RNase-resistant virus-like particles containing long chimeric RNA sequences produced by two-plasmid coexpression system
RNase-resistant virus-like particles containing long chimeric RNA sequences produced by two-plasmid coexpression system
复制标题
DOI：
10.1128/jcm.02248-07
复制
发表时间：
2008-05-01
影响因子：
9.4
通讯作者：
Li, Jinming
中科院分区：
文献类型：
--
作者：
Wei, Yuxiang;Yang, Changmei;Li, Jinming
含有外源性RNA序列（装甲RNA）的RNA耐药、非感染性病毒样颗粒是RNA病毒检测的良好对照和标准。然而，高效率的类病毒颗粒包装的RNA长度通常小于500个碱基。在这项研究中，我们描述了一种生产装甲L-RNA的方法。甲化L-RNA是一种由MS2噬菌体外壳蛋白和RNA组成的复合物，在大肠杆菌中通过诱导双质粒共表达系统产生，其中外壳蛋白和成熟酶在一个质粒中表达，而带有修饰的MS2茎环（pac位点）的靶RNA序列在另一个质粒中转录。通过双质粒共表达系统成功表达了含有6个基因片段(丙型肝炎病毒、严重急性呼吸综合征冠状病毒（SARS-CoV1、SARS-CoV2和SARS-CoV3）、禽流感病毒基质基因（M300）和H5N1禽流感病毒（HA300）、2248个碱基的3V装甲L-RNA，并证明其具有装甲RNA的所有特征。我们评估了3V装甲L-RNA作为多种病毒检测的校准物。我们使用WHO HCV RNA国际标准（NIBSC 96/790）校准嵌合装甲L-RNA，将其稀释10倍，获得含有10(6)至10(2)份的样品。综上所述，我们所采用的包衣L-RNA制备方法是可行的，可以减少多重RNA病毒检测的人工和质量控制成本。此外，我们可以分配嵌合装甲RNA与国际单位定量检测。
RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In this study, we describe a method for producing armored L-RNA. Armored L-RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of a two-plasmid coexpression system in which the coat protein and maturase are expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop (pac site) is transcribed from another plasmid. A 3V armored L-RNA of 2,248 bases containing six gene fragments-hepatitis C virus, severe acute respiratory syndrome coronavirus (SARS-CoV1, SARS-CoV2, and SARS-CoV3), avian influenza virus matrix gene (M300), and H5N1 avian influenza virus (HA300)-was successfully expressed by the two-plasmid coexpression system and was demonstrated to have all of the characteristics of armored RNA. We evaluated the 3V armored L-RNA as a calibrator for multiple virus assays. We used the WHO International Standard for HCV RNA (NIBSC 96/790) to calibrate the chimeric armored L-RNA, which was diluted by 10-fold serial dilutions to obtain samples containing 10(6) to 10(2) copies. In conclusion, the approach we used for armored L-RNA preparation is practical and could reduce the labor and cost of quality control in multiplex RNA virus assays. Furthermore, we can assign the chimeric armored RNA with an international unit for quantitative detection.