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

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ORF 3a 和ORF 7a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。

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

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 OK. 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 have the similar ability in triggering the expression of Luceferase gene, i.e 3a and 7a protein of SARS-CoV can 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.