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Biotechnology Letters

, Volume 39, Issue 7, pp 1091–1099 | Cite as

An efficient rapid system for assaying HBx-mediated transactivation

  • Xiaoling Zhou
  • Haijun Shi
  • Shaozhe Yang
  • Pingnan Sun
Original Research Paper

Abstract

Objectives

To develop a rapid and accurate assay system for screening inhibitors or enhancing agents targeting the transactivation capability of hepatitis B virus X protein (HBx) that activates cellular promoters in host cells to facilitate viral replication.

Results

We constructed a new GFP-based reporter system which was different from a luciferase-based reporter system. Firstly, a FLAG-tagged HBx gene was inserted into an expression plasmid, resulting in plasmid pHBx. Next, HBx-FLAG was linked to EGFP by the internal ribosome entry site resulting in plasmid pHBxE. The transactivation effect of HBx-flag on cytomegalovirus (CMV) promoter was verified by EGFP expression using fluorescence quantitation and qPCR. Furthermore, the transactivation ability of the HBx gene was quantified by flow cytometry. Finally, this assay system was tested by known regulators of HBx including DDB1, ID1, and P53. As expected, the GFP reporter level in 293T cells changed with the increasing of HBx regulators. Furthermore, the system modeling the function of transactivation repressor in Hep3B, a HBV-integrated cell line.

Conclusion

Collectively, the GFP-based reporter system provides a rapid and accurate approach for analyzing transactivation ability of HBx.

Keywords

Cytomegalovirus promoter GFP Hepatitus B virus X protein HBx gene Transactivation Virus X protein 

Notes

Acknowledgments

This work was supported by the National Nature Science Foundation of China (No. 81570567, 81571994), the Natural Science Foundation of Guangdong province, China (No. 2014A030313483, 2015A030313447), Guangdong Government under the Top-tier University Development Scheme for Research and Control of Infectious Diseases, and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. We would like to thank Dr. Stanley Lin for kindly revising this manuscript.

Supporting information

Supplementary Figure 1—Melting curves of qPCR showed good specificity of EGFP and HBx primers.

Supplementary Figure 2—ELISA standard curve for HBsAg (R square > 99%).

Supplementary material

10529_2017_2334_MOESM1_ESM.tif (234 kb)
Supplementary material 1 (TIFF 234 kb)
10529_2017_2334_MOESM2_ESM.tif (132 kb)
Supplementary material 2 (TIFF 131 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Xiaoling Zhou
    • 1
    • 2
    • 3
  • Haijun Shi
    • 1
    • 2
    • 3
  • Shaozhe Yang
    • 1
    • 2
    • 3
  • Pingnan Sun
    • 1
    • 2
    • 3
  1. 1.Stem Cell P2 LaboratoryShantou University Medical CollegeShantouPeople’s Republic of China
  2. 2.The Center for Reproductive MedicineShantou University Medical CollegeShantouPeople’s Republic of China
  3. 3.Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular ImmunopathologyShantou University Medical CollegeShantouPeople’s Republic of China

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