靶向鸭肠炎病毒NP基因RNAi对DEV增殖的影响

doi:10.11843/j.issn.0366-6964.2017.01.016

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (1): 132-139.doi: 10.11843/j.issn.0366-6964.2017.01.016

靶向鸭肠炎病毒NP基因RNAi对DEV增殖的影响

郑敏^1,2，罗乐³，熊朝丽⁴，吴良涛¹，华敏¹，万润¹，程振涛^1,2，周碧君^1,2，杨琦^1,2，文明^1,2*

（1.贵州大学动物科学学院，贵阳550025；2.贵州省动物疫病与兽医公共卫生重点实验室，贵阳550025; 3.遵义市动物卫生监督所，遵义 520300；4.遵义市兽药饲料监测站，遵义 520300）

收稿日期:2016-07-27 出版日期:2017-01-23 发布日期:2017-01-23
通讯作者: 文明，博士，教授，E-mail：as.mwen@gzu.edu.cn
作者简介:郑敏(1987-)，女，重庆开县人，硕士生，主要从事病毒学研究，E-mail：zhengmin916@163.com
基金资助:
国家自然科学基金项目(31260607；31560703)；贵州省优秀青年科学人才培养计划项目(黔科合人字［2013］25号)；贵州省百层次创新型人才项目(黔科合人才［2016］4009号)；贵州省科技创新人才团队建设项目(黔科合人才团队［2015］4016号)；贵州大学研究生创新基金项目(研农2016022)

Effection of Targeting DEV-NP Gene on DEV Proliferation by RNA Interference

ZHENG Min^1,2, LUO Le³, XIONG Chao-li⁴, WU Liang-tao¹, HUA Min¹, WAN Run¹, CHENG Zhen-tao^1,2, ZHOU Bi-jun^1,2, YANG Qi^1,2, WEN Ming^1,2*

（1.College of Animal Science, Guizhou University, Guiyang 550025, China; 2.Key Laboratory of Animal Diseases and Veterinary Public Health in Guizhou Province, Guiyang 550025, China; 3. Zunyi Animal Hygienic Supervision Institute, Zunyi 520300, China; 4.Zunyi Animal Feed Monitoring Station, Zunyi 520300, China）

Received:2016-07-27 Online:2017-01-23 Published:2017-01-23

摘要/Abstract

摘要：

为探究核衣壳蛋白(NP)对鸭肠炎病毒(DEV)增殖的影响，笔者根据GenBank上DEV-NP基因序列，设计并构建pSilencer-DEV-NP，采用三种不同方式处理转染鸭胚成纤维(DEF)细胞后，应用荧光显微镜法和FQ-PCR法分析pSilencer-DEV-NP对DEV增殖的影响，结果显示：经荧光显微镜法观察，pSilencer-DEV-NP-l~4在DEF细胞中均呈现绿色荧光；FQ-PCR法扩增并计算，pSilencer-DEV-NP-l~4对DEV增殖的沉默效率分别为81.10%、72.69%、77.35%和67.69%；采用三种方法处理，pSilencer-DEV-NP-1对DEF细胞中DEV的增殖均有沉默作用，但沉默效率不尽一致，其中先后转染后感染时pSilencer-DEV-NP-1的沉默效果最好，在处理60 h时沉默效率高达69.20%；同时转染和感染时pSilencer-DEV-NP-1的沉默效果次之，在处理48 h时沉默效率最好仅达63.79%；先感染后转染时pSilencer-DEV-NP-1的沉默效果较差，在处理60 h时沉默效率最高仅为52.58%。上述研究结果提示，核衣壳蛋白对鸭肠炎病毒增殖具有一定的影响，为阐明核衣壳蛋白在鸭肠炎病毒复制与增殖的作用机制奠定了理论基础。

Abstract:

The aim of the present study was to explore the influence of nucleocapsid protein (NP) to the proliferation of duck enteritis virus (DEV). We designed and constructed pSilencer-DEV-NP according to the DEV NP gene sequence in GenBank. After these pSilencers were transfected into duck embryo fibroblast (DEF) cells in three different ways, the expression of pSilencer DEV-NP to DEV proliferation in DEF cells was analyzed by fluorescence microscope and FQ-PCR. The results showed that the green fluorescence was observed by microscope in DEF cells transfected with pSilencer-DEV-NP-l, pSilencer-DEV-NP-2, pSilencer-DEV-NP-3 and pSilencer-DEV-NP-4, respectively. The silencing efficiency of pSilencer-DEV-NP-1 to 4 on DEV proliferation were 81.10%, 72.69%, 77.35% and 67.69%, respectively. FQ-PCR was used to amplify and calculate. The results showed that pSilencer-DEV-NP-1 could silence the DEV proliferation in DEF cells by three transfection ways, but the silencing efficiency was different. The silencing effect of pSilencer-DEV-NP-1 was the best at 60 h after transfection and its efficiency was 69.20%. The second highest efficiency occurred in the way of simultaneously DEV infection and pSilencer transfection, the best situation at 48 h was 63.79%. The worst efficiency was 52.58% at 24 h after the operation with the method of pSilencer-DEV-NP-1 transfection after DEV infection. These results suggest that the nucleocapsid protein has a certain effect on DEV proliferation, which lays a theoretical foundation for clarification of the mechanism of DEV replication and proliferation.

中图分类号:

S852.659.1

郑敏，罗乐，熊朝丽，吴良涛，华敏，万润，程振涛，周碧君，杨琦，文明. 靶向鸭肠炎病毒NP基因RNAi对DEV增殖的影响[J]. 畜牧兽医学报, 2017, 48(1): 132-139.

ZHENG Min, LUO Le, XIONG Chao-li, WU Liang-tao, HUA Min, WAN Run, CHENG Zhen-tao, ZHOU Bi-jun, YANG Qi, WEN Ming. Effection of Targeting DEV-NP Gene on DEV Proliferation by RNA Interference[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(1): 132-139.

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参考文献

［1］殷震，刘景华.动物病毒学［M］.北京：科学出版社，1997.
YIN Z， LIU J H. Animal virology ［M］. Beijing: Science Press, 1997. (in Chinese)
［2］甘孟侯.中国禽病学［M］.北京:中国农业出版社，2000.
GAN M H. The Chinese diseases of poultry［M］. Beijing: China Agriculture Press, 2000. (in Chinese)
［3］WITTER R L, SCHAT K. Diseases of poultry ［M］. 11th, Ames: Iowa State University Press, 2003.
［4］MONDAL B, RASOOL T J, RAM H, et al. Propagation of vaccine strain of duck enteritis virus in a cell line of duck origin as an alternative production system to propagation in embryonated egg ［J］.Biologicals, 2010, 38(3):401-406.
［5］文明.DEV基因文库构建、核衣壳蛋白基因的发现及克隆与表达［D］. 雅安：四川农业大学，2005.
WEN M. Construction of DEV gene library and discover and cloning and expression of its nucleocapsid protein gene ［D］. Ya’an: Sichuan Agricultural University, 2005.
［6］高颖，代淑燕，张荷，等. 鸭肠炎病毒核衣壳蛋白密码子偏爱性分析［J］. 山地农业生物学报，2011，30(2): 136-140.
GAO Y, DAI S Y, ZHANG H, et al. Analysis on codon preference of duck enteritis virus nucleocapsid protein ［J］. Journal of Mountain Agriculture and Biology, 2011, 30 (2): 136-140. (in Chinese)
［7］徐超，李欣然，信洪一，等.鸭瘟病毒gC基因的克隆及其分子特性分析［J］.中国兽医科学，2008, 38(12): 1038-1044.
XU C, LI X R, XIN H Y, et al.Cloning and molecular characterization of gC gene of duck plague virus［J］. Chinese Veterinary Science, 2008,38(12): 1038-1044. (in Chinese)
［8］代淑燕，寨鸿瑞，文明，等. 鸭肠炎病毒核衣壳蛋白基因的克隆与原核表达［J］. 生物技术，2008，18(4): 12-15.
DAI S Y, ZHAI H R, WEN M, et al. Cloning and prokaryotic expression of nucleocapsid protein gene of duck enteritis virus ［J］. Biotechnology, 2008, 18 (4); 12-15.(in Chinese)
［9］张荷，毛君婷，杨颖，等. 鸭肠炎病毒核衣壳蛋白受体的筛选与鉴定［J］. 病毒学报，2012，28(1): 63-66.
ZHANG H, MAO J T, YANG Y, et al. Screening and identification of receptor reacting with nucleocapsid protein of duck enteritis virus ［J］. Chinese Journal of Virology, 2012, 28 (1): 63-66. (in Chinese)
［10］BROWN J C, NEWCOMB W W. Herpesvirus capsid assembly: Insights from structural analysis ［J］. Curr Opin Virol, 2011, 1(2): 142-149.
［11］SRIKUMARAN S, KELLING C L, AMBAGALA A. Immune evasion by pathogens of bovine respiratory disease complex ［J］.Anim Health Res Rev,2007,8(2):215-229.
［12］HANNON G J. RNA interference ［J］. Nature, 2002, 418(6894): 244-251.
［13］SANGHVI V R, STEEL L F. A re-examination of global suppression of RNA interference by HIV-1 ［J］. PLoS One,2011,6(2):e17246.
［14］姜龙. 鸭瘟病毒UL49基因功能研究［D］. 雅安：四川农业大学，2012.
JIANG L. Study on the function of duck plague virus UL49 gene ［D］. Ya’an: Sichuan Agricultural University, 2012.
［15］XIANG J, ZHANG S C, CHENG A C, et al. Expression and characterization of recombinant VP19c protein and N-terminal from duck enteritis virus ［J］. Virol J, 2011, 8: 82.
［16］BRUMMELKAMP T R, BERNARDS R, AGAMI R. A system for stable expression of short interfering RNAs in mammalian cells ［J］. Science, 2002, 296(5567): 550-553.
［17］YUAN J, DUTTON C M, SCULLY S P. RNAi mediated MMP-1 silencing inhibits human chondrosarcoma invasion ［J］. J Orthop Res, 2005, 23(6): 1467-1474.
［18］FIRE A, XU S, MONTGOMERY M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans ［J］. Nature, 1998, 391(6669):806-811.
［19］ZENG Y, YI R, CULLEN B R. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms ［J］. Proc Natl Acad Sci U S A,2003, 100(17):9779-9784.
［20］CHAN C Y, CARMACK C S, LONG D D, et al. A structural interpretation of the effect of GC-content on efficiency of RNA interference ［J］. BMC Bioinformatics, 2009, 10(Suppl 1): S33.
［21］PATZEL V, RUTZ S, DIETRICH I,et al. Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency［J］. Nat Biotechnol, 2005, 23(11):1440-1444.
［22］KLEINMAN M E, YAMADA K, TAKEDA A, et al. Sequence- and target-independent angiogenesis suppression by siRNA via TLR3 ［J］. Nature, 2008, 452(7187): 591-597.
［23］REYNOLDS A, LEAKE D, BOESE Q, et al. Rational siRNA design for RNA interference［J］. Nat Biotechnol, 2004,22(3):326-330.
［24］ONIMARU M, OHUCHIDA K, EGAMI T, et al. Gemcitabine synergistically enhances the effect of adenovirus gene therapy through activation of the CMV promoter in pancreatic cancer cells ［J］. Cancer Gene Ther, 2010, 17(8): 541-549.
［25］PHAM H M, CHANG K S, MAST M, et al. Molecular characterization of the nucleocapsid protein gene of Newcastle disease virus strains in Japan and development of a restriction enzyme-based rapid identifying method［J］. Arch Virol, 2004, 149(8): 1559-1569.
［26］SEAL B S, CRAWFORD J M, SELLERS H S, et al. Nucleotide sequence analysis of the Newcastle disease virus nucleocapsid protein gene and phylogenetic relationships among the Paramyxoviridae［J］.Virus Res, 2002, 83(1-2):119-129.

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靶向鸭肠炎病毒NP基因RNAi对DEV增殖的影响

Effection of Targeting DEV-NP Gene on DEV Proliferation by RNA Interference

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