含SARS CoV RNA病毒样颗粒的构建和表达

Construction and expression of virus-like particles containing SARS CoV RNA

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、RT PCR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 30d ,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RT PCR检测、定量分析的有效阳性参考品 .

AIM: To construct and express RNase-resistant virus-like 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 RT-PCR. These cDNAs were purified with SABC's 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 virus-like particles were purified and tested by quantitative analysis, RT-PCR and stable experiment. RESULTS: The RNase-resistant virus-like particles containing the RNA fragments of SARS CoV was successfully constructed and remained stable for 30 days at 37℃. CONCLUSION: The new-type virus-like particles can be conveniently used as positive control of SARS clinical nucleic diagnostic reagents.