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目的构建EV71非结构蛋白及EV71 3C蛋白酶微复制子体系,研究微复制子的复制及表达情况。方法聚合酶链式反应扩增绿色荧光蛋白(EGFP),利用特定酶切位点及连接酶,将目的基因片段连接至p MD19-T载体,分别构建EV71非结构蛋白及EV71 3C蛋白酶微复制子体系。荧光显微镜下观察微复制子表达情况,实时荧光定量PCR观察微复制子复制情况。结果 EV71非结构蛋白微复制子及EV71 3C蛋白酶微复制子表达后,均可观察到特异性绿色荧光,荧光强度有明显差异;转染后12~72 h取样所测RNA含量几乎不变。结论非结构蛋白全长及3C蛋白酶启动微复制子转录强度不同,所构建微复制子不具备复制功能,提示结构蛋白编码区对病毒RNA复制有重要意义。
Objective To construct the EV71 3C protease micro-replicon system and to study the replication and expression of the micro-replicons. Methods The green fluorescent protein (EGFP) was amplified by polymerase chain reaction (PCR), and the target gene fragment was ligated into pMD19-T vector using specific restriction sites and ligase to construct EV71 non-structural protein and EV71 3C protease micro-replicon system. The expression of microduplex was observed under a fluorescence microscope and the replication of microduplex was observed by real-time fluorescence quantitative PCR. Results After the EV71 non-structural protein micro-replicon and the EV71 3C protease micro-replicon were expressed, the specific green fluorescence was observed and the fluorescence intensity was significantly different. Conclusion The full length of nonstructural protein and the intensity of transcription of microtubules initiated by 3C protease are different. The constructed microduplex does not have the replication function, suggesting that the structural protein coding region is of important significance for viral RNA replication.