禽传染性支气管炎病毒H52反向遗传株的构建
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摘要
IBV H52基因组全长27.6kb,分13个1.3~2.9kb的片段扩增H52全长cDNA,额外扩增N-3′片段,并克隆到载体PMD19-T上.利用No see′m技术在每个片段中引入BsaⅠ或BsmBⅠ酶切位点,D1片段中引入沉默突变位点,在5′UTR端和N-3′引入T7启动子,3′UTR端引入Poly(A)尾.将克隆好的13个片段酶切,纯化,体外无缝连接为全长cDNA.将全长cDNA及N-3′体外转录获得它们的转录体,将转录体共转染BHK-21细胞,48h后收获细胞液,将细胞液接种10日龄SPF鸡胚获得反向遗传株H52-R.在鸡胚中盲传5代后,通过RT-PCR对无义突变位点进行扩增及序列分析,在反向株H52-R中发现引入的无义突变位点.对反向株H52-R的一些生物学特性如HA、EID50等测定发现,H52-R的这些生物学特性与亲本株H52相似.
The full-length genomic cDNA was obtained by ligating thirteen 1.3~2.9kb fragments contiguously spanning the virus genome based on"No see'm"ligation strategy and the T7 promoter was incorporated to the 5′UTR and N′3fragments.Consequently,the cDNA and N′3fragment was transcribed and transfected into BHK-21 cells by electroporation to rescue the genetic modified virus.48 hpost transfection,the cultures of transfected BHK-21 cells was harvested and inoculated into 10 days old SPF embryonated eggs(ECE)to replicate the rescued virus.After five passages,the silentmutation site which was introduced artificially in the tenth(D1)fragment of the rescued virus(H52-R)was detectable,revealed that the H52-R virus was successfully rescued.The EID50 and HA titers of H52-R Were characterized.It was confirmed that they were similar to its parent strain H52.
The full-length genomic cDNA was obtained by ligating thirteen 1.3~2.9kb fragments contiguously spanning the virus genome based on"No see'm"ligation strategy and the T7 promoter was incorporated to the 5′UTR and N′3fragments.Consequently,the cDNA and N′3fragment was transcribed and transfected into BHK-21 cells by electroporation to rescue the genetic modified virus.48 hpost transfection,the cultures of transfected BHK-21 cells was harvested and inoculated into 10 days old SPF embryonated eggs(ECE)to replicate the rescued virus.After five passages,the silentmutation site which was introduced artificially in the tenth(D1)fragment of the rescued virus(H52-R)was detectable,revealed that the H52-R virus was successfully rescued.The EID50 and HA titers of H52-R Were characterized.It was confirmed that they were similar to its parent strain H52.
引文
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[14]Yount B,Denison M R,Weiss S R.Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59[J].Jouranl of Virology,2000,76:11065.
[15]Yount B,Curtis K M,Baric R S.Strategy for systematic assembly of large RNA and DNA genomes:transmissible gastroenteritis virus model[J].Journal of Virology,2000,74:10600.
[16]Hughes L A,Savage C,Naylor C.Genetically diverse cornaviruse in wild bird populations of northern England[J].Emerging infectious diseases,2009,15:1091.
[17]Armesto M,Evans S,Cavanagh D,et al.A recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the 4/91serotype[J].PloS one,2011,6:e24352.
[18]Bentley K,Armesto M,Britton P.Infectious bronchitis virus as a vector for the expression of heterologous genes[J].PolS one,2013,8:e67875.
[2]王红宁.禽传染性支气管炎综合防治[M].北京:中国农业科技出版社,2000.
[3]Tidona C,Darai G.The Springer Index of Viruses[M].2ed Edition.Berlin:Springer Verlag,2011:403.
[4]Cavanagh D,Coronavirus avian infectious bronchitis virus[J].Vet Res,2007,38:281.
[5]Yount B,Curtis K M,Fritz E A.Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus[J].Proc Natl Acad Sci U S A,2003,100:12995.
[6]Casais R,Thiel V,Siddell S G.Reverse genetics system for the avian coronavirus infectious bronchitis virus[J].J Virol,2001,75:12359.
[7]Casais R,Dove B,Cavanagh D.Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism[J].J Virol,2003,77:9084.
[8]Youn S,Leibowitz J L,Collisson E W.In vitro assembled recombinat infectious bronchitis viruses demonstrate that the 5aopen reading frame is not essential for replication[J].Virology,2005,332:206.
[9]Hodgson T,Britton P,Cavanagh D.Neither the RNA nor the proteins of open reading frames 3aand3bof the coronavirus infectious bronchitis virus are essential for replication[J].J Virol,80,296.
[10]Fang S,Chen B,Tay F P L,et al.An arginine-toproline mutation in a domain with undefined functions within the helicase protein(Nsp13)is lethal to the coronavirus infectious bronchitis virus in cultured cells[J].Virology,2007,358:136.
[11]Geilhausen H,Ligon F,Lukert P.The pathogenesis of virulent and avirulent avian infectious bronchitis virus[J].Arch Virol,1973,40:285.
[12]Bijlenga G,Cook J K A,Gelb Jr,et al.Development and use of the H strain of avian infectious bronchitis virus from the Netherlands as a vaccine:a review[J].Avian Pathol,2004,33:550.
[13]Zhou Y S,Zhang Y.Establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain H120[J].Veterinary Microbiology,2013,162:53.
[14]Yount B,Denison M R,Weiss S R.Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59[J].Jouranl of Virology,2000,76:11065.
[15]Yount B,Curtis K M,Baric R S.Strategy for systematic assembly of large RNA and DNA genomes:transmissible gastroenteritis virus model[J].Journal of Virology,2000,74:10600.
[16]Hughes L A,Savage C,Naylor C.Genetically diverse cornaviruse in wild bird populations of northern England[J].Emerging infectious diseases,2009,15:1091.
[17]Armesto M,Evans S,Cavanagh D,et al.A recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the 4/91serotype[J].PloS one,2011,6:e24352.
[18]Bentley K,Armesto M,Britton P.Infectious bronchitis virus as a vector for the expression of heterologous genes[J].PolS one,2013,8:e67875.