| 泡桐丛枝植原体pPaWBNy-2-ORF4编码蛋白的抗体制备和表达分析 |
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| 引用本文： | 耿显胜,田国忠,任争光,宋传生,林彩丽.泡桐丛枝植原体pPaWBNy-2-ORF4编码蛋白的抗体制备和表达分析[J].林业科学研究,2013,26(4):494-500. |
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| 作者姓名： | 耿显胜 田国忠 任争光 宋传生 林彩丽 |
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| 作者单位： | 中国林业科学研究院森林生态环境与保护研究所,国家林业局森林保护学重点实验室,北京100091 |
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| 基金项目： | 国家自然科学基金面上项目"泡桐丛枝植原体质粒蛋白p23及寄主互作蛋白功能研究"(3117062) |
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| 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1:4096,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.
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| 关 键 词： | 茶翅蝽 多克隆抗体 昆虫传播 植原体 |
| 收稿时间： | 2013/3/25 0:00:00 |
Preparation of the Polyclonal Antibody Against pPaWBNy-2-ORF4 of Paulownia Witches'-broom Phytoplasma and Its Expression Analysis |
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| GENG Xian-sheng,TIAN Guo-zhong,REN Zheng-guang,SONG Chuan-sheng and LIN Cai-li.Preparation of the Polyclonal Antibody Against pPaWBNy-2-ORF4 of Paulownia Witches'-broom Phytoplasma and Its Expression Analysis[J].Forest Research,2013,26(4):494-500. |
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| Authors: | GENG Xian-sheng TIAN Guo-zhong REN Zheng-guang SONG Chuan-sheng and LIN Cai-li |
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| Institution: | Research Institute of Forest Ecology, Environment and Protection, Key Laboratory of Forest Protection of State Forestry Administration, Chinese Academy of Forestry, Beijing 100091, China;Research Institute of Forest Ecology, Environment and Protection, Key Laboratory of Forest Protection of State Forestry Administration, Chinese Academy of Forestry, Beijing 100091, China;Research Institute of Forest Ecology, Environment and Protection, Key Laboratory of Forest Protection of State Forestry Administration, Chinese Academy of Forestry, Beijing 100091, China;Research Institute of Forest Ecology, Environment and Protection, Key Laboratory of Forest Protection of State Forestry Administration, Chinese Academy of Forestry, Beijing 100091, China;Research Institute of Forest Ecology, Environment and Protection, Key Laboratory of Forest Protection of State Forestry Administration, Chinese Academy of Forestry, Beijing 100091, China |
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| 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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| Keywords: | Halyomorpha halys (Stå l) polyclonal antibody insect transmission phytoplasma |
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