【作者】 方丽君；

【导师】 焦新安；

【作者基本信息】 扬州大学 ， 预防兽医学， 2010， 硕士

【摘要】 鸡白痢沙门菌呈世界范围分布。目前,在西方部分发达国家鸡白痢已经被消灭或基本消灭,而在国内该病的危害仍严重。鸡白痢沙门菌多造成2-3周龄雏鸡死亡,成年鸡则可带菌而无临床症状。感染后存活的鸡常表现生殖系统病变,引起蛋鸡产蛋量下降,并污染种蛋,造成巨大的经济损失。该病既可垂直传播,又可水平传播,造成严重的危害。探究鸡白痢沙门菌潜在的致病基因,了解其致病机理,对于寻求新的有效防治措施具有重要的意义。随着各种分子生物学和免疫学研究手段的出现,人们对病原体在感染宿主过程中转录的基因序列的研究也逐渐深入。选择性捕获转录序列(selective capture of transcribed sequences,SCOTS)技术在研究病原体体内转录序列方面有着独特的优势。本研究比较了鸡白痢沙门菌在鸡体内和体外巨噬细胞系中的存活消亡趋势,以鸡巨噬细胞系HD-11为侵染模型,用SCOTS技术捕获该菌在巨噬细胞中的转录序列,并初步分析了其中一个序列在侵染细胞过程中的转录水平。1鸡白痢沙门菌体内外侵染实验为了探究鸡白痢沙门菌S06004在与鸡体单核吞噬细胞系统和体外巨噬细胞系的相互作用的过程中,细菌定殖、复制的变化趋势,进行了细菌的体内侵染实验和体外巨噬细胞侵染实验。体内侵染实验中,以口服和肌注两种方式感染9日龄非免疫海兰白蛋鸡,感染后第三天吞噬细胞中的细菌量都达到最高点,第九天下降到每克组织中带菌量不足500 cfu,且这种状态能够持续至少5天。在体内侵染实验中,细菌的定殖趋势为先发生复制增殖、后消减至很低水平,并且低水平的带菌状态会维持一段时间。同时在体外侵染实验中,不论给予细菌1h还是2.5h的作用时间,进入巨噬细胞内的细菌量在增殖3h左右时都达到最高点,消减到较低的水平后,能够维持一段时间。在体外实验中胞内的细菌同样具有先增殖后消减,并维持一段低水平细菌携带率的平台期的存活趋势。鸡白痢沙门菌S06004株对鸡巨噬细胞系HD-11的体外侵染实验结果和体内吞噬细胞侵染实验结果基本吻合,故采用鸡白痢沙门菌侵染鸡巨噬细胞系HD-11可以在一定程度上模拟细菌体内感染时与内脏器官吞噬细胞系统的相互作用。2选择性捕获鸡白痢沙门菌在巨噬细胞内的转录序列应用SCOTS方法鉴定鸡白痢沙门菌S06004株在感染鸡巨噬细胞系HD-11过程中细菌表达的基因。根据细菌体内和体外侵染实验的研究结果,选用鸡白痢沙门菌S06004侵染鸡巨噬细胞系HD-11,以模拟细菌体内侵染吞噬细胞过程,通过SCOTS技术对转录序列cDNAs的选择性捕获,检测了细菌在与巨噬细胞相互作用过程中毒力基因以及相关调控基因的表达,共得到16个序列。除了一个未知序列,这些体内转录序列包括毒力相关的III型分泌系统中分泌素蛋白和Ⅰ型分泌系统中外膜蛋白的编码基因、代谢相关的敏感激酶和核苷水解酶的编码基因、运输功能基因、一些质粒编码基因以及环境适应性功能基因等。3实时荧光定量PCR动态分析鸡白痢沙门菌体内转录序列7-6在侵染过程中的转录水平根据SCOTS技术捕获得到的体内转录序列7-6,设计了针对其特异性检测的一对引物,同时设计鸡白痢沙门菌16sRNA基因的引物作为内参。提取鸡白痢沙门菌S06004株在侵染细胞过程中各个阶段的总RNA,反转录成cDNAs,利用荧光定量PCR比较基因在不同阶段的表达水平的差异。结果表明:捕获序列7-6在体外培养时不表达;在侵染初期表达量最高;在细菌胞内增殖阶段,基因的表达量先上升后下降,在增殖3h时表达量最高,在增殖5h后,7-6序列的表达下降到很低的水平。基因的转录变化与细菌的巨噬细胞侵染过程密切关联。更多还原

【Abstract】 Salmonella pullorum is distributed in worldwide. Nowadays, the pullorum disease has been eliminated or partially eliminated in the developed countries, however, there it is an important avain disease causing serious losses in the poultry industry in China. Salmonella pullorum mainly causes the death of 2-3 weeks old chickens, and the adult chickens appear as bacteria carrier without clinical symptoms. Most of the survival chickens show the reproductive disorders, including decreased egg production, production of contaminated eggs, which cause severe economic losses. The disease can spread vertically, as well as transmit horizontally, which is the main reason for the reproductive disorders. It is important to explore the novel potential virulence-related gene and develop new strategies for the control of the pullorum disease.With the development of various molecular biological and immunological research methods, the gene expression of pathogens in infection process can be evaluated. It is notable that identification of transcribed sequences of pathogens in vivo is very important for exploring the pathogenetic mechanism. SCOTS is applicable to many kinds of bacteria from which transcribed sequences can be isolated. In this study, it was carried out an invasion test both in chicken model and macrophage cell line to compare the growth property of Salmonella pullorum in vivo and in vitro. In avian macrophage cell line HD-11, the bacterial transcribed sequences of S. pullorum was captured using SCOTS and the expression develop captured sequence 7-6 was analyzed by real-time quantitative PCR.1 Comparison of invasion process in vivo and in vitro of Salmonella pullorumIn order to investigate the specific infection of Salmonella pullorum in chicken model and in the avian macrophages cell line HD-11 in vitro, 9-days-old chickens were inoculated orally or intramuscularly with S. pullorum S06004. The bacteria within macrophages of spleen and liver can reach the highest point at the third day after inocularion, and would be less than 500 cfu/g at the ninth day, which can be persist more than 5 days. The result of invasion in vivo indicatied that bacteria in macrophages were proliferated at first, then reduced to a lower level which can last some time. Meanwhile bacteria infected the macrophage in vitro, intracellular bacteria reached the highest point in 3h at proliferation stage, then reduced to a lower level which also lasted a few hours. So the in vitro invasion of HD-11 cells by S. pullorum can simulate the invasion in vivo in some extent.2 Identification of Salmonella pullorum genes expressed within macrophages by selective capture of transcribed sequencesThe selective capture of transcribed sequences(SCOTS) was used to identify Salmonella pullorum S06004 genes expressed during the process of infection of avian macrophage cell line HD-11. According to bacterial invasion in vivo and in vitro, the invasion of Salmonella pullorum S06004 in avian macrophage cell line HD-11 can simulate in vivo. The transcribed sequences were captured, and identified the virulence genes and related regulation genes expressed within macrophages. 16 sequences were obtained. Except an unknown sequence, these sequences included the coding genes of virulence-related secreted proteins in type III secretion system, outer membrane protein coding genes of typeⅠsecretion system, sensitive kinase related to metabolism and nucleoside hydrolase coding genes, transport genes, and some plasmid genes, environmental adaptability functional gene.3 Kinetic detection of the expression level of a captured sequence 7-6 during the infection process by real-time quantitative PCRIt was designed a pair of primers of the captured sequence 7-6 of S. pullorum for the specific detection and the primers of 16sRNA gene were used as internal control. The total RNA of infected cells at different time was extracted, and reverse transcribed into cDNAs, and then the gene expression levels of 7-6 was monitored using real-time PCR. The results showed: the captured sequence 7-6 was not expressed in vitro, and then there are the highest expression in early invasion, at the intracellular bacteria proliferation phase, the expression level reached to the highest at 3h proliferation, and then decreased to a very low level after 5h proliferation. The changes in transcriptional level of 7-6 were related to the infection process of macrophages.更多还原