[Expression of the metalloproteinase domain of von Willebrand factor-cleaving protease and preparation of its McAb].
The von Willebrand factor-cleaving protease (vWF-cp) is a newly identified plasma metalloproteinase and plays an important role in the pathogenesis of thrombotic microangiopathy. In the present study, the metalloproteinase domain of vWF-cp was expressed by IPTG-induced the recombinant engineered E.coli strain harbouring pET28a (+)-vWF-cp/MD and purified using chromatography on Ni-NTA column. Then the BALB/c mice were immunized with the recombinant protein to prepare the monoclonal antibodies (McAb) against vWF-cp and the obtained McAbs were characterized. Furthermore, the expression panels of vWF-cp in human normal tissues were investigated using immunohistochemistry. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 28% of total bacteria 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 belong to IgG(1). The concentration of them in ascites was 4 mg/ml, and their titers were as high as 1 x 10(-5). The data of ELISA showed that SZ-112 and SZ-113 recognized different epitopes of recombinant protein. The Western blot and immunoprecipitation data showed that the two monoclonal antibodies reacted not only with the recombinant protein, but also with a 200 kD protein in platelet lysate. Moreover, the vWF-cp was found to be present in the cytoplasm of many human tissues such as liver, prostate, ovary, etc. However, the protease could not be detected in brain tissue. In conclusion, the above-mentioned research work contributed not only to the further study of the structure and function of this protease, but also to the establishment of the method for quantifying the vWF-cp antigen in plasma.
Gao
Wei-Qiang
WQ
Jiangsu Institute of Hematology, The First Affiliated Hospital of Suzhou University, Suzhou 215006, China.
Shen
Fei
F
Su
Jian
J
Bai
Xia
X
Ruan
Chang-Geng
CG
D051722:ADAM Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000071120:ADAMTS13 Protein; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D014841:von Willebrand Factor / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human