Antisera-mediated in vivo reduction of Cry1Ac toxicity in Helicoverpa armigera.

Abstract

A functional assessment of Bacillus thuringiensis (Bt) toxin receptors in the midgut of lepidopteran insects will facilitate understanding of the toxin mode of action and provide effective strategies to counter the development of resistance. In this study, we produced anti-aminopeptidase (APN) and anti-cadherin sera with purified Cry1Ac toxin-binding APN or cadherin fragments from Heliocoverpa armigera. Antisera were evaluated for their effects on Cry1Ac toxicity through bioassays. Our results indicated that both the anti-APN and anti-cadherin sera reduced Cry1Ac toxicity in vivo, although cadherin antiserum reduced toxicity more than APN antiserum. These results suggest that both APN and cadherin are involved in Cry1Ac intoxication of H. armigera, evidence that the pore formation model may be representative of Cry1Ac toxin mode of action in this insect.

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Antisera-mediated in vivo reduction of Cry1Ac toxicity in Helicoverpa armigera
Chenxi Liua, Yulin Gaoa, Changming Ninga,b, Kongming Wua,*, Brenda Oppertc, Yuyuan Guoa
aState Key Laboratory of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Science, West Yuanmingyuan Road, Beijing 100193, China
bDepartment of Entomology, College of Agronomy and Biotechnology, China Agricultural University, West Yuanmingyuan Road, Beijing 100193, China
cUSDA ARS Grain Marketing and Production Research Center, 1515 College Avenue, Manhattan, KS 66502, USA
1. Introduction
Cry toxins from Bacillus thuringiensis are effective insect control
proteins, and several different models regarding the mechanism of
the action of Cry toxins have evolved. In one model, the Cry toxin
first binds to a cadherin-like protein, resulting in a conformational
change (Bravo et al., 2004). After further proteolytic activation and
oligomerization, toxin oligomers display increased binding affinity
for aminopeptidase N (APN), which facilitates the insertion of
toxins into the membrane with a concomitant pore formation that
results in cell death by osmotic shock. According to this model, the
two receptors interact sequentially with different structural
species of the toxin, resulting in efficient membrane insertion.
An alternative model has been proposed wherein Cry toxin
monomers bind to a cadherin-like receptor, through which a
protein kinase A-dependent oncotic signaling pathway is activat-
ed, leading to cell death (Zhang et al., 2005, 2006, 2008). In this
model, APN binding and pore formation are irrelevant to biological
activity.
Because of binding to Cry toxins, APN has been proposed as a
Cry toxin receptor in several lepidopteran species, including
Helicoverpa armigera. In the midgut epithelium of H. armigera
larvae, a 120 kDa APN was characterized as a Bt toxin receptor
(Liao et al., 2005; Ingle et al., 2001), similar to other lepidopteran
species, such as Heliothis virescens (Gill et al., 1995), Bombyx mori
(Yaoi et al., 1997), Lymantria dispar (Valaitis et al., 1995), Plutella
xylostella (Luo et al., 1997a), and Manduca sexta (Knight et al.,
1994). In addition, a second gene encoding another 120 kDa
isoform of APN was recently cloned from the midgut of H. armigera
(Wang et al., 2005a). Several in vitro studies have provided
additional evidence that APN is a toxin receptor (Lorence et al.,
1997; Luo et al., 1997b; Sangadala et al., 1994). Transgenic
Drosophila melanogaster expressing M. sexta APN demonstrated in
vivo that Cry toxin sensitivity could be induced and also supported
the hypothesis that APN is a functional receptor for the Cry1Ac
toxin (Gill and Ellar, 2002). Other studies have demonstrated the
functional relevance of midgut APNs of Spodoptera litura and H.
armigera to Cry1C and Cry1Ac toxins, respectively, by direct gene
silencing using a double-stranded RNA interference (RNAi)
(Rajagopal et al., 2002; Sivakumar et al., 2007). Additionally, the
siRNA-directed silencing of S. litura midgut APN, and RNAi of
midgut APN with a H. armigera transgene in cultured insect cell
lines, indicated functional interactions with Cry toxins (Agrawal
etal.,2004;Sivakumaretal.,2007).Thecompleteabsenceoftoxin-
binding APN expression in Cry1Ca-resistant S. exigua and deletion
mutation of an APN in Cry1Ac-resistant H. armigera also have been
reported (Herrero et al., 2005; Zhang et al., 2009).
A cadherin has been identified as putative Cry1A toxin-binding
receptor in midgut epithelial cells in many lepidopteran species,
including M. sexta (Vadlamudi et al., 1993, 1995), H. virescens
(Gahan et al., 2001; Jurat-Fuentes and Adang, 2006), B. mori
(Nagamatsu et al., 1998a, 1998b), H. amigera (Wang et al., 2005b),
Pectinophora gossypiella (Morin et al., 2003), and Ostrinia nubilalis
(Flannagan et al., 2005). Several studies have demonstrated that
Journal of Insect Physiology 56 (2010) 718–724
A R T I C L EI N F O
Article history:
Received 5 November 2009
Received in revised form 11 December 2009
Accepted 17 December 2009
Keywords:
Antiserum
Bacillus thuringiensis
Aminopeptidase N
Cadherin
Cry1Ac toxin
Toxicity
A B S T R A C T
AfunctionalassessmentofBacillusthuringiensis(Bt)toxinreceptors inthemidgut oflepidopteraninsects
will facilitate understanding of the toxin mode of action and provide effective strategies to counter the
development of resistance. In this study, we produced anti-aminopeptidase (APN) and anti-cadherin
serawith purified Cry1Ac toxin-binding APN or cadherin fragments from Heliocoverpa armigera. Antisera
were evaluated for their effects on Cry1Ac toxicity through bioassays. Our results indicated that both the
anti-APN and anti-cadherin sera reduced Cry1Ac toxicity in vivo, although cadherin antiserum reduced
toxicity more than APN antiserum. These results suggest that both APN and cadherin are involved in
Cry1Ac intoxication of H. armigera, evidence that the pore formation model may be representative of
Cry1Ac toxin mode of action in this insect.
? 2009 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +86 10 62815906; fax: +86 10 62896114.
E-mail address: wkm@caascose.net.cn (K. Wu).
Contents lists available at ScienceDirect
Journal of Insect Physiology
journal homepage: www.elsevier.com/locate/jinsphys
0022-1910/$ – see front matter ? 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2009.12.012

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the toxin-binding cadherin serves as a functional Cry toxin
receptor in vivo. One study demonstrated that the susceptibility
to Cry1Ab toxin was reduced by cadherin gene silencing with RNAi
in M. sexta (Sobero ´n et al., 2007), while other studies illustrated
that Cry1Aa toxicity against B. mori was reduced by anti-cadherin
serummixedwithartificialdiet(Nagamatsuetal.,1998b;Xieetal.,
2005). The toxicity of Cry1A toxins against some lepidopteran
larvae was reduced by premixing the toxin with a soluble peptide
containing the putative toxin-binding site of a toxin-binding
cadherin (Dorsch et al., 2002; Griko et al., 2004; Xie et al., 2005; Liu
et al., 2009). Alterations in toxin-bindingcadherin genes have been
found in laboratory-selected Cry1A-resistant strains of H. virescens
(Gahan et al., 2001; Jurat-Fuentes et al., 2004), field populations of
P. gossypiella (Morin et al., 2003), and laboratory-selected strains of
the H. armigera GYBT strain (Xu et al., 2005; Yang et al., 2007).
Likewise, site-directed mutagenesis of the H. virescens cadherin
gene reduced the ability of cadherin to bind to Cry1Ac toxin (Xie et
al., 2005).
The binding of Cry toxin to specific midgut receptors is
considered a key step in the mode of action of Cry1 toxins (Ferre ´
and van Rie, 2002). Disruption of the interaction between the Cry1
toxin and midgut receptors is a common mechanism of toxin
resistance in lepidopteran insects. Highly specific antibodies
against APN and cadherin from B. mori inhibited the binding of
Cry1Ab toxin to their respective candidate receptor proteins or
dissociated epithelial cells from the B. mori midgut (Ibiza-Palacios
et al., 2008). To further probe the role of the H. armigera Cry1Ac
toxin-binding APN and cadherin in Cry1Ac intoxication, we
investigated the effect of anti-APN and anti-cadherin sera in toxin
feeding bioassays.
2. Materials and methods
2.1. Insect
H. armigera strain 96S was originally collected from Xinxiang
County, located in the Henan Province of China, in 1996. Larvae
were reared on an artificial laboratory diet.
2.2. Preparation of Cry1Ac toxin
The Biotechnology Research Group of the Institute of Plant
Protection, Chinese Academy of Agricultural Science, provided the
Cry1Ac protoxin.For activation, Cry1Ac protoxinwas incubatedfor
2 h at 37 8C with a 1:125 mass ratio of bovine trypsin (Sigma), and
the soluble trypsinized toxin was purified by a Superdex 200 HR
10/30 column (Amersham Biosciences) on a fast protein liquid
chromatography (FPLC) system.
2.3. Cloning, expression, and purification of an aminopeptidase and a
cadherin fragment
ToobtainapartialpeptideofAPN,a270 bpDNAfragmentofthe
H. armigera APN1 (GenBank accession no. EU568875) was cloned
and expressed in Escherichia coli cells as a His-tag recombinant
protein. Total RNA was extracted from the midgut of fifth instar
larvae with TRIzol1
reagent (Invitrogen) according to the
manufacturer’s instructions and reverse-transcribed with Super-
Script III RNase H?reverse transcriptase (Invitrogen). The cDNA
fragments served as templates for subsequent PCR amplification
using APN-2719F (50-agtggatccgccacgtctaggtccaac-30) and APN-
2988R (50-cacaagcttgatgtttgctgagccagg-30) primers. Amplicons
were cloned into the T-Easy vector (Promega) following the
manufacturer’sinstructions.Therecombinantplasmidwasexcised
withBamHIandHindIII,subclonedintotheHis-taggedexpression
vector pET30a+ (Novagen), and transfected into E. coli BL21 (DE3)
cells. The transfected cells were cultured and expression-induced
with 0.2 mM IPTG for 6 h at 25 8C. For protein purification, the
pelleted cells were collected by centrifugation at 3000 ? g, 4 8C for
10 min, washed with ice-cold PBS buffer, re-suspended in PBS
buffer, and sonicated for 3 min on ice. After 25,000 ? g centrifuga-
tion for 20 min at 4 8C, the supernatant was subjected to affinity
purification using Ni-Sepharose beads (Amersham Biosciences).
After washing with 50 mM imidazole in PBS buffer, the recombi-
nant protein was eluted with 400 mM imidazole and dialyzed
against PBS buffer.
Toobtain a partial peptide of cadherin, a 1092 bp DNA fragment
of the H. armigera cadherin (GenBank accession no. AF519180) was
cloned into the His-tagged expression vector pET28a+ (Novagen)
andexpressed inE. colicellsasa His-tagrecombinantproteinusing
cadherin-F (50-agtcatatgacgattcgtgctacggac-30) and cadherin-R (50-
atactcgagtggctcgcgcctgcgcgt-30) primers. The purification of the
cadherin fragment was conducted as described for APN.
2.4. Production and determination of dose-response of antisera
Purified APN and cadherin fragments were used to raise
polyclonal antisera in rabbits. The APN or cadherin fragment was
emulsifiedwithan equalvolume ofFreund’s completeadjuvantfor
the first injection and incomplete adjuvant for subsequent
injections. Four injections were administered at 20-day intervals,
and antisera were collected after the initial and subsequent
immunizations. Antisera were aliquoted and stored at ?80 8C.
The dose-response of the antisera was measured using ELISA.
Briefly, microplates (96-wells, Corning Incorporated) were coated
with 100 ml APN or cadherin fragments (0.4 mg) in 0.05 M
carbonate buffer (pH 9.6) and incubated overnight at 4 8C. After
the incubation, the wells were washed three times with PBS
containing 0.1% Tween-20 (PBST). Afterwards, 200 ml of 3% dry
skim milk diluted in PBS were added to block nonspecific binding
sites.Theplateswerekeptatroomtemperaturefor1 handwashed
with PBST as described above, after which 100 ml of eight serial
dilutions (ranging from 1:10 to 1:1,562,500) of anti-APN or anti-
cadherin serum in PBS were added to the wells. After incubation at
37 8C for 1 h, the wells were washed five times with PBST. One
hundred microliters of horseradish peroxidase (HRP)-conjugated
secondary antibody (ZSGB-BIO, China) diluted 1:60,000 in PBST
were added. The plates were incubated at 37 8C for 1 h. At the end
of the incubation, plates were rinsed with PBST as above, after
which 100 ml of 1-step Ultra TMB-ELISA Substrate (Pierce,
Rockford, IL) were added to the wells. The reaction was stopped
with the addition of 100 ml 2 M H2SO4. Absorbance was read at
450 nm by a multi-mode microplate reader (Synergy HT, BioTek)
and was presented as optical density (OD) for each sample.
2.5. Preparation of brush border membrane vesicles
Midguts from fifth instar larvae of H. armigera were dissected
longitudinally, washed in ice-cold MET buffer (250 mM mannitol,
17 mM Tris–HCl, and 5 mM EGTA, pH 7.5), and stored at ?80 8C
until use. Brush border membrane vesicles (BBMV) were prepared
from midguts using the differential centrifugation method
(Wolfersberger et al., 1987). Briefly, frozen midguts were
mechanically homogenized in MET buffer. One volume of
24 mM MgCl2 was added, and the mixture was incubated for
15 min. Following centrifugation for 15 min, 2500 ? g at 4 8C, the
supernatant was again centrifuged for 30 min, 30,000 ? g at 4 8C,
andthefinalpelletwassuspendedinresuspensionbuffer (300 mM
mannitol, 1 mM DTT, 10 mM Hepes–Tris, pH 7.4) and stored at
?80 8C until use. The concentration of proteins in the BBMV
preparation was determined using bovine serum albumin as a
standard (Bradford, 1976).
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2.6. Ligand blotting
BBMV proteins (corresponding to 10 mg protein) were separat-
ed by 10% SDS-PAGE, transferred onto PVDF membranes, and
blocked with dry skim milk (5%). The membranes were incubated
in PBST buffer containing 10 nM Cry1Ac toxin for 2 h. Binding was
detectedusingpolyclonalanti-Cry1Acantibody(1:10,000,1 h)and
a horseradish peroxidase (HRP)-conjugated secondary antibody
(ZSGB-BIO, China) (1:20,000, 1 h). The blot was developed using
the Easysee Western Blot Kit (Transgen, China).
2.7. Immunoblotting
Immunoblotting analysis was used to determine the specificity
of the antiserum raised against the APN and cadherin proteins in H.
armigera BBMV. Briefly, BBMV proteins were separated by a 10%
SDS-PAGE and transferred onto a PVDF membrane which was
subsequently blocked with dry skim milk (5%). The membranes
were incubated with anti-APN antiserum (1:15,000, 1 h) or anti-
cadherin antiserum (1:15,000, 1 h), followed by incubation with a
horseradish peroxidase (HRP)-conjugated secondary antibody
(ZSGB-BIO, China) (1:20,000, 1 h). The blots were also developed
using the Easysee Western Blot Kit (Transgen, China).
2.8. Insect bioassays
The MVP II commercial formulation containing 19.7% of Cry1Ac
protoxin(DowAgroSciences)wasthoroughlymixedwithadefined
amount of artificial diet at the LC80for the H. armigera (0.8 mg/g for
MVP II). A disc of treated artificial diet (1.6-cm diameter) was
placed into a 24-well plate and made to fit into the inner wall and
bottom of the plate using gentle pressure. Different doses of the
anti-APN or anti-cadherin antisera diluted by PBS with a ratio of
1 ml antiserum: 10 ml PBS buffer were applied to the diet surface.
Controls included PBS and preimmune serum diluted as treat-
ments. One first instar larva of the H. armigera strain 96S was
placed in each well of the plate. Each treatment had three
replicates, and a total of 24 larvae were used for each replicate. The
environment for the bioassay was maintained at 28 8C and
75 ? 10% relative humidity (RH), with a photoperiod of 14:10 h
(L:D). The survival rates of larvae were measured after eight days.
2.9. Toxin labeling and direct binding assay
Trypsin-activated Cry1Ac toxin was labeled with the fluores-
cent Cy3 mono-reactive dye according to the manufacturer’s
protocol (Amersham Biosciences). Labeled Cry1Ac toxin was
separated from unconjugated dye by dialysis. A calibration curve
was made using fluorescence intensities measured at 545
(excitation) and 570 (emission) nm by a multi-mode microplate
reader (Synergy HT, BioTek).
H. armigera BBMV were pre-incubated with 2.5 ml anti-APN or
2.5 ml anti-cadherin serum, 2.5 ml of a mixture containing a half
dose of each antisera, preimmune serum, or PBS buffer, all diluted
1:10 in PBS buffer, and incubated for 30 min at 25 8C. After
incubation, 10 nM Cy3-labeled Cry1Ac toxin was added to each
treatment, and again incubated for 50 min in the dark at 25 8C.
BBMVwerepelletedby30,000 ? gcentrifugationfor30 minat4 8C
Fig. 1. SDS-PAGE and ligand blot analysis of the expression of the H. armigera APN and cadherin fragments. (A) Structure of H. armigera aminopeptidase N (APN) (1) and
cadherin (2). The arrows indicate the location of primers in the H. armigera APN and cadherin sequences used to obtain the toxin-binding APN or cadherin fragments. (B)
Expressed and purified proteins were Coomassie-stained following 12% SDS-PAGE. Lane M: protein marker; lane 1: IPTG-0 h (APN fragment); lane 2: IPTG-6 h (APN
fragment); lane 3: purified APN fragment; lane 4: IPTG-0 h (cadherin fragment); lane 5: IPTG-6 h (cadherin fragment); and lane 6: purified cadherin fragment. (C) Purified
proteins transferred to a PVDF membrane were probed with activated Cry1Ac toxin and detected using a polyclonal anti-Cry1Ac antibody.
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and rinsed twice with 500 ml of ice-cold resuspension buffer to
remove the unbound labeled toxins. The final BBMV pellet was
suspended with 300 ml of resuspension buffer and sonicated for
25 s on ice, and the fluorescence of Cy3-labeled Cry1Ac toxin
bound to BBMV was determined as described above. Nonspecific
binding was determined by adding a 500-fold excess of unlabeled
toxin to each reaction mixture, and specific binding was obtained
by subtracting nonspecific binding from the total binding.
2.10. Statistical analysis
Mortality data were transformed using an arcsine transforma-
tion and were subjected to analysis of variance (ANOVA) (SAS
Institute, 1998). Treatment differences were determined using
Duncan’s multiple range test. Statistical significance was assumed
at P < 0.05.
3. Results
3.1. Expression and purification of APN and cadherin fragments
cDNA fragments from toxin-binding regions of H. armigera APN
and cadherin were cloned (Fig. 1A). The two fragments were
expressed in E. coli BL21 (DE3) cells, and the peptides were
evaluatedbySDS-PAGE(Fig.1B).Theresultsdemonstratedthatthe
APN and cadherin fragments expressed in E. coli had the expected
size of 30 kDa and 47.5 kDa, respectively, and also were purified to
homogeneity (Fig. 1B). A ligand blot analysis indicated that both
fragments bound Cry1Ac toxin (Fig. 1C).
3.2. Titers of anti-APN and anti-cadherin sera
The dose-response of anti-APN and anti-cadherin sera was
measured using ELISA (Fig. 2). The amount of APN fragment added
Fig. 2. Dose-response of antisera. Dose-responses of the antisera were measured
using ELISA. Microplates were coated with a 0.4 mg of H. armigera APN fragment (A)
orcadherinfragment(B).Adilutionseries ofeither preimmuneseraorantiseratoH.
armigera APN (A) or cadherin (B) was applied to the appropriate plate, and binding
was detected by a secondary antibody linked to HRP, and colorimetric detection
with HRP substrate was made at 450 nm.
Fig. 3. SDS-PAGE, ligand blotting, and immunoblotting analysis of the H. armigera
BBMV proteins. (A) Ten micrograms of H. armigera BBMV proteins were separated
by a 10% SDS-PAGE and stained with Coomassie blue (Lane 1). Lane M is protein
marker. (B) BBMV proteins were transferred to a PVDF membrane and probed with
activated Cry1Ac toxin, and detected using a polyclonal anti-Cry1Ac antibody. (C)
BBMV proteins transferred to the PVDF membrane were detected by an anti-APN
serum (lane B) or anti-cadherin (lane A) serum.
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to the titer plates was approximately 13.3 pmol, approximately
1.6-fold more than that of the cadherin fragment. A dilution of
1:62,500 for anti-APN and 1:12,500 for anti-cadherin sera gave
approximately equal OD readings of 1.4, suggesting that the per
molar reaction was approximately 3.2-fold higher for anti-APN
than for anti-cadherin. However, a titer of 1:312,500 was
determined for both anti-APN and anti-cadherin sera, and the
HRP enzyme reaction was saturated at 1:2500 for both antisera.
3.3. Recognition of 210 kDa and 120 kDa H. armigera BBMV proteins
by Cry1Ac and anti-cadherin and anti-APN sera
Proteins from the H. armigera gut (Fig. 3A) were used to probe
for Cry1Ac toxin-binding or immunoreactive proteins. Immuno-
blotting demonstrated that a 210 kDa and 120 kDa H. armigera
BBMV proteins reacted with the anti-cadherin and anti-APN sera,
respectively(Fig.3C).The210 kDaBBMVproteinrecognizedbythe
cadherin antiserum and the 120 kDa protein recognized by the
APN antiserum were similar in migration to the Cry1Ac toxin-
binding proteins (Fig. 3B). The results suggested that each
antiserum specifically interacted with its antigen.
3.4. Effect of anti-APN and anti-cadherin sera on the binding of Cy3-
Cry1Ac toxin to H. armigera BBMV
The binding of Cy3-labeled Cry1Ac toxin to H. armigera BBMV
was reduced by pre-incubation with anti-APN or anti-cadherin
serum (Fig. 4). The reduction in binding was more with anti-
cadherin serum than with anti-APN serum. Controls using
preimmune serum or PBS were similar and demonstrated maximal
binding. These results further demonstrate that both APN and
cadherin in H. armigera BBMV bound Cry1Ac toxin.
3.5. Effect of APN and cadherin antisera in vivo
First instar larvae of H. armigera were fed an artificial diet
containing preimmune serum, or equal dilutions of anti-APN
serum or anti-cadherin serum. Treatments of H. armigera larvae
with anti-APN and anti-cadherin sera reduced the insecticidal
action of Cry1Ac toxin, but treatment with preimmune serum did
not affect Cry1Ac toxicity (F = 32.374; d.f. = 7, 23; P = 0.0001)
(Fig. 5). The results provided additional evidence that both APN
and cadherin are likely functional receptors of Cry1Ac toxin. These
data suggest that cadherin is the primary Cry1Ac toxin-binding
receptor in the toxin mode of action, particularly given that the
ELISA results suggested that the APN antisera may recognize APN
at a higher dilution. Because both antiserum affected toxicity, the
pore formation model is supported for Cry1Ac intoxication in H.
armigera larvae.
4. Discussion
The cotton bollworm, H. armigera, is one of the insect pests that
pose serious problems in cotton, corn, vegetables, and other crops
worldwide. Pest management tactics associated with Bt-expres-
sing cotton have resulted in a drastic reduction in insecticide use
(Wu and Guo, 2005) and cotton bollworm suppression in host
crops (Wu et al., 2008). However, resistance to Bt has emerged in
some insect species and has thus become a serious threat to
agricultural production (Ferre ´ and van Rie, 2002). Therefore,
elucidation of toxin receptors should facilitate improved under-
standing of the mechanism of the Bt toxin mode of action and
develop methods to counter Bt resistance.
A direct functional assessment of a putative receptor has been
employed using various strategies. Gene silencing of S. litura APN
and H. armigera APN transgenes expressed in cultured insect cell
lines revealed the role of APN in toxin mode of action (Agrawal
et al.,2004; Sivakumaret al.,2007). A knockdownofAPN inS. litura
and H. armigera (Rajagopal et al., 2002; Sivakumar et al., 2007) and
cadherin from H. armigera (Sobero ´n et al., 2007) with dsRNA
demonstrated that APN and cadherin serve as functional Cry toxin
receptors in vivo. In our study, we evaluated the functional role of
APN and cadherin using anti-APN and anti-cadherin sera in vivo.
Reduced Cry1Ac toxicity in bioassays with either antisera suggest
that both the H. armigera midgut epithelial APN and cadherin are
functional receptors of the Cry1Ac toxin, in agreement with
previous studies.
Antibodies are gamma globulin proteins that are found in the
blood or other bodily fluids of vertebrates. These molecules are
used by the immune system to identify and neutralize foreign
objects, such as bacteria and viruses. It is known that antibodies
raised against recombinant peptides react with sites in the parent
protein. In addition, there is a high degree of specificity in antigen-
Fig. 4. Reduction of the binding of Cry1Ac toxin using specific antisera. H. armigera
BBMV were pre-incubated for 30 min with anti-APN, anti-cadherin, or preimmune
sera or buffer (control) before adding Cy3-Cry1Ac labeled toxin (mean ? S.E., n = 3).
Fig. 5. Reduction of the toxicity of Cry1Ac by anti-APN or anti-cadherin serum.
Larvae of H. armigera were fed a diet containing Cry1Ac protoxin at the LC80for the
toxin and different doses of antisera; PBS buffer and preimmune serum were used
as controls. Survival was assessed after eight days. Mean with different letters are
considered statistically different (P < 0.05).
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