【作者】 潘巍巍；

【导师】 宋方洲；

【作者基本信息】 重庆医科大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 人乳头瘤病毒(human papillomavirus,HPV)是一种无包膜的环状闭合双链DNA病毒,在鳞状上皮细胞层进行病毒复制。迄今,已发现100多种HPV型,其中只有少数高危型的HPV易引起宫颈癌。高危型HPV(如HPV-16、18、31)与生殖道感染有关并且可以通过基因组整合入宿主细胞染色体中,引起细胞恶性转化,从而使生殖系统癌变。低危型HPV(如HPV6、11)主要引起尖锐湿疣等良性增生。HPV基因组的早期区编码干扰细胞周期调控的E6/E7癌蛋白,高危型HPV的E6/E7癌蛋白能够与宿主细胞靶向结合,并与转录活性有关,大多数研究者致力于研究HPV癌蛋白与靶细胞中蛋白质的相互作用。E6蛋白通过泛素途径降解p53,因此,在DNA受损伤时不能诱导细胞生长停滞或凋亡。E7结合pRB,p107,and p130,并使其降解,同时阻止Mi2组蛋白乙酰基转移酶,诱导c-Myc,抑制TGFβ信号转导通路。HPV-16 E6/E7的重要性已被人们所公认。因此,为了更多的了解HPV病毒感染、致癌机制、RNAi、临床治疗和疫苗的研制,我们需要从动物组织、细胞、分子等不同水平研究HPV。首先,用PCR方法从含有HPV-16全基因序列的质粒上扩增E6/E7基因,将E6/E7基因连接到pET-32a (+)质粒上,经酶切测序鉴定后转化到大肠杆菌BL21(DE3)细菌中。抗性生长的克隆用异丙基硫代-β-D-半乳糖苷(IPTG)诱导,溶解细胞,通过SDS-PAGE电泳检测E6/E7蛋白的大更多还原

【Abstract】 Human papillomaviruses (HPVs) are double-stranded covalently closed circular DNA (cccDNA) viruses, and the productive phase of the viral life cycle elaborated in the upper squamous epithelial cell layers. To date, there are more than 100 types of HPVs, but only a subset of“high-risk”viral types induced cerviral carcinogenesis. High-risk HPVs, such as HPV-16, 18, 31, have been directly linked to genital infection that can progress to malignancy. They integrate their genomes into the chromosome of the host cell to induce immortalization and transformation. In contrast, low-risk HPV types, which also infect genital epithelia, primarily induce benign lesions, e.g. condyloma acuminatum.HPVs coded two genes in their early region, E6 and E7, and high-risk viral types encode E6 and E7 oncoproteins with an enhanced affinity for host cellular targets.Whereas transcriptional activity has been associated with both E6 and E7, the major thrust of investigation has centered on protein-protein interactions with cellular targets. E6 degradated p53 protein via the ubiquitin pathway. Thus, impairing the ability of cells to either arrest the cell cycle or enter the apoptotic pathway in response to DNA damage. E7 binded, destabilized, and consequently destructed pRB,p107,and p130. At the same time ,it sequestrated Mi2 histone deacetylase,inducted c-Myc to abrogate transforming growth factorβinhibitory signaling.更多还原