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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。
Bacterial outer membrane vesicles (OMVs) are secreted by the gram-negative bacteria as a kind of liposome, which can be genetically engineered to become a carrier of exogenous macromolecular antigen protein. Porphyromonasin A (Cly A) can serve as a guide sequence for the C-terminal fusion of foreign proteins located in the bacterial cells and their secreted OMVs outer membrane. OMVs recombined with the Cly A fusion protein mediate antigen presentation and prepare vaccines. In order to validate this hypothesis, this study proposed to use enhanced green fluorescent protein (EGFP) as the target protein, recombinant Cly A-EGFP fusion protein expression vector was constructed, transformed E. coli DH5α, Cly A-EGFP fusion protein integration Of recombinant bacterial OMVs and explored the feasibility of integrating OMVs with Cly A / EGFP fusion proteins as a novel vector for vaccine production. Western blot analysis of recombinant OMVs treated with proteinase K and / or EDTA, respectively, showed that the Cly A-EGFP fusion protein was efficiently integrated in the recombinant OMVs and EGFP was present on the surface. Recombinant OMVs and mouse macrophage Raw264.7 co-incubated with EGFP as a marker, fluorescence microscopy showed that OMVs macrophages can be phagocytosed, uptake. Furthermore, fluorescence microscopy and flow cytometry analysis demonstrated that recombinant OMVs can be efficiently taken up by bone marrow-derived dendritic cells (BMDCs) and promote BMDCs to express CD86 and CD80, suggesting that recombinant OMVs activate dendritic cells and promote It is mature. Our results suggest that recombinant OMVs fused with a specific protein of Cly A can be used as a foreign protein carrier to mediate antigen presentation and have potential application in the design and preparation of vaccines.