人源腺病毒伴随病毒Ⅱ型单克隆抗体Fab段及其全抗体的研制

Generation of human recombinant antibody Fab fragment and its IgG to adeno-associated virus type Ⅱ from phage display library

目的 运用噬菌体表面表达技术 ,获得基因工程抗腺病毒伴随病毒抗体Fab、IgG。方法 从酶联免疫吸附试验阳性的人外周血中分离淋巴细胞 ,提取总RNA ,逆转录cDNA ,聚合酶链反应扩增人IgGFab轻、重链基因 ,利用pComb3载体构建噬菌体抗体库。用纯化的腺病毒伴随病毒为固相抗原筛选抗体Fab段 ,并在E coli中进行分泌性表达。通过ELISA和间接免疫荧光试验、筛选和鉴定Fab抗体 ,并进行序列测定。然后将其重链和轻链基因克隆到全抗体表达载体pAC L Fc上 ,转染昆虫sf 9细胞 ,利用杆状病毒 昆虫细胞系统实现全抗体的分泌型表达 ,免疫沉淀试验鉴定它的抗蛋白。结果 分离到一株Fab克隆AAVs 31,腺病毒伴随病毒抗原和抗Fab抗体直接ELISA检测阳性 ,间接免疫荧光试验呈阳性 ,序列分析结果表明是一新的序列 ,所获得的基因为人源IgGFab基因 ,由Gamma链和Kappa链组成。表达的全抗体ELISA反应、免疫荧光反应和间接免疫荧光试验均为阳性 ,免疫沉淀试验结果显示它结合病毒颗粒 ,不结合任一VP1、VP2、VP3单独衣鞘蛋白。结论 用噬菌体表面表达技术首次获得了抗腺病毒伴随病毒Ⅱ型单克隆抗体Fab段 ,并在真核系统中表达了其全抗体 。

Objective To acquire the recombinant human monoclonal antibodies and IgG to adeno associated virus type 2 (AAVs 2) Methods Construct and pan human Fab antibody library to AAVs 2 was established from normal volunteer donors by using phage display technology and secreted expression in E Coli system The positive Fab clones were selected and characterized through ELISA and immunofluorescent assay, and then the heavy and light chain were sequenced The gene of light chain and heavy chain Fd fragment of recombinant mAb were inserted into baculovirus expression vector pAC L Fc and construct expression vectors of intact IgG, then transfected insert sf 9 cell secreted expression in Baculovirus/Insert system Immunoprecipation test was used to detect its recognizing region Results One clone named AAVs 31 showed positive responses in ELISA and IFA, the Fab was composed of gamma chain and kappa chain IgG was positive in ELISA and IFA The IgG failed to detect nonassembled or denatured capsid proteins, but recognized the AAVs 2 stock from immunoprecipation test Conclusion The authors isolated a clone of Fab and IgG to adeno associated virus type 2 by phage display technology, they perhaps recognize an epitope which is formed during capsid assembly