Production of polyclonal antiserum specific to coat protein gene of Eggplant yellow mosaic virus (EYMV)

Abstract

Polyclonal antiserum of eggplant yellow mosaic virus (EYMV) was produced. The recombinant protein synthesized from clone EYMV coat protein obtained from the plasmid DNA extraction of diseased eggplant. The DNA was amplified using the primers EYMVCPF and EYMV-CPR which designed from the coat protein of EYMV. The obtained 777 bp DNA fragment was cloned into pUC57 plasmid DNA vector. The plasmids were extracted and ligated with the expression vector pET200/D-TOPO (Invitrogen). The recombinant plasmid was cloned into E. coli competent cell (Top10). The positive clone was selected and transformed into E. coli BL21 (DES 3). The bacteria were cultivated in 2xYT medium supplemented with 50 mg/l kanamycin, then protein synthesis was induced with IPTG. The highest protein production was obtained at 24 hours. The obtained 28 kDa protein was purified in Ni-NTA column for the antibody production in rabbit. The antiserum could be used for the detection of the recombinant protein for Indirect ELISA and Nitrocellulose membrane-Enzyme-linked immuno sorbent assay (NCM-ELISA) and conferred specificity. The IgG of EYMV-CP was used for the detection of the diseased plant with 1:10-1:80 concentrations.