Bt Research 2012, Vol.3, No.4, 20
-
28
22
(10
6
/
well) and infected with wild-type AcMNPV and
different recombinant viruses (10 pfu/cell). After 1 h
(
zero times p.i.), virus inoculum was removed and the
plates incubated at 27
℃
.
Ninety-six h.p.i, cells were
photographed (Figure 2), collected by centrifugation
(1,000
×g, 5 min) and stored at -80
℃
.
T. ni
cells
infected with vSyncry10Aa produced possible
Cry10Aa protein crystals inside the cells’ cytoplasm
as seen by light microscopy (Figure 2D). Sucrose
gradient purified samples from extracts of larvae
infected with recombinant vSyncry10Aa viruses
showed the presence of large cuboidal crystals and
baculovirus occlusion bodies (Figure 2E). The
cuboidal shape of these crystals are different from the
spherical shape of crystals found in
B. thuringiensis
subsp. israelensis
strains toxic to insects of Lepidoptera,
Coleoptera and Diptera orders (Praça et al., 2004).
Figure 2 Structural and ultra-structural analysis of putative
Cry10Aa crystals produced in insect cells and larvae
Note: A: Light microscopy of BTI-Tn5B1-4 cells not infected;
B: BTI-Tn5B1-4 cells infected with vSynVI-gal at 96 h.p.i, (C)
BTI-Tn5B1-4 cells infected with AcMNPV at 96 h p.i. (D).
BTI-Tn5B1-4 cells infected with vSyncry10Aa at 96 h.p.i. and
(
E) Scanning electron microscopy of purified viral occlusion
bodies (PIBs) and crystals (Cry) from
S. frugiperda
larvae
infected with the recombinant vSyncry10Aa. In each
photograph was added an inset which show a cell in detail.
Long arrows show viral occlusion bodies (OBs) short arrows
show putative Cry10a large cuboidal crystals. Scale bar in all
photographs = 20 µm, except in E
1.4
Heterologous protein expression analysis
Extracts of wild type and different recombinant
viruses-infected insects were then analyzed by
SDS-PAGE (Figure 3). The heterologous protein was
expressed in insect since we have detected a protein
band of around 85 kD by SDS-PAGE in vSyncry10Aa-
infected insect extracts that was not present in other
insect cells extracts infected with wild type AcMNPV and
other recombinant baculovirus (Figure 3).
1.5
Binding assay with isolated BBMV of
Antonomus grandis
Since we showed that the Cry10Aa recombinant protein
was toxic to
A. grandis
we decided to analyze the
binding of the toxin to the midgut of
A. grandis
larvae.
The binding assay showed that the toxin binds to
A.
grandis
BBMVs and that the binding was specific since
no binding was detected in the presence of a 100 fold
excess of unlabelled Cry10Aa toxin (Figure 4).
Figure 3
Recombinant Cry10Aa protein expression: 12%
SDS-PAGE of extracts from wild type and recombinant infected
insect extracts (120 h.p.i.)
Note: Lane 1
:
Molecular marker High-Range (GE); Lane 2:
AcMNPV-infected insect extract; Lane 3: vSynVI-gal-infected
insect extract; Lane 4: vSyncry10Aa-infected insect extract. The
arrows indicate the viral occlusion body protein polyhedrin
(
lower) and the recombinant Cry10A protein (upper),
respectively
Figure 4 Binding assays on BBMV isolated from
A. grandis
.
Note: Biotinylated trypsin-activated Cry toxins were incubated
with the BBMV in the absence or in the presence of a 100-fold
excess of unlabeled toxin. After 1 h post incubation, unbound
toxins were removed, and vesicles containing bound toxins were
run in an SDS-PAGE gel and blotted onto a nitrocellulose
membrane. Labeled proteins were visualized by incubating the
membrane with streptavidin-peroxidase conjugate and Luminol.
Lane 1: biotinylated protein marker (Biorad), Lane 2 and Lane 3:
biotinylated
