Elsevier

Thrombosis Research

Volume 109, Issues 2–3, 25 January 2003, Pages 137-144
Thrombosis Research

Regular Article
Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibα

https://doi.org/10.1016/S0049-3848(03)00152-XGet rights and content

Abstract

A recombinant single chain Fv (scFv) fragment with specific activity against platelet glycoprotein (GP) Ibα was developed and characterized. The scFv was generated from the SZ-2 hybridoma, which produced an anti-platelet antibody reactive to GPIbα. VH and VL gene segments were generated from the SZ-2 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR). After cloning into pUCm-T vector, the DNA sequences of both VH and VL genes were analyzed from two different clones, respectively, the same results were obtained. Comparison of SZ-2 variable region to the Kabat database showed that VH belonged to the mouse Ig heavy family XV while VL belonged to the mouse Ig kappa family XXVI. For assembly of the SZ-2 scFv, VH and VL fragments were cloned into pSW1-scFv successively. The scFv was arranged in VH–VL orientation, being joined together with a 15-amino-acid (Gly4Ser)3 linker. The scFv encoding sequence was amplified and cloned into pET22b vector in-frame with a pel B leader sequence to direct secretion of the protein. Escherichia coli strain BL-21(DE3)PlysS was transformed with the recombinant plasmid, and expression of the scFv was induced using isopropyl-β-d-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 31,000. By comparing band intensity on a Coomassie brilliant blue-stained SDS-PAGE, the production yield of SZ-2 scFv was about 25% of the total cellular proteins. The recombinant SZ-2 scFv antibody was successfully purified using Ni-NTA affinity chromatography with a yield of 120 mg/l. The SZ-2 scFv antibody could bind to platelets demonstrated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Analyzed by Western blot, it could bind to platelet GPIb. It retained the binding capacity of its parental SZ-2 monoclonal antibody (MoAb). In functional studies, SZ-2 scFv inhibited platelet agglutination and aggregation induced by ristocetin and thrombin, respectively, but had no effect on ADP-induced platelet aggregation. Therefore, SZ-2 scFv has the potential to be used as an antithrombotic agent.

Section snippets

Cells, vectors, bacterial strains, oligonucleotide primers and reagents

The hybridoma secreting the GPIb-specific monoclonal antibody SZ-2 was generated, as described previously [6], and cells were grown on DMEM, 10% fetal calf serum with 5% CO2 at 37 °C. Plasmid pSW1-scFv were kindly provided by Dr. Greg Winter (MRC Lab., University of Cambridge, Cambridge, UK). Plasmid pET22b and E. coli strain BL-21(DE3)PlysS were obtained from Novagen (Novagen, Darmstadt, Germany). Qiaex II gel extraction kit, Ni-NTA Resin and Penta-His antibody were purchased from QIAGEN

Cloning and sequencing of MoAb SZ-2 variable region genes

The amplified VH and VL fragment were approximately 360 and 320 bp, respectively (Fig. 2). Then VH and VL fragments were cloned into the pUCm-T vector and sequenced. The DNA sequences of VH or VL gene from two different clones were identical. Comparison of SZ-2 variable domains to the Kabat database showed that VH of SZ-2 antibody belonged to the mouse Ig heavy family XV and VL belonged to the mouse Ig kappa family XXVI. The DNA sequences of VH and VL have been submitted to GenBank, and the

Discussion

Engineered monoclonal antibodies have been proven of value in the therapy and diagnosis of a wide range of diseases [9], [12], [13]. This strategy has been successfully applied to thrombotic diseases. Abciximab, a chimeric Fab fragment, is effective in the prevention of ischemic complications in patients undergoing high-risk coronary angioplasty or atherectomy [14], [15]. Our previous studies showed that SZ-2 is a potent inhibitor of ristocetin-induced, vWF-dependent platelet agglutination [6]

Acknowledgements

This work was supported by a grant from the Natural Science Fund of Jiangsu Province (project BK 2002137).

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