Lin Shu-Mei, Cheng Jun, Lu Yin-Ying, Zhang Shu-Lin, Yang Qian, Chen Tian-Yan, Liu Min, Wang Lin
Department of Infectious Diseases, The First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, China.
World J Gastroenterol. 2006 Feb 21;12(7):1043-8. doi: 10.3748/wjg.v12.i7.1043.
To investigate the biological function of HBcAg in pathogenesis of HBV replication in peripheral blood mononuclear cells (PBMCs).
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-alpha-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.
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.
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.
研究乙肝核心抗原（HBcAg）在乙肝病毒（HBV）在外周血单个核细胞（PBMCs）中复制的发病机制中的生物学功能。
通过聚合酶链反应（PCR）扩增HBcAg区域，采用常规分子生物学方法构建HBV HBcAg诱饵质粒pGBKT7-HBcAg。然后将重组质粒DNA转化到酵母AH109中。在AH109酵母菌株中表达乙肝核心蛋白后（蛋白质免疫印迹分析），通过将AH109与含有白细胞cDNA文库质粒的Y187进行交配进行酵母双杂交筛选。将二倍体酵母细胞接种在合成缺陷营养培养基（SD/-Trp-Leu-His-Ade）（QDO）和合成缺陷营养培养基（SD/-Trp-Leu-His-Ade）（TDO）上。用LacZ报告基因进行第二次筛选（酵母细胞在含有X-α-半乳糖的QDO培养基中生长）。通过重复酵母双杂交进一步确认乙肝核心蛋白与从阳性菌落获得的蛋白质之间的相互作用。从蓝色菌落中提取质粒DNA并测序后，采用生物信息学方法分析结果。
获得18个菌落并进行测序，包括癌症高甲基化2（3个克隆）、真核翻译延伸因子2（2个克隆）、乙酰辅酶A合成酶3（1个克隆）、DNA聚合酶γ（1个克隆）、假定翻译起始因子（1个克隆）、趋化因子（C-C基序）受体5（1个克隆）、线粒体核糖体蛋白L41（1个克隆）、kyot结合蛋白基因（1个克隆）、RanBPM（1个克隆）、HBeAg结合蛋白3（1个克隆）、程序性细胞死亡2（1个克隆）。鉴定出4个功能未知的新基因。
成功克隆白细胞中与HBV核心蛋白相互作用的蛋白基因，可能为研究HBV核心蛋白的生物学功能提供一些新线索。
