FMDV replicons encoding green fluorescent protein are replication competent.
FMDV replicons encoding green fluorescent protein are replication competent.
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DOI：
10.1016/j.jviromet.2014.08.020
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发表时间：
2014-12
影响因子：
3.1
通讯作者：
Ryan MD
中科院分区：
文献类型：
--
作者：
Tulloch F;Pathania U;Luke GA;Nicholson J;Stonehouse NJ;Rowlands DJ;Jackson T;Tuthill T;Haas J;Lamond AI;Ryan MD
口蹄疫病毒复制可以用高度的疾病安全设施进行研究。口蹄疫病毒复制子基因组编码绿色荧光蛋白具有复制能力。这些口蹄疫病毒复制子系统可用于通过活细胞成像/图像分析来研究复制。对需要高度生物安全设施的病毒复制的研究可以通过使用非传染性“复制子”系统进行不严格的控制来完成。对文献报道的口蹄疫病毒复制子系统(PT7rep)进行了改造，将编码氯霉素乙酰转移酶(CAT)的序列替换为编码功能性L蛋白酶(LPRO)的序列，该酶与双功能荧光/抗生素抗性融合蛋白(绿色荧光蛋白/嘌呤霉素抗性，GFP-PAC)相连。将复制子衍生的转录本RNA导入细胞，并定量检测GFP荧光。转录RNA的复制很容易被荧光检测到，而来自复制能力不强的基因组形式的信号要低2倍。令人惊讶的是，缺少LPro的复制子形式显示出明显更强的荧光信号，但出现了轻微的延迟动力学。因此，复制可以简单地通过活细胞成像和图像分析来量化，提供了RT-qPCR或CAT分析的快速和简便的替代方法。
FMDV replication can be studied outwith high disease secure facilities. FMDV replicon genomes encoding GFP are replication competent. These FMDV replicon systems can be used to study replication by live-cell imaging/image analyses. The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious ‘replicon’ systems. The FMDV replicon system (pT7rep) reported by was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (Lpro) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the Lpro showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays.
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影响因子：
5.1
作者：
Ryan, MD;Donnelly, M;Gani, D
通讯作者：
Gani, D
影响因子：
3.8
作者：
Ellard, FM;Drew, J;King, AMQ
通讯作者：
King, AMQ
影响因子：
5.4
作者：
BELSHAM, GJ;BRANGWYN, JK
通讯作者：
BRANGWYN, JK
影响因子：
5.4
作者：
Mason, PW;Bezborodova, SV;Henry, TM
通讯作者：
Henry, TM
影响因子：
5.4
作者：
McKnight, KL;Lemon, SM
通讯作者：
Lemon, SM