Applying of recombinant protein technology for developing of specific antibody against transmitting agent of rhizomania in suger beet

Safarnejad, M.R; Basirat, M; Ebrahimi, M.A; Safarpour, H; Nazari, S; Mahmoudi, B; Ataie Kachoie, S

Applying of recombinant protein technology for developing of specific antibody against transmitting agent of rhizomania in suger beet

Document Type : Research Paper

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Abstract

Rhizomania is one of the most important Sugar beet diseases throughout the world. The disease is caused by beet necrotic yellow vein virus (BNYVV). The Polymyxa betae (Keskin) is the only natural transmitting agent of the disease between the plants. The fungus is an obligate parasite and could not be cultured in the media, then detection of fungus is done normally by microscopic observation. In order to facilitate detection process, present study is done to develop specific antibodies against this fungus by applying recombinant protein. The Glutathione-S-transferase (GST) as a specific immunogenic protein is a critical enzyme expressed in zoospores, sporangia and resting spores and could be a good candidate to develop a serological test for P. betae. For this aim, the DNA region encoding fungal GST gene was isolated and cloned into pET28a bacterial expression vector. Large scale expression of the recombinant protein was performed in Escherichia coli (Migula). Purification was carried out by applying immobilized metal affinity chromatography under native conditions. The purified recombinant GST protein was used for immunization of rabbit. Purification of immunoglobulin was performed by affinity chromatography using protein A column. The purified antibodies were applied for efficient detection of infected plant in serological assays.

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