通过构建轻链二级库对人源抗TNF-α抗体进行亲和力成熟

摘要

通过构建轻链二级库的方法，对人源抗TNF-α单抗Fab进行轻链置换，并筛选出具有更高亲和力的人源抗TNF-α单抗的Fab。首先用RT-PCR技术扩增正常人全套抗体轻链基因，并与已获得的人源抗TNF-α单抗的重链基因配对，构建人源抗TNF-α噬菌体抗体轻链二级库，然后筛选与TNF-α具有更高亲和力的克隆。经过三轮的生物淘筛（biopanning），获得了比原来的人源抗TNF-α单抗Fab具有更高亲和力的人源抗体Fab。轻链二级库筛选法能够有效使抗体的亲和力提高，为解决噬菌体抗体库法制备的人源抗体的亲和力较低这一难题提供了一种有效的方法。%Human anti-TNF-α antibody Fabs with higher affinity was obtained through replacing the light chain fragment facilitated by constructing a light chain second library. Firstly, amplify the repertoire of the human light chains genes from human peripheral blood lymphocytes using RT-PCR technique and assemble them randomly with the heavy chain gene of the anti-TNF-α antibody so as to construct a second light chain library which comprises single 　heavy chain　 gene of the antibody to TNF-α and all of the human light chain genes.Τhen select the antibody clones with higher binding affinity. Some new phage displayed antibodies, which had higher affinity for TNF-α, were obtained from the new library through three rounds of biopanning. It is conclued that constructing light chain second library could effectively improve the affinity of the antibody, and could be a valid method to overcome the disadvantage of the lower affinity of the human antibody from phage display.