利用单个B细胞分选方法获得HIV-1单克隆抗体基因及功能鉴定  

Single B cell sorting-based amplification and functional identification of HIV-1 monoclonal antibody genes

作　　者：鞠斌 郝彦玲[2] 李丹[2] 任莉[2] 梁华[2] 王硕[2] 侯佳利[2] 魏民[1] 邵一鸣[1,2] Ju Bin;Hao Yanling;Li Dan;Ren Li;Liang Hua;Wang Shuo;Hou Jiali;Wei Min;Shao Yiming(School of Medicine,Nankai University,Tianfin 300071,China;State Key Laboratory of Infectious Disease Prevention and Control,National Center for AIDS/STD Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]南开大学医学院,天津300071 [2]中国疾病预防控制中心性病艾滋病预防控制中心传染病预防控制国家重点实验室,北京102206

出　　处：《中华微生物学和免疫学杂志》2018年第7期481-487,共7页Chinese Journal of Microbiology and Immunology

基　　金：博士后创新人才支持计划（BX201600078）;国家自然科学基金项目（81571991,31600734）;传染病预防控制国家重点实验室项目（2016SKLID602）

摘　　要：目的从人类免疫缺陷病毒1型（human immunodeficiency virus-1，HIV-1）感染者中获得单克隆抗体基因及功能鉴定。方法单细胞流式分选得到抗原特异性单个B细胞，基因扩增和测序得到抗体可变区序列，BioEdit软件包中Sequence Identity Matrix程序分析基因序列一致性，免疫遗传学数据库分析抗体基因家系和突变率，酶联免疫吸附试验测定抗体结合能力，TZM-bl/假病毒试验检测抗体中和能力。结果从3个HIV-1感染者中分别获得了4个、7个和11个不同抗体的重链和轻链基因序列，其基因家系、CDR3长度和突变率广泛多样；验证了一个感染者的4个抗体基因序列，抗体A1和B3是HIV-1单克隆结合抗体，能同时与B亚型、CRF01_AE和CRF07_BC抗原蛋白结合；A1和B3也是HIV-1单克隆中和抗体，能够中和C亚型的MW965病毒，50%抑制浓度分别为0.04 μg/ml和37.34 μg/ml。结论本研究成功获得了大量的单克隆抗体基因，并对其中的部分序列进行了功能验证，为单克隆抗体的分离和鉴定等研究工作提供了基础。ObjectiveTo amplify and identify monoclonal antibody genes from HIV-1-infected patients.MethodsSingle cell sorting was used to isolate antigen-specific single B cells. Sequence Identity Matrix and the international ImMunoGeneTics information system were used to analyze antibody variable region genes. Binding abilities were detected by enzyme linked immunosorbent assay. Neutralizing activities were tested by TZM-bl/pseudovirus assay.ResultsThe heavy and light chain genes of four, seven, and eleven antibodies were amplified and sequenced from three HIV-1-infected patients, respectively. They were derived from various germline genes with flexible CDR3 lengths and somatic mutations. A1 and B3 antibodies bound to HIV-1 clade B, CRF01_AE, and CRF07_BC antigens. The half maximal inhibitory concentration values of A1 and B3 against MW965 virus were 0.04 μg/ml and 37.34 μg/ml.ConclusionIn this study, we acquired a lot of monoclonal antibody genes and two HIV-1 monoclonal binding and neutralizing antibodies, which would provide basic data for further research on monoclonal antibody identification.

关 键 词：单个B细胞分选 抗体基因 序列分析 结合能力 中和活性 

分 类 号：R392[医药卫生—免疫学]