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刺突蛋白(S)和核心蛋白(N)是SARS冠状病毒的主要结构蛋白.在病毒细胞受体结合和病毒包装过程起重要作用.重组融合表达这2种蛋白具有较高的诊断学价值.对SARS病毒N蛋白和S蛋白氨基酸序列进行计算机分析,选择含有优势抗原表位的N蛋白1～227位氨基酸片段和S蛋白450～650位氨基酸片段,采用序列重叠延伸策略(sequenceoverlappingextension,SOE)构建编码N1227LinkerS450650新型融合蛋白的基因片段,导入原核表达载体,实现融合蛋白在大肠杆菌的高效表达.利用组氨酸标签亲和层析的方法纯化,获得高纯度的融合蛋白.对该融合蛋白的结构特征模拟分析的结果显示,其免疫化学性质均无显著改变.采用ELISA和Western印迹方法对其识别SARS冠状病毒特异性抗体的能力进行初步鉴定,显示该融合蛋白具有较好的抗原性和特异性,可有效特异性地检测恢复期SARS病人血清中抗SARS冠状病毒结构蛋白的抗体,可以作为SARS冠状病毒感染的辅助诊断手段.
The spike protein (S) and core protein (N) are the major structural proteins of SARS-CoV, and play an important role in virus cell receptor binding and viral packaging. Recombinant fusion expression of these two proteins has high diagnostic value. The amino acid sequences of N protein and S protein of SARS virus were analyzed by computer. The amino acid fragment of amino acids 1-227 and the amino acid fragment of 450-650 of S protein containing the dominant epitopes were selected and sequenced using sequence overlap extension (SOE) The gene fragment encoding the novel fusion protein N1227LinkerS450650 was introduced into the prokaryotic expression vector to achieve high expression of the fusion protein in E. coli and purified by histidine tag affinity chromatography to obtain high purity fusion protein.The structure of the fusion protein The results of the characteristic simulation analysis showed that there was no significant change in the immunochemical properties of the SARS coronavirus, and the preliminary identification of its ability to recognize SARS-CoV-specific antibodies by ELISA and Western blotting showed that the fusion protein had good antigenicity and specificity , Which can effectively and specifically detect the anti-SARS coronavirus structural protein in serum of convalescent SARS patients The antibody can be used as means of diagnosis of SARS coronavirus infection.