3 | RESULTS
3.1 | Host DNA insertion and A insertion at the two A7
MNRs were prevented for AcP3-FPSDM during passage in Hi5 cells. We
hypothesized that elimination of the two A7 MNRs and the
10th TTAA site should prevent the mutations [17].
We then performed site-directed mutagenesis at the MNRs and the
10th TTAA site as illustrated in Fig. 1 and eventually
obtained a new virus AcP3-FPSDM. To test the stability of the engineeredfp25k of AcP3-FPSDM during passage in Hi5 cells, viral DNA was
extracted from BVs harvested from each passage followed by PCR
amplification with a primer pair specific to the AcMNPV fp25kgene (Fig. 2 A). Compared to AcP3 fp25k , AcP3-FPSDM fp25kthat lacked the 2 A7 MNRs and 10th TTAA site showed
that the 1.2 kbp PCR is the most dominant one in all the passages (Fig.
2 B b). This suggested that the 287 bp host DNA insertion did not occur
in the fp25k of AcP3-FPSDM. When the 1.2 kbp PCR product was gel
purified and sequenced, no mutations were found at the
10th TTAA site and the two A7 MNR sites (Data not
shown).