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[目的]构建含肠道病毒71型(EV71)VP1基因片段的抗核糖核酸酶的病毒样颗粒。[方法]利用PCR技术扩增大肠杆菌MS2噬菌体的外壳蛋白和成熟酶蛋白基因,将其克隆到pET32a中构建中间载体pET32a-MS2。将EV71VP1基因片段连接到中间载体噬菌体基因的下游,构建原核表达载体pET32a-MS2-EV71。将重组质粒pET32a-MS2-EV71转化宿主菌,经IPTG诱导表达获得病毒样颗粒,对病毒样颗粒进行透射电镜观察、RT-PCR检测和稳定性试验。[结果]重组质粒pET32a-MS2-EV71经PCR、双酶切鉴定和测序分析后证实构建成功;透射电镜观察到直径大约23nm的圆形病毒样颗粒;RT-PCR和稳定性试验结果表明,诱导产生的病毒样颗粒含EV71VP1基因片段,并且稳定性良好。[结论]成功构建了含EV71VP1基因片段的病毒样颗粒,稳定性良好,可作为病毒检测时标准品和质控品使用。
[Objective] To construct an anti-ribonuclease-like virus-like particle containing the VP1 gene fragment of enterovirus 71 (EV71). [Method] The coat protein and mature enzyme protein of Escherichia coli MS2 phage were amplified by PCR and cloned into pET32a to construct the intermediate vector pET32a-MS2. The EV71VP1 gene fragment was ligated to the downstream of the intermediate vector phage gene to construct the prokaryotic expression vector pET32a-MS2-EV71. The recombinant plasmid pET32a-MS2-EV71 was transformed into host bacteria and induced by IPTG to obtain virus-like particles. The virus-like particles were observed by transmission electron microscopy, RT-PCR and stability test. [Result] The recombinant plasmid pET32a-MS2-EV71 was confirmed by PCR, double enzyme digestion and sequence analysis. The results of RT-PCR and stability test showed that induction of induction The resulting virus-like particles contain the EV71VP1 gene fragment and are stable. [Conclusion] The virus-like particles containing the EV71VP1 gene fragment were successfully constructed and have good stability and can be used as standards and controls for virus detection.