IMMUNODIAGNOSIS OF CUCURBIT YELLOW STUNTING DISORDER VIRUS USING POLYCLONAL ANTIBODIES DEVELOPED AGAINST RECOMBINANT COAT PROTEIN
H. Hourani, Y. Abou-Jawdah
The coat protein (CP) gene of Cucurbit yellow stunting disorder virus (CYSDV) was amplified by reverse transcription – polymerase chain reaction (RT-PCR). The amplicon was cloned in pGEM-T, sequenced and subcloned into a bacterial expression vector (pQE-31). The recombinant CYSDV CP was expressed as a fusion protein with an N-terminal hexa-histidine tag, purified by affinity chromatography yielding 8 mg native protein per liter of bacterial culture, and used as an antigen to produce CYSDV CP antiserum in a rabbit. The resulting antiserum was successfully assayed in tissue blot immunoassay (TBIA), dot blot immunoassay (DBIA), indirect ELISA and DAS- ELISA, with a titer of about 103 for all methods. TBIA was very specific and showed the virus localization in the phloem tissue and is recommended for large-scale surveys.