中文摘要：

目的应用酵母双杂交及生物信息学（bioinformatics）技术获得HBxAg蛋白结合蛋白XBP1,为了解该基因的反式调节机制,利用抑制性消减杂交技术筛选并克隆XBP1蛋白反式激活的靶基因。方法以XBP1表达质粒pcD-NA3.1（-）-myc-his（A）-XBP1转染HepG2细胞,以空载体pcDNA3.1（-）-myc-his（A）为对照;制备转染后的细胞裂解液,提取mRNA并合成cDNA,经RsaⅠ酶切后将实验组cDNA分成两组,分别衔接两种不同接头,再与对照组cDNA进行两次消减杂交及两次PCR反应,产物与T/A克隆载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增,随机挑选克隆经PCR鉴定后进行测序及同源性分析。结果成功构建人XBP1蛋白反式激活相关基因差异表达的cDNA。扩增后得到80个200～1 000bp插入片段的克隆,随机挑选其中30个插入片段测序,并通过生物信息学分析获得其全长基因序列,结果共获得28个已知编码序列基因和2个未知的新基因。结论 XBP1在肝细胞内表达后能够上调HepG2细胞中许多不同基因表达的变化,这些基因与细胞信号转导、细胞增殖与分化、免疫应答、能量代谢、细胞凋亡、肿瘤发生等生物过程密切相关。

英文摘要：

Objective Human gene XBP1 interacting with HBxAg protein（HBXBP1） has been identified by yeast-two hybrid and bioinformatic technique.The purpose of this study was to screen genes transactivated by HBXBP1 with suppression subtractive hybridization（SSH）.Methods Su ppression subtractive hybridization and bioinformatics techniques were used for screening and cloning of the target genes transactivated by XBP1 protein.The mRNA was isolated from HepG2 cells transfected pcDNA3.1（-）-myc-his（A）-XBP1 and pcDNA3.1（-）-myc-his（A） empty vector,respectively;SSH method was employed to analyze the differentially expressed DNA sequence in the two groups.After restriction enzyme Rsa I digestion,small size cDNAs were obtained.Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2,respectively.After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR,it was subcloned into T/A plasmid vectors to set up the subtractive library.Amplification of the library was carried out with E.coli strain DH5α.The cDNA was sequenced and analyzed in GenBank with Blastn search after PCR.Results The subtractive librar y of genes transactivated by XBP1 was constructed successfully.The amplified library contained 100 positive clones.Colony PCR showed that 80 clones contained 200-1 000bp inserts.Sequence analysis was performed randomly in30 clones,and the full-length sequences were obtained with bioinformatics method.We obtained altogether 30 coding sequences,which consisted of 28 known and2 unknown ones.Conclusion XBP1 can u pgrade the changes of many genes in HepG2 cells after being expressed in liver cells.The genes are closely related to cell signal transmission,cell proliferation and differentiation,immune response,energy metabolism,cell necrosis,and oncogenesis.