Use of a recombinant protein for development of a DAS-ELISA serological kit for sensitive detection of witches' broom disease of lime

摘要

Witches' broom disease of lime (WBDL), associated with 'Candidatus Phytoplasma aurantifolia' is the most devastating disease of acid lime in Southern Iran. Lack of an efficient approach for control of the disease has resulted in application of quarantine measures for protection of healthy plants and to limit the spread of disease to uninfected areas. Toward this aim, development of a rapid and efficient method for detection of infected plants is a major focus. The present study introduces application of recombinant DNA technology for development of a sensitive serological technique (DAS-ELISA) for detection of infected plants. The immunodominant membrane protein (IMP), as a major protein present on the surface of phytoplasma cells, was selected as a target for generating specific antibody molecules. The gene encoding IMP of 'Ca. P. aurantifolia' was obtained from infected plants. The region encoding the IMP fragment was isolated by PCR amplification followed by insertion into the pZ57R/T cloning vector. Intact clones containing the right sequences were selected and sub-cloned into the pET28a bacterial expression vector. Large scale expression of recombinant protein was performed in E. coli and purification was carried out through affinity chromatography in Ni-agarose columns. To obtain specific polyclonal antibodies against WBDL, the purified recombinant IMP was used for rabbit immunization. The antisera titer was determined after each boosting via indirect ELISA. When the titer reached 1:100,000, the animal was sacrificed, blood was collected and serum was separated from blood cells. The IgG molecules were purified from serum content by affinity chromatography using protein A columns followed by conjugation to alkaline phosphatase (AP) enzyme. The purified specific antibodies and conjugate were used for detection of the corresponding antigen, IMP in infected plants in DAS-ELISA and dot-blot methods. The results confirmed the capability of this technique for efficient detection of infected plants, while no reaction was observed in negative controls. The detection limit of the DAS-ELISA method was determined at 70 μg IMP/ml leaf extract.