利用B细胞培养和RT-PCR技术从我国HIV-1感染者中筛选膜蛋白特异性单克隆抗体的初步研究  

Identification of Env-specific Monoclonal Antibodies from Chinese HIV-1 Infected Person by B cell Activation and RT-PCR Cloning

作　　者：汪慧敏[1,2] 徐柯[1] 余双庆[1] 丁林林[1] 罗海艳[1] Robin Flinko George K Lewis 冯霞[1] 邵继荣[2] 管永军 曾毅[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京100052 [2]四川农业大学生命科学与理学院,雅安625014 [3]马里兰州立大学人类病毒学研究所

出　　处：《病毒学报》2012年第4期358-365,共8页Chinese Journal of Virology

基　　金：国家重大科技专项(2008ZX10001-009)

摘　　要：本研究通过采集1型人类免疫缺陷病毒(Human immunodeficiency virus-1,HIV-1)感染者抗凝全血,分离出外周血单个核细胞,然后用磁珠分选纯化记忆性B细胞和体外活化记忆性B细胞,促使其分泌抗体,用ELISA法识别阳性B细胞克隆,并提取阳性B细胞的RNA,从中扩增抗体重链和轻链基因并克隆到表达载体中,再用携带重链基因的质粒和携带轻链基因的质粒共转染293T细胞,获得HIV-1特异性人单克隆抗体,进行抗体特性的鉴定。结果从1例HIV-1感染者的记忆性B细胞中筛选出了4株HIV-1包膜糖蛋白(Envelope glycoprotein,Env)特异性人单克隆抗体,其中2株具有较好的抗体依赖细胞介导的细胞毒作用活性,另有1株对HIV-1假病毒有较弱的中和活性。说明我们成功地引进了利用B细胞培养和RT-PCR技术从人体淋巴细胞中筛选特异性抗体基因的人单克隆抗体技术平台。用该技术可以成功获得HIV-1Env特异性单克隆抗体,为将来从能产生高滴度广谱中和抗体的感染者体内筛选广谱中和抗体打下了基础。To obtain protective human monoclonal antibody from HIV-1 infected person, we adapted a technology for isolating antigen specific monoclonal antibody from human memory B ceils through in vitro B cell activation coupled with RT-PCT and expression cloning. Human B cells were purified by negative sorting from PBMCs of HIV-1 infected individuals and memory B cells were further enriched using anti- CD27 microbeads. Two hundred memory B cells per well were cultured in 96-well round-bottom plates with feeder cells in medium containing EBV and CpG. Env-specific antibodies in supernatants were screened by ELISA after 1-2 weeks＇ culture. Cells from positive wells of Env-specific antibody were harvested and total RNA was isolated. Human VH and Vk or Vλ genes were amplified by RT-PCR and cloned into IgG1 and k or λ expressing vectors. Functional VH and Vk or Vλ were identified by cotransfecting 293T cells with individual heavy chain and light chain clones followed by analysis of culture supernatants by ELISA for Envspecifie antibodies, Finally, corresponding mAb was produced by transient transfection of 293T cells with the identified VH and Vk/λ pair and purified by protein A affinity ehromatography. Pu- rified monocolonal antibodies were used for HIV-1 specific antibody-dependent cell-mediated cytotoxicity （ADCC） and neutralizing aetivity assay. Four monoeolonal Env-specific antibodies were isolated from one HIV-1 subtype B＇ infected individual. Two of them showed strong ADCC activity and one showed weak neutralizing activity against HIV-1. Its further studies on their application in therapeutic or prophylactic vaccines against HIV-1 should be grounded.

关 键 词：人类免疫缺陷病毒 单克隆抗体 中和活性 抗体依赖细胞介导的细胞毒作用 

分 类 号：R373.9[医药卫生—病原生物学]