Basic HTML Version
Journal of Mosquito Research, 2013, Vol.3, No.4, 21
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32
ISSN 1927-646X
http://jmr.sophiapublisher.com
23
annealing temperature at 47 (1 min); (4) primer
℃
extension at 72 (1 min) and (5) final extension at
℃
72 (10min). PCR products were analyzed on 1%
℃
agarose gel in 1× Tris-borate EDTA (TBE) buffer (178
mM Tris base, 178 mM boric acid and 1 mM EDTA).
PCR results were analyzed by Alphainnotech gel
documentation
system
for
semiquantitative
determination of changes in transferrin transcripts in
control and treated insects upon induction by
nematodes and its harbored entomopathogenic
bacteria by determination of integrated display value
(IDV) for each sample from the various treatments.
2.4 Purification and Cloning of PCR product
The PCR product was extracted and purified from the
agarose gel with Wizard
®
Plus SV DNA purification
and clean up system (Promega, U.S.A., cat#, A9281).
The purity and integrity of the Tsf PCR product was
verified after extraction and purified by agarose gel
electrophoresis. The purified DNA fragments were
subcloned into pGEM-T easy vector system,
according to the manufacturer’s instructions, then the
(Tsf/pGEM-T) construct was introduced into The
Mach1™- T1
®
E. coli
chemically competent cells
(Invitrogen, cat. No. ATCC#9637). Screening and
selection of white/blue colonies was performed. The
plasmids containing (Tsf/pGEM-T) construct were
mini prepared using Wizard
®
Plus SV Mini Preps
DNA Purification System (Promega, cat #A1340),
according to the manufacturer’s instructions. Double
digestion of the prepared plasmids by
Bam
HI and
Hin
dIII was carried out at 37
℃
for 2 h to release the
Tsf fragment and to verify that the plasmids contains
Tsf insert.
2.5 DNA sequencing and analysis of the Tsf insert
DNA sequencing of Tsf insert was done to confirm the
sequence of the cloned insert and to compare the
sequence of Tsf with the sequence of homologues Tsf
from other organisms. Sequencing was done using
ready reaction Kit (Applied Bio-systems, USA) in
conjunction with ABI-PRISM and ABI-PRISM big
dye terminator cycler model 310 DNA automated
sequencer. The sequences were provided by
MACROGEN Company. The obtained sequences
were analyzed for similarities to other known
sequences found in the GenBank database using the
BLAST program of the National Center for
Biotechnology Information (NCBI) database (Altschul
et al., 1997).
2.6 Cloning and expression of the recombinant
Cx
quinquefasciatus
Tsf protein
Small scale preparation of over express plasmid was
carried out by using Gen Elute Plasmid Mini Prep Kit
(Promega), according to manufacturer’s instructions. The
partial transferrin cDNA of
Cx. quinquefasciatus
was
sub-cloned between
Bam
HI and
Hin
dIII sites of pET-28a
vector (Novagen, USA) through PCR Two specific primers
5’-CGGGATCCCAAGGGACTGTCGGATTTGT-3’
and 5’-CCAAGCTTAGAACTGCTGCAGCTTGAT-3’,
were designed to contain
Bam
HI and
Hin
dIII sites.
The conditions for PCR were 95 for 45 s, 47 for
℃
℃
45 s, and 72 for 2 min (29
℃
cycles) with a final
10-min extension at 72 . Both the insert and pET
℃
-28a
vector were double digested with
Bam
HI and
Hin
dIII.
Then, each was purified and both were ligated using
T4 DNA ligase (Promega), following the
manufacturer’s instructions. The ligation mix was then
used to transform chemically competent
BL21 (DE3)-
pLysS
E. coli
(Invitrogen) (Fritsch et al., 1980).
Successfully transformed
E. coli
cells containing the
Tsf/pET-28, a vector, were selected by plating the
transformed cells on Luria broth (LB)/agar plates
[1.0% (w/v) tryptone, 0.5 % (w/v) yeast-extract and
1.0% (w/v) NaCl at pH 7.5] and containing ampicillin
(50 μg/mL).
E. coli
cells were induced to express Tsf
protein by adding IPTG (1mM final concentration) to
the cultures when OD
600
reached 0.4, the cells were
induced for 4 h at 37 , and then were collected by
℃
centrifugation at 6 000 rpm for 5 min, the cells were
resuspended in 100 mL of 1× binding buffer (5 mM
imidazole, 0.5 mM NaCl, 20 mM Tris-HCl, pH 7.9)
and sonicated in ice bucket four times each for 30 s.
The extract was centrifuged at 10 000 rpm at 4
℃
for
10 min to separate the soluble proteins fraction
(supernatant) from the insoluble proteins fraction (pellet).
The insoluble protein fractions were analyzed for Tsf
protein content by SDS-PAGE (Laemmli et al., 1970).
2.7 Batch purification of 6× his-tagged recombinant
Tsf protein from
E. coli
under native condition
The cell pellet that was prepared as mentioned earlier
was resuspended in lysis buffer (50 mM NaH
2
PO
4
;