杀香鱼假单胞菌是大黄鱼内脏白点病的致病菌,感染该菌常会导致养殖大黄鱼很高的死亡率和严重的经济损失。病原株NB2011编码典型的Ⅲ型分泌系统,可能是该菌重要的毒力因子,ExsA是控制此分泌系统表达的重要调控蛋白。为确认ExsA在NB2011...杀香鱼假单胞菌是大黄鱼内脏白点病的致病菌,感染该菌常会导致养殖大黄鱼很高的死亡率和严重的经济损失。病原株NB2011编码典型的Ⅲ型分泌系统,可能是该菌重要的毒力因子,ExsA是控制此分泌系统表达的重要调控蛋白。为确认ExsA在NB2011致病过程中的作用,开发有效疫苗,实验采用双交换同源重组法构建了ExsA内部序列被卡那霉素基因替换的突变株,检测突变株与野生株对鼠巨噬细胞J774的黏附、内化和胞内增殖特性,并比较对大黄鱼的毒力变化,同时,通过透射电子显微镜观察人工感染后大黄鱼内脏组织的病理变化。研究表明,巨噬细胞对突变株的内化率降低,内化的细菌在12 h内被清除,野生株在内化后虽然一段时间内数量稍有下降,12~24 h期间数量急剧上升;突变株对大黄鱼的96 h LD_(50)为2.59×10~7/mL,比野生株高数百倍;电镜切片中未观察到组织内有菌体的存在,表明突变株的毒力明显减弱,可以作为弱毒疫苗的开发对象。展开更多
目的重组表达类鼻疽杆菌Ⅵ型分泌系统溶血素共调节蛋白1(hemolysin coregulated protein 1,Hcp1),并对其免疫学性质进行鉴定。方法利用DNA重组技术以pET-28a表达系统在E.coli BL21(DE3)中重组表达Hcp1;通过亲和层析制备高纯度Hcp1蛋白;...目的重组表达类鼻疽杆菌Ⅵ型分泌系统溶血素共调节蛋白1(hemolysin coregulated protein 1,Hcp1),并对其免疫学性质进行鉴定。方法利用DNA重组技术以pET-28a表达系统在E.coli BL21(DE3)中重组表达Hcp1;通过亲和层析制备高纯度Hcp1蛋白;采用腹腔注射免疫小鼠制备抗Hcp1血清,ELISA检测抗体滴度,免疫印迹及免疫荧光分析抗体的免疫反应性。结果成功获得纯度达95%的重组Hcp1蛋白,该蛋白免疫小鼠后获得的抗血清具有较好的特异性和较高的抗体滴度(1∶512000),表现出良好的免疫原性和免疫反应性。结论成功制备了具有生物学活性的Hcp1重组蛋白及其抗血清。展开更多
The general secretory(Sec)pathway represents a common mechanism by which bacteria secrete proteins,including virulence factors,into the extracytoplasmic milieu.However,there is little information about this system,as ...The general secretory(Sec)pathway represents a common mechanism by which bacteria secrete proteins,including virulence factors,into the extracytoplasmic milieu.However,there is little information about this system,as well as its associated secretory proteins,in relation to the fire blight pathogen Erwinia amylovora.In this study,data mining revealed that E.amylovora harbors all of the essential components of the Sec system.Based on this information,we identified putative Sec-dependent secretory proteases in E.amylovora on a genome-wide scale.Using the programs SignalP,LipoP,and Phobius,a total of 15 putative proteases were predicted to contain the N-terminal signal peptides(SPs)that might link them to the Sec-dependent pathway.The activities of the predicted SPs were further ttvreaaalicsdyeta otgepelda nsuesmsi inicng cprarenoa psEeesrtdcy h.s eiTrgirncaihfniicsa carcniotplltiyi-obwnahasle enad nEaa.ll kyaaslmiensye lo spvhohorowas epwdh atasht aaust sete h(deP theoo xipAnr)o ecgsueslinaotene f iuofms i1 om1 na tosufyr steth epe em1 a t5 rh s,ae txs tcuroagcngfyeitrsomtipnelagd s ttmhhieecii rr pperoox---xL?tential roles in plant infection.The results of this study support the suggestion that E.amylovora might employ the Sec system to secrete a suite of proteases to enable successful infection of plants,and shed new light on the interaction of E.amylovora with host plants.展开更多
文摘杀香鱼假单胞菌是大黄鱼内脏白点病的致病菌,感染该菌常会导致养殖大黄鱼很高的死亡率和严重的经济损失。病原株NB2011编码典型的Ⅲ型分泌系统,可能是该菌重要的毒力因子,ExsA是控制此分泌系统表达的重要调控蛋白。为确认ExsA在NB2011致病过程中的作用,开发有效疫苗,实验采用双交换同源重组法构建了ExsA内部序列被卡那霉素基因替换的突变株,检测突变株与野生株对鼠巨噬细胞J774的黏附、内化和胞内增殖特性,并比较对大黄鱼的毒力变化,同时,通过透射电子显微镜观察人工感染后大黄鱼内脏组织的病理变化。研究表明,巨噬细胞对突变株的内化率降低,内化的细菌在12 h内被清除,野生株在内化后虽然一段时间内数量稍有下降,12~24 h期间数量急剧上升;突变株对大黄鱼的96 h LD_(50)为2.59×10~7/mL,比野生株高数百倍;电镜切片中未观察到组织内有菌体的存在,表明突变株的毒力明显减弱,可以作为弱毒疫苗的开发对象。
文摘目的重组表达类鼻疽杆菌Ⅵ型分泌系统溶血素共调节蛋白1(hemolysin coregulated protein 1,Hcp1),并对其免疫学性质进行鉴定。方法利用DNA重组技术以pET-28a表达系统在E.coli BL21(DE3)中重组表达Hcp1;通过亲和层析制备高纯度Hcp1蛋白;采用腹腔注射免疫小鼠制备抗Hcp1血清,ELISA检测抗体滴度,免疫印迹及免疫荧光分析抗体的免疫反应性。结果成功获得纯度达95%的重组Hcp1蛋白,该蛋白免疫小鼠后获得的抗血清具有较好的特异性和较高的抗体滴度(1∶512000),表现出良好的免疫原性和免疫反应性。结论成功制备了具有生物学活性的Hcp1重组蛋白及其抗血清。
基金Project supported by the Key Technologies R&D Program of Xinjiang Production and Construction Corps(No.2018AB038)the National Natural Science Foundation of China(No.31970126)。
文摘The general secretory(Sec)pathway represents a common mechanism by which bacteria secrete proteins,including virulence factors,into the extracytoplasmic milieu.However,there is little information about this system,as well as its associated secretory proteins,in relation to the fire blight pathogen Erwinia amylovora.In this study,data mining revealed that E.amylovora harbors all of the essential components of the Sec system.Based on this information,we identified putative Sec-dependent secretory proteases in E.amylovora on a genome-wide scale.Using the programs SignalP,LipoP,and Phobius,a total of 15 putative proteases were predicted to contain the N-terminal signal peptides(SPs)that might link them to the Sec-dependent pathway.The activities of the predicted SPs were further ttvreaaalicsdyeta otgepelda nsuesmsi inicng cprarenoa psEeesrtdcy h.s eiTrgirncaihfniicsa carcniotplltiyi-obwnahasle enad nEaa.ll kyaaslmiensye lo spvhohorowas epwdh atasht aaust sete h(deP theoo xipAnr)o ecgsueslinaotene f iuofms i1 om1 na tosufyr steth epe em1 a t5 rh s,ae txs tcuroagcngfyeitrsomtipnelagd s ttmhhieecii rr pperoox---xL?tential roles in plant infection.The results of this study support the suggestion that E.amylovora might employ the Sec system to secrete a suite of proteases to enable successful infection of plants,and shed new light on the interaction of E.amylovora with host plants.
