人IFN-β基因启动子荧光表达载体的构建及鉴定  

Construction and identification of luciferase and EGFP expression vectors containing human IFN-β promoter

作　　者：徐永妮[1,2] 曹梦远 惠倩倩[1] 叶伟[1] 于澜[1] 张亮[1] 张芳琳[1] 

机构地区：[1]第四军医大学微生物学教研室,陕西西安710032 [2]解放军第323医院检验科,陕西西安710054

出　　处：《细胞与分子免疫学杂志》2014年第6期588-591,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点基础研究发展计划(973)(2012CB518905)

摘　　要：目的构建人IFN-β基因启动子萤火虫荧光素酶报告基因载体(pGL3-IFNB1)及增强型绿色荧光蛋白(EGFP)表达载体(pGE3-IFNB1),并在A549细胞中进行表达验证。方法采用PCR方法扩增出IFN-β启动子区片段,克隆入荧光表达载体pGL3 basic和pGE3 basic,并用脂质体瞬时转染A549细胞,6 h后用汉滩病毒(HTNV)感染转染后的细胞,24 h后检测重组载体在细胞内的表达情况。结果重组载体pGL3-IFNB1和pGE3-IFNB1分别经双酶切鉴定及序列测定证实载体构建正确;且在A549细胞内成功表达。经HTNV刺激后表达明显增加。结论成功构建了人IFN-β启动子荧光素酶报告基因载体(pGL3-IFNB1)及增强型绿色荧光蛋白表达载体(pGE3-IFNB1),为进一步研究病毒诱导产生IFN-β机制提供了有用的工具。Objective To construct a luciferase expression vector （pGL3-1FNB1） and an enhanced green fluorescent protein expression vector （pGE3-1FNB1） containing human IFN-β promoter, and verify the promoter activity of IFN-β in A549 cells. Methods IFN-β promoter was amplified with the human genome DNA by PCR, then the segment was cloned into the eukaryotic expression vectors pGL3-basic and pGE3-basic by PCR. The eukaryotic expression vectors were named pGL3- IFNB1 and pGE3-1FNB1, respectively. The recombinant vectors were then transiently transfected into A549 cells respectively, and 6 hours later, the transfected cells were infected with Hantaan virus （HTNV）. After 24 hours, the expressions of pGL3- IFNBI and pGE3-1FNB1 were tested in A549 cells. Results Double restriction enzyme digestion and sequence analysis showed that the recombinant vectors were successfully constructed and expressed in A549 ceils correctly. After HTNV infection, stronger expression was observed in these cells. Conclusion The recombinant vectors pGL3-1FNB1 and pGE3- IFNB1 containing IFN-β promoter have been successfully constructed. It provides useful tools for further study on the mechanism of IFN-I5 production induced by virus.

关 键 词：IFN-Β 启动子 表达载体 汉滩病毒 病毒感染 

分 类 号：Q786[生物学—分子生物学] R392.11[医药卫生—免疫学]