Isolation and expression of an immunodominant membrane protein gene of the ESFY phytoplasma for antiserum production

Our objective is to design an antibody that could be used for the highly specific diagnosis of a phytoplasma infection. Antibodies produced using immunogens extracted from phytoplasma infected plants often cross-reacted with plant antigens due to difficulties in separating phytoplasma and plant antigens. Another more promising method is the development of antibodies against immunogenic recombinant phytoplasma proteins expressed in Escherichia coli. In the present study, a sequence coding a membrane protein of the European Stone Fruit Yellows (ESFY) phytoplasma has been isolated. Primers amplifying the ESFY sequence were designed based on the sequence of the immunodominant membrane protein (IMP) of the Apple proliferation (AP) phytoplasma published by Berg et al. (1999). Sequencing of the amplified product showed the ESFY sequence to be significantly different from the AP sequence despite the phylogenetic proximity of the two phytoplasmas. Among the two phytoplasmas, there is a 66% similarity in the DNA sequences and 46% similarity in the sequence of amino acid residues (aa). Despite the high dissimilarity in their sequence, the ESFY and AP IMPs (164 aa and 165 aa, respectively with deduced molecular masses of 19 kDa) have analogous structures. The two proteins are most similar at both their N-terminal and a neighbouring short hydrophobic transmembrane segment; they differ the most in their long hydrophilic C-terminal exposed to the outer side of the cell. When compared to a major antigenic membrane protein of the sweet potato witches broom (SPWB) phytoplasma (Yu et al., 1998), only at the short transmembrane region has sequence homology been identified. Immediately downstream of the coding region, 102 and 72 base pair hairpin structures were found in the ESFY and AP sequences, respectively. The PCR amplified ESFY IMP gene was cloned into two different expression vectors, Pinpoint TA-vector (Promega) and pQE-40 (Qiagen) which were then transformed into E. coli. The isolated and purified ESFY protein product is being used for the production of monoclonal antibodies in mice.