FIGURE LEGEND
Fig. 1. Schematic diagram for the generation of AcP3-FPSDM
Fig. 2. PCR sequencing of the passaged wild type virus and the engineered virus. A) Schematic of AcNPV fp25k “hot” spots engineering. The ORF of the gene is shown in a shaded box with the translation termination site as an empty arrow head. Small circles above the ORF represent TTAA sites, the black small circle is the 10th TTAA site and the open circles are the rest of 12 TTAA sites. The “hot” spots are shown in the brackets and the mutated nucleotides are underlined and highlighted in bold. A pair offp25k gene specific primers is denoted by the arrows. B) PCR amplification of fp25k gene by using the DNA extracted from the passaged BVs. a. the wild type virus, b. the engineered virus. P0, the non-passaged virus stock; P1, passaged once; P2, passaged twice and until P10, passaged 10th time. C) Histogram of PCR products sequencing of the 1.5 kb band of the passage 5th virus showing the 10th TTAA site had a host DNA insertion. a. the non-passaged wild type virus, b. the passage 5th virus.
Fig. 3. Baculovirus budding virion occlusion assay. A) Comparison of BV budding of AcP3 and AcP13 in the Hi5 cell line infection. P values were calculated by the t-test using Excel. Vertical bars denote standard deviation, n=3. B and C) Comparison of FP formation and polyhedra yield between the wt virus (B) and the engineered virus (C). a. non-passaged virus stock; b, passaged once; c, passaged twice and until g, passaged 10th time. (D) Comparison of total polyhedra yield/well of the two passaged viruses. Open bars are the wild type virus and the black bars are the engineered virus. Vertical bars denote standard deviation, n=3. The p values were calculated by the t-test using Excel. * denotes statistical significant level at 95%.
Fig. 4. Comparison of virion occlusion of polyhedra of the passaged wt virus (A) and the engineered virus (B) by Western blot and SDS-PAGE. Upper panel, polyhedrin protein loading control by SDS-PAGE, P, polyhedrin. Lower panel, Western blot detection of a virion major capsid protein (VP39) with a monoclonal anti-VP39 antibody, V, VP39. (C). Quantitative virion occlusion assay by densitometry, n=4.
Fig. 5. Southern blot hybridization analysis. The 287bp host DNA inserts once into the fp25k locus of passaged AcMNPV. AcMNPV P3 strain (AcP3) was passaged ten times in Sf21 cells and viral DNA extracted from un-passaged (P0) and passage ten (P10) BV. A) Left panel, restriction fragment length polymorphism (RFLP) analysis on P0 and P10 AcP3 DNA digested with EcoRI and HindIII. Arrows denotes original REN fragments presented in AcP3-P0 and AcP3-P10, respectively. Arrowheads denote REN fragment that shifted in AcP3-P0 and AcP3-P10, respectively. Right panel, Southern blot analysis of P0 and P10 AcP3 DNA using the 287 bp host DNA as a probe. The relative positions of the Eco-F and Hind-I fragments, where the fp25k locus is located, are indicated on the right. B) A short agarose gel to capture potential small REN fragments that may contain the host DNA. Arrows denotes original REN fragments presented in AcP3-P0 and AcP3-P10, respectively. Arrowheads denote REN fragment that shifted in AcP3-P0 and AcP3-P10, respectively. Right panel, Southern blot analysis of P0 and P10 AcP3 DNA using the 287 bp host DNA as a probe. The relative positions of the Eco-F and Hind-I fragments, where the fp25k locus is located, are indicated on the right.