2.2kb乙型肝炎病毒基因组剪接变异体特异性新蛋白的功能研究

发布时间：2018-05-19 23:08

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

【摘要】： HBV基因组剪接变异体( spliced variants of hepatitis B virus genomes)是由HBV前基因组RNA (pregenomic RNA, pgRNA)经剪接并逆转录产生的亚基因组DNA。长度为2.2Kb的HBV剪接变异体占80%以上,分为双剪接型和单剪接型,它们可编码剪接特异性新蛋白并与病毒的持续性感染及致病性相关。本研究着眼于这两种剪接变异体编码的两种不同的剪接特异性新蛋白,拟用酵母双杂交的方法,寻找能够与它们相互作用的细胞蛋白,从而探索它们的致病性。本研究第一部分从临床标本中分离获得双剪接型与单剪接型2.2Kb乙型肝炎病毒基因组剪接变异体,在此基础上构建了双剪接型剪接变异体特异性新基因(TPds基因)与单剪接型剪接变异体特异性新基因(TPss基因)的酵母双杂交诱饵载体pGBKT7-TPds、pGBKT7-TPss。在酵母双杂交实验过程中,发现TPds具有反式激活GAL4反应元件的能力;而TPss则没有这一功能。本研究的第二部分旨在深入探讨TPds的反式激活作用。利用分段克隆的方法,确定了TPds蛋白反式激活的主要功能区域位于其中部的28个氨基酸,此28个氨基酸编码框符合preS1第30-57位氨基酸,从剪接体的角度阐明了具有致病意义的preS1相关蛋白的第二种来源。进一步构建TPds蛋白的哺乳动物细胞表达载体pcDNA3.1/HisC-TPds,证实了该蛋白在肝细胞中对CMV即刻早期启动子、SV40启动子/增强子也具有反式激活作用,同时还证实了该蛋白对HBV自身启动子/增强子也具有刺激作用。本研究的第三部分利用酵母双杂交系统筛查肝细胞中与TPss蛋白相互作用的细胞蛋白,获得四种候选蛋白:组织蛋白酶B、微粒体环氧化物水解酶、组织蛋白酶D、纤维蛋白原γ链;并通过哺乳动物细胞双杂交实验验证了它们与TPss蛋白在肝癌细胞Huh7中的相互作用;提示TPss可能影响肝细胞凋亡与肿瘤的侵袭转移,并可能影响肝组织的解毒功能与凝血功能。
[Abstract]:HBV genomic splicing variant (spliced variants of hepatitis B virus genomes) is a subgenomic DNA produced by splicing and reverse transcription of pregenomic RNA pregenomic RNAs (pgRNAs). More than 80% of HBV splicing variants with length of 2.2Kb can be divided into double splicing type and single splicing type. They can encode splicing specific new protein and are related to persistent infection and pathogenicity of virus. This study aims at two different splicing specific proteins encoded by these two splicing variants. We intend to explore their pathogenicity by using yeast two-hybrid method to search for cellular proteins that can interact with them. In the first part of this study, double splicing and single splicing 2.2Kb HBV genomic splicing variants were isolated from clinical specimens. The yeast two-hybrid bait vector pGBKT7-TPSS was constructed based on the novel splicing variant specific gene (TPDS gene) and the single splice variant gene (pGBKT7-TPSS gene) of yeast two hybrid bait vector pGBKT7-TPKT7-TPss. based on the above results, the yeast two-hybrid decoy vector pGBKT7-TPKT7-TPssis was constructed. In yeast two-hybrid experiments, it was found that TPds had the ability to transactivate GAL4 reaction elements, but TPss did not. The second part of this study aims to explore the transactivation of TPds. By using the method of segmental cloning, 28 amino acids located in the middle of TPds protein were identified as the main functional region of transactivation. The 28 amino acid coding frames were in accordance with the 30-57 amino acids of preS1. The second source of preS1 associated proteins with pathogenicity was elucidated from splicing point of view. Further construction of mammalian expression vector pcDNA3.1 / HisC-TPdsof TPds protein confirmed that the protein could also transactivate the CMV immediate early promoter SV40 promoter / enhancer in hepatocytes. It is also confirmed that the protein also stimulates the promoter / enhancer of HBV itself. In the third part of this study, yeast two-hybrid system was used to screen cellular proteins interacting with TPss protein in hepatocytes. Four candidate proteins were obtained: cathepsin B, microsomal epoxide hydrolase, cathepsin D, fibrinogen 纬 chain. The interaction between TPss and TPss protein in Huh7 cells was verified by two-hybrid assay of mammalian cells, suggesting that TPss may affect hepatocyte apoptosis and tumor invasion and metastasis, and may affect the detoxification function and coagulation function of liver tissue.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R373.21

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