【作者】 康名松；

【导师】 周锐；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae,APP）是巴氏杆菌科中重要的病原菌,它引起猪的一种高度接触性传染呼吸道疾病—猪传染性胸膜肺炎（porcinecontagious pleuropneumonia,PCP）。该病分布广泛,难以根除,给世界养猪业造成了巨大的经济损失。因此,预防和控制该疾病尤其重要。然而APP血清型众多（目前已经发现有15种血清型）,并且不同血清型之间的交叉保护力较低,对该病的预防控制和治疗带来了极大的困难。而且许多国家用大量不同的抗生素来预防和治疗猪传染性胸膜肺炎,致使许多病原菌（包括APP）产生了极强的抗药性,这对该病的预防和治疗更是雪上加霜。现阶段,为了解决APP的预防和治疗问题,需要对APP的毒力因子、毒力相关因子以及膜蛋白和鞭毛等进行分子生物学研究。但研究这些基因和相关因子的前提是要具备相应的遗传操作工具,如穿梭载体,高效的突变体构建平台等,为技术支持。本文主要对APP的遗传操作工具的研究,包括以下两个方面内容:1.胸膜肺炎放线杆菌内生质粒pHB0503分析质粒pHB0503分离于2005年的胸膜肺炎放线杆菌地方分离菌株HB0503。大小为15.079kb,含有16个ORF,其中7个是抗性基因（sul2,catA3,aacC2,strA,strB’,blaROB-1和aph（3）-I）。对这些抗性基因簇序列的生物信息分析,来研究该抗生素簇的进化和结构组成。实验数据分析表明该质粒可能在来源不同的质粒间,发生了同源重组而形成的。另外,RT-PCR实验证明:两处抗性基因簇的共转录,分别从sul2和bla_ROB-1启动子开始地。对该质粒的复制蛋白的分析,有可能在此基础上构建在APP中能稳定复制地穿梭载体和遗传操作系统,为进一步从分子生物学水平研究胸膜肺炎放线杆菌提供技术支持。而且对内生质粒抗性基因的研究,对临床抗生素的使用也有指导意义。2.胸膜肺炎放线杆菌温敏复制型质粒的构建PCR定向突变系统是新型的高效遗传操作体系,已经广泛应用于微生物学研究。构建该系统至少需要重组酶系统、抗性基因PCR模版、诱导型启动子、温敏复制型质粒等四大要件。本实验室已经拥有前三大要件,缺乏APP的温敏复制型质粒。为了构建一个APP的温敏复制型质粒,本研究以APP-E.coli穿梭质粒pJN105和pR318为出发质粒,采用低保真度PCR基因扩增和修复系统缺陷型E.coli感受态细胞基因克隆两种途径筛选温敏复制元件,为构建温敏复制载体奠定基础。更多还原

【Abstract】 Actinobacillus pleuropneumoniae（APP）,a member of the Pasteurellaceae family,is the causative agent of porcine contagious pleuropneumonia（PCP）,which is worldwide epidemic and difficult to eradicate,and is responsible for substantial economic losses in the pig industry worldwide.Therefore,it is essential to prevent,control and treat this disease,but due to at least 15 distinct serotpyes and lack of cross-protection among them, this disease is dfficult to prevent and control.Moreover,the widespread use of antimicrobial agents to treat or prevent PCP,which force many pathogenic bacteria to become multidrug resistant bacteria,including APP.So it is more difficult to prevent and treat animal diseases.At present,in order to prevent and treat APP,we need to understand some knowledge about virulence factors,virulence associated factors,membrance proteins and flagellas in the level of molecular biology.However,the prerequisite condition of research on these genes is to possess some genetic operation tools,such as shuttle vector or high-efficency technology of constructing mutant.This study mainly focused on genetic operation tool,including two parts as below:Isolation,sequencing and genetic characterization of A.pleuropneumoniae native plasmid pHB0503.Plasmid pHB0503 was extracted from Actinobacillus pleuropneumoniae HB0503,which was isolated in 2005.The complete 15.079 kb sequence of this plasmid,containing 16 ORFs,seven of which were associated with antibiotic resistance genes(sul2 catA3,aacC2,strA,strB’,bla_ROB-1 and aph（3）-I),was analyzed with regard to the organization and evolution.The experiment data indicated that pHB0503 arose through inter-plasmid recombination processes among other plasmids. In addition,co-transcription of the cluster of resistance genes from the promoter upstream of sul2 and bla_ROB-1 was confirmed by RT-PCR.Then,the analysis of Rep protein may help us to construct stable shuttle vector or genetic tools,which is very useful for molecular biology research on APP.Moreover,the detection of antibiotic resistance genes could instruct the clinical use of antibiotic.Construction of temperature-sensitive plasmids for A.pleuropneumoniae.PCR targeting system is a high-efficiency genetic modification system and has been widely used in research fields of microbiology.This system consists of four parts:recombinase systems,templates for PCR amplification of resistance gene,induced promoter and temperature- sensitive（TS） vector.At present,we already have possessed three parts.So the only element that we lack is TS vector.In order to construct a TS vector for APP,this study researched on a base of APP-E.coli shuttle vectors（pJN105 and pR318） and used two different kinds of methods（low-Fi PCR amplification method and the method involved in cloning genes into XL1-RED competence cell,which is deficient in primary DNA repair pathways） to construct it,which provided some supports to the further construction or research on TS vector.更多还原