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The Proximal Carboxyl-terminal Domains of ADAMTS13 Function Study under Flow Conditions

Author: SunZuo
Tutor: AiJiHui
School: Huazhong University of Science and Technology
Course: Obstetrics and Gynaecology
Keywords: ADAMTS13 COS7 cell transient transfection force-stress Flow Chamber system
CLC: R341
Type: Master's thesis
Year: 2011
Downloads: 4
Quote: 0
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Abstract


Objective: Under flow conditions, to compare the cleavage activity and influence on platelets adhesion between ADAMTS13 and its recombinants.Method:(1) ADAMTS13 and its recombinants was transformed into E.coli DH5αand amplified, then confirmed by double enzyme digestion and DNA agarose gel electrophoresis and DNA sequence.(2) Transient transfect confirmed plasmids of ADAMTS13 and its combinants into COS7 cell, collect the supernatant of cell culture media, then initial purify protein with Centrifugal Filter Devices and detect the protein by Western Blotting with anti-V5 IgG.(3) Culture HUVECs(Human Umbilical Vein Endothelial Cell) on the Flow Chamber system, and imitate the under flow condition in the veins of human by peristaltic pump, compare the cleavage activity of UL-VWF secreted by damaged HUVECs and ability of reduce the platelets adhesion and stay thrombosis formation between ADAMTS13 and its recombinants.Result:(1) Successfully transient express ADAMTS13 and its recombinants in eukaryotic cell and initial purify ADAMTS13 and variants proteins, and confirm the proteins by Western Blotting.(2) Imitate the under flow condition in the vein of human by Flow Chamber system, FL-ADAMTS13 and MDTCS proteins may reduce platelets aggregation in different degrees, but the ability to reduce platelets aggregation of delCUB protein significantly decrease.Conclusion:(1) Successfully transient express ADAMTS13 and its recombinants.(2) Tentative confirm that under flow conditions,the proximal carboxyl-terminal domain of ADAMTS13 play an important role in cleave VWF in order to inhibit platelets aggregation.

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