Hybrid tetanus toxin C fragment-diphtheria toxin translocation domain allows specific gene transfer into PC12 cells
Hybrid tetanus toxin C fragment-diphtheria toxin translocation domain allows specific gene transfer into PC12 cells
复制标题
DOI：
10.1006/exnr.2002.7999
复制
发表时间：
2002-09-01
影响因子：
5.3
通讯作者：
Rush, RA
中科院分区：
文献类型：
--
作者：
Barati, S;Chegini, F;Rush, RA
为了研究基因高效转移到特定细胞类型的机制，我们将破伤风毒素无毒的C片段和白喉毒素易位域与GAL4转录因子的DNA结合片段重组，构建了几种新的单链多组分蛋白，用于将质粒DNA转运到神经元细胞中。破伤风毒素的C片段提供神经元选择性，白喉毒素的易位结构域允许内体逃逸，GAL4结构域提供与DNA的结合。为了评估基因转移中每个组分的细胞任务，通过聚合酶链反应产生这些片段的不同组合，在大肠杆菌中表达，并在天然条件下从可溶性蛋白中纯化。我们发现，只有携带破伤风毒素C片段的融合蛋白才能与神经节苷脂结合，然后与分化的PC12细胞特异性结合，并在10分钟内内化。这些蛋白将绿色荧光蛋白基因传递到pc12细胞中，同时含有C片段和易位结构域的蛋白转染效率最高。添加氯喹提高了转染效率，并通过在传递系统中加入核定位信号进一步提高了转染效率。此外，还研究了不同dna凝聚材料(聚l -赖氨酸、鱼精蛋白、赖氨酸(n=8)-色氨酸（n=2)-赖氨酸（n=8））对基因转移的影响。(C) 2002 Elsevier Science （USA）。
To study the mechanism by which genes can efficiently be transferred into specific cell types, we have constructed several novel, single-chain multicomponent proteins by recombining the nontoxic C fragment of tetanus toxin and the translocation domain of diphtheria toxin together with the DNA-binding fragment of GAL4 transcription factor, for transportation of plasmid DNA into neuronal cells. The C fragment of tetanus toxin provided neuronal selectivity, the translocation domain of diphtheria toxin permitted endosomal escape, and the GAL4 domain provided binding to DNA. To assess the cellular tasks of each component in gene transfer, different combinations of these fragments were produced by polymerase chain reaction, expressed in Escherichia coli, and purified under native conditions from the soluble proteins. We show that only fusion proteins bearing the C fragment of tetanus toxin bind to gangliosides and, followed by their specific binding to differentiated PC12 cells, are internalized within 10 min. These proteins delivered the green fluorescence protein gene to PC 12 cells, with the highest transfection efficiency achieved with proteins containing both the C fragment and the translocation domain. Addition of chloroquine elevated the transfection efficiency, which was further increased by incorporation of a nuclear localization signal in the delivery system. In addition, the effect of different DNA-condensing materials (poly-L-lysine, protamine, lysine(n=8)-trytophan(n=2)-lysine(n=8)) on gene transfer was investigated. (C) 2002 Elsevier Science (USA).