Bt Research 2012, Vol.3, No.4, 20
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28
25
BACE 1000, Amersham Bioscience). DNA from the
recombinant plasmid (pGemcry10Aa) was digested
with
Eco
R
Ⅰ
and separated by electrophoresis in a
0.8%
agarose gel following standard protocols
(
Sambrook et al.. 2001). The 2,077 bp fragment was
gel-purified using the GFX Kit (GE) and cloned into
the transfer vector pSynXIVVI+X3 (Ribeiro and
Crook, 1993) previously digested with
Eco
R
Ⅰ
.
One
µg of the recombinant plasmid (pSyncry10Aa) DNA
and 0.5 µg of vSynVI
-
gal DNA, previously linearised
with the restriction enzyme
Bsu
36
I, were
co-transfected into a monolayer of BTI-TN5B1-4
cells (10
6
cells) in a 60-mm plate, using liposomes
(
Cellfectin
®
,
Invitrogen
®
)
following the manufacturer’s
instructions. The plate was incubated one week at 27
℃
until the appearance of viral occlusion bodies
(
OBs), when the supernatant was collected and used to
purify the recombinant virus through end-point
dilution in 96 well plates (O’Reilly et al., 1992). The
single
Bsu
36
Ⅰ
site of vSynVI
-
gal is located inside the
β-galactosidase
gene, and linearization makes it
non-infective
facilitating
recombinant
virus
purification (O’Reilly et al., 1992). Furthermore, the
pSyncry10Aa plasmid possesses, besides the
cry10Aa
gene, the
polyhedrin
gene (disrupted in vSynVI
-
gal).
Upon homologous recombination of plasmid and viral
DNA during co-transfection, vSynVI
-
gal regains
expression of polyhedrin, which is made evident by
the formation of OBs by the new recombinant virus
(
vSyncry10Aa). The latter was purified by three rounds
of serial dilution in 96 well plates (O’Reilly et al., 1992;
Jarvis, 1997).
3.3
Recombinant protein expression
BTI-Tn5B1-4 (10
6
cells/plate) cells were infected
with recombinant and wild-type AcMNPV viruses
(10
pfu/cell) and observed by light microscopy. At
120
h.p.i., cells were collected by centrifugation
(5,000
g/10 min) and stored at -80
℃
.
Third instar
S.
frugiperda
larvae were infected by injection of 10 µL
of viral stocks (10
8
pfu/mL for vSyncry10Aa and
10
7
pfu/mL for AcMNPV) into the hemolymph
.
Extracts of virus-infected larvae (120 h.p.i) were
analysed in a 12% SDS-PAGE (Mini-protean
Ⅱ
—
Biorad
®
)
following the manufacturer’s instruction. A
band around 85 kD on SDS-PAGE was quantified by
densitometry using the program Image Phoretix 2D
(
Pharmacia). The purification of protein crystals was
carried out by ultracentrifugation of virus-infected
S.
frugiperda
extracts on a discontinuous sucrose
gradient as described elsewhere (Chang et al., 1993).
The purified crystals from vSyncry10Aa infected
larvae were solubilized for 1 h at 4
℃
with 0.1 mol/L
NaOH. The pH of the solution containing the
solubilized protoxin was decreased to pH 9 by
addition of same volume of 1 mol/L Tris-HCl, pH 8.0,
the protoxin was then activated with trypsin (1:50 w/w)
for 2 h at 37
℃
,
and the reaction stopped by adding
trypsin inhibitor (Sigma). The molecular mass and
integrity of the recombinant protein were determined
by SDS-PAGE.
3.4
Structural and ultra-structural analysis of
putative Cry10Aa crystals produced in insect cells
and larvae
Insect cells were infected with AcMNPV and
vSyncry10Aa as described above and at 96 h.p.i.,
analyzed in a light microscope (Axiovert 100, Zeiss),
and photographed. One hundred third-instar
S.
frugiperda
larvae were infected with the recombinant
vSyncry10Aa by injection of virus as described above,
and at 120 h.p.i., OBs and protein crystals were
purified following the protocol described by (Praça et
al. 2004). The purified OBs and crystals were
processed for scanning electron microscopy and
analyzed under a Zeiss DSM962 scanning electron
microscope at 10 kV or 20 kV.
3.5
Preparation of BBMV of
Antonomus grandis
BBMVs were prepared from dissected midguts of
fourth instar larvae of
A. grandis
by differential
precipitation using MgCl
2
,
as previously reported
(
Wolfersberger et al., 1987), and stored at -80
℃
until
use. The purity of a preparation was checked by
evaluating the enrichment of the brush border
membrane marker enzymes aminopeptidase N and
alkaline phosphatase. Aminopeptidase N (EC 3.4.11.2)
and alkaline phosphatase (EC 3.1.3.1) activities in the
homogenate and in the BBMV preparation were
determined as described previously (Giordana et al.,
1982).
Protein concentrations in the BBMV
preparations were determined by the method of
Bradford, using BSA as the standard (Bradford, 1976).
3.6
Protein binding assays
Recombinant solubilized, trypsin-activated Cry10Aa
