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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 (EBNA 1) plays an important role in the maintenance of latent infection of Epstein-Barr virus. V-val variant EBNA1 and nasopharyngeal carcinoma are closely related. The aim of this study was to compare the differences in the functions of the EBNA1 gene in the prototype and V-val variants in epithelial cells. Methods: The full length of the prototype and V-val variant EBNA1 gene was amplified by PCR and cloned into pGFP vector. The expression of EBNA1 protein in two subtypes of HEK293 cells was detected by flow cytometry. The luciferase vector containing the Epstein-Barr virus enhancer FR sequence was used as a reporter gene to compare the transcriptional activation of plasmids between two subtypes of EBNA1 gene. RESULTS: The expression of prototype and V-val variant EBNA1 gene had no significant effect on the growth rate of HEK293 cells, but the clonogenic capacity of EBNA1-expressing cells was significantly lower than that of V-val subtype. In the nude mice tumorigenicity experiment, no tumor formation was observed in the experimental group inoculated with EBNA1 cells expressing the prototype and the V-val variant. However, in transient transfection experiments, the luciferase activity of HEK293 cells expressing V-val variant EBNA1 gene was significantly higher than that of the original EBNA1 gene. CONCLUSION: Neither the prototype nor the variant EBNA1 has the ability to transform cells, but the VNA variant EBNA1 protein has a significantly enhanced transcriptional activation compared with the prototype.