禽病原性大肠杆菌iro和tsh基因缺失株的构建  

Construction and Characterization of Avian Pathogenic Escherichia coli Mutants with iro and/or tsh Gene Mutation

作　　者：陈祥[1] 刘静[1] 高崧[1] 潘志明[1] 焦新安[1] 刘秀梵[1] 

机构地区：[1]扬州大学农业部畜禽传染病学重点开放实验室,江苏省人兽共患病学重点实验室,扬州225009

出　　处：《生物工程学报》2008年第3期401-408,共8页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30471281,30771604);国家“863计划”(No.2003AA222141)~~

摘　　要：通过SSH和SCOTS研究,铁系统(Iro)和温度敏感性血凝素(Tsh)在禽病原性大肠杆菌(APEC)的感染中可能发挥重要作用。基因检测发现,在243个禽源大肠杆菌分离株中,有205株为iro+菌株,其中高、中度和低致病株分别为89.8%(184/205)、8.8%(18/205)和1.5%(3/205);有167株为tsh+菌株,高、中度、低致病株分别为87.4%(146/167)、12.6%(21/167)和0%(0/167),结果显示iro+或tsh+株大多数为高致病株。为了确定iro和tsh基因在APEC致病力中的作用,以APECE037株为基础,通过自杀性载体分别构建了iro和tsh基因缺失突变株E037(Δiro)、E037(Δtsh)和E037(ΔiroΔtsh)。动物感染性试验表明,突变株在鸡体内的繁殖能力和致病性均明显下降,但两个基因的协同致病作用不显著。进一步证实Iro和Tsh为APEC重要的致病因子。Iro system and temperature-sensitive hemagglutinin （Tsh） genes were identified by suppression subtraetive hybridization （SSH） and selective capture of transcribed sequences （SCOTS）. To get more insights in the distribution and the occurrence of the iroC and tsh genes, we examined 243 avian E. coli strains for the presences of the these genes. Among 243 avian E. coli isolates, iroC gene was present in 84.4% strains （205/243）. Of the 205 iroC-positive isolates, iroC gene was found in 184 （89.8%）, 18（8.8%） and 3 （1.5%） isolates with high, intermediate and low pathogenicity, respectively. Of the 167 tsh-positive isolates, tsh gene was detected in 146 （87.4%）, 21 （12.6%） and 0 （0%） isolates with high, intermediate and low pathogenicity, respectively. Among tsh-positive isolates, 89.5 to 100% of the highly pathogenic isolates of O1, O2 or O78 serogroups had the tsh gene, while 53.3% of the highly pathogenic isolates of non-O1, O2 and O78 serogroups had the tsh gene （P〈0.01）. Suicide vectors for deletion of the iroBCDEN or tsh genes were constructed as follows. The 715-bp fragments of iroB and 603-bp fragment of the iroN were generated by PCR respectively. Both of these two fragments together with EGFP gene were cloned into pUC18, termed pUC18-iroBNEGFP. A resultant suicide vector containing the iroB-EGFP-iroN fragment was obtained and named pMEG375-iroBNEGFP. Similarly, both of the 685-bp fragment of tshF and the 692-bp fragment of the tshR together with gentamycin gene were cloned into pUC18, resulting in pUC18-tshFRGm. A resultant suicide vector containing the tshF-Gm-tshR fragment was named pMEG375-tshFRGm. Mutant derivatives of strain E037 were generated by allelic replacements and were named E037（△iro）, E037（△tsh） and E037（△iro△tsh）. The 50% lethal dose （LD50） of E037, E037（△iro）, E037（△tsh） and E037（△iro△tsh） in commercial day-old chickens experimentally inoculated via intratrachea were determined to be 10^5.6, 10^8.4, 10^9.

关 键 词：禽病原性大肠杆菌 铁系统 温度敏感性血凝素 缺失突变株 

分 类 号：S852.5[农业科学—基础兽医学] Q78[农业科学—兽医学]