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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 antibodies and compare their binding activity and neutralizing activity with IgG. Methods The heavy chain constant region gene of IgG1 antibody was modified to construct IgG1-tH of the truncated IgG1 heavy chain constant region gene and 2G12 of non-covalent bond in the CH2 and CH3 regions, respectively. Heavy Chain Gene 2G12-HM. 293T cells were co-transfected with the heavy chain and light chain gene expression plasmids of IgG1-tH and 2G12 antibody to express the monovalent antibody containing intact Fc. The 2G12-HM and light chain gene expression plasmids were co-transfected into 293T cells to express the semi-molecular Monovalent antibody. The size of the expressed antibody was identified by non-reducing SDS-PAGE and the binding and neutralizing activities of the prototype 2G12 antibody and the monovalent antibody were compared by ELISA and micro-neutralization experiments. Results Two kinds of monovalent antibodies were successfully expressed by genetically engineering HIV-1 neutralizing antibody 2G12, one consisting of a complete heavy chain, light chain and N-terminal truncated heavy chain molecule containing 1 Fab and 1 A monovalent antibody Fc of 2G12-tH; the other one is composed of a heavy chain and a light chain half-cell monovalent antibody 2G12-HM. Both antibodies could bind effectively with antigen, and there was no significant difference compared with IgG. Neutralization results showed that IgG antibodies were neutralized and that both monovalent antibodies to virus were also neutralized. Conclusion Two monovalent antibodies were successfully constructed. Compared with IgG, there was no significant change in their binding activity and neutralizing activity.