外膜蛋白C作为蛋白呈递平台将抗原定位至细菌外膜囊泡表面的可行性  

Feasibility of outer membrane protein C as a protein presenting platform targeting antigen to surface of outer membrane vesicle

作　　者：孙京京 黄建东[1] SUN Jing-jing;HUANG Jian-dong(Shenzhen Institute of Advanced Technology,Chinese Academy of Sciences,Shenzhen Institute of Synthetic Biology,Shenzhen 518055,Guangdong Province,China)

机构地区：[1]中国科学院深圳先进技术研究院深圳合成生物学创新研究院,广东深圳518055

出　　处：《中国生物制品学杂志》2023年第1期39-42,47,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(82003259);深圳市科技计划(KQTD2015033117210153)。

摘　　要：目的探讨外膜蛋白C(outer membrane protein C,OmpC)作为蛋白呈递平台将抗原定位至细菌外膜囊泡(outer membrane vesicle,OMV)表面的可行性。方法构建含有ompC基因片段及金黄色葡萄球菌EsxA抗原基因(esxA基因)的重组表达质粒,转化感受态E.coli BL21(DE3),IPTG诱导表达,表达产物进行12%SDS-PAGE分析。提取重组菌OMV,经12%SDS-PAGE分析OMV总蛋白,并采用Western blot法和纳米流式仪检测融合蛋白定位至OMV表面的情况。结果含ompC基因和esxA基因的重组表达质粒经测序证明构建正确。融合蛋白OmpC-EsxA经12%SDS-PAGE分析,可见相对分子质量约57000的目的蛋白条带,大小与预期相符。OMV总蛋白经12%SDS-PAGE分析,可见多条蛋白条带,表明成功提取重组菌OMV。重组菌OMV表面的融合蛋白OmpC-EsxA可与小鼠抗His-Tag抗体发生特异性结合,且纳米流式仪可检测到荧光抗体标记的OMV。结论OmpC可作为蛋白呈递平台将抗原定位至OMV表面,有望应用于抗原呈递疫苗的研发。Objective To investigate the feasibility of outer membrane protein C(OmpC)as a protein presenting platform targeting antigen to the surface of outer membrane vesicle(OMV).Methods The recombinant expression plasmid containing ompC gene fragment and Staphylococcus aureus EsxA antigen gene(esxA gene)was constructed,transformed to competent E.coli BL21(DE3),induced by IPTG,and analyzed for expressed product by 12%SDS-PAGE.The total protein of recombinant strain OMV was analyzed by 12%SDS-PAGE,and the localization of fusion protein on the surface of OMV was detected by Western blot and Flow NanoAnalyzer.Results The recombinant expression plasmid containing ompC gene and esxA gene was constructed correctly as proved by sequencing.12%SDS-PAGE showed that the fusion protein OmpC-EsxA had a relative molecular mass of about 57000,which was consistent with the expected size,while the total protein of OMV showed multiple target protein bands,indicating that recombinant strain OMV was successfully extracted.The fusion protein OmpCEsxA on the surface of recombinant strain OMV specifically bound to mouse antibody against His-Tag,and OMVs labeled with fluorescent antibody were detected by Flow NanoAnalyzer.Conclusion OmpC may be used as a protein presenting platform to locate antigen to OMV surface,which was expected to be applied in the development of antigen presentation vaccine.

关 键 词：外膜蛋白C 蛋白呈递 细菌外膜囊泡 

分 类 号：Q939.99[生物学—微生物学]