Liu Guozhen, Hu Shaohui, Hu Yongwu, Chen Peng, Yin Jianning, Wen Jie, Wang Jingqiang, Lin Liang, Liu Jinxiu, You Bo, Yin Ye, Li Shuting, Wang Hao, Ren Yan, Ji Jia, Zhao Xiaoqian, Sun Yongqiao, Zhang Xiaowei, Fang Jianqiu, Wang Jian, Liu Siqi, Yu Jun, Zhu Heng, Yang Huanming
College of Life Sciences, Hebei Agricultural University, Baoding 071001, China.
Genomics Proteomics Bioinformatics. 2003 Aug;1(3):193-7. doi: 10.1016/s1672-0229(03)01024-6.
In order to develop clinical diagnostic tools for rapid detection of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.
为了开发用于快速检测严重急性呼吸综合征相关冠状病毒（SARS-CoV）的临床诊断工具，并确定疫苗开发的候选蛋白，利用逆转录聚合酶链反应（RT-PCR）从SARS-CoV基因组中扩增出核衣壳（NC）基因的C末端部分，克隆到酵母表达载体（pEGH）中，并在诱导型启动子的控制下表达为谷胱甘肽S-转移酶（GST）和Hisx6双标签融合蛋白。对纯化蛋白进行的Western分析证实了酵母中NC融合蛋白的表达和纯化。为了确定其抗原性，用SARS患者和正常对照的血清样本对融合蛋白进行检测。NC融合蛋白表现出高抗原性和高特异性，因此，它在设计临床诊断工具方面具有巨大潜力，并可为疫苗开发提供有用信息。
