猪繁殖与呼吸综合征病毒N蛋白磷酸化位点鉴定及其作用研究

发布时间：2018-02-15 06:58

本文关键词： PRRSV N蛋白磷酸化转录调控　出处：《中国预防兽医学报》2017年07期 　论文类型：期刊论文

【摘要】：为了探索猪繁殖与呼吸综合征病毒(PRRSV)N蛋白磷酸化对于病毒复制和转录的影响,本研究利用Phos-tag和点突变技术实现了N蛋白磷酸化形式的分离并鉴定出磷酸化位点为第120位丝氨酸(Ser120)。通过反向遗传操作系统将N蛋白的磷酸化位点突变并拯救出病毒。空斑实验、IFA检测、病毒生长曲线结果显示,磷酸化位点突变对于病毒的感染性和N蛋白的核定位没有影响,但是病毒的复制能力减弱。进一步利用荧光定量RT-PCR方法检测突变病毒与亲本病毒在不同感染时间的基因组RNA和亚基因组RNA的转录水平,结果表明突变病毒的g RNA和长链sg RNAs转录水平明显低于亲本病毒,表明N蛋白Ser120磷酸化可能参与了病毒的转录调控过程。
[Abstract]:To investigate the effect of phosphorylation of PRRSVN protein on replication and transcription of porcine reproductive and respiratory syndrome virus. In this study, Phos-tag and point mutation techniques were used to isolate the phosphorylation of N protein and identify the phosphorylation site as serine serine serine Ser120. The phosphorylation site of N protein was mutated and the disease was saved by reverse genetic operating system. Virulence. Plaque test, IFA detection, The results of the virus growth curve showed that the phosphorylation site mutation had no effect on the infection of the virus and the nuclear localization of the N protein. However, the replication ability of the virus was weakened. The transcription levels of genomic RNA and subgenomic RNA of mutant virus and parent virus at different infection times were further detected by fluorescence quantitative RT-PCR. The results showed that the transcriptional levels of g RNA and long chain sg RNAs of mutant viruses were significantly lower than those of parental viruses, suggesting that the phosphorylation of N protein Ser120 might be involved in the transcriptional regulation of the virus.
【作者单位】：中国农业科学院上海兽医研究所;
【基金】：国家科技支撑项目(2015BAD12B01-1) 国家自然科学基金项目(31502072、31302098、31300140)
【分类号】：S852.651

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