摘要
【目的】制备基因I型草鱼呼肠孤病毒(GCRV)VP7蛋白合成肽抗体并验证其特异性,为研究VP7外衣壳蛋白在基因I型GCRV病毒与宿主间的相互作用及研制基因工程疫苗提供参考依据。【方法】以GCRV GZ1208株为模板进行RT-PCR扩增,运用在线生物信息学分析软件对VP7蛋白B细胞表位进行功能预测,选择位于VP7蛋白第26~39位氨基酸的多肽序列进行人工合成,与钥孔血蓝蛋白载体蛋白偶联后免疫新西兰白兔以获得VP7蛋白合成肽抗体,然后采用Western blotting和间接免疫荧光试验验证VP7蛋白合成肽抗体对基因I型GCRV的特异性识别能力。【结果】VP7蛋白氨基酸序列在基因I型GCRV中高度保守,其合成肽抗体效价约1∶256000。Western blotting分析结果显示,融合蛋白VP7约在55 kD处出现单一的特异性条带,而GZ1208株和JX0901株样品约在30 kD处出现对应的特异性条带,与预期结果基本一致;间接免疫荧光试验结果表明,VP7蛋白合成肽抗体可特异性识别感染基因I型GCRV的草鱼脑细胞(GCB),感染细胞中出现特异性绿色荧光信号,而感染其他基因型GCRV及正常未感染病毒的GCB细胞中均无特异性绿色荧光出现。【结论】制备获得的VP7蛋白合成肽抗体能特异性识别基因I型GCRV,而不识别其他基因型毒株,可用于草鱼出血病的临床诊断和基因I型GCRV的病原学研究。
【Objective】Anti-VP7 protein synthetic peptide antibody of grass carp reovirus(GCRV)genotype I was prepare and its specificity was identified to provide reference for the research on the interaction of VP7 capsid protein between GCRV genetype I and the host,and development of genetic engineering vaccine.【Method】GCRV GZ1208 was used as the template to conduct RT-PCR amplification. Online bioinformatics software was used to predict the function of the B cell epitopes of VP7 protein. The polypeptide sequences of amino acid located at 26-39 positions of VP7 protein were selected and synthesized artificially. The New Zealand white rabbits were immunized with the polypeptide of VP7 coupling linked with keyhole limpet hemocyanin carrier protein,and the synthetic peptide antibody against VP7 protein was obtained. Then,Western blotting and indirect immunofluorescence assays were used to test the specific identifiction af anti-VP7 synthetic peptide antibody to GCRV genotype I.【Result】The amino acid sequences of VP7 was highly conserved in genotype I GCRV,and the titer of synthetic peptide antibody was as high as 1∶256000. The results of Western blotting indicated that the fusion protein VP7 showed a single specific band at about 55 kD,while GZ1208 strain and JX0901 strain showed corresponding specific bands at about 30 k D,which was consistent with the expected results.The results of indirect immunofluorescence assays indicated that VP7 protein synthetic peptide antibody could specificallyidentify the grass carp's brain cells(GCB)infected with genetype I GCRV,and the specific green fluorescence appeared in the infected cells. While there was no specific green fluorescence appeared in the cells infected with other genotypes GCRV and in normal cells.【Conclusion】The synthetic peptide antibody against VP7 protein is successfully prepared,which can specifically recognize GCRV genotype I but cannot recognize other genotype strains. The present study can be applied in clinical diagnosis of grass carp hemorrhagic disease as well as pathogenic research of GCRV genotype I.
引文
高华义,吕传忠,任东兴,陈文娟,辛波,梁姗,付旭彬.2018.多肽合成去势疫苗免疫佐剂的筛选[J].黑龙江畜牧兽医,(7):169-172.[Gao H Y,LüC Z,Ren D X,Chen WJ,Xin B,Liang S,Fu X B.2018.The screening of immunoadjuvant in peptide synthesis castration vaccine[J].Heilongjiang Animal Science and Veterinary Medicine,(7):169-172.]
高岩,裴超,张超,李筝,孔祥会.2017.草鱼呼肠孤病毒疫苗的研发与应用[J].水产科学,36(2):237-242.[Gao Y,Pei C,Zhang C,Li Z,Kong X H.2017.A review of research and development of vaccine against grass carp reovirus[J].Fisheries Science,36(2):237-242.]
郝贵杰,潘晓艺,姚嘉赟,徐洋,尹文林,沈锦玉.2010.草鱼呼肠孤病毒衣壳蛋白VP7基因真核表达载体pCI-VP7的构建及鉴定[J].水产学报,34(5):807-813.[Hao G J,Pan X Y,Yao J Y,Xu Y,Yin W L,Shen J Y.2010.Construction and identification of recombinant eukaryotic expression vector pCI-VP7 containing GCRV VP7 gene[J].Journal of Fisheries of China,34(5):807-813.]
胡涛,安艳,吕群丹,徐英武.2015.拟南芥SPX1蛋白原核表达及纯化分析[J].生物技术通报,31(8):88-93.[Hu T,An Y,LüQ D,Xu Y W.2015.Prokaryotic expression and purification of AtSPX1 protein in Arabidopsis thaliana[J].Biotechnology Bulletin,31(8):88-93.]
黄剑波.2016.PRRSV SC株GP5蛋白合成肽抗体的制备及ADE作用研究[D].成都:四川农业大学.[Huang J B.2016.Preparation of GP5 protein synthetic peptide antibody of PRRSV SC strain and the research of ADE[D].Chengdu:Sichuan Agricultural University.]
黄谧,冯勇.2017.大鲵虹彩病毒主要衣壳蛋白的原核表达及多克隆抗体制备[J].湖北农业科学,56(21):4146-4150.[Huang M,Feng Y.2017.Prokaryotic expression and polyclonal antibody preparation of the major capsid protein of Chinese giant salamander iridovirus[J].Hubei A-gricultural Sciences,56(21):4146-4150.]
黄新新,袁辰刚,宁雪,顾鸣,蔡强,刘锐,陆承平.2015.桃拉综合征病毒VP1高变区蛋白抗体制备及初步特性分析[J].中国免疫学杂志,(2):221-224.[Huang X X,Yuan C G,Ning X,Gu M,Cai Q,Liu R,Lu C P.2015.Polyclonal antibody specific for hypervariable region of VP1capsid protein of Taura syndrome virus(TSV)prepartion and characteristic analysis[J].Chinese Journal of Immunology,(2):221-224.]
黄毅昌,雷燕,杨玉滔,闫秀英,简纪常,吴灶和.2016.草鱼出血病分子流行病学及GCRV多样性研究[J].安徽农业科学,44(11):120-125.[Huang Y C,Lei Y,Yang Y T,Yan X Y,Jian J C,Wu Z H.2016.Molecular epidemiology of hemorrhage disease of grass carp and the diversity research of GCRV[J].Journal of Anhui Agricultural Sciences,44(11):120-125.]
李丽,鞠文,殷德辉,孟日增,李娟,宋秀英,徐坤.2015.生物素标记的布鲁菌多克隆抗体的制备[J].中国生物制品学杂志,28(2):147-154.[Li L,Ju W,Yin D H,Meng RZ,Li J,Song X Y,Xu K.2015.Preparation of biotinlabeled polyclonal antibody against Brucella abortus[J].Chinese Journal of Biologicals,28(2):147-154.]
刘文慧,阚丽丽,崔永胜,谭李倩,梁雪雪,李新,赵微.2015.人星状病毒I型非结构蛋白nsP1a及其C末端蛋白nsP1a/4的原核表达及鉴定[J].病毒学报,31(1):46-50.[Liu WH,Kan L L,Cui Y S,Tan L Q,Liang X X,Li X,Zhao W.2015.Prokaryotic expression and identification of human astrovirus nonstructural proteins,nsP1a and nsP1a/4[J].Chinese Journal of Virology,31(1):46-50.]
刘学光,郑怀东,郭欣硕,罗靳,蔺翠翠,王秋雨.2013.传染性造血器官坏死病毒糖蛋白的原核表达及多克隆抗体的制备[J].大连海洋大学学报,28(3):254-258.[Liu X G,Zheng H D,Guo X S,Luo J,Lin C C,Wang Q Y.2013.Prokaryotic expression and polyclonal antibody preparation of glycoprotein gene in infectious hematopoietic necrosis virus[J].Journal of Dalian Ocean University,28(3):254-258.]
罗忠永,王印,杨泽晓.2017.PRRSV M蛋白基因片段的原核表达及间接ELISA检测方法的初步建立[J].中国兽医科学,47(1):95-99.[Luo Z Y,Wang Y,Yang Z X.2017.Establishment of an indirect ELISA using the prokaryotically-expressed M protein for the detection of antibodies against PRRSV[J].Chinese Veterinary Science,47(1):95-99.]
汤亚芳,曾伟伟,王庆,王英英,石存斌,冯茹,高辉,王承宝.2018.草鱼呼肠孤病毒Ⅱ型VP6蛋白多克隆抗体制备及其特异性分析[J].淡水渔业,48(1):9-14.[Tang Y F,Zeng W W,Wang Q,Wang Y Y,Shi C B,Feng R,Gao H,Wang C B.2018.Preparation and specification analysis of polyclonal antibody against grass carp reovirus type II VP6 protein[J].Freshwater Fisheries,48(1):9-14.]
王玉坤,李锦程,曹远银.2008.兔抗灭多威多克隆抗体的制备[J].江苏农业科学,(2):79-81.[Wang Y K,Li J C,Cao Y Y.2008.Preparation of rabbit anti-methomyl polyclonal antibody[J].Jiangsu Agricultural Sciences,(2):79-81.]
吴倩,张丽芬,陈复生.2015.超声波对蛋白质提取及改性影响的研究进展[J].食品与机械,31(4):256-259.[Wu Q,Zhang L F,Chen F S.2015.Research progress of ultrasonic effect on protein extraction and modification[J].Food&Machinery,31(4):256-259.]
杨映,于辉,古勇明.2015.草鱼呼肠孤病毒研究进展[J].广东农业科学,42(15):92-97.[Yang Y,Yu H,Gu Y M.2015.Research progress on grass carp reovirus[J].Guangdong Agricultural Sciences,42(15):92-97.]
曾伟伟,王庆,王英英,李莹莹,郝贵杰,石存斌,沈锦玉.2017.草鱼呼肠孤病毒HZ08株抗体IPMA检测方法的建立及应用[J].水产学报,41(1):142-149.[Zeng W W,Wang Q,Wang Y Y,Li Y Y,Hao G J,Shi C B,Shen JY.2017.Establishment and application of immunoperoxidase monolayer assay for detection of the antibody against grass carp reovirus HZ08[J].Journal of Fisheries of China,41(1):142-149.]
张敏利,盛佳璐,喻飞,王浩,吕立群.2018.草鱼Fibulin-4蛋白与草鱼呼肠孤病毒外衣壳蛋白相互作用的研究[J].病毒学报,34(4):557-564.[Zhang M L,Sheng J L,Yu F,Wang H,LüL Q.2018.Studies on the interaction of grass carp Fibulin-4 protein with grass carp reovious outer capsid proteins[J].Chinese Journal of Virology,34(4):557-564.]
张玉瑶,马秀丽,黄兵,李玉峰,于可响,李建亮,刘存霞,韩宏宇,崔言顺.2015.鸭甲肝病毒1型和3型间接ELISA方法的建立与应用[J].微生物学报,55(4):501-509.[Zhang Y Y,Ma X L,Huang B,Li Y F,Yu K X,Li J L,Liu CX,Han H Y,Cui Y S.2015.Indirect ELISA for simultaneous detection of antibodies against duck hepatitis a type 1 and 3 viruses[J].Acta Microbiologica Sinica,55(4):501-509.]
宗乾坤,张也,吕立群.2016.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,40(3):355-362.[Zong Q K,Zhang Y,LüL Q.2016.Preparation and immunogenicity of polyclonal antibodies against VP4,VP35 protein of typeⅡgrass carp reovirus[J].Journal of Fisheries of China,40(3):355-362.]
Al-Juboori R A,Yusaf T.2012.Identifying the optimum process parameters for ultrasonic cellular disruption of E.coli[J].International Journal of Chemical Reactor Engineering,10(1):1498-1502.
Andreolla A P,Erpen L M S,Frandoloso R,Kreutz L C.2018.Development of an indirect ELISA based on recombinant capsid protein to detect antibodies to bovine leukemia virus[J].Brazilian Journal of Microbiology,pii:S1517-8382(18)30043-1.doi:10.1016/j.bjm.2018.05.001.
Brudeseth B E,Wiulsr?d R,Fredriksen B N,Lindmo K,L?kling K E,Bordevik M,Steine N,Klevan A,Gravningen K.2013.Status and future perspectives of vaccines for industrialised fin-fish farming[J].Fish&Shellfish Immunology,35(6):1759-1768.
Chen C L,Sun X Y,Liao L J,Luo S X,Li Z Q,Zhang X H,Wang Y P,Guo Q L,Fang Q,Dai H.2013.Antigenic analysis of grass carp reovirus using single-chain variable fragment antibody against IgM from Ctenopharyngodon idella[J].Science China.Life Sciences,56(1):59-65.
Cheng Z Y,Zhang M,Ning R H,Hu B Q,Wen C G.2016.Molecular cloning,prokaryotic expression and functional characterization of superoxide dismutase gene from Siniperca chuatsi[J].Fish&Shellfish Immunology,53:116.doi:10.1016/j.fsi.2016.04.097.
Dong Z D,Zhang J,Ji X S,Zhou F N,F Y,Chen W Y,Zeng Y Q,Li T M,Wang H.2012.Molecular cloning,characterization and expression of cathepsin D from grass carp(Ctenopharyngodon idella)[J].Fish Shellfish Immunology,33(5):1207-1214.
Fan Y D,Rao S J,Zeng L B,Ma J,Zhou Y,Xu J,Zhang H.2013.Identification and genomic characterization of a novel fish reovirus,Hubei grass carp disease reovirus,isolated in 2009 in China[J].The Journal of General Virology,94(10):2266-2277.
Fang Q,Seng E K,Ding Q Q,Zhang L L.2008.Characterization of infectious particles of grass carp reovirus by treatment with proteases[J].Archives of Virology,153(4):675-682.
Gao S,Shi W,Wang Y T,Guo M T,Duan K X,Song A C,Lian G H,Ren T,Li Y J,Tang L J,Sun L,Liu M.2018.Establishment and evaluation of an indirect immunofluorescence assay for the detection of salmonid alphavirus[J].Letters in Applied Microbiology,66(4):293-299.
Hao K,Chen X H,Qi X Z,Yu X B,Du E Q,Ling F,Zhu B,Wang G X.2017.Protective immunity of grass carp induced by DNA vaccine encoding capsid protein gene(vp7)of grass carp reovirus using bacterial ghost as delivery vehicles[J].Fish&Shellfish Immunology,64:414-425.
He L B,Zhang A D,Pei Y Y,Chu P F,Li Y M,Huang R,Liao L J,Zhu Z Y,Wang Y P.2017.Differences in responses of grass carp to different types of grass carp reovirus(GCRV)and the mechanism of hemorrhage revealed by transcriptome sequencing[J].BMC Genomics,18(1):452.doi:10.1186/s12864-017-3824-1.
He Y X,Yang Q,Xu H X,Wu H,Wu F Y,Lu L Q.2011.Prokaryotic expression and purification of grass carp reovirus capsid protein VP7 and its vaccine potential[J].African Journal of Microbiology Research,5(13):1643-1648.
Ivanovic T,Agosto M A,Chandran K,Nibert M L.2007.Arole for molecular chaperone Hsc70 in reovirus outer capsid disassembly[J].Journal of Biological Chemistry,282(16):12210-12219.
Liu B,Gong Y,Li Z,Hu X L,Cao G L,Xue R Y,Gong C L.2016.Baculovirus-mediated GCRV vp7 and vp6 genes expression in silkworm and grass carp[J].Molecular Biology Reports,43(6):509-515.
Liu W S,Wang H,Yu F,Lu L Q.2017.Grass carp reovirus outer capsid proteins VP5 and VP7 interact in vitro[J].Archives of Virology,162(8):2375-2380.
Lu L,Xu H,He Y,Li J.2011.Protection of grass carp,Ctenopharyngon idellus(Valenciennes),through oral administration of a subunit vaccine against reovirus[J].Journal of Fish Diseases,34(12):939-942.
Luo S X,Yan L,Zhang X H,Yuan L,Fang Q,Zhang Y A,Dai H P.2015.Yeast surface display of capsid protein VP7 of grass carp reovirus:Fundamental investigation for the development of vaccine against hemorrhagic disease[J].Journal of Microbiology and Biotechnology,25(12):2135-2145.
Mahajan S,Mohapatra J K,Pandey L K,Sharma G K,Pattnaik B.2015.Indirect ELISA using recombinant nonstructural protein 3D to detect foot and mouth disease virus infection associated antibodies[J].Biologicals,43(1):47-54.
Qiu T,Lu R H,Zhang J,Zhu Z Y.2001.Complete nucleotide sequence of the S10 genome segment of grass carp reovirus(GCRV)[J].Diseases of Aquatic Organisms,44(1):69-74.
Rao Y L,Su J G.2015.Insights into the antiviral immunity against grass carp(Ctenopharyngodon idella)reovirus(GCRV)in grass carp[J].Journal of Immunological Research,2015:670437.doi:10.1155/2015/670437.
Shao L,Sun X Y,Fang Q.2011.Antibodies against outer-capsid proteins of grass carp reovirus expressed in E.coli are capable of neutralizing viral infectivity[J].Virology Journal,8:347.doi:10.1186/1743-422X-8-347.
Suwal S,Roblet C,Amiot J,Doyen A,Beaulieu L,Legault J,Bazinet L.2014.Recovery of valuable peptides from marine protein hydrolysate by electrodialysis with ultrafiltration membrane:Impact of ionic strength[J].Food Research International,65:407-415.
Tanzadehpanah H,Asoodeh A,Mahaki H,Mostajabodave Z,Chamani J,Mojallal-Tabatabaei Z,Emtenani S,Emtenani S,Moradi M R.2016.Bioactive and ACE binding properties of three synthetic peptides assessed by various spectroscopy techniques[J].Process Biochemistry,51(12):2067-2075.
Tian Y Y,Jiao Z Z,Dong J J,Sun C F,Jiang X Y,Ye X.2017.Grass carp reovirus-GD108 fiber protein is involved in cell attachment[J].Virus Genes,53(4):613-622.
Wang Q,Xie H L,Zeng W W,Wang L C,Liu C,Wu J X,Wang Y Y,Li Y Y,Bergmann S M.2018.Development of indirect immunofluorescence assay for TCID50measurement of grass carp reovirus genotype II without cytopathic effect onto cells[J].Microbial Pathogenesis,114:68-74.
Wang Q,Zeng W W,Liu C,Zhang C,Wang Y Y,Shi C B,Wu S Q.2012.Complete genome sequence of a reovirus isolated from grass carp,indicating different genotypes of GCRV in China[J].Journal of Virology,86(22):12466.
Wang Q,Zeng W W,Yin L,Wang Y Y,Liu C,Li Y Y,Zheng S C,Shi C B.2016.Comparative study on physicalchemical and biological characteristics of grass carp reovirus from different genotypes[J].Journal of The World Aquaculture Society,47(6):862-872.
Wu M L,Li H Y,Jiang H,Hou G,He J,Jiang Y,Chen H.2017.Structure and function of S9 segment of grass carp reovirus Anhui strain[J].Virusdisease,28(1):26-32.
Yan X Y,Wang Y,Xiong L F,Jian J C,Wu Z H.2014.Phylogenetic analysis of newly isolated grass carp reovirus[J].SpringerPlus,3:190.doi:10.1186/2193-1801-3-190.
Ye X,Tian Y Y,Deng G C,Chi Y Y,Jiang X Y.2012.Complete genomic sequence of a reovirus isolated from grass carp in China[J].Virus Research,163(1):275-283.
Yu F X,Du Y H,Huang X Y,Ma H,Xu B L,Adungo F,Hayasaka D,Buerano C C,Morita K.2015.Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA[J].Virology Journal,12:117.doi:10.1186/s12985-015-0350-0.
Zhang L L,Shen J Y,Lei C F,Li X M,Fang Q.2008.The high level expression of grass carp reovirus VP7 protein in prokaryotic cells[J].Virologica Sinica,23(1):51-56.