用蛋白Ⅷ在噬菌体表面展示抗体分子效果的观察

Evaluation of cpⅧ mediated phage display of antibody fragments

目的 观察抗体分子通过丝状噬菌体主要外壳蛋白Ⅷ（ｃｐⅧ）和次要外壳蛋白Ⅲ（ｃｐⅧ）在噬菌体表面呈现效果的差异。方法 分别构建通过ｃｐⅧ或ｃｐⅧ展示抗乙肝表面抗原（ＨＢｓ）Ｆａｂ、ＳｃＦＶ和抗角蛋白（Ｋｅｒ）Ｆａｂ的表达载体，制备噬菌体抗体，比较其抗原结合活性和Ｆａｂ呈现水平。用多种方法尝试提高ｃｐⅧ 介导的噬菌体展示效果。结果ｃｐⅧ对不同特异性抗体Ｆａｂ段和不同形式的小分子抗体（Ｆａｂ和ＳｃＦｖ）的展示效果均低于ｃｐⅧ的展示，增加 ｃｐⅧ-Ｆａｂ对野生型 ｃｐⅧ的表达比例、试用不同菌株、在Ｆａｂ和ｃｐⅧ之间插入间隔序列及换用控制更为严密的启动子等措施，均未能改善ｃｐⅧ介导的噬菌体展示。结论 以前所报道的通过ｃｐⅧ多价展示Ｆａｂ段不是普遍存在的现象，对有些抗体基因不能达到多价展示。

Aim To evaluate the cpⅧ- and cpⅢ-mediated phage display of antibody fragments Methods Vectors displaying anti-HBS Fab, scFv or anti-keratin Fab via cpⅧ or cpⅢ were constructed. Phage displayed antibod- ies were prepared. Antigen binding activity and Fab displaying level were tested by ELISA. Different ways were tried to improve the efficiency of cpⅧ meaiatea dis play Results Both Ag binding activity and Fab expression level were lower for cpⅧ constructs than those for cpⅧ. Efforts to increase the Fab display via cp Ⅷ, by increasing the ratio of cp Ⅷ-Fab fusion with wild type cpⅧ, using different E. coil strains, inserting spacer between Fab and cpⅧ, and using more tightly controlled promoter for cp Ⅷ-Fab fusion were tried without success. Conclusion Previously reported multi-valence displaying Fab fragment via cpⅧ is not applicable for all Abs. A more deep- going understanding of the structure and function of cpⅧ is necessary before multi-valence display can he obtained.