【作者】 严亚贤；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2003， 博士

【摘要】 大肠杆菌O157：H7是一种致病力很强的肠道致病菌，主要引起人和动物的出血性肠炎、溶血性尿毒综合征而致较高的死亡率，已在全球范围内引起关注。尽管O157的高致病力的机制尚未完全明了，但已确定该菌的重要毒力因子之一是志贺毒素（Shiga toxin,ST），也称为Vero毒素（VT）。编码VT的基因位于噬菌体（VT噬菌体）的染色体上。VT噬菌体的毒粒特征、VT噬菌体是否进行毒力基因的水平转移、基因水平转移的条件、VT噬菌体的溶源转换与细菌毒力的相关性，均直接关系到病原菌的致病力。因此对VT噬菌体的研究，有助于对大肠杆菌O157：H7的预防、诊断和治疗。 为掌握日常肉品中大肠杆菌O157的污染情况，本实验对超市和农贸市场的生牛肉进行大肠杆菌O157的调查。利用鉴别分离培养、形态特征观察，API 20E生化特性鉴定，玻片凝集试验、快速检测试剂盒等，对上海某地区5家超市及3个菜市场的生牛肉进行大肠杆菌O157的检测，并测定分离菌株对小鼠的致病力。结果从120份样本中检测出3株大肠杆菌O157：H7和2株大肠杆菌O157：NM。3株大肠杆菌O157：H7均能致死试验小鼠，表明超市、菜市场生牛肉中存在高致病力的大肠杆菌O157：H7的污染。 高致病力的大肠杆菌O157的存在，提示与该菌相关的VT噬菌体的存在。本试验利用敏感指示菌MC1061，经双层琼脂法纯化和PCR扩增VT2基因，分别从大肠杆菌O157菌株、牛粪、鸡粪和污水中分离到了5株VT2噬菌体。这些噬菌体蚀斑透明，直径为0.5-2mm，对指示菌的感染效价均在10₉PFU／mL以上。电镜下可见头部为六边形、尾部短而硬。对氯仿和56℃30min抵抗。噬菌体分离株SHφW1感染MC1061后产生溶源菌（MC1061／SHφW1），经检测谊溶源菌具有VT2基因，而亲本MC1061中不含VT2基因。分离到VT2噬菌体的菌株及溶源菌株的滤液均对Vero细胞产生病变效应。表明环境中普遍存在含有VT2基因的噬菌体，VT2噬菌体通过溶源可将毒力基因水平传递，证实了VT2基因与细菌毒力的相关。 大肠杆菌O157菌株、牛粪、鸡粪和污水中VT噬菌体的存在，造成了土壤的污染。本试验利用重组标记有Kan^r基因的VT噬菌体，壤土、粘土和砂土三种不同的土质建立了土壤模型，以检测土壤中噬菌体对土壤的吸附力以及在土壤中存活的差异，并测定VT噬菌体对不同菌株的敏感性。结果显示噬菌体在3种土质中的吸附能力不同，噬菌体能较好地吸附到粘土和壤土，但在砂土中的吸附力较差。在上述三种土质中，室温条件下 30d仍有一部分感染性的噬菌体粒子存在．M k土壤是 VT噬菌体存活的重要场所。噬菌体对不同菌株的敏感性不同，能使15株大肠杆菌中12株溶源或裂解，2株福氏痢疾杆菌溶源，但不能感染肠中的3株沙门氏菌，在溶源菌株中均检测到VTZ基因。可见VT噬菌体的感染具有宿主特异性，敏感菌由于获得了VT基因而增强了毒力。 VT噬菌体能感染多种宿主菌，简种属特异性，这种特异性与宿主菌表面的蛋白受体有关，噬菌体与特异性受体的结合是噬菌体感染的第一步。本试验对已克隆的但尚未明确功能的vpr基因进行了功能检测．利用含有卡那霉素抗性(Kan门基困的自杀性质粒 pJP5603和含 vpP全长基因的 pUC19phiOlllD质粒，分别采用粘性末端及平末端两种连接法。pJP5603用 Hn 11和 Xmal消化，回收 3kb的载体质粒，PUC中m用m。11和dg6I消化，回收778咖w目的基因。通过粘性末端连接，转化到感受态细菌 CClls人pir，利用 Kanr’进行筛选，获得的克隆经 ACC切鉴定和 PCR检测，确定得到重组质粒，RP pYYvp。将重组质粒的 DNA转化到含有全长叩r基因的感受态细胞 MC 06，筛选到的克隆经 PCR检测，得到一株克隆既扩增出 vpr基因，又扩增出 Kan’基困，进一步经 Southers blot狮，获得一个可靠的 MC1061的叩厂同源基因突变株，即MCm61 ovpr。该突变株与亲本MCm61具有相似的生长特征。噬菌体裂解试验表明，突变株对VTZ噬菌体C43b不敏感，而 MC1061敏感，表明 vpr基因与VTZ噬菌体的裂解敏感性有关。 在获得 IW同源基因突变株 MC 06 IW的基础上，通过基因转化获得了突变株的回复株。并采用6－His标于己的方法；纯化了Vpr蛋白，制备了兔的抗VPr抗体。噬菌体裂解试验显示，回复株和亲本株对＊D噬菌体的裂解敏感，而突变株＊C106］a vpr能抵抗 VTZ噬菌体的裂解。噬菌体吸附细胞壁试验中；93％以上的噬菌体能与亲本MC 06和回复株MC1061c 的细胞壁不可逆性结合，但85．4o的噬菌体不能与突变株MC1061 Lw的细胞壁结合．在蛋白表达中，Western blot的结果显示，＠复株和亲本株均能转印出特异性 90 000主带，而突变株没有，因此可以确定分子量为90 000的 Vpr蛋白为 VTZ噬菌体的受体。更多还原

【Abstract】 Escherichia coli O157 are important enteropathogens causing outbreaks of haemorrhagic colitis and haemolytic uraemic syndrome of human and animals with high mortality. So it is noticed worldwidely. Although the mechanism of which factors make toxigenic E.coli O157 more virulent is unclear, the one of important virulent factors of the pathogens is Shiga toxin, which can cause the CPE of Vero cells, also called Vero toxin (VT). The VT gene is encoded on the chromosome of bacteriophage which contains VT gene (VT phage). The characteristics of VT phage virion, the conditions of horizontal gene transmition, the effect on the bacterial virulence of lysogenic conversion, all these will be related to the virulence of pathogen. So it will be contributed to prevention, diagnosis, and treatment of E.coli O157 infection to research all above.To detect the contamination of E.coli O157 in market, the beeves from the five supermarkets and three food markets were collected and detected by selective culture, biochemical characteristic evaluation, serologic agglutinative test and BINAX NOW?EH E.coli test kit. Animal experiments were made to determine the virulence of the isolates. Results showed that 3 strains of E.coli O157: H7 and 2 strains of E.coli O157: NM were identified from 120 samples, and the isolates of E.coli O157: H7 had virulence to mice. It revealed that E.coli O157: H7 contamination existed in the market beeves and had potential risk to human being.Occuring high virulent E.coli O157: H7 in market beef revealed that there was verocytotoxigenic bacteriophage (VT phage) in E.coli. Five strains of VT2 phage were isolated and purified by two layers agar assay, and identified by amplifying VT2 gene. These phages were from E. coli O157 strains, cow and chicken feces, and sewage, respectively. Phages were 0.5-2mm diameter and had regular hexagonal heads and short stubby tails. The litre of phages with lucidity plaques against host strain MC1061 was more than 109 PFU/mL.A lysogen (MC1061/SH W1 ) with VT2 gene was gained after transfecting MC1061 by VT2 phage SH W1 from sewage.The cytopathic effect on Vero cells were produced after inoculating the filtrates from the E. coli strains isolated VT2 phages and the above lysogen. All these showed that VT2 phages were common inenvironment and VT2 virulent factor could transfer horizontally through the lysogenic infection of VT2 phage. VT2 phage was related to the virulence of bacteria,A recombinant verocytotoxigenic bacteriophage (VT phage) contained Kan was used to detect the difference of adsorbability to soils and survival in soils of VT phage. The soils model was set up used clay, loam and sandy soils, respectively. The susceptibility of different strains to VT phage was also researched. The results showed that VT phage adsorbed strongly to clay and loam soils, but not to sandy soils. A small proportion of infective VT phage particles could survive for more than 30 days in all three soils type. Soils were important place for survival of VT phages. Of 15 E. coli strains tested, 12 were susceptible to lysogenic or lytic infection. 2 Shigella flexneri strains were all susceptible to infection by the phage, while 3 Salmonella strains were not. VT2 gene was detected in all above lysogens. All theses indicated VT phage had specific host rang. The virulence of sensitivity bacteria was enhanced because of obtaining VT2 gene.VT phage had the specific host rang might be related to the receptor on the surface of bacteria. A cloned gene with not clear function, named vpr was studied. The pJP5603 plasmid is a suicide plasmid and contains the Kan resistance cassette, which was digested with Xmal. Hin II and the pUC19phi RID plasmid contains the vpr gene, digested with Age , Hin II to obtain 778bp piece of vpr aim gene. After cohesive ligation. the recombinant plasmid was transformed into CC118 pir. Colonies were selected on Kan plates. A recombinant plasmid, pYYvpr, was confirmed by PCR to amplify vpr gene and digestion with restriction enzyme Acc I The recombinant suici更多还原