摘要
目的 筛选、克隆乙型肝炎病毒 (HBV)X蛋白 (HBxAg)反式激活基因XTP4转染肝癌细胞后的反式调节基因 ,探索XTP4基因表达对肝细胞基因表达谱的影响。方法 常规的分子生物学技术构建真核表达载体pcDNA3 1( ) XTP4 ,应用抑制性消减杂交(SSH)技术对重组表达质粒pcDNA3 1( ) XTP4转染的HepG2细胞和空载体转染的相同细胞差异表达的mRNA进行研究 ,将富集的二次PCR产物与T/A载体连接 ,并转化大肠杆菌进行文库扩增 ,随机挑取克隆PCR扩增后进行测序及同源性分析。结果 消减文库扩增后得到 2 1个阳性克隆 ,PCR分析显示其中 16个克隆含有 2 0 0～ 10 0 0bp的插入片段。对插入片段进行测序及生物信息学分析 ,结果共获得 9种蛋白编码基因。这些差异表达的基因与肝细胞纤维化形成、肿瘤发生、线粒体氧化还原代谢、细胞生长调节密切相关。结论 应用SSH技术成功筛选了XTP4对肝癌细胞的反式调节基因 ,为进一步阐明HBxAg对肝细胞蛋白的反式调节作用提供了理论依据。
Objective To explore the influence of XTP4 on genomic expression profile of hepatocyte through screening and cloning of genes trans-regulated by XTP4-expressing plasmid. Methods The expressive vector pcDNA3.1(-)-XTP4 was constructed by routine molecular biological methods, and suppression subtractive hybridization (SSH) method was employed to detect the mRNA differentially expressed by the HepG2 cells transfected with pcDNA3.1(-)-XTP4 and pcDNA3.1(-), respectively, using lipofectamine. The twice enriched PCR products were subcloned into T/A vectors to set up the subtractive library. Amplification of the library was carried out in E. coli strain JM109. The screened cDNA were sequenced and analyzed in GenBank with Blast search after PCR. Results The amplified subtractive library containd 21 positive clones. Colony PCR analysis showed that there were 16 clones containing 200-1000bp inserts. Sequence analysis was performed and 9 kinds of encoding sequences were achieved. These genes trans-regulated by XTP4 protein involved in hepatic fibrogenesis, tumorgenesis, mitochondrial function, and cell growth regulation. Conclusions The findings obtained by SSH provide significant data for a preliminary understanding of the biological function of a new identified gene-XTP4. These results will pave the way for the study of the molecular mechanism of the transactivating effects of HBxAg and the development of new therapy for chronic hepatitis B.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2004年第10期887-888,共2页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金攻关项目(编号C0 30 1 1 4 0 2 0
C30 0 70 689)
军队"十五"科技攻关青年基金项目(编号 0 1Q1 38)
军队医学科研"十五"科技攻关面上项目(编号 0 1B1 35)
军队回国留学人员启动基金 (编号 98H0 38)资助课题
关键词
肝炎病毒
乙型
X蛋白
反式激活
抑制性消减杂交
hepatitis B virus
X protein
transactivation
suppression subtractive hybridization
