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目的 应用抑制性消减杂交 (SSH )技术及生物信息学技术 (bioinformatics)筛选并克隆乙型肝炎病毒 (HBV)e抗原(HBeAg)反式激活新型靶基因。方法 以HBeAg表达质粒pcDNA3 .1(-) HBeAg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 (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 ,利刚表达序列标签 (EST)序列的搜索和比对 ,进行电子拼接 ,根据基因起始密码子的KozaK规则和终止密码子下游保守的多聚腺苷酸信号序列 ,确定新型基因序列。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 ,命名为HBeAgTP ,在GenBank中注册 ,注册号为AY42 3 62 4。结果 HBeAgTP基因的编码序列全长为 3 2 4个核苷酸 (nt) ,编码产物由 10 7个氨基酸残基 (aa)组成。结论 HBeAg反式激活新型靶基因HBeAgTP的筛选与克隆 ,为进一步研究HBeAg在体内激活作用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 a novel target gene transactivated by hepatitis B virus e antigen (HBeAg) using SSH and bioinformatics. Methods HepG2 cells were transfected with HBeAg expression plasmid pcDNA3. 1 (-) HBeAg. The empty vector pcDNA3. 1 (-) was used as a parallel control. MRNA was extracted and subjected to SSH. The sequence analysis of the obtained gene fragments showed that one of them was a novel gene fragment, which was not homologous to the known functional gene sequence registered in GenBank, and the search and alignment of the sequence sequences of the Ligand expression sequence tags (EST) , A novel gene sequence was identified based on the KozaK rule of the gene start codon and the conserved polyadenylation signal sequence downstream of the stop codon. Total RNA was extracted from HepG2 cells and the full-length sequence of the new gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The full length of the new gene was confirmed by sequencing and named as HBeAgTP and registered in GenBank under the accession number of AY42 3 62 4 . Results The full-length coding sequence of HBeAgTP gene was 324 nucleotides (nt) and the coding product consisted of 107 amino acid residues (aa). Conclusion The screening and cloning of HBeAgTP, a new target gene of transactivation of HBeAg, lay the foundation for the further study of the molecular biological mechanism of HBeAg activation in vivo and exploration of new therapeutic techniques