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

1995 Fiscal Year Final Research Report Summary

• Project/Area Number: 05453163
• Research Category: Grant-in-Aid for General Scientific Research (B)
• Allocation Type: Single-year Grants
• Research Field: 応用微生物学・応用生物化学
• Research Institution: The University of Tokyo
• Principal Investigator: MUROFUSHI Noboru Department of Applied Biological Chemistry, The University of Tokyo, Professor, 農学部, 教授 (00011916)
• Co-Investigator(Kenkyū-buntansha): NAKAJIMA Masatoshi Department of Applied Biological Chemistry, The University of Tokyo, Research As, 農学部, 助手 (50237278) ; SUZUKI Yoshihito Department of Applied Biological Chemistry, The University of Tokyo, Research As, 農学部, 助手 (90222067) ; YAMAGUCHI Isomaro Department of Applied Biological Chemistry, The University of Tokyo, Associate P, 農学部, 助教授 (00012013)
• Project Period (FY): 1993 – 1995
• Keywords: Gibberellins / Anti-gibberellin antibody / Single chain Fv / Transgenic tobacco

Research Abstract

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.