Construction of libraries of artificial antibodies and their databases

该项目试图构建人工抗体库，其中包含选择性地与任何类型的抗原结合的抗体。抗体在M13噬菌体表面以Fab形式与cpl11分子融合表达。在分离噬菌体抗体后，Fab部分可以很容易地转化为与我们基因结构中的蛋白A融合的形式。为了构建文库，在V区骨架序列保持不变的情况下，只有形成抗原结合位点的复杂性决定区(CDR)的序列通过聚合酶链式反应(PCR)高度分化。在本研究中，我们构建了一个由3x10^8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。从文库中随机分离的20个克隆在没有筛选的情况下显示了CDR中的高度差异序列。用平皿法制备了与蛋清溶菌酶(HEL)结合的噬菌体抗体。噬菌体与HEL的结合-结合噬菌体的恢复-噬菌体的生长，这一过程一轮需要两天。经过三轮淘洗，从回收的噬菌体中分离到20个克隆。其中19个具有抗HEL活性，结合常数分布在10^6~10^7M；具有抗HEL活性的抗体的CDR序列分析表明，其中一半以上是独一无二的，其余的具有分歧残基。这表明有些残基是显示抗HEL活性所必需的，但有些残基不是必需的。由于本研究中构建的文库是基于抗HEL抗体D1.3的序列而分离的，因此该文库可能包含的抗原已被排除在外。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对CDR序列进行多样性。

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.