Preparation And Characterization Of A Single Chain Antibody Of MAb SZ-123Against VWF-A3Domain

Background:Thromboembolic diseases are life-threatening diseases that affect millions of people each year. Thrombosis research is a major subject in field of medical sciencs.90%myocardial infarction and80%stroke result from arterial thrombus. Arterial embolism cardiovascular diseases are always the first cause of death in developed countries, venous thromboembolism are the third cause of death of cardiovascular diseases after myocardial infarction and stroke in developed countries, of which platelet play a very important role in arterial thrombosis.As a result, antiplatelet therapies are crucial measure in prevention and treating of thromboembolic diseases. Classics antithrombus drugs like the first generation of aspirin and ticlopidine which are applied widespreadly in clinic can inhibit accordingly the single procedure mediated by TXA2and ADP with the effect of prevention thrombosis formation in the process of platelets activation. The mechanism of the second generation antithrombus drugs is mainly to block the binging of GPâ…¡b/â…¢a to Fg. Recently as the spotlight had been at the early stage of arterial thrombus, the mechanism of molecular interaction between Collagen-vWF-GP â…  b axis and new targets aiming at antithrombus therapies had become one of the hot spots and frontier subjects in the field of hemostasis and thrombosis.Owing to the earlier and better antithrombus activity to block the platelets adhesion and activation aiming at collagen-vWF-GP â…  b axis, new generation antithrombus drugs had more expansive application prospect.Zhao YM reported mAb SZ-123against vWF-A3domain which not only could specifically block the binging of vWF to collagen,but aslo inhibit platelet aggregation induced by ristocein(GP â…  b bings to vWF-A1domain).The result suggested that the binging site of vWF-A3 domain to collagen possibly modulateed vWF-A1domain.In addition, it had been demonstrated that mAb SZ-123had the antithrombotic effects in the monkeys. MAb SZ-123against vWF-A3domain which not merely specifically block the binging vWF-A3domain of to collagen, but aslo inhibit platelet aggregation induced by ristocein.So far, it had been ruled out the interaction between mAb SZ-123and vWF-A1domain,it was supposed that mAb SZ-123interacted vWF-A3domain as well as influenting space structure of vWF-A1domain, then changed its space structure,so that it influented the binging of vWF-A1domain to platelets and had the effects of antithrombus. Therefore, it was in favor of explaining the mechanism of mAb SZ-123in protein space structure to prepare small molecular ScFv which was the vital objective to develop mAb SZ-123.Objective:The study aimed to prepare a single chain antibody of mAb SZ-123against vWF-A3domain, the molecular weight of which was smaller than mAb SZ-123by gene engineering technique.The goal of this investigation was to test whether the characterization of SZ-123ScFv was similar to mAb SZ-123or not by prokaryotic and eukaryotic expression and characterization study,so that it was favorable for explaining the mechanism of the interaction between mAb SZ-123and different structural domains of vWF in protein space structure, providing the basis for humanizing mAb SZ-123and investigating antithrombus mechanism of mAb SZ-123.Meanwhile it was in favor of lying a foundation for developing antithrombus agents based on mAb SZ-123in future.Methods:(1)First of all, VH and VL of monoclonal antibody (mAb) SZ-123were amplified by RT-PCR,and concatenated with linker subsequence to SZ-123ScFv. The prokaryotic vector of SZ-123ScFv was constructed and transformed into E.coli.Then SZ-123ScFv was prokaryoticly expressed successfully and concentrated. At last, the immunogenicity of SZ-123ScFv was tested by ELISA and Western blot.(2))Firstly, VH and VL of monoclonal antibody (mAb) SZ-123were amplified by RT-PCR,and concatenated with linker subsequence to SZ-123ScFv. The eukaryotic vector pSectag-123ScFv of SZ-123ScFv was constructed and transfected into the CHO Cells. Then SZ-123ScFv was eukaryoticly expressed successfully and concentrated. Finally, the immunogenicity of SZ-123ScFv was tested by SDS-PAGE and Western blot.(3) To test characterization of SZ-123ScFv by inhibition of vWF binding to collagen assay:human collagen typeâ…¢ was immobilized onto microtiter plate wells. vWF was added before supernatant concentrated was added and incubated. Finally, SZ-123ScFv was detected by a polyclonal anti-vWF antibody conjugated with horseradish peroxidase (HRP). Visualisation was obtained with tetramethylbenzidine (TMB) and the absorbance was determined at450nm.(4) To test characterization of SZ-123ScFv by platelet aggregation induced by ristocein:health adult venous blood with sodium citrate anticoagulant was drawed and separated into PRP and PPP. Characterization of platelet aggregation was detected by platelet aggregation analyser after ristocein was added.Results:The prokaryotic expression of SZ-123ScFv:The VH and VL genes were homologous with the published gene sequences of mouse antibody variable region.Except for a little protein of secretory type, the recombinant protein was mostly in the form of inclusion bodies, and the yield was up to20%of the total amount of bacteria protein.The ScFv fragment which had biological activity were attained and had some effect of inhibiting platelet aggregation induced by ristocein (P<0.05).The eukaryotic expression of SZ-123ScFv:780bp ScFv after being concatenated was transferred into eukaryotic vector pSectag/SZ-123ScFv. After DNA sequencing test was right, pSectag-123ScFv was constructed success-fully.Then pSectag-123ScFv was transfected into CHO.After being tested by SDS-PAGEå'ŒWestern Blot, the molecular weight of interest protein attained was45kD.The results of the characterization study suggested that SZ-123ScFv could inhibit vWF binding to collagen typeâ…¢ with dose dependent,as well as inhibit platelet aggregation induced by ristocein. Comparing with mAb SZ-123, the effect of SZ-123ScFv descended Conclusions:(1)We constructed prokaryotic vector of SZ-123ScFv pET20b(+)-123ScFv successfully and transported it into. E.coli.After induction, we accomplished its prokaryotic expression and attained the interest protein which could bind specifically to vWF-A3domain.(2) We constructed eukaryotic vector of SZ-123ScFv pSectag-123ScFv successfully and screened monoclonal cells after transfecting it into CHO. We accomplished its eukaryotic expression of SZ-123ScFv.(3) Prokaryotic expression of SZ-123ScFv had weaker effect of inhibitting platelet aggregation induced by ristocein.And eukaryotic expression of SZ-123ScFv had the effect of inhibitting vWF binding to collagen as well as platelet aggregation induced by ristocein which descended compared with mAb SZ-123.(4) The goal of developing SZ-123ScFv and characterization study was to explain the mechanism of mAb SZ-123in protein space structure and further lay a fundation for developing antithrombus agents based on mAb SZ-123.In addition,it was use of investigating the mechanism of thrombosis formation furthermore and lying a foundation for developing new antithrombus drugs.