【摘要】 目的构建梅毒螺旋体(Tp)粘附素Tp0751的重组大肠埃希菌菌影(E.coli bacterial ghosts,EBG),为深入评价其在抗Tp感染中的潜在免疫保护作用奠定基础。方法将含有噬菌体Phi X174裂解基因E的质粒p HH43转化E.coli DH5α,42℃温控诱导使其形成EBG并计算裂解效率,用琼脂糖DNA电泳和透射电镜(TEM)鉴定EBG。构建含有裂解基因盒E-box、膜锚定序列E’-linker及Tp0751目的基因的原核双表达重组质粒p ET28a-E’-Tp0751-E-box,28℃诱导重组E.coli BL21表达Tp0751并用Western Blot鉴定;42℃诱导形成重组EBG(r EBG),计算裂解效率并用DNA电泳和TEM鉴定。结果构建的EBG裂解率为98.13%,DNA电泳未观察到DNA条带,TEM显示绝大部分细菌都裂解成缺乏胞浆成分的细胞空壳并保持活菌基本形态。r EBG在28℃时高效表达重组Tp0751蛋白,且仅与梅毒患者血清特异性结合;42℃下其r EBG裂解率为96.37%,电泳和TEM鉴定结果与EBG的相似。结论成功构建表达Tp粘附素Tp0751的r EGB,其所表达目的蛋白具有良好免疫反应性。更多还原

【Abstract】 Objective To construct the recombinant E.coli bacterial ghosts( EBG) expressing Treponema pallidum( Tp) adhesin Tp0751 and to further investigate its potential immunoprotection against Tp infection.Methods E.coli DH5α cells transformed with the plasmids p HH43,containing phagephi X174 lysis E gene,were induced at 42 ℃ to produce EBG. The lysis rate was calculated and EBG were identified by DNA agarose gel electrophoresis and Transmission Electron Microscope( TEM). E. coli BL21 cells with prokaryotic double expression recombinant plasmids p ET28a-E’-Tp0751-E-box,containing lysis gene cassette( E-box),membrane anchor sequence E’-linker and target gene Tp0751,were induced to express recombinant prtotein Tp0751 at 28 ℃ and to generate EBG at 42 ℃.Recombinant Tp0751 was identified by western blot.The lysis rate of recombinant EBG( r EBG) was calculated,DNA agarose gel electrophoresis and TEM were used to identify the EBG. Results The lysis rate of EBG was 98.13%. No DNA ladder was observed by DNA gel electrophoresis and TEM showed that the vast majority of bacteria were lysed into empty cell envelopes lacking cytoplasmic content yet retaining unaltered morphological features of their living counterparts. At 28 ℃,r EBG effectively expressed Tp0751 that were specifically reactive with syphilitic sera by Western blot.At 42 ℃,the lysis rate of r EBG was 96.37%.and similar to EBG tested by electrophoresis and TEM. Conclusion The recombinant EBG expressing Tp0751 are successfully constructed and expressed Tp0751 shows good immunoreactivity.更多还原