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1、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
2、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
3、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.Abstrac
4、t: ObjectiveTo prepare and characterize monoclonal antibodies (mAbs) against S1 protein of severe acute respirato-ry syndrome (SARS)-associated coronavirus (SARS-CoV). Methods6-His-tagged recombinant fragment at N-terminal residues 249 to 667 of SARS-CoV S1 protein including S-protein receptor-bindi
5、ng 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 immunofluo
6、rescence assay (IFA) and Western blotting, respectively. ResultsThree 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 othe
7、r was IgG1. ConclusionThis 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 study of the function of the S protein and for diagnosis of SARS-CoV infection.Key words: severe acute respiratory syndrome-associa
8、ted coronavirus; SARS-CoV; monoclonal antibody; spike glycoprotein; receptor-binding domainReceived: 2004-01-03This work is supported as the Key Medical Research Project for SARS Prevention sponsored by the Ministry of Science and Technology of China and by Guangdong ProvinceCorresponding author: CH
9、E Xiao-yan, M.D., Professor in the Central Laboratory of Zhujiang Hospital，Tel: 86-20-61643592, Fax: 86-20-61643592, E-mail: , The two authors, WEN Kun and MEI Ya-bo, have made equal contribu-tions to this work. A novel coronavirus has been identified as the ma-jor cause of sever acute respiratory s
10、yndrome (SARS)123, but the biological functions of this SARS-associated coronavirus (SARS-CoV) remain currently poorly char-acterized. Recent studies reported that angiotensin-con-verting enzyme 2 (AEC2) could be a functional receptor for SARS-CoV and the receptor-binding domain (RBD) was identified
11、 in the N-terminal residues 17 to 274 of the S1 spike protein of the virus 4. Subsequent studies demonstrated that the RBD was located in the N-termi-nal residues 303 to 5375 or residues 318 to 5106. The S1 protein is also found to be a target for inducing neu-tralizing antibody responses to SARS-Co
12、V infection7. In spite of the fact that the function of S protein is not fully understood, S1 protein seems to play a key role in the initial virus infection. Based on this understanding, the monoclonal antiodies(mAbs) targeted at S1 domain can be a potentially useful tool for the study of the func-
13、tion of S protein and for the diagnosis of SARS. Fur-thermore, the availability of blocking antibodies may be meaningful in the treatment of SARS.In this paper, we report the cloning, expression and antigenic characterization of the various fragments of S1 domain as well as the preparation and chara
14、cteriza-tion of mAbs against the recombinant protein S1c de-rived from the S1 protein containing the N-terminal residues 249 to 667. MATERIALS AND METHODSVirus and cell linesHuman coronavirus strains 229E (No. VR740), OC43 (No. VR759) and cell lines Vero E6 (No. CRL-1586), MRC-5 (No. CCL-171), BS-C-
15、1 (No. CCL-26) were all purchased from American Type Culture Collection (ATCC).PlasmidsThe plasmid encoding S1 protein gene, pET-28b (+)/S1 (40-2 001 bp, HKU-39849), was kindly provid-ed by the Department of Microbiology, University of Hong Kong. The expression vector pQE-30 and M15 strain of Escher
16、ichia coli were purchased from QIA-GEN. ReagentsThe restriction endonucleases BamHI and HindIII were purchased from New England Biolabs, and KpnI, PstI, ExTaq, dNTP and DNA ladder were from TaKaRa. Ni resin was obtained from QIAGEN. Freunds adju-vant, 50% PEG 1450 solution, HAT and HT were pur-chased from Sigma Aldrich. Goa
