联合应用错配PCR和交替延伸PCR法提高抗TNFα抗体亲和力  

Improving the affinity of an anti-TNFα scFv by error prone PCR and staggered extention process(StEP)

作　　者：汪保安[1] 陈晓穗[1] 乔媛媛[1] 曲佳[1] 王欲晓[1] 王琰[1] 

机构地区：[1]海军总医院中心实验科,北京100037

出　　处：《中国免疫学杂志》2004年第12期846-849,共4页Chinese Journal of Immunology

基　　金：解放军医药卫生"十五"重点项目 (0 1Z0 15 )资助课题

摘　　要：目的 :通过突变方法提高抗TNFα单链抗体pscTNF的亲和力。方法 :以pscTNF质粒为模板 ,应用错配PCR构建突变抗体库 ,筛选亲和力得到改善的变种 ,再以所获变种为模板 ,用交替延伸PCR技术 ,再次构建突变库 ,筛选活性得到改良的克隆。以斑点ELISA方法及硫氰酸盐洗脱ELISA法评估其亲和力的改善。结果 :从错配PCR抗体库中筛选得到 7个活性有所增强的变种 ,再通过交替延伸PCR使这 7个变种的突变位点交换重组 ,获得了亲和力进一步提高的克隆。结论 :联合应用错配PCR和交替延伸PCR法可提高单链抗体亲和力。Objective:To improve the affinity of an anti TNFα scFv.Methods:Starting from an anti TNFα scFv gene a mutant phage antibody library was generated by error prone PCR.Affinity improved clones were selected and subjected to staggered extension process to shuffle the mutated sites.Mutants with further improved affinity were selected by bio panning.Affinity was judged by dot blot ELISA and thiocyanate elusion ELISA.Results:Seven affinity improved mutants were obtained from library constructed by error prone PCR.By StEP mediated shuffling of these 7 clones and via bio panning,mutants with further improved affinity were obtained.Conclusion:Combination of error prone PCR and StEP could be used to improve the affinity of antibodies. [

关 键 词：错配PCR 交替延伸PCR 抗体亲和力 斑点ELISA 

分 类 号：R392-33[医药卫生—免疫学]