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

Dan,Zhaokui; Tan,Zui; Xia,Hongli; Wu,Gan

doi:10.3892/etm.2013.1132

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

Authors:
- Zhaokui Dan
- Zui Tan
- Hongli Xia
- Gan Wu
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Affiliations: Department of Vascular Surgery, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China, Department of Hepatobiliary Gastrointestinal Surgery, Xianning Central Hospital, Xianning, Hubei 437100, P.R. China, Centre of Inspection, Detection and Testing on GMO Environmental Biosafety, The Ministry of Agriculture, Wuhan, Hubei 430062, P.R. China
Published online on: May 30, 2013 https://doi.org/10.3892/etm.2013.1132
Pages: 552-556

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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.

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