| 论文摘要： |
Ranaviruses (family Iridoviridae, genus Ranavirus) have been recognized as emerging infectious pathogens and caused a great loss to the global biodiversity. Thymidine kinase (TK) and deoxyuridine triphosphatase (dUTPase, DUT, encoded by ORF 67R) are ubiquitous, existing in iridoviruses and other organisms. Previous studies showed that TK and DUT could be individually knocked out without impeding viral replication. In this study, we tried to insert two fluorescence genes into the above loci. We started with Delta 6.67R-RGV, a recently generated recombinant Rana grylio virus (RGV) with the whole DUT replaced by enhanced green fluorescence protein (EGFP) gene. Then, a red fluorescence protein (RFP) gene initiated by RGV immediate-early (IE) ICP18 gene promoter was inserted into TIC locus through homologous recombination. A novel recombinant virus, Delta DUT, TK-RGV, was generated by nine successive rounds of plaque isolation using RFP selection. All of the plaques produced by this recombinant virus could emit both green and red fluorescence. Furthermore, one-step and multiple-step growth curves of Delta DUT, TK-RGV were similar to those of wt-RGV and Delta 67R-RGV. In conclusion, a novel dual-fluorescence labeled recombinant iridovirus in which DUT and TK gene locus were simultaneously used for foreign gene expression was constructed. (C) 2016 Elsevier B.V. All rights reserved. |
