HIV-1特异性单抗2G12单价抗体的构建及活性分析  

Construction and activity analysis of HIV specific monovalent mAb 2G12

作　　者：冯一帆[1,2] 刘雪[1,2] 徐柯[2] 冯霞[2] 曾毅[2] 余双庆[2] 

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100022 [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,北京100052

出　　处：《中国病毒病杂志》2015年第3期171-175,共5页Chinese Journal of Viral Diseases

基　　金：国家"十二五"艾滋病和病毒性肝炎等重大传染病防治科技重大专项(2012ZX10001005)

摘　　要：目的构建单价抗体并与IgG比较结合活性及中和活性的差异。方法对IgG1抗体重链恒定区基因进行改造,分别构建N端截短型的IgG1重链恒定区基因IgG1-tH和去除绞链区二硫键及CH2、CH3区的稳定非共价键的2G12重链基因2G12-HM。将IgG1-tH与2G12抗体重链和轻链基因表达质粒共转染293T细胞,表达含有完整Fc的单价抗体;将2G12-HM和轻链基因表达质粒共转染293T细胞,表达半分子型的单价抗体。通过非还原SDS-PAGE对表达抗体的大小进行鉴定,并用ELISA和微量中和实验比较原型2G12抗体和单价抗体的结合活性及中和活性。结果以HIV-1中和抗体2G12为模型通过基因改造成功表达了两种单价抗体,一种为完整的重链、轻链以及N端截短型的重链分子组成的包含1个Fab和1个Fc的单价抗体2G12-tH;另一种为1条重链和1条轻链组成的半分子型单价抗体2G12-HM。两种抗体均可与抗原有效结合,且与IgG分子相比差异无统计学意义。中和实验结果发现IgG抗体能中和的,病毒两种单价抗体也均能中和。结论成功构建了两种单价抗体,与IgG相比其结合活性和中和活性无明显变化。Objective To construct monovalent form IgG antibodies and compare their binding and neutralizing activity with parent antibody. Methods To obtain the monovalent form HIV-1specific antibody 2G12 with 1Fab and 1Fc（2G12-tH）,an expression vector containing DNA encoding the IgG1 hinge,CH2and CH3 domain exons was constructed and co-transfected with the plasmids expressing 2G12 heavy and light chains.To obtain the half molecule form 2G12（2G12-HM）,the disulfide bond at hinge region and the stable non-covalent bonds in CH2,CH3 regions were removed by site-directed mutation on 2G12 heavy chain gene.The mutated heavy chain gene was co-transfected with light chain expressing plasmid.Purified 2G12-tH and 2G12-HM were analyzed by non-reducing SDS-PAGE.The antigen specific binding and neutralizing activity against HIV-1was compared with parent IgG by ELISA and micro-neutralizing assay. Results Two forms of monovalent 2G12 antibody,2G12-tH and 2G12-HM,were constructed and expressed correctly.Both 2G12-tH and 2G12-HM effectively combined with HIV-1Env.The results of neutralizing assay based on six pseudoviruses of HIV-1subtype B indicated that monovalent form antibodies had similar neutralizing activity with IgG. ConclusionsTwo forms of monovalent antibody were successfully constructed.The binding and neutralizing activity of monovalent form antibodies were similar to that of parent IgG antibody.

关 键 词：人类免疫缺陷病毒 单价抗体 中和活性 

分 类 号：R392[医药卫生—免疫学]