Yeast two-hybrid方法筛选VISA相互作用蛋白

doi:10.13523/j.cb.20170610

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

1. 江西师范大学功能有机小分子教育部重点实验室南昌 330022;
2. 江西师范大学生命科学学院南昌 330022

Screening of VISA Interacting Proteins by Yeast Two-hybrid System

WANG Dan-dan^1,2, CHEN Tian^1,2, XU Liang-guo^1,2

1. Key Laboratory of Small Functional Organic Molecule, Ministry of Education, Jiangxi Normal University, Nanchang 330022, China;
2. College of Life Sciences, Jiangxi Normal University, Nanchang 330022, China

全文: PDF(673 KB) HTML

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。

关键词： 酵母双杂交系统; 先天免疫; VISA; Sec13L1

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.

Key words: VISA Sec13L1 Innate immune Yeast two-hybrid system

收稿日期: 2017-02-27 出版日期: 2017-06-25

ZTFLH:

Q291

基金资助: 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

通讯作者: 许亮国 E-mail: xul@jxnu.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	王丹丹
	许亮国
	陈恬

引用本文:

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.

WANG Dan-dan, CHEN Tian, XU Liang-guo. Screening of VISA Interacting Proteins by Yeast Two-hybrid System. China Biotechnology, 2017, 37(6): 63-69.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170610 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I6/63

[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.

[1]	王涛, 杜丽, 马琼, 崔玉芳. 秀丽隐杆线虫先天免疫信号转导途径[J]. 中国生物工程杂志, 2011, 31(7): 121-125.
[2]	史春林,王云峰,石星明,王玫,孙妍,童光志. 抗菌肽在宿主防御中作用[J]. 中国生物工程杂志, 2008, 28(4): 82-86.
[3]	马海蓉, 李维琪. 酵母双杂交衍生系统[J]. 中国生物工程杂志, 2003, 23(2): 37-41.

Viewed

Full text

Abstract

Cited

Shared

Discussed