Immunodiagnosis of Cucurbit yellow stunting disorder virus using polyclonal antibodies developed against recombinant coat protein [Cucumis sativus L.]

• Published in: Journal of plant pathology 2003-11, Vol.85 (3), p.197-204
• Main Authors: Hourani, H, Abou-Jawdah, Y. (American Univ. of Beirut (Lebanon). Dept. of Plant Sciences)
• Format: Article
• Language: English
• Subjects: Antibodies; Antiserum; Capsid proteins; CLONACIÓN MOLECULAR; CLONAGE MOLÉCULAIRE; CLONAZIONE MOLECOLARE; Cucumbers; CUCUMIS SATIVUS; Cucurbit yellow stunting disorder virus; Enzyme linked immunosorbent assay; GENES; GENETIC MARKERS; GENI; Growth retardation; GÈNE; IDENTIFICACIÓN; IDENTIFICATION; IDENTIFICAZIONE; IMMUNOLOGICAL TECHNIQUES; MARCADORES GENÉTICOS; MARCATORI GENETICI; MARQUEUR GÉNÉTIQUE; MOLECULAR CLONING; PCR; Phytopathology; Plant diseases; PLANT VIRUSES; Reverse transcriptase polymerase chain reaction; TECHNIQUE IMMUNOLOGIQUE; TECNICHE IMMUNOLOGICHE; TÉCNICAS INMUNOLÓGICAS; VIRUS DE LAS PLANTAS; VIRUS DELLE PIANTE; VIRUS DES VÉGÉTAUX; Viruses
• ISSN: 1125-4653; 2239-7264

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 [Il gene della proteina di rivestimento (CP) del virus della malattia del nanismo giallo delle cucurbitacee (CYSDV) e' stato amplificato mediante reazione a catena della polimerasi a trascrizione inversa (RT-PCR). L'amplicone e' stato clonato in pGEM-T, sequenziato e subclonato in un vettore di espressione batterica (pQE-31). La CP ricombinante di CYSDV e' stata espressa come una proteina di fusione con un tag terminale di N costituito da sei istidine, purificato mediante cromatografia per affinita', producendo 8 mg di proteina nativa per litro di coltura batterica, e utilizzato come antigene per produrre un antisiero per CP di CYSDV in un coniglio. L'antisiero ottenuto e' stato sperimentato con successo in saggio immunitario tissue blot (TBIA), saggio immunitario dot blot (DBIA), ELISA indiretto e DAS-ELISA, con un titolo pari a 103 per tutti i metodi. TBIA risultava molto specifico ed evidenziava la localizzazione del virus nel tessuto floematico ed e' raccomandato per indagini su vasta scala]