Expression and characterization of the ScFv fragment of antiplatelet GPIIIa monoclonal antibody SZ-21.
Platelet thrombus formation is a major contributor to various cardiovascular diseases caused by vascular occlusion. Glycoprotein IIb/IIIa (GPIIb/IIIa) plays a key role in platelet aggregation and hence the formation of thrombi. In the present study, the genes encoding the light- and heavy-chain variable regions (V(H) and V(L)) of SZ-21, which is a monoclonal antibody (MoAb) against GPIIIa integrin have been cloned by RT-PCR from the SZ-21 hybridoma. The genes of V(H) and V(L) were attached to the oligonucleotide of the linker peptide and single-chain antibody fragment (ScFv) was constructed. The ScFv was ligated into phage display vector pHEN1, and the phagemid pHEN1-21ScFv was constructed. The high-affinity phage display technology was used to retain the SZ-21ScFv binding activity to platelets in great effort. After four rounds of enrichment, the screening clone of SZ-21ScFv gene with good reactivity to platelets was ligated into highly expressed vector pET20b and expressed in Escherichia coli BL21(DE3)PlysS for the fusion protein. Recombinant ScFv fragment was produced mostly in the form of inclusion bodies, with its yield up to 21% of the total amount of bacteria protein. The ScFv fragment with the similar binding activity to platelets as MoAb SZ-21 was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blot. ADP-induced platelet aggregation can be inhibited by ScFv fragment in a dose-dependent manner and the maximal inhibition rate was obtained at a concentration of 750 nM. In addition, the ScFv fragment has ability to inhibit the binding of fibrinogen to platelets and react with endothelial cells. In this study, we have successfully produced the SZ-21ScFv, which retained the binding affinity to platelets and antithrombotic ability of their murine counterparts.
An
Guangyu
G
Thrombosis and Haemostasis Research Unit, Jiangsu Institute of Hematology, First Affiliated Hospital of Suzhou University, Suzhou, China.
Dong
Ningzheng
N
Shao
Bojing
B
Zhu
Mingqing
M
Ruan
Changgeng
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics*; D001792:Blood Platelets / Q000276:immunology / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005340:Fibrinogen / Q000378:metabolism; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D066298:In Vitro Techniques; D039661:Integrin beta3 / Q000276:immunology*; D051379:Mice; D010974:Platelet Aggregation
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D039661:Integrin beta3; D005340:Fibrinogen