稳定表达H1N1流感病毒核糖核蛋白的Hela细胞株的建立

Stable expression of ribonucleoprotein of H1N1 influenza virus in Hela cell line

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 stable expression of ribonucleoprotein of H1N1 influenza virus of Hela cell line,with enhance green fluorescent protein as indicating protein. Methods The recombinant eukaryotic expressing plasmids pc DNA3.1 / Zeo-PB1,pc DNA3.1 / Hygro-PB2, pc DNA3.1 / Neo-PA and p EF6 / V5-His A-NP were constructed by DNA recombination technique, and confirmed by PCR and gene sequencing. The minimum lethal concentrations of Zeocin,Hygromycin B, Geneticin（G418）and Blasticidin S HCl for He La cells were determined. The Four types of recombinant plasmids were stably transfected into Hela cell using Lipofectamine 2000,and selected by corresponding antibiotics.Results The genes of ribonucleoprotein integrated into the host genome stably were proved by RT-PCR. The effective gene expression in Hela cells was confirmed by POL I system. Conclusion The stable ribonucleoprotein-expressing of Hela cells were constructed successfully, and could be used as high through put selection of target-specific anti-influenza virus drugs.