甲型H1N1流感病毒核蛋白NP的真核表达

发布时间：2018-01-03 13:41

本文关键词：甲型H1N1流感病毒核蛋白NP的真核表达　出处：《现代预防医学》2013年04期 　论文类型：期刊论文

【摘要】：目的构建甲型SWH1N1流感病毒核蛋白(NP)的杆状病毒真核表达载体,转染sf9细胞表达并纯化其编码蛋白。方法从江苏SWH1N1流感病毒毒株(A/Jiangsu/1/2009(H1N1))提取病毒RNA,采用RT-PCR技术扩增NP基因,将其克隆至pMD18-TSimple Vector中构建pMD18-T/NP质粒,双酶切pMD18-T/NP与p-fast后,连接构建真核表达载体p-fast-NP,转化DH10Bac细胞,转座构建杆状病毒NP-Bacmid,经酶切及测序鉴定后将NP-Bacmid转染到sf9细胞中,通过免疫印迹法鉴定NP蛋白的表达,采用亲和层析法纯化NP蛋白。结果经双酶切、测序鉴定证实NP基因的真核表达载体构建成功;免疫印迹法证实NP基因的表达;纯化获得了高纯度的NP蛋白。结论成功克隆了NP基因,并构建了其杆状病毒真核表达载体,该表达载体的构建为后期的流感病毒快速检测以及基因工程疫苗制备奠定了良好的基础。
[Abstract]:Objective to construct a baculovirus eukaryotic expression vector of influenza A SWH1N1 nucleoprotein (NPN). Methods RNA was extracted from Jiangsu SWH1N1 influenza virus strain A / Jiang / 1 / 2009 H _ 1 / H _ 1. NP gene was amplified by RT-PCR and cloned into pMD18-TSimple Vector to construct pMD18-T/NP plasmid. After double enzyme digestion of pMD18-T/NP and p-fast, the eukaryotic expression vector p-fast-NPN was constructed and transformed into DH10Bac cells. The baculovirus NP-Bacmida was constructed by transposing. After restriction endonuclease digestion and sequencing, NP-Bacmid was transfected into sf9 cells and the expression of NP protein was identified by Western blotting. NP protein was purified by affinity chromatography. Results the eukaryotic expression vector of NP gene was successfully constructed by double enzyme digestion and sequencing. The expression of NP gene was confirmed by Western blot. Conclusion the NP gene was cloned successfully and its eukaryotic expression vector was constructed. The construction of the expression vector laid a good foundation for rapid detection of influenza virus and preparation of genetic engineering vaccine.
【作者单位】：江苏省疾病预防控制中心病原微生物研究所卫生部肠道病原微生物重点实验室;
【基金】：江苏省自然科学基金资助项目(BK2009433;BK2009431) 江苏省卫生厅资助项目(Z200919;H200857)
【分类号】：R373.1
【正文快照】： 流行性感冒是一种最常见的病毒性急性呼吸道传染病,传染性强,并易导致肺炎等严重并发症,甚至死亡[1]。流感病毒是致流行性感冒的主要病原体。流感病毒属正黏病毒科,是一种带包膜并且分节段的单负链RNA病毒[2]。根据病毒核衣壳蛋白(NP)和基质蛋白(Matrix,M)抗原性的差异,流感

【相似文献】