【作者】 李求春；

【导师】 焦新安；

【作者基本信息】 扬州大学 ， 遗传学， 2007， 硕士

【摘要】 鸡白痢沙门氏菌多侵害20日龄以内幼雏,引起白色下痢,病死率极高,成年鸡则可带菌而无临床症状。该病既可垂直传播,又有严重的水平传播,所造成的危害和经济损失巨大。目前,在西方发达国家该病已经被消灭或基本消灭,然而在个别小的禽群中仍有发生,国内禽群鸡白痢的爆发仍较多。寻求鸡白痢沙门氏菌的致病基因对于了解其致病机理及有效地防治鸡白痢具有重要的意义。随着微生物基因组序列的大量测定,通过比较基因组差异分析致病微生物的分子致病机制已成为新近微生物基因组研究的一大热点。抑制性差减杂交方法(suppression subtractive hybridisation,SSH)的问世为微生物基因组学研究、致病基因和新基因的筛选提供了新技术。本研究利用SSH构建了鸡白痢沙门氏菌与肠炎沙门氏菌的差减文库,并对其中的部分序列进行了初步分析。1.鸡白痢沙门氏菌与肠炎沙门氏菌抑制差减杂交文库的构建应用抑制性差减杂交技术(SSH)对鸡白痢沙门氏菌533株与肠炎沙门氏菌50041株进行基因组片段的差异分析,构建了鸡白痢沙门氏菌的差减文库。经Dot-blot筛选,并对部分片段测序和同源性分析,结果表明这些序列主要分为4类:可移动遗传元件,III型分泌系统相关序列,质粒转移相关序列,未知功能序列。其中3个差减片段与编码福氏志贺菌侵入质粒抗原IpaJ蛋白基因的同源性达50%。本研究中构建的差减文库为研究鸡白痢沙门氏菌特异性致病基因和其致病机理提供了重要的材料。2.鸡白痢沙门氏菌ipaJ基因的克隆与表达鸡白痢沙门氏菌与肠炎沙门氏菌差减文库中的片段PEA2、PE31、PE44与猪霍乱沙门氏菌psfD10 ipaJ相应序列同源性分别达97%、99%和100%。将PEA2、PE31和PE44序列拼接后获得鸡白痢沙门氏菌完整的ipaJ序列,设计一对引物从鸡白痢沙门氏菌中克隆出ipaJ基因并将其构建到原核表达载体pET-30a(+)上,诱导表达出37KDa的蛋白。Western-blot试验显示鸡白痢沙门氏菌阳性血清能与体外表达的蛋白呈现特异性反应。特异性实验表明ipaJ是鸡白痢沙门氏菌特有的基因。对97株不同来源的鸡白痢沙门氏菌分离株的PCR结果反映了ipaJ广泛存在于鸡白痢沙门氏菌中。本研究在国际上首次证实了ipaJ基因存在于鸡白痢沙门氏菌中,但对其功能的了解还有待于更深入的研究。3.鸡白痢沙门氏菌mobA基因的克隆与表达鸡白痢沙门氏菌与肠炎沙门氏菌抑制差减杂交文库中片段PE30与猪霍乱沙门氏菌psfD10 mobA基因序列同源性达99%。根据GenBank上发表的mobA基因序列,设计了一对引物从鸡白痢沙门氏菌标准株533中扩增出片段约为1.6kb的序列,将此序列构建到原核表达载体pET-30a(+)和pGEMX6p-1上,再将重组质粒分别转化入宿主菌BL21(DE3)和BL21中,在37℃,0.5mmol/L IPTG诱导下,分别表达出分子量约为64KDa和84KDa的蛋白质。将BL21(DE3)中的重组蛋白表达产物经His纯化柱回收后免疫小鼠,Western-blot试验显示免疫小鼠的血清与BL21中表达的MobA蛋白呈现特异性反应,这说明了体外表达的蛋白具有免疫原性。mobA基因的成功克隆与表达为进一步研究提供了条件。更多还原

【Abstract】 Salmonella enterica serovar Pullorum is the causative agent of pullorum disease in poultry, an acute systemic disease that results in a high mortality rate in young chicks but rarely causes severe clinical disease in adult birds. Salmonella pullorum causes infection by vertical transmission and horizonal transmission. Although pullorum disease has been largely eliminated from many western countries, it still has been happenning in small flocks. The explosion of pullorum disease is popular in china. Searching for virulent genes is very important to understand the pathogenesis of Salmonella pullorum and find therapeutic measures to the disease.With the completion of genomic sequence of microbes, comparative analysis of complete genome provides a powerful tool for studying the molecular pathgenesis of pathegenic mirobes. Although SSH (suppression subtractive hybridization) was first developed for cDNA comparison, it has been applied in genomics study of microbes and screening virulent genes and new genes. In this study, the Salmonella pullorum subtractive library was constructed and analyzed by comparison of genomic differences between Salmonella ullorum 533 (tester) and Salmonella enteritidis 50041 (driver) by SSH, and further ipaJ、mobA gene were cloned and expressed. 1. Construction of suppression subtractive library between Salmonella pullorum and Salmonella enteritidisUsing suppression subtractive hybridization (SSH) technique, the Salmonella pullorum subtractive library was constructed by comparison of genomic differences between Salmonella pullorum strain 533 (tester) and Salmonella enteritidis strain 50041 (driver). Parts of subtracted fragments were sequenced and searched homologically in GenBank after identification by Dot-blot. The results showed four types of fragments were identified: mobile genetic element, type III secretion sequences, sequences for plasmid transfer, sequences with unknown function. The amino acid homology of three subtracted sequences with ipaJ of Shigella flexneri was 50%. The results suggested that the Salmonella pullorum subtracted library was very useful for searching pathogenicity- related sequences and analyzing pathogenesis of Salmonella pullorum.2. Cloning and expression of Salmonella pullorum ipaJ geneThe Salmonella pullorum subtracted sequences PEA2、PE31 and PE44 were blasted on the GenBank. The results of BLASTX showed that the homological ratio of PEA2、PE31 and PE44 was 97%、99% and 100% respectively to ipaJ gene fragment in plasmid psfD10 of Salmonella choleraesuis. The three subtracted sequences were spliced together into the whole sequences of ipaJ in Salmonella pullorum, then a pair of primers were synthesized to amplify ipaJ gene in Salmonella pullorum strain 533 by polymerase chain reaction (PCR). PCR products were cloned into prokaryotic expressive vector pET-30a(+). The recombinant was transformed into the host strain BL21(DE3) and expressed by 0.5mM IPTG induced at 37℃. The expression product was found to be 37KDa by SDS-PAGE. Specific reaction was found between Salmonella pullorum positive serum and expressed protein in vitro by Western-blot assay. It implied that ipaJ was immunogenic and expressed when Salmonella pullorum infected chicks, may be it was related to pathogenicity of Salmonella pullorum. The specificity test proved ipaJ was only amplified from Salmonella pullorum and this gene is carried by all 97 salmonella pullorum isolates tested. The function of ipaJ was remained to be further studied.3. Cloning and expression of Salmonella pullorum mobA geneThe homological ratio of Salmonella pullorum subtracted sequence PE30 was 99% to mobA in Salmonella choleraesuis plasmid psfD10. According to the published sequence of mobA gene in plasmid psfD10 of Salmonella choleraesuis, a pair of primers were designed and synthesized to amplify mobA gene (about 1.6kb) in Salmonella pullorum strain 533 by polymerase chain reaction (PCR). PCR products were cloned into prokaryotic expressive vectors pET-30a(+) and pGEMX6p-1, then the recombinants were transformed into the host strain BL21(DE3) and BL21 respectively. After induction at 37℃with 0.5mM IPTG for four hours, the expression products were found to be 64kDa in BL21(DE3) and 84kDa in BL21 by SDS-PAGE. The His-MobA fusion protein were purified and used to immunize mice. Specific reaction was found between GST-MobA fusion protein expressed in BL21 and positive serum from mice immunized with His-MobA by Western-blot assay. It proved the immunogenicity of His-MobA protein.更多还原