Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, Osong Health Technology Administration Complex, Cheongwon-gun, Chungbuk 363-951, South Korea.
Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, Osong Health Technology Administration Complex, Cheongwon-gun, Chungbuk 363-951, South Korea.
J Virol Methods. 2014 Mar;197:55-62. doi: 10.1016/j.jviromet.2013.11.015. Epub 2013 Dec 16.
Recombinant measles virus nucleoproteins (rMeV N) and fusion (F) proteins were characterized as major antigenic proteins expressed in insect cells mediated by recombinant baculoviruses (rBVs). Band intensities were analyzed by Western blotting to recognize IgG and IgM antibodies against the rMeV N and F proteins in human sera and cerebrospinal fluids (CSFs) from patients with measles infections. Positive results from the blots using the rMeV N were consistent with the results of enzyme-linked immunosorbent assays (ELISAs) in which whole viral proteins were used as antigens. Human sera and CSFs reacted more strongly with the rMeV N than with the rMeV F proteins prepared in an identical expression system. For efficient and reliable purification, ion-exchange chromatography using Source Q anion resin was applied, and high-purity rMeV N protein was harvested. To characterize the similarity with the native viral protein to purified N protein, structural mimicry of purified recombinant proteins with intact rMeV N was shown through transmission electron microscopy, and the truncation and the phosphorylation status of the expressed protein were analyzed. These results suggest that the rMeV N purified by ion-exchange chromatography has features similar to those of naïve N including a self-assembled structure, phosphorylation and antigenic function. Thus, these expression and purification methods can be applied to the large-scale production of the rMeV N, which is essential for the development of new diagnostic tools and vaccines for acute and chronic MeV infections.
重组麻疹病毒核蛋白（rMeV N）和融合（F）蛋白是由重组杆状病毒（rBVs）介导的昆虫细胞中表达的主要抗原蛋白。通过 Western blot 分析条带强度，以识别人类血清中和麻疹感染患者脑脊液（CSF）中针对 rMeV N 和 F 蛋白的 IgG 和 IgM 抗体。使用 rMeV N 的印迹的阳性结果与使用整个病毒蛋白作为抗原的酶联免疫吸附测定（ELISA）结果一致。人血清和 CSF 与在相同表达系统中制备的 rMeV F 蛋白相比，与 rMeV N 反应更强。为了实现高效可靠的纯化，应用了使用 Source Q 阴离子树脂的离子交换色谱法，并收获了高纯度的 rMeV N 蛋白。为了表征与天然病毒蛋白的相似性，通过透射电子显微镜显示了纯化的重组蛋白与完整 rMeV N 的结构模拟，分析了表达蛋白的截短和磷酸化状态。这些结果表明，通过离子交换色谱法纯化的 rMeV N 具有与天然 N 相似的特征，包括自组装结构、磷酸化和抗原功能。因此，这些表达和纯化方法可应用于 rMeV N 的大规模生产，这对于开发用于急性和慢性 MeV 感染的新诊断工具和疫苗至关重要。
