Selection of Single Chain Fragment Variable Monoclonal Antibody Against Papaya Ringspot Virus Coat Protein

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Date
2016-06
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University of Agricultural Science, Dharwad
Abstract
Papaya ringspot virus (PRSV) is an important virus causing the most devastating disease in papaya. The symptoms caused by PRSV are not visible at early stages. This necessitates early diagnosis of PRSV infections before planting papaya seedlings to main field. The most commonly used diagnostic tool for PRSV 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 requires pure protein therefore, PRSV-CP was expressed in bacteria. An 864 bp PCR product containing coat protein coding region of PRSV was amplified using gene specific primers and amplified product was cloned into the pTZ57R/T and further expressed in the pQE30. After transformation the clones were confirmed through PCR, restriction analysis and sequencing. Amplification with expected size of 864 bp and 94 % homology with other isolates showed integrity of the clone. Further, the coat protein appeared to be expressed at 3 hr after induction with 1 mM IPTG. A band of 32 KDa on the SDS-PAGE confirmed that coat protein was fused to the His-tag. The yield of coat protein about 1.2 mg/100 ml was purified using His-tag purification kit. Four rounds of biopanning were performed by coating purified PRSV-CP into the immunotube using Tomlinson library. The fourth biopan reading (1.46) shown higher binding specificity to PRSV-CP. These were subsequently used for production of scFv monoclones against PRSV-CP. Finally, the randomly selected scFv clones were screened by ELISA. The ELISA readings were observed that three clones had higher binding affinity to PRSV-CP. The sequencing of the clones showed 94% homology with the scFv antibody gene (KF732845.1). The selected clone is highly specific to PRSV-CP as there was no cross reaction with banana BBTV, cucumber CMV and groundnut GBNV. Further, the sensitivity test results imply that the developed scFv antibody can detect at the concentration of 10 µg/ml antigen.
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