通过随机突变提高抗TNF-α单链抗体的亲和力  

Improving the affinity of an anti-TNF-α scFv by random mutation

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

机构地区：[1]海军总医院中心实验科,北京100037

出　　处：《细胞与分子免疫学杂志》2005年第4期445-448,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：北京市自然科学基金资助项目(No.5052025)

摘　　要：目的:提高抗TNFα单链抗体(scFv)的亲和力。方法:应用错配PCR技术在抗TNFαscFv基因中引入随机突变,再通过DNA交换(shuffling)使引入的点突变进一步重排组合,构建突变噬菌体抗体库。采用硫氰酸盐洗脱法对抗体库进行淘筛。挑选活性得到改良的克隆,用斑点ELISA法及硫氰酸盐洗脱ELISA法评估其亲和力的改善。结果:对PCR错配的突变库筛选未得到亲和力明显改善的抗体变种,经DNA交换进一步构建变种库后,筛选获得1个亲和力提高的克隆,其相对亲和指数为1.37mol/L,较亲本抗体的相对亲和指数0.48mol/L有明显提高。结论:通过错配PCR和DNA交换引入随机突变构建抗体库,可有效地提高scFv的亲和力。AIM: To improve the affinity of an anti- TNF-α scFv. METHODS: A mutant phage antibody library derived from an anti-TNF-α scFv gene was generated by error-prone PCR. The mutated genes were then subjected to DNA shuffling. Mutants with improved affinity were selected by bio-panning. Affinity improvement of the selected mutants was verified by dot blot ELISA and thiocyanate elusion ELISA. RESULTS: One mutant was obtained with relative affinity index (1.37 mol/L) higher than that of the parent scFv (0.48 mol/L). CONCLUSION: Error-prone PCR plus DNA shuffling is effective in improving the affinity of antibodies.

关 键 词：致错PCR DNA重排 亲和力 

分 类 号：R392.11[医药卫生—免疫学]