Screen The Differential Expression Of Tyrosine-Phosphorylated Proteins Involved In Hepatocyte Transfected By HCV/NS3 Plasmid

Objective:To select and identify the differential expression of tyrosine-phosphorylated proteins in hepatocytes which transfected with HCV/NS3 plasmid.Methods:Firstly, the HCV/NS3 plasmid (pRcHCNS3-5') was transfected into human hepatocyte cell line QSG7701 and selected by G418, the blank plasmid (pRcCMV) was used as a negative control. The resulting cell lines were called QSG/NS3 and QSG/CMV respectively. Secondly, total proteins of each groups were extracted and separated by 2-D gels electrophoresis in paralled for on analytical as well as preparative. The proteins on analytical gel were transferred onto PVDF membrane, and incubated with anti-phosphotyrosine antibody. Then, maps of differential expression of tyrosine-phosphorylated proteins were acquired. Compared the electrophoresis map with Western Blot map, the corresponding points were found in preparative gels, and the differential expression tyrosine-phosphorylated proteins were identified by MALDI-TOF-MS analysis. The tyrosin-phosphorylation sites of identified proteins were predicted by NetPhos software.Results:There were 13 differential expressions tyrosin-phosphorylated proteins were screened in the QSG/NS3 and the QSG/CMV cells. We identified 8 of the proteins and found that four of which were up-regulated in the QSG/NS3 cells, while the other four were down-regulated. Each of the eight proteins has tyrosin phosphorylation sites.Conclusion:(1) Eight tyrosin-phosphorylated proteins were identified, which may be involved HCV/NS3 pathogenesis, and providing new clues to HCV/NS3 carcinogenic mechanism.(2) The function of identified proteins is involved in cell metabolism, apoptosis, proliferation and signal transduction.(3) Online software analysis forecasts that each protein has at least one tyrosine phosphorylation site.