Construction of libraries of artificial antibodies and their databases

本项目试图构建人工抗体文库，其中包含选择性结合任何抗原的抗体。抗体在M13噬菌体表面以Fab形式与cll分子融合表达。噬菌体抗体分离后，Fab部分可以很容易地转化为与蛋白A融合的形式。为了构建文库，在V区框架序列保持不变的情况下，利用聚合酶链反应（PCR），只有形成抗原结合位点的互补决定区（CDR）的序列高度分化。本研究构建了一个由3 × 10^8个独立克隆组成的文库，并对文库中克隆的特征进行了分析。未经筛选从文库中随机分离的20个无性系在CDR中显示出预期的高度分化序列。用筛分法分离了与蛋清溶菌酶结合的噬菌体抗体。噬菌体与hell结合-结合噬菌体的恢复-噬菌体的生长，一轮这一过程需要两天。经过三轮筛选，从回收的噬菌体中分离出20个克隆体。其中19个具有抗hel活性，结合常数分布在10^6 ~ 10^7 M^<-1>之间。具有抗hel活性的抗体的CDR序列表明，其中一半以上是唯一的，其余为发散残基。这表明一些残留物是显示抗hel活性所必需的，而一些残留物不是必需的。由于本研究构建的文库是根据抗hel抗体D1.3的序列进行分化的，因此该文库所能覆盖的抗原可能存在偏倚。现在，我们正在进行各种抗原特异性抗体的CDR序列的嫁接实验，并将以嫁接的克隆为模板进行CDR序列的多样化。

This project attempted to construct libraries of artificial antibodies which contain antibodies selectively bound to any kind of antigens. Antibodies are expressed on the surface of M13 phages as an Fab form fused with cplll molecules. After isolation of phage antibodies, Fab portions can easily be converted to the form fused with protein A in our gene construct. To make libraries, while the sequences of the framework in V regions are kept constant, only the sequences of the comprementarity-determining regions (CDR) that form antigenbinding sites are highly diverged by use of polymerase chain reaction (PCR). In the present study, we constructed a library composed of 3x10^8 independent clones and analyzed the characteristics of clones in the library. The 20 clones randomly isolated from the library without screening showed the highly diverged sequences in the CDR as expected. The phage antibodies bound to hen egg white lysozyme (HEL) were isolated by panning method. Binding of phages to HEL-recovery of the bound phage-growth of phages, one round of this process requires two days. After three rounds of the panning, 20 clones were isolated from the recovered phages. 19 of them had anti-HEL activity and the binding constnats distributed from 10^6 to 10^7 M^<-1> The suquences of the CDR of antibodies that had anti-HEL activities indicated that more than half of them were unique and the rest had diverged residues. This suggested that some residues are required for showing anti-HEL activities but that some residues are not essential. Since the library that were constructed in the present study was diverged based on the sequence of anti-HEL antibody, D1.3, the antigens that could be covered by this library may have been biassed. Now, we are performing the grafting experiments of the CDR seuquences of various antigen-specific antibodies and are going to diversity the CDR sequences using the grafted clones as templates.