Harper K, Kerschbaumer R J, Ziegler A, Macintosh S M, Cowan G H, Himmler G, Mayo M A, Torrance L
Scottish Crop Research Institute, Invergowrie, Dundee, UK.
J Virol Methods. 1997 Jan;63(1-2):237-42. doi: 10.1016/s0166-0934(96)02133-7.
A single chain Fv antibody fragment (scFv) was obtained from a synthetic phage-antibody library after four rounds of selection against purified preparations of potato leafroll luteovirus (PLRV). Nucleotide sequence analysis showed that the scFv belongs to the human V(H)3 family. DNA encoding the scFv was sub-cloned into pDAP2 such that a scFv-alkaline phosphatase fusion protein was produced by transformed bacteria following induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The fusion protein was obtained at concentrations of 10 mg/l of Escherichia coli culture medium and these fusion protein preparations were used directly in ELISA to detect PLRV in sap extracts from infected plants. Our work is the first report of the selection of a scFv specific for a luteovirus from a synthetic phage-display library and the production of a fusion protein with alkaline phosphatase for the detection of PLRV in infected plants. The results demonstrate the potential of scFv and enzyme-scFv fusion proteins in routine testing for plant virus infection.
经过四轮针对马铃薯卷叶黄化病毒（PLRV）纯化制剂的筛选，从合成噬菌体抗体文库中获得了单链Fv抗体片段（scFv）。核苷酸序列分析表明，该scFv属于人类V(H)3家族。将编码scFv的DNA亚克隆到pDAP2中，使得异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，转化细菌产生scFv-碱性磷酸酶融合蛋白。融合蛋白在大肠杆菌培养基中的浓度为10 mg/l时获得，这些融合蛋白制剂直接用于酶联免疫吸附测定（ELISA），以检测感染植物汁液提取物中的PLRV。我们的工作是首次报道从合成噬菌体展示文库中筛选出对黄化病毒特异的scFv，以及生产用于检测感染植物中PLRV的碱性磷酸酶融合蛋白。结果证明了scFv和酶-scFv融合蛋白在植物病毒感染常规检测中的潜力。
