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 GSTtagged 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 in Halyomorpha halys(Stl) 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.