【作者】 王斌；

【导师】 贝为成； 陈焕春； 金梅林； 何启盖； 周锐； 蔡旭旺； 谭臣； 徐晓娟；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2014， 硕士

【摘要】 猪传染性胸膜肺炎(Porcine Contagious Pleuropneumonia, PCP),是由猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae, APP)引起的一种猪的传染性呼吸道疾病,在世界范围内对养猪业造成巨大损失。磷是病原菌一种重要的营养元素,在信号传导和能量代谢中起重要的作用。糖类作为碳源,对于细菌的生长有着至关重要的作用。在肺脏磷和碳源缺乏的情况下,有效均衡的生理反应和各种糖类的摄取利用,对于APP在猪呼吸道与肺脏中的生存定植具有非常重要的意义。病原菌在感染宿主的过程中,能通过双组份调控系统(Two-Component Regulatory System, TCS)密切感受和响应体内外各种微环境的变化,进而调节各种基因表达以完成其致病的过程。APP血清3型JL03的全基因组测序分析,表明APP具有5对完整的双组份调控系统：ArcA/B, CpxA/R, NarP/Q, PhoB/R, QseB/C。众多研究者对PhoBR双组份转导系统的研究表明：在磷限制培养条件下,PhoBR参与调控磷、糖的代谢和相关毒力因子的表达,但当前未见PhoBR在猪传染性胸膜肺炎中调控致病机制的研究。鉴于以上背景,本研究选用APP血清1型强毒力菌株SLW01作为亲本菌株,构建了SLW01的双基因缺失突变株AphoBR,同时构建了AphoBR基因缺失突变株的互补菌株C△phoBR。对突变株△phoBR进行了生物学特性和对小鼠致病力的研究,并在磷限制与正常培养情况下,通过Agilent单通道表达谱芯片和qRT-PCR,筛选突变株AphoBR与亲本株SLW01差异表达基因,初步探讨了PhoBR调控APP的可能致病机制。主要研究内容如下：1.双基因缺失突变株AphoBR与互补菌株CAphoBR的构建与鉴定。参考APP血清3型JL03的全基因组序列,设计引物,以APP血清1型SLW01基因组为模板,扩增phoB/R基因的上下游同源臂片段,将上下游片段分别连接到pEMD-18T载体上,酶切鉴定回收,连接到自杀性质粒pEMOC2,经酶切验证,成功构建了插入2272bp外源片段的重组质粒pEM-phoBR。测序表明,该外源片段与模板序列100%同源,将重组质粒pEM-phoBR转化至大肠杆菌p2155,作为供体菌与亲本株SLW01做接合转移,获得整合了pEM-phoBR重组质粒的单交换菌株,通过蔗糖负向筛选和氯霉素正向筛选,结合PCR鉴定,筛选出对氯霉素敏感,具有蔗糖抗性的克隆,进一步通过测序和荧光定量证实双基因缺失突变株△phoBR构建成功。参考APP血清3型JL03的全基因组序列,设计引物,以APP血清1型SLW01基因组为模板,扩增phoB/R基因片段,将该目的片段依次连接pEMD-18T、APP-E.coli穿梭质粒pJFF224-XN,构建重组表达质粒pJFF-phoBR,酶切、测序鉴定,该外源片段与pho B/R基因序列100%同源。将该重组质粒电转化至双基因缺失突变株AphoBR,经PCR和RT-PCR鉴定,证实互补菌株C△phoBR构建成功。2.突变株AphoBR生物学特性研究对突变株AphoBR进行生物学特性研究的结果表明：AphoBR可在体外稳定遗传；在正常培养条件下,突变株AphoBR体外增殖能力相比较野生株生长速率明显变慢,活菌数下降。在磷限制培养条件下,突变株AphoBR体外增殖能力与野生株SLW01基本一致；小鼠LD5o实验表明：突变株AphoBR木目比于亲本株SLW01和互补菌株C△phoBR毒力分别上升1.73倍和2.05倍(Korbor寇氏法)。小鼠存活率实验表明：相同感染剂量,突变株AphoBR组14只仅存活一只,存活率为7.14%。亲本株SLW01组最终14只存活6只,存活率42.86%,毒力差异显著；小鼠载菌量实验表明：在入侵与感染初期,突变株AphoB/R肺与血液中的载菌量显著高于野生株SLW01。随着感染时间的延长至120h,两者在肺组织中的菌量基本趋于一致,而在血液中的突变株的载菌量还维持在一个较高的水平；中性粒细胞杀伤实验表明,突变株AphoBR抵抗PMN杀伤能力相比于亲本株SLW01增强,差异极显著。以上结果表明,突变株AphoBR毒力相比于亲本株毒力上升。3.PhoBR调控机制的探讨对突变株AphoBR与亲本菌株SLW01差异表达基因进行分析,结果显示,在正常培养条件下,突变株AphoBR相比于亲本株SLW01上调表达2倍以上基因5个下调表达基因7个,这12个基因主要与糖类转运代谢先关；在磷限制培养条件下,突变株AphoBR相比于野生株SLW01上调表达2倍以上基因119个,下调表达2倍以上基因60个。这些基因主要与能量代谢、氨基酸转运和代谢、糖类的转运和代谢、辅酶代谢、转录后修饰,蛋白折叠和分子伴侣、无机铁的转运与代谢相关。进一步通过荧光定量PCR试验证实芯片结果可靠。PhoB/R可能通过调控毒力相关因子来影响APP的致病力。更多还原

【Abstract】 Actinobacillus pleuropneumoniae(APP) is the causative agent of Porcine Contagious pleuropneumonia(PCP), a severe and often fatal respiratory disease of swine, causing serious economic losses worldwide in the swine industry. This organism cause sudden death and colonize the respiratory tracts, tonsils and lungs of pigs, causing chronic and persistent infections, lung lesions, and reduced growth. Phosphate is an essential nutrient for pathogen and has an important role in signal transduction and energy metabolism. Carbohydrate as a carbon source has a vital role in the growth of bacteria. In respiratory tracts and lung are generally thought to be limiting for phosphorus and Carbohydrate. Therefore, bacteria must actively pursue phosphate and Carbohydrate to ensure survival in porcine respiratory tracts and lung.In the process of the infection, the pathogen could sense and respond to extracellular signals through the two component regulatory system(TSC), and then regulate the expression of the various genes to complete its pathogenic process. Five TSC, ArcA/ArcB, CpxA/CpxR, NarP/NarQ, PhoB/PhoR and QseB/QseC, are found in APP based on the analysis of the genome of APP strain JL03. Many researchers study on PhoBR show that PhoBR involved in the regulation of phosphorus, sugar metabolism and the expression of virulence factors, But the function of PhoBR in the APP is still unknown.In view of the above background, this study selects the APP serotype1strain SLW01as the parent strain and use homologous recombination to constructure the double gene deletion mutant strain AphoBR. At the same time, the strain AphoBR of complementary strain CAphoBR is constructured. Biological characteristics and pathogenicity in mice of ΔphoBR is compared with the parent strain. In adition, using Agilent single channel expression profile chip and qRT-PCR, the differentially expressed genes between mutant strains AphoBR and parental strains SLW01are screened. The pathogenic mechanism of phoBR in APP is preliminarily investigated. The main research content are as follows:1. Construction and identification of the mutant strain AphoBR and complementary strain CΔphoBRRefer to the phoBR genes sequence in JL03Genbank genome, the upstream and downstream fragments of phoBR genes are amplified from the genomic DNA of APP serotype1SLW01. The upstream and downstream segments are cloned into the vector pMD-18T. After identification by restriction endonuclease analysis, the segments were further cloned into the vector pEMOC2, creating the recombinant suicide plasmid pEM-phoBR. β2155transferred with recombinant suicide plasmid pEM-phoBR is regarded as the donor strain. Then the donor strain was conjugal transfered with the receptor strain SLW01, obtaining single exchange strain. Through negative and positive selection, the double exchange strain which is Cm-sensitive and sucrose resistant was further verified with PCR, qRT-PCR and sequencing. The mutant strain was constructed successfully, and named it ΔphoBR.The phoBR genes were amplified from the genomic DNA of APP serotype1SLW01, and then cloned into the vector of pEMD-18and shuttle plasmid pJFF224-XN of APP-E. coli in succession. After being verified by PCR, enzyme digestion and sequencing, the recombinant plasmid pJFF-phoBR was then electrotransfered into double mutant strain ΔphoBR. The recombinants were verified by PCR and qRT-PCR to confirm that the complementary strains CΔphoBR was constructed successfully.2. Research on the basic biological characteristics of PhoBRThe research results of biological characteristics of mutant strain show that ΔphoBR could propagate stability in vitro. In normal culture condition, the mutant strain ΔphoBR behave a significantly slower growth rate compared to SLW01. In the condition of phosphorus limited, the growth rate of mutant strains ΔphoBR shows no difference with wild strains SLW01. LD50in mice infection experiment show that the virulence of mutant strains ΔphoBR compared to the parent strain SLW01and complementary strain CΔphoBR rose1.73times and1.73times respectively (Korbor method); Survival rate of mice infected with ΔphoBR was7.14%, while that with parent strain SLW01was42.86%. In early infectin phase, bacteria load in lung and blood of mice infected by AphoBR is siginificantly higher than that infected by SLW01. On120h post infection, the bacteria load in lung of mice infected with the two strains was almost the same while ΔphoBR still maintain a higher level in blood. PMN mediated killing experiments shows that the mutant strains AphoBR exhibits significantly greater resistance than the parent strain SLW01. The above results showed that the virulence of mutant strains ΔphoBR enhanced.3. The regulation mechanism and pathogenesis of PhoBRAnalysis of differentially expressed genes between mutant strains ΔphoBR and parent strain SLW01shows that in normal culture condition, the absence of phoBR led to12genes differently expressed (5upregulated and7down regulated). In phosphorus limited culture condition, the absence of phoBR genes led to up-regulation of119genes fold change more than2and60genes downregulated. These genes are mainly related to energy metabolism, amino acid transport and metabolism, sugar transport and metabolism, coenzyme metabolism, post-translational modifications, protein folding and molecular chaperone, inorganic iron transport and metabolism. Further, the chip results were confirmed by qRT-PCR. In conclusion, PhoB/R is verified to be involved in the pathogensis of APP via regulating virulence factores.更多还原