,产生持续的抗菌效果;噬菌体安全性高,其针对的是病原菌,对正常菌群不会造成影响;噬菌体产生耐药的速率远远低于抗生素,而且噬菌体的溶菌作用不受细菌耐药性的限制。另外,噬菌体筛选周期短,制备工艺简单,成本低廉。
本发明的技术方案具体如下:
一方面,本发明提供一种大肠杆菌噬菌体,命名为PD06,保藏号为CGMCCNo.16390,于2018年9月26日保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为:北京市朝阳区北辰西路1号院3号。
在电子显微镜下观察了噬菌体PD06的形态,该噬菌体具有呈正二十面体的头部和可伸缩的尾部,头部宽约100nm,长约100nm,尾长约110nm,属于肌尾噬菌体科。
本发明还提供另一种大肠杆菌噬菌体,命名为PD114,保藏号为 CGMCC No.18866,于2019年11月04日保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为:北京市朝阳区北辰西路1号院3号。在电子显微镜下观察到噬菌体PD114该噬菌体具有呈多面体的头部结构,头部长约60nm,宽约63nm,尾长约150nm,属于长尾噬菌体科。PD114噬菌体在双层琼脂培养基平板上可以形成透亮空斑,周围无晕环,边缘清晰规则,直径约0.5~1mm。
经测序和基因组分析,所述的噬菌体PD06全基因组由149506个核苷酸组成,为环形双链结构,G+C%含量为37.65%,含有238个开放阅读框(ORFs);噬菌体PD114全基因组由150354个核苷酸组成,为环形双链结构,G+C%含量为37.54%,含有239个开放阅读框(ORFs)。噬菌体PD06和PD114不含耐药基因及毒力基因,不含溶原性相关基因。
一方面,本发明还提供一种噬菌体组合物,其包括如上所述的大肠杆菌噬菌体PD06和大肠杆菌噬菌体PD114。所述噬菌体组合物可有效降低水线中大肠杆菌数量,净化水源;同时该噬菌体组合物能够产生多糖解聚酶特异地降解水线中由大肠杆菌形成的生物膜。
可选地,大肠杆菌噬菌体PD06与大肠杆菌噬菌体PD114的活体数量之比为1:1;噬菌体的总含量不低于1×108pfu/mL。
在前述基础上,所述噬菌体组合物还包括大肠杆菌噬菌体PD06和大肠杆菌噬菌体PD114的突变体中的一种或多种;所述突变体与对应的噬菌体的同源性不小于90%。
进一步地,所述杀菌组合物还包括辅料;所述辅料为SM缓冲液、海藻酸钠、蔗糖、麦芽糖糊精、葡萄糖中的一种或多种。
可选地,所述杀菌组合物还包括其他特定病原菌的噬菌体。
可选地,所述噬菌体组合物的剂型为粉剂、水剂、冻干剂、凝胶剂、霜剂、膏剂。
本发明还提供上述噬菌体组合物在防治鸡大肠杆菌病的药物中的应用。所述药物的活性成分为上述大肠杆菌噬菌体组合物,用法为:该药物可通过饮水或混合饲料的方式对鸡进行喂食,可以预防大肠杆菌引起的腹泻疾病。经过实验验证,在感染大肠杆菌5日龄左右的雏鸡中,109PFU/mL的噬菌体组合物在14天内可以有效降低患病雏鸡的85%左右的死亡率。
本发明还提供一种用于畜禽产品消毒的生物抑菌剂,其包括上述的噬菌体组合物,生物抑菌剂的使用方法为:对畜禽产品表面进行浸泡或者喷雾消毒,来抑制产品加工或保鲜过程中大肠杆菌的增殖。
本发明还提供一种可用于水线或养殖场环境消毒的消毒剂,其有效成分为如上所述的噬菌体组合物;优选地,每种噬菌体的浓度为1×108PFU/ml以上。消毒剂的使用方法为:通过喷雾消毒来抑制养殖环境中大肠杆菌增殖,或者稀释在水体中对水线进行冲洗消毒。
可选地,所述消毒剂还包含其他用于抑制或消灭水线中病毒、细菌的其他活性成分。
本发明还提供一种饲料添加剂,其特征在于,包括如上所述的噬菌体组合物;优选地,饲料中每种噬菌体的浓度至少为1×108PFU/g。作为饲料添加剂使用,不仅使用便利,并且便于预防及治疗大肠杆菌感染。
此外,本发明还提供一种检测试剂盒,其包括如上所述的噬菌体组合物。本领域技术人员可根据本发明公开内容和本领域常识利用上述噬菌体组合物制备检测试剂盒,用于检测其特异侵染的大肠杆菌、或用于防控由其宿主菌大肠杆菌侵染导致的疾病。
本发明实施例提供的大肠杆菌噬菌体组合物及其应用具有以下有益效果:
1、发明还提供一种能高效清洁水线并抑制大肠杆菌繁殖的大肠杆菌噬菌体组合物,该噬菌体组合物是通过大肠杆菌噬菌体PD06与大肠杆菌噬菌体 PD114的复配作为有效预防及治疗鸡大肠杆菌病药物的成分,也可用于制备水线消毒剂,可有效降解水线中因大肠杆菌富集形成的生物膜,以及净化饮水的特点;与单一裂解性大肠杆菌噬菌体相比,明显拓宽了其杀菌范围,增加其杀菌活性,可快速、高效、安全杀灭致病性大肠杆菌乃至耐药性大肠杆菌,有效降低发病雏鸡的死亡率。
2、本发明涉及的噬菌体组合物从自然界中获得,数量庞大,具有宿主专一性,使用安全,不会影响雏鸡体内正常菌群。经雏鸡安全试验证明,噬菌体是安全、无残留、没有毒副作用,因此可作为食品生物抑菌剂、环境消毒剂或者水线清洁剂来预防或治疗禽大肠杆菌病。
3、本发明设计的噬菌体组合物应用广泛,其不仅可用于环境消毒剂、水线消毒剂使用,还可与饲料混合及随水饮用,用于防治由大肠杆菌染导致的禽类疾病,还可作为生物抑菌剂,用于禽类产品的消毒,来抑制产品加工或保鲜过程中大肠杆菌的增殖。
附图说明
图1为大肠杆菌噬菌体PD06的噬菌斑图片;
图2为大肠杆菌噬菌体PD114的噬菌斑图片;
图3为大肠杆菌噬菌体PD06的电镜图片;
图4为大肠杆菌噬菌体PD114的电镜图片;
图5为噬菌体PD06的热稳定性;
图6为噬菌体PD114的热稳定性;
图7为噬菌体PD06的pH值稳定性;
图8为噬菌体PD114的pH值稳定性;
图9为噬菌体PD06的一步生长曲线;
图10为噬菌体PD114的一步生长曲线;
图11为噬菌体组合物对鸡肉表面的杀菌效果图;
图12为噬菌体组合物对鸡舍空气中肠杆菌科的消毒效果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1噬菌体的分离及制备
1、样品的采集
本发明中的鸡粪样品采自辽宁省盘锦市某鸡场。
2、宿主菌的复苏培养与增殖液的制备
宿主菌为本实验室分离鉴定保存的大肠杆菌,分离来自江苏省某鸡场的病鸡。用灭菌的接种环蘸取宿主菌菌液,在麦康凯琼脂培养基上划线接种, 37℃的恒温箱中培养16~24h,得到单菌落。挑取单菌落,接种于5mL的NB 肉汤试管中,37℃,170rpm/min震荡培养12~16h,即可得到新鲜的宿主菌增殖液备用。
3、粪样、污水等样品处理
取适量粪水放入泡样瓶中,加入宿主菌增殖液各取200μL,再加入适量的 NB肉汤,37℃的条件下,以170rpm的转速在摇床过夜培养,增殖富集噬菌体,得到培养液;取5ml上述培养液,在11000rpm的条件下离心10min,取上清液用0.22μm滤膜过滤除菌,收集滤液。
4、噬菌体的分离
以点斑法鉴定上述滤液是否有噬菌体,即取200μL宿主菌液加到上层琼脂 (琼脂浓度为0.7%),混匀后迅速倾倒下层琼脂(琼脂浓度为1.5%)平板上,待凝固后,滴上2μL上述滤液,37℃的恒温箱中培养4~6h,观察是否有透亮噬菌斑。
培养出现噬菌斑后,则采用双层平板法分离噬菌体:取噬菌体原液100μL,按照10倍比例稀释,取适当梯度稀释液120μL与120μL宿主菌增殖液混合均匀,37℃孵育5min后,加入约45℃上层琼脂(琼脂浓度为0.7%),混匀后迅速倾倒下层琼脂(琼脂浓度为1.5%)平皿上,摇匀平置至培养基凝固,置于37℃的恒温箱中倒置培养6~8h后,获得形成噬菌斑的双层平板,根据噬菌斑形态及宿主菌不同,最终获得两株大肠杆菌噬菌体,分别命名为噬菌体PD06和噬菌体PD114,结果如图1和图2所示。
实施例2噬菌体的增殖纯化
1、噬菌体的纯化
采用双层平板法,在形成噬菌斑的双层平板中挑取单个、边缘光滑且透亮的噬菌斑,接种于盛有1mlNB肉汤的无菌EP管中,37℃、170rpm条件下振荡30min,得到噬菌体浸出液;11000rpm离心5min,取100μL离心后的噬菌体浸出液与100μL宿主菌增殖液充分混合,37℃孵育5min后,加入预热融化的上层琼脂中,混匀,迅速倒入含有下层琼脂培养基平板上,待上层琼脂凝固后,倒置37℃恒温箱培养6~8h。重复上述步骤3-5次反复纯化噬菌体,直至得到大小、形态、分布均一的噬菌斑。
2、噬菌体增殖液的制备
从完成纯化的噬菌斑双层平板上,扣取单个噬菌斑接种于盛有1ml NB肉汤的无菌EP管中,37℃170rpm培养30min,11000rpm离心5min,取100μL 上清液与100μL宿主菌增殖液加入5ml肉汤中,37℃、170rpm/min的条件下,震荡培养,直至混合液摇清,然后再离心过滤,即可得到噬菌体增殖液。按照上述方法分别得到噬菌体PD06和噬菌体PD114的增殖液。
实施例3噬菌体的形态观察和鉴定
形态观察方法为:取20μL噬菌体样本滴在带有碳涂布膜的铜网上,待其自然沉淀15min,用滤纸稍吸干后,再用2%(W/V)磷钨酸(PTA)染色1-2min,滤纸稍吸干,待干燥后,采用透射电子显微镜进行观察拍照。
观测结果:噬菌体PD06如图3所示,头部呈正二十面体,头部宽约100nm,长约100nm,尾长约110nm,根据噬菌体分类,本研究噬菌体形态符合肌尾噬菌体科的特征,属于肌尾噬菌体科。
噬菌体PD114如图4所示,头部呈多面体结构,头部长约60nm,宽约63 nm,尾长约150nm,根据噬菌体分类,本研究噬菌体形态符合长尾噬菌体科的特征,属于长尾噬菌体科。
实施例4噬菌体基因组分析
噬菌体PD06全基因组大小为149506bp,为环形双链结构,G+C%含量为 37.65%;噬菌体PD114全基因组大小为150354bp,为环形双链结构,G+C%含量为37.54%。经RAST预测分析,PD06基因组含有238个开放阅读框 (ORFs),平均ORF长度为628bp,PD114基因组含有239个开放阅读框 (ORFs),平均ORF长度为629bp。
使用在线工具BLASTp和保守域数据库(CDD)分别对PD06的238个 ORFs和PD114的239个ORFs蛋白序列进行比对分析,得到:PD06含47 个已知编码功能蛋白及191个假想蛋白,PD114含74个已知编码功能蛋白以及165个假想蛋白。
PD06基因组结构蛋白主要包括噬菌体的结构和包装蛋白(尾蛋白、尾纤维蛋白、尾部装配伴侣、衣壳蛋白、支架蛋白等)、DNA复制和修饰有关的蛋白质(DNA聚合酶I、核糖核苷酸还原酶、DNA解旋酶、特异性DNA-甲基转移酶或甲基化酶)。其中,与噬菌体宿主识别相关的尾部纤维(tail fibers protein)蛋白相关的3个基因序列见序列表序列1、3和5,这三个基因表达的蛋白序列分别为序列2、4和6;高度保守的末端酶大亚基(terminase largesubunit) 蛋白基因的序列见序列表中序列7,其表达蛋白序列见序列表中序列8;DNA 聚合酶(DNA polymerase)基因的序列见序列表中序列9,其表达蛋白序列见序列表中序列10。
PD114基因组结构蛋白主要包括噬菌体的结构蛋白(尾纤维蛋白、末端酶大亚基等)、DNA复制和修饰有关的蛋白质(DNA聚合酶、胸苷激酶、1-磷酸葡萄糖胸苷基转移酶、DNA解旋酶、核苷酸外切酶、核糖核苷酸还原酶等)。噬菌体PD114的基因组中,与噬菌体宿主识别相关的尾部纤维(tail fibers protein) 蛋白相关3个基因序列见序列表序列11、13和15,这三个基因表达的蛋白序列分别为序列12、14和16;DNA聚合酶(DNA polymerase)基因的序列见序列表中序列17,其表达蛋白序列见序列表中序列18;与裂解能力相关的裂解酶(lysozyme)基因序列见序列表中序列19,其表达蛋白序列见序列表中序列20。上述基因具体信息见表1。
经软件tRNAscan-SE分析二者基因组不含tRNA基因。经PHASTER分析二者基因组不含溶原性相关基因。经在线工具CGE server分析二者基因组不含oxa1、aadAla、aadB、catB2、cmlA、dfrA1、dfrB1、TEM、SHV等耐药基因及不含Colv、Stxs、irp2、ompT、fyuA、tsh、fimD、fimF、fimG等毒力基因。
表1基因序列信息表
实施例5噬菌体的生物学特性
1、温度对噬菌体PD06和噬菌体PD114的影响
各取500μL噬菌体PD06和噬菌体PD114分装于无菌EP管中,分别于 40℃、50℃、60℃、70℃、80℃水浴中作用20min、40min和60min,每一温度设3个重复,待作用结束后取样,并立即将样品置于冰浴中冷却,按照10 倍比例进行稀释,采用双层平板法测定噬菌体效价。以温度为横坐标,以噬菌体效价的对数值为纵坐标,分别绘制噬菌体PD06和噬菌体PD114热稳定性曲线。
结果可见,噬菌体PD06(图5)在40~50℃中放置60min,依然保持较高活性,与起始效价基本无变化,在80℃中失活,在60℃中放置60min仅降低1个数量级;噬菌体PD114(图6)在40~50℃中放置60min,其活性稳定,在70℃中放置60min则失活;可见,该噬菌体PD06和PD114具有较强的耐热性。
3、pH对噬菌体PD06和噬菌体PD114的影响
于无菌试管加入不同pH值(2、3、4、5、6、7、8、9、10、11、12、13) 的NB肉汤4.5mL,然后将上述试管置于37℃的水浴锅中,待温度平衡后各加入500μL噬菌体增殖液混匀后,37℃水浴作用1h、2h和3h。采用双层平板法测定不同pH作用下的噬菌体效价,并且每个pH值设定3重复。以pH值为横坐标,噬菌体效价的对数值为纵坐标,绘制噬菌体PD06和噬菌体PD114的pH值稳定性曲线。
结果如图所示,噬菌体PD06(图7)在pH5~11范围内作用3h后,活性稳定,效价仍接近于起始效价;噬菌体PD114(图8)在pH为5~11时,与起始效价无显著性差异,在pH 3或者12时作用3h后,虽然效价明显降低,但噬菌体仍有活性;可见噬菌体PD06和PD114具有较强的耐酸碱,对碱性环境耐受力更为突出,对pH具有较广的适应范围。
4、噬菌体PD06和噬菌体PD114的一步生长曲线
将感染复数为10的噬菌体增殖液和宿主菌新鲜增殖液各1mL,充分混匀 (此时开始计时),37℃孵育5min,13000rpm离心30s,用微量移液器吸净上清,再用5mL NB肉汤洗涤1次(13000rpm离心30s),弃上清。用预热的NB肉汤混悬沉淀(总体积为5mL)并充分混匀,迅速置于37℃摇床中170 rpm/min振荡培养,在0时刻和每隔5min取出150μL,10000rpm离心1min,用NB肉汤做10倍倍比稀释后采用双层平板法测噬菌体效价,做3个平行,结果取平均值,以感染时间为横坐标,感染体系中噬菌体滴度的对数值为纵坐标,绘制一步生长曲线,得到噬菌体PD06和PD114的潜伏期、爆发期,计算出爆发量。
爆发量=噬菌体爆发末期的总个数/爆发初期细菌的总个数通过图9发现,噬菌体PD06感染宿主菌后,在10min内效价基本不变,效价稳定在106PFU/mL,表明噬菌体PD06潜伏期约为10min,噬菌体侵染宿主菌后的10~60min内,噬菌体数量逐渐增加,在70min时效价增长开始趋于平稳,此时效价可达1010PFU/mL,可见噬菌体PD06的爆发期约为50min,爆发量为95;通过图10的噬菌体PD114一步生长曲线发现,噬菌体感染宿主菌后,在15min内效价基本不变,效价稳定在105PFU/mL,表明噬菌体PD114 潜伏期约为15min,噬菌体侵染宿主菌后的15~90min内,噬菌体数量逐渐增加,在105min时效价增长开始趋于平稳,此时效价可达109PFU/mL,可见噬菌体PD114的爆发期约为75min,爆发量为104。
实施例6噬菌体组合物裂解谱测定
准备新鲜的噬菌体增殖液PD06、PD114和从山东、江苏、安徽、河南、广西等地临床分离的引起鸡眼炎(5)、输卵管炎(15)、腹膜炎(20)、心包炎(10)、肠炎(5)共55株鸡致病性大肠杆菌,这55株鸡致病性大肠杆菌分别为NABTD1-NABTD55(分类见表2)。
在96孔板中,试验组每孔中分别加入5μL噬菌体液和5μL细菌菌液,加入190μL灭菌营养肉汤,混匀;对照组中加入5μL细菌菌液,195μL灭菌营养肉汤,混匀;分别于0h、4h测定各孔中的OD600值;计算噬菌体对不同大肠杆菌OD值下降比例;以OD值下降>50%表示完全裂解此菌,即混合液是清亮的,统计噬菌体PD06、噬菌体PD114和噬菌体组合PD06+PD114对55 株大肠杆菌的裂解率;
OD值下降比例=(对照组OD值—试验组OD值)/对照组OD值
结果显示:噬菌体PD06共裂解33株大肠杆菌,裂解率为60.00%;噬菌体PD114共裂解27株大肠杆菌,裂解率为49.09%;而噬菌体组合物可裂解 50株大肠杆菌,裂解率为90.91%,其裂解能力被最大化,说明该噬菌体组合物是具有更强的裂解性和广谱性,可弥补噬菌体单一应用时宿主谱的局限性。
表2噬菌体的裂解谱
实施例7噬菌体的安全性试验
选取40只1日龄雏鸡,随机分为4组,每组10只;试验组1每只雏鸡肌肉注射6.8×1010PFU/mL噬菌体组合物1mL,试验组2每只雏鸡灌服 6.8×1010PFU/mL噬菌体组合物1mL,对照组1每只雏鸡肌肉注射1mL无菌生理盐水,对照组2每只雏鸡灌服1mL无菌生理盐水;将4组雏鸡在同等环境条件下饲养两周,观察临床变化;
结果发现,4组雏鸡在饲养两周内均未出现精神萎靡、腹泻、死亡等临床症状,生长状况良好,处死后剖检每只鸡未见组织脏器病理变化,说明该噬菌体组合物具有良好的安全性且无毒副作用。
实施例8噬菌体组合物对规模化鸡场水线清洁杀菌效果试验
以山东省潍坊某规模化鸡场中1栋肉鸡舍为研究对象,选取同一高度4条水线,其中一条作为对照组,3条作为消毒组。打开水线,彻底排空水线中的水,分别将噬菌体及其组合物组按照1:500灌入水线,直至水线内充满噬菌体溶液,关闭阀门;浸泡2h后,分别在消毒前和消毒后对水线前、中、后端3 处饮水乳头采集10ml水样,使用麦康培养基倾注法检测水中大肠杆菌数量。检测结果效果见表3。
实验发现:使用噬菌体组合物浸泡水线2h后,对照组前中后端大肠杆菌数量与消毒前基本无变化;PD06消毒组平均大肠杆菌杀灭率为81.06%;PD114 消毒组平均大肠杆菌杀灭率为75.46%;PD06+PD114消毒组平均大肠杆菌杀灭率为98.36%。结果表明,使用噬菌体组合物可有效降低饮水中大肠杆菌数量,达到净化水源的目的,其中PD06+PD114噬菌体组合杀菌率最高,消毒效果最好。
表3噬菌体浸泡水线后大肠杆菌检测效果(单位:cfu/ml)
实施例9噬菌体组合物对雏鸡大肠杆菌的防控作用
选择75只健康的5日龄的SPF鸡,分为5组,每组15只。将新鲜增殖培养的大肠杆菌菌浓度调整为7.10×107CFU/mL,按照表4对各个进行分组,攻毒。待鸡群50%出现精神萎靡,食欲减退,拉稀等发病症状,开始用噬菌体进行治疗。连续用药治疗5天后饲养观察14天,观察鸡只的死亡情况,活鸡剖检观察包心包肝病变情况。
表4分组及处理
如上表的结果显示,空白对照组鸡只无死亡,攻毒组死亡率100%,包心包肝率90%;PD06治疗组死亡率50%,包心包肝率75%;PD114治疗组死亡率70%,包心包肝率55%;PD06+PD114组死亡率20%,包心包肝率10%。可见感染大肠杆菌的雏鸡连续口服噬菌体组合物可以有效降低死亡率和包心包肝率,PD06+PD114噬菌体组合治疗效果最佳。
实施例10噬菌体组合物对食品消毒试验
以山东省某食品有限公司的鸡胸肉产品为研究对象,用灭菌刀在无菌砧板上取下表面积为3cm×3cm的肉块,要确保试验表面平整,肉块样重约4g,将肉样置于超净台紫外灯下照30min灭菌处理,得到无菌肉样;取100μL 1×105CFU/mL的宿主菌混合菌液随机滴加在鸡肉样品表面,待其干燥后;将样品放入1×106PFU/mL且等体积的PD06噬菌体液、PD114噬菌体液、PD06+ PD114噬菌体液以及PBS中,浸泡30min后取出,检测样品中的大肠杆菌数。
如图11的结果显示,在PD06+PD114噬菌体组合液中浸泡后,大肠杆菌的数量下降3个数量级,明显优于PD06噬菌体或PD114噬菌体的杀菌效果。
实施例11噬菌体组合物对鸡舍空气的消毒效果
在青岛某鸡场进行试验,随机选取种鸡日龄为90日龄的3栋鸡舍作为试验鸡舍;参照NY/T388-1999《畜禽场环境质量标准》中规定的采用平板自然沉降法分别于消毒前采样空气中的微生物:鸡舍内选择中央料线的中点,在鸡舍内取对角线上与两条料线交叉的四点共五点为采样点。采样点距离地面 60cm,采样点避开风口,离墙壁距离大于0.5m,每点放置3个麦康凯平板,同时移去3个平板的平皿盖,使培养基表面暴露在鸡舍空气中,5min后盖上平皿盖,将培养皿放37℃恒温培养箱中培养18~24h后记录培养皿中肠杆菌菌落数。按奥氏公式计算大肠杆菌菌落总数:
C=50000N/AT式中,C:每立方米菌落总数(cfu/m3);N:每皿菌落数(个);A:培养皿面积(cm2);T:采样时间(min)。
喷雾噬菌体消毒前将鸡舍门窗、进气孔和排气孔都关上,噬菌体与水1:500 比例(效价约1×108PFU/mL)混合均匀进行喷雾消毒,喷雾量为20ml/m3,喷雾噬菌体30min后自然沉降法采集空气微生物培养后计数。
如图12的结果显示:喷雾噬菌体消毒后,PD06对大肠杆菌的杀菌率为 76.99%;PD114对大肠杆菌的杀菌率为77.05%;PD06+PD114对大肠杆菌的杀菌率为90.84%。结果说明噬菌体在鸡舍空气喷雾消毒中对肠杆菌有明显的消毒效果,其中PD06+PD114噬菌体组合物的消毒效果最佳。
可以理解的是,对本领域普通技术人员来说,可以根据本发明的技术方案及本发明构思加以等同替换或改变,而所有这些改变或替换都应属于本发明所附的权利要求的保护范围。
序列表
<110> 青岛诺安百特生物技术有限公司
<120> 一种大肠杆菌噬菌体组合物及其应用
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1971
<212> DNA
<213> 噬菌体 PD06的尾纤蛋白(tail fibers protein of phage PD06)
<400> 1
gtgattttaa cacgtccaaa tagtgccatt gcagacatgg tagagtctcg ctatagaaag 60
cgtagaagtt ttgcaagtgg ttttacctta agagatgctc gtgaagcatt gctctttgaa 120
gctgacggta actactatca gtggaaaggt tctctaccta aagttgtacc tgctaactct 180
actcctacat ctactggagg gatttcagat acagcttgga aaaatgtggg taatgcagat 240
gacattgctc aaatagaatc agaattagat gctattgaag ctgaaattga agatcttaga 300
gaatccatcc cagaagctgg taaatcagct taccagattg ccgtagacaa tggtttccaa 360
ggtactgaga cacagtggtt ggcttcttta aaaggtgatc caggtgctcc attaaagcta 420
ttagggacac tatctaatac caaccaatta cctacaagtg gaaacaatgc aggggatgca 480
tggattgtag gcgagaatat gtatgcttgg gatggtactg cttggagaaa agtaaactca 540
caggggccag aaggtaaatc tgcttatgaa gtatggcttg cagcaggaaa tgaaggtaca 600
gaactagatt atcttgcttc aattaaaggt gataaagggg agcctggccc agaggggcca 660
caaggtgtgc caggtaataa tgctaatggt tttgattacc aaggcgcagc taattccgtt 720
tcagatcttc cttctgcatc tcatggaaac acatctcaag catggagtgt tggtacatat 780
ctatacgtgt caaacggtgt agaatggatc aatatgggtg ctatagtggg gccaaaagga 840
gataaaggcg acaaaggtga taagggtaac actggtgatt cagcttatca agtctggtta 900
aatgctggta acgttggtac tattgacgaa tatattgcct ctctgaaagg agaaaaaggt 960
gataagggag acaagggtga tcaaggtgca cagggtatac aaggcttgac ggggccaact 1020
ggtgcagggt tgaccattat tgacattctg actaacactt ctgaactacc acaaactgga 1080
gaagagggtg atggttatct gatcaataat gaactttggg tctggttgca aggagccaca 1140
gagtggacaa atgttggtcg tttagctggt actccaatca ctgcaaaagg acacgttgcg 1200
tctgcaagtg cgcttcctcc tacagccaat tacggtgatg cttatctgac ggaagataca 1260
ggtcatcttt acatttacac ctataatgaa caaactcaaa ctgctgaata tgtagatatg 1320
ggtagtttga aaggtgataa gggtgaaaaa ggtgagaaag gtgacgctgg tactaacggt 1380
acagacggtg caacttggat tgttaaaaca acagatccaa tctctagtga tggtaaacaa 1440
ggtgattatg cctttaatat ctccactggc gaggtgttct taaaaacatc tgcaactgtc 1500
tggacttctc aaggtacact acctagtatc acagatgcac ctagtgatgg taagacttat 1560
gctcgtaaag atggggcatg ggaagaactc actaattctg tagatggtca agatatagtt 1620
ccaacaaaag taacaactaa ttttatagag gttacagcat ataccaacaa ttctgttaca 1680
ggggactgga cacctccagg aacacataac atgaatgtgt tgaatcttac tggtaacgct 1740
actataaaag catggcctgg tgtcgaagat agtacaaaac ctaaaccatt ctctgcaata 1800
gtttatctta tccaagatgc cactggaggt cgtactgtta ctttagatcc atcttacaaa 1860
gtaattggtt ccagtgatat taatacagat cctaacggag taacaatact acaactaaca 1920
tattgtggtg ttggcgatta cgtggatgct attgttgcta aacgcacata a 1971
<210> 2
<211> 656
<212> PRT
<213> 噬菌体 PD06的尾纤蛋白(tail fibers protein of phage PD06)
<400> 2
Met Ile Leu Thr Arg Pro Asn Ser Ala Ile Ala Asp Met Val Glu Ser
1 5 10 15
Arg Tyr Arg Lys Arg Arg Ser Phe Ala Ser Gly Phe Thr Leu Arg Asp
20 25 30
Ala Arg Glu Ala Leu Leu Phe Glu Ala Asp Gly Asn Tyr Tyr Gln Trp
35 40 45
Lys Gly Ser Leu Pro Lys Val Val Pro Ala Asn Ser Thr Pro Thr Ser
50 55 60
Thr Gly Gly Ile Ser Asp Thr Ala Trp Lys Asn Val Gly Asn Ala Asp
65 70 75 80
Asp Ile Ala Gln Ile Glu Ser Glu Leu Asp Ala Ile Glu Ala Glu Ile
85 90 95
Glu Asp Leu Arg Glu Ser Ile Pro Glu Ala Gly Lys Ser Ala Tyr Gln
100 105 110
Ile Ala Val Asp Asn Gly Phe Gln Gly Thr Glu Thr Gln Trp Leu Ala
115 120 125
Ser Leu Lys Gly Asp Pro Gly Ala Pro Leu Lys Leu Leu Gly Thr Leu
130 135 140
Ser Asn Thr Asn Gln Leu Pro Thr Ser Gly Asn Asn Ala Gly Asp Ala
145 150 155 160
Trp Ile Val Gly Glu Asn Met Tyr Ala Trp Asp Gly Thr Ala Trp Arg
165 170 175
Lys Val Asn Ser Gln Gly Pro Glu Gly Lys Ser Ala Tyr Glu Val Trp
180 185 190
Leu Ala Ala Gly Asn Glu Gly Thr Glu Leu Asp Tyr Leu Ala Ser Ile
195 200 205
Lys Gly Asp Lys Gly Glu Pro Gly Pro Glu Gly Pro Gln Gly Val Pro
210 215 220
Gly Asn Asn Ala Asn Gly Phe Asp Tyr Gln Gly Ala Ala Asn Ser Val
225 230 235 240
Ser Asp Leu Pro Ser Ala Ser His Gly Asn Thr Ser Gln Ala Trp Ser
245 250 255
Val Gly Thr Tyr Leu Tyr Val Ser Asn Gly Val Glu Trp Ile Asn Met
260 265 270
Gly Ala Ile Val Gly Pro Lys Gly Asp Lys Gly Asp Lys Gly Asp Lys
275 280 285
Gly Asn Thr Gly Asp Ser Ala Tyr Gln Val Trp Leu Asn Ala Gly Asn
290 295 300
Val Gly Thr Ile Asp Glu Tyr Ile Ala Ser Leu Lys Gly Glu Lys Gly
305 310 315 320
Asp Lys Gly Asp Lys Gly Asp Gln Gly Ala Gln Gly Ile Gln Gly Leu
325 330 335
Thr Gly Pro Thr Gly Ala Gly Leu Thr Ile Ile Asp Ile Leu Thr Asn
340 345 350
Thr Ser Glu Leu Pro Gln Thr Gly Glu Glu Gly Asp Gly Tyr Leu Ile
355 360 365
Asn Asn Glu Leu Trp Val Trp Leu Gln Gly Ala Thr Glu Trp Thr Asn
370 375 380
Val Gly Arg Leu Ala Gly Thr Pro Ile Thr Ala Lys Gly His Val Ala
385 390 395 400
Ser Ala Ser Ala Leu Pro Pro Thr Ala Asn Tyr Gly Asp Ala Tyr Leu
405 410 415
Thr Glu Asp Thr Gly His Leu Tyr Ile Tyr Thr Tyr Asn Glu Gln Thr
420 425 430
Gln Thr Ala Glu Tyr Val Asp Met Gly Ser Leu Lys Gly Asp Lys Gly
435 440 445
Glu Lys Gly Glu Lys Gly Asp Ala Gly Thr Asn Gly Thr Asp Gly Ala
450 455 460
Thr Trp Ile Val Lys Thr Thr Asp Pro Ile Ser Ser Asp Gly Lys Gln
465 470 475 480
Gly Asp Tyr Ala Phe Asn Ile Ser Thr Gly Glu Val Phe Leu Lys Thr
485 490 495
Ser Ala Thr Val Trp Thr Ser Gln Gly Thr Leu Pro Ser Ile Thr Asp
500 505 510
Ala Pro Ser Asp Gly Lys Thr Tyr Ala Arg Lys Asp Gly Ala Trp Glu
515 520 525
Glu Leu Thr Asn Ser Val Asp Gly Gln Asp Ile Val Pro Thr Lys Val
530 535 540
Thr Thr Asn Phe Ile Glu Val Thr Ala Tyr Thr Asn Asn Ser Val Thr
545 550 555 560
Gly Asp Trp Thr Pro Pro Gly Thr His Asn Met Asn Val Leu Asn Leu
565 570 575
Thr Gly Asn Ala Thr Ile Lys Ala Trp Pro Gly Val Glu Asp Ser Thr
580 585 590
Lys Pro Lys Pro Phe Ser Ala Ile Val Tyr Leu Ile Gln Asp Ala Thr
595 600 605
Gly Gly Arg Thr Val Thr Leu Asp Pro Ser Tyr Lys Val Ile Gly Ser
610 615 620
Ser Asp Ile Asn Thr Asp Pro Asn Gly Val Thr Ile Leu Gln Leu Thr
625 630 635 640
Tyr Cys Gly Val Gly Asp Tyr Val Asp Ala Ile Val Ala Lys Arg Thr
645 650 655
<210> 3
<211> 552
<212> DNA
<213> 噬菌体 PD06的假定尾纤蛋白(putative tail fibers protein of phagePD06)
<400> 3
atgaaaaaag atgttatttt tacaggtttt gaattgtacg aaccggaatt aacagaggag 60
aacattcttt atctttctgt tggcggtggt tttttgcaag acaaggatgg taatgattgg 120
tatcaacttc aaaaaacttt gatggctgaa tatcccgacg cttatttcat taccttagat 180
gataataata ttgtacgtgg tgctactaaa gatgttacta cctatttccc tgccggacaa 240
tctattttag ttacatataa tgcaccagat aaaatagaag atgaaagtaa tattggctct 300
tggcaatatg ttgaaggtaa atttgttcct cacaatagtc ttgctattga agatgctact 360
cttaagttag aaaaggagtt aacttgggct acgaatcaag tgtctgcgat tcaagattta 420
gaggaattac aaggtttatc tcaagaacaa aaagattggt taggaaaagt taaactatat 480
cgtgcaacac tgattcaaat taacgttgaa gatgcaccta atataacttg gcctacaaga 540
cctaagaaat ag 552
<210> 4
<211> 183
<212> PRT
<213> 噬菌体 PD06的假定尾纤蛋白(putative tail fibers protein of phagePD06)
<400> 4
Met Lys Lys Asp Val Ile Phe Thr Gly Phe Glu Leu Tyr Glu Pro Glu
1 5 10 15
Leu Thr Glu Glu Asn Ile Leu Tyr Leu Ser Val Gly Gly Gly Phe Leu
20 25 30
Gln Asp Lys Asp Gly Asn Asp Trp Tyr Gln Leu Gln Lys Thr Leu Met
35 40 45
Ala Glu Tyr Pro Asp Ala Tyr Phe Ile Thr Leu Asp Asp Asn Asn Ile
50 55 60
Val Arg Gly Ala Thr Lys Asp Val Thr Thr Tyr Phe Pro Ala Gly Gln
65 70 75 80
Ser Ile Leu Val Thr Tyr Asn Ala Pro Asp Lys Ile Glu Asp Glu Ser
85 90 95
Asn Ile Gly Ser Trp Gln Tyr Val Glu Gly Lys Phe Val Pro His Asn
100 105 110
Ser Leu Ala Ile Glu Asp Ala Thr Leu Lys Leu Glu Lys Glu Leu Thr
115 120 125
Trp Ala Thr Asn Gln Val Ser Ala Ile Gln Asp Leu Glu Glu Leu Gln
130 135 140
Gly Leu Ser Gln Glu Gln Lys Asp Trp Leu Gly Lys Val Lys Leu Tyr
145 150 155 160
Arg Ala Thr Leu Ile Gln Ile Asn Val Glu Asp Ala Pro Asn Ile Thr
165 170 175
Trp Pro Thr Arg Pro Lys Lys
180
<210> 5
<211> 2160
<212> DNA
<213> 噬菌体 PD06的另一假定尾纤蛋白(another putative tail fibers proteinof phage PD06)
<400> 5
atgaaacagg ctatttttac tgtaactcga aatacgagag taaatgccag ttcattcagc 60
tataagaccg tcgatggaac ggcggtcgct ggtagagatt atgtagctaa acagggaaca 120
gtaacgttcc cttctgatgc agattctgct actgtggtgg ttgatgtata tgaacgtccg 180
gcaaactctt tagatcttta ttttactctt gagattacta gcataagtca aaataacatg 240
atgatcaatg aagaaattcg ttgtgttatt acgactaatc aatcgaatac aaccccgcta 300
acttggcctg ttgtgaagtc tagaatgttt gatcctaagt tttggacaat agattctcaa 360
gcaacagaaa gttgttctat tatctctaat ggtgattctt tcacagcaag aatggttaat 420
aggacagtgg ctggtcttgc tgggattatt tggtcaactt acgataaata tgatcacaaa 480
ggattaggct accaagaaca ttctgactta agtaaagaga aactttggtt taaaatggaa 540
cttagtcaag gtgttcctgg attaaaagaa gaaaaattat tgccatcctt aacagtgaca 600
ctgaaggatg aaacagttca ttatgtatct ctaaactttt atgcagaaga agtttcagag 660
gatggtaaaa cagcaacaat aaaattagat ttctcaaatt taaaatctgg gccagaaaat 720
gatattgttg ttgacactac acagatagat aatatgttct tctccttgat ctccacaagg 780
tatcaagaaa ttgaagatat tattcctgta gacaatgagg atctttcctt taagtttacc 840
atactggaac ctgatacggg ttatagtatg atgaatatgg gtaatttaaa tgttccagca 900
cacaccataa gaatgtgtac atcttatgat gatatgtata acctaacccc agagcgagtt 960
atttctaaca cctatagatt aggttataga gatatgatta accattataa tggtatgtca 1020
cactattatc aatttagttg ggatgctgga tctaataaat gggctttgaa tagaacagaa 1080
tatttaaacc cagcaacaga agcctggttt gataatttcc acgctaatgc aaaagctatg 1140
ggctacacag taatgaactc cttaagtttt gaacttatga gtacagtgtg tcctgtagaa 1200
tgggtacaac atacttggaa tgatgatctg gctgctacag gttatgagcc accaagttat 1260
gttttatctc cttcaattga tgaagggatg aattatttac aaggtgtaat aaataagata 1320
gcttctatat ccgctgccaa tgatcttcct gttattattc aggttggtga accgtggtgg 1380
tggtataaca cagctaccaa tctcccgtgt atttatgatt atgcaaccaa agttgcattt 1440
aataatgaaa ctggattata tgcacaagat gttggtacag ttaagaatta taaaactggt 1500
acaccatatg atgaatatta tgcattccta tctaaaacat taggactgcg tgttgctaaa 1560
tttgcaacag atactaaatt agcctatcct ggagctaaag ttacattatt accatttgtg 1620
ccatccattt taggtaacgg tatgatggag tttgttaact tccctaaaga ttattatatc 1680
ccagaaaact ttgactttta ttgttcagaa tgttatgact ggttgttgga aggtcagatg 1740
gaaagatctt tcgacgctat caaaatacct aatgagcaac taggttttag ttatgataag 1800
atacattatc ttgcagggtt tgttccagac gcaactctag caccattata tggctttgat 1860
cctgaatcag actacagaac atatttgtgg aaaatgatta taggtaatat tgtattgaat 1920
gatcagaagt ttgttggttt atatcaatac atatgggctt acccacaaat aatgcatgat 1980
tcaataactg ttcttaattc aaaatcccaa gtgttttata tgggtaatgt gcctttgaat 2040
tgctacttgc aagatatcac tcttaaacag gtaaatgttg agattgccac tataagtgga 2100
attacattag ccactaaatc tggagaagat ttagtaacaa cacaaaaatt ttatatatag 2160
<210> 6
<211> 719
<212> PRT
<213> 噬菌体 PD06的另一假定尾纤蛋白(another putative tail fibers proteinof phage PD06)
<400> 6
Met Lys Gln Ala Ile Phe Thr Val Thr Arg Asn Thr Arg Val Asn Ala
1 5 10 15
Ser Ser Phe Ser Tyr Lys Thr Val Asp Gly Thr Ala Val Ala Gly Arg
20 25 30
Asp Tyr Val Ala Lys Gln Gly Thr Val Thr Phe Pro Ser Asp Ala Asp
35 40 45
Ser Ala Thr Val Val Val Asp Val Tyr Glu Arg Pro Ala Asn Ser Leu
50 55 60
Asp Leu Tyr Phe Thr Leu Glu Ile Thr Ser Ile Ser Gln Asn Asn Met
65 70 75 80
Met Ile Asn Glu Glu Ile Arg Cys Val Ile Thr Thr Asn Gln Ser Asn
85 90 95
Thr Thr Pro Leu Thr Trp Pro Val Val Lys Ser Arg Met Phe Asp Pro
100 105 110
Lys Phe Trp Thr Ile Asp Ser Gln Ala Thr Glu Ser Cys Ser Ile Ile
115 120 125
Ser Asn Gly Asp Ser Phe Thr Ala Arg Met Val Asn Arg Thr Val Ala
130 135 140
Gly Leu Ala Gly Ile Ile Trp Ser Thr Tyr Asp Lys Tyr Asp His Lys
145 150 155 160
Gly Leu Gly Tyr Gln Glu His Ser Asp Leu Ser Lys Glu Lys Leu Trp
165 170 175
Phe Lys Met Glu Leu Ser Gln Gly Val Pro Gly Leu Lys Glu Glu Lys
180 185 190
Leu Leu Pro Ser Leu Thr Val Thr Leu Lys Asp Glu Thr Val His Tyr
195 200 205
Val Ser Leu Asn Phe Tyr Ala Glu Glu Val Ser Glu Asp Gly Lys Thr
210 215 220
Ala Thr Ile Lys Leu Asp Phe Ser Asn Leu Lys Ser Gly Pro Glu Asn
225 230 235 240
Asp Ile Val Val Asp Thr Thr Gln Ile Asp Asn Met Phe Phe Ser Leu
245 250 255
Ile Ser Thr Arg Tyr Gln Glu Ile Glu Asp Ile Ile Pro Val Asp Asn
260 265 270
Glu Asp Leu Ser Phe Lys Phe Thr Ile Leu Glu Pro Asp Thr Gly Tyr
275 280 285
Ser Met Met Asn Met Gly Asn Leu Asn Val Pro Ala His Thr Ile Arg
290 295 300
Met Cys Thr Ser Tyr Asp Asp Met Tyr Asn Leu Thr Pro Glu Arg Val
305 310 315 320
Ile Ser Asn Thr Tyr Arg Leu Gly Tyr Arg Asp Met Ile Asn His Tyr
325 330 335
Asn Gly Met Ser His Tyr Tyr Gln Phe Ser Trp Asp Ala Gly Ser Asn
340 345 350
Lys Trp Ala Leu Asn Arg Thr Glu Tyr Leu Asn Pro Ala Thr Glu Ala
355 360 365
Trp Phe Asp Asn Phe His Ala Asn Ala Lys Ala Met Gly Tyr Thr Val
370 375 380
Met Asn Ser Leu Ser Phe Glu Leu Met Ser Thr Val Cys Pro Val Glu
385 390 395 400
Trp Val Gln His Thr Trp Asn Asp Asp Leu Ala Ala Thr Gly Tyr Glu
405 410 415
Pro Pro Ser Tyr Val Leu Ser Pro Ser Ile Asp Glu Gly Met Asn Tyr
420 425 430
Leu Gln Gly Val Ile Asn Lys Ile Ala Ser Ile Ser Ala Ala Asn Asp
435 440 445
Leu Pro Val Ile Ile Gln Val Gly Glu Pro Trp Trp Trp Tyr Asn Thr
450 455 460
Ala Thr Asn Leu Pro Cys Ile Tyr Asp Tyr Ala Thr Lys Val Ala Phe
465 470 475 480
Asn Asn Glu Thr Gly Leu Tyr Ala Gln Asp Val Gly Thr Val Lys Asn
485 490 495
Tyr Lys Thr Gly Thr Pro Tyr Asp Glu Tyr Tyr Ala Phe Leu Ser Lys
500 505 510
Thr Leu Gly Leu Arg Val Ala Lys Phe Ala Thr Asp Thr Lys Leu Ala
515 520 525
Tyr Pro Gly Ala Lys Val Thr Leu Leu Pro Phe Val Pro Ser Ile Leu
530 535 540
Gly Asn Gly Met Met Glu Phe Val Asn Phe Pro Lys Asp Tyr Tyr Ile
545 550 555 560
Pro Glu Asn Phe Asp Phe Tyr Cys Ser Glu Cys Tyr Asp Trp Leu Leu
565 570 575
Glu Gly Gln Met Glu Arg Ser Phe Asp Ala Ile Lys Ile Pro Asn Glu
580 585 590
Gln Leu Gly Phe Ser Tyr Asp Lys Ile His Tyr Leu Ala Gly Phe Val
595 600 605
Pro Asp Ala Thr Leu Ala Pro Leu Tyr Gly Phe Asp Pro Glu Ser Asp
610 615 620
Tyr Arg Thr Tyr Leu Trp Lys Met Ile Ile Gly Asn Ile Val Leu Asn
625 630 635 640
Asp Gln Lys Phe Val Gly Leu Tyr Gln Tyr Ile Trp Ala Tyr Pro Gln
645 650 655
Ile Met His Asp Ser Ile Thr Val Leu Asn Ser Lys Ser Gln Val Phe
660 665 670
Tyr Met Gly Asn Val Pro Leu Asn Cys Tyr Leu Gln Asp Ile Thr Leu
675 680 685
Lys Gln Val Asn Val Glu Ile Ala Thr Ile Ser Gly Ile Thr Leu Ala
690 695 700
Thr Lys Ser Gly Glu Asp Leu Val Thr Thr Gln Lys Phe Tyr Ile
705 710 715
<210> 7
<211> 930
<212> DNA
<213> 噬菌体 PD06的末端酶大亚基(terminase large subunit of phage PD06)
<400> 7
ttgtcatact atgcaacaga gcaaggtgaa aaatatgatg gtcgtttcaa cttaggcgta 60
gtttctttaa acttacctat cattgcagcg gagtcccgtg aaaagggcat cttatttact 120
gaactgttgg aacaacatat ggagatggcg tataaggcac atatgctacg tgttaaccgt 180
attagacata ccaaagcatc tcaaaaccct acattgttta tggaaggggc tttagctcga 240
ctagaagcta atgaagagat tggtaaacta tttgaaggtg gttatgcctc tattagtatc 300
ggttatgtag gtttagcaga agctatggag attttatacc caaataacac caatccaaaa 360
gcaatgggta aaaacatcct tcaatatatg aaggatatct gttgcacatt taaagaacgt 420
tctggtttag cttttagttt gtatggaaca ccagcagaat ccttgtgcta taaatttgcc 480
aaagcgttaa atgatcgcta ccctggttct attgatcgtg actacttgac aaactctttc 540
catcaacctg tatggatgca cactaccccg ttccaaaaat gggattatga ggaaggtttt 600
gcacaaatta gtaatggtgg gaacataggt tatgtagaaa caccgaacct taaaaataac 660
ttgcaagcac tggaaactct tgtagattat gcctacaata aaattcctta ttttggcgtg 720
aaccaacctg tagatcagtg tttcaaatgt ggttttcatg gtgaatttaa agcaacggct 780
aaaggcttcg agtgtccaag ttgcggtaat catgaagaag aatccatatc tgtgatcaga 840
agagtgtcag gatacctttc tgcccctaac tctcgccctt ataacaaagg taaaatgcaa 900
gaggtactag aaagggagaa acatttttga 930
<210> 8
<211> 309
<212> PRT
<213> 噬菌体 PD06的末端酶大亚基(terminase large subunit of phage PD06)
<400> 8
Met Ser Tyr Tyr Ala Thr Glu Gln Gly Glu Lys Tyr Asp Gly Arg Phe
1 5 10 15
Asn Leu Gly Val Val Ser Leu Asn Leu Pro Ile Ile Ala Ala Glu Ser
20 25 30
Arg Glu Lys Gly Ile Leu Phe Thr Glu Leu Leu Glu Gln His Met Glu
35 40 45
Met Ala Tyr Lys Ala His Met Leu Arg Val Asn Arg Ile Arg His Thr
50 55 60
Lys Ala Ser Gln Asn Pro Thr Leu Phe Met Glu Gly Ala Leu Ala Arg
65 70 75 80
Leu Glu Ala Asn Glu Glu Ile Gly Lys Leu Phe Glu Gly Gly Tyr Ala
85 90 95
Ser Ile Ser Ile Gly Tyr Val Gly Leu Ala Glu Ala Met Glu Ile Leu
100 105 110
Tyr Pro Asn Asn Thr Asn Pro Lys Ala Met Gly Lys Asn Ile Leu Gln
115 120 125
Tyr Met Lys Asp Ile Cys Cys Thr Phe Lys Glu Arg Ser Gly Leu Ala
130 135 140
Phe Ser Leu Tyr Gly Thr Pro Ala Glu Ser Leu Cys Tyr Lys Phe Ala
145 150 155 160
Lys Ala Leu Asn Asp Arg Tyr Pro Gly Ser Ile Asp Arg Asp Tyr Leu
165 170 175
Thr Asn Ser Phe His Gln Pro Val Trp Met His Thr Thr Pro Phe Gln
180 185 190
Lys Trp Asp Tyr Glu Glu Gly Phe Ala Gln Ile Ser Asn Gly Gly Asn
195 200 205
Ile Gly Tyr Val Glu Thr Pro Asn Leu Lys Asn Asn Leu Gln Ala Leu
210 215 220
Glu Thr Leu Val Asp Tyr Ala Tyr Asn Lys Ile Pro Tyr Phe Gly Val
225 230 235 240
Asn Gln Pro Val Asp Gln Cys Phe Lys Cys Gly Phe His Gly Glu Phe
245 250 255
Lys Ala Thr Ala Lys Gly Phe Glu Cys Pro Ser Cys Gly Asn His Glu
260 265 270
Glu Glu Ser Ile Ser Val Ile Arg Arg Val Ser Gly Tyr Leu Ser Ala
275 280 285
Pro Asn Ser Arg Pro Tyr Asn Lys Gly Lys Met Gln Glu Val Leu Glu
290 295 300
Arg Glu Lys His Phe
305
<210> 9
<211> 963
<212> DNA
<213> 噬菌体 PD06的DNA 聚合酶(DNA polymerase of phage PD06)
<400> 9
ttgagtaaat taacagacga acagaaagcc ttgattgtat ctatgaaagc acaaggttat 60
tcttcgcgta agattgcaca agaggtgttg ggaagttcta ctaagaagag cacagtaaat 120
gattacttag ctcgtgtaaa ttcaccacat cgcggtgctc cgcaagataa aggtgggcca 180
gtaattaaaa ttattgacgt agagacagca cctgagattg tgtatagctt ctcacgtttc 240
aaagccttta ttgcacctga acaagttatt cgtcgaggtt acttgctttc ctattcatct 300
gctgatttag aaacaggaga agtagaaggt aagaacctta gcaattaccc tctgtttgac 360
atagatcata cagatgattg ggaattatgt caagatctgt ggcacttgct tgacgatgca 420
gatattgtga tcgcgcacaa cggtattaag tttgataagg cttatattaa ccaacgtttt 480
gcttttcacg gtatggagcc accatcacac tatgttgtaa ttgatacact caaggtagct 540
aagaagcagt ttgcactacc ttctaatgcg cttaaagaaa tgtgtttgta ttttgataca 600
gaaaacttta aacttgataa tgaaggtttt cctttgtgga aagcatgttg tgaaggagac 660
agacacgcct tcttccgtat gcaaacatac aacgatggtg atgttgtttc tctcagagat 720
ctttacatca aacttcgtgc gtggattcca caacatccta acgtatctgc attctatgac 780
gatgaaaaac ttcgttgtcc tcgttgcggt aaagagcacc ttgaagttgt tcctggtaag 840
gtttacacaa cagcggttag tacattccaa gtgctaagat gcacatcatg taatagcctg 900
tctcgtggac gtgttaattt gcgtagcaaa gagaagcgtg taaacactgt tattggaatt 960
taa 963
<210> 10
<211> 320
<212> PRT
<213> 噬菌体 PD06的DNA 聚合酶(DNA polymerase of phage PD06)
<400> 10
Met Ser Lys Leu Thr Asp Glu Gln Lys Ala Leu Ile Val Ser Met Lys
1 5 10 15
Ala Gln Gly Tyr Ser Ser Arg Lys Ile Ala Gln Glu Val Leu Gly Ser
20 25 30
Ser Thr Lys Lys Ser Thr Val Asn Asp Tyr Leu Ala Arg Val Asn Ser
35 40 45
Pro His Arg Gly Ala Pro Gln Asp Lys Gly Gly Pro Val Ile Lys Ile
50 55 60
Ile Asp Val Glu Thr Ala Pro Glu Ile Val Tyr Ser Phe Ser Arg Phe
65 70 75 80
Lys Ala Phe Ile Ala Pro Glu Gln Val Ile Arg Arg Gly Tyr Leu Leu
85 90 95
Ser Tyr Ser Ser Ala Asp Leu Glu Thr Gly Glu Val Glu Gly Lys Asn
100 105 110
Leu Ser Asn Tyr Pro Leu Phe Asp Ile Asp His Thr Asp Asp Trp Glu
115 120 125
Leu Cys Gln Asp Leu Trp His Leu Leu Asp Asp Ala Asp Ile Val Ile
130 135 140
Ala His Asn Gly Ile Lys Phe Asp Lys Ala Tyr Ile Asn Gln Arg Phe
145 150 155 160
Ala Phe His Gly Met Glu Pro Pro Ser His Tyr Val Val Ile Asp Thr
165 170 175
Leu Lys Val Ala Lys Lys Gln Phe Ala Leu Pro Ser Asn Ala Leu Lys
180 185 190
Glu Met Cys Leu Tyr Phe Asp Thr Glu Asn Phe Lys Leu Asp Asn Glu
195 200 205
Gly Phe Pro Leu Trp Lys Ala Cys Cys Glu Gly Asp Arg His Ala Phe
210 215 220
Phe Arg Met Gln Thr Tyr Asn Asp Gly Asp Val Val Ser Leu Arg Asp
225 230 235 240
Leu Tyr Ile Lys Leu Arg Ala Trp Ile Pro Gln His Pro Asn Val Ser
245 250 255
Ala Phe Tyr Asp Asp Glu Lys Leu Arg Cys Pro Arg Cys Gly Lys Glu
260 265 270
His Leu Glu Val Val Pro Gly Lys Val Tyr Thr Thr Ala Val Ser Thr
275 280 285
Phe Gln Val Leu Arg Cys Thr Ser Cys Asn Ser Leu Ser Arg Gly Arg
290 295 300
Val Asn Leu Arg Ser Lys Glu Lys Arg Val Asn Thr Val Ile Gly Ile
305 310 315 320
<210> 11
<211> 2877
<212> DNA
<213> 噬菌体 PD114的推定尾纤蛋白(putative tail fiber protein of phagePD114)
<400> 11
atgtcaagaa ttaagcacag aattgacaat gtttttcaac tgatcgatgg acatattcag 60
ttcttagatc gtaatacagg tttaactgta gatcctacag tgcaacacta tgtattaaac 120
ccacaaaacg ttctagctaa taacagacac tttataaact ggactgctca agaagggcaa 180
ccaaacgggg atgctacaac agaaggtcaa tccgtgctta ttcttggttg cgcttatgca 240
tacctggctt ccggcgatac aaagtattta gactctgcta aaaaatattg gcaagcctat 300
atagattact tctttggtgg tcaacctatc ccagatccgc cagcaaaata tcgccctaac 360
tggattatta acggtaaaga accacgtctt gcacactatc ctcttacaga tgatggctac 420
cctacacacg gaggttttaa gggtagctta atgacgtgga ctaacggtag aacagttatt 480
cctcatggtg ctcctcattg gggagaatac cttgacaaag catggtttgc ttttaatggt 540
aatctaggat ggaactctgt aaacgctaca gtatatgctg ttaatgcaga tggtacaaca 600
gattggtcta agaatggaga tcaatgggat gttgattgga tcattgatcg tctaggacgt 660
aaagttgatt gggatggaaa tatcttagaa gaaggttttc caacatctca atacggaaca 720
gttcaactaa aaaatacttc tgtaaacggt acatataaat ttaactacgc gacttgtaac 780
cctgttgaac acggtggtta tttaatggag cgtaacacta tgtggcataa cagaccagtt 840
aacgttccgg tagaaatggg ctttcaggat aatgcgtcag atgctgaaac ttggtggtgt 900
gatgctaatt acctgatgta ccaaattaca ggagaaagaa aatattggct atgctggcag 960
tcttcactgt tagtgtgtca agattactca aacattgata tgtttgataa attcttccgt 1020
aagtctacat atgcaactat cccgtttaca gatggcatct cttacgatta ttcgtatcca 1080
tctacagcaa tacctaaata ttcaagagat actttaggtt atattgttat tagacaagat 1140
gttgctgctc aaacaacatt agaacagcaa gccatttggt ttaaagttga tcagaactct 1200
aagttacgag ttcagttcca aggtgtagat gacgtaggac atggtatcct tttccgtcca 1260
gaattggagc taagtaagac caaagatgaa aataacacag taacttaccg ttgtggttta 1320
ccactaggta cagctaatat gacaagcatg gatattccat tatccagctt tatgagaagt 1380
acccctccga atggtggaac atatattatc gcagacccgc gaattattgt tgactggggt 1440
gacaatactg tggtacaata taagtatgta acaggtattg ctggaactaa taatgaccag 1500
attgttactg catcaatgga tacggatggt ggtgttactg ttggtttttg gttaactcct 1560
acatcaaaag ctaatgttaa gtctatcaca tatagaacat atgaagatga ctttaacatg 1620
actatcacgg atgctcaagg ttggagatgg tgggctatgc tgcctaaaac gcaaggggca 1680
tggagtacga agactttatc agcctctgct tttaagttaa gtgcttacca gcctaatcat 1740
ccagaaacag atcctaaacc ttcagcaata aatcttactg cggtagatca ggtaaattta 1800
gttctggata cggctcctgt caatggttta acaggaacaa tagatttcta ttgtattaac 1860
gatgtacctg aaagatattc atcatcttct ggtgatgatt ataccatgta cttccgtatc 1920
actgtacagg cttcaagttc tcatactgct aagttaggtg attgtactat tattgactat 1980
aagttaaata gtcttaatta cacaccagga ataattccat tctctaatat cagtgaccca 2040
aacactgcat tatacgatgg atggcgtggt attccatacc caggatacca gtatccttcc 2100
ttatactgtt tcgcaggtag ggatattgat tggacacgtt taaacaacag tattgatttc 2160
ttgtatgatg ctcaaatgtg gttttataac acgtttgatc ctgtgatgcc tgggcctatg 2220
gcacaagcat atgtgtggga tcgttgggat gcaagaaaat acggtgaacc taatacgttt 2280
acaatgaaac actggaatga aaaagcgtgg gatggttatg aagctcgtgc attctactgt 2340
gcttgtcatg ctgtatacga acttgtacaa agaggagaaa cacctcctga aaaactagta 2400
actgtttgta aaaattggat caactacctt aaatggttcc aagacaataa tgatggaaga 2460
acaccaacta tctttaaccc agatggtaca gttcttgcac ctatagatga ctttactagt 2520
catatgtctg cactgtttat ggcgggttgt tcaatcatgg gtttggcagg ttatgattca 2580
caaattccaa acattggtat tgtagcagac agatgttttg atctgattca gcaaaactat 2640
attgtgcttt cccctaacca tccgatgaat ggttcctgga gtgttgcacc acgcccagat 2700
tcagataacg gaatgttctt tggtttccat gctggagaac tattacgtgg ttttggttta 2760
tatgccttgt atcgtaaact aaaccctcaa aacgctttac caattttgga taatagtaac 2820
ataccaacac taactgttct aactatgaat gatattagtg ttgatgtaac caaataa 2877
<210> 12
<211> 958
<212> PRT
<213> 噬菌体 PD114的推定尾纤蛋白(putative tail fiber protein of phagePD114)
<400> 12
Met Ser Arg Ile Lys His Arg Ile Asp Asn Val Phe Gln Leu Ile Asp
1 5 10 15
Gly His Ile Gln Phe Leu Asp Arg Asn Thr Gly Leu Thr Val Asp Pro
20 25 30
Thr Val Gln His Tyr Val Leu Asn Pro Gln Asn Val Leu Ala Asn Asn
35 40 45
Arg His Phe Ile Asn Trp Thr Ala Gln Glu Gly Gln Pro Asn Gly Asp
50 55 60
Ala Thr Thr Glu Gly Gln Ser Val Leu Ile Leu Gly Cys Ala Tyr Ala
65 70 75 80
Tyr Leu Ala Ser Gly Asp Thr Lys Tyr Leu Asp Ser Ala Lys Lys Tyr
85 90 95
Trp Gln Ala Tyr Ile Asp Tyr Phe Phe Gly Gly Gln Pro Ile Pro Asp
100 105 110
Pro Pro Ala Lys Tyr Arg Pro Asn Trp Ile Ile Asn Gly Lys Glu Pro
115 120 125
Arg Leu Ala His Tyr Pro Leu Thr Asp Asp Gly Tyr Pro Thr His Gly
130 135 140
Gly Phe Lys Gly Ser Leu Met Thr Trp Thr Asn Gly Arg Thr Val Ile
145 150 155 160
Pro His Gly Ala Pro His Trp Gly Glu Tyr Leu Asp Lys Ala Trp Phe
165 170 175
Ala Phe Asn Gly Asn Leu Gly Trp Asn Ser Val Asn Ala Thr Val Tyr
180 185 190
Ala Val Asn Ala Asp Gly Thr Thr Asp Trp Ser Lys Asn Gly Asp Gln
195 200 205
Trp Asp Val Asp Trp Ile Ile Asp Arg Leu Gly Arg Lys Val Asp Trp
210 215 220
Asp Gly Asn Ile Leu Glu Glu Gly Phe Pro Thr Ser Gln Tyr Gly Thr
225 230 235 240
Val Gln Leu Lys Asn Thr Ser Val Asn Gly Thr Tyr Lys Phe Asn Tyr
245 250 255
Ala Thr Cys Asn Pro Val Glu His Gly Gly Tyr Leu Met Glu Arg Asn
260 265 270
Thr Met Trp His Asn Arg Pro Val Asn Val Pro Val Glu Met Gly Phe
275 280 285
Gln Asp Asn Ala Ser Asp Ala Glu Thr Trp Trp Cys Asp Ala Asn Tyr
290 295 300
Leu Met Tyr Gln Ile Thr Gly Glu Arg Lys Tyr Trp Leu Cys Trp Gln
305 310 315 320
Ser Ser Leu Leu Val Cys Gln Asp Tyr Ser Asn Ile Asp Met Phe Asp
325 330 335
Lys Phe Phe Arg Lys Ser Thr Tyr Ala Thr Ile Pro Phe Thr Asp Gly
340 345 350
Ile Ser Tyr Asp Tyr Ser Tyr Pro Ser Thr Ala Ile Pro Lys Tyr Ser
355 360 365
Arg Asp Thr Leu Gly Tyr Ile Val Ile Arg Gln Asp Val Ala Ala Gln
370 375 380
Thr Thr Leu Glu Gln Gln Ala Ile Trp Phe Lys Val Asp Gln Asn Ser
385 390 395 400
Lys Leu Arg Val Gln Phe Gln Gly Val Asp Asp Val Gly His Gly Ile
405 410 415
Leu Phe Arg Pro Glu Leu Glu Leu Ser Lys Thr Lys Asp Glu Asn Asn
420 425 430
Thr Val Thr Tyr Arg Cys Gly Leu Pro Leu Gly Thr Ala Asn Met Thr
435 440 445
Ser Met Asp Ile Pro Leu Ser Ser Phe Met Arg Ser Thr Pro Pro Asn
450 455 460
Gly Gly Thr Tyr Ile Ile Ala Asp Pro Arg Ile Ile Val Asp Trp Gly
465 470 475 480
Asp Asn Thr Val Val Gln Tyr Lys Tyr Val Thr Gly Ile Ala Gly Thr
485 490 495
Asn Asn Asp Gln Ile Val Thr Ala Ser Met Asp Thr Asp Gly Gly Val
500 505 510
Thr Val Gly Phe Trp Leu Thr Pro Thr Ser Lys Ala Asn Val Lys Ser
515 520 525
Ile Thr Tyr Arg Thr Tyr Glu Asp Asp Phe Asn Met Thr Ile Thr Asp
530 535 540
Ala Gln Gly Trp Arg Trp Trp Ala Met Leu Pro Lys Thr Gln Gly Ala
545 550 555 560
Trp Ser Thr Lys Thr Leu Ser Ala Ser Ala Phe Lys Leu Ser Ala Tyr
565 570 575
Gln Pro Asn His Pro Glu Thr Asp Pro Lys Pro Ser Ala Ile Asn Leu
580 585 590
Thr Ala Val Asp Gln Val Asn Leu Val Leu Asp Thr Ala Pro Val Asn
595 600 605
Gly Leu Thr Gly Thr Ile Asp Phe Tyr Cys Ile Asn Asp Val Pro Glu
610 615 620
Arg Tyr Ser Ser Ser Ser Gly Asp Asp Tyr Thr Met Tyr Phe Arg Ile
625 630 635 640
Thr Val Gln Ala Ser Ser Ser His Thr Ala Lys Leu Gly Asp Cys Thr
645 650 655
Ile Ile Asp Tyr Lys Leu Asn Ser Leu Asn Tyr Thr Pro Gly Ile Ile
660 665 670
Pro Phe Ser Asn Ile Ser Asp Pro Asn Thr Ala Leu Tyr Asp Gly Trp
675 680 685
Arg Gly Ile Pro Tyr Pro Gly Tyr Gln Tyr Pro Ser Leu Tyr Cys Phe
690 695 700
Ala Gly Arg Asp Ile Asp Trp Thr Arg Leu Asn Asn Ser Ile Asp Phe
705 710 715 720
Leu Tyr Asp Ala Gln Met Trp Phe Tyr Asn Thr Phe Asp Pro Val Met
725 730 735
Pro Gly Pro Met Ala Gln Ala Tyr Val Trp Asp Arg Trp Asp Ala Arg
740 745 750
Lys Tyr Gly Glu Pro Asn Thr Phe Thr Met Lys His Trp Asn Glu Lys
755 760 765
Ala Trp Asp Gly Tyr Glu Ala Arg Ala Phe Tyr Cys Ala Cys His Ala
770 775 780
Val Tyr Glu Leu Val Gln Arg Gly Glu Thr Pro Pro Glu Lys Leu Val
785 790 795 800
Thr Val Cys Lys Asn Trp Ile Asn Tyr Leu Lys Trp Phe Gln Asp Asn
805 810 815
Asn Asp Gly Arg Thr Pro Thr Ile Phe Asn Pro Asp Gly Thr Val Leu
820 825 830
Ala Pro Ile Asp Asp Phe Thr Ser His Met Ser Ala Leu Phe Met Ala
835 840 845
Gly Cys Ser Ile Met Gly Leu Ala Gly Tyr Asp Ser Gln Ile Pro Asn
850 855 860
Ile Gly Ile Val Ala Asp Arg Cys Phe Asp Leu Ile Gln Gln Asn Tyr
865 870 875 880
Ile Val Leu Ser Pro Asn His Pro Met Asn Gly Ser Trp Ser Val Ala
885 890 895
Pro Arg Pro Asp Ser Asp Asn Gly Met Phe Phe Gly Phe His Ala Gly
900 905 910
Glu Leu Leu Arg Gly Phe Gly Leu Tyr Ala Leu Tyr Arg Lys Leu Asn
915 920 925
Pro Gln Asn Ala Leu Pro Ile Leu Asp Asn Ser Asn Ile Pro Thr Leu
930 935 940
Thr Val Leu Thr Met Asn Asp Ile Ser Val Asp Val Thr Lys
945 950 955
<210> 13
<211> 2160
<212> DNA
<213> 噬菌体 PD114的另一推定尾纤蛋白(putative tail fiber protein of phagePD114)
<400> 13
atgaaacagg ctatttttac tgtaactcga aatacgagag taaatgccag ttcattcagc 60
tataagaccg tcgatggaac ggcggtcgct ggtagagatt atgtagctaa acagggaaca 120
gtaacgttcc cttctgatgc agattctgct actgtagtgg ttgatgtata tgaacgtccg 180
gcaaactctt tagatcttta ttttactctt gagattacta gcataagtca aaataacatg 240
atgatcaatg aagaaattcg ttgtgttatt acgactaatc aatcgaatac aaccccgcta 300
acttggcctg ttgtgaagtc tagaatgttt gatcctaagt tttggacaat agattctcaa 360
gcaacagaaa gttgttctat tatctctaat ggtgattctt tcacagcaag aatggttaat 420
aggacagtgg ctggtcttgc tgggattatt tggtcaactt acgataaata tgatcacaaa 480
ggattaggct accaagaaca ttctgactta agtaaagaga aactttggtt taaaatggaa 540
cttagtcaag gtgttcctgg attaaaagaa gaaaaattat tgccatcctt aacagtgaca 600
ctgaaggatg aaacagttca ttatgtatct ctaaactttt atgcagaaga agtttcagag 660
gatggtaaaa cagcaacaat caaattagat ttctcaaatt taaaatctgg gccagaaaat 720
aatattgttg ttgacactac acaggtagat aatatgttct tctccttgat ctccacaagg 780
tatcaagaaa ttgaagatat tattcctgta gagaatgagg atctttcctt taagtttacc 840
atactggaac ctgatacggg ttatagtatg atgaatatgg gtaatttaaa tgttccagca 900
cacaccataa gaatgtgtac atcttatgat gatatgtata accttacccc agagcgagtt 960
atttctaaca cctacagatt aggttatagg gatatgatta accactataa tggtatgtca 1020
cactattatc aatttagttg ggacgctggc tctaataaat gggctttgaa cagaacagaa 1080
tatttaaacc cagcaacaga agcctggttt gataacttcc acgctaatgc aaaagctatg 1140
ggctacacag taatgaactc cttaagtttt gaacttatga gtacagtgtg tcctgtagaa 1200
tgggtacaac atacttggaa tgatgatctg gctgctacag gttatgagcc accaagttat 1260
gttttatctc cttcaattga cgaagggatg aattatttac aaggtgtaat aaataagata 1320
gcttctatat ccgctgctaa tgatcttccg gttattattc aggttggtga accgtggtgg 1380
tggtataaca cagctaccaa tgtaccgtgt atttatgatt atacaaccaa agttgcattt 1440
aataatgaaa ctggattata tgcacaagat gttggtacag ttaaaaatta taaaactggt 1500
acaccatatg atgaatatta tgcattccta tctaaaacat taggactgcg tgttgctaaa 1560
tttgcaacag ataccagatt agcataccct ggagcgaaag ttacattgtt accatttgta 1620
ccatccatct taggtaatgg tatgatggag tttgttaact tccctaaaga ttattatatc 1680
ccagaaaact ttgactttta ttgttcagaa tgttatgact ggttgttgga aggtaaaatg 1740
gaaagatctt tcgacgctat cacaatacct aatgagcaac taggttttag ttacgataag 1800
atacattatc ttgcagggtt tgttccagat gcaactctag caccattata tggctttgat 1860
cctgaatcaa actatagaac atatttgtgg aaaatgatta taggcaatat tgtattgaat 1920
gaccagaagt ttgttggttt atatcaatac atatgggctt acccacaaat aatgcatgat 1980
tcaataactg ttcttaattc aaaatcccaa gtgttttata tgggtaatgt gcctttgaat 2040
tgctacttgc aagatatcac tcttaaacag gtaaatgttg agattgccac tataagtgga 2100
attacattag ccactaaatc tggagaagat ttagtaacaa cacaaaaatt ttatatatag 2160
<210> 14
<211> 719
<212> PRT
<213> 噬菌体 PD114的另一推定尾纤蛋白(putative tail fiber protein of phagePD114)
<400> 14
Met Lys Gln Ala Ile Phe Thr Val Thr Arg Asn Thr Arg Val Asn Ala
1 5 10 15
Ser Ser Phe Ser Tyr Lys Thr Val Asp Gly Thr Ala Val Ala Gly Arg
20 25 30
Asp Tyr Val Ala Lys Gln Gly Thr Val Thr Phe Pro Ser Asp Ala Asp
35 40 45
Ser Ala Thr Val Val Val Asp Val Tyr Glu Arg Pro Ala Asn Ser Leu
50 55 60
Asp Leu Tyr Phe Thr Leu Glu Ile Thr Ser Ile Ser Gln Asn Asn Met
65 70 75 80
Met Ile Asn Glu Glu Ile Arg Cys Val Ile Thr Thr Asn Gln Ser Asn
85 90 95
Thr Thr Pro Leu Thr Trp Pro Val Val Lys Ser Arg Met Phe Asp Pro
100 105 110
Lys Phe Trp Thr Ile Asp Ser Gln Ala Thr Glu Ser Cys Ser Ile Ile
115 120 125
Ser Asn Gly Asp Ser Phe Thr Ala Arg Met Val Asn Arg Thr Val Ala
130 135 140
Gly Leu Ala Gly Ile Ile Trp Ser Thr Tyr Asp Lys Tyr Asp His Lys
145 150 155 160
Gly Leu Gly Tyr Gln Glu His Ser Asp Leu Ser Lys Glu Lys Leu Trp
165 170 175
Phe Lys Met Glu Leu Ser Gln Gly Val Pro Gly Leu Lys Glu Glu Lys
180 185 190
Leu Leu Pro Ser Leu Thr Val Thr Leu Lys Asp Glu Thr Val His Tyr
195 200 205
Val Ser Leu Asn Phe Tyr Ala Glu Glu Val Ser Glu Asp Gly Lys Thr
210 215 220
Ala Thr Ile Lys Leu Asp Phe Ser Asn Leu Lys Ser Gly Pro Glu Asn
225 230 235 240
Asn Ile Val Val Asp Thr Thr Gln Val Asp Asn Met Phe Phe Ser Leu
245 250 255
Ile Ser Thr Arg Tyr Gln Glu Ile Glu Asp Ile Ile Pro Val Glu Asn
260 265 270
Glu Asp Leu Ser Phe Lys Phe Thr Ile Leu Glu Pro Asp Thr Gly Tyr
275 280 285
Ser Met Met Asn Met Gly Asn Leu Asn Val Pro Ala His Thr Ile Arg
290 295 300
Met Cys Thr Ser Tyr Asp Asp Met Tyr Asn Leu Thr Pro Glu Arg Val
305 310 315 320
Ile Ser Asn Thr Tyr Arg Leu Gly Tyr Arg Asp Met Ile Asn His Tyr
325 330 335
Asn Gly Met Ser His Tyr Tyr Gln Phe Ser Trp Asp Ala Gly Ser Asn
340 345 350
Lys Trp Ala Leu Asn Arg Thr Glu Tyr Leu Asn Pro Ala Thr Glu Ala
355 360 365
Trp Phe Asp Asn Phe His Ala Asn Ala Lys Ala Met Gly Tyr Thr Val
370 375 380
Met Asn Ser Leu Ser Phe Glu Leu Met Ser Thr Val Cys Pro Val Glu
385 390 395 400
Trp Val Gln His Thr Trp Asn Asp Asp Leu Ala Ala Thr Gly Tyr Glu
405 410 415
Pro Pro Ser Tyr Val Leu Ser Pro Ser Ile Asp Glu Gly Met Asn Tyr
420 425 430
Leu Gln Gly Val Ile Asn Lys Ile Ala Ser Ile Ser Ala Ala Asn Asp
435 440 445
Leu Pro Val Ile Ile Gln Val Gly Glu Pro Trp Trp Trp Tyr Asn Thr
450 455 460
Ala Thr Asn Val Pro Cys Ile Tyr Asp Tyr Thr Thr Lys Val Ala Phe
465 470 475 480
Asn Asn Glu Thr Gly Leu Tyr Ala Gln Asp Val Gly Thr Val Lys Asn
485 490 495
Tyr Lys Thr Gly Thr Pro Tyr Asp Glu Tyr Tyr Ala Phe Leu Ser Lys
500 505 510
Thr Leu Gly Leu Arg Val Ala Lys Phe Ala Thr Asp Thr Arg Leu Ala
515 520 525
Tyr Pro Gly Ala Lys Val Thr Leu Leu Pro Phe Val Pro Ser Ile Leu
530 535 540
Gly Asn Gly Met Met Glu Phe Val Asn Phe Pro Lys Asp Tyr Tyr Ile
545 550 555 560
Pro Glu Asn Phe Asp Phe Tyr Cys Ser Glu Cys Tyr Asp Trp Leu Leu
565 570 575
Glu Gly Lys Met Glu Arg Ser Phe Asp Ala Ile Thr Ile Pro Asn Glu
580 585 590
Gln Leu Gly Phe Ser Tyr Asp Lys Ile His Tyr Leu Ala Gly Phe Val
595 600 605
Pro Asp Ala Thr Leu Ala Pro Leu Tyr Gly Phe Asp Pro Glu Ser Asn
610 615 620
Tyr Arg Thr Tyr Leu Trp Lys Met Ile Ile Gly Asn Ile Val Leu Asn
625 630 635 640
Asp Gln Lys Phe Val Gly Leu Tyr Gln Tyr Ile Trp Ala Tyr Pro Gln
645 650 655
Ile Met His Asp Ser Ile Thr Val Leu Asn Ser Lys Ser Gln Val Phe
660 665 670
Tyr Met Gly Asn Val Pro Leu Asn Cys Tyr Leu Gln Asp Ile Thr Leu
675 680 685
Lys Gln Val Asn Val Glu Ile Ala Thr Ile Ser Gly Ile Thr Leu Ala
690 695 700
Thr Lys Ser Gly Glu Asp Leu Val Thr Thr Gln Lys Phe Tyr Ile
705 710 715
<210> 15
<211> 552
<212> DNA
<213> 噬菌体 PD114的另一推定尾纤蛋白(putative tail fiber protein of phagePD114)
<400> 15
atgaaaaaag atgttatttt tacaggtttt gaattgtacg aaccggaatt aacagaggag 60
aacattcttt atctttctgt tggcggtggt tttttgcaag acaaggatgg taatgattgg 120
tatcaacttc aaaaaacttt gatggctgaa tatcccgacg cttatttcat taccttagat 180
gataataata ttgtacgtgg tgctactaaa gatgttacta cctatttccc tgccggacaa 240
tctattttag ttacatataa tgcaccagat aaaatagaag atgaaagtaa tattggctct 300
tggcaatatg ttgaaggtaa atttgttcct cacaatagtc ttgctattga agatgctact 360
cttaagttag aaaaggagtt aacttgggct acgaatcaag tgtctgcgat tcaagattta 420
gaggaattac aaggtttatc tcaagaacaa aaagattggt taggaaaagt taaactatat 480
cgtgcaacac tgattcaaat taacgttgaa gatgcaccta atataacttg gcctacaaga 540
cctaagaaat ag 552
<210> 16
<211> 183
<212> PRT
<213> 噬菌体 PD114的另一推定尾纤蛋白(putative tail fiber protein of phagePD114)
<400> 16
Met Lys Lys Asp Val Ile Phe Thr Gly Phe Glu Leu Tyr Glu Pro Glu
1 5 10 15
Leu Thr Glu Glu Asn Ile Leu Tyr Leu Ser Val Gly Gly Gly Phe Leu
20 25 30
Gln Asp Lys Asp Gly Asn Asp Trp Tyr Gln Leu Gln Lys Thr Leu Met
35 40 45
Ala Glu Tyr Pro Asp Ala Tyr Phe Ile Thr Leu Asp Asp Asn Asn Ile
50 55 60
Val Arg Gly Ala Thr Lys Asp Val Thr Thr Tyr Phe Pro Ala Gly Gln
65 70 75 80
Ser Ile Leu Val Thr Tyr Asn Ala Pro Asp Lys Ile Glu Asp Glu Ser
85 90 95
Asn Ile Gly Ser Trp Gln Tyr Val Glu Gly Lys Phe Val Pro His Asn
100 105 110
Ser Leu Ala Ile Glu Asp Ala Thr Leu Lys Leu Glu Lys Glu Leu Thr
115 120 125
Trp Ala Thr Asn Gln Val Ser Ala Ile Gln Asp Leu Glu Glu Leu Gln
130 135 140
Gly Leu Ser Gln Glu Gln Lys Asp Trp Leu Gly Lys Val Lys Leu Tyr
145 150 155 160
Arg Ala Thr Leu Ile Gln Ile Asn Val Glu Asp Ala Pro Asn Ile Thr
165 170 175
Trp Pro Thr Arg Pro Lys Lys
180
<210> 17
<211> 963
<212> DNA
<213> 噬菌体 PD114的 DNA 聚合酶( DNA polymerase of phage PD114)
<400> 17
ttgagtaaat taacagacga acagaaagcc ttgattgtat ctatgaaagc acaaggttat 60
tcttcgcgta agattgcaca agaggtgttg ggaagttcta ctaagaagag cacagtaaat 120
gattacttag ctcgtgtaaa ttcaccacat cgcggtgctc cccaagataa aggtgggcca 180
gtaattaaaa ttattgacgt agagacagca ccggagattg tgtatagctt ctcacgtttc 240
aaagccttta ttgcacctga acaagttatt cgtcgaggtt acttgctttc ctattcatct 300
gctgatttag aaacaggaga agtagaaggt aagaacctta gcaattatcc tctgtttgac 360
atagatcata cagatgattg ggaattatgt caagatctgt ggcacttgct tgacgatgca 420
gatattgtga tcgcacacaa cggtattaag tttgataagg cttatattaa ccaacgtttt 480
gcttttcacg gtatggagcc accatcacac tatgttgtga ttgatacact caaggcagct 540
aagaagcagt ttgcactacc ttctaatgcg cttaaagaaa tgtgtttgta ttttgataca 600
gaaaacttta aacttgataa tgaaggtttt cctttgtgga aagcatgttg tgaaggagac 660
agacacgcct tcttccgtat gcaaacatac aacgatggtg atgttgtttc tcttagagat 720
ctttacatca aacttcgtgc gtggattcca caacatccta acgtatctgc attctatgac 780
gatgaaaaac ttcgttgtcc tcgttgcggt aaagagcacc ttgaagttgt tcctggtaag 840
gtttacacaa cagcggttag tacattccaa gtgctaagat gcacatcatg taatagcctg 900
tctcgtggac gtgttaattt gcgtagcaaa gagaagcgtg taaacactgt tattggaatt 960
taa 963
<210> 18
<211> 320
<212> PRT
<213> 噬菌体 PD114的 DNA 聚合酶( DNA polymerase of phage PD114)
<400> 18
Met Ser Lys Leu Thr Asp Glu Gln Lys Ala Leu Ile Val Ser Met Lys
1 5 10 15
Ala Gln Gly Tyr Ser Ser Arg Lys Ile Ala Gln Glu Val Leu Gly Ser
20 25 30
Ser Thr Lys Lys Ser Thr Val Asn Asp Tyr Leu Ala Arg Val Asn Ser
35 40 45
Pro His Arg Gly Ala Pro Gln Asp Lys Gly Gly Pro Val Ile Lys Ile
50 55 60
Ile Asp Val Glu Thr Ala Pro Glu Ile Val Tyr Ser Phe Ser Arg Phe
65 70 75 80
Lys Ala Phe Ile Ala Pro Glu Gln Val Ile Arg Arg Gly Tyr Leu Leu
85 90 95
Ser Tyr Ser Ser Ala Asp Leu Glu Thr Gly Glu Val Glu Gly Lys Asn
100 105 110
Leu Ser Asn Tyr Pro Leu Phe Asp Ile Asp His Thr Asp Asp Trp Glu
115 120 125
Leu Cys Gln Asp Leu Trp His Leu Leu Asp Asp Ala Asp Ile Val Ile
130 135 140
Ala His Asn Gly Ile Lys Phe Asp Lys Ala Tyr Ile Asn Gln Arg Phe
145 150 155 160
Ala Phe His Gly Met Glu Pro Pro Ser His Tyr Val Val Ile Asp Thr
165 170 175
Leu Lys Ala Ala Lys Lys Gln Phe Ala Leu Pro Ser Asn Ala Leu Lys
180 185 190
Glu Met Cys Leu Tyr Phe Asp Thr Glu Asn Phe Lys Leu Asp Asn Glu
195 200 205
Gly Phe Pro Leu Trp Lys Ala Cys Cys Glu Gly Asp Arg His Ala Phe
210 215 220
Phe Arg Met Gln Thr Tyr Asn Asp Gly Asp Val Val Ser Leu Arg Asp
225 230 235 240
Leu Tyr Ile Lys Leu Arg Ala Trp Ile Pro Gln His Pro Asn Val Ser
245 250 255
Ala Phe Tyr Asp Asp Glu Lys Leu Arg Cys Pro Arg Cys Gly Lys Glu
260 265 270
His Leu Glu Val Val Pro Gly Lys Val Tyr Thr Thr Ala Val Ser Thr
275 280 285
Phe Gln Val Leu Arg Cys Thr Ser Cys Asn Ser Leu Ser Arg Gly Arg
290 295 300
Val Asn Leu Arg Ser Lys Glu Lys Arg Val Asn Thr Val Ile Gly Ile
305 310 315 320
<210> 19
<211> 501
<212> DNA
<213> 噬菌体 PD114的 裂解酶(lysozyme of phage PD114)
<400> 19
atggctaaag tagttgatgt gtttgatatg ttacgttttg atgaaggact aaagctaact 60
gtatatactg ataccgaagg gtattggacg gttggtattg gacaccttct gacaaaactt 120
aaagataaat cagaagctat acgcattctt gataacttag taggtagaaa aactaatggg 180
gttattactg aagcggaagc tagaaaaatc tttgagggtg acgtaaagaa agcgatacaa 240
caaatccatt caagtaccat attatctcct atttacgata aagtaagtcc taatcgtaaa 300
atggctatta tcaatatggt atttcaaatg ggattgaaag gtgcagaatc tttcaaaaat 360
agcttgactt tagtgagtaa ttcatattat actcaagcct ctataaattt acgtaaaagt 420
aaatggtatc gccaaacgcc taatcgcgca gagcgtgtaa ttcaggtgct taaaactgga 480
acattagacg cttataacta a 501
<210> 20
<211> 166
<212> PRT
<213> 噬菌体 PD114的 裂解酶(lysozyme of phage PD114)
<400> 20
Met Ala Lys Val Val Asp Val Phe Asp Met Leu Arg Phe Asp Glu Gly
1 5 10 15
Leu Lys Leu Thr Val Tyr Thr Asp Thr Glu Gly Tyr Trp Thr Val Gly
20 25 30
Ile Gly His Leu Leu Thr Lys Leu Lys Asp Lys Ser Glu Ala Ile Arg
35 40 45
Ile Leu Asp Asn Leu Val Gly Arg Lys Thr Asn Gly Val Ile Thr Glu
50 55 60
Ala Glu Ala Arg Lys Ile Phe Glu Gly Asp Val Lys Lys Ala Ile Gln
65 70 75 80
Gln Ile His Ser Ser Thr Ile Leu Ser Pro Ile Tyr Asp Lys Val Ser
85 90 95
Pro Asn Arg Lys Met Ala Ile Ile Asn Met Val Phe Gln Met Gly Leu
100 105 110
Lys Gly Ala Glu Ser Phe Lys Asn Ser Leu Thr Leu Val Ser Asn Ser
115 120 125
Tyr Tyr Thr Gln Ala Ser Ile Asn Leu Arg Lys Ser Lys Trp Tyr Arg
130 135 140
Gln Thr Pro Asn Arg Ala Glu Arg Val Ile Gln Val Leu Lys Thr Gly
145 150 155 160
Thr Leu Asp Ala Tyr Asn
165
Claims (10)
1.一种大肠杆菌噬菌体组合物,其特征在于,包括大肠杆菌噬菌体PD06和大肠杆菌噬菌体PD114;其中,大肠杆菌噬菌体PD06的保藏号为CGMCC No.16390,于2018年9月26日保藏在中国微生物菌种保藏管理委员会普通微生物中心;大肠杆菌噬菌体PD114,保藏号为CGMCC No.18866,于2019年11月04日保藏在中国微生物菌种保藏管理委员会普通微生物中心。
2.根据权利要求1所述的噬菌体组合物,其特征在于,大肠杆菌噬菌体PD06与大肠杆菌噬菌体PD114的活体数量之比为1:1;噬菌体的总含量不低于109pfu/mL。
3.根据权利要求2所述的噬菌体组合物,其特征在于,所述噬菌体组合物还包括大肠杆菌噬菌体PD06和大肠杆菌噬菌体PD114的突变体中的一种或多种;所述突变体与对应的噬菌体的同源性不小于90%。
4.根据权利要求1所述的噬菌体组合物,其特征在于,所述杀菌组合物还包括辅料;所述辅料为SM缓冲液、海藻酸钠、蔗糖、麦芽糖糊精、葡萄糖中的一种或多种;
优选的,所述杀菌组合物还包括不同种类细菌的特定病原菌的噬菌体。
5.根据权利要求1-4中任一项所述的噬菌体组合物,其特征在于,所述噬菌体组合物的剂型为粉剂、水剂、冻干剂、凝胶剂、霜剂、膏剂。
6.如权利要求1-5中任一项所述的噬菌体组合物在防治鸡大肠杆菌病的药物中的应用。
7.一种用于畜禽产品消毒的生物抑菌剂,其特征在于,包括如权利要求1-5中任一项所述的噬菌体组合物,生物抑菌剂的使用方法为:对畜禽产品表面进行浸泡或者喷雾消毒,来抑制产品加工或保鲜过程中大肠杆菌的增殖。
8.一种可用于水线或养殖场环境消毒的消毒剂,其特征在于,有效成分包括如权利要求1-5中任一项所述的噬菌体组合物;优选地,每种噬菌体的浓度为5×108PFU/ml以上。
9.一种饲料添加剂,其特征在于,包括如权利要求1-5中任一项所述的噬菌体组合物;优选地,饲料中每种噬菌体的浓度至少为1×108PFU/g。
10.一种检测试剂盒,其特征在于,包括如权利要求1-5中任一项所述的噬菌体组合物。
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