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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。结论 构建的Fab抗体库可用于筛选特异性抗乙型肝炎病毒表面抗原Fab抗体分子
Objective To construct human phage antibody library against human hepatitis B virus surface antigen. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from human whole blood with a high titer (1:10 2 4) of anti-hepatitis B virus surface antibody. The light chain variable region and heavy chain were amplified by RT-PCR respectively (Cκ) and constant region of heavy chain (CH1) were amplified by using phage plasmid as template. The light chain variable region, light chain constant region (Cκ) and heavy chain variable region The 1st gene was spliced in the heavy chain constant region (CH1) to form the kappa light chain and the Fd heavy chain, respectively, and the second gene splicing was carried out using the kappa light chain and the Fd heavy chain as templates to form a complete Fab gene, which was ligated with pComb3H After the SS phage plasmid was ligated, E. coli XL1 Blue was transformed by electroporation. As a result, the phage antibody library having a total volume of 4 × 10 5 was obtained by multiple electroporation transformations. Conclusion The constructed Fab antibody library can be used to screen specific anti-hepatitis B virus surface antigen Fab antibody molecules