含SARSCoVRNA病毒样颗粒的构建和表达.doc

含SARS CoV RNA病毒样颗粒的构建和表达 作者：李振勇，景建洲，刘明霞，邵大晓，冯艳铭，李智涛，宋国英，王秋旗，王云龙，屈凌波，赵玉芬 【关键词】 病毒样颗粒 　　【Abstract】 AIM: To construct and express RNaseresistant viruslike particles containing the RNA fragments of SARS CoV. METHODS: The primers of MS2 assembly protein gene, coat protein gene and the fragment of SARS Cov RNA polymerase gene were synthesized according to their sequences from Genbank data and were used to amplify their cDNAs by RTPCR. These cDNAs were purified with SABCs ResincolumTM system for DNA purification from agarose gel. Expression vector pTrc99a and their cDNA fragments were ligated together with T4 DNA ligase after digestion with restriction enzymes. The prokaryotic expression was obtained by the transformation of the recombinant plasmid constructed above into E.coli JM109. The obtained viruslike particles were purified and tested by quantitative analysis, RTPCR and stable experiment. RESULTS: The RNaseresistant viruslike particles containing the RNA fragments of SARS CoV was successfully constructed and remained stable for 30 days at 37℃. CONCLUSION: The newtype viruslike particles can be conveniently used as positive control of SARS clinical nucleic diagnostic reagents. 　　【Keywords】 SARS; reverse transcriptase polymerase chain reaction; RNA, viruslike particles; ribonucleases 　　【摘要】 目的： 构建并表达含有SARS冠状病毒RNA片断的抗核糖核酸酶的病毒样颗粒. 方法： 通过克隆大肠杆菌噬菌体MS2的装配蛋白和壳蛋白基因以及SARS冠状病毒RNA聚合酶基因片段，将这些基因连接到载体 pTrc99a上表达，并进行纯化、定量分析、RTPCR检测和稳定性试验. 结果： 获得病毒样核蛋白颗粒，在37℃稳定性可达到30 d，能抵抗核糖核酸酶降解. 结论： 该病毒样核蛋白颗粒稳定、安全、可靠，可作为SARS冠状病毒RTPCR检测、定量分析的有效阳性参考品. 　　【关键词】 SARS； 逆转录聚合酶链反应； RNA，病毒样颗粒； 核糖核酸酶类 　　0引言 　　200211/200306 SARS［1,2］在全球流行，对全球尤其我国的经济发展带来了巨大影响，但在科技人员的努力下，很快研制成功了快速、准确的SARS病毒基因检测试剂. 临床检测中人们普遍关心的是检测试剂盒中使用的阳性参考品是否具有传染性、或者能否起到阳性全程参比的作用. 因此，研制稳定、无传染性的阳性参考品尤为重要. 我们根据Drosten amp; Collahan等［3,4］报道的方法，制备了含有SARS冠状病毒RNA聚合酶基因片段的抗核糖核酸酶病毒样核蛋白颗粒. 该病毒样颗粒能有效抵抗RNase引起的RNA降解，能作为SARS冠状病毒基因检测的阳性参考品，对SARS基因检测起到有效的全程监控作用. 　　1材料和方法 　　1.1材料 　　① 灭活的SARS冠状病毒样本由Robert Koch Institut（德国）提供，MS2噬菌体（ATCC 15597B1）购于