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