2.2kb乙型肝炎病毒基因组剪接变异体剪接特异性新蛋对Huh7肝细胞基因表达的影响

发布时间：2018-04-13 09:53

本文选题：乙型肝炎病毒 + RNA剪接　；参考：《福建医科大学》2007年硕士论文

【摘要】： 乙型肝炎病毒基因组剪接变异体( spliced variants of hepatitis B virus genomes)是一大类基因部分缺失的亚基因组DNA,由乙型肝炎病毒(Hepatitis B virus, HBV)前基因组RNA (pregenomic RNA, pgRNA)经剪接并逆转录产生[1]。在已经分离到的HBV剪接变异体中,长度约为2.2kb的剪接变异体占80%[2],此类变异体主要分为单剪接与双剪接两种类型。研究显示这两种HBV基因组剪接变异体编码的剪接特异性新蛋白与病毒的持续性感染及致病性相关[3,4],但其确切的致病机制迄今不明。本研究通过人类基因表达谱芯片研究分析这两种剪接特异性新蛋白对Huh7肝细胞基因表达的影响,探寻它们可能的致病机制。本研究第一部分旨在构建剪接特异性新蛋白真核表达载体,并证实其在肝细胞中的表达。为此将剪接特异性新蛋白编码区克隆到真核表达载体pcDNA3.1/HisC ,获得重组载体pcDNA3.1/HisC-TPds(双剪接型)、pcDNA3.1/HisC-TPss(单剪接型),瞬时转染Huh7肝细胞,以Anti-Xpress抗体进行Western Blot检测融合蛋白,证实剪接特异性新蛋白可在Huh7肝细胞中表达。本研究第二部分以pcDNA3.1/HisC、pcDNA3.1/HisC-TPds、pcDNA3.1/HisC-TPss分别瞬时转染Huh7肝细胞,提取转染细胞总RNA,进行人类基因表达谱芯片分析。同时以半定量RT-PCR验证芯片结果。芯片分析显示:2.2kb HBV基因组剪接变异体剪接特异性新蛋白可能从干扰肝细胞骨架的重塑、细胞内物质代谢等方面引起肝细胞的增生、迁移及代谢异常,促进肿瘤的发生。
[Abstract]:Spliced variants of hepatitis B virus genomes is a class of partially absent subgenomic DNAs, which is produced by splicing and reverse transcription of RNA pregenomic RNAs (pgRNAs) before hepatitis B virus (HBV).Among the isolated HBV splicing variants, about 80% of the splicing variants with length of 2.2kb are divided into two types: single splicing and double splicing.Studies have shown that the splicing specific proteins encoded by these two HBV genomic splicing variants are related to the persistent infection and pathogenicity of the virus.In this study, the effects of two splicing specific novel proteins on the gene expression of Huh7 hepatocytes were studied by using human gene expression microarray, and the possible pathogenetic mechanisms of these two novel splicing proteins were explored.The first part of this study was to construct a splicing specific eukaryotic expression vector and to confirm its expression in hepatocytes.Therefore, the splicing specific new protein coding region was cloned into eukaryotic expression vector pcDNA3.1/HisC, and the recombinant vector pcDNA3.1 / HisC-TPdswas obtained. The recombinant vector pcDNA3.1 / HisC-TPsss was transiently transfected into Huh7 hepatocytes, and the fusion protein was detected by Western Blot with Anti-Xpress antibody.It is confirmed that splicing specific new protein can be expressed in Huh7 hepatocytes.In the second part of the study, pcDNA3.1 / HisC-TPdsnpcDNA3.1 / HisC-TPSS pcDNA3.1 / HisC-TPss was used to transfect Huh7 hepatocytes, and the total RNAs were extracted and analyzed by human gene expression microarray.At the same time, semi-quantitative RT-PCR was used to verify the chip results.The microarray analysis showed that the splicing specific new protein of 2. 2kb HBV genomic splicing variant might cause hepatocyte proliferation, migration and abnormal metabolism, and promote the occurrence of tumor by interfering with the remodeling of hepatocyte cytoskeleton and intracellular substance metabolism.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373

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