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Molecular Pathogens
MP2011, Vol.2, No.2
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agarose gels running in 0.5x TBE buffer.
3.6 Amplification of a conserved region within the
coat protein gene of
FMV
Based on the amino acid conserved region of 67 virus
coat protein sequences of
potyviruses
available in
Genbank, two degenerate primers were designed and
used to amplify cDNA fragment within the coat
protein gene of the infected tissues. The forward and
reverse primers were designated as CP1 and CP2,
respectively. CP1 was (5'-ZAY GGX GAX GAZ CAZ
GTG-3') and CP2 was (5'AAZ GCX GCZ GCX ATY
AAY -3'). The PCR reaction conditions were initial
denaturation at 95°C for 5 min, followed by 34 cycles
at 95°C for 1 min, 60°C for 1 min and 72°C for 1 min.
Final extension at 72°C was done for 10 min.
Amplification products were visualized in 1.5%
agarose gel run in 0.5x TBE buffer.
3.7 PCR purification, sequencing and sequencing
analyses
PCR products were purified using PCR clean up
column kit (Maxim biotech INC, USA) according to
manufacturer's instructions and then sequenced using
forward primer. Sequencing was performed using
BigDye® Terminator v3.1 Cycle Sequencing kit
(Applied Biosystems, Foster City, CA, USA) and
model 3130xl Genetic Analyzer (Applied Biosystems,
Foster City, CA, USA). Analysis of nucleotide
sequences was carried out using Blast search. But for
sequence alignment, the obtained sequence was
aligned with the published ones using ClustalW (1.83)
according to Thompson et al., 1994. Phylogenetic
analysis was performed using MEGA4 program
(Tamura et al., 2007).
3.8 Cloning, subcloning and gene expression of the
amplified CP
To examine if the extracted viral RNA is a negative
strand or a positive one, the purified PCR product
corresponding to the
CP
gene fragment was cloned in
PCR-TOPO vector with TOPO TA cloning kit
(Invitrogen, USA). Subcloning was performed into
pPROEX HTa expression vector using pPROEXTM
HTa Prokaryotic Expression System kit (Life
technologies, USA). Bacteria were grown at 37 ºC for
3 h, induced by the addition of 1 mM IPTG, incubated
at 37ºC for 5 h and then the cells were harvested,
hydrolyzed and partial protein was obtained according
to Mellor et al. (1983). Sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) was
carried out using the discontinuous buffer system as
described by Sambrook et al. (1989) in order to
demonstrate the expression level of the coat protein
gene in the induced and non induced recombinant
bacteria.
4 Conclusion
In this paper, the biological, ultrastructural, and
molecular characterization of Egyptian isolate of FMV
was described. Furthermore, cloning and sequencing
of a conserved region in
CP
gene were performed.
Further studies to classify the isolate as putative
potyvirus were done. The availability of CP products
would be helpful in studies concerning ELISA, PCR,
and other related molecular techniques. Additionally, a
novel primer set was developed to amplify 374 bp
within the
CP
gene from both infected tissue and
partial purified virus of an Egyptian FMV isolate. We
suggest that it may be useful to monitor the
distribution of FMV, and the release of wild type as
well as the genetically engineered FMV.
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