CDR3突变提高抗TNF-αFab段的亲和力  

CDR3 mutagenesis improves the affinity of anti-TNF-α antibody Fab

作　　者：汪保安[1] 陈晓穗[1] 王琰[1] 王欲晓[1] 曲佳[1] 周丽君[1] 

机构地区：[1]海军总医院中心实验科,北京100037

出　　处：《免疫学杂志》2005年第5期388-392,共5页Immunological Journal

基　　金：北京市自然科学基金资助项目(5052025)

摘　　要：目的通过抗体库技术在CDR3区引入限定性突变以提高抗TNF-α抗体Fab的亲和力.方法首先合成含CDR3区突变的寡核苷酸,限定突变率以保持50%～70%的亲本序列,通过重叠延伸PCR的方法引入突变,构建突变库,再以硫氰酸铵溶液作洗涤液,筛选亲和力得到提高的特异性克隆,用非竞争性ELISA法测定亲和常数.结果从轻链突变库中筛选得到1个活性提高的特异性克隆K8,在第93位发生N→R突变,其亲和常数为2.8×108 L/mol,较亲本抗体(0.1×108 L/mol)提高了近28倍;从重链突变库中筛选得到多个活性增高的克隆,部分克隆测序显示96、97和100 d位置发生R→K、Y→Q、M→L突变的几率最高,且对亲和力提高贡献大.测定了10个变种的亲和常数,分别为0.2×108、0.22×108、1.8×108、3.2×108、3.3×108、3.5×108、3.7×108、4.0×108、 6.3×108和6.5×108 L/mol,最大的提高了近65倍.结论 CDR3区的限定性突变可显著提高抗体亲和力.Objective To improve the affinity of anti-TNF-α antibody Fab by phage display technique through parsimonious mutagenesis. Methods Mutant phage antibody libraries were generated by introducing mutations through synthesized oligonucleotides, which contained 50% - 70% parental sequences within CDR3 by PCR splice overlap extension. Several phage antibody libraries were constructed and mutants with improved affinity were selected by biopanning. The sequences of mutants were analyzed and their affinity constants were determined by noncompetitive ELISA. Results One mutant was selected from mutated library with a 28-fold higher Ka（2.8×10^8L/mol） than its parental Fab. Ten mutants were selected from Fd mutated libraries with 2 - 65-fold higher Ka （0.2-6.5×10^8L/mol）. Conclusion The parsimonious mutagenesis of CDR3 could significantly improve the affinity of antibody.

关 键 词：亲和力成熟 CDR3突变 肿瘤坏死因子Α 

分 类 号：R392[医药卫生—免疫学]