猪肿瘤坏死因子受体相关因子6的克隆、表达及对伪狂犬病病毒的抑制效应

doi:10.11843/j.issn.0366-6964.2017.02.012

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (2): 289-296.doi: 10.11843/j.issn.0366-6964.2017.02.012

猪肿瘤坏死因子受体相关因子6的克隆、表达及对伪狂犬病病毒的抑制效应

李永涛^1,2，李红杰¹，王晓雪¹，贾广敏³，陈陆¹，赵军¹，王川庆¹，刘红英^1*

（1. 河南农业大学牧医工程学院，郑州 450002； 2. 华中农业大学农业微生物学国家重点实验室，武汉 430070；3. 河南省动物卫生监督所，郑州 450002）

收稿日期:2016-09-05 出版日期:2017-02-23 发布日期:2017-02-23
通讯作者: 刘红英，副教授，E-mail:ndlhy@163.com
作者简介:李永涛(1985-)，男，河南周口人，讲师，博士，主要从事畜禽天然免疫研究，Tel：0371-63555516，E-mail: yongtaole@126.com
基金资助:
国家自然科学基金（31602033）；河南省高校科技创新团队支持计划（14IRTSTHN015）

Cloning, Expression and Antiviral Effect against Pseudorabies Virus of Porcine Tumor Necrosis Factor Receptor-associated Factor 6

LI Yong-tao^1,2, LI Hong-jie¹, WANG Xiao-xue¹, JIA Guang-min³, CHEN Lu¹, ZHAO Jun¹, WANG Chuan-qing¹, LIU Hong-ying^1*

（1. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China; 2. State Key Laboratory of Agriculture Microbiology, Huazhong Agricultural University, Wuhan 430070, China; 3. Animal Health Supervision Institute of Henan Province, Zhengzhou 450002, China）

Received:2016-09-05 Online:2017-02-23 Published:2017-02-23

摘要/Abstract

摘要：

肿瘤坏死因子受体相关因子6（TRAF6）是天然免疫信号通路中重要的接头分子，笔者旨在探索猪TRAF6的抗病毒和免疫调控功能。首先利用RT-PCR从猪外周血单核细胞克隆该基因，并进行序列比对分析；其次利用Piggybac转座子系统建立猪TRAF6稳定表达的PK-15细胞系；最后在细胞水平检测猪TRAF6对伪狂犬病病毒复制的影响以及对猪IFN-β等启动子活性的影响。结果显示，猪TRAF6基因为1 623 bp；将猪TRAF6基因连接到Piggybac转座子质粒成功构建真核表达载体PB-TRAF6，然后将重组质粒转染PK-15细胞，经药物处理和荧光筛选获得稳定表达TRAF6的细胞系；通过荧光定量PCR和Western blot确定稳定细胞系TRAF6的高效表达；体外抗病毒试验表明，TRAF6稳定细胞系能够显著抑制伪狂犬病病毒的复制；双荧光报告基因法检测发现，TRAF6可以促进poly(I:C)诱导的Ⅰ型干扰素应答。结果提示猪TRAF6具有显著的抗病毒和免疫调节功能，为研究TRAF6的免疫学功能奠定基础。

Abstract:

This study aimed to explore the antiviral effect and immunomodulatory functions of porcine tumor necrosis factor receptor-associated factor 6 (TRAF6), an important adaptor molecule in innate immune signaling pathway. Firstly, porcine TRAF6 gene was cloned by RT-PCR from porcine peripheral blood mononuclear cells and gene sequence was analyzed by sequence alignment. Secondly, stably expressing porcine TRAF6 cell line was established by Piggybac transposon system. And finally, we detected the impacts of TRAF6 on Pseudorabies virus (PRV) replication and porcine IFN-β promoter activity at the cellular level. The results showed that porcine TRAF6 gene was 1 623 bp. It was connected to Piggybac transposon (PB) plasmid and successfully constructed the eukaryotic expression vector PB-TRAF6, and then the recombinant plasmid was transfected into PK-15 cells to establish stably-expressed cell line by drug treatment and fluorescence screening. The high-expression of TRAF6 was confirmed by real time PCR and Western blot in the cell line. In vitro antiviral tests showed that the replication of PRV could be significantly inhibited in the cell line. Dual luciferase reporter system showed that porcine TRAF6 significantly enhanced poly(I:C)-mediated type Ⅰ interferon responses. These results indicated that porcine TRAF6 possesses significant anti-PRV activity and immunomodulatory functions, which will provide the basis for further research on immunology functions of TRAF6.

中图分类号:

S858.285.3

李永涛，李红杰，王晓雪，贾广敏，陈陆，赵军，王川庆，刘红英. 猪肿瘤坏死因子受体相关因子6的克隆、表达及对伪狂犬病病毒的抑制效应[J]. 畜牧兽医学报, 2017, 48(2): 289-296.

LI Yong-tao, LI Hong-jie, WANG Xiao-xue, JIA Guang-min, CHEN Lu, ZHAO Jun, WANG Chuan-qing, LIU Hong-ying. Cloning, Expression and Antiviral Effect against Pseudorabies Virus of Porcine Tumor Necrosis Factor Receptor-associated Factor 6[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(2): 289-296.

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猪肿瘤坏死因子受体相关因子6的克隆、表达及对伪狂犬病病毒的抑制效应

Cloning, Expression and Antiviral Effect against Pseudorabies Virus of Porcine Tumor Necrosis Factor Receptor-associated Factor 6

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