Preparation And Characterization Study Of Gene Engineering Antibody Against Platelets GP

Platelet thrombus formation is a major contributor to various cardiovascular diseases caused by vascular occlusion. Glycoprotein GPIIb/IIIa on the surface of platelets plays a key role in the final common pathway of platelet aggregation. Blocking the binding of GPIIb/IIIa to fibrinogen receptor could inhibit platelet aggregation potently and effectively, hence inhibit the formation of thrombi. SZ-2 1 is a murine monoclonal antibody reactive with the GPIIIa. Its anti-thrombotic effects were confirmed in rabbit carotid-vein thrombus formation models. It suggested that SZ-2 1 is a potential candidate for treating cardiovascular diseases. To reduce the human anti-mouse antibody response (HAMA) and the potential complement梞ediated lysis of platelets caused by Fe region of the Ab, we produced and purified the fragments of SZ-2 1 antibody. The anti-thrombotic effects of the fusion protein was characterized in vitro. 1 Cloning and sequences of the variable regions genes of SZ-21 monoclonal antibody The genes encoding the light- and heavy-chain variable regions(V~ and VL) have been cloned by RT-PCR from the SZ-2 1 hybridoma of the anti-platelets GPIIIa murine monoclonal antibody and sequenced. VH gene is 342 bp and VL gene is 306 bp, the genes consist of 114 and 102 amino acid residues respectively. Accession numbers of the VH and VL in genebank are AF3 54053 and AF3 54054. 2.. Screening and expression of a single chain antibody fragment (ScFv) III Cf The DNA encoding the variable regions were attached to the oligonucleotide of the linker peptide by means of recombinant DNA technique and single chain antibody fragment (ScFv) was obtained. Then we ligated the ScFv into phage display vector pHENI and constructed the phagemid pHJENI-2 1 ScFv. The high affinity phage display technology was used to retain the SZ-2 1 ScFv binding activity to platelets in great effort. Purified GPIIIa was coated on the tissue culture flask, affinity panning was used to select antigen-positive recombinant phage antibody. Clone of antibody fragment with good reactivity to platelets was isolated after 4 round of enrichment and soluble ScFv was produced in the E. coil HB2 151. The ScFv fragment with the similar binding activity to platelets as the mAb SZ-2 1 was confirmed by ELISA and Western blot. 3. Construction and high expression of single chain antibody fragment of mAb SZ-21 The screening ScFv gene was ligated into highly expressed vector pET2Ob, and the fusion protein was expressed in Eschrichia coli BL21(DE3)PlysS by IPTG induced. The induced band was in the MW 27000 distance. The recombinant ScFv fragment was mostly produced in the form of inclusion bodies and its yield was up to 21 % of the total amount of bacteria protein. A series steps, including cell breakage, inclusion body dissolution, and protein refolding were used to obtain fusion protein in an active form. The ScFv fragment remaining binding activity to platelet was confirmed by ELISA and Western blot. 4.. Purification and characterization of the SZ-2lScFv fragment The SZ-2 1 ScFv purity was over 95% through affinity chromatography. It was shown that the ScFv fragment reacted with endothelial cells and platelets by flow cytometry. It inhibited ADP-induced platelets aggregation in a dose-dependent manner and t...