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Preparation of the Polyclonal Antibody Against pPaWBNy-2-ORF4 of Paulownia Witches’-broom Phytoplasma and Its Expression Analysis

  • Received Date: 2013-03-25
  • 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 in Halyomorpha 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.
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Preparation of the Polyclonal Antibody Against pPaWBNy-2-ORF4 of Paulownia Witches’-broom Phytoplasma and Its Expression Analysis

  • 1. Research Institute of Forest Ecology, Environment and Protection, Key Laboratory of Forest Protection of State Forestry Administration, Chinese Academy of Forestry, Beijing 100091, China

Abstract: 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 in Halyomorpha 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.

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