V-val突变型EB病毒核抗原1的功能研究

Functions of V-val Subtype of Epstein-Barr Nuclear Antigen 1

背景与目的:EB病毒核抗原1(Epstein-Barr viral nuclear antigen 1,EBNA 1)对于维持EB病毒的潜伏感染有重要作用。V-val变异型的EBNA1与鼻咽癌有密切的相关性。本研究旨在比较原型和V-val变异型的EBNA1基因在上皮细胞中的功能的差异。方法:用PCR方法扩增出原型和V-val变异型EBNA1基因的全长并克隆到pGFP载体上,转染HEK293细胞,检测两种亚型的EBNA1蛋白的表达对细胞生物学性状的影响。以含有EB病毒增强子FR序列的荧光素酶载体作为报告基因,比较两种亚型的EBNA1基因对质粒的转录激活能力。结果:原型和V-val变异型EBNA1基因的表达对HEK293细胞的生长速度没有明显影响,但表达原型EBNA1的细胞的克隆形成能力明显低于V-val亚型。在裸鼠致瘤实验中,接种表达原型和V-val变异型EBNA1细胞的实验组均未见肿瘤形成。但是在瞬时转染实验中,表达V-val变异型EBNA1基因的HEK293细胞的荧光素酶活性明显高于原型EBNA1基因。结论:原型和变异型EBNA1均未发现有明显的转化细胞的能力,但是V-val变异型EBNA1蛋白与原型相比,其转录激活的功能明显增强。

BACKGROUND ＆ OBJECTIVE： Epstein-Barr viral nuclear antigen 1 （EBNA1） plays a crucial role in the latency of Epstein-Barr virus （EBV）. A close relation of V-val subtype of EBNA1 with nasopharyngeal carcinoma （NPC） was suggested by its preference to infect NPC cells. This study was to investigate the functional difference between prototype and V-val EBNA1 in epithelial cell line HEK293. METHODS： The coding sequences of prototype and V-val EBNA1 were amplified by polymerase chain reaction and cloned into pGFP vector, then transfected into HEK293 cells respectively. The biological consequences of EBNA1 gene expression were examined. The transcriptional activation ability was compared between prototype and V-val subtype of EBNA1 using luciferase reporter system containing family of repeats （FR） sequence of EBV. RESULTS= Prototype and V-val EBNA1 showed no effect on cell proliferation, while the cloning efficiency of prototype EBNA1-expressing cells was obviously lower than that of V-val EBNA1-expressing cells. No tumor formed in nude mice after injection of prototype or V-val EBNA1-trasfected HEK293 cells. However, the luciferase activity was significantly higher in V-val EBNA1-expressing HEK293 cells than in prototype EBNA1-expressing HEK293 cells in transient transfection assay. CONCLUSION： Prototype and V-val EBNA1 have no direct transforming activity on cells, whereas the transactivation activity of V- val EBNA1 in FR-containing plasmid is significantly higher than that of prototype EBNAI.