【作者】 李曼；

【导师】 冯德云； 何琼琼；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2011， 硕士

【摘要】 目的：通过差异磷酸化蛋白质组学研究,筛选和鉴定出HCV/NS3质粒转染肝细胞后差异表达的酪氨酸磷酸化蛋白。方法：首先建立细胞系,将HCV/NS3质粒(pRcHCNS3-5’)和空白质粒(pRcCMV)分别转染入人源性肝细胞系QSG7701,通过G418筛选,构建稳定表达HCV/NS3蛋白的细胞系(QSG/NS3)和阴性对照组(QSG/CMV)。通过RT-PCR检测转染细胞HCV/NS3表达情况。然后进行蛋白质组学分析,分别抽提两组细胞的总蛋白,每组蛋白平行进行2次双向凝胶电泳,其中一块作为制备胶,另一块作为分析胶,分析胶中将蛋白质转移至PVDF膜后,与抗酪氨酸磷酸化抗体孵育,进行Western Blot分析,获得差异表达酪氨酸磷酸化蛋白质的反应图谱。将制备胶的电泳图谱和Western Blot的反应图谱进行比对分析,在制备胶上找到相应的差异酪氨酸磷酸化蛋白质点,经脱色、还原、酶解,通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析,获得肽质量指纹图(PMF),在Http://www.matrixscience.com/上搜索,匹配蛋白质,应用在线软件Netphos预测蛋白质酪氨酸磷酸化位点。结果：在QSG/NS3和QSG/CMV两组细胞系中,识别了13个差异表达的酪氨酸磷酸化蛋白质,其中鉴定了8个差异表达蛋白。4个蛋白质的酪氨酸磷酸化水平在QSG/NS3细胞系中表达上调,另4个蛋白质的酪氨酸磷酸化水平表达下调。软件预测每个蛋白质均存在酪氨酸磷酸化位点。结论：(1)鉴定出8个可能与HCV NS3致病相关的酪氨酸磷酸化蛋白质,为揭示HCV NS3致癌机制提供新的科学理论线索。(2)鉴定出的差异蛋白功能涉及细胞代谢、凋亡增殖及信号转导等方面。(3)应用在线软件分析预测蛋白质的酪氨酸磷酸化位点,鉴定出的8个蛋白均有至少一个酪氨酸磷酸化位点。更多还原

【Abstract】 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.更多还原