Production of polyclonal and monoclonal antibodies specific against membrane proteins of ‘Candidatus Phytoplasma asteris’, chrysanthemum yellows isolate
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چکیده
Three genes encoding membrane proteins of chrysanthemum yellows (CY) phytoplasma were cloned and sequenced: SecY, Amp and ArtI encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively. Alignments of CY-specific SecY sequences with the corresponding genes of other phytoplasmas confirmed the 16SrDNA-based classification, while Amp sequences resulted highly variable. Five CY partial sequences were cloned into pRSetC expression vector and two (Amp 64-224 and ArtI 131-512) were expressed as fusion antigens in Escherichia coli for the production of CY-specific polyclonal antisera (A416 against Amp 64-224; A407 against ArtI 131-512) and monoclonal antibodies. A416 recognized, in Western blots, the full length Amp from CY-infected plants (periwinkle, daisy) and insect vectors (Euscelidius variegatus, Macrosteles quadripunctulatus). A416 also reacted to European aster yellows (EAY) and primula yellows (PY) phytoplasmas and to Northern Italian strains of ‘Candidatus Phytoplasma asteris’ from lettuce (LY163) and gladiolus (GLA), but not to American aster yellows (AAY) and clover phyllody (CPh) phytoplasmas.
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