干扰素诱导蛋白Nmi与人巨细胞病毒皮层蛋白UL23相互作用区域的研究

目的:探究干扰素诱导蛋白N-Myc相互作用因子(Nmi)与人巨细胞病毒(HCMV)皮层蛋白UL23相互作用的关键区域。方法:根据前期实验结果,分别将10个不同截短突变型的Nmi构建到原核表达载体p GEX-4T-1上,转化到大肠杆菌Rosetta(DE3)感受态细胞中,表达并纯化出带有GST标签的融合蛋白,利用GST-pulldown的方法探究Nmi与UL23相互作用的区域。根据GST-Pulldown实验结果,用同源重组的方法在真核表达载体pc DNA4-Myc上分别构建3个缺失突变型Nmi。将实验组和对照组的Nmi分别与含有Flag标签的UL23质粒共转染至He La细胞中,通过免疫共沉淀法进一步研究Nmi与UL23的相互作用区域。结果:(1)10个截短突变型Nmi与GST基因融合的原核表达载体构建成功;(2)3个缺失突变型Nmi与Myc基因融合的真核表达载体构建成功;(3)GST-pulldown实验证明Nmi与UL23相互作用位点位于Nmi上的第192~202位氨基酸区域;(4)免疫共沉淀法确认Nmi的第192~202位氨基酸区域是与UL23相互作用的区域,与GST-pulldown实验结果一致。结论:Nmi与UL23相互作用的区域位于Nmi上第192~202位氨基酸区域。这为阐明UL23帮助HCMV在宿主体内潜伏的分子机理提供了基础。

Key amino acids of interactions between interferon-induced protein N-Myc interactor and human cytomegalovirus tegument protein UL23

AIM:To investigate the key amino acids of interferon-induced protein N-Myc interactor (Nmi) that interacts with human cytomegalovirus (HCMV) tegument protein UL23. METHODS:According to the previous results, 10 groups of the truncated Nmi fragment were constructed on plasmid pGEX-4T-1. Recombinant plasmids were transformed into E.coli Rosetta (DE3) competent cells, and fusion proteins with GST tag were expressed and purified. The domains on Nmi that mediated the interaction with UL23 were identified by GST-pulldown test. Based on the results of GST-pulldown test, 3 groups of deleted Nmi fragments were inserted into the eukaryotic expression plasmid pcDNA4-Myc by homologous recombination. The plasmid with Flag-tagged UL23 plasmid and wild-type Nmi or deletion mutants were co-transfected into HeLa cells to reconfirm the key amino acids on Nmi that interacted with UL23 by the method of co-immunoprecipitation (Co-IP). RESULTS:The prokaryotic expression vectors of 10 groups of truncated Nmi mutants fused with GST gene were successfully constructed. Three groups eukaryotic expression vectors of Nmi deletion mutants fused with Myc gene were also successfully constructed. The results of GST-pulldown test indicated that the region of 192~202 aa located on Nmi was a key area to interact with UL23. The UL23 binding domain of Nmi in the amino acids 192~202 aa was confirmed by Co-IP, which was consistent with the GST-pulldown results. CONCLUSION:The 192~202 aa of Nmi are key amino acids in the interaction with UL23. This may provide the basis for clarifying the molecular mechanisms by which UL23 plays an important role in the latency of HCMV in host.