摘要
目的:为了阐明乙型肝炎病毒(HBV)感染相关疾病的发病机制,筛选并克隆丙型肝炎病毒非结构蛋白HBx反式激活新型靶基因. 方法:以HBV X蛋白(HBx)表达质粒pcDNA3.1(-)-X转染HepG2细胞,以空载体pcDNA3.1(-)为平行对照,提取mRNA并进行SSH分析.对于所获基因片段序列分析表明, 其中之一为新型基因片段,与GenBank中注册的已知功能基因序列没有同源性,利用表达序列标签(EST)序列的搜索和比对,确定新型基因序列并命名为XTP5.从HepG2细胞提取总RNA,以逆转录多聚酶链反应(RT-PCR)技术扩增获得该新基因的全长序列. 结果:XTP5基因的编码基因序列全长为1 527个核苷酸(nt),编码产物由508个氨基酸残基(aa)组成. 结论:HBx是一种由病毒基因组编码的具有反式激活作用的蛋白.应用SSH技术发现了HBx反式激活作用的新的靶基因,这一发现,为阐明HBx蛋白的反式激活作用及其机制, 开辟了新的研究方向.
AIM: To explore the new target genes transactivated by HBx, suppression subtractive hybridization (SSH) method and to pave the way for elucidating the pathogenesis mechanism of HBV infection. METHODS: The mRNA was isolated from HepG2 cells trans-fected with pcDNA3.1(-)-X and pcDNA3.1(-) empty vector, respectively, using SSH and bioinformatics technique, and the differentially expressed DNA sequence between the two groups was analyzed. The obtained sequences were searched for homologous DNA sequence from GenBank, one of which was a novel gene with unknown function. The new gene with no homology with known genes in this database was confirmed and electric polymerase chain reaction (PCR) was conducted for the cloning of the full-length DNA for the new gene and in conjunction with Kozak role and the exist of polyadenyl signal sequence. The reverse transcription PCR (RT-PCR) was used to amplify the new gene, named as XTP5, from the mRNA of HepG2 cells transfected. RESULTS: The new gene was cloned in combination of molecular biological and bioinformatics methods. CONCLUSION: HBx is a potential transactivator. A new gene has been recognized as the new target transactivated by HBx protein. These results pave the way for study on the transactivation of HBx protein.
出处
《世界华人消化杂志》
CAS
2004年第1期74-77,共4页
World Chinese Journal of Digestology
