Construction of Libraries of antibodies and its application

抗体文库的构建及其应用

• 批准号: 09557030
• 负责人: KUROSAWA Yoshikazu
• 金额: $ 6.91万
• 依托单位: Fujit Health University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09557030/
• 关键词: recombinant DNA; antigen antibody interaactions; CDR framework region; antibody library; phage-display system; ファージ抗体; 抗体レパートリー; ヒト抗体; 抗原抗体反応; error-prone PCR; 抗体-抗体反応; 人工抗体; ステロイド抗原; チトクロームC抗原; γ-グロブリン製剤; error-pronePCR; ヒト型抗体

We examined the conditions for constructing libraries of antibodies from which antibodies can be isolating only by screening with various antigens. It has been well known that antibodies bind specifically to antigens through the structures formed by six CRDs (complementarity-determining regions) that are located in V regions of H and L chains. Our basic design for construction of libraries of antibodies is diversification of amino acid sequences of six CDRs. We used the phage -display system. First, we compared the form of antibody expressed on the phage surface among Fab form, single chain Fv form and Fv form and we adopted the Fab form of antibody. Next, we have developed the strategy for changing antigen specificity using steroid antigens and antibodies. Thirdly, we performed CDR-grafting experiments and found that amino acids at several positions in the framework regions and their combinations gave substantial effects on the structures formed by the CDRs. Thus, we obtained various precious informations upon antigen-antibody interactions and the characteristics of phage-display system. For construction of libraries of antibodies. however, we adopted completely different strategies later on.

我们研究了构建抗体文库的条件，仅通过用各种抗原筛选就可以从中分离抗体。众所周知，抗体通过位于H链和L链V区的6个CRD（互补决定区）形成的结构与抗原特异性结合。我们构建抗体文库的基本设计是六个CDR的氨基酸序列的多样化。我们使用噬菌体展示系统。首先，我们比较了噬菌体表面表达的抗体的Fab形式、单链Fv形式和Fv形式，并采用了Fab形式的抗体。接下来，我们开发了使用类固醇抗原和抗体改变抗原特异性的策略。第三，我们进行了CDR移植实验，发现框架区多个位置的氨基酸及其组合对CDR形成的结构有显着影响。因此，我们获得了有关抗原抗体相互作用和噬菌体展示系统特征的各种宝贵信息。用于构建抗体库。但后来我们采取了完全不同的策略。

项目成果

H. Yasui: "Temperature dependency of the thermodynamic parameters in interactions between he egg-white lysozyme (HEL) and anti-HEL antibody"J. Biochem.. 123. 827-831 (1998)

H. Yasui：“蛋清溶菌酶 (HEL) 和抗 HEL 抗体之间相互作用的热力学参数的温度依赖性”J。

Hisashi Yasui: "Temperature dependency of the thermodynamic parameters in interactions between hen egg-white lysozyme (HEL) and anti-HEL antibody." J.Biochemistry. 123. 827-831 (1998)

Hisashi Yasui：“鸡蛋清溶菌酶 (HEL) 和抗 HEL 抗体之间相互作用的热力学参数的温度依赖性。”

Chie Miyazaki: "Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis." Protein Engineering. in press.

Chie Miyazaki：“通过位点特异性诱变和随机诱变来改变抗体的特异性。”

Y.Iba: "Expression vectors for introduction of highly diverged sequences into the six complementarity-determining regions of antibody." Gene. 194. 35-46 (1997)

Y.Iba：“用于将高度分化的序列引入抗体的六个互补决定区的表达载体。”

Y.Iba: "Comparison of strategies for the construction of libraries of artificial antibodies"Immunol.and Cell Biol.. 75. 217-221 (1997)

Y.Iba：“人工抗体文库构建策略的比较”Immunol.and Cell Biol.. 75. 217-221 (1997)