Epstein-Barr病毒潜伏性膜蛋白1 CTAR3缺失突变体的构建与功能分析  

Construction and function analysis of the Epstein-Barr Virus-encoded latent membrane protein-1 of CTAR_3 region

作　　者：张志伟[1] 张琼[2] 余艳辉[2] 欧阳咏梅[2] 贺智敏[2] 

机构地区：[1]南华大学医学院肿瘤研究所,衡阳421001 [2]中南大学湘雅医学院肿瘤研究所,长沙410078

出　　处：《微生物学报》2008年第10期1308-1313,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30470668);湖南省卫生厅科研基金(B2006-100)~~

摘　　要：【目的】探讨Epstein-Barr病毒潜伏性膜蛋白1(LMP1)促细胞转化的主要活性部位及其作用机制。【方法】采用PCR方法重组LMP1羧基末端活化域3(aa232-aa351)对应密码子缺失突变体(LMP1△232-351),将突变型LMP1△232-351和野生型LMP1(LMP1WT)分别导入永生化的鼻咽上皮细胞NP69中,比较二者对细胞的转化作用。同时,构建含JAK3启动子序列的荧光素酶表达质粒(pGL-2/JAK3-LUC),将LMP1△232-351与LMP1WT分别与含有JAK3启动子序列或NF-κB结合序列启动子的荧光酶表达质粒共转染293细胞(用pLNSX质粒作对照),比较二者活化JAK3启动子或转录因子NF-κB的功能。【结果】(1)LMP1△232-351促NP69细胞转化的能力较LMP1WT显著降低(n=3,p<0.01)。(2)LMP1WT能明显呈浓度依赖性活化JAK3启动子,而LMP1△232-351上调能力几乎丧失。【结论】LMP1羧基末端活化域3(aa232-aa351)是LMP1的重要活性部位之一,其促细胞转化的作用与JAK3蛋白表达调节有关。[Objective] The role of carboxyl terminal activating region of latent membrane proteinl （LMP1） in Epstein-Barr virus infection and oncogenesis is unclear. In this study, we investigated the activating sites and functionary mechanism of LMP1. [Methods] We recombined a deletion mutant type LMP1 （LMP1△232-351）, deleted the amino acid residues including 232-351 codons in carboxyl terminal activating region-3 by PCR. Then we compared mutant type LMP1△232-351 with wild type LMP1 （LMP1wT） to alter biological effect in Nasopharyngeal Epithelial Cell line NP69. Moreover, we constructed a Janus Kinase 3 （JKA3） promoter luciferase reporter system （pGL-2/JAK3-LUC）. We respectively cotransfected the LMP1△232-351 and LMP1^WT with promoter including NF-kB binding sequence or JAK3 promoter luciferase reporter into 293 cells （controlled with pLNSX vector）, and compared their actions to activating promoters by results of luciferase activity assay. [Results] （1） The colony forming number （CFN） of NP69-LMP1△232-351 cells significantly decreased to compare with CFN of NP69-LMP1^WT （n=3, p〈0.01）. （2） LMP1^WT was able to up-regulate the transcription activity of JAK3 promoter and the level of up-regulation was correlated with its concentration in Human em- bryonic kidney 293 cell line; while LMP1△232-351 was almost defective ability to activate the promoter. [Conclusion] The carboxyl terminal activating region-3 may be one of the most important function sites of LMP1, which involved in activating the JAK3 promoter and regulating the expression of JAK3 protein.

关 键 词：EB病毒 潜伏性膜蛋白 缺失突变体 NF-KB JAK3启动子 

分 类 号：R373[医药卫生—病原生物学]