丙型肝炎病毒核心蛋白反式激活基因TAHCCP2的克隆

Cloning and identification of human gene 2 transactivated by hepatitis C virus core protein

目的:筛选与克隆 HCV 核心蛋白反式激活新型靶基因.方法:以 HCV 核心蛋白表达质粒 pcDNA3.1(-)-core 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,从转染 pcDNA3.1(-)-core 的 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:新基因命名为 TAHCCP2,编码序列全长为429个核苷酸(nt),编码产物由142个氨基酸残基(aa)组成,测序证实 TAHCCP2克隆正确.在 GenBank 中注册,注册号为 AY039043.结论:筛选与克隆 HCV 核心蛋白反式激活新型靶基因TAHCCP2,为进一步研究 HCV 核心蛋白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 clone and identify of human gene 2 transactivated by hepatitis C virus core protein. METHODS:To screen and clone the target genes transactivated by hepatitis C virus(HCV)core protein,we used suppression subtractive hybridization(SSH)and bioinformatics techniques.The mRNA was isolated from HepG2 cells transfected pcDNA3.1(-)-core 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 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 TAHCCP2,from the mRNA of HepG2 cells transfected.The sequence for the TAHCCP2 gene was deposited into GenBank,and the accession number is AY039043. RESULTS:The full-length of TAHCCP2 was of 429 bp,and it was composed of 142 amino acid residues.The cloning of TAHCCP2 was correct and confirmed by sequencing. CONCLUSION:The cloning and identification of human gene 2 transactivated by HCV core protein will pave the way for the study of the molecular mechanism of the transactivating effects of HCV core protein and the devel- opment of new therapy for chronic hepatitis C.