Cloning and identification of human gene 1 transactivated by nonstructural protein 4A of hepatitis C virus

Objective:To screen and clone the target genes transactivated by nonstructural protein 4A（NS4A） of hepatitis C virus（HCV）.Methods:The mRNA was isolated from HepG2 cells transfected with pcDNA3.1（-）-NS4A and pcDNA3.1（-） empty vector,respectively,and suppression subtractive hybridization（SSH） method was employed to analyze the differentially expressed DNA sequence between the two groups.On the base of subtractive cDNA library of genes transactivated by HCV NS4A,the coding sequence of a new gene was obtained by bioinformatics methods.The new gene with no homology with known genes in GenBank database was confirmed.The reverse transcription PCR（RT-PCR） was used to amplify the new gene using mRNA from HepG2 cell as the template.The coding sequence for the new gene was deduced according to the nucleotide sequence.Results:A new gene with unknown function is named as NS4ATP1.The nucleotide sequence of the NS4ATP1 gene and the corresponding protein-encoding amino acid have been determined,which contain 963 nt and 321 aa.The sequence of the NS4ATP1 gene was deposited into GenBank,and the accession number is AY 740521.Conclusions:NS4ATP1 gene transactivated by HCV NS4A protein was cloned and identified successfully by combining molecular biological technology and bioinformatics technique.These results will pave the way for the study of the molecular mechanism of the transactivating effects of HCV NS4A protein and the development of new therapy for chronic hepatitis C.