Protein expression and function of gene 2 transregulated by hepatitis B virus pre-s1 protein and its cloning / 中华肝脏病杂志
Chinese Journal of Hepatology
; (12): 88-92, 2008.
Article
em Zh
| WPRIM
| ID: wpr-277596
Biblioteca responsável:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To screen proteins in leukocytes interacting with PS1TP2 by yeast-two hybrid and to view their subcellular localization in HepG2 cells.</p><p><b>METHODS</b>The function and structure of PS1TP2 were studied by bioinformatic analysis. PS1TP2 gene was amplified and cloned into plasmid pET32a (+) and pGBKT7 to construct recombinant expression vectors pET32a (+)-PS1TP2 and pGBKT7-PS1TP2. They were transduced into E. coli Rosetta strain and yeast AH109. The transformed yeast mated with yeast Y187 containing leukocyte cDNA library plasmid in a 2xYPDA medium. Diploid yeast cells were plated on a synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) for selecting twice and then screening. Then a green fluorescent protein (GFP) expression vector pEGFP-C1-PS1TP2 was established, transduced into HepG2, and its subcellular localization was studied by fluorescence microscopy and confocal microscopy.</p><p><b>RESULTS</b>Bioinformatic analysis showed that the PS1TP2 gene was located at 6q24.1, the protein was unstable and the aliphatic index was very high. After transformation of the E. coli and yeast AH109, the expression protein showed (1) the molecular weight of the expressed product was about 41000 Da, and (2) PS1TP2 existed within the cells. Diploid yeast cells were plated on the synthetic dropout nutrient medium containing X-a-gal for selecting twice and then screening. Twenty-six colonies from blue colonies were sequenced, pEGFP-C1-PS1TP2 was successfully expressed in the HepG2 cells, and PS1TP2 was located in the cell plasma.</p><p><b>CONCLUSION</b>A prokaryotic expression vector pET32a(+)-PS1TP2 was constructed successfully and the PS1TP2 was successfully expressed in the yeast system. Genes of PS1TP2 interact with leukocyte proteins. These results bring some new clues for studying the biological functions of HBV.</p>
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Índice:
WPRIM
Assunto principal:
Precursores de Proteínas
/
Sequência de Bases
/
Proteínas
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Expressão Gênica
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Clonagem Molecular
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Técnicas do Sistema de Duplo-Híbrido
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Dipeptídeos
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Células Hep G2
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Genética
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Antígenos de Superfície da Hepatite B
Limite:
Humans
Idioma:
Zh
Revista:
Chinese Journal of Hepatology
Ano de publicação:
2008
Tipo de documento:
Article