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目的构建稳定表达H1N1流感病毒核糖核蛋白的Hela细胞株,为抗病毒药物筛选提供细胞模型。方法利用基因重组技术构建四种重组真核表达载体pc DNA3.1/Zeo-PB1、pc DNA3.1/Hygro-PB2、pc DNA3.1/Neo-PA、p EF6/V5-His A-NP,进行PCR和DNA测序鉴定;使用Hela细胞依次进行四种抗生素Zeocin、Hygromycin B、Geneticin(G418)、Blasticidin S HCl最小致死浓度实验,获得其最佳筛选浓度;利用Lipofectamine 2000将四种重组真核表达载体依次转染Hela细胞,并添加相应抗生素筛选稳定表达核糖核蛋白细胞株;RT-PCR以及POL I系统鉴定Hela细胞株是否稳定表达H1N1流感病毒核糖核蛋白。结果 RT-PCR初步鉴定流感病毒核糖核蛋白基因片段稳定整合在宿主基因组中,POL I系统证实核糖核蛋白在Hela细胞中已经获得了功能性表达。结论成功构建了Hela-核糖核蛋白细胞株,为以流感病毒RNA聚合酶复合体为靶点的抗流感病毒药物的研究提供了细胞模型。
Objective To construct a Hela cell line stably expressing the H1N1 influenza virus ribonucleoprotein and to provide a cell model for antiviral drug screening. METHODS: Four recombinant eukaryotic expression vectors pcDNA3.1 / Zeo-PB1, pcDNA3.1 / Hygro-PB2, pcDNA3.1 / Neo-PA and p EF6 / V5-His A-NP were constructed by gene recombination technique. PCR and DNA sequencing were carried out. The Hela cells were used to carry out the minimum lethal concentration test of four antibiotics, namely, Zeocin, Hygromycin B, Geneticin (G418) and Blasticidin S HCl, and the optimal screening concentration was obtained. Four recombinant eukaryotic expression vectors The vector was transfected into Hela cells in succession, and the corresponding antibiotics were selected to screen stable expression of ribonucleoprotein cell lines. RT-PCR and POL I system were used to identify whether Hela cells stably expressed H1N1 influenza virus ribonucleoprotein. Results The results of RT-PCR showed that the influenza virus ribonucleoprotein gene fragment was stably integrated in the host genome. POL I system confirmed that ribonucleoprotein has been successfully expressed in Hela cells. Conclusion The Hela-ribonucleoprotein cell line was successfully constructed and provided a cell model for the study of anti-influenza virus drugs targeting the influenza virus RNA polymerase complex.