高级检索+

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

  • 摘要: 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。

     

    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.

     

/

返回文章
返回