The Biological Functions Of Gene P7TP2 Transregulated By Hepatitis C Virus Protein P7

At present there were 170 million people infected hepatitis C virus(HCV),however nowadays there weren't specific treatment techniques and methods.Because the pathogenesis of hepatitis C virus hadn't been completed known. HCV p7TP2 was the target gene down-regulated by HCV p7 screening with microarray assay,its many functions were still unknown and needed further research.p7TP2 gene was amplified by PCR,consists of 495bp. Bioinformatic analysis showed that the new gene was located at chromosome 8q24.3, its estimated half-life was 30 hours and the aliphatic index was very high. Some tools showed that p7TP2 protein may be globular,not as compact as a domain and had signal peptide and two transmembrane regions.Realtime PCR was used to check that HCV p7 down-regulated the expression of p7TP2, suppression subtractive hybridization technique was used to screening and setting up the subtractive library down-regulated by p7TP2, The cDNA subtractive library was sequenced, analyzeds and showed that the down-regulated genes mainly located in mitochondrion and were involved in cell proliferation,differentiation amd apoptosis signal transduction. Prokaryotic expression techniques were used to induce p7TP2 fusion protein.The Mr being 33000 appeared on SDS-PAGE gel. The fusion protein was purified and immunized New Zealand rabbits to prepare a polyclonal antibody. The antiserun was obtained and characterized by ELISA,Western blot and immunohistochemistry. Results showed that the antibody had high titer,affinity and specificity. Immunohistochemistry showed that p7TP2 protein were localized in cytoplasm and expressed more in the normal tissues than the pathological tissues.The subcellular localization of p7TP2-GFP fusion protein were detected in cytoplasm.p7TP2 bait plasmid was transformed into AH109, and the transformed yeast cells were amplified and mated with yeast cells Y187 containing liver cDNA library plasmid. Plasmids of positive blue colonies were extracted and analysed by DNA sequencing. Results Showed that p7TP2 protein may play a role of inhibiting blood clotting by the unknown way.The sequence of p7TP2 promoter was amplified from HepG2 genome,which was cloned into pCAT3 reporter vector and luciferase reporter.The CAT expression system and the luciferase reporter system both showed that the promoter sequence had higher transcription activities.The co-transfection experiments showed that HCVp7 down-regulate the promoter activity of p7TP2.All these conclusions brought some new clues for studying the structure and biological functions of p7TP2 gene and the pathogenesis of HCV.