Yeast two-hybrid方法筛选VISA相互作用蛋白

王丹丹, 陈恬, 许亮国

中国生物工程杂志 ›› 2017, Vol. 37 ›› Issue (6) : 63-69.

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中国生物工程杂志 ›› 2017, Vol. 37 ›› Issue (6) : 63-69. DOI: 10.13523/j.cb.20170610
技术与方法

Yeast two-hybrid方法筛选VISA相互作用蛋白

  • 王丹丹1,2, 陈恬1,2, 许亮国1,2
作者信息 +

Screening of VISA Interacting Proteins by Yeast Two-hybrid System

  • WANG Dan-dan1,2, CHEN Tian1,2, XU Liang-guo1,2
Author information +
文章历史 +

摘要

病原微生物感染对人类健康构成巨大威胁,先天免疫系统是生物体在长期进化过程中建立起来的天然保护系统。VISA(MAVS,CARDIF,IPS-1 )是连接胞浆dsRNA受体RIG-I与下游信号转导通路的一个接头蛋白,研究结果表明VISA无论在TLR3非依赖的或者TLR3介导的抗病毒IFN信号途径中都具有关键作用,但目前对VISA信号转导的详细机制还不十分清楚。更多VISA相互作用蛋白的发现以及它们之间的作用机制的研究能完善我们对VISA参与的信号转导机制的认识。利用酵母双杂交系统,用VISA蛋白的全长做诱饵(Bait)筛选293 T细胞cDNA表达文库,并结合免疫共沉淀实验,双荧光素酶报告基因系统验证筛选到的阳性克隆和VISA作用的真实性。通过酵母双杂交系统成功筛选到 Sec13L1 候选基因并验证了其与VISA的全长蛋白相互作用,在293 T细胞中过表达该基因,研究结果显示 Sec13L1 过表达能促进VISA对IFNβ的诱导,并存在剂量效应。

Abstract

The infection of a virus is a great threat to host; the innate immune system is set up as the natural protection system of an organism. VISA (also know as MAVS, CARDIF, IPS-1), is an critical adapter protein required for virus-triggered IFN-β signaling, recruiting RIG-I-like receptors(including RIG-I and MDA5) and downstream signaling proteins to VISA signalosome at mitochondria upon virus infection, leading to activation of transcriptional factor IRF3 and NF-κB, and resulting in the induction of IFNs and other inflammatory cytokines. However, the mystery of VISA for the antiviral function is still not completely figured out. VISA was used as bait for screening a human 293T cell cDNA library in a yeast two-hybrid screening, trying to identify the interacting proteins of VISA. Then, coimmunoprecipitation experiments were carried out to verify the association between VISA and candidates proteins screened from the yeast two-hybrid system assay. It is indicated that Sec13L1 associated with VISA. Overexpression of Sec13L1 in 293 cells facilitated to enhance the activation of IFNβ promoter induced by VISA and virus infection in a dose-dependent manner.

关键词

酵母双杂交系统 / 先天免疫 / VISA / Sec13L1

Key words

VISA / Sec13L1 / Innate immune / Yeast two-hybrid system

引用本文

导出引用
王丹丹, 陈恬, 许亮国. Yeast two-hybrid方法筛选VISA相互作用蛋白[J]. 中国生物工程杂志, 2017, 37(6): 63-69 https://doi.org/10.13523/j.cb.20170610
WANG Dan-dan, CHEN Tian, XU Liang-guo. Screening of VISA Interacting Proteins by Yeast Two-hybrid System[J]. China Biotechnology, 2017, 37(6): 63-69 https://doi.org/10.13523/j.cb.20170610
中图分类号: Q291   

参考文献

[1] Xu L G, Wang Y Y, Han K J, et al. VISA is an adapter protein required for virus-triggered IFN-β signaling. Mol. Cell, 2005, 19(6):727-740.
[2] Seth R B, Sun L J, Ea C K, et al. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-κB and IRF3. Cell, 2005, 122(5):669-682.
[3] Kawai T, Takahashi K, Sato S, et al. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nature Immunol, 2005, 6(10):981-988.
[4] Belgnaoui S M, Paz S, Hiscott J. Orchestrating the interferon antiviral response through the mitochondrial antiviral signaling (MAVS) adapter. Immunology, 2011, 23(5):564-572.
[5] Moreira T G, Zhang L, Shaulovet L, et al. Sec13 regulates expression of specific immune factors involved in inflammation in vivo. Scientific Reports, 2015, 5:17655.
[6] He Y, Yang F, Wang F, et al. The upregulation of expressed proteins in HepG2 cells transfected by the recombinant plasmid-containing HBx gene. Scandinavian Journal of Immunology, 2007, 65(3):249-256.

基金

国家自然科学基金(31370876,31570876)、江西省自然科学基金重大项目(20143ACB20004)资助项目

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