禽病原性大肠杆菌tsh突变株的构建及分离株tsh基因的检测  

Construction of Defined Mutations of Avian Pathogenic Escherichia coli Strain and Detection of tsh Gene in Avian Isolates

作　　者：陈祥[1] 刘静[1] 苗晓青[1] 王晓泉[1] 高崧[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

出　　处：《畜牧兽医学报》2007年第5期493-499,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(30471281);国家杰出青年科学基金(30425031);国家"863"计划基金(2006AA10A206)

摘　　要：运用基因重组方法将庆大霉素（Gentamycin,GM）抗性基因连接到PCR扩增的tsh两端区域产生的2个目的基因片段之间,并共同插入到pUC18载体的多克隆位点中,构建出带GM标志的载体pUC18-tshFRGM,从中切下此tshF-GM-tshR片段,再将之克隆到pMEG-375自杀性载体中,构建出自杀性载体pMEG375-tshFRGM,将突变载体转化到含tsh基因的受体APEC E037株中,根据同源重组原理,筛选出tsh基因缺失的E037突变株,命名为E037（Δtsh）。E037和E037（Δtsh）株对1日龄鸡的LD50分别为105.6CFU和109.0CFU,动物感染性试验表明E037（Δtsh）株在内脏器官和血液中的感染能力和大肠杆菌病变程度均有明显下降。在243株禽源分离株中,有167株为tsh＋菌株,其中高致病株、中等致病株和低致病株分别为87.4%（146/167）、12.6%（21/167）和0%（0/167）;O1、O2和O78血清型的高致病株占所在血清型分离株的89.5%-100%,而其它血清型的高致病株仅占其它分离株的53.3%,差异极显著（P〈0.01）。结果显示tsh＋株大多数为高致病性菌株,且其致病性与血清型的种类有一定的相关性,温度敏感性血凝素为APEC重要的致病因子。Gentamycin-resistant gene （GM） was ligated with the flanking fragments of tsh gene amplified by PCR, and then the product was transferred into MCS of pUC18 via DNA recombination techniques. The resulting plasmid was named pUC18-tshFRGM. The fragment of interest was digested and subcloned into suicide vector pMEG375 to generate the plasmid pMEG375- tsh- FRGM. The plasmid was transformed into APEC E037 used as recipient strain containing tsh gene. △Tsh-deleted E037 mutant strain was screened by homologous recombination. The animal infection results revealed that the pathogenicity of E037 （△tsh） strain reduced. Successful construction of △tsh-deleted E037 mutant strain has an important value and significance for elucidating the function of tsh gene and the role of tsh gene playing in the pathogenicity. The 50% lethal dose （LD50） of E037 and E037 （△tsh） in commercial 1 day-old chickens experimentally inoculated via intratrachea were 10^5.6 CFU and 10^9.0 CFU, respectively. In the chicken challenge model, the mutants were tested to determine the individual role of this system for virulence and persistence in chickens. Among 243 avian E. coli isolates, tsh was present in 68.7% （167 strains）. 146 （87.4%）, 21 （12.6%） and 0 （0%） of 167 tsh^＋ isolates were high, intermediate and low in pathogenicity, respectively. Among tsh^＋ isolates, 89. 5%-100% of those high pathogenic isolates of 01, 02 or 078 serogroups hold tsh, while 53.3% in other isolates except 01, 02 and 078 serogroups possessed tsh （P〈0. 01）.

关 键 词：禽病原性大肠杆菌 同源重组 温度敏感性血凝素 突变体 

分 类 号：S852.6[农业科学—基础兽医学]