基于单克隆抗体的三联抗体夹心酶联免疫吸附法和免疫捕获逆转录聚合酶链反应检测齿舌兰环斑病毒。
Monoclonal antibody-based triple antibody sandwich-enzyme-linked immunosorbent assay and immunocapture reverse transcription-polymerase chain reaction for Odontoglossum ringspot virus detection.
机构信息
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, China.
出版信息
J Virol Methods. 2011 Jan;171(1):40-5. doi: 10.1016/j.jviromet.2010.09.027. Epub 2010 Oct 7.
Odontoglossum ringspot virus (ORSV) infects numerous commercially important orchids and causes significant losses worldwide. The coat protein (CP) gene of ORSV was cloned and expressed in Escherichia coli by using the pET-32a expression vector, and the expression of recombinant protein was confirmed by Western blotting using anti-ORSV antibodies. The recombinant protein was purified using Ni-NTA agarose, and the purified protein was used as an immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Five murine MAbs against ORSV CP were obtained. Among them, two MAbs (6B4 and 1D1) also reacted with TMV CP. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) methods using the MAb (8A5) were then developed for sensitive, specific, and rapid detection of ORSV. TAS-ELISA and IC-RT-PCR could detect ORSV in the infected leaf saps with dilutions of 1:10,240 and 1:81,920 (w/v, g mL(-1)), respectively. TAS-ELISA and IC-RT-PCR detections indicated that ORSV was prevalent in orchids in the Zhejiang Province of China.
齿舌兰环斑病毒（ORSV）感染了许多具有商业价值的兰花，在全球范围内造成了巨大的损失。我们利用 pET-32a 表达载体在大肠杆菌中克隆并表达了 ORSV 的外壳蛋白（CP）基因，并使用抗 ORSV 抗体通过 Western blot 验证了重组蛋白的表达。利用 Ni-NTA 琼脂糖纯化了重组蛋白，并将其作为免疫原制备单克隆抗体（MAb）和多克隆抗体（PAb）。获得了针对 ORSV CP 的 5 株鼠源 MAb。其中，2 株 MAb（6B4 和 1D1）也与 TMV CP 发生反应。然后，我们利用 MAb（8A5）建立了三重抗体夹心酶联免疫吸附测定法（TAS-ELISA）和免疫捕获逆转录-聚合酶链反应（IC-RT-PCR）方法，用于 ORSV 的灵敏、特异和快速检测。TAS-ELISA 和 IC-RT-PCR 分别可以检测到稀释度为 1:10、240 和 1:81,920（w/v，g mL(-1)）的感染叶片汁液中的 ORSV。TAS-ELISA 和 IC-RT-PCR 的检测结果表明，ORSV 在浙江省的兰花中普遍存在。
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