人IFN-β启动子基因报告质粒的构建及SARS-CoV3a和7a蛋白对IFN-β诱生作用的初步研究  

The SARS-CoV 3a and 7a Protein May Enhance the Induction of IFN-β

作　　者：徐春娥[1] 付玲[1] 侯丽华[1] 翁少洁[1] 来大志[1] 李健民[1] 于婷[1] 于长明[1] 陈薇[1] 

机构地区：[1]军事医学科学院微生物流行病学研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《中国生物工程杂志》2006年第12期6-10,共5页China Biotechnology

基　　金：中德科学基金资助项目(GZ239-202/12)

摘　　要：3a蛋白和7a蛋白是SARS-CoV的非结构蛋白,分别主要由SARS基因组中ORF3a和ORF7a所编码。在体内和体外感染病毒的细胞中均发现了有3a蛋白的表达。首先将pGL3-Control载体进行了改构,除去了SV40启动子基因,获得了pGL3-Enhancer载体,将获得的IFN-β启动子基因连入载体中,构建了带有人IFN-β启动子基因的荧光素酶报告质粒IP-21,并且通过实验证明所构建的质粒在干扰素的诱导剂NDV的作用下能够表达荧光素酶活性,照度计检测荧光素酶活性增强,从而验证了所构建的重组质粒的有效性。为了观察SARS-CoV非结构蛋白3a和7a对干扰素诱生途径的影响,将IP-21瞬转入稳定表达有3a和7a蛋白的CHO细胞,通过荧光素酶的信号强弱对3a和7a的作用进行分析,结果表明SARS-CoV的3a和7a蛋白能够刺激荧光素酶的表达,即在体外的细胞实验中,3a和7a能有效地激活IFN-β的启动子部分。此结果对进一步研究3a和7a的功能以及对SARS-CoV的致病机制的进一步研究有一定意义。3a and 7a are nonstructural proteins of SARS-CoV, which are encoded separately by ORF 3a and ORF 7a in SARS-CoV genome. The expression of 3a has been founded in cells infected by virus in vivo or in vitro. Firstly, the pGL3-Control vector was reconstructed , the pGL3-Enhancer vector deletious of SV40 promoter gene was obtained . Then the IFN-β promoter gene was cloned into the pGL3 -Enhancer vector and pGL-IP21, the Luciferase reporter plasmid with IFN-β promoter was established. The availability of pGL-IP21 was verified by NDV ,the inductor of IFN-β, the Luciferase activity was assayed in cells transfected with pGL-IP21 by Luminometer. In order to see the function of 3a and 7a protein of SARS-CoV,CHO cells expressing 3a or 7a protein were transfected with pGL-IP21, the intensity of luciferase activity was analyzed. By analysis, in vitro, 3a and 7a protein of SARS -CoV had the similar ability in triggering the expression of Luceferase gene, i. e 3a and 7a protein of SARS-CoV could effectively activate the promoter fragment of IFN-β gene. This result will help studying the function of 3a and 7a protein and provide a method to study the nosogenesis mechanism of SARS-CoV.

关 键 词：SARS-COV 3a蛋白7a蛋白 IFN-β启动子 荧光素酶报告系统 

分 类 号：Q819[生物学—生物工程]