人源抗乙肝表面抗原基因工程抗体的研究

目的 研制人源抗乙肝表面抗原(HBsAg)基因工程抗体.方法 采集多个HBsAg加强免疫后表面抗体阳性(HBsAb+)志愿者的外周血,分离淋巴细胞,构建人源抗HBsAg Fab噬菌体抗体基因文库.用纯化的HBsAg对抗体库进行富集筛选,经过序列测定确定抗体轻重链型别,分别克隆入真核细胞表达载体pAc-FcR和HL51-14,转染昆虫Sf9细胞和293T细胞,利用杆状病毒/昆虫细胞系统和哺乳动物细胞系统实现全抗体的分泌型表达.结果 成功筛选出20株抗HBsAg的人源Fab抗体并制备出其中6株的IgG全抗体,竞争性ELISA结果显示6株全抗体针对的是HBsAg 3个不同表位.结论 成功筛选并获得了6株针对3个不同表位的抗HBsAg的IgG全抗体,为治疗性抗体和新疫苗的研制奠定了基础.

Research on recombinant human antibodies against hepatitis B surface antigen

Objective To obtain recombinant human anti-HBsAg genetic engineering antibodies from volunteer-derived antibody phage library. Methods A combinatorial human Fab library against HBsAg was constructed with antibody genes harvested from peripheral blood of HBsAb ＋ volunteers after HBsAg booster immunization. The library was panned with phage display and selected by purified HBsAg. After that the selected antibodies were converted to full human IgG antibody with recombinant baculovirus/insect cell system and mammal cell system. Results 20 different human Fab antibodies specific for HBsAg were obtained by ELISA and determination of subtype and subclass of antibodies after gene sequencing. Six specific anti-HBsAg human Fab antibodies were converted to full human IgG antibody. Competitive ELISA showed three of the six full human IgG antibodies had different epitopes against HBsAg. Conclusion Three monoclonal antibodies with different epitopes against HBsAg were panned and obtained successfully and laid the foundation for the therapeutic antibodies and new vaccines.