小鼠抗中东呼吸综合征冠状病毒刺突蛋白受体结合区单克隆抗体的筛选  

Screening of mouse-derived monoclonal antibodies against the receptor binding domain of Middle East respiratory syndrome coronavirus （MERS-CoV） spike protein

作　　者：王慧娟[1] 王文玲[1] 蓝佳明[1] 邓瑶[1] 谭文杰[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,卫生和计划生育委员会医学病毒学与病毒病重点实验室,北京102206

出　　处：《中华微生物学和免疫学杂志》2016年第2期88-92,共5页Chinese Journal of Microbiology and Immunology

摘　　要：目的制备抗中东呼吸综合征冠状病毒（Middle East respiratory syndromecoronavirus，MERS—CoV）刺突蛋白（s）受体结合区（receptor binding domain，RBD）的单克隆抗体（单抗），并对其特性进行鉴定。方法将昆虫-杆状病毒表达的MERS—CoVS蛋白RBD片段纯化后免疫BALB／c小鼠，取小鼠脾细胞与骨髓瘤细胞Sp2／0融合，通过有限稀释法筛选阳性克隆，用酶联免疫吸附试验（ELISA）、免疫印迹法（Westernblot）和MERS—CoV假病毒中和试验对所获得的细胞克隆进行筛选和鉴定。结果共筛选出12株杂交瘤细胞，ELISA证实其分泌的单抗与MERS—CoVS蛋白RBD片段有特异反应；双抗体夹心法鉴定其中两株单抗2E4和2F3为免疫球蛋白IgM型，其余10株均为IgGl型；MERS—CoV假病毒中和试验结果显示4株单抗：1F1、2E4、3C3、3E6有中和活性；Westernblot分析表明单抗1F1、2E4、3C3、3E6能特异识别MERS—CoVS蛋白，而不与SARS—CoVS蛋白的RBD反应。结论我们制备了12株针对MERS-CoYs蛋白RBD的鼠源单抗，所获杂交瘤细胞株特异性强，分泌抗体性能稳定，其中4株具有中和活性。Objective To prepare and screen out monoclona] antibodies against the receptor bind- ing domain （ RBD ） of Middle East respiratory syndrome coronavirus （ MERS-CoV ） spike （ S ） protein in mice. Methods The RBD of MERS-CoV S protein expressed in the inseet-baculovirus system was purified and then used to immunize the female BALB/c mice. The spleen cells collected from the mice were fused with myeloma Sp2/0 cells. The positive hybridoma cells were obtained by using limited dilution method. Enzyme-linked immunosorbent assay （ELISA）, Western blot assay and neutralization test based on the MERS-CoV pseudovirus were performed for further screening and identification. Results Twelve strains of hybridoma cells that produced the monoclonal antibodies against RBD of MERS-CoV S protein were screened out. All of the 12 monoclonal antibodies （McAbs） could have specific reaction with the RBD of MERS-CoV S protein as indicated by the results of ELISA. Of the 12 MeAbs, two were identified as the immunoglobulin M （IgM） isotype and the rest were lgG1 isotype by using double antibodies sandwich ELISA. Four McAbs including 1F1, 2FA, 3C3 and 3E6 were identified as having neutralizing activity by the neutralization test based on MERS-CoV pseudovirus. Results of the Western blot assay showed that the four McAbs （ 1F1, 2FA, 3C3 and 3E6） could have specific reaction with the RBD of MERS-CoV S protein, but no cross-reaction with that of SARS-CoV S protein. Conclusion Twelve mouse-derived MeAbs against the RBD of MERS-CoV S protein were obtained. The prepared hybridoma cells showed the characteristics of high speci- ficity and stability in antibody secretion. Four out of the 12 MeAbs were proved to have neutralizing activity.

关 键 词：中东呼吸综合征 冠状病毒 S蛋白 RBD 单克隆抗体 

分 类 号：R373.1[医药卫生—病原生物学]