生物工程學(xué)報(bào) Chin J Biotech 2008, March 25; 24(3): 401-408

journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061

[email protected] . 2008 Institute of Microbiology, CAS & CSM, All rights reserved

陳祥, 劉靜, 高崧, 潘志明, 焦新安, 劉秀梵

揚(yáng)州大學(xué) 農(nóng)業(yè)部畜禽傳染病學(xué)重點(diǎn)開放實(shí)驗(yàn)室/江蘇省人獸共患病學(xué)重點(diǎn)實(shí)驗(yàn)室, 揚(yáng)州 225009

摘 要: 通過SSH 和SCOTS 研究, 鐵系統(tǒng)(Iro)和溫度敏感性血凝素(Tsh)在禽病原性大腸桿菌(APEC)的感染中可能發(fā)揮重要作用?；驒z測發(fā)現(xiàn), 在243 個(gè)禽源大腸桿菌分離株中, 有205 株為iro+菌株, 其中高,、中度和低致病株分別為89.8%(184/205)、8.8%(18/205)和1.5%(3/205); 有167 株為tsh+菌株, 高,、中度,、低致病株分別為87.4%(146/167)、12.6%(21/167)和0%(0/167), 結(jié)果顯示iro+或tsh+株大多數(shù)為高致病株,。為了確定iro 和tsh 基因在APEC 致病力中的作用, 以APEC E037 株為基礎(chǔ), 通過自殺性載體分別構(gòu)建了iro 和tsh 基因缺失突變株E037(Δiro),、E037(Δtsh)和E037(ΔiroΔtsh)。動物感染性試驗(yàn)表明, 突變株在雞體內(nèi)的繁殖能力和致病性均明顯下降, 但兩個(gè)基因的協(xié)同致病作用不顯著,。進(jìn)一步證實(shí)Iro 和Tsh 為APEC 重要的致病因子,。

關(guān)鍵詞: 禽病原性大腸桿菌, 鐵系統(tǒng), 溫度敏感性血凝素, 缺失突變株

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

Xiang Chen, Jing Liu, Song Gao, Zhiming Pan, Xin’an Jiao and Xiufan Liu

Animal Infectious Disease Laboratory of Ministry of Agriculture, Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou 225009, China

Abstract: Iro system and temperature-sensitive hemagglutinin (Tsh) genes were identified by suppression subtractive 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(Δiro), E037(Δtsh) and E037(ΔiroΔtsh). The 50% lethal dose (LD50) of E037, E037(Δiro), E037(Δtsh) and E037(ΔiroΔtsh) in commercial day-old chickens experimentally inoculated via intratrachea were determined to be 105.6, 108.4, 109.0 and 109.5CFU, respectively. In the chicken challenging model, the mutants were tested to determine the individual role of this system for virulence and persistence in chickens. The result suggested that Iro system and Tsh were important in the pathogenicity of APEC.

Keywords: avian pathogenic Escherichia coli, iro system, temperature-sensitive hemagglutinin, mutant

禽大腸桿菌病(Avian colibacillosis)指由禽病原性大腸桿菌(Avian pathogenic Escherichia coli, APEC)所引起的傳染病, 常引起局部或全身感染, 是雞、鴨,、鵝和火雞等禽類的一種常見傳染病,。部分APEC菌株基因組與人的尿道致病性大腸桿菌(Uropathogenic E. coli, UPEC)非常相似, 提示部分APEC 菌株可能是一種食源性病原微生物及人泌尿系統(tǒng)感染UPEC 的來源, 顯示其重要的公共衛(wèi)生意義[1]。禽大腸桿菌血清型復(fù)雜多樣, 世界許多地方都開展了禽病原性大腸桿菌血清型的調(diào)查工作, O78 為最優(yōu)勢的血清型[2], 近年來從我國分離的1351 株禽源大腸桿菌中, O78 血清型有271 株, 占分離菌株的20.1%, 為最主要的血清型[3],。禽大腸桿菌病致病機(jī)理復(fù)雜, 許多毒力因子功能尚不清楚, 本文以我國分離的APEC E037 株(O78血清型)獲得的SSH 差異片段和SCOTS 體內(nèi)轉(zhuǎn)錄序列為基礎(chǔ)[4,5], 選取鐵系統(tǒng)(Iro system, Iro)和溫度敏感性血凝素(Temperature-sensitive hemagglutinin,Tsh)為研究對象, 分別構(gòu)建了iro 和tsh 基因缺失突變E037(Δiro),、E037(Δtsh)和E037(ΔiroΔtsh), 并初步研究了突變株的致病性, 為深入研究iro 和tsh基因的功能及研制腸道外感染大腸桿菌基因工程疫苗提供了有益嘗試。

全文下載：禽病原性大腸桿菌iro 和tsh 基因缺失株的構(gòu)建

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