재조합 대장균을 이용한 곰팡이 독소 deoxynivalenol에 대한 특이적인 재조합 항체의 생산

Thesis (master`s)--서울대학교 대학원 :농생명공학부,2003.The variable region genes of a monoclonal antibody specific for trichothecene mycotoxin deoxynivalenol (DON) were cloned, characterized and expressed in recombinant Escherichia coli. The sequence analysis revealed that the variable regions belong to the mouse heavy chain subgroup I and light-chain subgroup ¥ë-2, respectively. The variable heavy (VH) and variable light (VL) genes were combined by overlap-extension PCR using a flexible DNA linker encoding polypeptide (Gly4Ser)3 as single-chain variable fragment (scFv). In order to express the funtional scFv antibody in recombinant E. coli, various expression strategies were introduced. Coexpression of molecular chaperone (DnaK-DnaJ-GrpE) with the anti-DON scFv antibody increased the soluble fraction of the scFv. The scFv antibody containing the His 6-tag was purified by immobilized metal affinity chromatography (IMAC). The kinetic analysis for interactions between ligand (Ag) and analyte (Ab) were carried out by the BIAcore system using surface plasmon resonance (SPR). The KD-value of the anti-DON scFv antibody was found to be approximately 200-fold lower than that of the anti-DON monoclonal antibody.Maste