HBV X基因的表达及在真核细胞中对内质网压力的作用

Expression of HBV X Gene and Its Function in Endoplasmic Reticulum Stress in Eukaryotic Cell Lines

运用PCR技术获得HBx基因,分别克隆到原核表达载体pET-his和真核表达载体pcDNA3.1(-)上。重组质粒pET-his-HBx转化大肠杆菌BL21(DE3)后,IPTG诱导表达,利用Ni柱纯化后的蛋白免疫家兔,获得特异性的抗-HBx兔抗血清。重组质粒pcDNA3.1(-)-HBx分别转染HepG2和Hep3B细胞系后,经RT-PCR和Westernblot检测,证明HBx可以在这两种细胞系中表达。通过报告基因的表达研究了HBx对XBP1和GRP78启动子的激活活性,结果表明瞬时转染HBx的细胞系中,XBP1和GRP78启动子介导的荧光素酶活性比相应的对照细胞增加了3～7倍。通过RT-PCR分析证明,转染了HBx的细胞中XBP1mRNA发生了剪切。因此,可以初步推断HBx在HepG2和Hep3B细胞中的表达可以引起内质网压力反应,为进一步阐明HBx表达对内质网的影响和肝脏病原发生机制奠定了基础。

HBV X gene was amplified by PCR and cloned into the prokaryotic expressing vector pET-his and the eukaryotic expressing vector pcDNA3.1（-）. E.coli BL21 （DE3） were transformed by the recombinant plasmid pET-his-HBx.. HBx protein was expressed by IPTG induction and formed insoluble inclusion bodies, which were dissolved in urea, dialyzed against PBS, and then applied onto Ni column. Anti HBx antibodies were generated in rabbits by immunization with the purified HBx protein. The recombinant plasmid pcDNA3. 1（-）-HBx was transfected into HepG2 and Hep3B cell lines, in which HBx expression was detected by RT-PCR and Western blots. In this study, luciferase activities of XBP1 promoter and GRV78 promoter in HBx expressing HepG2 and Hep3B cells were increased by 3-7 folds compared with that of cells mock-transfected with pcDNA3.1 （-）. RT-PCR showed that XBP1 mRNA was spliced in HBx-expressing HepG2 and Hep3B cells compared with negative controls. Our results demonstrated that expression of HBx induced UPR in HepG2 and Hep3B cell lines and provided insights into the effects of HBx on ER and the mechanisms underlying liver pathogenesis.