摘要
目的 应用抑制性消减杂交 (SSH)技术及生物信息学 (bioinformatics)技术筛选并克隆乙型肝炎病毒 (HBV)DNA聚合酶(DNAPolymerase)逆转录酶 (RT)反式调节新型靶基因 ,进一步阐明HBV感染相关疾病的发病机制。方法 以HBVDNA聚合酶逆转录酶表达质粒pcDNA3 .1( ) RT转染HepG2细胞 ,以空载体pcDNA3 .1( )为平行对照 ,提取mRNA并进行抑制性消减杂交分析。并应用分子生物学技术 ,结合生物信息学技术 ,克隆RT反式调节作用的新的靶基因。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HBVDNA聚合酶逆转录酶反式调节 ,故命名为DNA多聚酶反式调节蛋白 1(DNAPTP1) ,已在GenBank中注册 ,注册号AY45 0 3 89。DNAPTP1基因的编码序列全长为 43 5个核苷酸 (nt) ,编码产物由 14 4个氨基酸残基 (aa)组成。结论 逆转录酶反式调节新靶基因DNAPTPl的发现 ,为进一步研究RT蛋白的分子生物学机制和探索新型治疗技术奠定基础。
Objective To screen and clone the target genes transaetivated by hepatitis B virus (HBV) DNA polymerase reverse transcriptase (RT) and to pave the way for elucidating the pathogenesis of HBV infection. Methods Suppression subtractive hybridization (SSH) technique and bioinformatics technique were used. The mRNA from HepG2 cells transfected with pcDNA3. 1(-)-RT and pcDNA3. 1(-) empty vector was isolated,respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. The coding gene transactivated by RT was cloned by bioinformatics methods.Results The obtained sequences were searched for homologous DNA sequences from GenBank, one of which was a new gene with unknown function. The new gene with no homology with known genes in this database was confirmed and electronic polymerase chain reaction (PCR) was conducted for the cloning of the full-length DNA and in conjunction with Kozak role and existance of polyadenylate signal sequence. The reverse transcription PCR (RT-PCR) was used to amplify the new gene, named DNAPTP1, from the mRNA of HepG2 cells. The sequence for the DNAPTP1 gene has been deposited into GenBank, the access number is AY450389. Conclusion These results will pave the way of the molecular mechanism of the transactivating effects of RT protein and the development of new therapy for chronic hepatitis B.
出处
《胃肠病学和肝病学杂志》
CAS
2005年第1期23-26,共4页
Chinese Journal of Gastroenterology and Hepatology
基金
国家自然科学基金资助项目 (C0 30 1 1 4 0 2
30 371 2 88
C30 0 70 689)
军队"九
五"科技攻关项目 (98D0 63)
军队回国留学人员启动基金项目(98H0 38)
军队"十
五"科技攻关青年基金项目 (0 1Q1 38)
军队"十
五"科技攻关面上项目 (0 1MB1 35)。
