Identification of Protein-protein Interaction of Hepatitis B Virus X Protein and Tab1 <i>in Vivo</i> and <i>in Vitro</i>

doi:10.13523/j.cb.20180701

China Biotechnology

2018, Vol. 38

Issue (7): 1-6 DOI: 10.13523/j.cb.20180701

Identification of Protein-protein Interaction of Hepatitis B Virus X Protein and Tab1 in Vivo and in Vitro

Li-li YU,Bo HU,Xue LI,Nai-shuo ZHU(

)

College of Life Science, State Key Laboratory of Genetic Engineering, Laboratory of Microbial and Molecular Immunology, Fudan University, Shanghai 200438, China

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Abstract

With the help of analysis of mass spectrometry and data analysis according to the preliminary laboratory basic research, Co-IP and GST pull-down methods was used to prove the interaction of HBV X protein with Tab1. In order to study the carcinogenic mechanism of HBx protein, Some experimental evidence for further study of the role of HBx in the carcinogenic mechanism of HBV chronic infection were provided. pGEX-2TK-GST-HBx plasmid was successfully constructed, GST-HBx fusion protein was induced and incubated with GST-beads. pcDNA3.1/myc-His (-) B-Tab1 plasmid was constructed,then was transfected 293T cells to express Myc-Tab1. Finally the interaction of GST-HBx and Myc-Tab1 was verified in GST pull-down test. Moreover, the eukaryotic expression plasmids including pcDNA3.1/myc-His (-) B-Tab1 and pcDNA3.1-3×flag-HBx were successfully constructed, then were co-transfected into human embryonic kideny 293T cells and HepG2 cells to express fusion protein, further Co-IP test was demonstrated that the anti-Myc antibody could precipitate HBx from the cell lysate, and confirmed their intracellular interaction in two human cell lines. In conclusion, all results show that HBx and Tab1 could interact in vitro and in vivo, also established foundation to reveal the function and mechanism of HBV X protein.

Key words： HBV X protein Tab1 Co-IP GST pull-down Interaction

Received: 27 February 2018 Published: 13 August 2018

ZTFLH:

Q816

Corresponding Authors: Nai-shuo ZHU E-mail: nzhu@fudan.edu.cn

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Cite this article:

Li-li YU,Bo HU,Xue LI,Nai-shuo ZHU. Identification of Protein-protein Interaction of Hepatitis B Virus X Protein and Tab1 in Vivo and in Vitro. China Biotechnology, 2018, 38(7): 1-6.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180701 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I7/1

Fig.1 PCR products of human Tab1 M1:Trans 15 000 DNA marker,Tab1 of PCR;M2: Trans 2 000 DNA marker

Fig.2 Restriction enzyme digestion and colony PCR identification of pcDNA3.1/myc-His(-)B-Tab1(a)Double enzyme digestion identification M1: Trans 15 000 DNA marker; 1:Empty vector;2: Double digestion products;M2:Trans 2 000 DNA marker (b)Colony PCR identification M1:Trans 15 000 DNA marker; Clone 1-5: Selecting five monoclonal colonies;M2:Trans 2 000 DNA marker

Fig.3 Expression identification of pcDNA3.1/myc-His(-)B-Tab1

Fig.4 PCR products of HBxM1:Trans 2 000 DNA marker; HBx: Products of PCR

Fig.5 Restriction enzyme digestion and colony PCR identification of pGEX-2TK-GST-HBx (a)Double enzyme digestion identification M1: Trans 15 000 DNA marker; 1:Empty vector;2: Double digestion products;M2:Trans 2 000 DNA marker (b)Colony PCR identification M1: Trans 15 000 DNA marker; Clone 1-6: Selecting five monoclonal colonies;M2:Trans 2 000 DNA marker

Fig.6 IPTG Induction of GST-HBx fusion protein expressionM:Protein marker; 1-3:IPTG induction of GST-HBx; 4-6: IPTG induction of GST

Fig.7 SDS-PAGE test of GST-HBx fusion protein purification by Agarose BeadsM:Protein marker; 1: GST-HBx; 2: GST

Fig.8 Interaction between Tab1 and HBx in 293T cells by Co-IP

Fig.9 Interaction between Tab1 and HBx in HepG2 cells by Co-IP

Fig.10 Interaction between Tab1 and HBx in vitro


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