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The Establishment of a High-throughput Screening Model for HCV NS3 Serine Protease Inhibitors

Author: ChenNa
Tutor: FuXueQi；ZhaoZhiHu
School: Jilin University
Course: Microbial and Biochemical Pharmacy
Keywords: HCV NS3 serine protease inhibitors HTS NS5A / B EGFP
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 104
Quote: 1
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Abstract


Hepatitis C virus (HCV) infection has become a global public health problem , there is no specific drug . Is a novel method to screen inhibitor drugs by measuring the activity of the enzyme molecules . Due to the drug candidate is preferably play a role in the cells , therefore , the establishment of the enzymatic activity in a cell level detection system for high throughput screening of the significance of anti-HCV drugs . HCV NS3 serine protease is a common target for the development of anti-HCV drugs . Based on this, initially established a cell level of the NS3 serine protease activity detection method , and to prepare for further high - throughput screening for inhibitors . The experiments selected two models of E.coli and HeLa cells . In prokaryotic systems , plasmids , co-expression of two proteins . In eukaryotic systems , by introducing F2A polypeptide sequence , the two target gene in series together, transiently transfected cells for achieving co-expression of two proteins . First, make sure the reporter gene . At 172-173aa of EGFP insert NS5A/5B sequence recorded as EGFP5AB . Achieve EGFP5AB separate expression in prokaryotic and eukaryotic systems , the co-expression of the N terminus of EGFP (1-172aa) and C-terminal (173-238aa) fragment . Observed under fluorescence microscope green fluorescent , EGFP5AB expression alone , while no expression of EGFP in the N-terminal and C-terminal fragment of green fluorescence . Prokaryotic and eukaryotic systems , EGFP5AB can be used as a reporter gene . Next, the detection of the activity of the NS3 serine protease . , Respectively, to achieve the the NS3/4A and EGFP NS3/4A and EGFP5AB co-expressed in prokaryotic and eukaryotic systems . The results showed that green fluorescent NS3/4A and EGFP expression ; while the NS3/4A and EGFP5AB co-expression of green fluorescence . Description NS3 serine protease specificity and cut NS5A/5B sites . Shows that green fluorescent reporter molecules reflect the NS3 serine protease activity , and laid the foundation for further screening NS3 protease inhibitor .

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