Generation and characterization of a recombinant single chain Fv antibody to von Willebrand factor A1 domain from phage display library.
In this study, cDNA of von Willebrand factor domain A1 (VWF-A1) was cloned from human umbilical vein endothelial cells, and expressed in E. coli. The expressed VWF-A1 was used as antigen to immunize Balb/c mice. Reverse transcribed-polymerase chain reaction (RT-PCR) was used to amplify VH and VL gene segment from the RNA isolated from the immunized mouse spleens. Single chain Fv (ScFv) genes were generated by splicing overlap extension and subcloned into a phagemid vector pHEN-1. Phage display library of repertoire single chain antibodies of anti-VWF-A1 was then constructed and screened for ScFvs that interact with VWF-A1. The ScFv gene of phage clone 28 that has best binding activity to VWF-A1 was cloned into pET20b vector for higher expression in E. coli strain BL-21(DE3) pLysS. The recombinant ScFv specifically reacted with VWF, rVWF-A1 and rVWF-A1/A3, but not with rVWF-A3, P-selectin and BSA. It inhibited ristocetin-induced platelet aggregation with an IC50 of 0.75 micromol/l, and its maximal extent of inhibition is 62.5%. The ScFv had no effect on thrombin-induced platelet aggregation. These data show that the ScFv is specific against VWF-A1 and could have a potential to be used as an antithrombotic agent.
Zhu
Huaiping
H
Thrombosis and Hemostasis Research Unit, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China.
Wang
Yingchun
Y
Jiang
Miao
M
Ji
Shundong
S
Bai
Xia
X
Ruan
Changgeng
C
D000818:Animals; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D010974:Platelet Aggregation / Q000187:drug effects / Q000276:immunology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012310:Ristocetin / Q000494:pharmacology; D014841:von Willebrand Factor / Q000737:chemistry / Q000276:immunology*
D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D014841:von Willebrand Factor; D012310:Ristocetin; D004247:DNA