禽致病性大肠杆菌Ⅲ型分泌系统2 EivC点突变的构建及其ATPase活性分析  

CONSTRUCTION OF EIVC POINT MUTATION AND ANALYSIS OF ITS ATPASE ACTIVITY IN AVIAN PATHOGRNIC ESCHERICHIA COLI TYPE Ⅲ SECRETION SYSTEM 2

作　　者：许漩 王少辉[2] 刘新[2] 王栋[2] 梁华[2] 黄彩兰[2] 吴晓君[2] 丁铲[2] 王桂军[1] 于圣青[2] XU Xuan WANG Shao-hui LIU Xin WANG Dong LIANG Hua HUANG Cai-lan WU Xiao-jun DING Chan WANG Gui-jun YU Sheng-qing(College of Animal and Technology, Anhui Agricultural University, Hefei 230036, China Shanghai Veterinary Research Institute, CAAS, Shanghai 200241, China)

机构地区：[1]安徽农业大学动物科技学院,合肥230036 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2017年第3期47-53,共7页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金项目(No.31572523);公益性农业(科研)专项项目(No.201303044)

摘　　要：为了分析禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)Ⅲ型分泌系统2(E.coli TypeⅢsecretion system2,ETT2)Eiv C ATPase关键活性位点,本研究通过比对分析病原菌ATPase序列,筛选APEC ETT2 Eiv C的关键位点。然后设计点突变引物,通过PCR方法构建Eiv C点突变片段,构建Eiv C点突变重组表达质粒。经IPTG诱导表达后纯化相应蛋白,并检测其ATPase活性。结果显示,Eiv C蛋白包含F0F1 ATPase的保守序列,且具有ATPase活性。测序结果显示Eiv C第175、199、201、233位氨基酸突变成功,融合蛋白获得成功表达与纯化。然而,点突变与野生型Eiv C的ATPase活性差异不具有显著统计学意义。本研究结果表明第175、199、201、233位氨基酸不影响Eiv C的ATPase活性。To determine the catalytic sites of Type Ⅲ secretion system 2 （ETT2） ATPase EivC in Avian pathogenic Escherichia coli （APEC）, we screened the key points of APEC ETT2 EivC by comparing the ATPase sequences of other pathogenic bacteria. The site- directed mutagenesis primers were designed and mutant recombinant plasmids were constructed using PCR method. The fusion protein was expressed with induction of isopropyl-beta-D-thiogalactopyranoside （IPTG）. Then, the ATPase activity of the fusion protein was determined using an ATPase/GTPase assay kit. The results indicated that EivC contained conserved region FOF1 ATPase and possessed ATPase activity. The DNA sequencing showed that the mutations at 175, 199, 201 and 233 amino acids of EivC were constructed. However, there was no significant difference in the ATPase activity between the recombinant mutant and wild-type EivC proteins, suggesting that 175, 199, 201 and 233 amino acids did not affect ATPase activity of EivC.

关 键 词：禽致病性大肠杆菌 Ⅲ型分泌系统2 eivC基因 ATPASE 

分 类 号：S852.612[农业科学—基础兽医学]