Preparation of the polyclonal antibody against pPaWBNy-2-ORF4 of paulownia witches'-broom phytoplasma and its expression analysis

The pPaWBNy-2-ORF4 was amplified from genome DNA extracted from infected paulownia plantlets by PCR. The amplified DNA fragments were inserted into the prokaryotic expression vector pGEX-4T-3. The recombinant plasmid pGEX-p2ORF4 was transformed into the Escherichia coli Rosseta (DE3) strain. The 38 kDa GST-tagged p2ORF4 fusion protein was expressed efficiently in E. coli Rosseta (DE3) induced by IPTG. The fusion protein was purified and injected into a rabbit to raise antiserum. The titer of the antiserum was 1:4 096 determined by indirect ELISA. Western blot analysis showed that the obtained polyclonal antibody could react with GST-tagged p2ORF4 protein but had no reaction with pPaWBNy-1-ORF5 protein expressed in E. coli. Western blot analysis also revealed a specific18 kDa protein band inHalyomorpha halys (Stål) exposure to PaWB-infected paulownia, but not in non-infected H. halys and PaWB-infected paulownia. It was inferred that pPaWBNy-2-ORF4 might be involved in the transmission of H. halys.