抗SARS-CoV N蛋白单克隆抗体的特异性及其识别抗原表位的初步鉴定

为了明确抗SARS-CoVN蛋白单克隆抗体的特异性,并鉴定其识别表位,首先在E.coli中表达了人类冠状病毒229E(HCoV-229E)和OC43(HCoV-OC4)N蛋白,用Westernblotting和间接免疫荧光方法分别检测了4株抗SARS-CoVN蛋白单克隆抗体(1-1C2、1-1D6、2-8F11和2-2E5)与HCoV-OC43和HCoV-229E及其N蛋白的交叉反应情况,而后应用12种重组截短型SARS-CoVN蛋白对上述4种单克隆抗体的识别表位进行了初步定位。结果显示:(1)在4株抗N蛋白单克隆抗体中,1-1C2、1-1D6和2-2E5不与HCoV-OC43和HCoV-229E及其N蛋白发生交叉反应,为SARS-CoVN蛋白特异性抗体;(2)2-8F11、1-1D6和2-2E5针对的抗原表位位于SARS-CoVN蛋白的aa30-60,1-1C2针对的抗原表位则位于SARS-CoVN蛋白的aa170-184。这一研究为阐明SARS-CoVN蛋白的免疫学特征,建立特异性免疫诊断技术和研究其致病机制提供了必要的依据和材料。

Specificity and Epitope Mapping of Four Monoclonal Antibodies against SARS-CoV Nucleocapsid Protein

In order to characterize the specificity of the monoclonal antibodies (McAbs) against SARS- associated coronavirus (SARS-CoV) nucleocapsid protein and to identify the epitopes recognized by the McAbs,the nucleocapsid proteins of human coronavirus OC43 (HCoV-OC43) and 229E (HCoV-229E) was expressed in E.coli. The specificities of four McAbs (1-1C2, 2-2E5, 1-1D6, and 2-8F11) were examined by Western blotting as well as indirect fluorescence assay. Twelve different recombinant truncated N proteins were then used to identify the epitopes recognized by the McAbs by Western blotting. The results showed that: (1) McAbs 1-1C2, 2-2E5, and 1-1D6 recognized neither human coronviruses HCoV-OC43 and HCoV-229E nor their nucleocapsid proteins, implying a possible specificity of these 3 McAbs to SARS-CoV; (2)The epitopes recognized by McAbs 2-8F11, 2-2E5, and 1-1D6 were located between the 30th and 60th amino acid (a.a.) residues of the SARS-CoV N protein, while the epitopes recognized by the McAb1-1C2 were located between 170th and 184 th a.a. residues. The identification of the specific epitopes of SARS-CoV N protein is paramount for the immunological characterization, the development of accurate immunological assay as well as for exploring the pathogenesis of SARS-CoV.