Characterization of cucurbit yellow stunting disorder virus and development of polyclonal antibodies using recombinant coat protein
Characterization of cucurbit yellow stunting disorder virus and development of polyclonal antibodies using recombinant coat protein
A. A. Rezk1,3, K. A. Alhudaib2 and A. M. Soliman2,3
ABSTRACT
Cucurbit yellow stunting disorder virus (CYSDV) was isolated from cucurbit plants growing in Eastern Province area in Saudi Arabia and characterized using reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide sequence for the coat protein (CP) gene was carried out and submitted in GenBank under accession number JN083790. The phylogenetic tree showed that there are two big clusters and the identity between them 90%. The isolated CYSDV in this study is located in the second cluster with the isolates from Sudan, Iran and the other isolate from Saudi Arabia. The analysis showed that the highest nucleotide identities were 100% with other isolates that isolated from Saudi Arabia and was 98% with other isolates from Iran and Sudan. While the identity was 90% with all other members in the first cluster. The data refers to the isolated CYSDV virus in this study is more related to the isolates from Iran and Sudan. CP gene of CYSDV (CYSDV-CP) was cloned into the expression vector upon induction; the viral protein was expressed as 6XHis-tagged CYSDV fusion protein in Escherichia coli cells BL21 strain. The purified protein was immunized by injecting to New Zealand white rabbit using 6 injections at weekly intervals intravenously and subcutaneous. The rabbit blood was collected by bleeding and the antiserum was obtained. IgG was purified and conjugated to alkaline phosphatase. The conjugated antiserum was evaluated by indirect enzyme-linked immunosorbent assay (ELISA). The conjugated antisera that prepared in this study were succeeded to detect the virus in infected plants.
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