丙型肝炎病毒非结构蛋白NS5A反式激活基因NS5ATP5的克隆

Cloning and identification of human gene 5 transactivated by nonstructural protein 5A of hepatitis C virus

目的:应用抑制性消减杂交技术及生物信息学技术筛选并克隆丙型肝炎病毒非结构蛋白5A(HCV NS5A)反式激活新型靶基因.方法:以 HCV NS5A 表达质粒 pcDNA3.1(-)-NS5A 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,利用表达序列标签(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.结果:NS5ATP5基因的编码序列全长为1191个核苷酸(nt),编码产物由396个氨基酸残基(aa)组成.命名为NS5ATP5,在 GenBank 中注册,注册号为 AF529366.结论:HCV NS5A 反式激活新型靶基因 NS5ATP5的筛选与克隆,为进一步研究 HCV NS5A 反式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.

AIM:To screen and clone the target genes transactivated by nonstructural protein 5A(NS5A)of hepatitis C virus (HCV)using suppression subtractive hybridization(SSH) and bioinformatics techniques. METHODS:The mRNA was isolated from HepG2 cells trans fected with pcDNA3.1(-)-NS5A and pcDNA3.1(-)empty vector,respectively,and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups.The obtained sequences were searched for homologous DNA sequence 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 electric polymerase chain reaction was con- ducted for the cloning of the full-length DNA in conjunction with Kozak role and the exist of polyadenyl signal sequence. The reverse transcription PCR(RT-PCR)was used to am- plify the new gene,named NS5ATP5,from the mRNA of HepG2 cells.The sequence for the NS5ATP5 gene was deposited into GenBank,and the accession number is AF529366. RESULTS:The full-length DNA sequence for the NS5ATP5 gene was of 1191 nudeotides,and its coding product con- sisted of 396 amino acid(aa)residues. CONCLUSION:These results will gave the way for the study of the molecular mechanism of the transactivating effects of NS5A protein of HCV and the development of new therapy for chronic hepatitis C.