噬菌体展示抗乙型肝炎病毒表面抗原Fab抗体分子的筛选和序列分析  

Screening and Gene Sequencing of Fab Antibodies Against HBsAg from Phage-displayed Library

作　　者：张爱华[1] 端义坤[1] 闭兰[1] 赵亚杰[1] 张智[1] 阎莹[1] 王志友[1] 余模松[1] 

机构地区：[1]武汉生物制品研究所,武汉430060

出　　处：《中国生物制品学杂志》2005年第1期1-4,共4页Chinese Journal of Biologicals

摘　　要：目的　从自建的免疫噬菌体展示Fab抗体库中获得抗乙型肝炎病毒表面抗原Fab抗体分子 ,并进行基因序列分析。方法　采用纯化的乙型肝炎病毒表面抗原作为包被抗原 ,对自建的 4× 10 5Fab噬菌体抗体库进行富集筛选 ,从阳性强的次级库中挑选单个克隆菌 ,分别进行噬菌体ELISA、PCR和限制性内切酶鉴定后 ,再选取三者均为阳性的克隆菌进行基因序列分析。结果　共挑选了 6 0个单克隆菌株 ,其中噬菌体ELISA阳性有 2 7个 ,阳性率为 4 5 % ;PCR鉴定均为相应大小片段 ;限制性内切酶鉴定表明 2 7个阳性克隆菌中有 13个具有约 15 0 0bp的片段 ,其余均为约 75 0bp大小 ;BstOI酶切鉴定表明各克隆菌的酶切方式不同 ;选取 5个含约 15 0 0bp片段和 2个含约75 0bp片段的克隆菌株 ,经测序均为人免疫球蛋白基因 ,且重链分别属于VH3、VH4、VH5家族 ,轻链分别属于L5、L6、L8和 0 12 /0 2家族。结论　从自建的免疫噬菌体展示Fab抗体库中成功地获得了抗乙型肝炎病毒表面抗原Fab抗体分子。表明所构建的抗乙型肝炎病毒表面抗原的Fab抗体库的抗体基因具有多样性。Objective To screen Fab antibodies against HBsAg from immune phage-displayed library constructed by the authors and analyze their gene sequence.Methods Screen specific Fab antibodies from Fab library containing 4×105 clones using purified HBsAg as a coating antigen.After five rounds of screening, single clones were selected out from the strong positive secondary library and identified by phage-ELISA, PCR and restriction analysis, and those positive in all the three identification tests were sequenced.Results A total of 60 single clones were selected from the strong positive secondary library, of which 27 (45%) were positive in phage-ELISA.The lengths of restriction fragments of 13 phage-ELISA positive clones were about 1 500 bp, and those of the rest 14 were about 750 bp.The fragments at corresponding lengths were also amplified by PCR.Five 1 500 bp and two 750 bp fragments were sequenced.The result showed that all the genes of Fab antibodies were human imunoglobulin genes belonging to VH3, VH4 and VH5 families of heavy chain or L5, L6, L8 and 012/02 families of light chain.Conclusion Fab antibodies against HBsAg were successfully screened from immune phage-displayed library constructed by the authors.The result showed gene diversity of the antibody library.

关 键 词：FAB抗体 抗乙型肝炎病毒 表面抗原 阳性克隆 噬菌体展示 克隆 ELISA 限制性内切酶 包被 片段 

分 类 号：Q78[生物学—分子生物学] R512.62[医药卫生—内科学]