Production of antisera for detection of the important Solanaceae-infecting potyviruses in Taiwan

Autor: Akbar Adjie Pratama
Rok vydání: 2013
Druh dokumentu: 學位論文 ; thesis
Popis: 101
Solanaceae plants such as tomato, chili pepper and bell pepper, are economic crops planted around 2,333 ha area in central and southern Taiwan. In 2012, a field survey for virus diseases of tomato and peppers was conducted, and a high incidence of potyviruses was detected using Potyvirus genus-degenerate primers by reverse transcription-polymerase chain reaction (RT-PCR). These potyviruses were further identified as Potato virus Y (PVY), Pepper mottle virus (PepMoV) and Pepper veinal mottle virus (PVMV) by virus isolation and sequencing of the coat protein (CP) genes. The aim of this study is to produce antisera against the CPs of PVY, PepMoV and PVMV for quick virus detection in field by enzyme-linked immunosorbent assay (ELISA). The CP open reading frames of PVY, PepMoV and PVMV were amplified by RT-PCR and cloned into the bacterial pET-28b(+) vector for protein expression. All three CPs expressed as insoluble fusion proteins containing a histidine tag were purified through a series of centrifugation to remove large impurities, following a final gel elution procedure from 15 % SDS-PAGE. The purified CPs were used as immunogens to produce antisera from New Zealand white rabbits. The end-point dilutions of antisera to PVY, PepeMoV and PVMV were determined as 1/256,000, 1/64,000 and 1/128,000 respectively, by indirect ELISA. All antisera were used at a dilution of 1/2,000 for tests. Serological analyses in indirect ELISA and western blotting revealed PVY is related to PepMoV and distantly to PVMV. PVMV is serological related to PepMoV. These antisera produced could be potential tools for virus detection in field. Sequence alignment and phylogenetic analysis of PVY, PepMoV and PVMV based on the aa sequences of CPs revealed that Taiwanese isolates (in this study) of PVY may have closer relation (95.5% aa identity) to PVY strain LYE84.2 from Canary Islands, Spain, which is classified in the C strain of PVY. The isolates of PepMoV and PVMV used in this study have the closest relations (98.2% and 99.6% aa identity, respectively) to the Korean isolates. Keywords: Potyvirus, pepper, antibody, detection
Databáze: Networked Digital Library of Theses & Dissertations
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