cmv-3′LTR嵌合启动子对逆转录病毒载体MFG滴度及外源基因表达的影响  

Study of the Effect of cmv-3′LTR C himerical Promoter on the Titer an d Expression of Trans-gene of Retr oviral Vector MFG

作　　者：戴冰冰[1] 梅文瀚[1] 王家敏[1] 杨蓉[1] 钱关祥[1] 卢健[1] 

机构地区：[1]上海第二医科大学生物化学教研室分子生物学实验室,上海200025

出　　处：《病毒学报》2003年第3期235-239,共5页Chinese Journal of Virology

基　　金：国家自然科学基金项目(编号:39970311);上海市科委重点项目(编号:01JC14025)

摘　　要：为减轻逆转录病毒载体介导的外源基因的沉默,进一步提高逆转录病毒载体MFG介导的外源基因的表达水平,同时探讨逆转录病毒3′端长末端重复序列(long terminalrepeat,LTR)内U3区对病毒基因表达的影响,将逆转录病毒载体3′端LTR内的U3区用cmv核心增强子、启动子序列替代,同时去除了3个与逆转录病毒载体启动子甲基化有关的序列NCR、DR,并以egfp为报告基因,构建了MFG egfp和MFG egfp cmv表达载体。结果显示:利用cmv启动子替代MFG载体3′端LTR的U3启动子序列,会显著降低MFG载体的病毒滴度及其介导的外源报告基因的表达。提示利用cmv启动子替代病毒载体的3′端LTR内的U3区,并不是提高MoMLV(moloneymurineleukemivirus)逆转录病毒载体介导外源基因的表达及其病毒滴度的理想策略。结果也同时提示:在MoMLV逆转录病毒3′端LTR的U3区,可能存在与病毒RNA加工、成熟及稳定性有关的信号序列。In order to relieve the r etroviral vector mediated gene sil encing and further to enhance the exp ression of MFG retroviral vector m ediated transgenes,and at the same time to study the influence of the U3 region of 3′ long te rminal repeat(LTR) of retroviral v ector on the expression of retrovi ral gene,we substituted the promot er of the U3 region in the 3′LTR o f retroviral vector MFG with promo ter and enhancer of cmv; the ne gative control region(NCR) and the dir ect repeats(DR) which are associat ed with the promoter methylation w ere also deleted.Then we constructed th e expression vector MFG-egfp and M FG-egfp-cmv.The results showed tha t the expression of report gene wi thin the MFG-egfp-cmv vector was s ignificantly decreased both in NIH- 3T3 cell and in PA317 packaging ce ll.And the titer of MFG-egfp-cmv v ector was also decreased about 2-f old compared with the MFG-egfp.The se results suggest that it is not a good strategy to use the cmv-LTR chimerical promoter to enhance and prolong the expression of the MoML V retroviral mediated trans-gene.An d also,it suggests that there may be some important signals in the U 3 region of 3′LTR for processi ng,maturation and stabilization of the MoMLV retroviral transcribed R NA.

关 键 词：嵌合启动子 逆转录病毒载体 外源基因 基因表达 

分 类 号：Q782[生物学—分子生物学]