Slibinskas Rimantas, Samuel Dhanraj, Gedvilaite Alma, Staniulis Juozas, Sasnauskas Kestutis
Institute of Biotechnology, Vilnius LT-2028, Graiciuno, Lithuania.
J Biotechnol. 2004 Jan 22;107(2):115-24. doi: 10.1016/j.jbiotec.2003.10.018.
The development of a simple, efficient and cost-effective system for generation of measles virus nucleoprotein might help to upgrade reagents for measles serology. The gene encoding measles nucleoprotein was successfully expressed in two different yeast genera, Pichia pastoris and Saccharomyces cerevisiae, respectively. Both yeast genera synthesized a high level of nucleoprotein, up to 29 and 18% of total cell protein, in P. pastoris and S. cerevisiae, respectively. This protein is one of most abundantly expressed in yeast. After purification nucleocapsid-like particles (NLPs) derived from both yeast genera appeared to be similar to those detected in mammalian cells infected with measles virus. A spontaneous assembly of nucleoprotein into nucleocapsid-like particles in the absence of the viral leader RNA or viral proteins has been shown. Compartmentalisation of recombinant protein into large compact inclusions in the cytoplasm of yeast S. cerevisiae by green fluorescence protein (GFP) fusion has been demonstrated. Sera from measles patients reacted with the recombinant protein expressed in both yeast genera and a simple diagnostic assay to detect measles IgM could be designed on this basis.
开发一种简单、高效且经济高效的麻疹病毒核蛋白生成系统，可能有助于升级麻疹血清学检测试剂。编码麻疹核蛋白的基因分别在两种不同的酵母属，即巴斯德毕赤酵母和酿酒酵母中成功表达。两种酵母属均合成了高水平的核蛋白，在巴斯德毕赤酵母和酿酒酵母中分别高达总细胞蛋白的29%和18%。这种蛋白是酵母中表达量最高的蛋白之一。纯化后，来自两种酵母属的核衣壳样颗粒（NLP）似乎与在感染麻疹病毒的哺乳动物细胞中检测到的颗粒相似。已证明在没有病毒前导RNA或病毒蛋白的情况下，核蛋白能自发组装成核衣壳样颗粒。通过绿色荧光蛋白（GFP）融合，已证明重组蛋白在酿酒酵母细胞质中被分隔成大的致密包涵体。麻疹患者的血清与两种酵母属中表达的重组蛋白发生反应，并可在此基础上设计一种简单的检测麻疹IgM的诊断方法。
