Detection of the nonstructural p21 protein of beet yellows closterovirus in vivo with polyclonal antibodies to the bacterially expressed proteinстатья
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Аннотация:In the positive-sense RNA genome of beet yellows closterovirus (BYV), the 3'-terminal ORF 8 codes for a nonstructural protein of 21 kDa. The cDNA of ORF 8 was inserted into an E. coli expression vector pQE-30 under the control of bacteriophage T5 promoter. The recombinant protein having an N-terminal tag of six histidines, was overexpressed in bacteria, purified, and used for antiserum production in rabbits. Using the IgG fraction from a polyclonal anti-p21 serum preadsorbed with the extract of healthy Tetragonia expansa plants, the p21 was detected in BYV-infected plants. Analysis of subcellular fractions of BYV-infected T. expansa indicated that the bulk of p21 is in the fractions of cytoplasm and cell walls. The p21 was readily detected by immunoblotting in the BYV-infected plant samples taken 20 to 120 days post inoculation, thus indicating steady accumulation of the protein in the BYV infection and/or its stability.