传染性法氏囊病病毒VP4酵母双杂交诱饵载体的构建及互作宿主细胞蛋白的筛选

doi:10.16431/j.cnki.1671-7236.2015.02.012

›› 2015, Vol. 42 ›› Issue (2): 317-323.doi: 10.16431/j.cnki.1671-7236.2015.02.012

传染性法氏囊病病毒VP4酵母双杂交诱饵载体的构建及互作宿主细胞蛋白的筛选

王念, 陈玉明, 张礼洲, 高立, 卢珍, 高玉龙, 王永强, 高宏雷, 刘长军, 崔红玉, 王笑梅, 祁小乐

中国农业科学院哈尔滨兽医研究所, 兽医生物技术国家重点实验室, 禽传染病研究室, 哈尔滨 150001

收稿日期:2014-08-13 出版日期:2015-02-20 发布日期:2015-02-13
通讯作者: 王笑梅, 祁小乐 E-mail:xmw@hvri.ac.cn;qxl@hvri.ac.cn
作者简介:王念(1991-),男,四川内江人,硕士生,研究方向:动物病毒学,E-mail:1582671471@qq.com
基金资助:
现代农业产业技术体系建设专项基金(nycytx-42-G3-01);国家重点实验室基本科研业务费(SKLVBP201303)

Construction of Bait Vector of Infectious Bursal Disease Virus VP4 and Screening of the Interacted Proteins of Host Cell Using Yeast Two-hybrid System

WANG Nian, CHEN Yu-ming, ZHANG Li-zhou, GAO Li, LU Zhen, GAO Yu-long, WANG Yong-qiang, GAO Hong-lei, LIU Chang-jun, CUI Hong-yu, WANG Xiao-mei, QI Xiao-le

Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agriculture Sciences, Harbin 150001, China

Received:2014-08-13 Online:2015-02-20 Published:2015-02-13

摘要/Abstract

摘要： VP4蛋白(viral protein 4)是传染性法氏囊病病毒(infectious bursal disease virus,IBDV)编码的一种重要非结构蛋白,具有蛋白水解酶活性,剪切多聚蛋白pVP2-VP4-VP3释放出pVP2、VP4、VP3,对病毒蛋白成熟具有重要的作用。为了研究VP4蛋白与宿主细胞的相互作用,本研究将IBDV VP4基因克隆入pGBKT7,构建诱饵载体pGBGtVP4,自激活试验证明其无自激活活性,对酵母细胞没有毒性。运用酵母双杂交技术,从鸡胚成纤维细胞(CEF)文库中筛选VP4的互作蛋白,经初筛、测序和回转验证,获得22个候选互作宿主细胞蛋白,为深入研究IBDV VP4与宿主互作的效应和机制奠定了基础。

关键词: 传染性法氏囊病病毒; VP4; 酵母双杂交; 诱饵载体; 互作蛋白

Abstract: Viral protein 4 (VP4) is an important non-structural protein with proteolytic enzyme activity encoded by infectious bursal disease virus (IBDV) genome that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form viral proteins pVP2,VP4,VP3,which plays an important role in the process of viral proteins maturity.To study the interaction between VP4 and the host cell,VP4 full-length cDNA sequence was amplified and inserted into pGBKT7 vector to construct a bait vector (pGBGtVP4).The result of autoactivation assay showed that pGBGtVP4 vector had no autoactivation and virulence in Y2H gold yeast cell.After screening,sequencing and confirming interaction,22 kinds of potential interacted proteins were obtained from the expression library of chicken embryo fibroblasts (CEF) using the matchmaker gold yeast two-hybrid system.This study laid the foundation for further researches on the effect and mechanism of the interaction of IBDV VP4 and its host cell.

Key words: infectious bursal disease virus (IBDV); VP4; yeast two-hybrid; bait vector; interacted protein

中图分类号:

Q785

王念, 陈玉明, 张礼洲, 高立, 卢珍, 高玉龙, 王永强, 高宏雷, 刘长军, 崔红玉, 王笑梅, 祁小乐. 传染性法氏囊病病毒VP4酵母双杂交诱饵载体的构建及互作宿主细胞蛋白的筛选[J]. , 2015, 42(2): 317-323.

WANG Nian, CHEN Yu-ming, ZHANG Li-zhou, GAO Li, LU Zhen, GAO Yu-long, WANG Yong-qiang, GAO Hong-lei, LIU Chang-jun, CUI Hong-yu, WANG Xiao-mei, QI Xiao-le. Construction of Bait Vector of Infectious Bursal Disease Virus VP4 and Screening of the Interacted Proteins of Host Cell Using Yeast Two-hybrid System[J]. , 2015, 42(2): 317-323.

参考文献

[1] Muller H,Islam M R,Raue R.Research on infectious bursal disease-The past,the present and the future[J].Vet Microbiol,2003,97(1):153-165.
[2] Azad A A,Jagadish M N,Brown M A,et al.Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus[J].Virology,1987,161(1):145-152.
[3] Granzow H,Birghan C,Mettenleiter T C,et al.A second form of infectious bursal disease virus-associated tubule contains VP4[J].J Virol,1997,71(11):8879-8885.
[4] Li Z,Wang Y,Li X,et al.Critical roles of glucocorticoid-induced leucine zipper in infectious bursal disease virus (IBDV)-induced suppression of type Ⅰ interferon expression and enhancement of IBDV growth in host cells via interaction with VP4[J]. J Virol,2013,87(2):21-31.
[5] 祁小乐,陈玉明,张礼洲,等.鸡胚成纤维细胞cDNA 酵母表达文库的构建与鉴定[J].中国家禽,2013,35(8):14-16.
[6] Fromont-Racine M,Rain J C,Legrain P.Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens[J]. Nat Genet,1997,16(1):277-282.
[7] 陈玉明,张礼洲,任宪刚,等.不同毒力传染性法氏囊病病毒衣壳蛋白酵母双杂交诱饵载体的构建及鉴定[J].中国畜牧兽医,2014,41(1):30-33.
[8] Gietz R D,Woods R A.Screening for protein-protein interactionsin the yeast two-hybrid system[J]. Methods Mol Biol,2002,185:471-486.
[9] White M A.The yeast two-hybrid system: Forward and reverse[J]. Proc Natl Acad Sci USA,1996,93(19):10001-10003.
[10] Browning C L,Culberson D E,Aragon I V,et al.The developmentally regulated expression of serum response factor plays a key role in the control of smooth muscle-specific genes[J].Developmental Biology,1998,194(1):18-37.
[11] Birghan C,Mundt E,Gorbalenya A E.A non-canonical lon proteinase lacking the ATPase domain employs the Ser-Lys catalytic dyad to exercise broad control over the life cycle of a double-stranded RNA virus[J]. Embo J,2000,19(1):114-123.
[12] Lejal N,Da Costa B,Huet J C,et al.Role of Ser-652 and Lys-692 in the protease activity of infectious bursal disease virus VP4 and identification of its substrate cleavage sites[J].J Gen Virol,2000,81(4):983-992.
[13] Sanchez A B,Rodriguez J F.Proteolytic processing in infectious bursal disease virus: Identification of the polyprotein cleavage sites by site-directed mutagenesis[J].Virology,1999,262(1):190-199.
[14] Chang G R,Wang M Y,Liao J H,et al.Endopeptidase activity characterization of E.coli-derived infectious bursal disease virus protein 4 tubules using flow cytometry[J].Arch Virol,1998,143(12):2327-2341.
[15] Manel N,Hogstad B,Wang Y,et al.A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells[J]. Nature,2010,467(7312):214-217.
[16] Takemura T,Kawamata M,Urabe M,et al.Cyclophilin A dependent restriction to capsid N121K mutant human immunodeficiency virus type Ⅰ in a broad range of cell lines[J].J Virol,2013,87(7):4086-4090.
[17] Liu Z,Yang F,Robotham J M,et al.Critical role of cyclophilin A and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis C virus replication complex[J].J Virol,2009,83(13):6554-6665.
[18] Liu X,Sun L,Yu M.Cyclophilin A interacts with influenza A virus M1 protein and impairs the early stage of the viral replication[J].Cellular Microbiology,2009,11(5):730-741.
[19] Liu X L,Zhao Z D,Liu W J.Insights into the roles of cyclophilin A during influenza virus infection[J].Viruses,2013,5(1):182-191.
[20] Stephen D,Emily L,Hengli T,et al.Cyclophilins as modulators of viral replication[J].Viruses,2013,5(7):1684-1701.
[21] Luban J,Goff S P.The yeast two-hybrid system for studying: Protein-protein interaction[J].Curr Opin Biotech,1995,6(1):59-64.

传染性法氏囊病病毒VP4酵母双杂交诱饵载体的构建及互作宿主细胞蛋白的筛选

Construction of Bait Vector of Infectious Bursal Disease Virus VP4 and Screening of the Interacted Proteins of Host Cell Using Yeast Two-hybrid System

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