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. 2005 Jun 14;11(22):3473-8.
doi: 10.3748/wjg.v11.i22.3473.

Molecular epidemiological study on pre-X region of hepatitis B virus and identification of hepatocyte proteins interacting with whole-X protein by yeast two-hybrid

Qian Yang  1 , Jun Cheng, Jing Dong, Jian Zhang, Shu-Lin Zhang
Affiliations

Molecular epidemiological study on pre-X region of hepatitis B virus and identification of hepatocyte proteins interacting with whole-X protein by yeast two-hybrid

Qian Yang et al. World J Gastroenterol. .

Abstract

Aim: To identify the pre-X region in hepatitis B virus (HBV) genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.

Methods: The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for alpha-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics.

Results: After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4-CD81 (TM4SF4).

Conclusion: The pre-X gene exists in HBV genome. Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.

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Figures

Figure 1
Figure 1
Nucleotide sequence of the pre-X gene.
Figure 2
Figure 2
Expression of whole-X protein in yeast demonstrated by Western blotting. Lane 1: whole-X protein, lane 2: negative control.
Figure 3
Figure 3
Colonies cut by EcoRI on 0.9% agarose/EtBr gel.
Figure 4
Figure 4
Positive clones interactive with the whole-X protein growing on media containing X-α-Gal and lacking leucine, tryptophan, histidine and adenine.
See this image and copyright information in PMC

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References

    1. Bertoletti A, Ferrari C. Kinetics of the immune response during HBV and HCV infection. Hepatology. 2003;38:4–13. - PubMed
    1. Besisik F, Karaca C, Akyüz F, Horosanli S, Onel D, Badur S, Sever MS, Danalioglu A, Demir K, Kaymakoglu S, et al. Occult HBV infection and YMDD variants in hemodialysis patients with chronic HCV infection. J Hepatol. 2003;38:506–510. - PubMed
    1. Bock CT, Schwinn S, Locarnini S, Fyfe J, Manns MP, Trautwein C, Zentgraf H. Structural organization of the hepatitis B virus minichromosome. J Mol Biol. 2001;307:183–196. - PubMed
    1. Sterneck M, Kalinina T, Günther S, Fischer L, Santantonio T, Greten H, Will H. Functional analysis of HBV genomes from patients with fulminant hepatitis. Hepatology. 1998;28:1390–1397. - PubMed
    1. Kim KH, Seong BL. Pro-apoptotic function of HBV X protein is mediated by interaction with c-FLIP and enhancement of death-inducing signal. EMBO J. 2003;22:2104–2116. - PMC - PubMed

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