大肠埃希菌外膜囊泡作为新型外源质粒递送载体的研究
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摘要
目的探讨大肠埃希菌外膜囊泡(outer membrane vesicles,OMVs)作为新型DNA疫苗递送载体的潜力及价值。方法从大肠埃希菌DH5α的培养上清中经过滤、超滤、超速离心等步骤收集天然分泌的OMVs,采用密度梯度离心法纯化OMVs。将纯化后的OMVs与哺乳动物吞噬细胞及非吞噬细胞进行体外孵育,观察不同细胞对OMVs的摄取效率。通过电击转化方式将带绿色荧光蛋白的真核表达质粒pIRES2-EGFP转入OMVs中,并与RAW264. 7细胞共孵育,观察OMVs携带真核表达质粒进入细胞并表达绿色荧光蛋白的情况。结果密度梯度离心法可有效纯化OMVs,纯化后的OMVs进入不同哺乳动物细胞的效率不同,进入吞噬细胞的效率高于非吞噬细胞,经电击转化后,质粒DNA可被导入OMVs中,并随吞噬细胞摄取及表达。结论约5 300 bp的真核表达质粒可用电击方式导入OMVs中,并通过OMVs介导该质粒在吞噬细胞中表达,提示OMVs具有作为新型质粒DNA递送载体的潜力。
Objective To investigate the potential and significance of E. coli outer membrane vesicles(OMVs) as a novel delivery vector for exogenous plasmid. Methods OMVs were collected from the supernatant of E. coli DH5α strain by filtration, ultrafiltration and ultracentrifugation, then purified by density gradient centrifugation. The purified OMVs were co-incubated with phagocytic cells and non-phagocytic cells, from animal origin, in vitro to observe the uptake rate of OMVs in different cells. OMVs were transformed with the eukaryotic expression plasmid p IRES2-EGFP with green fluorescent protein by electroporation, and incubated with RRAW264. 7 cells to observe the entrance of OMVs carrying eukaryotic expression plasmids into phagocytic cells and expression of green fluorescent protein. Results OMVs were purified effectively by density gradient centrifugation. The efficiencies of purified OMVs in entering various mammalian cells were different, which was higher in entering phagocytic cells than in entering non-phagocytic cells. After electrotransformation, plasmid DNA was introduced into OMVs, and ingested and expressed with phagocytic cells. Conclusion The eukaryotic expression plasmid at a length of about 5 300 bp may be introduced to OMVs by electroporation and express in phagocytic cells, indicating the potential of OMVs as a novel delivery vector of plasmid DNA.
Objective To investigate the potential and significance of E. coli outer membrane vesicles(OMVs) as a novel delivery vector for exogenous plasmid. Methods OMVs were collected from the supernatant of E. coli DH5α strain by filtration, ultrafiltration and ultracentrifugation, then purified by density gradient centrifugation. The purified OMVs were co-incubated with phagocytic cells and non-phagocytic cells, from animal origin, in vitro to observe the uptake rate of OMVs in different cells. OMVs were transformed with the eukaryotic expression plasmid p IRES2-EGFP with green fluorescent protein by electroporation, and incubated with RRAW264. 7 cells to observe the entrance of OMVs carrying eukaryotic expression plasmids into phagocytic cells and expression of green fluorescent protein. Results OMVs were purified effectively by density gradient centrifugation. The efficiencies of purified OMVs in entering various mammalian cells were different, which was higher in entering phagocytic cells than in entering non-phagocytic cells. After electrotransformation, plasmid DNA was introduced into OMVs, and ingested and expressed with phagocytic cells. Conclusion The eukaryotic expression plasmid at a length of about 5 300 bp may be introduced to OMVs by electroporation and express in phagocytic cells, indicating the potential of OMVs as a novel delivery vector of plasmid DNA.
引文
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[3]BEVERIDGE T J.Structures of gram-negative cell walls and their derived membrane vesicles[J].J Bacteriol,1999,181(16):4725-4733.
[4]HORSTMAN A L,KUEHN M J.Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles[J].J Biol Chem,2000,275(17):12489-12496.
[5]DEATHERAGE B L,LARA J C,BERGSBAKEN T,et al.Biogenesis of bacterial membrane vesicles[J].Mol Microbiol,2009,72(6):1395-1407.
[6]KULP A,KUEHN M J.Biological functions and biogenesis of secreted bacterial outer membrane vesicles[J].Ann Rev Microbiol,2010,64(1):163-184.
[7]AMANO A,TAKEUCHI H,FURUTA N.Outer membrane vesicles function as offensive weapons in host-parasite interactions[J].Microbes Infect,2010,12(11):791-798.
[8]BLANDER J M,MEDZHITOV R.Toll-dependent selection of microbial antigens for presentation by dendritic cells[J].Nature,2006,440(7085):808-812.
[9]SCHNARE M,BARTON G M,HOLT A C,et al.Toll-like receptors control activation of adaptive immune responses[J].Nat Immunol,2001,2(10):947-950.
[10]ADEREM A,ULEVITCH R J.Toll-like receptors in the induction of the innate immune response[J].Nature,2000,406(6797):782-787.
[11]TAKEDA K,KAISHO T,AKIRA S.Toll-like receptors[J].Annu Rev Immunol,2003,21(12):335-376.
[12]LIAUDET L,DEB A,PACHER P,et al.The flagellin-TLR5axis:therapeutic opportunities[J].Drug News Perspect,2002,15(7):397-409.
[13]SALEM M L,DIAZ-MONTERO C M,EL-NAGGAR S A,et al.The TLR3 agonist poly(I:C)targets CD8+T cells and augments their antigen-specific responses upon their adoptive transfer into naive recipient mice[J].Vaccine,2009,27(4):549-557.
[14]FIGUEIREDO R T,BITTENCOURT V C,LOPES L C,et al.Toll-like receptors(TLR2 and TLR4)recognize polysaccharides of Pseudallescheria boydii cell wall[J].Carbohydr Res,2012,356(7):260-264.
[15]SPARWASSER T,KOCH E S,VABULAS R M,et al.Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells[J].Eur JImmunol,1998,28(6):2045-2054.
[16]LOR? K,BETTS M R,BRENCHLEY J M,et al.Toll-like receptor ligands modulate dendritic cells to augment cytomegalovirus-and HIV-1-specific T cell responses[J].J Immunol,2003,171(8):4320-4328.
[17]DUBENSKY T W Jr,REED S G.Adjuvants for cancer vaccines[J].Semin Immunol,2010,22(3):155-161.
[18]GUJRATI V,KIM S,KIM S H,et al.Bioengineered bacterial outer membrane vesicles as cell-specific drug-delivery vehicles for cancer therapy[J].ACS Nano,2014,8(2):1525-1537.
[19]O'DONOGHUE E J,KRACHLER A M.Mechanisms of outer membrane vesicle entry into host cells[J].Cell Microbiol,2016,18(11):1508-1517.