随机化CDR3抗体库的构建及不经免疫制备抗体的初步探索

By passing immunization:Construction of a phage antibody library with randomized CDR3

为探索利用噬菌体抗体库技术不经免疫制备抗体，在已有的抗乙肝病毒表面抗原（ＨＢｓ）人Ｆａｂ段基因的基础上，通过ＰＣＲ引入突变，对其ＶＨＣＤＲ３中１２个氨基酸残基（９５～１００Ｊ）进行了完全随机化，对９４和１００Ｋ位残基进行了有限随机化，构建了半合成噬菌体抗体库，库容为２×１０８，５８％含有随机化ＣＤＲ３序列，用无关抗原小鼠ＩｇＧ对该抗体库进行筛选，获得了三个可与小鼠ＩｇＧ结合而不与ＨＢｓ反应的克隆，经测定其ＣＤＲ３的ＤＮＡ序列，发现这三个克隆具有相同的ＣＤＲ３，证明单纯改变ＶＨＣＤＲ３可以得到新的特异性抗体，从而绕过免疫制备抗体。

In order to prepare antibodies by passing immunization we constructed a semisynthetic phage antibody library with randomized CDR3.Using anti HBs human Fab gene as starting material,12 amino acid residues in V H CDR3(95￣100J) were totally randomized and residues 94 and 100K were limited randomized by PCR.A phage antibody library of 2×10 8 with 58% of the clones containing randomized CDR3 was obtained.Panning of the library by unrelated Ag,mouse IgG,yielded 3 clones that could bind to mouse IgG but not HBs.Sequence analysis revealed that the 3 clones contained identical CDR3 sequence indicating they were derived from specific enrichment during panning,thus CDR3 randmization was feasible for preparing antibodies by passing immunization.