Optimizing apoptotic activity of a modified caspase3 in cells infected by Hepatitis C virus
Date Issued
2005
Date
2005
Author(s)
Chang, Chi-Chih
DOI
en-US
Abstract
To develop anti- Hepatitis C virus therapy, we take a new strategy through modifying the pro-apoptotic molecule which could be specific activated by virus NS3 serine protease and then eliminating infected cells by apoptosis. Cofilin and caspase3 are selected as our targets and modified according to the specificity of HVC NS3 protease. Several plasmids encoding modified Cofilin and modified Caspase3 have been generated through introducing different peptide sequence recognized by HCV NS3 protease (NS3/4A, NS4A/4B, NS5A/5B site)(figure2,3). They are further examined on in-vitro mimetic system through co-transfecting with HCV NS3 protease into 293T cell.
Some modified cofilin which carry NS5A/5B site or NS4A/4B site could be cleaved by HCV NS3/4A protease, but they failed to induce apoptosis in 293T cell after processing.
Some modified caspase3 could be activated by HCV NS3 protease and induce cell apoptosis. After analyzing the protein expression level and the apoptosis makers through western immunoblotting, two clones - F88 (caspase3-NS5A/5B-88) and M (Caspase3-modified 2) were selected as our candidates. We examined the relationship between transfection dose, potency and toxicity by quantitating the degree of apoptosis with flow cytometry. After analysis, both clone could induce apoptosis efficiently. However, F88 clone showed certain extent of toxicity with higher transfection dose.
M clone (caspase3-modifed 2) was further examined in HCV subgenomic replicon (HCV type 1b NS3-NS5B in Huh7).It induced apoptosis at small population of cells, but the apoptotic effect was not significant. The reason may be low transfection efficiency and/or low efficient cleavage by NS3/4A protease in HCV replicon.
Subjects
C型肝炎
Hepatitis C virus
SDGs
Type
other
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