A型鸭肝炎病毒微基因组的构建

doi:10.11843/j.issn.0366-6964.2014.04.015

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (4): 609-613.doi: 10.11843/j.issn.0366-6964.2014.04.015

A型鸭肝炎病毒微基因组的构建

梁瑞英¹，胡文²,李宁³,缪秋红¹，毕庄莉³，孟春春¹，李传峰¹，陈宗艳¹，刘光清^1*

（1.中国农业科学院上海兽医研究所，上海 200241； 2.甘肃农业大学，兰州 730050； 3.安徽农业大学，合肥 230036)

收稿日期:2013-11-06 出版日期:2014-04-23 发布日期:2014-04-22
通讯作者: 刘光清(1968-),男，安徽砀山人，研究员，主要从事病毒分子生物学研究，E-mail:liugq@shvri.ac.cn
作者简介:梁瑞英（1989-），山东菏泽人，硕士生，主要从事动物病毒分子生物学研究，E-mail:ruiyingliang89@163.com
基金资助:
国家自然科学基金项目(31270194；31101848；31300141)；公益性农业科研专项(201003012；201303046；2014JB14)；“863”计划(2011AA10A200)

Construction of the Mini-genome of Duck Hepatitis A Virus

LIANG Rui-ying¹，HU Wen²，LI Ning³，MIAO Qiu-hong¹，BI Zhuang-li³，MENG Chun-chun¹，LI Chuan-feng¹，CHEN Zong-yan¹， LIU Guang-qing^1*

(1.Shanghai Veterinary Institution， Chinese Academy of Agricultural Sciences，Shanghai 200241， China； 2.Gansu Agricultural University,Lanzhou 730050, China; 3.Anhui Agricultural University,Hefei 230036, China)

Received:2013-11-06 Online:2014-04-23 Published:2014-04-22

摘要/Abstract

摘要：

为研究A型鸭肝炎病毒（DHAV)非编码区的结构和功能，笔者拟构建该病毒的微基因组并对其进行初步鉴定。采用酶切的方法将萤火虫荧光素酶报告基因（fLuc）与A型鸭肝炎病毒的ORF进行替换；并在5′UTR上游插入海肾荧光素酶报告基因Rluc作为内参基因；同时还在Rluc上游引入锤头状核酶，3′UTR下游引入丁型肝炎核酶，至此得到了含有双报告基因的A型鸭肝炎病毒微基因组（pRluc-fLuc）。将pRluc-fLuc转染DF-1细胞，8 h便可以检测到报告基因的表达，24 h表达量达到峰值。上述结果表明，已经成功构建了A型鸭肝炎病毒微基因组，这为进一步研究病毒非编码区的结构和功能以及病毒的翻译或复制的调控机理提供了良好的技术平台。

关键词: A型鸭肝炎病毒, 微基因组, 非编码区

Abstract:

In this study we constructed and identified the mini-genome of Duck Hepatitis A virus (DHAV), for analysis of the functional and structural properties of DHAV un-translated region (UTR).To construct a bicistronic of DHAV mini-genome (pRluc-fLuc)，the ORF of DHAV was replaced by firefly luciferase (fLuc) gene through enzyme digestion and the Renilla luciferase (Rluc) gene was inserted before 5′UTR.We also added the hammerhead ribozyme ahead Rluc gene and the hepatitis delta virus ribozyme behind 3′UTR.We transfected the pRluc-fLuc into DF-1 cells，the fLuc gene expression could be detected at 8 hours post transfected (h.p.t.) and with the levels peaking was at 24 h.p.t.The results showed that the DHAV mini-genome was constructed successfully and can be used for explore the role of UTR in virus transcription and translation.

Key words: duck hepatitis a virus, mini-genome, un-translated region

中图分类号:

S852.659.6

梁瑞英，胡文,李宁,缪秋红，毕庄莉，孟春春，李传峰，陈宗艳，刘光清. A型鸭肝炎病毒微基因组的构建[J]. 畜牧兽医学报, 2014, 45(4): 609-613.

LIANG Rui-ying，HU Wen，LI Ning，MIAO Qiu-hong，BI Zhuang-li，MENG Chun-chun，LI Chuan-feng，CHEN Zong-yan， LIU Guang-qing. Construction of the Mini-genome of Duck Hepatitis A Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(4): 609-613.

参考文献

［1］LEVINE P P，HOFSTAD M S.Duck disease investigation ［M］.Annual Report of the New York State Veterinary College，Ithaca，1945: 55-56.
［2］刘兆宇，程安春.雏鸭病毒性肝炎的研究进展［J］.中国家禽，2002，24(10)：6-9.
［3］PAN M，YANG X，ZHOU L，et al.Duck Hepatitis A virus possesses a distinct type IV internal ribosome entry site element of picornavirus ［J］.J Virol，2012，86(2): 1129-1144.
［4］YAMASHITA T，SAKAE K，TSUZUKI H，et al.Complete nucleotide sequence and genetic organization of Aichi virus，a distinct member of the Picornaviridae associated with acute gastroenteritis in humans［J］.J Virol，1998，72(10): 8408-8412.
［5］TSENG C H，KNOWLES N J，TSAI H J.Molecular analysis of duck hepatitis virus type 1 indicates that it should be assigned to a new genus ［J］.Virus Res，2007，123(2): 190-203.
［6］DING C，ZHANG D.Molecular analysis of duck hepatitis virus type 1 ［J］.Virology，2007，361(1): 9-17.
［7］HOENEN T，GROSETH A，DE KOK-MERCADO F，et al.Minigenomes，transcription and replication competent virus-like particles and beyond: reverse genetics systems for filoviruses and other negative stranded hemorrhagic fever viruses ［J］.Antiviral Res，2011，91(2):195-208.
［8］EMONET S E，URATA S，DE LA TORRE J C.Arenavirus reverse genetics: new approaches for the investigation of arenavirus biology and development of antiviral strategies ［J］.Virology，2011，411(2):416-425.
［9］ZHAO W，HU H，ZSAK L，et al.Application of the ligation-independent cloning (LIC) method for rapid construction of a minigenome rescue system for Newcastle disease virus VG/GA strain［J］.Plasmid，2013，70(3): 314-320.
［10］LIU G，YÁNGÜEZ E，CHEN Z，et al.The duck hepatitis virus 5′-UTR possesses HCV-like IRES activity that is independent of eIF4F complex and modulated by downstream coding sequences［J］.Virol J，2011.8:147.
［11］PALMENBERG A C，SGRO J Y.Topological organization of picornaviral genomes: statistical prediction of RNA structural signals ［J］.Semin Virol，1997，8(3):231-241.
［12］DORNER A J，SEMLER B L，JACKSON R J，et al.In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate ［J］.J Virol，1984，50(2):507-514.
［13］GLASS M J，SUMMERS D F.Identification of a trans-acting activity from liver that stimulates hepatitis A virus translation in vitro ［J］.Virology，1993，193(2):1047-1050.
［14］CHARD L S，KAKU Y，JONES B，et al.Functional analyses of RNA structures shared between the internal ribosome entry sites of hepatitis C virus and the picornavirus porcine teschovirus 1 Talfan ［J］.J Virol，2006，80(3):1271-1279.

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A型鸭肝炎病毒微基因组的构建

Construction of the Mini-genome of Duck Hepatitis A Virus

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