PCV2 Rep蛋白N-糖基化位点突变对病毒复制的影响
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摘要
N-糖基化对病毒的感染与增殖均具有重要作用,与PCV2复制相关的Rep蛋白含有三个N-糖基化位点。为了分析PCV2Rep蛋白N-糖基化位点突变对病毒复制的影响,本试验构建了三个双拷贝突变体感染性克隆2M23、2M256、2M286,并成功拯救病毒。通过间接免疫荧光检测病毒的拯救效果,TCID50测定病毒的感染力,荧光定量PCR检测细胞病毒的载量。结果显示,PCV2Rep蛋白的23~25aa、256~258aa N-糖基化位点突变后降低病毒的复制能力,而286~288aa突变后增强病毒的复制能力,为进一步阐明PCV2的复制及致病机制提供参考。
N-glycosylation site is critical for the infection and proliferation of most virus.The Rep protein of PCV2 involved in virus replication contains three N-glycosylation sites.In order to analyze the PCV2 Rep protein N-glycosylation site mutation effect on virus replication,three double copy mutant infectious clone 2M23,2M256,2M286 were constructed and successfully rescued virus.Immune flurescent assay(IFA)、TCID50and fluorescent quantitative RT-PCR to detect the virus rescue,the infectivity of virus and the virus load of cell.The results showed that mutation of the N-glycosylation site in the PCV2 Rep protein in 23~25aa,256~258aa reduced virus replication and in 286~288aa enhanced virus replication.This study arises a reference for the further elucidation of the mechanism of PCV2 replication and pathogenesis.
N-glycosylation site is critical for the infection and proliferation of most virus.The Rep protein of PCV2 involved in virus replication contains three N-glycosylation sites.In order to analyze the PCV2 Rep protein N-glycosylation site mutation effect on virus replication,three double copy mutant infectious clone 2M23,2M256,2M286 were constructed and successfully rescued virus.Immune flurescent assay(IFA)、TCID50and fluorescent quantitative RT-PCR to detect the virus rescue,the infectivity of virus and the virus load of cell.The results showed that mutation of the N-glycosylation site in the PCV2 Rep protein in 23~25aa,256~258aa reduced virus replication and in 286~288aa enhanced virus replication.This study arises a reference for the further elucidation of the mechanism of PCV2 replication and pathogenesis.
引文
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[2]Joaquim Segalés,Carles Rosell,Mariano Domingo,et al.Pathological findings associated with naturally acquired porcine circovirus type 2associated disease[J].Veterinary Microbiology,2004,98(2):137-149.
[3]Liu J,Chen1,Kwang J,et al.Characterizations of a previously unidentified viral protein of porcine circovirus type2infected cells and its role in virusinduced apoptosis[J].J.Virol,2005(79):8262-8274.
[4]Husa P,Chalupa P,Husova L,et al.The SEN virus-will there be another letter in the alphabet of viral hepatitis[J].Vnitr Lek,2002,48(8):763-766.
[5]Jinyan Gu,Ruibin Cao.Deletion of the single putative N-glycosylation site of the porcine circovirus type 2Cap Protein enhances specific immune responses by DNA immunisation in mice[J].The Veterinary Journal,2012(192):385-389.
[6]Fenaux M,Opriessnig T,Halbur P G,et al.Cloned genomic DNA of type 2porcine circovirus is infectious when injected directly into the liver and lymph nodes of pigs:characterization of clinical disease,virus distribution,and pathologic lesions[J].J Virol,2002,76(2):541-551.
[7]王鹏,张熙艳,时建立,等.PCV2Rep蛋白N-糖基化位点突变及其真核表达载体的构建[J].中国兽医杂志,2014,50(1):24-26.
[8]李俊,时建立,于周,等.猪圆环病毒2型双拷贝感染性DNA的构建及体外拯救[J].生物工程学报,2009,25(11):1633-1 638.
[9]Li J,Shi J I,Wu X Y,et al.Differentiation of PCV1and PCV2by a multiplex real-time PCR assay[J].Vet Rec,2013,173(14):346.
[10]Opriessnig T,Meng X J,Halbur P G,et al.Porcine circoviruses type 2-associated disease:update on current terminology,clinical manifestations,pathogenesis,diagnosis,and intervention strategies[J].J Vet Diagn Investig,2007,19(6):591-615.
[11]Li W,Wang X,Ma T,et al.Genetic analysis of porcine circovirus type 2(PCV2)strains isolated between 2001and2009:genotype PCV2bpredominate in postweaning multisystemic wasting syndrome occurrences in eastern China[J].Virus Genes,2010,40(2):244-251.
[12]Gibbs M J,Weiller G F.Evidence that a plant virus switched hosts to infect a vertebrate and then recombined with a vertebrate-infecting virus[J].Proc.Natl.Acad.Sci.U.S.A.1999,96(14):8 022-8 027.
[13]Vigerust D J,Shepherd V L.Virus glycosylation:Role in virulence and immune interactions[J].Trends in Microbiology,2007,15(5):211-218.
[14]Dowling W,Thompson E,Badger C,et al.The influences of glycosylation on the antigenicity,immunogenicity,and protective efficacy of ebola virus GP DNA vaccine[J].Virol,2007,81(4):1 821-1 837.