CN107904214A - Vibrio alginolyticus bacteriophage and the bactericidal composition comprising the bacteriophage - Google Patents
Vibrio alginolyticus bacteriophage and the bactericidal composition comprising the bacteriophage Download PDFInfo
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Abstract
本发明涉及溶藻弧菌噬菌体及包含该噬菌体的杀菌组合物。该噬菌体可在短时间内快速抑制弧菌的生长，且抑制作用维持时间长。此外，该噬菌体的杀菌组合物安全、有效，且特异性强，生产成本低，在致病菌的控制过程中弥补了单一噬菌体疗法的不足。
The invention relates to a bacteriophage of Vibrio alginolyticus and a bactericidal composition comprising the phage. The phage can quickly inhibit the growth of Vibrio in a short time, and the inhibitory effect lasts for a long time. In addition, the bactericidal composition of the phage is safe, effective, and highly specific, and the production cost is low, which makes up for the deficiency of single phage therapy in the process of controlling pathogenic bacteria.
Description
技术领域technical field
本发明涉及生物技术领域，具体而言，涉及一种溶藻弧菌噬菌体及包含该噬菌体的杀菌组合物。The invention relates to the field of biotechnology, in particular to a Vibrio alginolyticus phage and a bactericidal composition containing the phage.
背景技术Background technique
我国是水产养殖大国。近年来，我国的水产养殖业获得迅猛发展。随着海水养殖规模的日益扩大和集约化养殖方式的不断推广，鱼类疾病的频繁发生对海水鱼类规模化养殖的健康发展造成了严重的威胁。鱼类的病原体主要包括细菌、病毒、寄生虫等，其中细菌性疾病是对鱼类危害程度较为严重的一类疾病。my country is a big country in aquaculture. In recent years, my country's aquaculture industry has developed rapidly. With the increasing scale of marine aquaculture and the continuous promotion of intensive farming methods, the frequent occurrence of fish diseases poses a serious threat to the healthy development of large-scale marine fish farming. Fish pathogens mainly include bacteria, viruses, parasites, etc. Among them, bacterial diseases are a type of disease that is more harmful to fish.
弧菌是能运动的芽孢短杆状细菌，广泛分布于近海岸、河口海区海水及生物体内，是海洋中最常见的细菌类群之一，同时也是引起海水养殖鱼虾细菌性疾病最重要的病原菌之一。弧菌的致病强度与宿主的生理状态及水质环境条件等综合因素有关，属于条件性致病菌，其主要通过口或伤口感染，产生毒素，致使伤口肌肉溃烂化脓以及内脏器官的严重病变从而导致鱼虾类的死亡。由弧菌引起的疾病又称弧菌病，具有“流行面积广，发病率高，致死性强”等特点，是水产养殖动物的主要疾病，同时也被确定为是阻碍水产养殖业发展的重要限制性因素，给养殖业造成了巨大的危害。其中，以鳗弧菌(Vibrio anguillarum)、溶藻弧菌(Vibrio alginolyticus)、哈维氏弧菌(Vibrio harveyi)、副溶血弧菌(Vibrioparahaemolyticus)和杀鲑弧菌(Vibrio salmonicida)等弧菌引起的弧菌病被认为是鱼虾类养殖中最为严重的病害之一。目前，针对水产养殖相关动物疾病的预防和控制方法主要以化学抗生素法和免疫疫苗法，其中以化学抗生素使用为主。作为当前控制水产动物疾病的主要手段，化学抗生素法具有使用方便、见效快和疗效好等优点。但是，大批量频繁使用抗生素在控制病原菌的同时，极大了助长了病原菌的耐药性，并加速了广谱耐药菌的产生。这使得致病菌的耐药性问题日益严重。此外，化学药物残留也是导致人类食源性疾病产生的重要原因之一，对人类健康造成严重危害。Vibrio is a short rod-shaped spore-shaped bacterium that can move. It is widely distributed in seawater and organisms in coastal and estuary sea areas. It is one of the most common bacterial groups in the ocean, and it is also the most important pathogenic bacteria that cause bacterial diseases in seawater cultured fish and shrimp. one. The pathogenicity of Vibrio is related to comprehensive factors such as the host's physiological state and water quality environment conditions. It is an opportunistic pathogenic bacteria. It mainly produces toxins through mouth or wound infection, resulting in festering of wound muscles and serious lesions of internal organs. lead to the death of fish and shrimp. Diseases caused by Vibrio, also known as vibriosis, have the characteristics of "widespread area, high morbidity, and strong lethality". Restrictive factors have caused great harm to the aquaculture industry. Among them, Vibrio anguillarum (Vibrio anguillarum), Vibrio alginolyticus (Vibrio alginolyticus), Vibrio harveyi (Vibrio harveyi), Vibrio parahaemolyticus (Vibrio parahaemolyticus) and Vibrio salmonicida (Vibrio salmonicida) etc. Vibriosis is considered to be one of the most serious diseases in fish and shrimp farming. At present, the prevention and control methods for animal diseases related to aquaculture are mainly chemical antibiotics and immunization vaccines, among which the use of chemical antibiotics is the main method. As the main means of controlling aquatic animal diseases at present, the chemical antibiotic method has the advantages of convenient use, quick effect and good curative effect. However, the frequent use of antibiotics in large quantities has greatly promoted the drug resistance of pathogenic bacteria and accelerated the emergence of broad-spectrum drug-resistant bacteria while controlling pathogenic bacteria. This makes the problem of drug resistance of pathogenic bacteria increasingly serious. In addition, chemical drug residues are also one of the important causes of human foodborne diseases, causing serious harm to human health.
噬菌体又称细菌性病毒，是一类专一性裂解细菌的病毒，在环境中分布广泛，种类多样，结构简单，具有很强的宿主特异性。最重要的一点，噬菌体裂解细菌的机制不受细菌耐药性的影响，其通过受体识别从而吸附在特定宿主菌表面，将自身遗传物质注入宿主体内进行自我复制组装，并最终通过裂解宿主菌释放子代从而实现自我的生长繁殖。利用噬菌体控制水产致病菌早在噬菌体被发现之前就有应用，但由于对噬菌体的研究不够充分，而抗生素在当时具有广谱抑菌效果，且疗效好见效快，这使得噬菌体应用被大大削弱。近年来，由于抗生素滥用引发的一系列耐药菌问题，使得噬菌体重新回归人们的视野，应用噬菌体控制致病菌的研究也在科学界引起了广泛兴趣。作为应对抗生素抗性的重要武器之一，噬菌体的临床应用潜力巨大，但就其当前单一噬菌体疗法的应用而言，其在应用过程中仍存在一些潜在的缺点，如裂解宿主谱窄，裂解能力持续时间短，宿主菌易产生抗性等，其中以宿主菌易产生抗性为单株噬菌体应用的主要缺陷。Bacteriophage, also known as bacterial virus, is a kind of virus that specifically lyses bacteria. It is widely distributed in the environment, has various types, simple structure, and strong host specificity. The most important point is that the mechanism of phage lysis of bacteria is not affected by bacterial drug resistance. It adsorbs on the surface of a specific host bacterium through receptor recognition, injects its own genetic material into the host for self-replication assembly, and finally lyses the host bacterium. Release offspring to realize self-growth and reproduction. The use of phages to control aquatic pathogens has been used long before the discovery of phages, but due to insufficient research on phages, antibiotics had broad-spectrum antibacterial effects at that time, and the curative effect was good and quick, which greatly weakened the application of phages . In recent years, due to a series of drug-resistant bacteria problems caused by the abuse of antibiotics, phages have returned to people's vision, and the research on the application of phages to control pathogenic bacteria has also aroused widespread interest in the scientific community. As one of the important weapons to deal with antibiotic resistance, phage has great potential for clinical application, but as far as its current application of single phage therapy is concerned, there are still some potential disadvantages in the application process, such as narrow lytic host spectrum and lytic ability. The duration is short, and the host bacteria are easy to develop resistance, etc. Among them, the host bacteria are easy to develop resistance as the main defect of the application of single phage.
针对以上单一噬菌体疗法存在的不足，将多种噬菌体混合使用的鸡尾酒疗法应运而生。其通过多种噬菌体之间的复配既扩大了宿主谱范围，同时有效的抑制宿主菌抗性的产生，具有快速高效的裂解作用，目前在临床应用的研究中已取得较好的效果。此外，噬菌体鸡尾酒通过噬菌体之间的协同作用降低了在相等条件下单株噬菌体的添加剂量，起到了节约成本的作用。To address the shortcomings of the above single phage therapy, a cocktail therapy that uses a variety of phages in combination has emerged as the times require. It not only expands the scope of the host spectrum through the compounding of various phages, but also effectively inhibits the generation of host bacterial resistance, and has a fast and efficient lysis effect. It has achieved good results in clinical application research. In addition, the phage cocktail reduces the dosage of a single phage under equal conditions through the synergistic effect between phages, which saves costs.
有鉴于此，特提出本发明。In view of this, the present invention is proposed.
发明内容Contents of the invention
本发明涉及一种专一性裂解弧菌的溶藻弧菌噬菌体，保藏名为ValDsh1-1，于2017年9月14日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO:M 2017499，分类命名为Vibrio phage。The present invention relates to a Vibrio alginolyticus phage that specifically lyses Vibrio, and the preservation name is ValDsh1-1, which was preserved in the China Center for Type Culture Collection on September 14, 2017, and the preservation number is: CCTCC NO: M 2017499 , taxonomically named Vibrio phage.
该噬菌体均保藏于中国典型培养物保藏中心(CCTCC)，保藏地址为：湖北省武汉市武昌区八一路珞珈山，武汉大学中国典型培养物保藏中心；保藏时间为：2017年9月14日。经保藏中心于2017年9月14日检测为存活菌株。The phages are all preserved in the China Center for Type Culture Collection (CCTCC), and the preservation address is: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Type Culture Collection Center of Wuhan University; preservation time: September 14, 2017 day. It was detected as a surviving strain by the preservation center on September 14, 2017.
附图说明Description of drawings
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work.
图1为本发明实施例中的噬菌体筛选流程图；Fig. 1 is the flow chart of phage screening in the embodiment of the present invention;
图2为透射电镜下的各弧菌噬菌体的形态；Fig. 2 is the morphology of each Vibrio phage under the transmission electron microscope;
a：噬菌体ValDsh1-1；b：VpaSw-1；c：VpaSw-2；d：ValSw3-1；e：ValSw3-2；f:ValSw3-3；a: phage ValDsh1-1; b: VpaSw-1; c: VpaSw-2; d: ValSw3-1; e: ValSw3-2; f: ValSw3-3;
图3为基于16s DNA的宿主菌进化树；Figure 3 is the phylogenetic tree of host bacteria based on 16s DNA;
3-副溶血弧菌(Vibrio.parahaemolyticus)；18-溶藻弧菌(Vibrio.alginolyticus)；F1-金黄色弧菌(Vibrio.azureus)；F3-溶藻弧菌(Vibrio.alginolyticus)；F4-弧菌(Vibrio.sp)；F10-副溶血弧菌(Vibrio.parahaemolyticus)；3-Vibrio.parahaemolyticus; 18-Vibrio.alginolyticus; F1-Vibrio.azureus; F3-Vibrio.alginolyticus; F4- Vibrio (Vibrio.sp); F10-Vibrio parahaemolyticus (Vibrio.parahaemolyticus);
图4为不同感染复数(MOI)下单株噬菌体对溶藻弧菌V.alginolyticus的生长曲线作用图；在不同的MOI(10，1，0.1，0.01，0.001)下，3株噬菌体分别对溶藻弧菌生长曲线(OD600)抑制效果，control组(菌液+培养基)表示不加噬菌体，Blank组(培养基)表示空白对照；Fig. 4 is a figure showing the effect of a single phage on the growth curve of Vibrio alginolyticus at different multiplicity of infection (MOI); under different MOI (10, 1, 0.1, 0.01, 0.001), the three phages were respectively on the lysate Vibrio algal growth curve (OD600) inhibitory effect, control group (bacteria liquid+medium) means no phage, Blank group (medium) means blank control;
f4-1：ValSw3-1；f4-2：ValSw3-2；f4-3：ValSw3-3；f4-1: ValSw3-1; f4-2: ValSw3-2; f4-3: ValSw3-3;
图5为当MOI＝1时不同噬菌体鸡尾酒组合对6株病原菌生长曲线的作用图；Fig. 5 is when MOI=1, different phage cocktail combination is to the action figure of 6 strains of pathogenic bacteria growth curve;
a—副溶血弧菌(Vibrio.parahaemolyticus)；b—溶藻弧菌(Vibrio.alginolyticus)；c—金黄色弧菌(Vibrio.azureus)；d—溶藻弧菌(Vibrio.alginolyticus)；e—弧菌(Vibrio.sp)；f:—副溶血弧菌(Vibrio.parahaemolyticus)；a—Vibrio.parahaemolyticus; b—Vibrio.alginolyticus; c—Vibrio.azureus; d—Vibrio.alginolyticus; e— Vibrio (Vibrio.sp); f:—Vibrio parahaemolyticus (Vibrio.parahaemolyticus);
cock-1：ValSw3-1，ValDsh1-1；cock-2：ValSw3-1，VpaSw-1；cock-3：ValSw3-1，VpaSw-2；cock-4：ValSw3-1，ValSw3-2，ValSw3-3，VpaSw-1；cock-5：ValSw3-1，ValSw3-2，ValSw3-3，VpaSw-2，ValDsh1-1；control：菌液+培养基(不加噬菌体)；Blank：培养基。cock-1: ValSw3-1, ValDsh1-1; cock-2: ValSw3-1, VpaSw-1; cock-3: ValSw3-1, VpaSw-2; cock-4: ValSw3-1, ValSw3-2, ValSw3- 3. VpaSw-1; cock-5: ValSw3-1, ValSw3-2, ValSw3-3, VpaSw-2, ValDsh1-1; control: bacterial solution + medium (without phage); Blank: medium.
本发明所提供的溶藻弧菌噬菌体ValDsh1-1(Vibrio phage ValDsh1-1)，保藏号为CCTCC NO:M 2017499；The vibrio alginolyticus phage ValDsh1-1 (Vibrio phage ValDsh1-1) provided by the present invention has a preservation number of CCTCC NO: M 2017499;
本发明所提供的副溶血弧菌噬菌体VpaSw-1(Vibrio phage VpaSw-1)，保藏号为CCTCC NO:M 2017500；The Vibrio parahaemolyticus phage VpaSw-1 (Vibrio phage VpaSw-1) provided by the present invention has a preservation number of CCTCC NO: M 2017500;
本发明所提供的副溶血弧菌噬菌体VpaSw-2(Vibrio phage VpaSw-2)，保藏号为CCTCC NO:M 2017501；The Vibrio parahaemolyticus phage VpaSw-2 (Vibrio phage VpaSw-2) provided by the present invention has a preservation number of CCTCC NO: M 2017501;
本发明所提供的溶藻弧菌噬菌体ValSw3-1(Vibrio phage ValSw3-1)，保藏号为CCTCC NO:M 2017502；The Vibrio alginolyticus phage ValSw3-1 (Vibrio phage ValSw3-1) provided by the present invention has a preservation number of CCTCC NO: M 2017502;
本发明所提供的溶藻弧菌噬菌体ValSw3-2(Vibrio phage ValSw3-2)，保藏号为CCTCC NO:M 2017503；The Vibrio alginolyticus phage ValSw3-2 (Vibrio phage ValSw3-2) provided by the present invention has a preservation number of CCTCC NO: M 2017503;
本发明所提供的溶藻弧菌噬菌体ValSw3-3(Vibrio phage ValSw3-3)，保藏号为CCTCC NO:M 2017504；The Vibrio alginolyticus phage ValSw3-3 (Vibrio phage ValSw3-3) provided by the present invention has a preservation number of CCTCC NO: M 2017504;
上述菌株的保藏地址均为：湖北省武汉市武昌区八一路珞珈山，武汉大学中国典型培养物保藏中心；保藏时间均为：2017年9月14日。均经保藏中心于2017年9月14日检测为存活菌株。The preservation addresses of the above strains are: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Typical Culture Collection Center of Wuhan University; the preservation time is September 14, 2017. All were detected as viable strains by the preservation center on September 14, 2017.
具体实施方式Detailed ways
本发明针对由弧菌引起的水产养殖上鱼虾类疾病大肆传播的问题上，在抗生素过度使用引起弧菌耐药性菌株产生的基础上，从患病鱼虾病灶中分离致病性弧菌，并通过现场实验确定了该弧菌病原菌的致病性，以该致病性弧菌为宿主，从水环境中分离得到6株新的裂性弧菌噬菌体，通过形态观察，确定了该弧菌噬菌体的分类地位，并通过全基因组测序确定了该6株噬菌体的基因型。此外，通过平板点滴法，根据透明斑的有无，测定了弧菌噬菌体在29株不同种属菌株下的宿主谱。通过认识单一噬菌体对宿主菌的侵染特性，进一步评估了单株噬菌体在不同梯度感染复数(MOI)下的抑菌能力，进而根据各个噬菌体最病原性弧菌的裂解能力组合了5个噬菌体鸡尾酒组合，通过在单株噬菌体条件下确定的MOI从而进一步评估了该5个组合对病原性弧菌的生长抑制作用，由此我们了确定最佳的弧菌噬菌体鸡尾酒组合。我们该噬菌体在控制由弧菌引起的水产动物致病菌具有一定的潜力，应用空间广泛。The invention aims at the problem of widespread spread of fish and shrimp diseases caused by Vibrio in aquaculture, and isolates pathogenic Vibrio from diseased fish and shrimp lesions on the basis of the generation of Vibrio drug-resistant strains caused by excessive use of antibiotics , and determined the pathogenicity of the Vibrio pathogenic bacteria through field experiments, using the pathogenic Vibrio as the host, isolated 6 new strains of Vibrio phage from the water environment, and determined the Vibrio phage through morphological observation. The taxonomic status of bacteriophages was determined, and the genotypes of the six phages were determined by whole genome sequencing. In addition, the host spectrum of Vibrio phages in 29 strains of different species was determined according to the presence or absence of transparent spots by plate spot method. By understanding the infection characteristics of a single phage to host bacteria, the antibacterial ability of a single phage at different gradient multiplicity of infection (MOI) was further evaluated, and then five phage cocktails were combined according to the lysing ability of each phage to the most pathogenic Vibrio Combinations, the growth inhibitory effect of the five combinations on pathogenic Vibrio was further evaluated through the MOI determined under the condition of a single phage, so we determined the best Vibrio phage cocktail combination. Our phage has certain potential in controlling pathogenic bacteria in aquatic animals caused by Vibrio, and has a wide range of applications.
本发明一方面涉及一种溶藻弧菌噬菌体ValDsh1-1，于2017年9月14日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO:M 2017499，分类命名为Vibrio phage。One aspect of the present invention relates to a Vibrio alginolyticus phage ValDsh1-1, which was preserved in the China Center for Type Culture Collection on September 14, 2017, with the preservation number: CCTCC NO: M 2017499, and the classification name is Vibrio phage.
本发明所提供的溶藻弧菌噬菌体ValDsh1-1在治疗弧菌感染性疾病中具有很多优势，首先其可对耐药菌也具有很好的防治效果；其次，由于噬菌体的杀菌谱较窄，在复杂细菌的环境中——例如动物肠道——发生杀菌作用时，不会对益生菌造成伤害；第三，其效价较高，防治效果好。The alginolytic Vibrio phage ValDsh1-1 provided by the present invention has many advantages in the treatment of Vibrio infectious diseases, first of all it can also have a good control effect on drug-resistant bacteria; secondly, due to the narrow bactericidal spectrum of the phage, In the environment of complex bacteria - such as animal intestines - when the bactericidal effect occurs, it will not cause harm to the probiotics; third, its potency is high and the control effect is good.
然而细菌能通过自然筛选或人工筛选对抗生素产生抗药性，同理，其也能对单一的噬菌体逐渐耐受。有鉴于此，本发明通过认识单一噬菌体对于侵染宿主的特性，评估单一噬菌体的感染能力，采用一定的混合方法构建噬菌体鸡尾酒，最后通过特定指标确定最佳的鸡尾酒组成，该噬菌体鸡尾酒既具有噬菌体的专一性特征，又弥补了单一噬菌体疗法易导致宿主菌抗性的不足，应用前景广泛。However, bacteria can develop resistance to antibiotics through natural selection or artificial selection, and similarly, they can also gradually tolerate a single phage. In view of this, the present invention evaluates the infection ability of a single phage by understanding the characteristics of a single phage for infecting a host, adopts a certain mixing method to construct a phage cocktail, and finally determines the optimal cocktail composition through specific indicators. The phage cocktail has both phage The specificity of the phage therapy makes up for the deficiency that the single phage therapy can easily lead to the resistance of the host bacteria, and has a broad application prospect.
根据本发明的一方面，本发明还涉及含有如上所述的溶藻弧菌噬菌体ValDsh1-1的杀菌组合物。According to one aspect of the present invention, the present invention also relates to a bactericidal composition containing the above-mentioned Vibrio alginolyticus phage ValDsh1-1.
在本发明的一些实施方式中，所述杀菌组合物含有所述溶藻弧菌噬菌体ValDsh1-1以及其它弧菌噬菌体中的至少一种；优选为裂性弧菌噬菌体。In some embodiments of the present invention, the bactericidal composition contains at least one of the Vibrio alginolyticus phage ValDsh1-1 and other Vibrio phages; preferably the Vibrio phage.
由于同种病毒(噬菌体)之间的感染途径、分子基础均具有很大的相似性，因此当同种噬菌体共同感染宿主时彼此间可能会促进感染效率，发挥协同作用。Since the infection pathways and molecular basis of the same type of viruses (phages) are very similar, when the same type of phages co-infect the host, they may promote the infection efficiency and play a synergistic effect.
在本发明的一些实施方式中，所述杀菌组合物还包括副溶血弧菌噬菌体VpaSw-1，保藏号为：CCTCC NO:M 2017500；In some embodiments of the present invention, the bactericidal composition further includes Vibrio parahaemolyticus phage VpaSw-1, the preservation number is: CCTCC NO: M 2017500;
副溶血弧菌噬菌体VpaSw-2，保藏号为CCTCC NO:M 2017501；Vibrio parahaemolyticus phage VpaSw-2, the preservation number is CCTCC NO:M 2017501;
溶藻弧菌噬菌体ValSw3-1，保藏号为CCTCC NO:M 2017502；Vibrio alginolyticus phage ValSw3-1, the preservation number is CCTCC NO:M 2017502;
溶藻弧菌噬菌体ValSw3-2，保藏号为CCTCC NO:M 2017503；以及Vibrio alginolyticus phage ValSw3-2, the preservation number is CCTCC NO:M 2017503; and
溶藻弧菌噬菌体ValSw3-3，保藏号为CCTCC NO:M 2017504中的一种或多种；Vibrio alginolyticus bacteriophage ValSw3-3, the preservation number is one or more of CCTCC NO:M 2017504;
上述噬菌体均于2017年9月14日保藏于中国典型培养物保藏中心，分类命名均为Vibrio phage。The above-mentioned phages were all preserved in the China Center for Type Culture Collection on September 14, 2017, and all of them were named Vibrio phage.
本发明提供的噬菌体杀菌组合物可在短时间内快速抑制弧菌的生长，并抑制该病原菌噬菌体抗性的产生。此外，该噬菌体鸡尾酒安全、有效，且特异性强，生产成本低，在致病菌的控制过程中弥补了单一噬菌体疗法的不足。The phage bactericidal composition provided by the invention can quickly inhibit the growth of Vibrio in a short time and inhibit the generation of phage resistance of the pathogenic bacteria. In addition, the phage cocktail is safe, effective, and highly specific, with low production cost, and makes up for the deficiency of single phage therapy in the process of controlling pathogenic bacteria.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1以及VpaSw-1。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1 and VpaSw-1.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1以及VpaSw-2。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1 and VpaSw-2.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1以及ValSw3-1。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1 and ValSw3-1.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1以及ValSw3-2。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1 and ValSw3-2.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1、VpaSw-2以及ValSw3-1。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1, VpaSw-2 and ValSw3-1.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1、VpaSw-2以及ValSw3-2。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1, VpaSw-2 and ValSw3-2.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1、VpaSw-2以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1, VpaSw-2 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1、ValSw3-1以及ValSw3-2。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1, ValSw3-1 and ValSw3-2.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1、ValSw3-1以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1, ValSw3-1 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1、ValSw3-2以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1, ValSw3-2 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1、VpaSw-2、ValSw3-1以及ValSw3-2。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1, VpaSw-2, ValSw3-1 and ValSw3-2.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1、VpaSw-2、ValSw3-1以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1, VpaSw-2, ValSw3-1 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1、VpaSw-2、ValSw3-2以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1, VpaSw-2, ValSw3-2 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1以及VpaSw-2。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1 and VpaSw-2.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-2以及ValSw3-1。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-2 and ValSw3-1.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-2以及ValSw3-2。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-2 and ValSw3-2.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-2以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-2 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-2、ValSw3-1以及ValSw3-2。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-2, ValSw3-1 and ValSw3-2.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-2、ValSw3-1以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-2, ValSw3-1 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-2、ValSw3-2以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-2, ValSw3-2 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1以及ValSw3-1。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1 and ValSw3-1.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1以及ValSw3-1。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1 and ValSw3-1.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、ValSw3-1以及ValSw3-2。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, ValSw3-1 and ValSw3-2.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、ValSw3-1以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, ValSw3-1 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、ValSw3-1、ValSw3-2以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, ValSw3-1, ValSw3-2 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1以及ValSw3-2。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1 and ValSw3-2.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、ValSw3-2以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, ValSw3-2 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物中的有效成分主要为弧菌噬菌体ValDsh1-1、VpaSw-1、VpaSw-2、ValSw3-1、ValSw3-2以及ValSw3-3。In some embodiments of the present invention, the active ingredients in the bactericidal composition are mainly Vibrio phages ValDsh1-1, VpaSw-1, VpaSw-2, ValSw3-1, ValSw3-2 and ValSw3-3.
在本发明的一些实施方式中，所述杀菌组合物还包括ValDsh1-1的突变体、VpaSw-1的突变体、VpaSw-2的突变体、VpaSw-3-1的突变体、VpaSw-3-2的突变体以及VpaSw-3-3的突变体中的一种或多种。In some embodiments of the present invention, the bactericidal composition also includes mutants of ValDsh1-1, mutants of VpaSw-1, mutants of VpaSw-2, mutants of VpaSw-3-1, mutants of VpaSw-3- 2 mutants and one or more of VpaSw-3-3 mutants.
优选的，所述突变体序列至少90％与相应的噬菌体的天然序列相同。Preferably, the mutant sequence is at least 90% identical to the corresponding native sequence of the phage.
由于病毒在复制过程中非常容易发生突变，因而优选的，上述噬菌体的突变体也在本申请请求保护的范围内。通过现有技术中公知的计算机程序可以适当地测定同源性，例如GCG程序包中提供的缺口程序(威斯康星软件包程序手册，第8版，1994年8月，遗传学计算机小组，575科学道，麦迪逊市，威斯康星州，美国53711)(Needleman，S.B.与Wunsch，C.D.，(1970)《分子生物学杂质》48,443-453)。通过用于DNA序列比较的下列设置来使用缺口程序：缺口产生罚分5.0，缺口延伸罚分0.3。ValDsh1-1、VpaSw-1、VpaSw-2、ValSw3-1、ValSw3-2以及ValSw3-3的突变体至少90％与所述噬菌体的天然序列相同；且所述突变体具有与最初的噬菌体大致相同的灭杀病原菌的功能。更优选的，突变体有92％、94％、95％、96％、97％、98％或99％与各自对应的噬菌体的天然序列相同。其中，ValDsh1-1、VpaSw-1、VpaSw-2、ValSw3-1、ValSw3-2以及ValSw3-3的序列可根据本发明保藏的生物材料通过公知的方法测序得到。Since the virus is very prone to mutations during the replication process, preferably, the mutants of the above-mentioned phages are also within the protection scope of the present application. Homology can be suitably determined by computer programs known in the art, such as the Gap program provided in the GCG package (Wisconsin Package Program Manual, 8th Edition, August 1994, Genetics Computer Group, 575 Science Lane , Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, C.D., (1970) Impurities in Molecular Biology 48, 443-453). The Gap program was used with the following settings for DNA sequence comparison: gap creation penalty 5.0, gap extension penalty 0.3. The mutants of ValDsh1-1, VpaSw-1, VpaSw-2, ValSw3-1, ValSw3-2, and ValSw3-3 are at least 90% identical to the native sequence of the phage; and the mutants have approximately the same The function of killing pathogenic bacteria. More preferably, the mutants are 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the native sequence of the respective corresponding phage. Among them, the sequences of ValDsh1-1, VpaSw-1, VpaSw-2, ValSw3-1, ValSw3-2 and ValSw3-3 can be sequenced according to the biological materials preserved in the present invention by known methods.
噬菌体ValDsh1-1、VpaSw-1、VpaSw-2、ValSw3-1、ValSw3-2以及ValSw3-3的突变体可为点突变、缺失突变或添加突变，相对于最初的噬菌体序列，有1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或更多碱基可发生变化。对于本领域技术人员来说，根据本发明提供的噬菌体筛选出与其性状相似的突变体并不需要付出创造性的劳动。The mutants of phage ValDsh1-1, VpaSw-1, VpaSw-2, ValSw3-1, ValSw3-2 and ValSw3-3 can be point mutations, deletion mutations or addition mutations, relative to the original phage sequence, there are 1, 2 1, 3, 4, 5, 6, 7, 8, 9, 10 or more bases may vary. For those skilled in the art, screening out mutants with similar traits from the phage provided by the present invention does not require creative work.
如上所述，噬菌体可以以足够高的浓度存在以诱导溶菌作用，当混配成混合物时，优选的，如上所述的杀菌组合物，所述组合物中每种弧菌噬菌体的含量≥106PFU/mL；As mentioned above, the phage can be present in a sufficiently high concentration to induce bacteriolysis. When mixed into a mixture, preferably, the above-mentioned bactericidal composition, the content of each Vibrio phage in the composition is ≥ 10 6 PFU/mL;
优选的，如上所述的杀菌组合物，所述组合物中每种弧菌噬菌体的含量为106PFU/mL～1010PFU/mL，更优选为106PFU/mL～109PFU/mL，更优选为106PFU/mL～108PFU/mL，更优选为106PFU/mL～107PFU/mL，还可以取2×106PFU/mL，3×106PFU/mL，4×106PFU/mL，5×106PFU/mL，6×106PFU/mL，7×106PFU/mL，8×106PFU/mL，9×106PFU/mL，5×107PFU/mL，5×108PFU/mL，5×109PFU/mL等中间数值。Preferably, the above-mentioned bactericidal composition, the content of each Vibrio phage in the composition is 10 6 PFU/mL～10 10 PFU/mL, more preferably 10 6 PFU/mL～10 9 PFU/mL , more preferably 10 6 PFU/mL-10 8 PFU/mL, more preferably 10 6 PFU/mL-10 7 PFU/mL, and can also be 2×10 6 PFU/mL, 3×10 6 PFU/mL, 4×10 6 PFU/mL, 5×10 6 PFU/mL, 6×10 6 PFU/mL, 7×10 6 PFU/mL, 8×10 6 PFU/mL, 9×10 6 PFU/mL, 5× 10 7 PFU/mL, 5×10 8 PFU/mL, 5×10 9 PFU/mL and other intermediate values.
优选的，当ValDsh1-1与VpaSw-1、VpaSw-2、ValSw3-1、ValSw3-2以及ValSw3-3中的一种或多株进行混配时，按感染复数等比的方式进行混合。Preferably, when ValDsh1-1 is mixed with one or more strains of VpaSw-1, VpaSw-2, ValSw3-1, ValSw3-2 and ValSw3-3, the mixture is carried out in a manner of equal multiplicity of infection.
优选的，如上所述的杀菌组合物，所述杀菌组合物还包括辅料；Preferably, the bactericidal composition as described above, the bactericidal composition also includes auxiliary materials;
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；The auxiliary material is one or more of SM buffer, sodium alginate, sucrose, maltodextrin, and glucose;
SM缓冲液的配制方法为常规方法，例如：NaCl 5.8g，MgSO4·7H2O 2g，1mol/L的Tris·HCl 50mL(pH＝7.0)，5mL 2％gelatin，加入纯净水补足至1000mL。The preparation method of SM buffer is a conventional method, for example: NaCl 5.8g, MgSO 4 ·7H 2 O 2g, 1mol/L Tris·HCl 50mL (pH=7.0), 5mL 2% gelatin, add purified water to make up to 1000mL.
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。Preferably, the bactericidal composition further includes phages of specific pathogenic bacteria of different types of bacteria.
上述杀菌组合物可作为病毒制剂使用，所用剂型可为各种常见剂型，例如粉剂、水剂、冻干剂、凝胶剂、霜剂、膏剂等。The above-mentioned bactericidal composition can be used as a virus preparation, and the dosage forms used can be various common dosage forms, such as powder, water, lyophilized agent, gel, cream, ointment and the like.
优选的，如上所述的杀菌组合物在杀灭和/或预防弧菌属微生物中的应用；所述应用为治疗用途或非治疗用途；Preferably, the application of the above-mentioned bactericidal composition in killing and/or preventing Vibrio microorganisms; the application is a therapeutic use or a non-therapeutic use;
优选的，所述弧菌属微生物包括溶藻弧菌V.alginolyticus、鳗弧菌V.anguillarum、霍乱弧菌V.cholerae、非O1群霍乱弧菌non-O1V.cholerae、费氏弧菌V.fischeri、河流弧菌V.fluvialis、哈维氏弧菌V.harveyi、病海鱼弧菌V.ordalii、副溶血弧菌V.parahaemolyticus、杀鲑弧菌V.salmonicida、拟态弧菌V.mimicus、灿烂弧菌V.splendidus、金黄色弧菌V.azureus、产气弧菌V.gazogenes、创伤弧菌V.vulnificus、竹荚鱼弧菌V.trachuri、雀鲷弧菌V.damsela、漂浮弧菌V.natriegen、沙蚕弧菌V.nereis、鱼肠道弧菌V.ichthyoenteri、鲨鱼弧菌V.carchariae、坎氏弧菌V.campbellii以及杀对虾弧菌V.penaeicida中的一种或多种；Preferably, the Vibrio microorganisms include Vibrio alginolyticus V.alginolyticus, Vibrio anguillarum V.anguillarum, Vibrio cholerae V.cholerae, non-O1 group Vibrio cholerae non-O1V.cholerae, Vibrio fischeri V. fischeri, V.fluvialis, V.harveyi, V.ordalii, V.parahaemolyticus, V.salmonicida, V.mimicus, splendid V. splendidus, V. azureus, V. gazogenes, V. vulnificus, V. trachuri, V. damsela, V. floating One or more of .natriegen, V. nereis, V. ichthyoenteri, V. carchariae, V. campbellii and V. penaeicida;
更优选的，所述弧菌属微生物包括副溶血弧菌V.parahaemolyticus、溶藻弧菌V.alginolyticus、金黄色弧菌V.azureus。More preferably, the Vibrio microorganisms include V.parahaemolyticus, V.alginolyticus, and V.azureus.
优选的，如上所述的溶藻弧菌噬菌体、或杀菌组合物在制备用于治疗和/或预防动物弧菌病的药物中的应用；Preferably, the above-mentioned Vibrio alginolyticus phage, or the application of the bactericidal composition in the preparation of medicines for the treatment and/or prevention of vibriosis in animals;
优选的，如上所述的应用，所述动物包括：温血性动物和部分冷血性动物；Preferably, in the above application, the animals include: warm-blooded animals and some cold-blooded animals;
优选的，如上所述的应用，所述动物为人、鱼类、虾或软体动物(例如贝类)。Preferably, in the above-mentioned application, the animal is human, fish, shrimp or mollusk (such as shellfish).
一种防治动物弧菌病的方法，将如上所述的杀菌组合物作为药物添加到动物饲料中，或对动物体表喷雾，或给动物灌服，或给动物注射，或将所述杀菌组合物溶于水中再与动物接触。A method for preventing and treating vibriosis in animals, comprising adding the above-mentioned bactericidal composition to animal feed as a medicine, or spraying on the animal body surface, or feeding the animal, or injecting the animal, or adding the bactericidal composition dissolved in water before contacting animals.
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.
实施例Example
本发明通过采集水产养殖厂患病的鱼虾类样品，从其病灶中分离得到6株致病性弧菌，本发明噬菌体的筛选流程图如图1所示。通过现场实验证实了分离弧菌的致病性，确定了其中5株致病性极显著，LD50<104CFU/mL，作用时间<100h。其次，以致病性弧菌为宿主菌，从环境中分离得到6株噬菌体，裂解直径范围为0.3mm～1mm，其中最大为0.89mm。通过全基因组测序得到本次分离得到的噬菌体为弧菌噬菌体，与基因组数据库比对结果(相似度低于70％，覆盖度低于4％，两两之间比对相似度低于90％)显示该噬菌体均为新的弧菌噬菌体。采用磷钨酸负染法，透射电子显微镜下观察确定了该弧菌噬菌体属于有尾噬菌体目(Caudovirales)下的长尾噬菌体科(Siphoviridae)，弧菌噬菌体形态如图2所示。In the present invention, 6 strains of pathogenic Vibrio are obtained by collecting diseased fish and shrimp samples from aquaculture plants, and the screening flow chart of the bacteriophage of the present invention is shown in FIG. 1 . The pathogenicity of the isolated Vibrio was confirmed by field experiments, and 5 of them were determined to be extremely pathogenic, with LD 50 <10 4 CFU/mL and action time <100h. Secondly, with pathogenic Vibrio as the host bacteria, 6 strains of phage were isolated from the environment, and the cleavage diameter ranged from 0.3mm to 1mm, and the largest was 0.89mm. Through whole genome sequencing, the phage isolated this time is a Vibrio phage, and compared with the genome database (similarity is less than 70%, coverage is less than 4%, and the similarity between pairs is less than 90%) It was shown that the phages were all new Vibrio phages. Using the phosphotungstic acid negative staining method, it was confirmed under the transmission electron microscope that the Vibrio phage belonged to the long-tailed phage family (Siphoviridae) under the order Caudovirales. The morphology of the Vibrio phage is shown in Figure 2.
通过平板点滴法，根据透明斑的有无，测定了该弧菌噬菌体在29株不同种属菌株下的宿主谱(表1)，得出该噬菌体具有很强的宿主专一性，为弧菌属专一性裂性噬菌体。Through the plate spot method, according to the presence or absence of transparent spots, the host spectrum of the Vibrio phage under 29 strains of different genus was measured (Table 1), and it was concluded that the phage had strong host specificity and was a Vibrio phage. A specific split phage.
表1宿主谱Table 1 host spectrum
备注：“+”表示能裂解，“-”表示不能裂解；以上菌株均从水产养殖鱼虾类动物体及养殖水中分离得到；ValDsh1-1，VpaSw-1，VpaSw-2，ValSw3-1，ValSw3-2和ValSw3-3表示弧菌噬菌体编号；“*”表示经检测具有致病性的宿主菌，其进化树如图3所示。Remarks: "+" means it can be lysed, "-" means it can't be lysed; the above strains are all isolated from aquaculture fish and shrimp animals and aquaculture water; ValDsh1-1, VpaSw-1, VpaSw-2, ValSw3-1, ValSw3 -2 and ValSw3-3 indicate the number of Vibrio phage; "*" indicates the detected pathogenic host bacteria, and its evolution tree is shown in Figure 3.
通过认识单一噬菌体对宿主菌的侵染特性，从而评估了宿主谱最广的3株噬菌体在不同梯度感染复数(MOI＝100，10，1，0.1，0.01)下的单株噬菌体对宿主菌的抑菌能力，得出单株条件下当MOI＝10时，其对病原性弧菌的抑制作用显著且持续最长，在15h内OD600低于对照组，当MOI≤1时抑制效果不显著，由此确定了该弧菌噬菌体的应用条件MOI＝10。根据6株噬菌体的对致病性弧菌的裂解作用及宿主谱特性，确定了5个噬菌体鸡尾酒组合：A(ValSw3-1,ValDsh1-1)；B(ValSw3-1,VpaSw-1)；C(ValSw3-1,VpaSw-2)；D(ValSw3-1,ValSw3-2，ValSw3-3，VpaSw-1)；E(ValSw3-1,ValSw3-2，ValSw3-3，VpaSw-2，ValDsh1-1)，各组合之间以1:1比例添加。并在MOI＝10条件下评价上述5个组合对致病性弧菌的生长抑制作用。综合考虑各噬菌体鸡尾酒组合对6株致病性弧菌的抑制效果，确定了组合D(ValSw3-1,ValSw3-2，ValSw3-3，VpaSw-1)为最优组合，与对照组比较，对相应致病菌的浓度(OD600)起到了抑制作用，并维持该水平10h以上。由此，我们认为由以上6株噬菌体组成的噬菌体鸡尾酒在控制由弧菌引起的水产动物致病菌具有一定的潜力，应用空间广泛。By understanding the infection characteristics of a single phage to the host bacteria, the three phages with the broadest host spectrum were evaluated at different gradient multiplicity of infection (MOI=100, 10, 1, 0.1, 0.01). Bacteriostatic ability, it can be concluded that under single plant conditions, when MOI=10, its inhibitory effect on pathogenic Vibrio is significant and lasts the longest, and the OD600 is lower than that of the control group within 15 hours. When MOI≤1, the inhibitory effect is not significant. Thus, the application condition MOI=10 of the Vibrio phage was determined. According to the lysis of pathogenic Vibrio and host spectrum characteristics of six phage strains, five phage cocktail combinations were determined: A (ValSw3-1, ValDsh1-1); B (ValSw3-1, VpaSw-1); C (ValSw3-1, VpaSw-2); D (ValSw3-1, ValSw3-2, ValSw3-3, VpaSw-1); E (ValSw3-1, ValSw3-2, ValSw3-3, VpaSw-2, ValDsh1-1 ), each combination is added at a ratio of 1:1. And under the condition of MOI=10, the growth inhibitory effect of the above five combinations on pathogenic Vibrio was evaluated. Comprehensively considering the inhibitory effect of each phage cocktail combination on six pathogenic Vibrio strains, the combination D (ValSw3-1, ValSw3-2, ValSw3-3, VpaSw-1) was determined to be the optimal combination. The concentration of the corresponding pathogenic bacteria (OD600) played an inhibitory role and maintained this level for more than 10h. Therefore, we believe that the phage cocktail composed of the above six phages has certain potential in controlling the pathogenic bacteria in aquatic animals caused by Vibrio, and has a wide range of applications.
具体的，本发明所提供的溶藻弧菌噬菌体及杀菌组合物通过以下方法筛选得到：Specifically, the Vibrio alginolyticus phage and the bactericidal composition provided by the present invention are screened by the following methods:
致病性弧菌的筛选：采集水产养殖厂患病的鱼虾类样品，采用选择性培养基从发病的鱼虾病灶中分离纯化得到6株致病性弧菌，试剂盒法(Omega细菌DNA提取试剂盒)提取单株菌株的基因组DNA，通过16S测序，与最大相似序列构建进化树，进而确定该6株弧菌的分类地位。Screening of pathogenic Vibrio: collect diseased fish and shrimp samples from aquaculture plants, use selective medium to isolate and purify 6 strains of pathogenic Vibrio from diseased fish and shrimp lesions, kit method (Omega bacterial DNA extraction kit) to extract the genomic DNA of a single strain, through 16S sequencing, construct a phylogenetic tree with the most similar sequence, and then determine the taxonomic status of the six Vibrio strains.
弧菌致病性检测：通过现场实验，以中期对虾为实验对象，评价了8株弧菌对对虾的致死能力。每组设定30只对虾，每24h观察一次，共观察1周。Detection of Vibrio pathogenicity: through field experiments, the lethality of 8 strains of Vibrio to prawns was evaluated with medium-term prawns as the experimental object. Set 30 prawns in each group and observe once every 24 hours for a total of 1 week.
噬菌体筛选：以步骤1、2中确认的6株致病性弧菌为宿主，采用双层平板法并结合样品特征从水环境中分离噬菌体，通过多次感染确定透明斑，经分离纯化后，单株噬菌体于-80℃甘油(20％)保存。Phage screening: take the 6 strains of pathogenic Vibrio identified in steps 1 and 2 as hosts, isolate phages from the water environment by using the double-layer plate method combined with sample characteristics, and determine the transparent spots through multiple infections. After separation and purification, Individual phages were stored in glycerol (20%) at -80°C.
基因组测序：通过对单株噬菌体的富集培养(1012PFU/mL)后，在4℃条件下8000g离心15min，加入10％PEG8000和0.5M NaCl静置过夜，再加入等量氯仿混匀，静置分层后5000g下离心10min，去除氯仿层和PEG层后，加入限制性核酸内切酶(Dnase I和Rnase A)消化处理，并采用梯度密度氯化铯条件下悬浮噬菌体，后期采用TM缓冲液透析3次，每次30min，最后将透析好的噬菌体一部分预留做电镜观察，一部分采用λ噬菌体基因组DNA快速提取试剂盒(Aidlab Biotechnologies Co.,Ltd)进行噬菌体基因组DNA的抽提，将抽提好的基因组DNA通过Nano Drop测定浓度后，用不同限制性内切酶(Takara，宝生物工程有限公司)酶切，电泳检测观察酶切图谱，确定噬菌体基因组非环状后，样品送华大基因生物测序公司进行全基因组测序。Genome sequencing: after the enrichment culture of a single phage (10 12 PFU/mL), centrifuge at 8000g for 15min at 4°C, add 10% PEG8000 and 0.5M NaCl and let stand overnight, then add an equal amount of chloroform to mix. After static layering, centrifuge at 5000g for 10min, remove the chloroform layer and PEG layer, add restriction endonuclease (DNase I and Rnase A) for digestion, and use gradient density cesium chloride to suspend the phage, and then use TM The buffer solution was dialyzed 3 times, each time for 30 min. Finally, part of the dialyzed phage was reserved for electron microscope observation, and part of the phage genomic DNA was extracted using the lambda phage genomic DNA rapid extraction kit (Aidlab Biotechnologies Co., Ltd). After the concentration of the extracted genomic DNA was measured by Nano Drop, it was digested with different restriction endonucleases (Takara, Takara Bioengineering Co., Ltd.), detected by electrophoresis to observe the restriction map, and after confirming that the phage genome was non-circular, the sample was sent to China Big Genome Biosequencing Company conducts whole genome sequencing.
形态观察：对步骤4中透析好的噬菌体，选用磷钨酸进行负染处理后于透射电镜下观察噬菌体大小和形态。Morphological observation: For the dialyzed phages in step 4, use phosphotungstic acid for negative staining treatment, and then observe the size and shape of the phages under a transmission electron microscope.
宿主谱测定：采用点滴法，通过观察宿主平板上透明斑形成有无，确定了其对该菌株的裂解性。Determination of host spectrum: the lysis of the strain was determined by observing the formation of transparent spots on the host plate by spot method.
感染能力评价：以不同梯度MOI(MOI＝10，1，0.1，0.01，0.001)混合噬菌体和对数期宿主菌(OD600＝0.3)，采用生长测定仪检测18h内宿主菌的OD600变化，每隔10min记录一次。其中，不同感染复数(MOI)下ValSw3-1、ValSw3-2、ValSw3-3对溶藻弧菌V.alginolyticus的生长曲线作用结果如图4所示。Evaluation of infectivity: Mix phage and logarithmic phase host bacteria (OD600 = 0.3) with different gradient MOI (MOI = 10, 1, 0.1, 0.01, 0.001), and use a growth analyzer to detect the OD600 change of the host bacteria within 18 hours. Record once every 10 minutes. Among them, the results of the effects of ValSw3-1, ValSw3-2, and ValSw3-3 on the growth curve of Vibrio alginolyticus V. alginolyticus at different multiplicity of infection (MOI) are shown in Fig. 4 .
弧菌噬菌体鸡尾酒组合：根据6株噬菌体对致病性弧菌的裂解谱，得到5个组合(图5)，分别为：A(ValSw3-1,ValDsh1-1)；B(ValSw3-1,VpaSw-1)；C(ValSw3-1,VpaSw-2)；D(ValSw3-1,ValSw3-2，ValSw3-3，VpaSw-1)；E(ValSw3-1,ValSw3-2，ValSw3-3，VpaSw-2，ValDsh1-1)。图5中af、bd属于同种下的不同株，对动物的致病性不同。各组合的宿主谱均能覆盖筛选得出的致病性弧菌，组合中各噬菌体之间的比例为1:1。Vibrio phage cocktail combinations: According to the lysis profiles of 6 strains of phages against pathogenic Vibrio, 5 combinations were obtained (Figure 5), respectively: A(ValSw3-1,ValDsh1-1); B(ValSw3-1,VpaSw -1); C(ValSw3-1, VpaSw-2); D(ValSw3-1, ValSw3-2, ValSw3-3, VpaSw-1); E(ValSw3-1, ValSw3-2, ValSw3-3, VpaSw- 2, ValDsh1-1). In Figure 5, af and bd belong to different strains of the same species, and have different pathogenicity to animals. The host spectrum of each combination can cover the pathogenic Vibrio obtained by screening, and the ratio of each phage in the combination is 1:1.
最佳噬菌体鸡尾酒组合的筛选：通过在单株噬菌体条件下确定的MOI，评估了各组合对病原性弧菌的生长抑制作用，以短时间内抑制弧菌生长，抑制效果持续时间最长以及弧菌抗性出现时间最晚为要求，得出最佳的弧菌噬菌体鸡尾酒组合。Screening of the best phage cocktail combination: The growth inhibitory effect of each combination on pathogenic Vibrio was evaluated through the MOI determined under the condition of a single phage. The latest requirement for the emergence of bacterial resistance is to obtain the best Vibrio phage cocktail combination.
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。It should be noted that at last: above each embodiment is only in order to illustrate technical scheme of the present invention, and is not intended to limit; Although the present invention has been described in detail with reference to foregoing each embodiment, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. range.
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