鉴定西瓜银斑驳病毒血清群中的 Tospovirus 中 NSs 蛋白保守区的共同表位。
Identification of Common Epitopes on a Conserved Region of NSs Proteins Among Tospoviruses of Watermelon silver mottle virus Serogroup.
出版信息
Phytopathology. 2006 Dec;96(12):1296-304. doi: 10.1094/PHYTO-96-1296.
ABSTRACT The NSs protein of Watermelon silver mottle virus (WSMoV) was expressed by a Zucchini yellow mosaic virus (ZYMV) vector in squash. The expressed NSs protein with a histidine tag and an additional NIa protease cleavage sequence was isolated by Ni(2+)-NTA resins as a free-form protein and further eluted after sodium dodecyl sulfate-polyacrylamide gel electrophoresis for production of rabbit antiserum and mouse monoclonal antibodies (MAbs). The rabbit antiserum strongly reacted with the NSs crude antigen of WSMoV and weakly reacted with that of a high-temperature-recovered gloxinia isolate (HT-1) of Capsicum chlorosis virus (CaCV), but not with that of Calla lily chlorotic spot virus (CCSV). In contrast, the MAbs reacted strongly with all crude NSs antigens of WSMoV, CaCV, and CCSV. Various deletions of the NSs open reading frame were constructed and expressed by ZYMV vector. Results indicate that all three MAbs target the 89- to 125-amino-acid (aa) region of WSMoV NSs protein. Two indispensable residues of cysteine and lysine were essential for MAbs recognition. Sequence comparison of the deduced MAbs-recognized region with the reported tospoviral NSs proteins revealed the presence of a consensus sequence VRKPGVKNTGCKFTMHNQIFNPN (denoted WNSscon), at the 98- to 120-aa position of NSs proteins, sharing 86 to 100% identities among those of WSMoV, CaCV, CCSV, and Peanut bud necrosis virus. A synthetic WNSscon peptide reacted with the MAbs and verified that the epitopes are present in the 98- to 120-aa region of WSMoV NSs protein. The WSMoV sero-group-specific NSs MAbs provide a means for reliable identification of tospoviruses in this large serogroup.
摘要 本研究利用南瓜曲叶病毒（ZYMV）载体表达了西瓜银斑驳病毒（WSMoV）的 NSs 蛋白。表达的 NSs 蛋白带有组氨酸标签和额外的 NIa 蛋白酶切割序列，通过 Ni(2+)-NTA 树脂进行分离，作为游离形式的蛋白，然后在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后洗脱，用于生产兔抗血清和鼠单克隆抗体（MAbs）。兔抗血清与 WSMoV 的 NSs 粗抗原强烈反应，与辣椒褪绿斑驳病毒（CaCV）高温恢复分离株（HT-1）的 NSs 粗抗原弱反应，但与马蹄莲黄斑驳病毒（CCSV）的 NSs 粗抗原不反应。相比之下，MAbs 与 WSMoV、CaCV 和 CCSV 的所有 NSs 粗抗原均强烈反应。构建并表达了 NSs 开放阅读框的各种缺失突变体。结果表明，三种 MAbs 均靶向 WSMoV NSs 蛋白的 89-125 个氨基酸（aa）区域。半胱氨酸和赖氨酸这两个必需残基对 MAb 的识别至关重要。与报道的 Tospovirus NSs 蛋白相比，推导的 MAb 识别区域的序列比较显示，在 NSs 蛋白的 98-120-aa 位置存在一个保守序列 VRKPGVKNTGCKFTMHNQIFNPN（表示为 WNSscon），在 WSMoV、CaCV、CCSV 和花生芽坏死病毒的 NSs 蛋白中具有 86-100%的同一性。一个合成的 WNSscon 肽与 MAb 反应，证实表位存在于 WSMoV NSs 蛋白的 98-120-aa 区域。WSMoV 血清群特异性 NSs MAb 为鉴定该大血清群中的 Tospovirus 提供了一种可靠的方法。
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