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The aims of this study were to (i) produce a green
fluorescent protien (GFP) recombinant virus for detection of
viral infection, (ii) optimize the condition of tetracycline-
controlled gene expression system in insect cells.
Construction of the recombinant GFP virus was achieved by
transferring a GFP gene from jellyfish (Aequorea victoria) into
a polyhedrin gene-containing transfer vector pAcUW21 under the
control of a strong p10 promoter of Autographa californica
multiple nuclear polyhedrosis virus (AcMNPV). The transfer
vector and AcMNPV DNA were cotransfected into Spodoptera
frugiperda (Sf21) cells. The recombinant virus was isolated by
detecting the emission of green fluorescence from the infected
cells. The optimal conditions used for tetracycline-
controlled gene expression were 1 ug pTRE-LUC /2×105 cells at
viral m.o.i. 0.1. Expression of luciferease activity was
inhibited when tetracycline (Tc) was added to the mixtures at 1
ug Tc/ml.Contruction of an active-recombinant virus, vAcP10tTA,
was completed by transferring a fusion gene (tTA) containing a
tetracycline repressor and a VP16 transcription active domain
into a transfer vector pAcUW21. After identification, 10
recombinant viral colonies were selected. To obtain the best
active-recombinant virus, three cell lines (TN368, Sf9 and Sf21)
were used and transfected with pTRE-LUC. After 24 h of
transfection, the cells were infected with the 10 vAcP10tTAs,
respectively. Luciferase activity was assayed from each
infectant after 72 h of infection. The results showed that a
highest luciferase activity expressed in Sf9 was vAcP10tTA-1, in
Sf21 was vAcP10tTA-5 and in TN368 was vAcP10tTA-4.
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