CytoTrap细胞质酵母双杂交筛选与2447nt-489nt乙型肝炎病毒剪接特异蛋白相结合的肝细胞蛋白

发布时间：2018-06-28 00:06

本文选题：乙型肝炎病毒 + RNA剪接　；参考：《福建医科大学》2011年硕士论文

【摘要】：HBV基因组剪接变异体(spliced variants of hepatitis B virus genomes)是一大类由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2.2 kb的HBV剪接变异体占80%以上,其中2447nt-489nt剪接变异体可编码剪接特异蛋白HBSP (Hepatitis B spliced protein),并与病毒的持续性感染及致病性相关,但其机制尚未完全阐明。 CytoTrap细胞质酵母双杂交系统将相互作用的蛋白锚定在细胞膜上、激活Ras信号通路,从而使温度敏感突变株酵母细胞cdc25H能够在37°C生长。与经典的酵母细胞核双杂交相比,该系统更有助于筛查仅发生于细胞质的蛋白相互作用;且更适用于诱饵蛋白定位于细胞质并需要转录后翻译修饰的情况。本研究利用CytoTrap细胞质酵母双杂交系统筛选,获得与HBSP相互作用的肝细胞蛋白—转化生长因子β1诱导蛋白(transforming growth factor beta 1 induced transcript 1 ,TGFβ1I1),并通过GST-pull down和内源性免疫共沉淀在体内外初步证实,该蛋白能够与HBSP相互作用。提示HBSP可能通过与TGFβ1I1相互作用,影响肝癌细胞侵袭能力、凋亡以及雄激素诱导的肿瘤发生,为深入阐明该剪接变异体致病性奠定基础。
[Abstract]:The HBV genomic splicing variant (spliced variants of hepatitis B virus genomes) is a large class of sub genomic RNA (pregenomic RNA, pregenomic RNA), which is produced by splicing and reverse transcriptase, and is produced by the subgenome of 2.2. Is B spliced protein) is associated with persistent infection and pathogenicity of virus, but its mechanism has not yet been fully elucidated.
The CytoTrap cytoplasmic yeast two hybrid system anchors the interacting proteins on the cell membrane and activates the Ras signaling pathway so that the temperature sensitive mutant yeast cell cdc25H can grow at 37 degree C. Compared with the classical yeast cell nuclear double hybrid, the system is more helpful to screen the protein interaction only in the cytoplasm; and it is more suitable. The bait protein is located in the cytoplasm and needs post transcriptional modification.
In this study, CytoTrap cytoplasmic yeast two hybrid system was used to obtain the hepatocyte protein transforming growth factor beta 1 induced protein (transforming growth factor beta 1 induced transcript 1, TGF beta 1I1) interacting with HBSP, and it was preliminarily confirmed by GST-pull down and endogenous pestilence co precipitation in vivo and in vitro. BSP interaction suggests that HBSP may affect the invasiveness, apoptosis and androgen induced tumorigenesis of hepatoma cells through interaction with TGF beta 1I1, which lays the foundation for further elucidating the pathogenicity of the splicing variant.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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