鉴定与疱疹病毒蛋白 ORFV024 相互作用的宿主细胞蛋白 LAGE3 和 IGFBP6。
Identification of host cellular proteins LAGE3 and IGFBP6 that interact with orf virus protein ORFV024.
机构信息
Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou 510515, China.
Department of Laboratory Medicine, School of Stomatology and Medicine, Foshan University, 5 Hebin Road, Chancheng District, Foshan, Guangdong Province 528000, China.
出版信息
Gene. 2018 Jun 30;661:60-67. doi: 10.1016/j.gene.2018.03.089. Epub 2018 Mar 29.
OBJECTIVE
Orf virus (ORFV) is the pathogen causing contagious pustular dermatitis in goats, sheep and herdsmen. Evidence has confirmed that ORFV can be used as a preventive and therapeutic immunomodulatory agent in several animal models. Our previous data demonstrated that ORFV024 is able to inhibit activation of the NF-κB signaling pathway and act as an important modulator for early immune responses against viral infection. However, the molecular mechanism by which ORFV024 exerting biological function remains unclear. In the present study, we explored and analyzed the function of host cellular proteins that interact with ORFV024.
METHODS
The yeast two-hybrid (Y2H) assay was performed to screen proteins interacting with ORFV024 using a cDNA library derived from primary ovine fetal turbinate cells (OFTu). Two of the screened proteins were further confirmed by confocal microscopy, His-tag pull-down assay and CO-Immunoprecipitation (CO-IP) assay. In addition, the ORFV024 interaction network was constructed using the STRING database.
RESULTS
In this study, 11 ovine cellular proteins were found to interact with ORFV024. In view of the importance of LAGE3 and IGFBP6 in the ORFV024 functional analysis, we further constructed LAGE3 and IGFBP6 interaction networks. The interactions between ORFV024 and LAGE3 or IGFBP6 were confirmed by confocal microscopy, LAGE3 was further confirmed in the His-tag pull-down assay and CO-IP assay.
CONCLUSIONS
Our findings indicate that ORFV024 can interact with ovine cellular proteins LAGE3 and IGFBP6.
目的
口疮病毒（ORFV）是引起山羊、绵羊和牧民传染性脓疱性皮炎的病原体。有证据证实，ORFV 可作为几种动物模型中的预防和治疗性免疫调节剂。我们先前的数据表明，ORFV024 能够抑制 NF-κB 信号通路的激活，并作为针对病毒感染的早期免疫反应的重要调节剂。然而，ORFV024 发挥生物学功能的分子机制尚不清楚。在本研究中，我们探索和分析了与 ORFV024 相互作用的宿主细胞蛋白的功能。
方法
使用源自原代绵羊胎儿鼻甲细胞（OFTu）的 cDNA 文库，通过酵母双杂交（Y2H）试验筛选与 ORFV024 相互作用的蛋白质。通过共聚焦显微镜、His 标签下拉测定和 CO-免疫沉淀（CO-IP）测定进一步证实了其中的两种蛋白质。此外，使用 STRING 数据库构建了 ORFV024 相互作用网络。
结果
本研究发现 11 种绵羊细胞蛋白与 ORFV024 相互作用。鉴于 LAGE3 和 IGFBP6 在 ORFV024 功能分析中的重要性，我们进一步构建了 LAGE3 和 IGFBP6 相互作用网络。通过共聚焦显微镜证实了 ORFV024 与 LAGE3 或 IGFBP6 之间的相互作用，进一步通过 His 标签下拉测定和 CO-IP 测定证实了 LAGE3。
结论
我们的研究结果表明，ORFV024 可以与绵羊细胞蛋白 LAGE3 和 IGFBP6 相互作用。
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