830 Q. Feng et al. Insect Biochemistry and Molecular Biology 30 2000 829–837
mRNA. The N-terminal end of the deduced amino acid sequence of this cDNA shows similarity with the amino
acid sequence of the 21-kDa and 19-kDa proteins ident- ified from the locust Kanost et al., 1988; Zhang et al.,
1993, but the amino acids at the C-terminal end are different. Expression of the mRNA for this protein is
tissue-, development- and sex-specific.
2. Materials and methods
2.1. Animals African migratory locusts Locusta migratoria were
taken from a large, gregarious colony and reared under a 12-h light12-h dark cycle, day temperature of 36
° C,
night temperature of 22 °
C, and 70 relative humidity. The sexes were separated at the fifth larval stage and the
adults were fed for 16 days after emergence. 2.2. cDNA library construction and screening
A cDNA library was constructed using mRNA iso- lated from the locust vitellogenic ovaries and the fat bod-
ies of mated females that had oviposited. The synthe- sized cDNAs were cloned into Uni-ZAP XR vectors at
the EcoR
I and
Xho I
sites using
a cDNA
synthesiscloning kit from Stratagene La Jolla, CA, USA, following the manufacturer’s instructions.
Screening of the cDNA library was performed using an immuno-screening kit from Stratagene using poly-
clonal antibodies produced by immunizing a rabbit with partially purified 35-kDa JH membrane receptor Kim et
al., 1999. The phage was plated in E. coli XL1-Blue MRF
9, transferred to a nitrocellulose filter, and then screened with the polyclonal antibodies at a dilution of
1:2000. Sheep anti-rabbit antibodies conjugated with alkaline phosphatase Sigma Chemicals Company, USA
were used as the secondary antibody at a dilution of 1:5000.
2.3. Sequencing and analysis of sequence The inserts of the cDNA clones that reacted positively
to the antibodies were sequenced on both strands using Cy5
AutoRead
Sequencing Kit and ALFexpress
DNA Sequencer Pharmacia Biotech Inc., Piscataway, NJ, USA. Sequence analysis was performed using the
MacVector DNA
Analysis Program
International Biotechnologies Inc., New Haven, CT, USA. The
sequences were then compared to the sequences in the GenBank database at the National Center for Biotechnol-
ogy Information using the BLAST network services Altschul et al., 1990. Amino acid sequences were
aligned using the Clustal Alignment Program Higgins and Sharp, 1988.
2.4. Northern blot Total RNA was extracted from the fat body and
ovaries of adults aged 0, 2, 4, 6 and 8 days using the guanidinium
thiocyanate–phenol–chloroform method
Chomczynski and Sacchi, 1987. Ten micrograms of total RNA samples were separated on a 1 formal-
dehyde–agarose gel and transferred to a Hybond N nylon membrane. The Northern blot was hybridized with a
cDNA probe labeled with [
32
P]dATP. Hybridization and washes were conducted as described in Palli et al.
1998. 2.5. SDS–PAGE and Western blot
Proteins were extracted from cells infected with recombinant viruses expressing the cloned cDNA using
homogenization buffer 20 mM Tris, 50 mM KCl, 300 mM sucrose, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1
µ gml leupeptin, 1
µ gml pepstatin, pH 7.5. The protein
samples were denatured at 100 °
C for 5 min in a denatur- ing buffer 0.1 M Tris, pH 6.8, 2 SDS, 0.5
β -mercap-
toethanol, 12 glycerol, and 0.002 bromphenol blue. SDS–PAGE sodium dodecyl sulfate polyacrylamide gel
electrophoresis was performed on 12 polyacrylamide gels in Tris–glycine–SDS buffer 10 mM Tris, 50 mM
glycine, 0.1 SDS, pH 8.0. Gels were stained with 0.5 Coomassie Blue R-250 for 1 h and then destained
with destaining solution containing 7 acetic acid and 10 MeOH in H
2
O. After electrophoresis, proteins were transferred from
gels to Hybond C nylon membranes in transfer buffer 10 Tris–glycine–SDS buffer and 20 MeOH in
H
2
O, and immunodetection was performed using the primary and secondary antibodies as described in Sec-
tion 2.2. 2.6. In vitro translation
In vitro translation of cDNA was performed using the TNT
Coupled Reticulocyte Lysate System from Pro-
mega Madison, WI, USA. The reaction mixture con- tained 25
µ l of rabbit reticulocytes lysate, 2
µ l of
10 ×
reaction buffer, 1 µ
l of T3 RNA polymerase, 0.02 mM amino acid mixture minus methionine, 0.3 mCiml
[
35
S]-methionine, 40 units RNase inhibitor, and 1 µ
g of DNA template in a total volume of 50
µ l. The reaction
mixture was incubated at 30 °
C for 2 h. The translation products were resolved on an SDS–PAGE gel 12 and
[
35
S]-labeled protein products were detected using an InstantImager
Electronic Autoradiography System
Packard Instrument Company, Meriden, CT, USA. 2.7. Construction of recombinant baculovirus
Recombinant baculovirus expressing the 25K cDNA was constructed using BAC-TO-BAC
Baculovirus
831 Q. Feng et al. Insect Biochemistry and Molecular Biology 30 2000 829–837
Expression System
from Life
Technologies Gaithersburg, MD, USA according to the manufac-
turer’s protocol. The cDNA insert was first cloned into the mini-Tn7 element of a pFASTBAC donor plasmid
at the sites of EcoR I and Xho I. The recombinant plas- mid was then transformed into DH10BAC cells contain-
ing helper plasmid and Autographa californica multicap- sid nucleopolyhedrovirus AcMNPV genomic DNA.
The mini-Tn7 element carrying the cDNA insert was transposed into the AcMNPV genome, resulting in a
recombinant virus. The recombinant baculovirus DNA was selected by disruption of the lacZ
9 gene and the recombinants were confirmed by PCR followed by
Southern blotting. Spodoptera frugiperda cells SF-21, Vaughn et al.,
1977 were cultured in Grace’s medium Grace, 1962 in six-well plates at a concentration of 10
6
cellswell. The cells were incubated for 5 h at 28
° C with a transfec-
tion mixture containing 300 ng of the recombinant DNA and 10
µ l of CellFectin
reagent Life Technologies.
After removal of the transfection mixture, the cells were cultured at 28
° C for 4 days. The recombinant baculo-
virus was harvested and the titer was determined. The virus was then used at an MOI of 0.04 to infect SF-21
cells cultured in 15-ml flasks. The infected cells were harvested at 0, 1, 6, 12, 24, 48, 72, and 96 h post-inocu-
lation h.p.i. for analysis of mRNA and protein.
2.8. JH binding assay SF-21 cells inoculated with the recombinant virus
were homogenized
in the
homogenization buffer
described in Section 2.5. The homogenate was centri- fuged at 1000g for 10 min to pellet cell debris. The
supernatant was centrifuged again at 30 000g for 60 min to pellet cell membranes. The membrane pellet was sus-
pended in 20 mM Tris–HCl buffer, pH 7.5. For specific binding, the binding mixture contained 50 mM Tris–HCl
pH 7.5, 0.1 mgml membrane protein, 15 nM [
3
H]-JH III in a total volume of 200
µ l. For determination of non-
specific binding, unlabeled JH III at 1.5 µ
M was added to the above mixture. The binding reaction was perfor-
med at 28 °
C for 60 min. The mixture was centrifuged
at 30 000g for 30 min to pellet the membranes. The pel- let was rinsed twice with 500
µ l of 50 mM Tris–HCl
binding buffer. The top portion of the tubes was cut off right above the membrane pellet and the bottom portion
was dropped into 10 ml of scintillation cocktail and the radioactivity was counted.
3. Results
