摘要
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 aim of this study is to construct a SARS-CoV S protein-specific phage displayed antigen li- brary for the epitope characterization of anti-S monoclonal antibodies （mAbs）. First, the full-length gene of SARS-S protein was PCR amplified, purified and then digested with DNase I to obtain DNA fragments in the size range of 50-500 bp. The resulting fragments were blunt-end ligated to the modified phage dis- play vector pComb3XSS. The reactions were electrotransformed into XL1-Blue and infected with VCSM13 helper phage. The SARS-CoV S protein-specific phage displayed antigen library was biopanned and screened against two anti-S mAbs, S-M1 and S-M2. The results showed that we successfully constructed the phage displayed antigen library with a size of 5.7× 106. After three-rounds of biopanning, 14 positive phage clones for S-M1 and 15 for S-M2 were respectively identified. Sequence analyses revealed the possi- ble epitopes of two mAbs. Therefore, the S protein-specific phage displayed antigen library provides a cru- cial platform for the epitope characterization of anti-S antibodies and it is highly valuable for development of SARS vaccines and diagnostics.
出处
《病毒学报》
CAS
CSCD
北大核心
2013年第3期280-286,共7页
Chinese Journal of Virology
基金
国家十二五艾滋病和病毒性肝炎等重大传染病防治专项(2012ZX10001-008)
国家杰出青年基金(81025009)
