泡桐丛枝植原体pPaWBNy-2-ORF4编码蛋白的抗体制备和表达分析

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

以感病泡桐组培苗提取的DNA为模板,采用PCR方法扩增pPaWBNy-2-ORF4的部分片段。将目的片段克隆到原核表达载体pGEX-4T-3,重组质粒pGEX-p2ORF4转化大肠杆菌Rosseta(DE3)菌株。IPTG诱导表达,分子量约为38 kDa的含GST标签的融合蛋白得到表达。切胶回收目的蛋白,免疫大白兔制备抗血清。间接ELISA测定抗血清的效价约为1∶4 096,免疫印迹实验显示:抗血清能够与原核表达的GST融合蛋白发生特异的免疫反应,与pPaWBNy-1-ORF5的原核表达蛋白无明显的交叉反应。利用制备的抗血清,在感病泡桐饲毒的茶翅蝽中检测到分子量约为18 kDa的蛋白条带,而在无菌茶翅蝽和感病泡桐组培苗中均未检测到,表明pPaWBNy-2-ORF4在饲毒的茶翅蝽中表达,而在感病泡桐组培苗中未表达或表达量低于检测水平。据此推测,该基因参与茶翅蝽传播泡桐丛枝植原体。

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.