[1]温贵兰*,张升波,李昌红,等.稳定表达猪繁殖与呼吸综合征病毒NSP2细胞株的建立及其对p65核转位影响的研究[J].中国预防兽医学报,2018,(09):851-853.[doi:0.3969/j.issn.1008-0589.201801044]
 WEN Gui-lan*,ZHANG Sheng-bo,LI Chang-hong,et al.Establishment of cell line stably expressing nonstructured protein 2 of porcine reproductive and respiratory syndrome virus[J].Chinese journal of preventive veterinary medicine,2018,(09):851-853.[doi:0.3969/j.issn.1008-0589.201801044]
点击复制

稳定表达猪繁殖与呼吸综合征病毒NSP2细胞株的建立及其对p65核转位影响的研究()
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年09
页码:
851-853
栏目:
研究简报
出版日期:
2018-09-17

文章信息/Info

Title:
Establishment of cell line stably expressing nonstructured protein 2 of porcine reproductive and respiratory syndrome virus
文章编号:
1008-0589(2018)9-0851-03
作者:
 

温贵兰*张升波李昌红徐 丽

 (贵州大学 动物科学学院,贵州 贵阳 550025)
Author(s):
 

WEN Gui-lan* ZHANG Sheng-bo LI Chang-hong XU Li

 ( College of Animal Sciences, Guizhou University, Guiyang 550025, China)
关键词:
猪繁殖与呼吸综合征病毒非结构蛋白NSP2稳定表达
Keywords:
  PRRSV  NSP2  consistent expression
分类号:
S852.65
DOI:
0.3969/j.issn.1008-0589.201801044
文献标志码:
A
摘要:
为建立一株稳定表达猪繁殖与呼吸综合征病毒(PRRSV)非结构蛋白2(NSP2)的细胞株,本研究通过脂质体2000介导的方法将携带有PRRSV NSP2基因序列的真核表达载体(pcDNA3.1-5’Flag-Nsp2)瞬时转染于HEK293细胞,应用G418筛选、间接免疫荧光(IFA)与western blot方法进行鉴定,获得了1株稳定表达NSP2的阳性细胞株,命名为NSP2-11C1-HEK293;将TNF-α作用于该细胞株,采用IFA分析稳转细胞内NSP2对p65入核的影响。IFA结果显示NSP2主要在细胞浆中围绕细胞核表达,在细胞中的表达率达95 %以上;western blot结果显示出现120 ku的蛋白条带,与预期相符。结果表明本研究获得了一株稳定表达NSP2的阳性细胞株,在该稳转细胞内NSP2对p65的入核并没有影响,这为进一步研究揭示PRRSV的致病机理奠定基础。
  
Abstract:
To establish a permissive cell line for porcine reproductive and respiratory syndrome virus (PRRSV), the recombinant plasmid of pcDNA3.1-5’Flag-Nsp2 was transfected into HEK293 cells and a cell line stably expressing the NSP2, named NSP2-11C1-HEK293, was generated using colony-purification selection in the present of 800 μg/mL G418. The expression of NSP2 in the cells was confirmed by indirect immunofluorescence assay (IFA) and western blot, indicating that porcine NSP2 was stably expressed in the HEK293 cell lines. The NSP2-11C1-HEK293 was treated with TNF-alpha and the effect on translocation of p65 was analyzed by IFA. The results showed that the NSP2 was 120ku mainly expressed in the HEK293 cytoplasm and rounded the nucleus. The rate of the NSP2 expression in the cell was over 95%. Indditionly, there was no effect on translocation of p65 from cytoplasm to nucleus by NSP2 in the NSP2-11C1-HEK293, which would facilitate further study on the mechanism of interaction between virus and host protein.

参考文献/References:

[1] Music N, Gagnon C A. The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis [J]. Anim Health Res Rev, 2010, 11(2): 135-163.
[2]Fang Ying, Fang Liu-rong, Wang Yang, et al. Porcine reproductive and respiratory syndrome virus nonstructural protein 2 contributes to NF-kappaB activation [J]. Virol J, 2012, 9: 83.
[3]Kappes M A, Miller C L, Faaberg K S. Highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions [J]. J Virol, 2013, 87(24): 13456-13465.
[4]Jin Huan, Zhou Lei, Ge Xin-na, et al. Cellular DEAD-box RNA helicase 18 (DDX18) promotes the PRRSV replication via Interaction with virus nsp2 and nsp10 [J]. Virus Res, 2017, 238: 204-212.
[5]Li Y, Shang P, Shyu D, et al. Nonstructural proteins nsp2TF and nsp2N of porcine reproductive and respiratory syndrome virus (PRRSV) play important roles in suppressing host innate immune responses [J]. Virology, 2018, 517: 164-176.
[6]Pires B R B, Silva R C M C, Ferreira G M, et al. NF-kappaB: Two sides of the same coin [J]. Genes (Basel), 2018, 9(1): 24.
[7]温贵兰,张涵淞,扈鸿霞,等. 猪繁殖与呼吸综合征病毒感染细胞中非结构蛋白Nsp2表达分析[J]. 畜牧兽医学报, 2013,44(7):1109-1116.
[8]Han J, Rutherford M S, Faaberg K S. The porcine reproductive and respiratory syndrome virus nsp2 cysteine protease domain possesses both trans- and cis-cleavage activities [J]. J Virol, 2009, 83(18): 9449-9463.
[9] Sun Zhi, Chen Zhen-hai, Lawson S R, et al. The cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions [J]. J Virol, 2010, 84(15): 7832- 7846.
[10]Fang Ying, Fang Liu-rong, Wang Yang. Porcine reproductive and respiratory syndrome virus nonstructural protein 2 contributes to NF-κB activation [J]. Virol J, 2012, 9: 83.
(本文编辑:李   爽)

相似文献/References:

[1]周艳君,田志军,刘金霞,等.猪繁殖与呼吸综合征病毒M蛋白B细胞抗原表位的鉴定[J].中国预防兽医学报,2009,(04):251.
 ZHOU Yan-jun,TIAN Zhi-jun,LIU Jin-xia,et al.Identification of a B cell epitope on matrix protein of porcine reproductive and respiratory syndrome virus[J].Chinese journal of preventive veterinary medicine,2009,(09):251.
[2]曹宗喜,潘全会,张原江,等.猪繁殖与呼吸综合征病毒各结构蛋白的融合表达[J].中国预防兽医学报,2009,(02):85.
 CAO Zong-xi,PAN Quan-hui,ZHANG Yuan-jiang,et al.Fusion expression of structural proteins of porcine reproductive and respiratory syndrome virus[J].Chinese journal of preventive veterinary medicine,2009,(09):85.
[3]马 平,蔡雪辉*,刘永刚,等.猪繁殖与呼吸综合征病毒GD3株的分离鉴定及其分子特征 [J].中国预防兽医学报,2009,(01):28.
 MA Ping,CAI Xue-hui*,LIU Yong-gang,et al.Isolation and characterization of PRRSV GD3 strain isolated in China [J].Chinese journal of preventive veterinary medicine,2009,(09):28.
[4]宋艳华,王海燕,李文良,等.PRRSV NSP2基因在重组杆状病毒中的表达与鉴定 [J].中国预防兽医学报,2009,(07):509.
 SONG Yan-hua,WANG Hai-yan,LI Wen-liang,et al.Expression and identification of NSP2 ofPRRSV in recombinant baculovirus [J].Chinese journal of preventive veterinary medicine,2009,(09):509.
[5]李宏宇,孙 元,常天明,等.共表达猪繁殖与呼吸综合征病毒GP5基因和猪瘟病毒E2基因的重组腺病毒的构建及其在小鼠模型上的免疫原性分析[J].中国预防兽医学报,2010,(03):161.
 LI Hong-yu,SUN Yuan,CHANG Tian-ming,et al.Immunogenicity of a recombinant adenovirus co-expressing porcine reproduction and respiratory syndrome virus GP5 protein and classical swine fever virus E2 protein in mice[J].Chinese journal of preventive veterinary medicine,2010,(09):161.
[6]庞海洋,赵焕云,张文东,等.云南高致病性猪繁殖与呼吸综合征病毒Nsp2基因变异分析[J].中国预防兽医学报,2008,(12):934.
 PANG Hai-yang,ZHAO Huang-yun,ZHANG Wen-dong,et al.Gentic variation of Nsp2 genes of highly pathogenic porcine reproductive and respiratory syndrome viruses in Yunnan province[J].Chinese journal of preventive veterinary medicine,2008,(09):934.
[7]徐明明,蔡雪辉*,刘永刚,等.高致病性PRRSV ORF3基因在昆虫细胞中的表达与免疫原性研究[J].中国预防兽医学报,2010,(06):455.
 XU Ming-ming,CAI Xue-hui*,LIU Yong-gang,et al.Expression of highly pathogenic PRRSV ORF3 gene in insect cells[J].Chinese journal of preventive veterinary medicine,2010,(09):455.
[8]邓雨修#,招丽婵#,苏润环,等.猪繁殖与呼吸综合征病毒TM 株的分离鉴定及其Nsp2序列分析[J].中国预防兽医学报,2010,(07):503.
 DENG Yu-xiu#,ZHAO Li-chan#,SU Run-huan,et al.Isolation and Nsp2 gene sequence analysis of the PRRSV TM isolate[J].Chinese journal of preventive veterinary medicine,2010,(09):503.
[9]石文达,刘永刚,王洪峰,等.猪繁殖与呼吸综合征病毒CH-1R株全基因组序列测定及遗传变异分析[J].中国预防兽医学报,2010,(07):517.
 SHI Wen-da,LIU Yong-gang,WANG Hong-feng,et al.Sequencing analysis of full length genome of porcine reproductive and respiratory syndrome virus CH-1R strain[J].Chinese journal of preventive veterinary medicine,2010,(09):517.
[10]韦天超,田志军,安同庆,等.猪繁殖与呼吸综合征病毒Taq Man-MGB荧光定量RT-PCR方法的建立和应用[J].中国预防兽医学报,2008,(12):944.
 WEI Tian-chao,TIAN Zhi-jun,AN Tong-qing,et al.Development and application of Taq Man-MGB fluorescence quantitative RT-PCR assay for detection of porcine reproductive and respiratory syndrome virus[J].Chinese journal of preventive veterinary medicine,2008,(09):944.

更新日期/Last Update: 2018-10-19