恙虫病东方体Karp株56-kDa外膜抗原基因片段的原核表达及免疫原性研究

Prokaryotic expression and immunogenicity of major outer membrane protein 56-kDa antigen of Orientia tsutsugamushi strain Karp

目的探讨恙虫病东方体56-kDa蛋白在大肠埃希菌中的表达及作为诊断性抗原的可行性。方法用PCR扩增恙虫病东方体Karp菌株编码56-kDa蛋白长约1 100bp的DNA,克隆至表达载体pET30a(+)。表达产物经SDS-PAGE、质谱分析及western blot鉴定分析。重组蛋白质纯化后作为包被抗原,用方阵实验确定最佳浓度包被,ELISA法检测其与立克次体目16种成员及10种致病菌阳性血清的反应性。用此重组蛋白质免疫小鼠后制备抗血清,分别用ELISA及间接免疫荧光法(IFA)检测抗血清的效价。结果重组质粒经PCR及测序分析证明,56-kDa外膜抗原的基因片段以正确的读码框连入到pET30a(+)质粒载体中,SDS-PAGE结果表明重组蛋白质在约46-kDa处有表达的目的条带,western blot分析显示该条带可与1∶1 600稀释的兔抗Karp血清反应,证实其具有免疫活性。质谱分析鉴定该蛋白质为恙虫病东方体56-kDa蛋白;方阵滴定确定选择抗原的最佳稀释度为1∶1 600,特异性分析结果表明,重组蛋白质除与查菲埃立克体、嗜吞噬细胞无形体及五日热巴尔通体存在弱交叉反应外,与其他细菌均无交叉反应。IFA及ELISA检测表明抗血清效价可达1∶1 600。结论构建的重组蛋白质可以作为恙虫病东方体快速诊断的抗原。

Objective To express the 56-kDa protein of Orientia tsutsugamashi strain Karp and evaluate the application of the recombi- nant protein in immunological diagnosis. Methods The fragment of 56-kDa gene was cloned into pET30a（ ＋ ） and then transformed to host bacteria Escherichia coli Rossetta. Specificity of the recombinant protein was assessed by ELISA with rabbit antisera against common member of order Rickettsiae and ten kinds of other clinical pathogens. Immunogenicity of the recombinant protein was studied in BALB/c mice. Results After induction with IPTG and analysis by SDS-PAGE, an approximately 46-kD band was observed and the test via western blot. Mass-spectrum proved that the recombinant protein was expressed successfully. Specificity analysis demonstrated that all sera except antisera against O. tsutsugamashi strain TA763, TH817 and Ehrlichia, B. Quintana, A. phagocytophilum were nega- tive. The purified protein was used to immunize BALB/c male mice and then the antisera （polyclonal antibody） were harvested. Ac- cording to IFA and ELISA test, the titer of the polyclonal antibody reaches to 1 ： 1 600. Conclusion The recombinant 56-kDa protein in this study exhibited excellent antigenicity and specificity, and should prove valuable for developing a simple and rapid diagnostic test and preparing the vaccine for O. tsutsugamushi.