Study on the Domains of von Willebrand Factor and the Generation of Single Chain Fv to Its Domain A1

Thrombotic disease is a serious threat to human health, the mechanism of thrombosis is one of the major topics of the field of medical science. von Willebrand factor (vWF) by megakaryocytes, endothelial cell adhesion function of the large molecular weight glycoprotein, plays an important role in thrombus formation process, both with the platelet membrane glycoprotein (GP) I b / IX / Ⅴ, GP Ⅱ b / Ⅲ a combination, can be combined and collagen-mediated platelet adhesion and aggregation in the subcutaneous tissue of the damaged blood vessels, leading to thrombosis. In this process, the interaction between the collagen-vWF-GP I b is the early events of platelet activation, vWF is a bridge between the platelets and the vessel wall collagen molecules, thus blocking the role of vWF in this process, can effectively inhibit the formation of blood clots . Based on collagen-vWF-GP Ⅰ b interaction, vWF molecules. Umbilical vein endothelial cells as experimental material, the first application of recombinant DNA technology to clone the gene fragment of vWF functional domains, and expression in prokaryotic cells, to observe the biological function of the recombinant protein; immune mice and then the recombinant protein, phage display library screening specific blocking platelet adhesion, aggregation antibody, trying to get the superior performance of antithrombotic agents. vWF-A1 and platelet GP I b of Reese NS (ristocetin), botrocetin-binding site, heparin and collagen binding sites, structural areas vWF play important biological function. vWF-A3 region is a collagen binding site. By RT-PCR from human umbilical vein endothelial cells cloned vWF-A1, vWF-A3 region gene fragment and vWF-A1 / 3 chimeric gene. Sequencing the cloned gene sequence is completely correct, and then were constructed expression vector pQE-31-vWF-A1, pET-20b-vWF-A3 and pET-22b-vWF-A1 / 3 induced expression in E. coli, the The expressed protein were 30%, 46.7%, and 12.6% of the total bacterial proteins. Nickel nitrilotriacetic acid agarose (Ni-NTA agarose) after column purification, the purity of the target protein in more than 95%. The purified recombinant target protein by dialysis refolded the study of biological functions. The reorganization of the vWF-A1 (rvWF A1) having good biological activity. Analyzed by flow cytometry prove rvWF-A1 GP Ⅰ b of transfected CHO-K1 cells and platelets, the positive rate was 96.90% and 78.60%, respectively, indicating that the rvWF-A1 with the combination of GP Ⅰ b. Platelet aggregation was measured, the results show: the rvWF-A1 can not directly cause platelet aggregation, but can inhibit human plasma vWF ristocetin-induced platelet aggregation, and its inhibitory effect is dose-dependent manner, the IC ₅₀ the the rvWF - A1 concentration 0.56μmol / L, 1.4μmol / L when the concentration of the highest inhibition rate reached 84.70%. These results indicate that the expression in prokaryotic cells v recognized under the functional domains and its Al District genetically engineered antibody preparation English summary recognized the T Al District proteins competitive inhibition of plasma vWF and platelet GPIb binding, inhibition of ristocetin-induced platelet aggregation, having antithrombotic application prospects. E.coli recombinant v recognized the next A3 protein (d recognize the next A3), collagen binding assays and competitive inhibition experiments to analyze the biological activity of shaking recognize the next A3 protein refolding. 4 Put the next A3 and I, m, type VI collagen have binding activity, but there is a significant difference in binding capacity among Bu 12,198, p lt; 0.001, wherein m collagen binding highest rate, I type Secondly, VI lowest. And can competitive inhibition of vWF binding to collagen, inhibition rate of 54%. These results indicate that the four Put the next A3 has good biological function, can be used as a blocking agent for the intervention v Put T mediated platelet adhesion process. Restructuring v recognize T Al 3 (potassium recognized T Al has), four T-A3 recognized by indirect immunofluorescence flow cytometry, and the results show the eight recognized TAI/A3 chimera binding to platelets positive rate 70.4% of State recognized the next A3 can not be combined with platelet further recognized that v T and platelet glycoprotein GPIb Jiang X, binding sites located in the Al district. The four recognized the T Al/A3 chimeric not cause platelet aggregation, but that is recognized T Al/A3 chimera platelet incubation can block Rist. . In the induced plasma v stock T platelet aggregation, and showed a dose-dependent, ICS. Four recognized AI T 3 concentration. The .76 the PMO ratio, when the concentration of 1 .17 pm. Than the highest inhibition rate of 76.8%. The collagen ELISA binding assay results indicate that four Put T AI/A3 of combination and I, m, type VI collagen, but there is a statistically significant difference in the binding rate among F = n .259, p LT; 0.001 which m collagen combined with the highest rate, followed by the type I, VI minimum; at the same time it can be competitively inhibits v recognized with a combination of collagen type m, inhibition rate of 76%. To show that rvWF AI from 3 W recognize the T Al rvWF A3 dual function, while blocking v from T and GPIB collagen combined, and has good application prospects. Since the anti-GPD seven river lla monoclonal antibody 7E3 clinical treatment approved by the FDA used in patients with ischemic heart the PTCA postoperative reinfarction prevention, achieved satisfactory results, the antithrombotic antibody drug development has become the field research hot spots. V recognized feet GPIb.L. V receptor is activated platelet adhesion, aggregation and lead to thrombosis of the most important part, so v recognize GPIb binding domain antibody inhibition of platelet adhesion , aggregation and thrombosis. Order to reduce immunogenicity and side effects of the intact antibody, this study, phage display technology to build anti-human v T Al recognized set of single-chain antibody phage display library, screened and v that the next Al high-affinity single-chain antibody , this technique is the exogenous protein expressed by fusion with the phage coat protein on the phage particle surface, the genotype and phenotype of specific molecules in a phage, by repeated panning process to filter out high vw million. functional domains and its Al District genetically engineered antibody preparation Chinese Abstract affinity single-chain antibody, which not only expanded screening capacity, while avoiding the traditional hybridoma technique of cell fusion process, but also to overcome the hybridoma cells secrete antibodies unstable disadvantages. Therefore, the technology will soon be widely used for the development of genetically engineered antibody provides a quick and efficient technical means