Narayan MogerShilpa B.S.2016-11-222016-11-222013http://krishikosh.egranth.ac.in/handle/1/87105Banana 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.Thesis