Modulation of plumbagin production in Plumbago zeylanica using a single-chain variable fragment antibody against plumbagin

A single-chain variable fragment antibody (scFv) against plumbagin (PL) accumulated the PL production in the hairy roots of Plumbago zeylanica. Recombinant Agrobacterium rhizogenes (ATCC 15834) containing an scFv gene against PL (PL-scFv) were obtained through triparental mating and transformed into P. zeylanica to induce PL-scFv protein in the hairy roots. Up to 40 μg recombinant PL-scFv were expressed per milligram of soluble protein in transgenic P. zeylanica hairy root cultures. The mean PL content obtained from transgenic hairy roots (12.24 μg/100 mg dry weight) exhibited 2.2 times higher than those obtained from wild-type (5.48 μg/100 mg dry weight). The high correlation between the PL-scFv expression level and PL content of the recombinant plants suggested that the PL biosynthesis pathway had been modulated by the expression of PL-scFv protein in the hairy roots of P. zeylanica.     

Effect of linker length between variable domains of single chain variable fragment antibody against daidzin on its reactivity

The peptide linker between variable domains of heavy (V_H) and light (V_L) chains is one of important factors that influence the characteristics of scFv, including binding activity and specificity against target antigen. The scFvs against daidzin (DZ-scFvs) with different linker lengths were constructed in the format of V_H-(GGGGS)_n-V_L (n = 1, 3, 5, and 7). They were expressed in the hemolymph of silkworm larvae using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system, and their reactivity against daidzin and related compounds were evaluated using an indirect competitive enzyme-linked immunosorbent assay (icELISA), which is applicable for quantitative analysis of daidzin. The results showed that the reactivity of scFvs against daidzin was increased, whereas specificity slightly decreased when their peptide linker was lengthened. These results suggested that the linker length of DZ-scFvs contributes to its reactivity. In addition, the results emphasize that the linker length could control the reactivity of DZ-scFvs.     

A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay

A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv.