Construction, expression and functional characterization of Single-Chain Variable Fragment (ScFv) against human plasminogen
Mahboobeh Saifi Abolhassan Department of Biochemistry School of Biological Science,
Tarbiat Modares University Tehran, Iran
Abstract:
Circulatory disorders such as ischemic stroke, pulmonary embolism, deep-vein thrombosis and myocardial infarction are increasingly leading to mortality in modern societies worldwide. Thus, over the past decade, fibrinolysis therapy has revolutionized the treatment of these circulatory disorders. Important mechanism in the initiation and propagation of local fibrinolytic activity is the activation of plasminogen to plasmin. We have been able to characterize a monoclonal antibody (mAb A1D12) that enhances the activation of plasminogen 3-50-fold in the presence of plasminogen activators. Total RNA was isolated from the hybridoma cell line (A1D12) and was used as a template to make the first strand of cDNA. Based on the FR1 (framework region 1) and FR4 conserved regions of A1D12 gene, the variable regions of heavy chain (VH) and light chain (VL) were amplified and were connected to a single chain Fv (scFv) by SOE-PCR (splice overlap extension PCR). The DNA fragments containing sequences encoding VL- linker- VH were cloned into a bacterial pET26b (+) expression vector. An active form of a single-chain antibody (scFv) was successfully produced in Escherichia coli. The size of VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively.Following transformation of Escherichia coli cells, a recombinant ScFv protein was successfully expressed in the periplasmic space. The purity of the scFv protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as a 33 kDa homogenous single band. The plasminogen binding ability of the scFv was determined by an ELISA assay. The immunological properties of scFv were identical to those of the parental murine MAb, specifically in recognition of the human plasminogen. The fibrinolytic activity of the protein was determined to be similar to the parent monoclonal antibody which highlights its potential for fibrinolysis therapy.
