Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards
Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards
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DOI：
10.1128/jcm.36.12.3590-3594.1998
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发表时间：
1998-12-01
影响因子：
9.4
通讯作者：
DuBois, DB
中科院分区：
文献类型：
--
作者：
Pasloske, BL;Walkerpeach, CR;DuBois, DB
病毒和细胞RNA序列检测的灵敏测定法的广泛使用产生了对稳定的、充分表征的对照品和标准品的需求。我们描述了一个通用的，新的系统，用于创建RNA酶抗性RNA的发展。“装甲RNA”是通过诱导编码外壳蛋白的表达质粒和RNA标准序列在埃希氏菌属中产生的MS 2噬菌体外壳蛋白和RNA的复合物，RNA序列在噬菌体样复合物内被完全保护免于RNA酶消化，作为原型，合成来自人免疫缺陷病毒I型（HIV-I）gag基因的一部分的172个碱基的共有序列，并将其克隆到用于产生噬菌体样颗粒的包装载体中。在生产和纯化后，所得HIV-1装甲RNA颗粒显示出对人血浆中的降解具有抗性，并且当在-20 ℃下储存180天或在4 ℃下储存60天时，在Amplicor HIV-I Monitor测定中产生可重现的结果。此外，Armored RNA制备物是均质的和非感染性的。
The widespread use of sensitive assays for the detection of viral and cellular RNA sequences has created a need for stable, well-characterized controls and standards. We describe the development of a versatile, novel system for creating RNase-resistant RNA. "Armored RNA" is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia call by the induction of an expression plasmid that encodes the coat protein and an RNA standard sequence, The RNA sequences are completely protected from RNase digestion within the bacteriophage-like complexes, As a prototype, a 172-base consensus sequence from a portion of the human immunodeficiency virus type I (HIV-I) gag gene was synthesized and cloned into the packaging vector used to produce the bacteriophage-like particles. After production and purification, the resulting HIV-1 Armored RNA particles were shown to be resistant to degradation in human plasma and produced reproducible results in the Amplicor HIV-I Monitor assay for 180 days when stored at -20 degrees C or for 60 days at 4 degrees C. Additionally, Armored RNA preparations are homogeneous and noninfectious.