FUNCTIONAL ASPECTS OF HEPATITIS B VIRUS X-PROTEIN

HBV X蛋白已被证明对HBV DNA有反式激活作用。 多种病毒和细胞基因的表达。的表达 在瞬时表达系统中研究了肿瘤抑制基因RB， 将HBV X基因转染Hep G2细胞。 HBV X开放阅读框架 通过PCR从含有野生型HBV DNA的质粒产生， 插入到表达载体中。用质粒pHBV-X进行转染， Hep G2，人肝母细胞瘤细胞系。提取蛋白质48小时 DNA转染后。内源性RB蛋白的水平通过以下方法分析： 使用RB单克隆抗体的western免疫印迹。RB蛋白 未转染的Hep G2细胞和单独用载体转染的Hep G2细胞， 也作为对照进行分析。pHBV-X转染细胞RB蛋白水平 Hep G2细胞比对照细胞增加近10倍。 RB蛋白与HSP 70之间的初步体外免疫共沉淀研究 HBVX蛋白不提示RB与HBVX之间存在直接相互作用 proteins. HBV源性X基因可改变HBV DNA表达， RB抑癌基因提示X基因在HBV中可能起作用 介导的肝癌发生。 工作仍在继续， X蛋白的反式激活机制。宿主介导 将通过以下方法分析涉及X基因活性的转录因子： X蛋白在不同肝细胞和非肝细胞中的反式激活作用 线

HBV X protein has been shown to have a transactivating effect on the expression of a variety of viral and cellular genes. The expression of tumor suppressor gene RB was studied in a transient expression system by transfecting HBV X gene into Hep G2 cells. The HBV X open reading frame was generated by PCR from plasmid containing wild type HBV DNA and was inserted into expression vector. The plasmid pHBV-X was used to transfect Hep G2, a human hepatoblastoma cell line. Protein was extracted 48 hours after DNA transfection. The level of endogenous RB protein was analyzed by western immunoblotting using RB monoclonal antibody. RB protein untransfected Hep G2 cells and Hep G2 transfected with vector alone were also analyzed as controls. The level of RB protein in pHBV-X transfected Hep G2 cells increased nearly 10-fold compared to the control cells. Preliminary in vitro co-immunoprecipitation studies between RB protein and HBV X protein do not indicate direct interaction between RB and HBV X proteins. The finding that HBV derived X gene can alter the expression of RB tumor suppressor gene suggest a possible role for the X gene in HBV mediated hepatocarcinogenesis. Work continues in an effort to determine the mechanism of transactivation by X protein. Host mediated transcriptional factor(s) involved in X-gene activities will be analyzed by X-protein transactivation effect in different hepatic and non hepatic cell lines.