National Center for Biotechnology (NCB), 13/5, Qorghalzhyn Hwy., Astana, 010000, Kazakhstan.
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA.
J Virol Methods. 2018 May;255:1-7. doi: 10.1016/j.jviromet.2018.01.016. Epub 2018 Feb 2.
Plants offer a unique combination of advantages for the production of valuable recombinant proteins in a relatively short time. For instance, a variety of diagnostic tests have been developed that use recombinant antigens expressed in plants. The envelope glycoprotein gp51 encoded by Bovine leukemia virus (BLV) is one of the essential subunits for viral infectivity. It was indicated that the recombinant gp51 (rgp51) of BLV сan be used as an synthetic alternative antigen useful in the diagnosis of BLV infection in cattle. Here we evaluate the potential for using a viral vector based on the genome of Tomato bushy stunt virus (TBSV) for the efficient expression of BLV envelope glycoprotein rgp51 in Nicotiana benthamiana plants. The codon-optimized gene encoding rgp51 was synthesized by the de novo DNA synthesis to replace the GFP gene in the TBSV-derived viral vector that was then delivered into 4-5 week old N. benthamiana plants by agroinfiltration. Expression of recombinant his-tagged rgp51 was verified by protein extraction followed by western blot procedures, and by purification using Ni-affinity chromatography. The molecular weight of this plant-expressed rgp51 ranged from 43 to 55 kDa and it was shown to be glycosylated. Important for potential use in diagnostic tests, purified rgp51 specifically reacted with BLV infected bovine sera while no reaction was observed with the negative serum samples.
植物在相对较短的时间内生产有价值的重组蛋白方面具有独特的优势组合。例如，已经开发出了多种使用植物中表达的重组抗原的诊断测试。牛白血病病毒 (BLV) 编码的包膜糖蛋白 gp51 是病毒感染力的必需亚基之一。有研究表明，BLV 的重组 gp51 (rgp51) 可作为一种有用的合成替代抗原，用于诊断牛 BLV 感染。在这里，我们评估了基于番茄丛矮病毒 (TBSV) 基因组的病毒载体在提高烟草原生质体中 BLV 包膜糖蛋白 rgp51 表达效率方面的潜力。通过从头 DNA 合成合成了经密码子优化的编码 rgp51 的基因，以取代 TBSV 衍生的病毒载体中的 GFP 基因，然后通过农杆菌浸润将其递送至 4-5 周龄的烟草原生质体中。通过蛋白提取和 Western blot 程序以及使用 Ni 亲和层析进行纯化，验证了重组 his 标记的 rgp51 的表达。这种在植物中表达的 rgp51 的分子量范围为 43 至 55 kDa，并且显示出糖基化。对于潜在的诊断测试用途很重要的是，纯化的 rgp51 特异性地与 BLV 感染的牛血清反应，而与阴性血清样本无反应。
