摘要
为了观察A型流感病毒NS1蛋白对细胞生长调控的影响,构建了2009、1918A型H1N1流感病毒NS1基因带HA、GFP融合标签的真核重组表达载体,用脂质体法转染A549细胞,应用荧光显微镜、An-nexinⅤ染色、流式细胞术、Western-blot检测NS1蛋白对细胞生长的影响。结果显示,构建的NS1基因真核表达载体在A549细胞中均成功表达了NS1蛋白;显微镜下观察到,A549细胞出现皱缩,体积变小,变圆,细胞核固缩,染色质边缘化等细胞凋亡现象。AnnexinⅤ染色法检测表明,试验组染色率均高于空白对照组和阴性对照组,且A549/NS11918的染色率显著(P<0.05)高于A549/NS12009;流式细胞仪检测表明,试验组细胞周期发生G2/M期阻滞,且A549/NS11918的G2/M期与Apoptotic期的细胞比例均大于A549/NS12009;Western-blot检测结果表明,NS1蛋白对p53、p-p53-S15(p53 15位Ser磷酸化)、p21具有上调作用,且NS11918蛋白对这些蛋白表达的上调作用大于NS12009蛋白。结果表明,NS1蛋白能够上调p53、p-p53-S15、p21蛋白表达,使细胞周期阻滞于G2/M期,同时诱导宿主细胞凋亡,NS11918蛋白的作用强于NS12009蛋白。
To observe the influence of NS1 protein on eukaryotic cells growths,recombinant eukaryotic expression vectors containing NS11918 and NS12009 genes with HA or GFP tag were constructed.The recombinant vector was transfected in A549 cells by Lipofectamine 2000 and the expression of NS1 protein was detected by Western-blot method.Transient transfection of the eukaryotic expression vector with NS12009 or NS11918 occured in A549 cells,then the A549 cells morphology was observed by microscopy.Shrunken,smaller sized,rounded,shrinkage and chromatin marginalization that are typical phenomenon of cell apoptosis were found.AnnexinⅤ staining was applied to detecting the cells apoptosis,and the staining ratio of cells was significant higher in the experimental group than in the blank and the negative control groups.Meanwhile,A549/NS11918 staining ratio was significant higher than that of A549/NS12009（P0.05）.The arrest in G2/M phase is detected by flow cytometry,and the ratio of G2/M phase in A549/NS11918 is greater than in A549/NS12009.The p53,p-p53-Ser15 and p21 are key proteins in cell cycle,their expression level was increased in the Western-blot,which indicated NS1 up-regulated expression of these proteins,and the up-regulation is greater in A549/NS11918 than in A549/NS12009.These results showed that NS1 protein played an important role in cell cycle arrest at G2/M phase and induced host cell apoptosis in A549 cell,and the possible mechanisms were by increasing the p53,p-p53-Ser15 and p21 protein expression levels to regulate cell cycle and apoptosis.The influence induced by NS11918 is greater than NS12009,which might be related on the PL domain deletion in NS12009.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2012年第12期1242-1248,共7页
Chinese Veterinary Science
基金
中国检验检疫科学研究院基本科研业务费专项(2011JK004)
国家重点基础研究发展计划项目(2011CB504704)
