Jones Kathryn L, Drane Debbie, Gowans Eric J
Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia.
Biotechniques. 2007 Nov;43(5):675-81. doi: 10.2144/000112593.
RNA replicons represent potential vaccine delivery vehicles, but are considered too unstable for such use. This study examined the recovery, integrity and function of in vitro transcribed replicon RNA encoding hepatitis C virus (HCV) proteins. To remove residual template DNA, the RNA was digested with TURBO DNase followed by RNeasy DNase set and purified through an RNeasy column. The RNA was freeze-dried in distilled water or trehalose, stored under nitrogen gas for up to 10 months and analyzed at different time points. The recovery of RNA stored at < or = 4 degrees C that was freeze-dried in distilled water varied between 66% to zero of that recovered from RNA freeze-dried in 10% trehalose, a figure that depended on the duration of storage. In contrast, the recovery of the RNA stored in trehalose was consistently high for all time points. After recovery, both RNAs were translationally competent and expressed high levels of proteins after transfection, although the level of expression from the trehalose-stored RNA was consistently higher. Thus the addition of trehalose permitted stable storage of functional RNA at 4 degrees C for up to 10 months and this permits the development of RNA vaccines, even in developing countries where only minimum storage conditions (e.g., 4 degrees C) can be achieved.
RNA复制子代表了潜在的疫苗递送载体，但被认为用于此用途时过于不稳定。本研究检测了体外转录的编码丙型肝炎病毒（HCV）蛋白的复制子RNA的回收率、完整性和功能。为去除残留的模板DNA，先用TURBO DNase消化RNA，再用RNeasy DNase试剂盒处理，然后通过RNeasy柱进行纯化。RNA在蒸馏水或海藻糖中冻干，在氮气下储存长达10个月，并在不同时间点进行分析。在≤4℃储存且在蒸馏水中冻干的RNA的回收率，与在10%海藻糖中冻干的RNA的回收率相比，在66%至零之间变化，该数值取决于储存时间。相比之下，在所有时间点，储存在海藻糖中的RNA的回收率始终很高。回收后，两种RNA都具有翻译活性，转染后都能高水平表达蛋白质，尽管储存在海藻糖中的RNA的表达水平始终更高。因此，添加海藻糖可使功能性RNA在4℃稳定储存长达10个月，这使得RNA疫苗的研发成为可能，即使在只能实现最低储存条件（如4℃）的发展中国家也是如此。
