利用酵母双杂交技术筛选草鱼Ⅲ型呼肠孤病毒VP6和VP38相互作用蛋白的研究
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摘要
为探究草鱼Ⅲ型呼肠孤病毒VP6和VP38蛋白的生物学功能,利用酵母双杂交Gal4系统分别筛选了能与GCRV104毒株编码的VP6和VP38相互作用的宿主蛋白。利用RT-PCR从感染GCRV104的草鱼肾脏细胞中扩增GCRV104 s8和s10基因组片段的编码基因后,通过酶切与连接分别克隆至载体p GBKT7中,构建诱饵质粒p GBKT7-VP6和p GBKT7-VP38。对这些重组质粒进行细胞毒性和自激活检测后,分别以VP6和VP38为诱饵在草鱼酵母文库中筛选与其相互作用的宿主蛋白,对筛选得到的阳性酵母菌落进行序列分析。结果表明,两个诱饵质粒p GBKT7-VP6和p GBKT7-VP38均无自激活作用;诱饵质粒p GBKT7-VP6筛选到7株阳性克隆,分别编码β肌动蛋白、augmin样复合体亚基2、甘露糖苷酶α2b1亚基、程序性细胞死亡蛋白6、真核翻译延长因子1γ和一个未知功能蛋白;诱饵质粒p GBKT7-VP38筛选到4株阳性克隆,分别编码剪切与多聚腺苷酸化特异性因子5、高迁移率组蛋白核小体结合结构域2、葡萄糖转运体X和蛋白酶体亚基β7。结果为进一步探究GCRV104毒株编码的VP6、VP38与宿主蛋白及其涉及的信号通路的相互作用奠定了基础。
To screen and identify the proteins that interact with VP6 and VP38 of type III reovirus of grass carp,GCRV104 strain,the complete ORFs encoded by the s8 and s10 genome fragments were amplified by RT-PCR from infected cells and used to generate the bait plasmids for yeast two-hybrid screen,p GBKT7-VP6 and p GBKT7-VP38,respectively. The c DNA library plasmid of Ctenopharyngodon idella kidney cells was screened to reveal potential interaction partners in yeast AH109 transformed with p GBKT7-VP6 or p GBKT7-VP38. The positive clones were analyzed by plasmid sequencing and nucleotide sequence blasting. The results showed that both bait plasmid p GBKT7-VP6 and p GBKT7-VP38 demonstrated no self-activation in yeast; 7 host proteins were suggested to bind VP6 including beta-actin,augmin-like complex subunit 2,zinc finger FYVE domain containing 21,mannosidase alpha class 2 B member 1,programmed cell death 6,eukaryotic translation elongation factor 1 gamma and an unknown protein; while,4 host proteins were suggested to bind VP38 that included cleavage and polyadenylation specific factor 5,high mobility group nucleosomal binding domain 2,glucose transporter X( glut X) gene,and proteasome subunit beta7. This study may pave the way for understanding functions of VP6 and VP38 proteins encoded by genotype Ⅲ reovirus of grass carp and present some information for the exploration of GCRV replication mechanism.
To screen and identify the proteins that interact with VP6 and VP38 of type III reovirus of grass carp,GCRV104 strain,the complete ORFs encoded by the s8 and s10 genome fragments were amplified by RT-PCR from infected cells and used to generate the bait plasmids for yeast two-hybrid screen,p GBKT7-VP6 and p GBKT7-VP38,respectively. The c DNA library plasmid of Ctenopharyngodon idella kidney cells was screened to reveal potential interaction partners in yeast AH109 transformed with p GBKT7-VP6 or p GBKT7-VP38. The positive clones were analyzed by plasmid sequencing and nucleotide sequence blasting. The results showed that both bait plasmid p GBKT7-VP6 and p GBKT7-VP38 demonstrated no self-activation in yeast; 7 host proteins were suggested to bind VP6 including beta-actin,augmin-like complex subunit 2,zinc finger FYVE domain containing 21,mannosidase alpha class 2 B member 1,programmed cell death 6,eukaryotic translation elongation factor 1 gamma and an unknown protein; while,4 host proteins were suggested to bind VP38 that included cleavage and polyadenylation specific factor 5,high mobility group nucleosomal binding domain 2,glucose transporter X( glut X) gene,and proteasome subunit beta7. This study may pave the way for understanding functions of VP6 and VP38 proteins encoded by genotype Ⅲ reovirus of grass carp and present some information for the exploration of GCRV replication mechanism.
引文
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[22]HUSI H,SKIPWORTH R J,CRONSHAW A,et al.Programmed cell death 6 interacting protein(PDCD6IP)and Rabenosyn-5(ZFYVE20)are potential urinary biomarkers for upper gastrointestinal cancer[J].Proteomics Clinical applications,2015,9(5/6):586-596.
[23]ACHILONU I,SIGANUNU T P,DIRR H W.Purification and characterisation of recombinant human eukaryotic elongation factor 1 gamma[J].Protein Expression&Purification,2014,99(7):70-77.
[24]XU R,ZHAO H,DINKINS R D,et al.The 73 k D subunit of the cleavage and polyadenylation specificity factor(CPSF)complex affects reproductive development in Arabidopsis[J].Plant Molecular Biology,2006,61(4/5):799-815.
[25]TRSAUGUES L,STENMARK P,SCHLER H,et al.The crystal structure of human cleavage and polyadenylation specific factor-5 reveals a dimeric Nudix protein with a conserved catalytic site[M].Proteins:Structure,Function,and Bioinformatics,2008,73(4):1047-1052.
[26]DENG L X,WU G X,CAO Y,et al.The chromosomal protein HMGN2 mediates the LPS-induced expression ofβ-defensins in mice[J].Inflammation,2012,35(2):456-473.
[27]FENG Y,HE F,ZHANG P,et al.Inhibitory effect of HMGN2 protein on human hepatitis B virus expression and replication in the Hep G2.2.15 cell line[J].Antiviral Research,2009,81(3):277-282.
[28]ZHANG Z,WU R S,MOK H O,et al.Isolation,characterization and expression analysis of a hypoxia-responsive glucose transporter gene from the grass carp,Ctenopharyngodon idellus[J].Febs Journal,2003,270(14):3010-3017.
[29]YANG W,LI L G,LIU R D,et al.Molecular identification and characterization of Trichinella spiralis proteasome subunit beta type-7[J].Parasit Vectors,2015,8:18.
[30]NGUYEN V T,FUSE Y,TAMAOKI J,et al.Conservation of the Nrf2-Mediated gene regulation of proteasome subunits and glucose metabolism in zebrafish[J].Oxidative Medicine&Cellular Longevity,2016,18:5720574.
[2]陈燕燊,江育林.草鱼出血病病毒形态结构及其理化特性的研究[J].科学通报,1983,28(18):1138.
[3]NIBERT M L,DUNCAN R.Bioinformatics of recent aqua-and orthoreovirus isolates from fish:evolutionary gain or loss of FAST and fiber proteins and taxonomic implications[J].Plos One,2013,8(7):e68607.
[4]PEI C,KE F,CHEN Z Y,et al.Complete genome sequence and comparative analysis of grass carp reovirus strain 109(GCRe V-109)with other grass carp reovirus strains reveals no significant correlation with regional distribution[J].Archives of Virology,2014,159(9):2435-2440.
[5]FAN Y,RAO S,ZENG L,et al.Identification and genomic characterization of a novel fish reovirus,Hubei grass carp disease reovirus,isolated in 2009 in China[J].Journal of General Virology,2013,94(Pt 10):2266-2277.
[6]WANG Q,ZENG W,LIU C,et al.Complete genome sequence of a reovirus isolated from grass carp,indicating different genotypes of GCRV in China[J].Journal of Virology,2012,86(22):12466.
[7]FIELDS S,SONG O.A novel genetic system to detect protein-protein interactions[J].Nature,1989,340(6230):245-246.
[8]ZHOU Y,FAN Y D,ZENG L B,et al.Prokaryotic expression and immunoassay of grass carp reovirus capsid VP6 protein[J].Acta Virologica,2013,57(4):456-461.
[9]WANG H,SHEN X B,XU D,et al.Lipopolysaccharide-induced TNF-alpha factor in grass carp;(Ctenopharyngodon idella):Evidence for its involvement in antiviral innate immunity[J].Fish&Shellfish Immunology,2013,34(2):538-545.
[10]YE X,TIAN Y Y,DENG G C,et al.Complete genomic sequence of a reovirus isolated from grass carp in China[J].Virus Research,2012,163(1):275-283.
[11]LIU W,WANG H,YU F,et al.Grass carp reovirus outer capsid proteins VP5 and VP7 interact in vitro.[J].Archives of Virology,2017,162(8):2375-2380.
[12]FANG Q,SENG E K,DING Q Q,et al.Characterization of infectious particles of grass carp reovirus by treatment with proteases[J].Archives of Virology,2008,153(4):675-682.
[13]喻飞,王浩,宗乾坤,等.草鱼呼肠孤病毒GCRVVP56相互作用蛋白的鉴定[J].水产学报,2016,40(3):371-378.
[14]YU F,WANG H,LIU W,et al.Grass carp ctenopharyngodon idella fibulin-4 as a potential interacting partner for grass carp reovirus outer capsid proteins[J].Fish&Shellfish Immunology,2016,48:169-174.
[15]丁燏,李杰,闫秀英,等.草鱼(Ctenopharyngodon idellus)呼肠孤病毒(GCRV)VP6相互作用蛋白的筛选[J].海洋与湖沼,2014,45(6):1295-1299.
[16]XIE X,YANG F.Interaction of white spot syndrome virus VP26 protein with actin[J].Virology,2005,336(1):93-99.
[17]LAWO S,BASHKUROV M,MULLIN M,et al.HAUS,the 8-subunit human Augmin complex,regulates centrosome and spindle integrity[J].Current Biology,2009,19(10):816-826.
[18]DU L,XU J,LI X,et al.Rumba and Haus3 are essential factors for the maintenance of hematopoietic stem/progenitor cells during zebrafish hematopoiesis[J].Development,2011,138(4):619-629.
[19]FU B Q,LIU M Y,Kapel C M,et al.Cloning and analysis of a novel c DNA from Trichinella spiralis encoding a protein with an FYVE zinc finger domain[J].Veterinary Parasitology,2005,132(1-2):27-30.
[20]VENKATESAN M,KUNTZ D A,ROSE D R.Human lysosomal alpha-mannosidases exhibit different inhibition and metal binding properties[J].Protein Science,2009,18(11):2242-2251.
[21]LEE J H,RHO S B,CHUN T.Programmed cell death 6(PDCD6)protein interacts with death-associated protein kinase 1(DAPk1):additive effect on apoptosis via caspase-3 dependent pathway[J].Biotechnology Letters,2005,27(14):1011-1015.
[22]HUSI H,SKIPWORTH R J,CRONSHAW A,et al.Programmed cell death 6 interacting protein(PDCD6IP)and Rabenosyn-5(ZFYVE20)are potential urinary biomarkers for upper gastrointestinal cancer[J].Proteomics Clinical applications,2015,9(5/6):586-596.
[23]ACHILONU I,SIGANUNU T P,DIRR H W.Purification and characterisation of recombinant human eukaryotic elongation factor 1 gamma[J].Protein Expression&Purification,2014,99(7):70-77.
[24]XU R,ZHAO H,DINKINS R D,et al.The 73 k D subunit of the cleavage and polyadenylation specificity factor(CPSF)complex affects reproductive development in Arabidopsis[J].Plant Molecular Biology,2006,61(4/5):799-815.
[25]TRSAUGUES L,STENMARK P,SCHLER H,et al.The crystal structure of human cleavage and polyadenylation specific factor-5 reveals a dimeric Nudix protein with a conserved catalytic site[M].Proteins:Structure,Function,and Bioinformatics,2008,73(4):1047-1052.
[26]DENG L X,WU G X,CAO Y,et al.The chromosomal protein HMGN2 mediates the LPS-induced expression ofβ-defensins in mice[J].Inflammation,2012,35(2):456-473.
[27]FENG Y,HE F,ZHANG P,et al.Inhibitory effect of HMGN2 protein on human hepatitis B virus expression and replication in the Hep G2.2.15 cell line[J].Antiviral Research,2009,81(3):277-282.
[28]ZHANG Z,WU R S,MOK H O,et al.Isolation,characterization and expression analysis of a hypoxia-responsive glucose transporter gene from the grass carp,Ctenopharyngodon idellus[J].Febs Journal,2003,270(14):3010-3017.
[29]YANG W,LI L G,LIU R D,et al.Molecular identification and characterization of Trichinella spiralis proteasome subunit beta type-7[J].Parasit Vectors,2015,8:18.
[30]NGUYEN V T,FUSE Y,TAMAOKI J,et al.Conservation of the Nrf2-Mediated gene regulation of proteasome subunits and glucose metabolism in zebrafish[J].Oxidative Medicine&Cellular Longevity,2016,18:5720574.