Rigg R J, Dando J S, Escaich S, Plavec I, Böhnlein E
Progenesys, Palo Alto, CA 94304, USA.
J Immunol Methods. 1995 Dec 27;188(2):187-95. doi: 10.1016/0022-1759(95)00237-5.
The Rev trans-activator protein plays a pivotal role in human immunodeficiency virus type 1 (HIV-1) replication by allowing expression of the viral structural proteins. We have developed a protocol to quantitatively assay intracellular steady state levels of Rev Ag (Rev wild type and RevM10 proteins) by flow cytometry. Three fixation and permeabilization techniques were compared. These protocols varied in the magnitude of the signal which could be detected, and in the ability to distinguish between Rev Ag positive and negative populations. This technology is applicable to a variety transduced or transfected cell types (species, lineage), and for cell lines and primary cells acutely infected with HIV-1. The assay is therefore a valuable tool both to analyze Rev protein expression levels in HIV-infected cells and to optimize delivery of the dominant-negative RevM10 gene for clinical gene therapy applications. In addition, a second, independent intracellular protein (HIV-Tat) has been detected using the same approach.
Rev反式激活蛋白通过促进病毒结构蛋白的表达，在1型人类免疫缺陷病毒（HIV-1）复制中发挥关键作用。我们已经开发出一种通过流式细胞术定量检测细胞内Rev抗原（Rev野生型和RevM10蛋白）稳态水平的方法。比较了三种固定和透化技术。这些方法在可检测信号的强度以及区分Rev抗原阳性和阴性群体的能力方面存在差异。该技术适用于多种转导或转染的细胞类型（物种、谱系），以及急性感染HIV-1的细胞系和原代细胞。因此，该检测方法是分析HIV感染细胞中Rev蛋白表达水平以及优化用于临床基因治疗的显性负性RevM10基因递送的宝贵工具。此外，使用相同方法还检测到了第二种独立的细胞内蛋白（HIV-Tat）。
