抗人CD4单克隆抗体制备及其抗HIV-1功能鉴定  

Preparation and identification of anti-CD4 monoclonal antibodies against HIV-1

作　　者：李岚 史宣玲 江立玮 殷昌斌[1] 李家伟[1] 刘巍[1] LI Lan;SHI Xuan-ling;JIANG Li-wei;YIN Chang-bin;LI Jia-wei;LIU Wei(Department of Laboratory Medicine,Beijing Luhe Hospital,Capital Medical University,Beijing 101149,China)

机构地区：[1]首都医科大学附属北京潞河医院检验科,北京101149 [2]清华大学医学院艾滋病综合研究中心,北京100084

出　　处：《中国病毒病杂志》2018年第3期194-199,共6页Chinese Journal of Viral Diseases

基　　金：北京市通州区科技计划项目(KJ2017CX039-14)

摘　　要：目的制备具有抗HIV-1功能的抗人CD4单克隆抗体。方法构建人CD4真核表达质粒pcDNA3.1-hCD4,将该质粒DNA免疫BALB/c小鼠。免疫前血清作为阴性对照,通过HIV-1假病毒中和实验和ELISA方法检测多克隆抗体血清。通过杂交瘤技术制备鼠抗CD4单克隆抗体,HIV-1假病毒中和实验进行筛选。通过不同亚型假病毒中和实验和CD4D1D2蛋白ELISA方法分别检测腹水纯化单克隆抗体的中和活性和特异性结合力。结果 DNA免疫后第6周的多抗血清100倍稀释后对HIV-1假病毒的抑制率可达75%,第8周其抑制率可上升至95%。CD4D1D2蛋白ELISA结果显示,免疫后第4周多抗血清就显示出同CD4蛋白较强的特异性结合。共获得160余株对HIV-1假病毒B′、BC、AE亚型具有中和活性的单克隆抗体。选择其中中和活性较高的8株制备纯化腹水单抗,其中5株单抗IC50小于130μg/ml。ELISA检测结果显示腹水纯化单抗与CD4D1D2蛋白可以特异性结合。结论获得了具有抗HIV-1假病毒的抗CD4单克隆抗体。具有HIV-1广谱中和活性的抗CD4抗体可用于研发有效的被动免疫制剂,控制艾滋病传播。Objective To prepare and evaluate anti-CD4 monoclonal antibodies against HIV-1. MethodspcDNA3.1-hCD4 eukaryotic plasmid expressing human CD4 was constructed and BALB/c mice were inoculated.Sera prior to immunization were used as negative control.The polyclonal antibodies in sera were tested by ELISA and pseudovirus neutralization assays.Monoclonal antibodies were obtained by hybridoma technique and screened by pseudovirus neutralization assays.The neutralizing activities of the monoclonal antibodies purified from ascites were determined by using pseudotyped HIV-1 viruses.CD4 D1 D2 protein ELISA was used to confirm the specific binding of the monoclonal antibodies. Results At the sixth week after immunization,1∶100 diluted sera showed up to 75%inhibition.The inhibition reached 95% at the eighth week.ELISA with CD4 D1 D2 domain protein showed that anti-sera at the fourth week after immunization had strong binding capacity to CD4.More than 160 monoclonal antibodies against HIV-1 subtype B′,BC and AE were obtained.Eight monoclonal antibodies with higher neutralizing activity were selected to be purified from ascites.IC50 values of five monoclonal antibodies were less than 130μg/ml.ELISA results also demonstrated that the purified monoclonal antibodies could bind CD4 D1 D2 protein specifically. Conclusions Anti-CD4 monoclonal antibodies against HIV-1 pseudovirions were obtained.The potent and broad inhibitory activity of antiCD4 antibodies against HIV-1 may be used to develop effective passive immunization agent to control the spread of AIDS.

关 键 词：人类免疫缺陷病毒Ⅰ型 CD4 DNA免疫 多克隆抗体 单克隆抗体 中和抗体 

分 类 号：R512.91[医药卫生—内科学]