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幽门螺杆菌cheA基因在细菌体外趋化和体内定植过程中的作用

Role of Helicobacter pylori cheA gene in chemotaxis in vitro and colonizationin vivo
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摘要 目的 了解cheA基因在幽门螺杆菌体外趋化和体内定植中的作用.方法 从幽门螺杆菌NCTC11637株基因组DNA中扩增并克隆全长cheA和cheY基因.构建该两个基因原核表达系统,Ni-NTA亲和层析法提取目的 重组蛋白rCheA和rCheY.rCheA和rCheY免疫家兔制备抗血清,采用硫酸铵沉淀法及DEAE-52柱层析法制备rCheA-IgG和rCheY-IgG.构建cheA基因自杀质粒,根据同源重组交换原理利用该自杀质粒构建cheA基因敲除突变株(cheA-),采用PCR及测序对cheA-突变株进行鉴定.采用rcheA-IgG和rCheY-IgG锚定靶蛋白及蛋白磷酸化检测试剂盒,测定cheA-突变株与野生株CheA和CheY分子磷酸化水平.采用幽门螺杆菌趋化模型及BALB/c小鼠感染模型,比较cheA-突变株与野生株体外趋化及体内定植能力的差异.结果 PCR及测序结果证实cheA-突变株基因组中cheA基因被敲除.0.001~0.1 mol/L盐酸作用10 min,野生株CheA和CheY磷酸化水平分别从(59.6±11.5)和(55.5±10.2)μmol迅速下降至(10.8±2.6)和(5.5±1.2)μmol(P<0.05),cheA-突变株CheY磷酸化水平均较低且无明显变化(P>0.05).cheA-突变株对盐酸、硫酸和乙酸趋化聚集环直径[(10~20)±(2~3)mm]明显小于野生株[(16~24)±(2~3)mm],P<0,05.野生株感染小鼠胃黏膜标本中幽门螺杆菌分离阳性率(90%)明显高于cheA-突变株(40%),P<0.05 荧光定量PCR结果也显示野生株感染小鼠胃黏膜标本中幽门螺杆菌数量(6.3×103±2.1×103拷贝/mg)也明显高于突变株的(8.3×101±3.1 ×101拷贝/mg),P<0.05.结论 cheA基因在幽门螺杆菌体外趋化和体内定植中有促进作用. Objective To determine the effect of cheA gene of Helicobacter pylori in the bacterial chemotaxis in vitro and colonization in vivo. Methods The entire cheA and cheY genes were amplified and cloned from genomic DNA of H. pylori NCTC11637 strain. Subsequently, the prokaryotic expression systems of cheA and cheY genes were generated and the target recombinant proteins rCheA and rCheY were extracted by Ni-NTA affinity chromatography. Rabbits were immunized with either rCheA or rCheY for obtaining antisera, and rCheA-IgG and rCheY-IgG in the antisera were prepared using ammonium sulfate precipitation plus DEAE-52 column chromatography. A suicide plasmid of cheA gene was constructed and then a cheA gene knock-out mutant ( cheA - ) was generated based on homologous recombinant exchange using the suicide plasmid. The cheA- mutant was identified using PCR and sequencing. The phosphorylation levels of CheA and CheY molecules of cheA - and wild-type strain were determined by using rCheA-IgG and rCheY-IgG anchoring the target proteins and protein phosphorylation detection kit. The differences of chemotaxis in vitro and colonization in vivo between cheA- mutant and wild-type strain were compared using chemotactic model and BALB/c infection model of H. pylori. Results The cheA gene knock-out in genome of cheA- mutant was confirmed by the results of PCR and sequencing. After treated with 0. 001-0. 1 mol/L HCI for 10 min, the phosphorylation levels of CheA and CheY molecules of wild-type strain were rapidly descended from ( 59.6 ±11.5) μmol and (55.5 ± 10.2) μmol to ( 10.8 ± 2.6) and (5. 5 ± 1.2) μmol (P 〈 0.05 ), while the phosphorylation of CheY molecule of cheA - mutant was no markedly changed with a persistent lower level ( P 〉0.05). The diameters [(10-20) ± (2-3) mm] of chemotactic aggregative rings of cheA- mutant were significantly less than those [(16-24) ± (2-3)mm] of wild-type strain (P 〈0.05). The positive isolation rate (90%) of H. pylori in gastric
作者 简丽娟 陈一强 温红侠 孔晋亮 闫萍 张东伟 CHEN Guang(Department of Medical Microbiology and Parasitology , College of Medicine, Zhejiang University, Hangzhou 310058, China) YAN Jie(Department of Medical Microbiology and Parasitology , College of Medicine, Zhejiang University, Hangzhou 310058, China) XU Li-hui WU Sheng-hai WANG Xian-jun
出处 《中华微生物学和免疫学杂志》 CAS CSCD 北大核心 2010年第11期1020-1024,共5页 Chinese Journal of Microbiology and Immunology
基金 基金项目:国家自然科学基金(30760084)
关键词 幽门螺杆菌 cheA基因 cheY基因 基因敲除 趋化 Helicobacter pylori cheA gene cheY gene Gene knock-out Chemotaxis
作者简介 通信作者:陈一强,Email:ehenyql63@tom.com,电话:0771.5350993
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