期刊
JOURNAL OF VIROLOGICAL METHODS
卷 209, 期 -, 页码 35-40出版社
ELSEVIER
DOI: 10.1016/j.jviromet.2014.08.020
关键词
FMDV; Replicon; Fluorescence; Replication
资金
- Wellcome Trust [089209/Z/09/Z]
- Biological and Biotechnological Sciences Research Council (BBSRC) [BB/K003801/1]
- Biotechnology and Biological Sciences Research Council [BB/L004526/1, BB/K003801/1, BBS/E/I/00001716, C20035, BB/E010709/1, BB/E014321/1, BB/H007849/1] Funding Source: researchfish
- Biotechnology and Biological Sciences Research Council
- Medical Research Council [977084] Funding Source: researchfish
- Medical Research Council [G0901002, 1065327] Funding Source: researchfish
- BBSRC [BB/K003801/1, BBS/E/I/00001411, BB/E014321/1, BBS/E/I/00001716, BB/E010709/1, BB/H007849/1, BB/L004526/1] Funding Source: UKRI
- MRC [G0901002] Funding Source: UKRI
- Wellcome Trust [089209/Z/09/Z] Funding Source: Wellcome Trust
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 McInerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (L-pro) 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 L-pro 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. (C) 2014 The Authors. Published by Elsevier B.V.
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