Construction of armored RNA containing long-size chimeric RNA by increasing the number and affinity of the pac site in exogenous RNA and sequence coding coat protein of the MS2 bacteriophage
Construction of armored RNA containing long-size chimeric RNA by increasing the number and affinity of the pac site in exogenous RNA and sequence coding coat protein of the MS2 bacteriophage
复制标题
通过增加外源RNA和MS2噬菌体序列编码外壳蛋白中Pac位点的数量和亲和力构建含有长尺寸嵌合RNA的装甲RNA
DOI：
10.1159/000141707
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发表时间：
2008-01-01
期刊：
影响因子：
4.6
通讯作者：
Li, Jinming
中科院分区：
文献类型：
--
作者：
Wei, Baojun;Wei, Yuxiang;Li, Jinming
目的：通过增加pac位点的数量和亲和力，构建含长嵌合RNA的装甲RNA单质粒表达体系。方法：先构建含有1个c -变体pac位点的质粒pET-MS2-pac，再构建含有1891 bp嵌合序列和2个c -变体pac位点的质粒pM-CR-2C。同时，获得三个质粒（pM-CR-C、pM-CR-2W和pM-CR-W）作为平行对照，它们具有不同数量和不同亲和力的pac位点。最后，表达和纯化装甲RNA。结果：具有两个C-variant pac位点的单质粒表达系统成功表达了含有1891个碱基的甲化RNA靶RNA，而对于一个pac位点，无论是否改变亲和性，都只包装了1200个碱基的靶RNA。我们还发现c变异体pac位点可以提高铠装RNA的表达效率。我们研究的1891 bp外源RNA的装甲RNA在不同时间点和温度条件下显示了核糖核酸酶抗性和稳定性的表征。结论：构建了1891个碱基外源RNA的装甲RNA，该表达系统可作为制备长序列装甲RNA的平台。
Objectives: To construct a one-plasmid expression system of the armored RNA containing long chimeric RNA byincreasing the number and affinity of the pac site. Methods: The plasmid pET-MS2-pac was constructedwith one C-variant pac site, and then the plasmid pM-CR-2C containing 1,891-bp chimeric sequences and two C-variant pac sites was produced. Meanwhile, three plasmids (pM-CR-C, pM-CR-2W and pM-CR-W) were obtained as parallel controls with a different number and affinity of the pac site. Finally, the armored RNA was expressed and purified. Results: The armored RNA with 1,891 bases target RNA was expressed successfully by the one-plasmid expression system with two C-variant pac sites, while for one pac site, no matter whether the affinity was changed or not, only the 1,200 bases target RNA was packaged. It was also found that the C-variant pac site could increase the expression efficiency of the armored RNA. The armored RNA with 1,891-bp exogenous RNA in our study showed the characterization of ribonuclease resistance and stability at different time points and temperature conditions. Conclusions: The armored RNA with 1,891 bases exogenous RNA was constructed and the expression system can be used as a platform for preparation of the armored RNA containing long RNA sequences.