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Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters
Published on: November 26, 2013
An efficient rapid system for assaying HBx-mediated transactivation
Xiaoling Zhou1,2,3, Haijun Shi1,2,3, Shaozhe Yang1,2,3
1Stem Cell P2 Laboratory, Shantou University Medical College, Shantou, 515041, People's Republic of China.
Biotechnology Letters
|April 8, 2017
Summary
A new GFP reporter assay effectively measures the transactivation ability of hepatitis B virus X protein (HBx). This rapid and accurate system aids in screening for HBx inhibitors and enhancers, crucial for understanding viral replication.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Hepatitis B virus X protein (HBx) plays a critical role in viral replication by transactivating cellular promoters.
- Existing assay systems for HBx transactivation may lack speed or accuracy.
- Developing a robust screening platform is essential for identifying therapeutic targets.
Purpose of the Study:
- To establish a novel, rapid, and accurate reporter assay for screening agents targeting HBx transactivation.
- To characterize the performance of this new assay system using known HBx regulators.
Main Methods:
- A Green Fluorescent Protein (GFP)-based reporter system was constructed using plasmids expressing HBx-FLAG and linked to EGFP via an internal ribosome entry site.
- Transactivation by HBx was quantified using fluorescence microscopy, quantitative PCR (qPCR), and flow cytometry.
- The assay's efficacy was validated by testing known HBx regulators (DDB1, ID1, P53) and modeling repressor function in Hep3B cells.
Main Results:
- The GFP reporter system successfully demonstrated and quantified the transactivation effect of HBx on the cytomegalovirus (CMV) promoter.
- Flow cytometry provided a quantitative measure of HBx transactivation ability.
- The assay accurately reflected changes in GFP levels in response to known HBx regulators, confirming its reliability.
Conclusions:
- The developed GFP-based reporter system offers a rapid and accurate method for analyzing the transactivation capability of HBx.
- This assay is a valuable tool for screening potential inhibitors or enhancers of HBx activity.
- The system facilitates further research into HBx function and its role in hepatitis B virus pathogenesis.
