【作者】 张雷；

【导师】 余旭平；

【作者基本信息】 浙江大学 ， 预防兽医学， 2007， 硕士

【摘要】 鸡白痢沙门氏菌(S. pullorum)又称雏沙门氏菌,引起雏鸡急性败血症,多侵害20日龄以内幼雏,日龄较大的雏鸡可表现白痢,发病率和致死率相当高。对成年鸡主要感染生殖器官,表现为慢性局部炎症,常导致母鸡产蛋明显下降,并经卵传染,所产蛋的孵化率和出雏率也明显降低。还可感染火鸡,对其他禽和鸟类偶能感染。必需基因是维持一个细胞的生命活动所必不可少的基因,必需基因所编码的蛋白质的功能被认为是生命活动的基础,必需基因的总和构成了维持一个细胞生命活动的最小基因集合。从必需基因的定义看,去除一个必需基因会使一个生物体无法继续存活,这是非常具有实际意义的。例如,由于大部分抗生素作用于细胞的基本过程,微生物必需基因的翻译产物就有可能成为抗细菌药物的靶点,这对人类开发抗菌药物是非常有意义的。pIDM1质粒是一类专属于革兰氏阴性细菌的温度敏感质粒。因为它含有编码温度敏感复制蛋白RepAts的基因,所以在30℃时该质粒复制,在高于37℃不复制。该质粒具有四环素抗性。本实验是通过插入复制突变(insertion duplication mutagenesis)来鉴定鸡白痢沙门氏菌必需基因。首先,设计含有KpnⅠ酶切位点rpcr引物扩增长度为350-450bp的片段。用温度敏感质粒pIDM1构建鸡白痢沙门氏菌的基因文库。首先用KpnⅠ酶切pIDM1,脱磷,然后与酶切后的rPCR产物连接,然后转化中间宿主细胞大肠杆菌TG1,构建基因文库。基因文库的克隆数在5×105。鉴定基因文库的插入率为90%。然后提取基因文库所有克隆的质粒,转化鸡白痢沙门氏菌,构建以鸡白痢沙门氏菌为宿主菌的基因文库。复核基因文库插入率为90%。基因文库的细菌内所含的pIDM1的克隆片段与细菌染色体对应的片段,发生同源重组的的几率为10-5。通过转换不同的生长温度,导致部分克隆发生同源重组,若克隆子上必需基因片段插入鸡白痢沙门氏菌的必需基因,导致细菌死亡,而非必需基因的插入的突变体则仍旧在抗生素平板上生长,然后通过负筛选找出含有必需基因的克隆。PCR扩增必需基因片段,测序,测序结果送BlastN分析,我们共得到5个必需基因,分别为：btuE、bglA、gltA、cheV、metG。其中btuE已被发现是Escherichia coli的必需基因,该基因编码的蛋白为：vitamin B12 ABC transport ATP-binding protein。metG已被发现是Escherichia coli、Haemophilus influenzae、Mycoplasma genitalium、Salmonella typhimurium共同的必需基因,该基因编码的蛋白为methionyl-tRNA synthetase。bglA、gltA、cheV是本实验最新发现的鸡白痢沙门氏菌C79-11-S2的必需基因。更多还原

【Abstract】 Salmonella pullorum is a species-specific salmonellae only found in chickens, causing pullorum disease. Pullorum disease is a bacterial infection of chickens and turkeys caused by Salmonella pullorum. The organism can be transmitted in fertile eggs from infected hens. Such passage results in offspring which chronically shed organisms in feces. S. pullorum is a highly host adapted bacterium that is pathogenic in domesticate gallinaceous birds (chickens and turkeys).Essential genes are genes that are indispensable to support cellular life. These genes constitute a minimal gene set required for a living cell.One important class of yet uncharacterized genes encodes proteins that are indispensable for bacterial growth and survival under defined laboratory conditions as many of them contribute to the minimal gene set and are thus likely to prove to be suitable for target-based drug discovery.pIDMl is specific for Gram-negative bacteria. That was derived from the Gram-positive plasmid pVE6007. pIDM1 harbouring the temperature sensitive replication protein RepAts and tetR gene encoding a tetracycline resistance protein.A novel screening approach based on insertionduplication mutagenesis (IDM) was established to efficiently screen for essential genes of Salmonella pullorum under laboratory conditions.Small, randomly generated genomic fragments were cloned into a conditionally replicating vector, and the resulting library of single Salmonella clones was grown under permissive conditions. Upon switching to non-permissive temperature, discrimination between lethal and non-lethal insertions following homologous recombination allowed the trapping of genes with essential functions.We find five essential genes:btuE、bglA、gltA、cheV、metG. btuE gene is the essential gene of Escherichia coli. metG is the essential gene of Escherichia coli、Haemophilus influenzae、Mycoplasma genitalium、Salmonella typhimurium.更多还原