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单抗(8E/9)H链和L链可变区的编码区。将编码H链Fv的DNA与编码L链Fv的DNA通过间隔区(Gly4Ser)串联，构建成单链Fv-DNA。用含单链Fv-DNA的载体转化大肠杆菌，回收单链Fv包涵体，变性后用尿素缓冲液提取。复性是通过透析缓冲液中的蛋白质以降低尿素浓度来进行的。在复性过程中，有单链Fv的析出。用放射免疫分析法检测含有单链抗体的乳剂具有轻微的GA结合活性。利用单链Fv基因转化烟草。Northern杂交分析表明，转化植株中产生了单链Fv的mRNA，但没有观察到形态上的变化。

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.