Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance
Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance
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DOI：
10.1007/s11248-010-9378-5
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发表时间：
2010-12-01
影响因子：
3
通讯作者：
Cambra, Mariano
中科院分区：
文献类型：
--
作者：
Cervera, Magdalena;Esteban, Olga;Cambra, Mariano
柑橘衰退病毒（Citrus tristeza virus，CTV）是世界上危害柑橘最严重的病毒病之一。通过基因工程培育抗病柑橘基因型是控制CTV的一个很好的途径。为了研究柑橘中单链可变片段（scFv）抗体的产生是否可以干扰和免疫调节CTV感染，产生了分别和同时表达两种不同scFv构建体的转基因墨西哥酸橙植物。这些构建体衍生自良好参考的单克隆抗体3DF 1和3CA 5，特异性针对CTV p25主要外壳蛋白，其混合物能够检测到目前为止表征的所有CTV分离株。单链抗体积累水平低，可以很容易地检测到只有在四个转基因株系。12个同质和充满活力的线进行繁殖和CTV的挑战与侵略性CTV株嫁接接种。在大多数转基因株系中观察到明显的保护作用，其在高达40-60%的繁殖中显示抗性。此外，与对照植物相比，在感染的转基因植物中观察到症状出现的延迟和症状强度的减弱。这种效应在携带3DF 1 scFv转基因的品系中更为明显，可能与该抗体识别的表位的生物学功能有关。这是第一个报告描述了成功的保护对病原体在木本转基因植物的异位表达的scFv重组抗体。
Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.