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本研究旨在构建SARS冠状病毒S蛋白特异性噬菌体抗原库,并用于鉴定抗S蛋白单克隆抗体的抗原表位。首先采用PCR技术扩增出SARS冠状病毒S蛋白的全基因,以DNaseI将其随机酶切成50～500bp不同大小的DNA片段。然后将DNA片段平末端化并连接到经过改造的噬菌体表达载体pComb3XSS,经电转化大肠杆菌XL1-Blue和辅助噬菌体感染获得S蛋白的特异性噬菌体抗原库。利用两个抗S蛋白单克隆中和抗体(S-M1和S-M2)对S蛋白抗原库进行富集和筛选。结果表明,我们成功构建库容量为5.7×106的S蛋白噬菌体抗原库。通过对S-M1和S-M2的有效富集和筛选,分别得到14个和15个阳性克隆,序列分析初步揭示了抗体的抗原表位。因此,S蛋白噬菌体抗原库的构建为鉴定S蛋白的抗原表位提供了重要的技术平台,对研发SARS疫苗和诊断试剂具有重要的科学意义和应用价值。
The purpose of this study was to construct SARS-CoV S protein-specific phage antigen library and to identify the epitope of anti-S protein monoclonal antibody. Firstly, the whole gene of S protein of SARS coronavirus was amplified by PCR and randomly digested with DNaseI into DNA fragments of 50 ~ 500bp in size. The DNA fragments were then blunt-ended and ligated into the engineered phage expression vector pComb3XSS to obtain a specific phage antigen pool of S protein by electroporation of E. coli XL1-Blue and helper phage infections. S protein antigen pool was enriched and screened using two anti-S protein monoclonal neutralizing antibodies (S-M1 and S-M2). The results showed that we successfully constructed the S protein phage antigen library with a capacity of 5.7 × 106. Through the enrichment and screening of S-M1 and S-M2, 14 and 15 positive clones were obtained respectively, and the sequence analysis revealed the epitope of the antibody. Therefore, the construction of the S protein phage antigen library provides an important technical platform for identifying the antigenic epitopes of the S protein, and has important scientific significance and application value for the research and development of SARS vaccines and diagnostic reagents.