改进型外部引导序列抑制人巨细胞病毒UL49基因的表达(英文)  

Mini External Guide Sequences (miniEGSs) Inhibit Expression of HCMV UL49

作　　者：周琪[1] 曾志锋[1] 李弘剑[1] 李月琴[1] 崔延伟[1] 杨丹[1] 邹奕[1] 杨光[1] 周天鸿[1] 

机构地区：[1]暨南大学广东省生物工程药物重点实验室,广州510632

出　　处：《中国生物化学与分子生物学报》2009年第6期522-527,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：Supported by National Natural Science Foundation of China (No.90608024 and 303707762);Key Science and Technology Project of Guangzhou (No.2006J1-C0111);Planned Science and Technology Project of Guangdong Province (No.2006B35502002);Key Program of Natural Science Foundation of Guangdong Province (No.36703);China Postdoctoral Science Foundation Funded Project (No.20080430845)~~

摘　　要：外部引导序列(EGS)技术(The EGS-based technology)是一种新型的基因沉默技术,能诱导内源性的核酶P(RNase P)对靶mRNA进行有效切割.以人类巨细胞病毒(HCMV)UL49基因mRNA片段为靶序列,基于前人实验基础上设计出更为精简与高效的改进型EGS(miniEGS),DNA片段长度仅为12bp.构建稳定表达HCMVUL49的细胞系,通过应用荧光定量PCR及Western印迹分别鉴定miniEGS对内源性UL49的抑制效率.结果显示,miniEGS能在HeLa细胞中能达到很高的转染效率(97.9%),并且在转染稳定表达UL49的HeLa细胞系后,发现UL49基因的mRNA与蛋白表达水平都出现明显下降(50%).研究表明,改进型的EGS序列不仅能有效抑制目的基因的表达,同时因其序列设计的精简性与高效性,可更好地应用到以后的抗病毒研究中.The technology involving external guide sequence （EGS） is a new approach for gene inactivation, since the induced endogenous RNase P cleaves the target RNA with high specificity and efficiency. Compared with the conventional designs, a more economical 12 bp optimized EGSs （miniEGSs） was constructed to target human cytomegalovirus （HCMV）. To investigate its inhibitory effects, quantitative real-time PCR and Western blot was used to detect the level of UL49 mRNA and protein in a cell line stably express HCMV UL49. The transfection efficiency of FITC labeled miniEGSs was above 97 % as assayed by flow eytometry. A significant reduction of UL49 mRNA （50%） was observed by quantitative real-time PCR assay following the miniEGS treatment, and the Western blot analyses confirmed a similar result. The data suggested a potential application to use miniEGSs for HCMV antiviral therapy.

关 键 词：外部引导序列(EGS) 核酶P 人巨细胞病毒 基因沉默 

分 类 号：R346[医药卫生—基础医学]