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Cloning of apoB intrabodies: specific knockdown of apoB in HepG2 cells |
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| Authors: | Liao Wei Strube Randall W Milne Ross W Chen Si-Yi Chan Lawrence |
| Institution: | a Department of Medicine and Molecular & Cellular Biology, Baylor College of Medicine, St. Luke’s Episcopal Hospital, Houston, TX, USA b Department of Molecular & Human Genetics and Center for Gene Therapy, Baylor College of Medicine, Houston, TX, USA c Department of Pathology and Laboratory Medicine, University of Ottawa Heart Institute, Ottawa, Ont., Canada d St. Luke’s Episcopal Hospital, Houston, TX, USA |
| Abstract: | Apolipoprotein (apo) B is essential for the assembly and secretion of triglyceride-rich lipoproteins made by the liver. As the sole protein component in LDL, apoB is an important determinant of atherosclerosis susceptibility and a potential pharmaceutical target. Single-chain antibodies (sFvs) are the smallest fragment of an IgG molecule capable of maintaining the antigen binding specificity of the parental antibody. In the present study, we describe the cloning and construction of two intracellular antibodies (intrabodies) to human apoB. We targeted these intrabodies to the endoplasmic reticulum for the purpose of retaining nascent apoB within the ER, thereby preventing its secretion. Expression of the 1D1 intrabody in the apoB-secreting human hepatoma cell line HepG2 resulted in marked reduction of apoB secretion. This study demonstrates the utility of an intrabody to specifically block the secretion of a protein determinant of plasma LDL as a therapeutic strategy for the treatment of hyperlipidemia. |
| Keywords: | Apo apolipoprotein ER endoplasmic reticulum ALLN l-leucinyl-l-leucinyl-l-norleucinal" target="_blank">N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal NEM N-ethylmaleimide PAGE polyacrylamide gel electrophoresis sFvs single-chain antibodies |
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