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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 perform in vitro affinity maturation of a human anti-keratin Fab monoclonal antibody by strand replacement technique. Methods Diversified human immunoglobulin light chain genes and IgG IgM heavy chain Fd gene were isolated and amplified to construct light and heavy chain libraries respectively. First, the light chain was replaced by a random combination of the heavy chain and the light chain of the original clone, and the affinity-enhanced clone was selected by the off ratesection on the resulting chain exchange library. Then, the light chain gene Heavy chain library gene recombination for heavy chain replacement, further screening of affinity improved clone and affinity determination, variable region gene sequence analysis and a series of identification. As a result, the affinity of the antibody obtained after the replacement of the light chain was increased by 10 times; the affinity of the replacement heavy chain was further improved to reach 7.9 × 10 10 mol L, nearly 23 times that of the original clone. Conclusion The technique of strand replacement is an effective means to improve the affinity of Fab antibody in antibody library.