Preparation Of Monoclonal Antibodies Against Von Willebrand Factor-Cleaving Protease And Studies On The Association Of The Protease With Thrombosis And Tumor

von Willebrand factor-cleaving protease, also designated as ADAMTS13 (A Disintegrin And Metalloprotease with Thrombo.spondin type I motif 13). is a newly characterized plasma metalloproteinase that degrades unusually large von Willebrand factor multimers (UL-vWF) derived from endothelial cells (ECs) and megakaryocytes (MCs) into small peptides circulating in blood, von Willebrand factor (vWF) is a matrix glycoprotein that plays a key role in primary hemostasis through its anchoring action of platelets onto the denuded subendothelial matrices under high shear stress. The proteolysis of vWF multimers by vWF-cp is critical in regulating vWF-platelet interaction since the ability of vWF to promote platelet binding to subendothelium depends upon its molecular size, as the highly polymeric forms of vWF are the most active in interacting with platelets. Deficiency of vWF-cp activity might result in abnormal accumulation of the UL-vWF in plasma, and hence, contribute to thrombosis in microcirculation, such as thrombotic thrombocytopenic purpura (TTP). In addition, the pathological statuses of arterial thrombosis, malignant tumors, and liver disorders are often accompanied by the increasing of vWF antigen levels. The reasons responsible for the abnormal of vWF in the pathological statuses above are unclear and the activity of vWF-cp is also decreased in these statuses. In the present studies, the metalloproteinase domain of vWF-cp, which was cloned and expressed in E. Coli, was used as the antigen to prepare the monoclonal antibodies against the protease. Moreover, the association of vWF-cp with thrombosis and tumor and their clinical significance were also investigated.Preparation of monoclonal antibodies against the metalloproteinase domain of vWF-cp with recombinant proteinIn order to prepare monoclonal antibodies against vWF-cp, the gene of metalloproteinase domain of human vWF-cp was cloned into pUC18 and its accuracy was verified by sequencing. The gene was subsequently inserted into the multiclone site of pET28a(+) and included a 6 X His Tag at its amino terminal. After induced by IPTG, the recombinant protein was purified using a Ni-NTA column. High-level expression of the recombinant protein was yielded after 5-hour induction, which amounted to 28% of the total bacteria protein in inclusion body. Western blot demonstrated that it possessed highspecificity. Then the Bal b/c mice were immunized by the recombinant protein. Three monoclonal antibodies against the metalloproteinase domain of vWF-cp were obtained and two of them, SZ-112 and SZ-113, were further evaluated. Both of them belonged to IgG1 subclass. The quantity of them in ascites were 4 mg/ml, and their titers were as high as 1X 10-5. The data of competitive ELISA showed that SZ-112 and SZ-113 recognized different epitopes of the recombinant protein. Western blot and immunoprecipitation results demonstrated that the two antibodies not only reacted against the recombinant protein, but also reacted to a 200 KDa protein in platelet lysate. Moreover, the expression panels of vWF-cp in human normal tissues were investigated using immunohistochemistry. And the protease was found to be present in many kinds of tissues such as liver, spleen, prostate and ovary, etc. Our data indicated that two novel specific monoclonal antibodies against the metalloproteinase domain of human vWF-cp were successfully prepared, which might be contributing to the further study of the structure and function of this protease.Additionally, we have constructed the eukaryotic expression plasmid pcDNA3.1-vWF-cp that contained the full-length vWF-cp cDNA with polymerase chain reaction-splicing overlap extension (PCR-SOE) and enzyme digestion. The correctness of the plasmid was confirmed by PCR, enzyme digestion and sequencing. Then the pcDNA3.1-vWF-cp, mediated by liposome, was transfected into CHO cell line. Western blot results showed that the vWF-cp protein was present in both supernatant and cell lysate of the transfected cell. Thus our initiatory research work settled a solid fou...