Development of Single Chain Fragment Variable Monoclonal Antibody Against Banana Bunchy Top Virus Coat Protein
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Date
2013
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Journal ISSN
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Publisher
UAS, Dharwad
Abstract
Banana bunchy top virus (BBTV) is a destructive pathogen in banana cultivating
areas worldwide. The symptoms induced by BBTV are similar to those caused by abiotic
factors and other vascular diseases. This lack of unambiguous symptoms necessitates early
diagnosis of BBTV infections. The most commonly used diagnostic tool for BBTV detection
is immunological assays, which is dependent on the availability of highly specific antibody to
differentiate the viruses. Production of antibody using phage display technology needs pure
protein therefore, BBTV/CP was bacterially expressed. A 531 bp PCR product containing
coat protein coding region of BBTV was amplified using BBTVCPF and BBTVCPR primers
and the amplified product was cloned into the pTZ57R/T and further subcloned in to the
pQE30. After transformation the clones were confirmed through PCR and sequencing.
Amplification with expected size of 531 bp and 100% homology with other isolates showed
integrity of the clone. Further, the coat protein appeared to be expressed at 3hr after induction
with 1 mM IPTG. A band of 21 kDa on the gel confirmed that coat protein was really fused
to the His-tag. Further, 10mg/litre of the coat protein were purified using His-tag purification
kit.
Four round of biopanning was performed by coating purified BBTV/CP in to the
immunotube using Tomlinson library. The fourth biopan reading (1.9) showed higher binding
specificity to BBTV/CP. These were subsequently used for scFv monoclone for BBTV/CP.
Finally, the randomly selected scFv clones were screened with ELISA. ELISA reading
showed that two clones had higher binding affinity to BBTV/CP. The sequencing of the
clones showed 85% homology with the scFv antibody gene (JN887438.1). The selected clone
is highly specific to BBTV/CP as there was no cross reaction with banana CMV and
groundnut GBNV. Further, the sensitivity test results imply that the developed scFv antibody
can detect at the concentration of 20 ıg/ml.