新型茄科雷尔氏菌噬菌体及其组合物和应用的制作方法

本发明属于微生物
技术领域：
，具体涉及新的茄科雷尔氏菌噬菌体、其组合物以及它们的制备方法和应用。
背景技术：
：茄科雷尔氏菌(ralstoniasolanacearum)是β-变形菌亚门的一类滋生于土壤的革兰氏阴性细菌。该病菌通常可从植物根部或茎部的伤口或是从没有受伤的次生根的根冠部位侵入皮层细胞间隙，通过破坏细胞间中胶层,使细胞壁分离,变形,形成空腔，进而侵染木质部薄壁组织使导管附近的小细胞受刺激形成侵填体，并移入侵填体。待侵填体破裂后，茄科雷尔氏菌继续被释放进入导管,并在导管内大量繁殖和快速传播扩张,从而引起植株萎蔫死亡(何礼远,康耀卫.植物青枯菌(pseudomonassolanacearum)致病机理。这种毁灭性的土传病害被称为作物青枯病，全世界每年因此病害所造成的经济损失数以亿计。茄科雷尔氏菌宿主广泛,能侵染包括生姜、烟草、番茄、马铃薯、花生、桑树以及木麻黄在内的50多科200多种作物。该病主要发生在亚热带和热带地区，并逐渐向温带地区扩散，其发病情况逐年加重。目前，对作物青枯病的防治方法主要包括化学防治、生物防治、种植抗病品种、作物轮作等，虽然对青枯病起到了一定程度的控制作用，但依然无法有效地控制该病发。噬菌体是一种通过与细菌细胞表面特异位点结合后杀灭特异性细菌的病毒，具有杀菌特异性强，对人畜无害，利用致病菌为宿主不断自我增殖等显著的优点，极具作为新型生物农药有效成分的开发潜质。迄今为止，国内外已报道的茄科雷尔氏菌噬菌体包括肌尾噬菌体фrsa1、фrsl1和∈rs-1；短尾科噬菌体фrsb1和rsk1；丝尾噬菌体фrsm1和фrss1，目前所发现的上述短尾科噬菌体均属于t7-likevirus属,还未见短尾科其它属噬菌体的报道。从应用的角度考虑，用于预防和治疗作物青枯病的茄科雷尔氏菌噬菌体应具备较宽的裂解谱(能够裂解来源于不同宿主作物和不同农业区的病原菌)，较强的稳定性(能适应田间温度以及不同的土壤ph值)以及可靠的安全性(应为烈性噬菌体，其基因组不能与宿主菌的基因组整合)等几个基本的特点。因此、有必要开发新的具有上述裂解谱宽、稳定性好、安全性高等特性的新型噬菌体。技术实现要素：针对以上技术现状，本发明提供了3株茄科雷尔氏菌的噬菌体以及它们的组合物，本发明噬菌体具有较强的裂解不同作物来源的茄科雷尔氏菌的能力。本发明所述茄科雷尔氏菌噬菌体包括以下噬菌体：茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)、保藏编号为cctccm2016533，保藏单位为中国典型培养物保藏中心，保藏日期为2016年11月10日；茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)、保藏编号为cctccm2016534，保藏单位为中国典型培养物保藏中心，保藏日期为2016年11月10日；或茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)、保藏编号为cctccno：m2016635，保藏单位为中国典型培养物保藏中心，保藏日期为2016年11月10日；本发明中，作为实施方案之一，所述茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)为烈性噬菌体，其形态为蝌蚪状，具有呈正多面体的头部结构和较短的尾部，头部直径约为60～70nm，尾部长约为15～20nm，且在ph3～ph11、在4℃～40℃条件下具有耐性，moi＝0.0000001条件下培养24h，其效价为4.3×1010pfu/ml；本发明中，作为实施方案之一，所述茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)为烈性噬菌体，其形态为蝌蚪状，其具有呈正多面体的头部结构和较短的尾部，头部直径约为60～70nm，尾部长约为15～20nm，且在ph3～ph11、在4℃～40℃条件下具有耐性，moi＝0.0000001条件下培养24h，其效价为2.9×1010pfu/ml；本发明中，作为实施方案之一，所述茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)为烈性噬菌体，其形态为蝌蚪状，其具有呈正多面体的头部结构和较短的尾部，头部直径约为60～70nm，尾部长约为15～20nm，且在ph3～ph11、在4℃～40℃条件下具有耐性，moi＝0.0000001条件下培养24h，其效价为6.7×1010pfu/ml；本发明中，作为实施方案之一，所述茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)具有seqidno.1所示的核苷酸序列；本发明中，作为实施方案之一，所述茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)具有seqidno.2所示的核苷酸序列；本发明中，作为实施方案之一，所述茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)具有seqidno.3所示的核苷酸序列；本发明中，作为实施方案之一，所述茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)的主要结构蛋白相对分子量为17kda和45kda；本发明中，作为实施方案之一，所述茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)的主要结构蛋白相对分子量为17kda和45kda；本发明中，作为实施方案之一，所述茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)的主要结构蛋白相对分子量为17kda和45kda；噬菌体gp1的生物学特征：以来源于姜瘟病的茄科雷尔氏菌“3-2-1”为宿主菌，采用双层平板法于30℃下过夜培养，gp1可形成平均直径为1～2mm的噬菌斑(图1-a)；在电子显微镜下观察gp1噬菌体形态发现其形态为蝌蚪状，具有呈正多面体的头部结构和较短的尾部，头部直径约为60～70nm，尾部长约为15～20nm(图2-a)。gp1噬菌体全基因组序列大小为61125bp，为双链线状dna。本发明所述gp1属于有尾噬菌体目(caudovirales)，短尾噬菌体科(podoviridae)，bcep22likevirus噬菌体属，为是新种噬菌体；基因组序列分析表明，gp1噬菌体全基因组无毒力基因或不良基因。噬菌体的溶原性鉴定试验结果表明gp1为烈性噬菌体。噬菌体gp2的生物学特征：以来源于姜瘟病的茄科雷尔氏菌“5-2-2”为宿主菌，采用双层平板法于30℃下过夜培养，gp2可形成平均直径为1～2mm的噬菌斑(图1-b)；在电子显微镜下观察gp2噬菌体形态发现其形态为蝌蚪状，具有呈正多面体的头部结构和较短的尾部，头部直径约为60～70nm，尾部长约为15～20nm(图2-b)。gp2噬菌体全基因组序列大小为61130bp，为双链线状dna。本发明所述gp2属于有尾噬菌体目(caudovirales)，短尾噬菌体科(podoviridae)，bcep22likevirus噬菌体属，是新种噬菌体；基因组序列分析表明，gp2噬菌体全基因组无毒力基因或不良基因。噬菌体的溶原性鉴定试验结果表明gp2为烈性噬菌体。噬菌体gp3的生物学特征：以来源于姜瘟病的茄科雷尔氏菌“zk15-2”为宿主菌，采用双层平板法于30℃下过夜培养，gp3可形成平均直径为1～2mm的噬菌斑(图1-c)；在电子显微镜下观察gp3噬菌体形态发现其形态为蝌蚪状，具有呈正多面体的头部结构和较短的尾部，头部直径约为60～70nm，尾部长约为15～20nm(图2-c)。gp3噬菌体全基因组序列大小为57402bp，为双链线状dna。本发明所述gp3属于有尾噬菌体目(caudovirales)，短尾噬菌体科(podoviridae)，bcep22likevirus噬菌体属，是新种噬菌体；基因组序列分析表明，gp3噬菌体全基因组无毒力基因或不良基因。噬菌体的溶原性鉴定试验结果表明gp3为烈性噬菌体。本发明还进一步提供了茄科雷尔氏菌噬菌体的组合物，作为实施方案之一，所述组合物包括茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)，茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)和茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)中的两种或两种以上；作为实施方案之一，所述组合物包括科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)和茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)的组合、或科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)和茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)的组合，或科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)和茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)的组合；所述组合物中的各噬菌体量在约101pfu/ml～108pfu/ml的效价范围内均能有效抑制茄科雷尔氏菌的生长，其中在108pfu/ml效价条件下抑菌率可达约95％；作为实施方案之一，所述组合物中，茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)和茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)的效价比为1：1；科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)和茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)的效价比为1：1，或科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)和茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)的效价比为1：1；作为本发明实施方案之一，所述组合物含有茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)，茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)和茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)。作为实施方案之一，所述组合物中，以单位体积中所含噬菌体效价(pfu/ml)计，茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)，茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)和茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)的比例为1：1：1。作为实施方案之一，本发明还提供一种含有上述噬菌体或噬菌体组合物的试剂或试剂盒。本领域技术人员可以根据本发明公开内容和本领域常识来制备本发明含有上述噬菌体或噬菌体组合物的试剂或试剂盒。本发明提供一种上述的噬菌体、噬菌体组合物或含有噬菌体或噬菌体组合物的试剂或试剂盒在杀灭或鉴定茄科雷尔氏菌中的应用。作为实施方案之一，本发明还提供一种所述的噬菌体、噬菌体组合物或含有噬菌体或噬菌体组合物的试剂或试剂盒制备在制备抗由茄科雷尔氏菌导致的姜瘟病、番茄青枯病、马铃薯青枯病、烟草青枯病、木麻黄青枯病、棉花青枯病、桑树青枯病或花生青枯病的药剂的应用。本发明还提供一种茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)，茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)和茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)的分离纯化方法。作为实施方案之一，所述方法包括：所述噬菌体gp1分离自山东省安丘市姜田土样，噬菌体gp2分离自安徽省阜阳市姜田土样分离出的，本发明中所述噬菌体gp3分离自四川省崇州市姜田土样。样品前处理：将采集的土样30g浸泡于100ml灭菌的生理盐水中，4℃冰箱中过夜静置后室温条件下4000rpm离心取上清液备用。噬菌体的分离：取20ml上清液，用0.22μm滤膜过滤，加入至20ml2×tsb液体培养基中，同时加入宿主菌，置于30℃摇床中200rpm过夜摇培。吸取2ml培养物，用0.22μm滤膜过滤上清液，备用。取对数期宿主菌加入到半固体tsb培养基中混匀，倾倒于tsa平板上，制备双层平板。将过滤后的上清液，点滴于含有宿主菌的已凝固的双层平板上，无菌条件下风干后，放置于30℃培养24h后观察噬菌斑。噬菌体的纯化：挑取噬菌体点滴斑至sm缓冲液中震荡，进行梯度稀释，取稀释液分别加入对数期宿主菌，混合均匀静置后，加入半固体tsb培养基,立刻倾倒于tsa平板上，摇匀平置待其凝固，置于30℃温箱培养，获得含有单个噬菌斑的双层平板。挑起单个噬菌斑至sm缓冲液中，按照上述方式纯化多次，最终在形成噬菌斑的平板上挑取形态大小一致的单个噬菌斑，置于含有对数期宿主菌菌液的tsb培养基中，30℃条件下摇培。取培养物离心、滤膜过滤上清液，即为得到的纯化噬菌体溶液。本发明上述3株噬菌体均属于短尾科bcep22likevirus属噬菌体，具有解谱宽，稳定性强，生物安全性可靠的烈性，将其组成噬菌体鸡尾酒组合物，其具有为研制新型生物农药提供了优质的候选噬菌体。实验证明：本发明所述的茄科雷尔氏菌噬菌体gp1,gp2和gp3所组成的噬菌体鸡尾酒“gp1+gp2”,“gp1+gp3”,“gp2+gp3”和“gp1+gp2+gp3”对32株来源于山东，四川，重庆，广东，安徽以及河北等6个省的姜瘟病的茄科雷尔氏菌的裂解率可达100％。实验证明：本发明所述的茄科雷尔氏菌噬菌体gp1,gp2和gp3所组成的噬菌体鸡尾酒“gp1+gp3”和“gp1+gp2+gp3”对21株来源于番茄，马铃薯，木麻黄，棉花，桑树，花生和烟草的茄科雷尔氏菌的裂解率可达100％，“gp1+gp2”和“gp2+gp3”的对上述茄科雷尔氏菌的裂解率为95.2％。实验证明：本发明所述的茄科雷尔氏菌噬菌体gp1,gp2和gp3均为烈性噬菌体。实验证明：噬菌体鸡尾酒组合在不足1×101pfu/ml的剂量下即可有效抑菌杀菌。与现有技术相比，本发明所述的茄科雷尔氏菌噬菌体gp1,gp2和gp3具有的优势包括：为严格的烈性噬菌体；对正常微生物菌群无毒害作用；其dna无法编码毒力基因；有较宽的裂解谱，可以杀灭不同地区来源以及不同物种来源的茄科雷尔氏菌。需重点申明的是，本发明未对供试噬菌体进行任何遗传修饰。因此，本发明的3株茄科雷尔氏菌噬菌体gp1,gp2和gp3可防治包括姜瘟病在内的由茄科雷尔氏菌引起的作物细菌病害。附图说明图1：实施例1中所得的3株噬菌体在来源于姜瘟病的茄科雷尔氏菌jw-20菌苔上产生的单一噬菌斑；图2：实施例2中所得的3株噬菌体透射电子显微镜下的形态特征；图3：试验例6中检测实施例1中所得3株噬菌体结构蛋sds-page胶分析；图4：试验例11中噬菌体溶原性鉴定中的双层平板点滴实验结果；图5：试验例12中噬菌体混合物对姜瘟病茄科雷尔氏菌的抑菌率毒力回归方程。茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)的保藏单位为中国典型培养物保藏中心，地址为中国.武汉.武汉大学，邮编430072；保藏日期为2016年11月10日、保藏编号为cctccno:m2016633；茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)的保藏单位为中国典型培养物保藏中心，地址为中国.武汉.武汉大学，邮编430072；保藏日期为2016年11月10日、保藏编号为cctccno:m2016634；茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)的保藏单位为中国典型培养物保藏中心，地址为中国.武汉.武汉大学，邮编430072；保藏日期为2016年11月10日、保藏编号cctccno：m2016635。具体实施方式以下实施例和试验例用于进一步阐述本发明，但不以任何的方式限制本发明的有效范围。以下实施例和实验例中，tsb液体培养基的配方为：胰蛋白胨15g，大豆蛋白胨5g，氯化钠5g，蒸馏水1000ml；tsa固体培养基的配方为：胰蛋白胨15g，大豆蛋白胨5g，氯化钠5g，琼脂15g，蒸馏水1000ml；tsb半固体琼脂培养基配方为：胰蛋白胨15g，大豆蛋白胨5g，氯化钠5g，琼脂7g，蒸馏水1000ml；sm液配方为：氯化钠8.5g，硫酸镁2g，1mol/ltrishcl50ml，明胶0.25g，蒸馏水1000ml。实施例1：噬菌体的分离，纯化样品前处理：分别取来源于山东省安丘市，安徽省阜阳市和四川省崇州市的姜田土样各30g，浸泡于100ml灭菌的0.9％nacl中，4℃冰箱中过夜静置后室温条件下4000rpm离心取上清液备用。噬菌体的分离：分别从上述3份材料中取20ml上清液，用0.22μm滤膜过滤，加入至20ml2×tsb液体培养基中。向含有来源于山东省安丘市的材料中加入200μl处于对数期的茄科雷尔氏菌“3-2-1”(来源于姜瘟病的茄科雷尔氏菌，由四川农业大学提供)菌液，向含有来源于安徽省阜阳市的材料中加入200μl处于对数期的茄科雷尔氏菌“5-2-2”(来源于姜瘟病的茄科雷尔氏菌，由四川农业大学提供)菌液，向含有来源于四川省崇州市的材料中加入200μl处于对数期的茄科雷尔氏菌“zk15-2”(来源于姜瘟病的茄科雷尔氏菌，由四川农业大学提供)菌液，分别置于30℃摇床中200rpm过夜摇培。吸取2ml培养物，用0.22μm滤膜过滤上清液，备用。取处于对数期的代号分别为“3-2-1”，“5-2-2”和“zk15-2”的姜瘟病来源的茄科雷尔氏菌分别加入到半固体tsb培养基中混匀，倾倒于tsa平板上，制备成分别含有“3-2-1”，“5-2-2”和“zk15-2”的双层平板。将用茄科雷尔氏菌“3-2-1”富集的过滤后的上清液，点滴于含有“3-2-1”的已凝固的双层平板上；将用茄科雷尔氏菌“5-2-2”富集的过滤后的上清液，点滴于含有“5-2-2”的已凝固的双层平板上；将用茄科雷尔氏菌“zk15-2”富集的过滤后的上清液，点滴于含有“zk15-2”的已凝固的双层平板上。在无菌条件下风干后，放置于30℃培养24h，形成噬菌体点滴斑。噬菌体的纯化：挑取噬菌体点滴斑至1mlsm缓冲液中震荡1min，进行10倍梯度稀释，取102,104和106稀释液分别加入对数期宿主菌0.5ml，混合均匀静置15min后，加入5ml,40℃半固体tsb培养基,立刻倾倒于tsa平板上，摇匀平置5min，待其凝固，置于30℃温箱培养16h后观察，获得含有单个噬菌斑的双层平板。挑起单个噬菌斑至1mlsm缓冲液中，按照上述方式纯化至少3次以上，最终在形成噬菌斑的平板上挑取形态大小一致的单个噬菌斑，置于含有1ml对数期宿主菌菌液的50mltsb培养基中，30℃条件下180rpm摇培16h。取培养物在8000rpm条件下离心10min，用0.22μm滤膜过滤上清，即为得到的纯化噬菌体溶液。分别将获得的3株茄科雷尔氏菌噬菌体命名为茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)(宿主为来源于姜瘟病的茄科雷尔氏菌“3-2-1”，由四川农业大学提供。)(简称“噬菌体gp1”或“gp1”),茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)(宿主为来源于姜瘟病的茄科雷尔氏菌“5-2-2”，由四川农业大学提供。)(简称“噬菌体gp2”或“gp2”),茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)(宿主为来源于姜瘟病的茄科雷尔氏菌“zk15-2”，由四川农业大学提供。)(简称“噬菌体gp3”或“gp3”)。上述各噬菌体的单一噬菌斑参见图1。实施例2：噬菌体的电镜观察分别取实施例1制得的各噬菌体作电镜观察：取20μl样本滴于铜网上，待其自然沉淀15min，用滤纸从侧面吸收多余液体，加1滴2％磷钨酸(pta)于铜网上，染色10min，用滤纸从侧面吸去染液，干燥后做电镜观察：结果如图2所示，gp1噬菌体具有呈正多面体的头部结构和较短的尾部，头部直径约为60～70nm，尾部长约为15～20nm(图2-a)；gp2噬菌体具有呈正多面体的头部结构和较短的尾部，头部直径约为60～70nm，尾部长约为15～20nm(图2-b)；gp3噬菌体具有呈正多面体的头部结构和较短的尾部，头部直径约为60～70nm，尾部长约为15～20nm(图2-c)。实施例3：噬菌体基因组的提取与测序分别取实施例1制得的各噬菌体100ml，加入终浓度1μg/ml的dnasei，rnasea，37℃条件孵育60min后加入5.84gnacl(终浓度1mol/l),溶解后置于冰浴中1h。4℃条件下，11000rpm离心10min，将上清转移至新的离心管中。加入固体peg8000(终浓度10％，即100ml加入10g),完全溶解后，冰浴至少1h。4℃条件下，11000rpm离心20min，用少量sm液重悬沉淀。加入等体积的氯仿异戊醇抽提，温和震荡30s，8000rpm离心1min后，吸取上清，重复抽提至澄清。再次加入dnasei、rnasea至终浓度1μg/ml，37℃反应30～60min。加入edta至终浓度20mmol/l(即1mlsm液加入40μl0.5mol/ledta),同时每1mlsm液中加入10μl10％sds。从加入蛋白酶步骤开始，使用dp315-rtianampvirusrnakit病毒rna提取试剂盒(tiangen)试剂盒提取。凝胶电泳验证dna后用eppendorfbiophotometerplus测定其浓度和纯度。产物送至军事医学科学院测序得：茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)具有seqidno.1所示的核苷酸序列；茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)具有seqidno.2所示的核苷酸序列；茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)具有seqidno.3所示的核苷酸序列。实施例4噬菌体鸡尾酒的制备以500ml，od值为0.1的茄科雷尔氏菌jw-20(来源于姜瘟病的茄科雷尔氏菌，由菲吉乐科(南京)生物科技有限公司提供)为发酵宿主菌，分别加入1ml效价为1×104pfu/ml的实施例1制备的茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)，茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)和茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)(由实施例1制得)，200rpm,30℃条件下摇培48h。富集得到的噬菌体溶液分装至50ml离心管中，每管40ml；加入固体peg8000终浓度10％，即40ml加入4g),完全溶解后，冰浴至少3h；将噬菌体4℃，12000rpm离心20min；弃去上清液，使用等体积的sm缓冲液充分溶解沉淀；将噬菌体sm洗脱液装入1lcorningpes0.22μm真空过滤系统过滤除去宿主菌后，进行效价检测，分别获得≧2×1010pfu/ml茄科雷尔氏菌噬菌体gp1(ralstoniasolanacearumphagegp1)，茄科雷尔氏菌噬菌体gp2(ralstoniasolanacearumphagegp2)和茄科雷尔氏菌噬菌体gp3(ralstoniasolanacearumphagegp3)，然后4℃保藏备用。用sm液分别将上述3种噬菌体效价调制2×1010pfu/ml后，按表1进行体积混合，最终获得效价为2×1010pfu/ml的噬菌体鸡尾酒混合液“gp1+gp2”，“gp1+gp3”，“gp2+gp3”和“gp1+gp2+gp3”。表1不同噬菌体组合物及比例和杀菌范围试验例5：噬菌体的毒力基因或不良基因缺失检测试验本发明选取44种经鉴定源自病原细菌体内溶源性噬菌体的毒力基因(表2)，通过测定噬菌体gp1,gp2和gp3的全基因组并对其进行生物信息学分析，以确定其是否含有上述毒力基因。结果显示，本发明3株供试噬菌体均不含有下列毒力基因。表2病原细菌体内溶源性噬菌体的主要已知毒性基因试验例6噬菌体sds-page分析取实施例1制备的噬菌体溶液，按照质量体积比1：10加入peg8000,溶解后冰浴3h后，4℃条件下11000rpm离心20min后，弃上清，用适量无菌水重悬沉淀，即获得该噬菌体浓缩液。取浓缩后的含噬菌体颗粒的液体15μl与3μl的6×上样缓冲液混匀后100℃水浴5min使蛋白变性，将处理后的样品和蛋白marker加入凝胶加样孔并做好标记，接通电源后90v约100min后待测样品电泳至浓缩胶和分离胶交界处，然后将电压升高至150v，继续电泳至分离胶下缘，停止电泳，将整块分离胶放到玻璃平皿中，用常规考马斯亮蓝染色方法进行染色，待蛋白条带显色后取出并扫描,结果如图3。结果表明：蛋白marker分子量标准依次为170kda、130kda、100kda、70kda、55kda、40kda、35kda、25kda、15kda、10kda；gp1,gp2和gp3均可观察到至少16个蛋白条带，说明上述3株噬菌体颗粒均至少含有16个结构蛋白，且主要结构蛋白均由约17kda和45kda大小的两种蛋白组成。试验例7：噬菌体moi及效价的测定取50μl处于对数生长期茄科雷尔氏菌jw-20，接种到盛有5mltsb培养液的试管中，30℃摇床中220rpm振荡培养24h，得到宿主菌悬液，菌液浓度1.0×109cfu/ml。将试验例4中制备的备用噬菌体gp1，gp2和gp3依次梯度分别稀释至1.0×108pfu/ml，1.0×107pfu/ml，1.0×106pfu/ml，1.0×105pfu/ml，1.0×104pfu/ml，1.0×103pfu/ml，1.0×102pfu/ml和1.0×101pfu/ml。将1ml浓度为1.0×109cfu/ml的宿主菌与1ml稀释后各梯度噬菌体依次按1:10，1:100，1:1000，1:1万，1:10万，1:100万，1:1000万(moi＝噬菌体数量pfu/细菌数量cfu)的比例接于灭菌的50mltsb培养基中，30℃条件下220rpm培养24h。培养完毕后10000g离心l0min并收集上清，按如下方法测定噬菌体效价：用sm液做稀释液，将待测噬菌体液10倍梯度稀释至l07倍。分别取l05，l06及l07稀释度的噬菌体培养液l00μl与其宿主菌茄科雷尔氏菌液300μl均匀混合，静置15min使其与细菌表面的受体充分结合。将上述混合液加入4ml冷却至40℃的半固体琼脂培养基中，混匀后立即铺于已凝固的固体琼脂平板上，待琼脂凝固后于30℃倒置培养24h。每个稀释度需做三个平行样，计数时取此稀释度的三个平行样的平均数。其中，噬菌体效价(pfu/ml)＝平均噬菌斑数×稀释倍数×10，以产生最高噬菌体效价的moi为最佳感染复数。结果如表3所示。结果表明，培养24h条件下，噬菌体gp1效价达到最高(4.3×1010pfu/ml)时，其moi＝0.0000001；噬菌体gp2效价达到最高(2.9×1010pfu/ml)时，其moi＝0.0000001；噬菌体gp3效价达到最高(6.7×1010pfu/ml)时，其moi＝0.0000001。表33株噬菌体moi值moigp1(pfu/ml)gp2(pfu/ml)gp3(pfu/ml)1001.1×1081.4×1082.5×108102.4×1085.4×1086.3×10814.5×1087.5×1088.1×1080.13.1×1092.7×1092.2×1090.014.2×1093.2×1094.2×1090.0017.2×1093.6×1097.1×1090.00011.4×10101.1×10102.4×10100.000012.5×10101.5×10104.1×10100.0000014.3×10102.9×10106.7×10100.00000012.1×10101.0×10103.2×1010试验例8：噬菌体及噬菌体鸡尾酒组合物ph和温度稳定性的测定8-1：不同ph条件下噬菌体的稳定性将1ml效价为1×107pfu/mlgp1，gp2,及gp3等3种单一噬菌体(由实施例4制得噬菌体经过稀释后得到)和1ml效价为1×107pfu/ml的噬菌体鸡尾酒混合物“gp1+gp2”,“gp1+gp3”,“gp2+gp3”和“gp1+gp2+gp3”(由实施例4制得噬菌体鸡尾酒混合物稀释后得到)各7份分别置于9mlph＝1,3,5,7,9,11和13的sm溶液中，制成效价为1×106pfu/ml的噬菌体溶液，4℃培养24h后，再检测其效价。结果如表4所示，在ph5～ph9之间，3株噬菌体gp1，gp2,及gp3及其噬菌体鸡尾酒“gp1+gp2”,“gp1+gp3”,“gp2+gp3”和“gp1+gp2+gp3”的效价均无显著变化，表明其在中性，微酸和微碱条件下有较好的稳定性。在ph＝3的酸性条件下和ph＝11的碱性条件下，3株噬菌体及其噬菌体鸡尾酒的效价有一定程度的下降，但是与ph＝7条件下相比，效价仍为下降1个数量级，表明其在酸性和碱性条件下仍有一定程度的耐受性。在ph＝1的极酸性条件下和ph＝13的极碱性条件下，3株噬菌体及其噬菌体鸡尾酒的效价1个小时内均下降为0。表43株单一噬菌体及其鸡尾酒混合物在不同ph条件下的稳定性8-2：不同温度条件下噬菌体的稳定性将效价为1.0×107pfu/mlgp1，gp2,及gp3等3种单一噬菌体(由实施例4制得备用噬菌体经过稀释后得到)，以及总效价为1.0×107pfu/ml的噬菌体鸡尾酒混合物“gp1+gp2”,“gp1+gp3”,“gp2+gp3”和“gp1+gp2+gp3”(由实施例4制得噬菌体鸡尾酒混合物稀释后得到)分别置于4℃，25℃以及37℃条件下，检测其效价。结果表明，在4℃条件下，gp1，gp2,及gp3等3种单一噬菌体，及其噬菌体鸡尾酒混合物“gp1+gp2”,“gp1+gp3”,“gp2+gp3”和“gp1+gp2+gp3”都具有较好的稳定性，存放3个月后，效价并无明显下降；存放12个月后，效价并无明显下降在25℃条件下；gp1，gp2,及gp3等3种单一噬菌体，及其噬菌体鸡尾酒混合物“gp1+gp2”,“gp1+gp3”,“gp2+gp3”和“gp1+gp2+gp3”在1周之内效价无明显下降，4周后，效价均大约仅仅下降1个数量级；在37℃条件下，gp1，gp2,及gp3等3种单一噬菌体，及其噬菌体鸡尾酒混合物“gp1+gp2”,“gp1+gp3”,“gp2+gp3”和“gp1+gp2+gp3”在24小时内效价没有明显下降，72小时后，效价均大约仅仅下降1个数量级。由此表明，由上述3种噬菌体组成的鸡尾酒混合物“gp1+gp2”,“gp1+gp3”,“gp2+gp3”和“gp1+gp2+gp3”在不同温度条件下都具有较好的稳定性，参见表5～7。表53株噬菌体及其鸡尾酒混合物在4℃条件下的稳定性表63株噬菌体及其鸡尾酒混合物在25℃条件下的稳定性表73株噬菌体及其鸡尾酒混合物在40℃条件下的稳定性试验例9茄科雷尔氏菌裂解范围试验9-1噬菌体对来源姜瘟病的茄科雷尔氏菌的裂解能力采用双层平板点滴法来测定噬菌体的裂解谱。分别挑取从山东，四川，重庆，安徽，广东和河北等6省姜瘟病样品上分离获得的32株茄科雷尔氏菌的单菌落，将其接种于盛有3mltsb的试管中，160rpm培养8h，制得各株细菌菌液。取300μl菌悬液分别与半固体培养基混合铺于普通琼脂平板上，分别取5μl各噬菌体及其鸡尾酒混合物溶液(由实施例4制得)滴于平板的不同位置上，加样时各噬菌体培养液间不可接触，以免影响试验结果。待自然风干后30℃培养24h，观察结果。试验重复三次。结果如表8所示，gp1,gp2和gp3均具有较宽的宿主范围，其裂解率分别可达84.37％，81.25％和87.5％。其噬菌体鸡尾酒混合物“gp1+gp2”,“gp1+gp3”,“gp2+gp3”和“gp1+gp2+gp3”的裂解率为100％。由此表明，采用上述3种噬菌体的鸡尾酒混合物对茄科雷尔氏菌裂解谱范围要远远高于单株噬菌体。表8噬菌体对来源姜瘟病的茄科雷尔氏菌的裂解能力注：“+++”完全透亮，“++”中等透亮，“+”轻微透亮，不裂解的为“-”9-2噬菌体对来源于其他作物的茄科雷尔氏菌的裂解能力采用双层平板点滴法来测定噬菌体的裂解谱。分别挑取来源于番茄青枯病，马铃薯青枯病，花生青枯病，木麻黄青枯病，桑树青枯病，棉花青枯病和烟草青枯病的21株茄科雷尔氏菌的单菌落，将其接种于盛有3mltsb的试管中，160rpm培养8h，制得各株细菌菌液。取300μl菌悬液分别与半固体培养基混合铺于普通琼脂平板上，分别取5μl各噬菌体及其鸡尾酒混合物溶液(由实施例4制得)滴于平板的不同位置上，加样时各噬菌体培养液间不可接触，以免影响试验结果。待自然风干后30℃培养24h，观察结果。试验重复三次。结果如表9所示，gp1,gp2和gp3均具有较宽的宿主范围，其裂解率分别可达61.9％，76.2％和71.4％。其噬菌体鸡尾酒混合物“gp1+gp2”和“gp2+gp3”的裂解率为95.2％，“gp1+gp3”和“gp1+gp2+gp3”的裂解率为100％。由此表明，采用上述3种噬菌体的鸡尾酒混合物对茄科雷尔氏菌裂解谱范围要远远高于单株噬菌体。表9噬菌体对茄科雷尔氏菌裂解谱的测定结果注：“+++”完全透亮，“++”中等透亮，“+”轻微透亮，不裂解的为“-”试验例10：噬菌体对非致病性有益细菌的裂解试验挑取5株非致病性根瘤菌，5株非致病性枯草芽孢杆菌分别接种于盛有3mltsb的试管中，160rpm培养8h，制得各株细菌菌液。取300μl菌悬液分别与半固体培养基混合铺于普通琼脂平板上。分别取5μl噬菌体培养液(由实施例4制得)滴于平板的不同位置上，加样时各噬菌体培养液间不可接触，以免影响试验结果。待自然风干后30℃培养24h，观察结果。试验重复三次。结果如表10所示，在本研究中，3株噬菌体及其鸡尾酒混合物均无法识别上述10株非致病性细菌。说明供试噬菌体具有极强的特异性，且对微生物群落无损伤作用。表103株噬菌体及其鸡尾酒混合物对非致病性有益菌的裂解试验注：“+++”完全透亮，“++”中等透亮，“+”轻微透亮，不裂解的为“-”试验例11：噬菌体溶原性鉴定分别将100μl(0pfu/ml,1.0×104pfu/ml,1.0×105pfu/ml,1.0×106pfu/ml,1.0×107pfu/ml)的噬菌体以及100μl效价为1.0×108cfu/ml的茄科雷尔氏菌jw-20接种于含有10mltsb的50ml离心管中，置于30℃摇床中培养48h；将所获混浊培养液(选一组最浑浊的梯度做，同时对照组一定要做)在tsa平板上梯度稀释涂布，置于30℃培养箱中培养48h；在平板上挑取30～50个单菌落(注意尽量挑去菌落中心部位，不可挑到菌落边缘，容易污染到残留的噬菌体)，置于含有200μltsb的2mlep管中，在30℃摇床中培养24h；加入终浓度为0.5μg/ml的丝裂霉素c于ep管中，继续培养12-16h；将所获培养液用0.22μm滤膜过滤除菌后，点滴在jw-20双层平板上，置于30℃培养；同时将效价为1.0×107pfu/ml的各噬菌体(由实施例4制得)点滴在双层平板上作为阳性对照。24h后观察双层平板，如果出现噬菌斑，证明噬菌体为溶原性。观察结果显示，对照平板均有噬菌斑出现(如图4a所示)而所有的试验平板均无噬菌斑出现(如图4b所示)，这表明，3株噬菌体均不具有溶原性，都是烈性噬菌体。试验例12：浊度法检测噬菌体混合物对姜瘟病茄科雷尔氏菌的抑菌率取40支50ml离心管，分别装量10mltsb，封口灭菌待用；宿主菌jw-2030℃160r/min过夜培养至浑浊，待用；噬菌体为gp1,gp2和gp31:1:1混合，效价为2×1010pfu/ml(由实施例4制得)。将噬菌体逐步梯度稀释至各处理组效价(如表11所示)，取100μljw-20菌液加入各组，再各取100μl各稀释的噬菌体加入各处理组中，30℃，120r/min培养24h，用浊度仪测各处理组浑浊度值。以抑菌率为y轴，噬菌体浓度的对数值为x轴，得出的抑菌率曲线(图5)。根据抑菌率回归方程y＝6.2494x+46.174(r2＝0.9983)，计算ec50＝0.612，也就是说不到101pfu/ml的噬菌体即对jw-20的抑菌率即可达到50％，这表明噬菌体鸡尾酒组合在极低的剂量下即可有效抑菌杀菌。表11噬菌体鸡尾酒对姜瘟病茄科雷尔氏菌的毒力序列表<110>菲吉乐科（南京）生物科技有限公司<120>新型茄科雷尔氏菌噬菌体及其组合物和应用<130>lac170529p<160>3<170>patentinversion3.5<210>1<211>61125<212>dna<213>ralstoniasolanacearumphagegp1<400>1atcgctatccttttcgagaatcgtctatgatcatggtaatgaaatcggaacactttcgca60accagagtggttgcttttcccgtcatgtggtgttgtatgcgaaccgcaatgaagcgaagt120aaaatgaaatgacagaagtaaaccgctcttatttcgagggcctgatggcacagcggaaaa180tttctctgcggcagctcgcggagaagatgggcctcgcgcactcagctctcagcatcacct240tcagcgggtcgcggcgcatgcagctggaggaggccgcccagctatccaacatcttcggtg300tgcctatccatgagatcatcgaaaacgccggcgtgacagcgcgcccggtcagcagctcgc360gcgtgaacgttgtcggcgcgctgcgcggcgacggccacgtcgagaaaatctcggggaagc420acgtcgagcgcacatctgcgccactggagtcgccggacggcaccgaggcgatccagtgcc480gcaccgccgacacgccactgtcgtggctcgatggggcggtcatgttcttcgtcccgtccg540acagggtcgaggcggaggccgtggggcgattgtgctacctgcaaatccacggcggcgagc600tggttgtcgccaccatgaagcgagggtatcgcgacagtacctacaacctctccggaccgc660gcactgctgagagcgtcaaagtggattgggcgacacctattcgcatgaccaggaattagc720cgaacgacatctgatcaggccgccctggcaaactctccgtggagcttgtttgcctcggcc780gcgtaggcttcatgcgccccttctgcggtatcaaaatatcctatatgccgcgtcttccca840tcccgatgaatcacggcgcgccatctcccttttactttgacgactccctttagcccggat900accgagcggcctctttggttcacaagattttgctgaatggtcgcttcacgcaggttgaag960aacctgttgtccgccttgtcgagattgatgtgatcaatcacatccgcaggccacttccca1020gtcatgtatagccacgcgagacggtgagccataagcctgaccccgtcgatccttatcgag1080acgtacccatacgaaattgtgccggcacgacaccccgcaagcacacccttgcggttaaaa1140cgccacgtgaactcgcccgtctcagggtcgtagttgagaagctccttcagccgctgttgc1200gtaagccccatgattcgagatggattggttggtcatgatggggaattctagcctcattca1260gcccatgatggccgataccggcgtcgtcgccgcgcaactgcgtggcgctttttacgaaca1320tagacgaaaaagcggtttgattttcgtaccactggtgctattctgcatgcacgccgcata1380gggtggcgcatggagataatattgtgagcattgagcgagtgacacttaccccggcaagcg1440aagcggaatggcttgcgatgcgagcgcaggatttgaccagcacggaagtcgccgcgctgt1500tcggctgctccccgtactcgacggaatacgagctgttcatgcgcaagaccaagcgcatcg1560tggccgagttcaaggaaaacgagcgcatgacctggggcaagcgcctggaaagtgccatcg1620ccgccggcatcgccgaggactacggcctgatcgtcgagccgttcaagacgtacatgcgca1680tcccggcgctgcgcatgggcgcgtcgttcgacttcaggatcatcggcacggttgccggcc1740acccgtccaacgaagcccgccgcatgttcgatcgccacggcgacggcatcatggagatca1800agaacgtcgacggcctgcaattccgccgcgcctggatcgacgatggcgatgacatcgaag1860cgccggcgcacatagaatttcaggcgcaatcgcagctcgaagtcgcgggcctgggctggt1920cgatgatcgccccgctggtcggtggcaacacgccgaaggtgtcgttgcgcgagcgcgaca1980cggaagtcggcgcgctgatccgcgagaaggtggcgcagttctggatgcgcgtcgacgccg2040gcgccgcgccgcagccggactacacccgcgacggcgacacgatcaagcagctctaccgcg2100acaacgacggcagcagcgtcgacctgtccgacgagccgcgccttgccgagttgtgcgcag2160cgtacaaggcggccggcgccgaggagaaggctgccgagggccgcaagaaagcggcgcagg2220ccgagatcatcacgatcatcgagcatgccaagagcatccgcgccgatggcttcaacatct2280cggccggaaccaacaaggagagctatcgcgtctacgatcgcgcggccagcaagcgctaca2340ccatcagcgtgagcgacatcccggccacgacgatcgaggccacggtggcgccgtttcgaa2400acgtccgtatcacgcccgacaaggccgcgtgagccgcaccattccgcaccataccgattg2460agtgagcatcatgaccaatcagacccaacagcagcagccgatgaccgaggccgaatggcg2520caagcgcgagctgtccgacatccgcaacaccctggagcggcagaagcaggagatcggcaa2580ggccctgcccgaggacatcccggcagagcgcttcatccgcacggcactgaccgccgtgca2640ggtgaaccccgatctgctcgacgctgaccgtcaatccctgttcatcgcctgcatgaaggc2700cgcccaagacggcctcctgccggacggccgcgaggccgtgctgaacgtctacaacaccaa2760gacaaaggtgtgggaaggcaatcggcaggtcgagaagtgggtgccgaccgtgcagtacct2820gcccatggtgcgcggcctgctgaagctgatccgcaactccggcgagatcggccacgtcga2880cgccgccgccgtgtacgagcgcgaccagttccggttcgtgcgcggcgacgatccgcgcct2940ggagcacgagccgtacctgggcgaggacgaccccggaaagatcgtcgcggcctacatcgt3000cgtgcgcttcaccaacggcgaagtgcaccgcgaggtcatgccgcgccgtgacatcgagaa3060ggtgcgcggcgcgtccaagtccggcgacggcgccaacagcccgtggacgaagtggtacga3120ccagatggcgatcaagtccgtcatcaagcgcgccgccaagttgctgcccaacagctcgaa3180gcgcctggagagcgcgatcggccacgacaacgaggactacgacggcttcaaccagcgcgg3240cgaagtgctcgacctgggcgccacggcggcagtggcgggcgcggcgccggcggccctgac3300ggacggccgcgccgaacaggaagcgcgccgcccttcccgcttcggcggcatcgtcaacaa3360ggccaagcaggaagccgtgcgccagccggccaccgacgcccaggcccaggcgcaggcagc3420ggagtgacgggcatgggccaccacctgaccgaaactgatctcgcccagcgcttcggcgtg3480acggagcgcacgcttcagaagtggcgccggcagaagaccggccccgcctggatcgtgatc3540ggcaagaacaccatccgttaccgcgaagaagacatcaggcaatacgaggaggattgcctt3600cgcaataactcgactcaaacagggaggagcgatgcatgacgaagacgtcgcaaagcgctt3660ctacgcgaaggttcgcattacaccaggatgctggatttggactgcctcgacacgcagatt3720tggatacggccaattccagtttcagggcaggccacaggctgcgcaccgagtctcctacat3780catcaacgttggcccggtgccggatgcgattctcgtgctgcacagatgcgacaacccagg3840ttgcgtgaaccctgaccaccttttccttggcactgccgcagacaacatggacgacatgtc3900gcgcaaaggccgaggaaagacgaaagaccagagaggcatcggcaacggcaatgctcggct3960agatcagcgacatgtggtggcgatcttcttcgacccgaggccatctgctgaaatcgcaat4020cgaatacggcatcagccagtttcacgtaaggcgcatcaaacgcggcgaggcttggcccca4080tctcggcctcatcaaaaaccaagcggcataaattccaccgacatcatgatctctttcgac4140accaaagactttgagcgcgcgctgtccaccgtctcgcgcgtgatccagcgcagcaccatc4200ccgg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