铜绿假单胞菌裂解性噬菌体YH6的分离鉴定及其生物学特性研究

发布时间：2018-04-13 01:14

本文选题：铜绿假单胞菌 + 噬菌体　；参考：《吉林大学》2015年硕士论文

【摘要】：铜绿假单胞菌是一种在环境中广泛存在的革兰氏阴性致病菌。该菌可以引起水貂严重的出血性肺炎，给水貂养殖业带来了巨大的损失。近些年，由于多重耐药铜绿假单胞菌的出现，使该菌引起感染的控制变得越来越困难。噬菌体是可以特异性感染细菌的病毒，可以作为一种新型的治疗制剂，在治疗多重耐药菌株感染方面具有很大的应用潜力。本研究以实验室前期在貂场患病水貂体内分离的致病性铜绿假单胞菌菌株D9作为宿主菌，从污水中分离获得了一株新的噬菌体，将其命名为YH6。对噬菌体YH6的生物特性进行了研究，表明该噬菌体是可以特异性的裂解铜绿假单胞菌的烈性噬菌体，噬菌斑清晰透亮、无晕环；该噬菌体属于有尾病毒目短尾病毒科，头部呈正二十面体，头部直径大约是65nm，尾部较短，，大约是25nm；其潜伏期为30min，裂解周期为60min；最佳感染复数为0.01-0.001，噬菌体滴度能达到1010PFU/mL；该噬菌体对乙醚、氯仿不敏感；在温度40°C、50°C环境中和pH值6.0~10.0时保持稳定；YH6可以裂解82.61%的铜绿假单胞菌，表现出了广谱的裂解活性。对噬菌体YH6进行了全基因组序列测序和分析，表明该噬菌体的遗传物质为双链线性DNA，长度为73,050bp，具有90个开放阅读框（ORFs），G+C%含量为54.88%，其基因编码率为92.64%，与铜绿假单胞菌的噬菌体LUZ7，vB_PaeP_C2-10_Ab09，PA26和LIT1的基因组同源性较近。YH6基因组中不含有细菌毒力和溶源性相关的ORF，更利于该噬菌体作为治疗制剂应用。通过序列比对和分析确定了噬菌体YH6的裂解酶基因（LysYH6），对该基因进行表达和纯化，活性测定实验表明裂解酶LysYH6在EDTA（25mM/L）存在的条件下表现出显著的杀菌活性。动物实验表明，对小鼠进行鼻腔接种铜绿假单胞菌D9（2×108CFU），能够成功构建小鼠的出血性肺炎模型。治疗实验表明一次性鼻腔应用噬菌体YH6（2×107PFU，MOI=0.1）可以有效保护感染铜绿假单胞菌造成的出血性肺炎小鼠模型。对治疗组小鼠血液、脾脏、肺脏进行了菌落计数，表明噬菌体YH6应用10h以后可以使脏器内的菌含量降低1.5个对数单位，2d以内可以将体内的铜绿假单胞菌完全清除；与此相反地，未治疗组脏器内菌含量升高4个对数单位，并在2d内引起死亡。另外，对治疗组小鼠的肺脏进行了组织病理学切片观察，表明YH6能有效的改善铜绿假单胞菌造成的小鼠肺损伤。综上，本研究所分离到的噬菌体YH6在治疗铜绿假单胞菌引起的小鼠出血性肺炎模型方面表现出很大的应用潜力。为后续研究将该噬菌体应用到水貂感染铜绿假单胞菌的治疗奠定了坚实的基础。
[Abstract]:Pseudomonas aeruginosa is a gram-negative pathogenic bacteria widely found in the environment.The bacteria can cause severe hemorrhagic pneumonia in minks and bring huge losses to mink breeding.In recent years, the emergence of multidrug resistant Pseudomonas aeruginosa makes it more and more difficult to control the infection caused by Pseudomonas aeruginosa.Bacteriophage is a virus that can infect bacteria specifically. It can be used as a new therapeutic agent and has great application potential in the treatment of multidrug resistant strains infection.In this study, a new bacteriophage was isolated from waste water of pathogenic Pseudomonas aeruginosa strain D9, which was isolated from mink farm in laboratory and named YH6.The biological characteristics of bacteriophage YH6 were studied. The results showed that the bacteriophage was a potent bacteriophage capable of specific cleavage of Pseudomonas aeruginosa, and the plaque of phage was clear and without halo ring, and the phage belonged to the family Caudoviridae.The head has a positive icosahedron, the head diameter is about 65 nm, the tail is short (about 25 nm), the incubation period is 30 min, the cleavage period is 60 min, the optimal number of infection is 0.01-0.001, the phage titer can reach 1010 PFU / mL, the phage is insensitive to ether and chloroform.At the temperature of 40 掳C ~ (50 掳C) and pH value of 6.0 ~ 10.0, the stable YH6 could decompose 82.61% of Pseudomonas aeruginosa, showing broad-spectrum pyrolysis activity.The whole genome sequence of phage YH6 was sequenced and analyzed.There was no ORF associated with bacterial virulence and lysogenesis in the group, which was more favorable for the application of the bacteriophage as a therapeutic agent.The lyase gene of bacteriophage YH6, LysYH6, was identified by sequence alignment and analysis, and the gene was expressed and purified. The activity test showed that the lyase LysYH6 exhibited significant bactericidal activity in the presence of EDTA 25 mm / L.Animal experiments showed that inoculation of Pseudomonas aeruginosa D9 2 脳 10 8 CFU through nasal cavity could successfully construct hemorrhagic pneumonia model in mice.The therapeutic experiment showed that the one-off nasal cavity application of phage YH6(2 脳 107 PFU moi 0.1) could effectively protect the hemorrhagic pneumonia model of mice infected with Pseudomonas aeruginosa.Colony count in blood, spleen and lung of mice in treatment group showed that bacteriophage YH6 could completely eliminate Pseudomonas aeruginosa within 2 days after application of bacteriophage YH6.In the untreated group, the content of bacteria in viscera increased by 4 logarithmic units and caused death within 2 days.In addition, histopathological sections of the lungs of the treated mice showed that YH6 could effectively improve the lung injury induced by Pseudomonas aeruginosa.In conclusion, the phage YH6 isolated in this study showed great potential in the treatment of hemorrhagic pneumonia model induced by Pseudomonas aeruginosa in mice.The results laid a solid foundation for further study on the application of this phage to the treatment of mink infected Pseudomonas aeruginosa (Pseudomonas aeruginosa).
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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