抗SARS冠状病毒S1蛋白N端249至667的单克隆抗体的制备与.docVIP

抗SARS冠状病毒S1蛋白N端249至667的单克隆抗体的制备与鉴定 作者：温坤1；梅亚波1；丘立文1；廖志勇1；袁国勇2；车小燕1　　(1第一军医大学珠江医院中心实验室，广东?? 广州 510282； 2香港大学微生物学系，香港) 摘要：目的??在获得了具有免疫原性的SARS冠状病毒S1蛋白片段的基础上，制备和鉴定特异性抗该段S1蛋白单克隆抗体(mAb)。方法??原核表达含S蛋白受体结合区的SARS冠状病毒S1蛋白片段S1c(N端249-667氨基酸残基)，其免疫原性经SARS病人恢复期血清鉴定后免疫BALB/c小鼠，按常规方法制备单克隆抗体，并采用ELISA间接法、免疫荧光和免疫印迹进行筛选和鉴定。结果??筛选出3株特异性针对SARS冠状病毒S1蛋白N端249-667的mAb杂交瘤细胞株，IgG亚类鉴定1株为IgG1，2株为IgG2a，经免疫荧光鉴定与人冠状病毒株229E和OC43无交叉反应。结论??获得3株抗SARS冠状病毒S蛋白受体结合区特异性单克隆抗体，为建立新的SARS冠状病毒检测方法的和进一步研究S蛋白的功能奠定了基础。 关键词：SARS冠状病毒；单克隆抗体；刺突蛋白；受体结合区 Preparation and characterization of monoclonal antibodies against S1 domain at N-terminal residues 249 to 667 of SARS-associated coronavirus S1 proteinWEN Kun1； MEI Ya-bo1； QIU Li-wen1； LIAO Zhi-yong1； Yuen Kwok-yung2； CHE Xiao-yan11Central Laboratory, Zhujiang Hospital, First Military Medical University, Guangzhou 510282, China; 2Department of Microbiology, University of Hong Kong, Hong Kong, China.Abstract: Objective??To prepare and characterize monoclonal antibodies (mAbs) against S1 protein of severe acute respirato-ry syndrome (SARS)-associated coronavirus (SARS-CoV). Methods??6-His-tagged recombinant fragment at N-terminal residues 249 to 667 of SARS-CoV S1 protein including S-protein receptor-binding domain was expressed in E.coli. The im-munogenicity of this S1 domain was identified and used to immunize BALB/c mice for the production of hybridomas. The identification of the mAbs against this S1 domain was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA) and Western blotting, respectively. Results??Three hybridomas producing mAbs spe-cific to the S1 domain was obtained, with a relative molecular mass of 48 500. None of the 3 mAbs were reactive with human coronaviruses 229E and OC43. Two of the mAbs were IgG2a isotype, and the other was IgG1. Conclusion??This is the first re-port of mAbs produced against S-protein receptor-binding domain of SARS-CoV. The 3 S1-specific mAbs may be useful for further