乙型肝炎病毒X蛋白反式激活基因XTP12的克隆化

Cloning of human gene XTP12 transaetlvated by X protein of hepatitis B virus

目的:应用基因表达谱芯片技术及生物信息学技术筛选并克隆乙型肝炎病毒(HBV)X蛋白反式激活新型靶基因,进一步阐明HBV感染相关性疾病的发病机制. 方法:以HBV X蛋白表达质粒pcDNA3.1(-)-X转染HepG2 细胞,以空载体pcDNA3.1(-)为平行对照,提取总RNA 进行逆转录,对产物行基因表达谱芯片分析.应用分子生物学技术,结合生物信息学技术,克隆HBV X反式激活作用的新的靶基因. 结果: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.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,并测序证实,因其可以被X蛋白反式激活,故命名为X蛋白反式激活蛋白12(XTPl2),已在GenBank中注册,注册号: AY598792.PS2TP1基因的编码序列全长为731个核苷酸(nt),编码产物由230个氨基酸残基(aa)组成. 结论:HBV X反式激活新靶基因被成功克隆,为进一步研究HBV X蛋白的分子生物学机制和探索新型治疗技术奠定基础.

AIM: To screen and clone the target gene transactivated by hepatitis B virus (HBV) X protein and to pave the way for further elucidating the pathogenesis of HBV infection. METHODS: The HBV X coding DNA fragment was amplified with polymerase chain reaction (PCR) technique using pCP10 plasmid containing the full length of HBV genome as the template. The expressive vector of pcDNA3.1 X was constructed by routine molecular biological methods. The HepG2 cells were transfected by pcDNA3.1(-) and pcDNA3.1-X respectively. The total mRNA was isolated and reversely transcribed. The cDNA was analyzed by DNA microarray and then target gene transactivated by hepatitis B virus (HBV) X protein was cloned by molecular biological technique. RESULTS: After searching for homologous DNA sequences from GenBank, we found that one of the obtained sequences was a new gene with unknown function. Its full length was comfirmed by PCR method. The new gene, amplified from the mRNA of HepG2 cells, consisted of 731 nucleotides (nt) and encoded 230 amino acids, and it was named as XTP12 and registered in GenBank with the accession number AY598792. CONCLUSION: The target gene is successfully cloned and it will pave the way for further study of the molecular mechanism of the transactivating effects of HBV X protein and the new therapy for chronic hepatitis B.