摘要
目的人乳头瘤病毒（Ｈｕｍａｎｐａｐｉｌｏｍａｖｉｒｕｓ，ＨＰＶ）１６型早期启动子Ｐ９７控制着病毒癌基因的表达。为了观测长控制区（Ｌｏｎｇｃｏｎｔｒｏｌｒｅｇｉｏｎ，ＬＣＲ）上ＹＹ１结合位点的破坏对病毒转化能力的影响。方法构建了带有自然发生突变ＬＣＲ序列的ＨＰＶ１６全基因质粒，利用ＥＭＳＡ和荧光素酶实验检测小鼠纤维细胞胞核内源性ＹＹ１蛋白存在和功能状态；将标准及突变的ＨＰＶ１６毒株转染至Ｃ１２７细胞进行软琼脂糖培养基生长实验。结果与上皮类细胞相似，在Ｃ１２７胞核提取物中可检出ＹＹ１蛋白，并且具有良好的ＤＮＡ结合功能和Ｐ９７活性抑制作用。转化实验显示ＹＹ１位点突变ＨＰＶ１６毒株可在软琼脂糖培养基上形成２～１０倍量多的生长集落。结论细胞转录调节因子ＹＹ１存在于啮齿类动物纤维细胞胞核中；ＬＣＲ上ＹＹ１结合位点的破坏可在完整基因组范围内明显增强病毒对小鼠纤维细胞的转化能力。
Objective To study the effect of removal of YY1 binding sites within the LCR region of HPV 16 on viral transforming activity.Methods Previously we had generated new plasmids carrying HPV 16 whole genome, which contained naturally occurred mutated LCR sequences. The viral transforming abilities on mouse fibroblasts were evaluated in anchorage independent assays, while the expression and activity of YY1 protein in fibrobalsts were tested with EMSA and luciferase assays.Results YY1 protein was expressed in mouse fibroblasts C127, with ability for DNA binding and repression on P97 activity. Both HPV 16 wild type DNA and mutated DNAs were transfected into C127 cells and spread to the soft agarose mediums after selecting with G 418. The growth numbers of the cells transfected with mutated HPV 16 DNAs were 2 10 fold more than that with wild type HPV 16 DNA.Conclusion Like in epithelial cells or cell lines, transcription regulator YY1 is widely expressed in rodent fibroblasts. Removal of YY1 binding motifs can elevate in the context of the whole genome, the anchorage independent growth ability and the transforming capacity of HPV 16 on the mouse fibroblasts.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
1999年第1期5-8,共4页
Chinese Journal of Experimental and Clinical Virology
