利用磁珠分选和单细胞RT—PCR筛选HIV-1Env特异性单克隆抗体的研究  

Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by magnetic beads separating B cells and single cell RT-PCR cloning

作　　者：黄湘滢[1] 余双庆[2] 程湛[1] 叶景荣[2] 徐柯[2] 冯霞[2] 曾毅[1] 

机构地区：[1]北京工业大学生命科学与生物工程学院,100124 [2]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

出　　处：《中华实验和临床病毒学杂志》2013年第2期123-125,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项课题：艾滋病治疗型疫苗研制（2012ZX10001005）

摘　　要：目的建立一种简单易行的从HIV-1感染者中筛选包膜糖蛋白（envelope glycoprotein，Env）特异性单克隆抗体的方法。方法采集HIV-1感染者抗凝全血，分离外周血单个核细胞，利用生物素标记的HIVGp120抗原与B细胞膜上的IgG特异性结合，然后用链霉亲和素标记的磁珠分选出能够产生Env特异性抗体的记忆性B细胞。用单细胞RT-PCR法从记忆性B细胞中扩增抗体的重链可变区基因与轻链可变区的基因并克隆到表达载体中，将携带重链基因的质粒与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，并进行抗体结合活性的鉴定。结果从1例我国HIV-1感染者记忆性B细胞中筛选出3株对HIV-1Env抗原有结合活性的单克隆抗体。结论利用生物素标记抗原与记忆性B细胞特异结合，并用亲和素标记的磁珠对细胞进行分选，结合单细胞RT-PCR技术可以成功筛选出抗原特异性的单克隆抗体。Objective To establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals. Methods Human B cells were purified by negative sorting from PBMCs and memory B cells were further enriched using anti-CD27 microbeads. Gpl20 antigen labbled with biotin was incubated with memory B cells to specifically bind IgG on cells membrane. The memory B cells expressing the Env-specific antibody were harvested by magnetic beads separating, counted and diluted to the level of single cell in each PCR well that loading with catch buffer containing RNase inhibitor to get RNAs. The antibody genes were amplified by single cell RT-PCR and nested PCR, cloned into eukaryotic expression vectors and transfected into 293T cells. The binding activity of recombinant antibodies to Env were tested by ELISA. Results Three monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual. Conclusion We can obtain Env-specific antibody by biotin labbled antigen, magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.

关 键 词：HIV-1 抗原 GP120 抗体 单克隆 聚合酶链反应 

分 类 号：R3[医药卫生—基础医学]