热点突变法提高人源抗TNF-α抗体的亲和力  

In vitro affinity maturation of a human ScFv against TNF-α by mutating the hot spots in somatic hypermutation

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

机构地区：[1]海军总医院,北京100037

出　　处：《中华微生物学和免疫学杂志》2004年第8期644-648,共5页Chinese Journal of Microbiology and 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　通过热点突变法对一株人源抗TNF αScFv进行体外亲和力成熟。方法　根据体内抗体亲和力成熟时V基因体细胞突变的热点 ,设计引物进行多步重叠PCR ,在本株ScFv的H CDR3和L CDR3热点部位引入随机突变 ,构建噬菌体抗体库 ,采用硫氰酸盐洗脱法对抗体库进行筛选 ,对选出的克隆测定其相对亲和力 ,并进一步将 4株转换表达成为可溶性Fab ,以非竞争ELISA法测定其亲和常数。结果　构建了库容为 1.8× 10 7的热点突变抗体库 ,筛选得到了多株亲和力明显提高的抗TNF α抗体的突变体 (最多达 10倍 )。结论　热点突变法是体外提高抗体亲和力的有效和可行的方法。ObjectiveTo improve the affinity of a human ScFv against TNF-α by mutating the hot spots in somatic hypermutation. MethodsBased on the hot spots in somatic hypermutation,primer-mediated mutations were introduced into H-CDR3 and L-CDR3 of the ScFv gene by overlap PCR and a phage antibody library was constructed. The mutation library was screened by bio-panning with thiocyanate washing and clones with improved activity were selected. Their relative affinities were determined by thiocyanate elution ELISA. Four selected ScFv mutants were converted to Fab and their affinity constants were determined by non-competitive ELISA. ResultsA mutation phage antibody library with 1.8×10 7 in size was constructed and several mutants with improved affinity were obtained,the best one had a 10-fold improvement of affinity than that of control. ConclusionThe method of hot spots mutation can be used to improve the affinity of antibodies.

关 键 词：热点突变法 抗TNF-α抗体 亲和力 人源抗体 抗体库 

分 类 号：R392[医药卫生—免疫学]