RNA标准物病毒样颗粒表达系统的构建及其特性研究

Construction of Standard RNA Virus-like Particle Expression System and Its Characteristic

为了构建RNA病毒样颗粒(virus-like particles,VLP)表达系统pET28a(+)-MS,将插入的外源基因诱导表达获得相应的RNA标准物质病毒样颗粒,将MS2噬菌体包膜蛋白和成熟蛋白基因插入表达质粒pET-28a(+),构建pET28a(+)-MS重组子,将带有包装位点序列的外源RNA对应的cDNA插入成熟蛋白基因的下游,经过诱导表达含外源RNA病毒样颗粒。结果表明,pET28a(+)-MS能表达活性的包膜蛋白(49.8ku)和成熟蛋白(14.4ku),能将外源基因RNA包装成病毒样颗粒,该VLP长度约1 500bp,RT-PCR反应性优良,能耐受RNase的降解,在血清中37℃经1个月未有明显降解。

To construct virus-like particles（VLP）expression platform pET28a（＋）-MS,the inserted foreign gene is induced to express the VLP containing corresponding RNA standard material.The envelope and mature protein genes of MS2 bacteriophages were inserted into plasmid pET-28a（＋）.The cDNA corre-sponding an exogenous RNA with package sequence was inserted into mature protein gene downstream, and the recombinant pET28a（＋）-MS was induced to express exogenous RNA containing VLP.The results showed that,pET28a（＋）-MS can express active envelope protein （49.8 ku）and mature protein （14.4 ku）,the foreign RNA was packaged into VLP.the VLP is about 1 500 bp long,RT-PCR has excellent re-activity,can withstand the degradation by RNase in serum 3 7 ℃ 1 month and no significant degradation de-tected.