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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。
In order to clarify the specificity of anti-SARS-CoVN monoclonal antibody and to identify its epitopes, human coronavirus 229E (HCoV-229E) and OC43 (HCoV-OC4) N protein were first expressed in E.coli and expressed by Western blotting And indirect immunofluorescence were used to detect the cross-reactivity of four anti-SARS-CoVN monoclonal antibodies (1-1C2, 1-1D6, 2-8F11 and 2-2E5) with HCoV-OC43 and HCoV-229E and their N proteins After that, 12 kinds of recombinant truncated SARS-CoVN proteins were used to locate the epitopes of the above four kinds of monoclonal antibodies. The results showed that: (1) 1-1C2, 1-1D6 and 2-2E5 did not cross-react with HCoV-OC43 and HCoV-229E and their N protein among the four anti-N monoclonal antibodies, which were SARS-CoVN protein (2) The antigenic epitopes against 2-8F11, 1-1D6 and 2-2E5 are located on aa30-60 of SARS-CoVN protein and the epitopes against 1-1C2 of SARS-CoVN protein are located on the aa170- 184. This study provided the necessary basis and material for elucidating the immunological characteristics of SARS-CoVN protein, establishing specific immunodiagnostic techniques and studying its pathogenesis.