Cloning and functions of the HBxAg-binding protein XBP1

Fan,Wanhu; Cheng,Jun; Zhang,Shulin; Liu,Xiaojing

doi:10.3892/mmr.2012.1232

Cloning and functions of the HBxAg-binding protein XBP1

Authors:
- Wanhu Fan
- Jun Cheng
- Shulin Zhang
- Xiaojing Liu
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Affiliations: Department of Infectious Diseases, The First Affiliated Hospital of Medical School, Xi'an Jiaotong University, Xi'an 710061, P.R. China
Published online on: December 13, 2012 https://doi.org/10.3892/mmr.2012.1232
Pages: 618-622

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Abstract

In the present study the hepatitis B virus X antigen binding protein 1 (XBP1) was cloned by inducing its expression, and its subcellular localization and function were examined. Total RNA was extracted from HepG2 cells and XBP1 was amplified using reverse transcription polymerase chain reaction (RT-PCR), followed by restriction enzyme digestion of the pGBKT7 yeast plasmid and identification by enzyme digestion. The plasmid was transformed into AH109 yeast via the lithium acetate method and protein extracts were prepared. XBP1 protein expression in the eukaryotic cells was determined using polyacrylamide gel electrophoresis and western blot analysis. The gene encoding the XBP1-binding protein was screened in liver cells using yeast two-hybrid technology. We transfected a human hepatocellular carcinoma cell line and observed the intracellular localization of the gene expression protein using a fluorescence microscope, followed by prokaryotic expression and XBP1 gene identification. A 921-bp XBP1 gene fragment was obtained via RT-PCR amplification and 20 proteins with known functions that interact with XBP1 were screened, including metallothionein, smooth muscle cell-related protein, asialoglycoprotein receptor, pyruvate dehydrogenase kinase 1 and a sequence with unknown functions. A green fluorescent protein expression plasmid pEGFP-C1-XBP1 of XBP1 was constructed successfully and its expression protein was localized in the cytoplasm. A 56-kDa recombinant protein was successfully obtained via prokaryotic expression and was demonstrated to have good specificity using western blot analysis. The XBP1 gene, which expresses the XBP1 protein, is located in the cytoplasm and plays a role in the intracellular structure, cell growth, intracellular metabolism and signal transduction pathway, as well as DNA duplication, transcription, recombination and repair.

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