新型抗原递送载体:细胞溶素A融合目的蛋白整合的重组外膜囊泡的构建与鉴定  

A Novel Model for Antigen Delivery Carrier: Construction and Identification of Cytolysin A(-EGFP) Fusion Protein-integrated Recombinant Outer Membrane Vesicles

作　　者：王世杰[1,2,3] 黄惟巍[1,2,3] 舒聪妍 黎奎[1,2,3] 夏烨[1,2,3] 李扬[1,2,3] 李慧[1,2,3] 孙鹏艳 杨旭[1,2,3] 白红妹[1,2,3] 孙文佳[1,2,3] 刘存宝[1,2,3] 褚晓杰[1,2,3] 冯雪军[1,2,3] 马雁冰[1,2,3] WANG Shi-Jie HUANG Wei-Wei SHU Cong-Yan LI Kui XIA Ye LI Yang LI Hui SUN Peng-Yan YANG Xu BAI Hong-Mei SUN Wen-Jia LIU Cun-Bao CHU Xiao-Jie FENG Xue-Jun MA Yan-Bing(Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118, China Yunnan Key Laboratory of Research and Development on Severe Infection Disease, Kunming 650118, China Yunnan Engineering Research Center of Vaccine Research and Development on Severe Infectious Disease, Kunming 650118, China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,昆明650118 [2]云南省重大传染病疫苗研发重点实验室,昆明650118 [3]云南省重大传染病疫苗工程技术研究中心,昆明650118

出　　处：《中国生物化学与分子生物学报》2016年第11期1272-1278,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：云南省科技计划项目(No.2016FA049);北京协和青年基金和中央高校基本科研业务费专项资金(No.3332015197;No.3332016117);北京协和医学院研究生创新基金项目(No.10023-0710-1003);北京协和医学院病原生物研究所基本科研业务费项目(No.2014IPB107)资助~~

摘　　要：细菌外膜囊泡(OMVs)是革兰氏阴性菌以出芽方式分泌的一种蛋白脂质体,可通过基因工程改造成为外源大分子抗原蛋白的载体。成孔蛋白细胞溶素A(Cly A)可作为引导序列将C-端融合的外源蛋白质定位在菌体及其所分泌的OMVs外膜上。利用Cly A融合蛋白重组的OMVs可介导抗原呈递,制备疫苗。为验证这一假说,本研究拟以增强型绿色荧光蛋白(EGFP)为目的蛋白,采用重组技术,构建Cly A-EGFP融合蛋白表达载体,转化E.coli DH5α,获得Cly A-EGFP融合蛋白整合的重组细菌OMVs,探索Cly A/EGFP融合蛋白整合的OMVs作为一种制备新型疫苗载体的可行性。重组OMVs分别经蛋白酶K和/或EDTA处理后,蛋白质印迹分析显示,Cly A-EGFP融合蛋白有效地整合在重组的OMVs中,且EGFP呈现于表面。重组OMVs与小鼠巨噬细胞Raw264.7共同孵育后,以EGFP作为标记,荧光显微镜观察显示,OMVs可被巨噬细胞吞噬、摄取。进而,荧光显微镜及流式细胞术分析证明,重组的OMVs可被骨髓来源的树突状细胞(BMDCs)有效摄取,并促进BMDCs表达CD86和CD80,说明重组的OMVs可激活树突状细胞,促进其成熟。本研究结果提示,利用Cly A融合特异目的蛋白整合的重组OMVs,可作为外源蛋白质的载体,介导抗原呈递作用,在设计与制备疫苗中具有潜在的应用价值。The bacterial outer membrane vesicles （OMVs） are proteoliposomes, budding from Gramnegative bacteria, and will become a carrier for exogenous protein antigens via genetic engineering modifications. The pore-forming cytolysin A （ClyA） is able to guide the recombinant protein, which is genetically fused with the C-terminus of ClyA, on the surface of the bacteria and their derived OMVs. Therefore, we hypothesize that vaccine can be prepared by making use of ClyA fushion for the recombinant OMVs to mediate antigen presentation. To test the hypothesis, the enhanced green fluorescence protein （EGFP）was used as a model protein and genetically fused with ClyA. Then, the constructed recombinant plasmid was transformed into E. coli DH5α and the recombinant OMVs delivering EGFP-ClyA fusion protein were acquired. Finally, the application possibility of a novel vaccine carrier based on the ClyA/EGFP integrated recombinant OMVs was investigated. Western-blot analysis indicated that the fusion protein ClyA/EGFP was successfully integrated on the surface of OMVs after treating them with protein K and/or EDTA. Using EGFP as a label, incubation between the recombinant OMVs and murine macrophages 264.7 showed that OMVs were uptaken by macrophages effectively. Moreover, fluorescent microscope observation and flow cytometry analysis showed that OMVs were uptaken by bone marrow dendritic cells （BMDCs） and mounted CD80 and CD86 expression, which revealed that the recombinant OMVs activated DCs and promoted their maturation. These results indicated that the engineered OMVs with integration of ClyA fused specific protein could become an exogenetic antigen delivery carrier, which mediate antigen presentation and will provide values in vaccine design and preparation.

关 键 词：外膜囊泡 疫苗 增强绿色荧光蛋白 抗原递呈细胞 

分 类 号：R392.11[医药卫生—免疫学]