Abstract:

The variable light and heavy chain genes from anti-malachite green (MG) monoclonal cells were cloned and
linked with peptide (Gly4Ser)3 to construct a single chain variable fragment (scFv) antibody gene. The gene was inserted into
the vector plip6/GN containing alkaline phosphatase (PhoA) gene to construct the recombinant plasmid plip6/GN-MG-scFv.
After the induction of E. coli BL21 containing the recombinant plasmid with isopropyl β-D-thiogalactoside (IPTG), the
protein from the periplasm was detected by SDS-PAGE and Western blotting. The results demonstrated that the fusion
protein scFv-PhoA (about 72 kD) was expressed successfully. A one-step enzyme-linked immunosorbent assay (ELISA)
was developed (IC50 = 9.81 ng/mL) by using scFv-PhoA fusion protein with disodium 4-nitrophenyl phosphate (pNPP) as
the substrate. These results demonstrate that fusion protein scFv-PhoA can be a homologous reagent which simplifies and
accelerates the detection of malachite green.

Key words: malachite green, single chain antibody, alkaline phosphatase, fusion expression, direct competitive enzyme-linked immunosorbent assay