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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。
In this study, peripheral blood mononuclear cells were isolated from anticoagulant whole blood collected from HIV-1 infected persons and then purified by magnetic beads to purify memory B cells and activated in vitro Memory B cells to promote the secretion of antibodies, ELISA positive B cell clones identified and positive RNA extracted from B cells from which to amplify antibody heavy and light chain genes and cloned into the expression vector, and then carry heavy chain gene 293T cells were co-transfected with plasmids carrying light chain genes and plasmids carrying light chain genes to obtain HIV-1 specific human monoclonal antibodies for antibody characterization. Results Four human HIV-1 envelope glycoprotein (Env) specific human monoclonal antibodies were screened from memory B cells of one HIV-1 infected patient. Two of them had better antibody-dependent cells Mediated cytotoxic activity, while another strain has weaker neutralizing activity against HIV-1 pseudoviruses. Indicating that we successfully introduced a human monoclonal antibody technology platform for screening specific antibody genes from human lymphocytes using B cell culture and RT-PCR technology. HIV-1 Env-specific monoclonal antibodies can be successfully obtained by this technique, which will lay the foundation for the future screening of broad-spectrum neutralizing antibodies in those infected with high-titer and broad-spectrum neutralizing antibodies.