禽致病性大肠杆菌毒力基因F11分布的检测及其表达  

Distribution detection and expression of virulence gene F11 of avian pathogenic Escherichia coli

作　　者：孟庆美[1] 王少辉[2] 任建鸾[1] 诸葛祥凯 陆承平[1] 戴建君[1] 

机构地区：[1]南京农业大学动物医学院农业部动物细菌病重点实验室,江苏南京210095 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国兽医科学》2013年第10期1011-1015,共5页Chinese Veterinary Science

基　　金：科技基础性工作专项(2012FY111000)

摘　　要：为研究禽致病性大肠杆菌F11毒力基因的功能,根据禽致病性大肠杆菌IMT5155毒力基因F11的序列,设计合成特异性引物,通过PCR方法检测了毒力基因F11在禽致病性大肠杆菌中的分布,并以IMT5155基因组DNA为模板扩增F11基因片段,将其定向克隆到pET-28a(+)载体中构建原核表达质粒pET-28a-F11,转化入大肠杆菌BL21(DE3)感受态细胞进行IPTG诱导表达,纯化表达产物,制备免疫血清进行黏附抑制试验。结果显示,F11基因在禽致病性大肠杆菌中的分布率为22.2%(10/45),主要分布于大肠杆菌B2进化群中。利用F11免疫血清进行的Western-blot分析表明,F11可以在实验室培养条件下正常表达。黏附抑制试验显示,F11免疫血清可以抑制禽致病性大肠杆菌IMT5155对宿主细胞的黏附,表明F11可能在禽致病性大肠杆菌感染过程中发挥作用。This study was performed to determine the role of Fll gene in avian pathogenic Escherichia coli（APEC）. Primers for Fll amplification were designed according to the sequence of Fll in APEC IMT5155. Then,PCR was used to detect the distribution of putative virulence gene Fll in APEC strains. A Fll gene fragment was amplified from genomic DNA of APEC IMT5155 strain by polymerase chain reaction（PCR）, which was cloned into pET-28a （＋） vectors. The resulting recombinant expression plasmid pET-28a-Fll was transformed into E. coli BL21（DE3） competent cells and the F11-His fusion protein was expressed by isopropyl-beta-D-thiogalactopyranoside（IPTG）. The fusion protein F11-His was purified and its antisera were prepared in rabbits and used for adhesion inhibition experiments. The results showed that virulence gene Fll was present in 22.2~ （10/45） APEC isolates. Moreover, the isolates positive for putative virulence genes were grouped into E. coli phylogenetic group B2. Western-blot analyses indicated that Fll was expressed under laboratory conditions. The anti-Fll antisera could significantly inhibit the adhesion of APEC IMT5155 ,indicating that Fll may play a role in the pathogenicity of APEC.

关 键 词：禽致病性大肠杆菌 毒力基因F11 黏附抑制试验 

分 类 号：S852.612[农业科学—基础兽医学]