用于检测人巨细胞病毒感染细胞中立即早期1、晚期pp67以及免疫逃逸基因US3、US6和US11的mRNA表达水平的定量竞争性核酸序列扩增法。

Quantitative competitive NASBA for measuring mRNA expression levels of the immediate early 1, late pp67, and immune evasion genes US3, US6 and US11 in cells infected with human cytomegalovirus.

作者信息

Greijer A E, Adriaanse H M, Kahl M, Tacken N M, Oldenburg N, Sijlmans A, van de Crommert J M, Dekkers C A, Sillekens P T, Middeldorp J M

机构信息

Organon Teknika, Boxtel, The Netherlands.

出版信息

J Virol Methods. 2001 Aug;96(2):133-47. doi: 10.1016/s0166-0934(01)00317-2.

DOI:10.1016/s0166-0934(01)00317-2

PMID:11445144

Abstract

Different cell types were infected with human cytomegalovirus (HCMV) and RNA expression dynamics were analyzed by quantitative NASBA assays for IE1 (UL123), pp67 (UL65) and the immune evasion genes (US3, US6 and US11). The quantitative NASBA assays gave reproducible quantification in the range of 10(3)-10(6) RNA copies for IE1 and pp67 RNA, from 3x10(3) to 1x10(6) RNA copies for US6 and US11 RNA, and from 1x10(4) to 1x10(6) RNA copies for US3 RNA. SMC, HAEC and HUVEC cells infected with an, in endothelial cells, propagated HCMV strain (VHL/E) showed similar RNA expression dynamics for the analyzed genes. Expression of all genes studied was observed within the first 4 h post-infection. The first gene for which expression could be detected was IE1, followed by US3, US11, pp67 and US6. Fibroblasts infected with HCMV strain AD169 showed a different RNA expression pattern for US3. Translation of the mRNA studied was demonstrated by detection of the proteins 48 h post-infection by immunofluorescence.

摘要

用人类巨细胞病毒（HCMV）感染不同细胞类型，并通过定量核酸序列扩增技术（NASBA）分析IE1（UL123）、pp67（UL65）以及免疫逃逸基因（US3、US6和US11）的RNA表达动态。定量NASBA分析在10³ - 10⁶个RNA拷贝范围内对IE1和pp67 RNA进行了可重复定量，对US6和US11 RNA在3×10³至1×10⁶个RNA拷贝范围内，对US3 RNA在1×10⁴至1×10⁶个RNA拷贝范围内。用在内皮细胞中传代的HCMV毒株（VHL/E）感染的平滑肌细胞（SMC）、人主动脉内皮细胞（HAEC）和人脐静脉内皮细胞（HUVEC），对所分析基因显示出相似的RNA表达动态。在感染后的最初4小时内观察到所有研究基因的表达。首先能检测到表达的基因是IE1，随后是US3、US11、pp67和US6。用HCMV毒株AD169感染的成纤维细胞对US3显示出不同的RNA表达模式。通过感染后48小时免疫荧光检测蛋白质，证明了所研究mRNA的翻译。