SARS-CoV单克隆抗体的制备及抗原表位的初步鉴定

参照已发表的SARS冠状病毒BJ01株基因序列 ,利用计算机软件预测并选取该病毒S、M、N三种主要结构蛋白部分抗原性优势区域 ,以编码Gly-Pro-Gly序列相连接合成两段嵌合基因A和B。并分别克隆于pGEX -6p- 1载体上用IPTG进行诱导表达 ,以纯化的嵌合蛋白A和B为抗原 ,分别免疫BALB c小鼠制备单克隆抗体。利用单克隆抗体亚型检测试剂盒和SARS CoV商品化ELISA检测试剂盒对其进行亚型和特异性鉴定。结果表明融合表达两段嵌合基因产物 ,其大小分别为 34kD和35kD ,Westernblot分析证实两种表达产物都能被SARS病人康复期血清所识别。获得了 6株能稳定分泌特异性抗体的阳性细胞克隆株。亚型鉴定结果除D3C5为IgG2a外其他单抗均为IgG1,而且所有单抗的轻链均为κ链。特异性鉴定发现除D3D1外 ,其余的 5株单抗均能与SARS CoV商品化ELISA检测试剂盒发生特异性反应。将D3D1与灭活后经超声波裂解的SARS CoV进行Westernblot分析 ,发现它能特异性识别 180kD的蛋白带。分别融合表达了 6个S蛋白的寡肽 (S1- S6 ) ,并对筛选出的单克隆…

Development of Monoclonal Antibodies Against SARS CoV and Identification of Antigenic Epitopes

Based on the genomic sequence of SARS-CoV strain BJ101, antigenic immu nodominant genes coding for the structure proteins of SARS-CoV were predicted b y bio-informatics methods, and two chimeric genes A and B with multi-immunodomi na nts lined up by Gly-Pro-Gly linker were synthesized. The chimeric genes were c lo ned into plasmid pGEX-6p-1 and expressed in E.coli with IPGT inducing. BAL B/c m ice were immunized with the purified recombinant fusion protein. The specificity of monoclonal antibodies were tested with a commercial ELISA kit for detecting antibody against SARS-CoV. The results showed that two peptides with molecular w eights of 34kD and 35kD expressed by the two chimeric genes could be recognized by SARS patient convalescent serum in Western blot. Six positive hybridoma cell lines stably secreting monoclonal antibodies were selected. The subtype of monoc lonal antibody D3C5 is IgG2a, and subtypes of all other five monoclonal antibodi es are IgG1. Light chains of all monoclonal antibodies are kappa. With a commer c ial SARS-CoV antibodies detection ELISA kit, five out of six monoclonal antibod i es were positively recognized. In western blot analysis with inactived virus cul tures, D3D1 specifically recognized a band of about 180 kD. To further analyse t he epitopes corresponding to the monoclonal antibodies, six oligoes (S1-S6) fro m S gene were synthesized and expressed. The results showed that the monoclonal a ntibodies D3D1 and D3C5 specifically recognized expression product of S2 and S5 oligoes, respectively. The S2 and S5 oligoes are corresponding to 447-458aa and 789-799aa of SARS-CoV S protein respectively.