采用基因拼接方法构建人源特异性抗乙型肝炎病毒表面抗原Fab噬菌体抗体库  

Construction of Human Specific Fab Phage Antibody Library Against HBsAg by SOE

作　　者：张爱华[1] 闭兰[1] 端义坤[1] 张智[1] 赵亚杰[1] 孙可芳[1] 闫莹[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》2004年第5期257-260,共4页Chinese Journal of Biologicals

摘　　要：目的　构建人源特异性抗乙型肝炎病毒表面抗原Fab噬菌体抗体库。方法　从抗乙型肝炎病毒表面抗体高滴度 (1:10 2 4 )的人全血中分离外周血单个核细胞 (PBMC) ,经RT PCR分别扩增出轻链可变区和重链可变区 ,再以噬菌体质粒为模板分别扩增出轻链恒定区 (Cκ)和重链恒定区 (CH1) ,将轻链可变区和轻链恒定区 (Cκ)及重链可变区和重链恒定区 (CH1)进行第 1次基因拼接 ,分别形成κ轻链和Fd重链 ,再以κ轻链和Fd重链作为模板进行第 2次基因拼接 ,形成完整的Fab基因 ,与pComb3H SS噬菌体质粒连接后 ,电穿孔转化大肠杆菌XL1 Blue。结果　通过多次电穿孔转化 ,获得总容量为 4× 10 5库容的噬菌体抗体库。Objective To construct a human specific Fab phage antibody library for screening Fab against HBsAg.Methods Amplify the cDNAs encoding variable regions of light and heavy chains, by RT-PCR, from the PBMCs of patients with high titer (1∶1 024 by ELISA) antibody to HBsAg. Isolate the genes of constant regions of light and heavy chains from phagmid carrying the templates respectively. Link κ light chain and Fd heavy chain by the first SOE, and create a full-length Fab gene by the second SOE. Ligate the Fab gene to phagmid pComb3H-SS and transformed to E.coli XL1-Blue (F+) by electroporation.Results A phage antibody library, with a gross capacity of 4×10~5,was constructed.Conclusion The constructed Fab antibody library may be used for screening specific Fab antibodies against HBsAg.

关 键 词：基因拼接 乙型肝炎病毒 表面抗原 噬菌体抗体库 

分 类 号：R512.62[医药卫生—内科学] R392-33[医药卫生—临床医学]