利用原核及真核表达系统体外表达草鱼Ⅱ型呼肠孤病毒外突VP56蛋白的研究
|
摘要
草鱼Ⅱ型呼肠孤病毒是当前引起我国草鱼出血病的主要流行毒株,VP56是其外层衣壳上的唯一突起蛋白并有可能在病毒入侵阶段发挥核心作用,但该蛋白的具体功能尚不清楚。为了探讨VP56的生物学功能,分别利用大肠杆菌原核表达系统和杆状病毒真核表达系统研究了VP56的体外表达特性。首先从GCRVJX02W株的细胞感染混合物中抽提总RNA,并通过RT-PCR方法获得目的基因VP56,分别克隆至pGEX-4T-3及pFastBacHTA载体上,将质粒pGEX-4T-3-VP56转化至BL21(DE3),经IPTG诱导后,成功表达融合蛋白GST-VP56,大小为83ku,以包涵体形式存在;蛋白经纯化后免疫老鼠,制备了VP56的多克隆抗体,可以和rVP56产生特异性免疫结合。将pFastBacHTA-VP56转化至DH10Bac,成功转座后,提取重组杆粒DNA并鉴定且测序正确后转染SF9昆虫细胞。结果表明,自转染96h起,转染杆粒的细胞生长停滞,不断裂解。Westernblot结果显示,细胞表达重组蛋白His-VP56,大小约为62ku,为可溶蛋白。本研究利用原核表达系统和真核表达系统体外表达了VP56蛋白,制备了抗VP56多克隆抗体,为深入研究VP56蛋白在病毒入侵时的生物学功能奠定了基础。
Type Ⅱ reovirus of grass carp represents the pandemic strain for grass carp hemorrahagic disease in China. VP56 is the outer most protein on the virion of type Ⅱ reovirus,and is suggested to play a key role during viral entry. To facilitate the study on biological functions of VP56, here, we investigated the prokaryotic and eukaryotic expression of VP56 gene of type Ⅱ reovirus. The VP56 ORF encoded by the S7 genomic fragment was amplified by RT-PCR technology from the c DNA of GCRV-JX02 W infected CIK cells,and it was cloned into the p GEX-4 T-3 and p Fast Bac HTA,respectively. After confirmation of the clones by sequencing analysis,the p GEX4 T-3-VP56 was transformed into E. coli BL21(DE3) strain to express fusion protein GST-VP56. SDS-PAGE and Western Blot assays indicated that GST-VP56 was successfully expressed in E. coli BL21(DE3) strain with the molecular weight of about 83 ku. The recombinant protein mainly existed in the form of inclusion bodies. Polyclonal antibody was generated by immunization of mices with the purified recombinant VP56 proteins and the specificity of antibodies was determined by Western Blot.p Fast Bac HTA-VP56 was transformed into E. coli DH10 Bac strain to get recombinant bacmid DNA that was analyzed by PCR. Then,the recombinant bacmid DNA was transfected into SF9 cells. Positive SF9 cells transfected with bacmid DNA stopped growth and lysed at 96 h post transfection. Western Blot results showed that the His-tag antibody could be specifically bound to His-VP56 fusion protein with the molecular weight of about 62 ku, which was soluble protein. In this paper, recombinant VP56 proteins were successfully expressed by both prokarotic and eukaryotic expression systems,and polyclonal antiserum against VP56 was generated,which laid a foundation for characterizing the function of VP56 protein during viral infection.
Type Ⅱ reovirus of grass carp represents the pandemic strain for grass carp hemorrahagic disease in China. VP56 is the outer most protein on the virion of type Ⅱ reovirus,and is suggested to play a key role during viral entry. To facilitate the study on biological functions of VP56, here, we investigated the prokaryotic and eukaryotic expression of VP56 gene of type Ⅱ reovirus. The VP56 ORF encoded by the S7 genomic fragment was amplified by RT-PCR technology from the c DNA of GCRV-JX02 W infected CIK cells,and it was cloned into the p GEX-4 T-3 and p Fast Bac HTA,respectively. After confirmation of the clones by sequencing analysis,the p GEX4 T-3-VP56 was transformed into E. coli BL21(DE3) strain to express fusion protein GST-VP56. SDS-PAGE and Western Blot assays indicated that GST-VP56 was successfully expressed in E. coli BL21(DE3) strain with the molecular weight of about 83 ku. The recombinant protein mainly existed in the form of inclusion bodies. Polyclonal antibody was generated by immunization of mices with the purified recombinant VP56 proteins and the specificity of antibodies was determined by Western Blot.p Fast Bac HTA-VP56 was transformed into E. coli DH10 Bac strain to get recombinant bacmid DNA that was analyzed by PCR. Then,the recombinant bacmid DNA was transfected into SF9 cells. Positive SF9 cells transfected with bacmid DNA stopped growth and lysed at 96 h post transfection. Western Blot results showed that the His-tag antibody could be specifically bound to His-VP56 fusion protein with the molecular weight of about 62 ku, which was soluble protein. In this paper, recombinant VP56 proteins were successfully expressed by both prokarotic and eukaryotic expression systems,and polyclonal antiserum against VP56 was generated,which laid a foundation for characterizing the function of VP56 protein during viral infection.
引文
[1]张超,王庆,石存斌,等.草鱼呼肠孤病毒HZ08株的分离与鉴定[J].中国水产科学,2010,17(6):1257-1263.ZHANG C,WANG Q,SHI C B,et al.Isolation and identification of a grass carp reovirus isolate GCRV HZ08[J].Journal of Fishery Sciences of China,2010,17(6):1257-1263.
[2]王方华,李安兴.草鱼病毒性出血病研究进展[J].南方水产科学,2006,2(3):66-71.WANG F H,LI A X.Advances in research of hemorrhage of grass carp[J].South China Fisheries Science,2006,2(3):66-71.
[3]马贵华,陈道印,刘六英,等.草鱼出血病的免疫学研究进展[J].渔业现代化,2008,35(1):45-49.MA G H,CHEN D Y,LIU L Y,et al.The study situation of the immunology of Grass carp's Toxic Bleeding Disease[J].Fishery Modernization,2008,35(1):45-49.
[4]ZHANG Q Y,RUAN H M,LI Z Q,et al.Detection of Grass Carp Hemorrhage Virus(GCHV)from Vietnam and comparison with GCHV strain from China[J].High Technology Letters,2003,9(2):7-13.
[5]WANG Q,ZENG W 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.
[6]曾伟伟,王庆,王英英,等.草鱼呼肠孤病毒三重PCR检测方法的建立及其应用[J].中国水产科学,2013,20(2):419-426.ZENG W W,WANG Q,WANG Y Y,et al.Establishment of multiplex PCR for detection of grass carp reovirus and its application[J].Journal of Fishery Sciences of China,2013,20(2):419-426.
[7]RAO Y L,SU J G.Insights into the antiviral immunity against grass carp(Ctenopharyngodon idella)reovirus(GCRV)in grass carp[J].Journal of Immunology Research,2015(2):670437.
[8]徐洋,郝贵杰,沈锦玉,等.两株草鱼呼肠孤病毒江西株的分离与鉴定[J].淡水渔业,2010,40(3):44-49.XU Y,HAO G J,SHEN J Y,et al.Isolation and identification of two grass carp reovrius strains in Jiangxi province[J].Freshwater Fisheries,2010,40(3):44-49.
[9]曾伟伟,王庆,刘永奎,等.一株草鱼呼肠孤病毒弱毒株的分离、鉴定及免疫原性初步分析[J].水生生物学报,2011,35(5):790-795.ZENG W W,WANG Q,LIU Y K,et al.Isolation and identification of new GCRV strain and primary study on its immunogenicity[J].Acta Hydrobiologica Sinica,2011,35(5):790-795.
[10]张超.草鱼呼肠孤病毒HZ08株的分离鉴定与全基因组分子特征分析[D].上海:上海海洋大学,2010.ZHANG C.Isolation,identification of grass carp reovirus HZ08 and molecular characteristics of its complete genome[D].Shanghai:Shanghai Ocean University,2010.
[11]ZHANG C,WANG Q,SHI C B,et al.Molecular analysis of grass carp reovirus HZ08 genome segments 1-3 and 5-6.[J].Virus Genes,2010,41(1):102-104.
[12]王土,许丹,吕利群.应用dsRNA测序技术检测草鱼呼肠孤病毒的混合感染[J].上海海洋大学学报,2012,21(5):756-762.WANG T,XU D,LL Q.Detection of the co-infection of different grass carp reovirus strains using dsRNA sequencing technology[J].Journal of Shanghai Ocean University,2012,21(5):756-762.
[13]NIBERT M L,DUNCAN R.Bioinformatics of recent aquaand orthoreovirus isolates from fish:evolutionary gain or loss of FAST and fiber proteins and taxonomic implications.[J].PLo S One,2013,8(7):e68607.
[14]喻飞,王浩,宗乾坤,等.草鱼呼肠孤病毒GCRV VP56相互作用蛋白的鉴定[J].水产学报,2016,40(3):371-378.YU F,WANG H,ZONG Q K,et al.Screening of potential host proteins interacting with VP56 of typeⅡgrass carp reovirus by yeast two-hybrid system[J].Journal of Fisheries of China,2016,40(3):371-378.
[15]YU F,WANG H,LIU W S,et al.Grass carp Ctenopharyngodon idella Fibulin-4 as a potential interacting partner for grass carp reovirus outer capsid proteins[J].Fish&Shellfish Immunology,2015,48:169-174.
[16]LIU B,GONG Y C,LI Z,et al.Baculovirus-mediated GCRV vp7 and vp6 genes expression in silkworm and grass carp[J].Molecular Biology Reports,2016,43(6):509-515.
[17]ATTOUI H,FANG Q,JAARAR M F,et al.Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus,Grass carp reovirus,Striped bass reovirus and golden ide reovirus(genus Aquareovirus,family Reoviridae).[J].Journal of General Virology,2002,83(8):1941-1951.
[18]HE Y X,XU H X,YANG Q,et al.The use of an in vitro microneutralization assay to evaluate the potential of recombinant VP5 protein as an antigen for vaccinating against Grass carp reovirus[J].Virology Journal,2011,8:132.
[19]HE Y X,YANG Q,XU H X,et al.Prokaryotic expression and purification of grass carp reovirus capsid protein VP7 and its vaccine potential[J].African Journal of Microbiology Research,2011,5(13):1643-1648.
[20]王杭军,叶星,田园园,等.草鱼呼肠孤病毒GCRVGD108株VP5蛋白的原核表达[J].华中农业大学学报,2013,32(2):97-102.WANG H J,YE X,TIAN Y Y,et al.Prokaryotic expression of GCRV-GD108 VP5 protein[J].Journal of Huazhong Agricultural University,2013,32(2):97-102.
[21]王杭军,叶星,田园园,等.草鱼呼肠孤病毒GCRVGD108株VP5蛋白功能及免疫原性分析[J].水产学报,2013,37(1):109-116.WANG H J,YE X,TIAN Y Y,et al.Analysis of function and immunogenicity of GCRV-GD108 VP5[J].Journal of Fisheries of China,2013,37(1):109-116.
[22]宗乾坤,张也,吕利群.Ⅱ型草鱼呼肠孤病毒VP4、VP35蛋白多克隆抗体制备及其免疫原性分析[J].水产学报,2016,40(3):355-362.ZONG Q K,ZHANG Y,LL Q.Preparation and immunogenicity of polyclonal antibodies against VP4,VP35protein of typeⅡgrass carp reovirus[J].Journal of Fisheries of China,2016,40(3):355-362.
[23]苏岚,曾令兵,周勇,等.草鱼呼肠孤病毒VP6蛋白在毕赤酵母中表达的初步研究[J].淡水渔业,2012,42(6):38-42.SU L,ZENG L B,ZHOU Y,et al.Preliminary study on the expression of Grass carp reovirus VP6 protein in Pichia pastoris[J].Freshwater Fisheries,2012,42(6):38-42.
[2]王方华,李安兴.草鱼病毒性出血病研究进展[J].南方水产科学,2006,2(3):66-71.WANG F H,LI A X.Advances in research of hemorrhage of grass carp[J].South China Fisheries Science,2006,2(3):66-71.
[3]马贵华,陈道印,刘六英,等.草鱼出血病的免疫学研究进展[J].渔业现代化,2008,35(1):45-49.MA G H,CHEN D Y,LIU L Y,et al.The study situation of the immunology of Grass carp's Toxic Bleeding Disease[J].Fishery Modernization,2008,35(1):45-49.
[4]ZHANG Q Y,RUAN H M,LI Z Q,et al.Detection of Grass Carp Hemorrhage Virus(GCHV)from Vietnam and comparison with GCHV strain from China[J].High Technology Letters,2003,9(2):7-13.
[5]WANG Q,ZENG W 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.
[6]曾伟伟,王庆,王英英,等.草鱼呼肠孤病毒三重PCR检测方法的建立及其应用[J].中国水产科学,2013,20(2):419-426.ZENG W W,WANG Q,WANG Y Y,et al.Establishment of multiplex PCR for detection of grass carp reovirus and its application[J].Journal of Fishery Sciences of China,2013,20(2):419-426.
[7]RAO Y L,SU J G.Insights into the antiviral immunity against grass carp(Ctenopharyngodon idella)reovirus(GCRV)in grass carp[J].Journal of Immunology Research,2015(2):670437.
[8]徐洋,郝贵杰,沈锦玉,等.两株草鱼呼肠孤病毒江西株的分离与鉴定[J].淡水渔业,2010,40(3):44-49.XU Y,HAO G J,SHEN J Y,et al.Isolation and identification of two grass carp reovrius strains in Jiangxi province[J].Freshwater Fisheries,2010,40(3):44-49.
[9]曾伟伟,王庆,刘永奎,等.一株草鱼呼肠孤病毒弱毒株的分离、鉴定及免疫原性初步分析[J].水生生物学报,2011,35(5):790-795.ZENG W W,WANG Q,LIU Y K,et al.Isolation and identification of new GCRV strain and primary study on its immunogenicity[J].Acta Hydrobiologica Sinica,2011,35(5):790-795.
[10]张超.草鱼呼肠孤病毒HZ08株的分离鉴定与全基因组分子特征分析[D].上海:上海海洋大学,2010.ZHANG C.Isolation,identification of grass carp reovirus HZ08 and molecular characteristics of its complete genome[D].Shanghai:Shanghai Ocean University,2010.
[11]ZHANG C,WANG Q,SHI C B,et al.Molecular analysis of grass carp reovirus HZ08 genome segments 1-3 and 5-6.[J].Virus Genes,2010,41(1):102-104.
[12]王土,许丹,吕利群.应用dsRNA测序技术检测草鱼呼肠孤病毒的混合感染[J].上海海洋大学学报,2012,21(5):756-762.WANG T,XU D,LL Q.Detection of the co-infection of different grass carp reovirus strains using dsRNA sequencing technology[J].Journal of Shanghai Ocean University,2012,21(5):756-762.
[13]NIBERT M L,DUNCAN R.Bioinformatics of recent aquaand orthoreovirus isolates from fish:evolutionary gain or loss of FAST and fiber proteins and taxonomic implications.[J].PLo S One,2013,8(7):e68607.
[14]喻飞,王浩,宗乾坤,等.草鱼呼肠孤病毒GCRV VP56相互作用蛋白的鉴定[J].水产学报,2016,40(3):371-378.YU F,WANG H,ZONG Q K,et al.Screening of potential host proteins interacting with VP56 of typeⅡgrass carp reovirus by yeast two-hybrid system[J].Journal of Fisheries of China,2016,40(3):371-378.
[15]YU F,WANG H,LIU W S,et al.Grass carp Ctenopharyngodon idella Fibulin-4 as a potential interacting partner for grass carp reovirus outer capsid proteins[J].Fish&Shellfish Immunology,2015,48:169-174.
[16]LIU B,GONG Y C,LI Z,et al.Baculovirus-mediated GCRV vp7 and vp6 genes expression in silkworm and grass carp[J].Molecular Biology Reports,2016,43(6):509-515.
[17]ATTOUI H,FANG Q,JAARAR M F,et al.Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus,Grass carp reovirus,Striped bass reovirus and golden ide reovirus(genus Aquareovirus,family Reoviridae).[J].Journal of General Virology,2002,83(8):1941-1951.
[18]HE Y X,XU H X,YANG Q,et al.The use of an in vitro microneutralization assay to evaluate the potential of recombinant VP5 protein as an antigen for vaccinating against Grass carp reovirus[J].Virology Journal,2011,8:132.
[19]HE Y X,YANG Q,XU H X,et al.Prokaryotic expression and purification of grass carp reovirus capsid protein VP7 and its vaccine potential[J].African Journal of Microbiology Research,2011,5(13):1643-1648.
[20]王杭军,叶星,田园园,等.草鱼呼肠孤病毒GCRVGD108株VP5蛋白的原核表达[J].华中农业大学学报,2013,32(2):97-102.WANG H J,YE X,TIAN Y Y,et al.Prokaryotic expression of GCRV-GD108 VP5 protein[J].Journal of Huazhong Agricultural University,2013,32(2):97-102.
[21]王杭军,叶星,田园园,等.草鱼呼肠孤病毒GCRVGD108株VP5蛋白功能及免疫原性分析[J].水产学报,2013,37(1):109-116.WANG H J,YE X,TIAN Y Y,et al.Analysis of function and immunogenicity of GCRV-GD108 VP5[J].Journal of Fisheries of China,2013,37(1):109-116.
[22]宗乾坤,张也,吕利群.Ⅱ型草鱼呼肠孤病毒VP4、VP35蛋白多克隆抗体制备及其免疫原性分析[J].水产学报,2016,40(3):355-362.ZONG Q K,ZHANG Y,LL Q.Preparation and immunogenicity of polyclonal antibodies against VP4,VP35protein of typeⅡgrass carp reovirus[J].Journal of Fisheries of China,2016,40(3):355-362.
[23]苏岚,曾令兵,周勇,等.草鱼呼肠孤病毒VP6蛋白在毕赤酵母中表达的初步研究[J].淡水渔业,2012,42(6):38-42.SU L,ZENG L B,ZHOU Y,et al.Preliminary study on the expression of Grass carp reovirus VP6 protein in Pichia pastoris[J].Freshwater Fisheries,2012,42(6):38-42.