Screening And Identification Of Genes Transactived By P7TP3 Spliced Variant 1 By Suppression Subtractive Hybridization

Hepatitis C virus (HCV) p7 protein is a small hydrophobic protein of 63 amino acids between structural proteins and nonstructural proteins. Recombinant HCV p7 could form ion channels in artificial lipid membranes. HCV p7 is essential for infectivity of HCV. Its actual role in the virus life cycle has not been determined. In order to explore the pathogenesis of HCV p7 protein infection,we conducted microarray assay on the hepatoblastoma cell HepG2 with transfected by p7 expressive vector, identified and cloned a novel gene p7TP3. In the course of study the function of p7TP3, we find its spliced variant, named p7TP3 spliced variant 1. The coding sequence of the p7TP3 spliced variant 1 is obtained by bioinformatics methods. In the purpose of to explicit the molecular biological mechanisms of p7TP3 spliced variant 1 after HCV infection, We design such experiments.1. Polymerase chain reaction(PCR)technique was employed to amplify the sequence of p7TP3 spliced variant 1 using HepG2 cell total RNA as template. The PCR product was cloned into pGEM-T vector, then the recombinant vector was identified by restriction enzyme digestion and sequencing.2. The p7TP3 spliced variant 1 gene was cut from pGEM-T-p7TP3 spliced variant 1 by EcoR I and BamH I, and then was cloned into pEGFP-C1, named pEGFP-C1- p7TP3 spliced variant 1. pEGFP-C1- p7TP3 spliced variant 1 was transiently transfected into the HepG2 cell line, and pEGFP-C1 empty vector was used as control, it was showed that green fluorescence protein was distributed in the cytoplasm.3. Recombinant pcDNA3.1/myc-his-p7TP3 spliced variant 1 was transiently transfected into the HepG2 cell line, and pcDNA3.1/myc-his empty vector was used as control. Suppression subtractive hybridization (SSH) technique was employed to analyze the differentially expressed cDNA sequence between the two groups. The products were cloned into pGEM-Teasy vector, amplification of the cDNA library was carried out with E. coli strain DH5αin random. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. From the SSH results, it was found 14 genes were up-regulated by p7TP3 spliced variant 1 gene.The results showed as follows: (1)p7TP3 spliced variant 1 protein is a potential transregulator. (2)The obtained sequences may be target genes transactivated by p7TP3 spliced variant 1 protein, among which some genes coding proteins involved in cell signal transduction, cycle regulation, translation and synthesis of protein, metabolism,apoptosis, immunoregulation, and correlated with tumor. These results provide a new evidence to explain the molecular biological mechanisms of p7TP3 spliced variant 1 protein in hepatocyte.