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目的通过抗原特异性记忆B细胞(Memory B)分选、抗体基因克隆表达和鉴定方法,从艾滋病病毒1型(HIV-1)感染者中获得HIV-1中和抗体,并进一步探讨获得抗体的特征。方法通过检测血浆中和抗体活性和表位筛选,挑选出1例含有CD4结合位点(CD4bs)特异性抗体的慢性HIV-1感染者。采用一对含有/缺失CD4-bs的探针RSC3/ΔRSC3进行Memory B分选,获得抗体可变区基因后进行抗体的表达纯化,并检验所得抗体的结合能力和中和能力。结果从9×106外周血单核淋巴细胞(PBMCs)中分离得6个特异性Memory B。通过反转录聚合酶链反应(RT-PCR)分别扩增出3对重链和轻链可变区基因,配对得到3个抗体,其中有一个抗体具有HIV-1CD4bs结合能力。中和实验表明,这个抗体能中和毒株SF162[50%抑制浓度(IC50=0.89μg/mL]。抗体可变区基因家系分析表明,该抗体属于IGHV1-18家系,重链可变区(VH)自体突变率为12%,低于广谱中和抗体VRC01VH的自体突变率(32%)。结论建立的单克隆特异性Memory B分选方法,可以获得有中和能力的抗体。该平台有希望获得具有我国自主知识产权的广谱中和抗体,并为开发抗体药物和设计新型免疫原提供技术支持。
OBJECTIVE: To obtain HIV-1 neutralizing antibodies from HIV-1 infected patients through the selection of antigen-specific memory B (B) and the cloning and expression of antibody genes. feature. Methods One patient with chronic HIV-1 infection with CD4-specific antibody was selected by detecting plasma neutralizing antibody activity and epitope screening. Memory B was sorted by using a pair of probes RSC3 / ΔRSC3 containing / missing CD4-bs. After antibody variable region genes were obtained, the antibodies were expressed and purified, and the binding ability and neutralization ability of the obtained antibodies were examined. Results Six specific Memory Bs were isolated from 9 × 106 peripheral blood mononuclear lymphocytes (PBMCs). Three pairs of heavy chain and light chain variable region genes were amplified by reverse transcription-polymerase chain reaction (RT-PCR). Three antibodies were obtained by pairing. One of them had HIV-1CD4bs binding ability. Neutralization experiments showed that this antibody neutralized the SF162 [50% inhibitory concentration (IC50 = 0.89μg / mL] .Analysis of antibody variable region gene families showed that the antibody belongs to the IGHV1-18 family and the heavy chain variable region VH) was 12%, which was lower than that of broad-band neutralizing antibody VRC01VH (32%) .Conclusion The established monoclonal-specific Memory B method can obtain neutralizing antibody. It is hoped to obtain broad-spectrum neutralizing antibodies with independent intellectual property rights in China and to provide technical support for the development of antibody drugs and the design of novel immunogens.