Identification of Single Chain Fragment Variable Monoclonal Antibody Against Coat Protein of Tomato Leaf Curl Virus Using Phage Display Technology
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Date
2017-06
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University of Agricultural Science, Dharwad
Abstract
An investigation was carried out to identify single chain fragment variable monoclonal antibody against Tomato leaf curl virus (ToLCV) coat protein using phage display technology, conducted at department of biotechnology, University of Agricultural Sciences, Dharwad during 2015-16. ToLCV is a major Geminivirus which causes serious loss to tomato production in tropical and subtropical regions of the world. The most commonly used diagnostic tool for ToLCV 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, ToLCV/CP was bacterially expressed. A 786 bp PCR product containing coat protein coding region of ToLCV was amplified using ToLCV CPF and ToLCV CPR primers and the amplified product was cloned into the pTZ57R/T and further subcloned in to the pQE30. The transformed clones were confirmed through PCR and sequencing.
The coat protein was expressed using 1 mM IPTG. A band of 31 kDa on the gel confirmed that coat protein was really fused to the His-tag. Further, the coat protein was purified using His-tag purification kit. Four rounds of biopanning was performed by coating purified ToLCV /CP in to the immunotube using Tomlinson library. The fourth biopan reading (1.37) showed higher binding specificity to ToLCV/CP. These were subsequently used for scFv monoclone for ToLCV/CP. Finally, the randomly selected scFv clones were screened with ELISA. ELISA reading showed that one clone had higher binding affinity to ToLCV/CP. The sequencing of the clone showed 80% similarity with the scFv antibody gene (DQ375454.1). The selected clone is highly specific to ToLCV/CP as there was no cross reaction with PRSV and groundnut GBNV. Further, the sensitivity test results imply that the developed scFv antibody can detect ToLCV coat protein at the concentration of 20 g/ml.