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Screening and identification of interacting proteins with hepatitis B virus core protein in leukocytes and cloning of new gene C1

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World Journal of Gastroenterology 2006年第7期12卷 1043-1048页

作者： Shu-Mei Lin Jun Cheng Yin-Ying Lu Shu-Lin Zhang Qian Yang Tian-Yan Chen Min Liu Lin Wang Department of Infectious Diseases The First Affiliated Hospital of Medical College of Xi'an Jiaotong University Xi'an 710061 Shaanxi Province China Institute of Infectious Diseases Ditan Hospital 13 Ditan Park Anwai Street Dongcheng District Beijing 100011 China

AIM： To investigate the biological function of HBcAg in pathogenesis of HBV replication in peripheral blood mononuclear cells （PBMCs）.METHODS： HBcAg region was amplified by polymerase chain reaction （PCR） and HBV HBcAg bait plasmid pGBKT7-HBcAg was constructed by routine molecular biological methods. Then the recombinant plasmid DNA was transformed into yeast AH109. After the HBV core protein was expressed in AH109 yeast strains （Western blot analysis）, yeast-two hybrid screening was performed by mating AH109 with Y187 containing leukocyte cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His- Ade）（QDO） and synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade）（TDO）. The second screening was performed with the LacZ report gene （ yeast cells were grown in QDO medium containing X-a-gal）. The interaction between HBV core protein and the protein obtained from positive colonies was further confirmed by repeating yeast-two hybrid. After plasmid DNA was extracted from blue colonies and sequenced, the results were analyzed by bioinformatic methods.RESULTS： Eighteen colonies were obtained and sequenced, including hypermethylated in cancer 2 （3 colones）, eukaryotic translation elongation factor 2 （2 colones）, acetyl-coenzyme A synthetase 3 （1 colone）, DNA polymerase gamma （1 colone）, putative translation initiation factor （1 colone）, chemokine （C-C motif） receptor 5 （1 colone）, mitochondrial ribosomal protein L41 （1 colone）, kyot binding protein genes （1 colone）, RanBPM （1 colone）, HBeAg-binding protein 3 （1 colone）, programmed cell death 2 （1 colone）. Four new genes with unknown function were identified.CONCLUSION： Successful cloning of genes of HBV core protein interacting proteins in leukocytes may provide some new clues for studying the biological functions of HBV core protein.

关键词： Hepatitis B virus Core protein yeast twohybrid system

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