半合成抗体库的构建及鉴定

Construction of a semisynthetic phage antibody library

目的构建半合成抗体库，不经免疫制备抗体。方法通过ＰＣＲ法，从人胎肝ＤＮＡ扩增基因组ＶＨ段基因，从Ｆｄ基因制备含有不同长度随机化ＣＤＲ３的ＦＲ３－ＣＤＲ３－Ｊ－ＣＨ１片段，然后用重叠ＰＣＲ法构建Ｆｄ库，与人轻链基因库组合构建成半合成抗体库。结果通过各种组合多次建库，获得总容量为４×１０８的半合成抗体库，其Ｆｄ段和轻链基因的重组率均大于９０％，Ｆａｂ表达率为５０％。挑选２０个克隆测定ＣＤＲ３序列符合所设计的随机序列，用三种不同抗原进行筛选均得到了特异性噬菌体抗体。

Objective To construct a semisynthetic phage antibody library for by passing immunization. Methods The genomic V H segment genes were amplified from fetus liver cell DNA and spliced to synthetic random CDR3 of different lengths, forming an Fd library. This Fd library was then combined with a human κ gene library to form a semisynthetic phage antibody library. Results An antibody library containing 4×10 8 different clones was obtained, with more than 90% containing Fd and κ chain genes. 50% of the clones expressed Fab fragment. Specific Fab could be selected after panning against 3 different antigens. Conclusion This semisynthetic antibody library is capable of cloning antibodies by passing immunization.