Construction of libraries of artificial antibodies and their databases

人工抗体文库及其数据库的构建

基本信息

批准号：
06557026
负责人：
KUROSAWA Yoshikazu
金额：
$ 13.25万
依托单位：
Institute for Comprehensive Medical Science, Fujita Health University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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.

该项目试图构建人工抗体的库，这些抗体含有选择性地与任何类型的抗原结合的抗体。抗体在M13噬菌体的表面表达为与CPLLL分子融合的Fab形式。分离噬菌体抗体后，可以轻松地将FAB部分转换为与我们的基因构建体中蛋白A融合的形式。为了制作文库，虽然V区域中的框架序列保持恒定，但仅使用聚合酶链反应（PCR），形成抗凝集位点的元素决定区域（CDR）的序列高度分歧。在本研究中，我们构建了一个由3x10^8独立克隆组成的库，并分析了库中克隆的特征。从图书馆随机分离而没有筛选的20个克隆表明CDR中的高度差异序列如预期的。通过平移方法分离与母鸡白色溶菌酶（HEL）结合的噬菌体抗体。噬菌体结合噬菌体的结合噬菌体增长，这一过程的一轮需要两天。三轮平移后，从回收的噬菌体中分离了20个克隆。其中19个具有抗螺旋活性，分布于10^6到10^7 m^<-1>的结合常数，具有抗螺旋活性的抗体的CDR的糖浆表明其中一半以上是独特的，其余的残留物分开。这表明表现出抗螺旋活性需要一些残基，但某些残基并不是必需的。由于本研究中构建的库是根据抗铁抗体D1.3的序列分歧的，因此该文库可以覆盖的抗原可能是有偏见的。现在，我们正在进行各种抗原特异性抗体的CDR癫痫发作的嫁接实验，并将使用移植的克隆作为模板进行多样性CDR序列。