Synthesis of DNA coding the amino acid sequence in an anti-GA4-antibody responsible for the recognition of GA and its application.

编码负责识别 GA 的抗 GA4 抗体中氨基酸序列的 DNA 的合成及其应用。

• 批准号: 05453163
• 负责人: MUROFUSHI Noboru
• 金额: $ 4.67万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05453163/
• 关键词: Gibberellins; Anti-gibberellin antibody; Single chain Fv; Transgenic tobacco; トランスジェニック タバコ; 抗ジベレリン抗体遺伝子; scFv; 植物の形質転換

The cDNA coding variable regions of H and L chain of monoclonal anti-GA4-antibody (8E/9) was cloned. The single chain Fv-DNA was constructed by combining the DNAs coding Fv of H chain and that of L chain in tandem via a spacer DNA coding (Gly4Ser). E.coli was transformed with plasmids containing the single chain fv-DNA.Single chain Fv was recovered as insoluble inclusion body, which was denatured and extracted with buffer containing urea. The refolding was perfoumed by dialyzing the protein in buffers reducting the concentration of urea. During the refolding process, the single chain Fv was precipitated. The emulsion containing the single chain Fv showed slight GA binding activity when examined by radio immunoassay. Tobacco was transformed with the single chain Fv. Northern blot analysis showed that the mRNA of the single chain Fv was produced in the transformed plants, although no morphological change was observed.

克隆了单克隆抗ga4抗体（8E/9） H链和L链的cDNA编码可变区。将编码H链Fv和L链Fv的DNA通过间隔DNA编码（Gly4Ser）串联组合，构建单链Fv-DNA。用含有单链fv-DNA的质粒转化大肠杆菌。单链Fv作为不溶性包涵体回收，用含尿素的缓冲液变性提取。通过在缓冲液中透析蛋白质，降低尿素浓度来进行再折叠。在再折叠过程中，单链Fv析出。含单链Fv的乳状液经放射免疫分析显示有轻微的GA结合活性。烟草被改造成单链Fv。Northern blot分析表明，转化植株中产生了单链Fv mRNA，但未观察到形态变化。