鸡源性大肠杆菌噬菌体的分离鉴定及其抗菌性初步研究

发布时间：2018-01-06 17:23

本文关键词：鸡源性大肠杆菌噬菌体的分离鉴定及其抗菌性初步研究　出处：《锦州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的本实验旨在从蛋鸡粪便中分离出一株强裂解性,宽裂解谱的大肠杆菌噬菌体,初步了解其生物学特性,建立雏鸡感染大肠杆菌动物模型,应用该噬菌体进行抗菌性实验,以PCR-DGGE分析不同组别雏鸡肠道菌群的变化,为今后噬菌体治疗雏鸡大肠杆菌病奠定一定的实验及理论基础。方法对26株鸡源性大肠杆菌进行种系分类群鉴定及其分子间的多态性分析,根据大肠杆菌种系及亲缘关系选择具有代表性的多株大肠杆菌作为分离噬菌体的宿主菌。利用宿主菌从不同地区鸡场粪便中分离噬菌体,对分离的噬菌体进行纯化,增殖及效价与裂解谱的确定,选择一株高效价、裂解性强、裂解谱宽的噬菌体,对其热稳定性、p H值稳定性、乙醚及氯仿稳定性,最佳感染复数(MOI)和一步生长曲线等生物学特性进行测定。通过确定雏鸡感染大肠杆菌动物疾病模型致病剂量(绝对致死量),建立了动物感染模型。应用PCR-DGGE技术对不同组别(噬菌体作用组、大肠杆菌感染阳性组和空白对照组)雏鸡的粪便菌群16S r DNA V6区段进行分离,得到其指纹图谱及聚类分析关系图,分析评价噬菌体对雏鸡大肠杆菌感染的抗菌作用效果。结果根据RAPD、种系分类群结果及大肠杆菌血清型,确定了14株鸡源性大肠杆菌为宿主菌。应用宿主菌从鸡场粪便中初步分离了9株噬菌体,分别命名为SP1~SP9。噬菌体SP2经纯化后效价最高为3.6×1010pfu/m L,噬菌斑大于2mm,边缘比较清晰;经交叉裂解试验确定SP2噬菌体的裂解率为46.1%,其中完全裂解率为33.3%,相比于另外8株所分离到的噬菌体,裂解率最高,裂解谱最广,故选择SP2作为后续实验的研究对象。对噬菌体SP2的生物学特性进行研究,该噬菌体的最佳感染复数为0.1,感染宿主菌的潜伏期为20min,裂解期70min,裂解量为36,70℃作用60min后仍可保留活性,在p H值为7时,噬菌体SP2的效价最高,酸性环境对其影响较大,乙醚及氯仿对其影响较小。电镜观察可见噬菌体头部呈正二十面体,可见尾部结构。应用鸡源性大肠杆菌(O78)建立雏鸡大肠杆菌感染动物疾病模型,绝对致死量确定为3×109CFU。DGGE图谱显示健康雏鸡1-10日龄肠道菌群多样性逐日增加,第10日龄最为丰富;雏鸡感染大肠杆菌后,图谱多样性出现变化,菌群明显失调;噬菌体作用组图谱显示个别条带恢复正常,此外,整体可见有些菌群不受大肠杆菌影响,始终存在于各组各日龄不变,聚类分析图中显示噬菌体作用第二天后肠道菌群多样性开始恢复。结论应用鸡源性大肠杆菌从蛋鸡粪便中分离出一株裂解性强,噬菌谱宽的大肠杆菌噬菌体,命名为SP2。热稳定性良好,适宜在中性或微碱性环境生长发育,具有相对较长的裂解期及较大的裂解量,噬菌体SP2对雏鸡大肠杆菌感染具有较好抗菌作用。
[Abstract]:Objective to isolate a Escherichia coli phage with strong cleavage and wide cleavage spectrum from the feces of laying hens, to understand its biological characteristics and to establish an animal model of Escherichia coli infection in chicks. The bacteriophage was used to analyze the changes of intestinal microflora in different chicks by PCR-DGGE. Methods 26 strains of Escherichia coli were identified and analyzed by molecular polymorphism. Several representative strains of Escherichia coli were selected as host bacteria to isolate bacteriophages according to the strains and relatives of Escherichia coli, and phage was isolated from feces of chicken farms in different areas. The isolated phage was purified, proliferated, titer and cleavage spectrum was determined. A bacteriophage with high titer, strong cleavage and wide cleavage spectrum was selected, and the thermal stability of phage was stable. The biological characteristics such as the stability of ether and chloroform, the optimum number of infected moi) and the one-step growth curve were determined. The pathogenic dose (absolute lethal dose) was determined by determining the pathogenic dose (absolute lethal dose) of chicken infected with Escherichia coli disease model. The animal model of infection was established. Different groups (phage group) were treated by PCR-DGGE technique. The 16s r DNA V6 region of fecal flora was isolated from the chicks infected with E. coli and the control group. The fingerprint and cluster analysis were obtained. Analysis and evaluation of bacteriophage bacteriophage to chicken Escherichia coli infection effect. Results according to RAPD, species classification group results and Escherichia coli serotype. Fourteen strains of Escherichia coli from chicken were identified as host bacteria and 9 strains of bacteriophage were isolated from feces of chicken farm. After purification, the titer of bacteriophage SP2 was 3.6 脳 10 ~ (10) pFu / m ~ (-1), the plaque of phage was more than 2 mm, and the edge of phage was clear. The cleavage rate of SP2 phage was 46.1, and the complete cleavage rate was 33.3%. Compared with the other 8 strains, the cleavage rate was the highest and the cleavage spectrum was the most extensive. The biological characteristics of phage SP2 were studied. The optimal number of phage infection was 0.1, and the incubation period of host bacteria was 20 minutes. The activity of bacteriophage SP2 was the highest when pH value was 7:00, and the effect of acid environment on the activity of bacteriophage SP2 was higher than that of bacteriophage SP2. The effect of ether and chloroform on it was small. The positive eicosahedron and tail structure were observed in the head of phage by electron microscope. The animal model of E. coli infection in chicks was established by using chicken Escherichia coli O78). The absolute lethal dose was determined to be 3 脳 10 9 CFU. DGGE map showed that the diversity of intestinal flora of healthy chicks increased from day 1 to day 10, and was the most abundant at the 10th day. After chickens were infected with Escherichia coli, the diversity of the map was changed, and the bacterial flora was obviously out of balance. Phage group map showed that individual bands returned to normal. In addition, some bacteria groups were not affected by Escherichia coli and remained unchanged at all ages in each group. Cluster analysis showed that the diversity of intestinal flora began to recover after the second day of bacteriophage interaction. Conclusion A bacteriophage with strong cleavage and wide spectrum of phage was isolated from the feces of laying hens by chicken Escherichia coli. It is named SP2. It has good thermal stability and is suitable for growth and development in neutral or slightly alkaline environment. It has a relatively long pyrolysis period and a large amount of pyrolysis. Bacteriophage SP2 has a good antibacterial effect on Escherichia coli infection in chicks.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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