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

克隆了抗GA_4单克隆抗体（8 E/9）H和L链可变区的cDNA。将编码H链Fv和L链Fv的DNA通过编码（Gly 4Ser）的间隔区DNA串联起来，构建单链Fv DNA。将含有单链抗体DNA的质粒转化大肠杆菌，回收单链抗体，得到不溶性包涵体，变性后用含尿素的缓冲液提取。通过在降低尿素浓度的缓冲液中透析蛋白质来进行重折叠。在复性过程中，单链Fv被沉淀。用放射免疫分析法检测，含有单链抗体的乳剂显示出轻微的GA结合活性。用单链Fv转化烟草。北方印迹分析表明，转化植株中产生了单链抗体的mRNA，但未观察到形态学变化。

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.