论文部分内容阅读
参照已发表的SARS冠状病毒BJ0 1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抗体相对应的抗原表位进行了初步鉴定。结果发现单克隆抗体D3D1特异性识别S2基因表达产物 (S蛋白的 4 4 7 4 5 8aa) ,D3C5特异性识别S5基因表达产物 (S蛋白的 789 799aa)。
With reference to the published sequence of one strain of SARS coronavirus BJ0, the predominant regions of antigenicity of three main structural proteins of S, M and N of the virus were predicted and selected by using computer software to synthesize two segments of Gly-Pro-Gly sequences Chimeric genes A and B. And were respectively cloned into pGEX-6p-1 vector and induced with IPTG. The purified chimeric proteins A and B were used as antigen to immunize BALB c mice respectively to prepare monoclonal antibodies. Subtypes and specificities were identified using the monoclonal antibody subtype detection kit and the SARS CoV commercial ELISA kit. The results showed that the two chimeric gene products were fused and expressed in the sizes of 34 kD and 35 kD respectively. Western blot analysis confirmed that both expressed products were recognized by the convalescent sera of SARS patients. Six positive clones that can stably secrete specific antibodies were obtained. The result of subtype identification was IgG1 except that D3C5 was IgG2a, and the light chains of all McAbs were all κ chain. Specific identification found that in addition to D3D1, the remaining five monoclonal antibodies were able to react specifically with the SARS CoV commercial ELISA kit. Western blot analysis of the D3D1 and the sonicated SARS CoV after inactivation revealed that it specifically recognizes 180 kD protein bands. The oligopeptides (S1-S6) expressing six S proteins were fused respectively, and the epitopes corresponding to the selected monoclonal antibodies were preliminarily identified. The results showed that the monoclonal antibody D3D1 specifically recognized the S2 gene expression product (447445aa) of S protein and D3C5 specifically recognized the S5 gene expression product (789779aa of S protein).