免疫沉淀筛选与双剪接型2.2Kb HBV剪接变异体特异性蛋白HBDSP相互作用的肝细胞蛋白

发布时间：2018-06-19 22:14

本文选题：乙型肝炎病毒 + RNA　；参考：《福建医科大学》2012年硕士论文

【摘要】：乙型肝炎病毒（hepatitis B virus, HBV）是一种部分双链环状嗜肝DNA病毒，在医学和公共卫生上具有重要的意义。从世界范围来看，大约有350000000的人感染乙肝病毒，其中亚洲地区占了75%。HBV是慢性肝炎、肝硬化和肝癌的主要致病原因，每年导致1000000人死亡。 HBV基因组剪接变异体(spliced variants of hepatitis B virus genomes)是由3.5Kb前基因组RNA (pregenomic RNA, pgRNA)经剪接后逆转录而成的一类亚基因组DNA。其中长度为2.2kb的HBV剪接变异体占了80%以上，，其剪接类型分为单剪接型和双剪接型，对2.2Kb HBV剪接变异体的功能研究显示，此类变异体可能导致肝细胞凋亡并与HBV的持续性感染密切相关，但其确切致病机制迄今未明。本研究旨在利用免疫沉淀技术探索能够与双剪接型2.2Kb剪接变异体编码的剪接特异性新蛋白（Hepatitis B Doubly Spliced Protein, HBDSP）相互作用的肝细胞蛋白，以此深入了解其致病性。本课题第一部分，我们运用免疫沉淀（Immunoprecipitation）及基质辅助激光解吸电离飞行时间质谱鉴定（MALDI-TOF-MS）的方法获得了四个与HBDSP相互作用的肝细胞蛋白，分别为：通用转录因子II,i (General transcription factor III, GTF2I或TFII-I)、葡萄糖调节蛋白78(Glucose-regulated protein, GRP78)、热休克蛋白70(Heat shock protein70kDa, HSP70)和钾离子通道(Potassiumvoltage-gated channel, KVβ.2)。本课题第二部分，首先通过免疫共沉淀（Co-Immunoprecipitation, Co-IP）实验来作验证，结果表明在肝细胞中HBDSP与TFII-I蛋白存在相互作用，而HBDSP与GRP78和KVβ.2没有相互作用。进一步通过GST pull-down实验证实HBDSP与TFII-I蛋白在体外具有相互作用。TFII-I蛋白是一个多功能转录因子，不仅能够促进染色质重塑，同时在细胞有丝分裂周期中具有重要作用。HBDSP与TFII-I的相互作用，可能影响肝癌细胞的分裂和增殖。本研究为深入探讨HBDSP的致病性奠定基础。
[Abstract]:Hepatitis B virus (HBV) is a partial double stranded circular hepatitis virus (HBV), which is of great significance in medicine and public health. Worldwide, about 350000000 people are infected with hepatitis B virus, of which 75. HBV is the leading cause of chronic hepatitis, cirrhosis and liver cancer in Asia. The splicing variant of variants of hepatitis B virus genomeses is a subgenome DNA, which is composed of 3.5kb pregenomic RNAs (pgRNAs) spliced and reverse transcribed. Among them, the length of 2.2kb splicing variants accounted for more than 80%, and the splicing types were divided into single splicing type and double splicing type. The function of 2.2 Kb HBV splicing variant was studied. Such variants may lead to hepatocyte apoptosis and are closely related to HBV persistent infection, but its exact pathogenesis has not been clarified. The aim of this study was to explore the hepatocyte proteins which can interact with the splicing specific protein (HBDSPN) encoded by double splicing variant 2.2Kb splicing variants by immunoprecipitation technique in order to understand the pathogenicity of Hepatitis B Doubly splicing protein (HBDSP). In the first part of this study, we obtained four hepatocyte proteins interacting with HBDSP by using immunoprecipitation and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). GTF2I, GTF2I, GTF2I, Glucose-regulated Glucose-regulated protein, GRP78A, heat shock protein 70heat shock 70kDa, HSP70) and Potassiumvoltage-gated channel, KV 尾. In the second part of this thesis, the co-immunoprecipitation (Co-IPT) experiment was carried out to verify the interaction between HBDSP and TFII-I protein in hepatocytes, but no interaction between HBDSP and GRP78 and KV 尾 .2 in hepatocytes. Furthermore, it was proved by GST pull-down that HBDSPs interact with TFII-I protein in vitro. TFII-I protein is a multifunctional transcription factor, which can not only promote chromatin remodeling, but also promote chromatin remodeling. The interaction between HBDSP and TFII-I may affect the division and proliferation of hepatoma cells. This study lays a foundation for further discussing the pathogenicity of HBDSP.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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