Purification and function of the liver sinusoidal endothelial cell specific antigen, SE-1
Grant-in-Aid for Scientific Research (C)
|Research Institution||Akita University|
森田 真守 秋田大, 医学部, 助手 (20282163)
ENOMOTO Katsuhiko Akita Univ.School of Medicine, Professor, 医学部, 教授 (20151988)
|Project Fiscal Year
1997 – 1998
Completed(Fiscal Year 1998)
|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1998 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1997 : ¥2,600,000 (Direct Cost : ¥2,600,000)
|Keywords||liver sinusoidal endothelial cell / SE-1 antigen / 肝類洞内皮細胞 / SE-1抗原 / 膜抗原|
We previously generated a monoclonal antibody, SE-1, which reacts with the unknown antigen expressed on the cell membrane of rat sinusoidal endothelial cells (SEC). Our previous results suggested that the antigen may participate in the specific function of SEC.In this study. therefore, we tried to purify and characterize the antigen molecule.
1. Isolation of rat sinusoidal endothelial cells
The rat liver was perfused with 0.05% collagenase containing Hanks buffer through the portal vein, and the collagenase digested cell mixed solution was centrifuged at 700 rpm to remove the parenchymal cells. Five cell layers were obtained after ultracentrifugation of the non-parenchymal cells. The third layer from the top was confirmed as SEC rich fraction by the immuno blot and electron microscopy.
2. Affinity purification of SE-1 antigen
Protein A affinity column was used to purify the antigen. The detergent treated SEC fraction was applied to the column which was preloaded with the antibody. After elution, presence of SE-1 antigen in the protein rich fraction was demonstrated by the immuno blot.
3. Analysis of the protein sequence
The purified SE-1 antigen was electrophoreted and blotted on the PVDF membrane. The band of SE-1 antigen was analyzed by Protein sequencer (ABI 491). However, final sequences of the antigen molecule were not yet determined. Since some N-terminal modification is suggested, the further characterization of the sample protein is currently progressed.
Research Output (13results)