鲤疱疹病毒Ⅱ型ORF72基因克隆、蛋白原核表达和抗体制备
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摘要
异育银鲫作为我国重要的淡水经济鱼类,其鳃出血病为主要病害,并造成重大经济损失,该病病原为鲤疱疹病毒Ⅱ型(cyprinid herpesvirusⅡ,CyHV-2)。目前,研究主要在该病的检测和病理方面,对于免疫学检测方面研究较少。本研究根据CyHV-2 ORF72基因序列设计了1对特异性引物,利用患病异育银鲫肾脏DNA作为模板,通过PCR扩增得到CyHV-2 ORF72基因全长,该基因序列全长为1 113 bp,可编码370个氨基酸的蛋白。该基因与pET-28a表达载体连接构建重组原核表达载体pET-ORF72转入大肠杆菌中,经IPTG诱导表达得到了分子量约为45 ku的重组蛋白,与预测的蛋白大小基本一致。将纯化的重组蛋白皮下多点注射免疫新西兰大耳兔制备了ORF72的多克隆抗体,经Western Blotting检测抗体能与病毒粗提液有良好反应,特异性较好,本结果可为下一步建立疫苗和免疫检测方法奠定基础。
引文
[1]廖红,林华,郝中香,等.鲤疱疹病毒2型ORF5截短基因的克隆表达及免疫原性研究[J].中国兽医科学,2016(11):1394-1400.
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[7]Xu J,Zeng L,Zhang H,et al.Cyprinid herpesvirus 2 infection emerged in cultured gibel carp,Carassius auratus gibelio,in China[J].Veterinary Microbiology,2013,166(1/2):138-144.
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[14]薛中仪,朱春艳,王瑶,等.一种鲤科疱疹病毒Ⅱ型的PCR快速检测方法[J].水产养殖,2015,36(7):45-48.
[15]彭俊杰,张琪,贾路路,等.鲤疱疹病毒Ⅱ型(CyHV-2)ORF25蛋白的原核表达及单克隆抗体的制备[J].华中农业大学学报,2017,36(2):96-101.
[16]李振言.鱼类细菌性败血症及其防治技术[J].科学养鱼,2004(1):46-46.
[17]张书俊,杨先乐,李聃,等.施氏鲟水霉病病原的初步研究[J].中国水产科学,2009,16(1):89-96.
[18]徐海圣,王淑霞,吴惠仙,等.异育银鲫寄生黏孢子虫病的研究[J].浙江大学学报(理学版),1997(3):259-264.
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[22]罗丹,梁利国,谢骏,等.2型鲤疱疹病毒双重PCR快速检测方法的建立及应用[J].中国预防兽医学报,2014,36(5):379-382.
[23]于力,王津津,史秀杰,等.鲤科疱疹病毒2型Taqman实时荧光PCR方法的建立[J].中国动物检疫,2015(10):80-84.
[24]周勇,曾令兵,张辉,等.鲤疱疹病毒Ⅱ型TaqMan real-time PCR检测方法的建立及应用[J].水产学报,2013,37(4):607-613.
[25]Ma J,Jiang N,Lapatra S E,et al.Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (CyHV-2)[J].Veterinary Microbiology,2015,177(3/4):315-325.
[26]乔宁,李美芹,刘永光,等.番茄黄化曲叶病毒CP基因的原核表达载体构建选择[J].安徽农业科学,2012(7):3901-3902,3905.
[27]王世会,贾智英,李池陶,等.锦鲤疱疹病毒ORF72基因克隆及结构功能分析[J].水产学杂志,2016,29(3):9-15.
[2]Wu T,Ding Z F,Ren M,et al.The histo- and ultra-pathological studies on a fatal disease of Prussian carp (Carassius gibelio) in mainland China associated with cyprinid herpesvirus 2 (CyHV-2)[J].Aquaculture,2013,412(6):8-13.
[3]周勇,范玉顶,徐进,等.鲤疱疹病毒Ⅱ型ORF4基因的克隆、表达与免疫学检测方法[J].淡水渔业,2017,47(1):61-65.
[4]Goodwin A E,Khoo L,Lapatra S E,et al.Goldfish hematopoietic necrosis herpesvirus(cyprinid herpesvirus 2)in the USA:molecular confirmation of isolates from diseased fish[J].Journal of Aquatic Animal Health,2006,18(1):11-18.
[5]Stephens F J,Raidal S R,Jones B.Haematopoietic necrosis in a goldfish (Carassius auratus) associated with an agent morphologically similar to herpesvirus[J].Australian Veterinary Journal,2004,82(3):167-169.
[6]Luo Y Z,Lin L,Liu Y,et al.Haematopoietic necrosis of cultured Silver prussian carp(Carassius gibelio)associated with cyprinid herpesvirus[J].Journal of Fish Diseases,2013,36(12):1035-1039.
[7]Xu J,Zeng L,Zhang H,et al.Cyprinid herpesvirus 2 infection emerged in cultured gibel carp,Carassius auratus gibelio,in China[J].Veterinary Microbiology,2013,166(1/2):138-144.
[8]Chang P H,Lee S H,Chiang H C,et al.Epizootic of herpes-like virus infection in goldfish,Carassius auratus in Taiwan[J].Fish Pathology,1999,34(4):209-210.
[9]Jung S J,Miyazaki T.Herpesviral haematopoietic necrosis of goldfish,Carassius auratus (L.)[J].Journal of Fish Diseases,1995,18(3):211-220.
[10]Jeffery K R,Bateman K,Bayley A,et al.Isolation of a cyprinid herpesvirus 2 from goldfish,Carassius auratus (L.),in the UK[J].Journal of Fish Diseases,2007,30(11):649-656.
[11]Wang L,He J,Liang L,et al.Mass mortality caused by cyprinid herpesvirus 2 (CyHV-2) in prussian carp (Carassius gibelio) in China[J].Bulletin of the European Association of Fish Pathologists,2012,32(5):164-173.
[12]李莉娟,罗杨志,刘学芹,等.金鱼鲤疱疹病毒Ⅱ型的分子诊断[J].华中农业大学学报,2013,32(1):92-96.
[13]Jung S J,Miyazaki T.Herpesviral haematopoietic necrosis of goldfish,Carassius auratus (L.)[J].Journal of Fish Diseases,2010,18(3):211-220.
[14]薛中仪,朱春艳,王瑶,等.一种鲤科疱疹病毒Ⅱ型的PCR快速检测方法[J].水产养殖,2015,36(7):45-48.
[15]彭俊杰,张琪,贾路路,等.鲤疱疹病毒Ⅱ型(CyHV-2)ORF25蛋白的原核表达及单克隆抗体的制备[J].华中农业大学学报,2017,36(2):96-101.
[16]李振言.鱼类细菌性败血症及其防治技术[J].科学养鱼,2004(1):46-46.
[17]张书俊,杨先乐,李聃,等.施氏鲟水霉病病原的初步研究[J].中国水产科学,2009,16(1):89-96.
[18]徐海圣,王淑霞,吴惠仙,等.异育银鲫寄生黏孢子虫病的研究[J].浙江大学学报(理学版),1997(3):259-264.
[19]Ma J,Jiang N,Lapatra S E,et al.Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (CyHV-2)[J].Veterinary Microbiology,2015,177(3/4):315-325.
[20]Zhang H,Zeng L B,Fan Y D,et al.A Loop-Mediated isothermal amplification assay for rapid detection of cyprinid herpesvirus 2 in gibel carp (Carassius auratus gibelio)[J].The Scientific World Journal,2014(1):1-6.
[21]郝中香,林华,佘容,等.鲤疱疹病毒2型微滴式数字PCR快速检测方法的建立[J].中国兽医科学,2016(2):167-173.
[22]罗丹,梁利国,谢骏,等.2型鲤疱疹病毒双重PCR快速检测方法的建立及应用[J].中国预防兽医学报,2014,36(5):379-382.
[23]于力,王津津,史秀杰,等.鲤科疱疹病毒2型Taqman实时荧光PCR方法的建立[J].中国动物检疫,2015(10):80-84.
[24]周勇,曾令兵,张辉,等.鲤疱疹病毒Ⅱ型TaqMan real-time PCR检测方法的建立及应用[J].水产学报,2013,37(4):607-613.
[25]Ma J,Jiang N,Lapatra S E,et al.Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (CyHV-2)[J].Veterinary Microbiology,2015,177(3/4):315-325.
[26]乔宁,李美芹,刘永光,等.番茄黄化曲叶病毒CP基因的原核表达载体构建选择[J].安徽农业科学,2012(7):3901-3902,3905.
[27]王世会,贾智英,李池陶,等.锦鲤疱疹病毒ORF72基因克隆及结构功能分析[J].水产学杂志,2016,29(3):9-15.