抗人AFP抗体噬菌体呈现文库的构建筛选及基因序列测定

从人ＡＦＰ免疫小鼠脾细胞ｍＲＮＡ中扩增出全套抗体Ｖ区基因并随机拼接为ＳｃＦｖ基因，构建全套ＳｃＦｖ基因噬菌体呈现文库，经两轮ｐａｎｎｉｎｇ筛选富集，一次从９４个单个重组噬菌体克隆中筛选到１４个具有ＡＦＰ结合活性的克隆，测定两个阳性克隆中ＳｃＦｖ基因的核苷酸序列，获得一个Ｖ＿Ｈ基因和两个Ｖ＿Ｋ基因，其基因序列分别与鼠ＩｇＶ＿ＨＪ５５８、Ｖ＿ｋ－ＯＸ１和Ｖ＿ｋ４／５家族同源性最高；推导出的氨基酸序列中均含有抗体Ｖ区特征性的两个恒定的半胱氨酸残基、具有明确的三个ＣＤＲ和四个ＦＲ序列，表明这三个基因均系新发现的功能性鼠抗体Ｖ区基因序列。

Generation of a Phage Display Library of the Immunoglobulin Repertoire from Human AFP-lmmunized Mouse and Sequencing of V-region Genes of Anti-AFP Antibody

A preliminary study on the construction of a phage display library of immunogolbulin repertoire from human AFP-immunized mouse and sequencing of the V-region genes of generated anti-AFP antibody clones was reported. The V_H and V_k gene repertoire of immunoglobulin were separately amplified from the spleen cell mRNA of a human AFP-immunized mouse, by RT-PCR.The ScFv repertoire was assembled in vitro and cloned into a phagemid vector.So, a phage display library of the immunoglobulin repertoire(ScFv) was constructed. This library was then selected by binding to solid phase antigen AFP, followed by elution and re-infection. After two rounds of panning,14 AFP-binding clones were identified from 94 enriched individual phagemid clones.Then, the nucleotide sequences of ScFv recombined in two positive phagemid clones were determined by Sangers method, and one V_H gene and two V_k genes were obtained.. The sequences of these three genes were confirmed as the mouse immunoglobulin V-region genes. By comparing with the published data in EMBL Gene Bank 1994, they appear to be total new genes. There are four framework regions (FRs),three complementarity determining regions (CDRs)and two invariant cysteine residues in each of these genes. The results indicate that these three genes ofmouse immunoglobulin variable regions are all rearranged and functional.