Generation and characterization of functional recombinant antibody fragments against tomato yellow leaf curl virus replication-associated protein.
Generation and characterization of functional recombinant antibody fragments against tomato yellow leaf curl virus replication-associated protein.
复制标题
针对番茄黄曲叶病毒复制相关蛋白的功能性重组抗体片段的生成和表征。
DOI：
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发表时间：
2008
期刊：
影响因子：
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通讯作者：
U. Commandeur
中科院分区：
文献类型：
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作者：
M. Safarnejad;R. Fischer;U. Commandeur
番茄黄化曲叶病毒（Tomato yellow leaf curl virus，TYLCV）是一种广泛分布于热带和亚热带地区的双生病毒复合体，严重危害番茄等重要经济作物。传统的疾病管理策略收效甚微，需要考虑基于基因工程的新方法。我们产生了两个单链可变片段抗体（scFv-ScRep 1和scFv-ScRep 2），其强烈结合TYLCV复制相关蛋白（Rep）内的连续表位。TYLCV-Ir C1基因（编码Rep）表达为谷胱甘肽-S-转移酶（GST）和麦芽糖结合蛋白（MBP）融合体。纯化的MBP-Rep用于免疫小鼠，从而允许构建初始和预免疫的scFv噬菌体展示文库。免疫分析显示scFv-ScRep 1识别Rep的N-末端表位，而scFv-ScRep 2识别更中心的表位。这是第一次成功生产针对双生病毒Rep的scFv抗体，这是生产具有TYLCV抗性的转基因植物的第一步。
Tomato yellow leaf curl virus (TYLCV) is a complex of geminivirus species prevalent in the tropics and sub-tropics, which causes severe diseases in economically important crops such as tomato. Conventional strategies for disease management have shown little success and new approaches based on genetic engineering need to be considered. We generated two single-chain variable fragment antibodies (scFv-ScRep1 and scFv-ScRep2) that bound strongly to continuous epitopes within the TYLCV replication-associated protein (Rep). The TYLCV-Ir C1 gene (encoding Rep) was expressed as glutathione-S-transferase (GST) and maltose-binding protein (MBP) fusions. Purified MBP-Rep was used to immunize mice allowing the construction of naïve and pre-immunized scFv phage display libraries. Immunoassays showed that scFv-ScRep1 recognized an N-terminal epitope of Rep, whereas scFv-ScRep2 recognized a more central epitope. This is the first successful production of scFv antibodies against a geminivirus Rep, the initial step in the production of transgenic plants with resistance to TYLCV.