| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Antibodies are widely used as a key reagent in various bioanalytical systems. However, conventional antibodies elicited in animals not only possess several disadvantages due to their large molecular weight (ca. 150000), but also sometimes lack enough affinity nor specificity. Thus, antibody-mimetic peptides (mini-antibodies) with a smaller molecular weight, which would be prepared by gene manipulation, are expected to be a novel and useful tools for developing highly sensitive and specific bioanalytical systems. From these points of view, we prepared two kinds of single-chain Fv fragments (scFvs ; MW ca. 25000) against 11-deoxycortisol (11-DC ; the biosynthetic precursor of cortisol) or (1R)-bufuralol (aβ-blocking agent), linking V_H and V_L domains of the corresponding mouse antibody via a flexible linker peptide.
The V_H-and V_L-genes were cloned by RT-PCR using the total RNA extracted from the relevant antibody-secreting hybridoma. These DNA fragments were spliced by overlap extension PCR introducing the linker sequence, which was then subcloned into an expression plasmid vector. E.coli cells were transformed with the recombinant, and the expressed scFv was prepared from the cells as a periplasmic extract. An RIA or ELISA analysis revealed that these scFvs possess excellent affinity and specificity to the relevant antigen, which are very similar to those of the original mouse antibodies, from which the V_H-and V_L-sequence has been derived. It is expected that a novel molecular-recognizing module with even smaller molecular weight would be generated based on the amino acid sequence of the complementarity-determining regions of these scFvs.
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