Construction and expression of D-dimer and GPIIb/IIIa single-chain bispecific antibody

Abstract

The aim of this study was to construct a plasmid expressing glycoprotein IIb-IIIa (GPIIb/IIIa) and D-dimer single-chain bispecific antibody for the targeted therapy of thrombosis. The phosphorylated gene encoding the anti-GPIIb/IIIa single-chain variable fragment (scFv) and the gene encoding the anti-D-dimer scFv were amplified by PCR and linked in tandem by blunt-end ligation. The recombinant plasmid was transfected into the competent cell line HB2151 and identified by PCR and DNA sequencing. Then, the soluble recombinant antibody in bacterial lysates was purified by an NTA column and molecular sieve chromatography in turn. Finally, the binding specificity of the purified antibody was tested by enzyme-linked immunosorbent assay (ELISA). Results demonstrated that the construction of the expression plasmid was successful and the purified recombinant protein, which had a molecular weight of ∼56 kDa, was specific to GPIIb/IIIa and D-dimer. In conclusion, a plasmid expressing a bispecific antibody was constructed by a new method of blunt-end ligation. The soluble recombinant protein is a promising platform for target-oriented thrombolytic therapy.

Keywords: D-dimer; glycoprotein IIb-IIIa; single-chain bispecific antibody.

Figure 1.

Map of vector pIT2 harboring a single-chain variable fragment (scFv)-encoding insert. RBS, ribosome binding site; PelB, signal sequence; VH-linker-VL, scFv; His-tag, immunopurification tag. A TAG amber stop codon was present at the junction of the scFv gene and gIII. The presence of an amber stop codon allows the production of scFv molecules as soluble antibody molecules instead of scFv-pIII fusion proteins. The size of the empty vector pIT2 was 4.2 kb, while the size of the scFv insert was ∼750 bp.

Figure 2.

Design and various cloning steps leading to the final bispecific construct.

Figure 3.

Agarose gel electrophoresis of purified construct I. Lane 1, linear construct I (∼4,950bp); lane 2, duplicate; lane M, O’GeneRuler™ 1 kb DNA Ladder.

Figure 4.

Agarose gel electrophoresis of purified construct II. Lane 1, construct II (∼750 bp); lane M, DL2000 DNA Marker.

Figure 5.

Expression and purification of the diabody (10% SDS-PAGE). Lane 1, total protein of bacterial pIT2-A1G9 prior to induction; lane 2, total protein of bacterial pIT2-A1G9 induced with IPTG; lane 3, periplasmic lysates of bacterial pIT2-A1G9 induced with IPTG; lane 4, cell lysate precipitate of bacterial pIT2-A1G9 induced with IPTG; lane 5, the Ni-NTA column flow-through; lane 6, the eluate washed at a concentration of 30 mM imidazole; lane 7, the eluate washed at a concentration of 500 mM imidazole; lane M, protein marker. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IPTG, isopropylthio-β-galactoside.

Figure 6.

Purification of the diabody by molecular sieve chromatography (10% SDS-PAGE). Lane 1, target protein prior to purification by molecular sieve chromatography; lane 2, target protein purified by molecular sieve chromatography; lane M, protein marker. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Figure 7.

Binding specificity of the recombinant protein. Data are presented as mean ± SD. The statistical comparisons were made by t-test and differences were considered to be significant at P<0.05. BSA, bovine serum albumin.