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Human cytomegalovirus UL50 with 1.6 kb DNA fragment encoding GFP gene was cloned into BAC-vector pALB02 as phagemid. The recombinant virus UL50/GFP was established by transfecting to CEF cells. The green fluorescence was observed in virion stained by anti-CMV-gB mAb. The infective rate of UL50/GFP was as high as 2% in CEF cells. The recombinant virus was stable at 36 degrees C but not at 42 degrees C, and the expression of GFP was retained as the temperature went down. The infected cells became GFP+ at 24 h after infection and at 72 h after infection, the green fluorescence of the cell was at least 10 times stronger than that of parental virus (CMV-UL50). No change was observed in lytic cycle of infection. UL50/GFP was able to prevent the appearance of plaque of CMV-UL50 on multi-well plate. The replication of UL50/GFP in CEF cells was much slower than that of CMV-UL50. The green fluorescence of UL50/GFP in CEF cell was observed and maintained for at least 6 days. At 36 degrees C, the expression of GFP was kept for at least 1 week. Our results showed that the GFP was able to be expressed both in the infected cells and in the progeny virus. The CMV recombinant virus containing GFP gene can be used as a reporter of CMV gene expression and a marker of cell lineage. It can also be used to study the formation of recombination and parental recombinant viruses.Q:
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