摘要
目的 通过链替换技术对一株人源性抗角蛋白Fab单抗进行体外亲和力成熟。方法分离、扩增多样性人免疫球蛋白轻链基因和IgG IgM重链Fd段基因 ,分别构建轻链库和重链库。先以原始克隆的重链与轻链库随机搭配进行轻链替换 ,对形成的换链库采用解离速率筛选法 (off ratese lection)选取亲和力提高的克隆 ;然后再将获得的轻链基因与重链库基因重组进行重链替换 ,筛选亲和力进一步提高的克隆并进行亲和力测定、可变区基因序列分析等一系列鉴定。结果 替换轻链后所获抗体的亲和力提高到原来的 10倍 ;替换重链后亲和力进一步提高达到 7.9× 10 1 0 mol L ,是原始克隆的近 2 3倍。结论 链替换技术是提高抗体库中所获Fab抗体亲和力的有效手段。
Objective To improve the affinity of a human anti keratin Fab. Methods Vκ and the Fd fragment of the anti keratin Fab were replaced sequentially with diverse Vκ genes and Fd fragments derived from lymphocytes of healthy donors. The shuffled libraries were panned using off rate selection process to isolate antibodies with increased affinity. Clones thus obtained were analyzed by ELISAs and sequence analysis. Results By shuffling the Vκ, antibodies with a 10 fold improved affinity were isolated. And a further improvement was obtained by Fd fragment shuffling, resulted in an affinity of 7.9×10 10 /mol/L, nearly 23 fold higher than that of the original Fab. Conclusion Chain shuffling is an effective method for affinity maturation of antibodies isolated from phage antibody library.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2001年第2期160-163,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目!( 3 9670 679)
全军医药卫生重点课题资助项目!( 96Z0 0 4)
关键词
亲和力成熟
噬菌体抗体库
单克隆抗体
角蛋白
Affinity maturation
Phage antibody library
Monoclonal antibody
Keratin
