蓝舌病病毒10质粒反向遗传操作系统的建立
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摘要
为建立蓝舌病病毒(BTV) 10质粒反向遗传操作系统,本研究将BTV16型BN96/16株10个基因节段分别克隆至pS111载体相应的酶切位点,构建10个重组质粒转染BSR细胞,拯救获得重组病毒,命名为rBTV-16。测序结果显示,拯救病毒rBTV-16与其亲本病毒wtBTV-16的核苷酸序列完全相同;经电镜观察,可见典型形态的环状病毒属病毒粒子;间接免疫荧光试验结果显示,rBTV-16能够感染细胞;病毒基因组检测结果显示,rBTV-16各个基因节段的长度与亲本病毒完全一致;病毒生长曲线的测定结果显示,rBTV-16与亲本病毒具有一致的复制特性。本研究建立的10质粒系统比传统体外转录拯救系统的操作过程简便,提高了拯救病毒的效率,为进一步深入研究BTV的致病机理奠定了基础。
To establish an entirely plasmid-based reverse genetics system for Bluetongue virus(BTV), ten reverse genetics plasmids were constructed by 10 segments of BTV-16 BN96/16 strain genome cloning into vector pS111. The recombinant BTV-16(rBTV-16) was successfully rescued from BSR cells after transfecting with the 10 plasmids. The nucleotide sequences of rBTV-16 were identical to wt BTV-16, which was confirmed by sequencing the respective genome segments and the typical Orbivirus virions were observed by electron microscopy. The cells were infected with rBTV-16 confirmed by indirect immunofluorescent assay. The genome dsRNA was extracted and analyzed by non-denaturing polyacrylamide gel electrophoresis.The growth curves indicated that rBTV-16 had similar replication characteristics to its parental virus of wtBTV-16. The 10 plasmid-based system greatly simplifies the complex progress of in vitro transcription system, and also improves the efficiency of virus rescue, which provides a promising technique support for further study on the pathogenic of BTV.
To establish an entirely plasmid-based reverse genetics system for Bluetongue virus(BTV), ten reverse genetics plasmids were constructed by 10 segments of BTV-16 BN96/16 strain genome cloning into vector pS111. The recombinant BTV-16(rBTV-16) was successfully rescued from BSR cells after transfecting with the 10 plasmids. The nucleotide sequences of rBTV-16 were identical to wt BTV-16, which was confirmed by sequencing the respective genome segments and the typical Orbivirus virions were observed by electron microscopy. The cells were infected with rBTV-16 confirmed by indirect immunofluorescent assay. The genome dsRNA was extracted and analyzed by non-denaturing polyacrylamide gel electrophoresis.The growth curves indicated that rBTV-16 had similar replication characteristics to its parental virus of wtBTV-16. The 10 plasmid-based system greatly simplifies the complex progress of in vitro transcription system, and also improves the efficiency of virus rescue, which provides a promising technique support for further study on the pathogenic of BTV.
引文
[1]Roy P.Bluetongue virus structure and assembly[J].Curr Opin Virol,2017,24:115-123.
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[5]Maan S,Rao S,Maan N S,et al.Rapid cD NA synthesis and sequencing techniques for the genetic study of bluetongue and other dsR NA viruses[J].J Virol Methods,2007,143:132-139.
[6]Boyce M,Celma C C P,Roy P.Development of reverse genetics systems for bluetongue virus:Recovery of infectious virus from synthetic RNA transcripts[J].J Virol,2008,82(17):8339-8348.
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[8]杨涛,徐青元,孙恩成,等.蓝舌病病毒反向遗传操作系统的建立[J].中国预防兽医学报,2014,36(2):85-89.
[9]Yang Tao,Zhang Ji-kai,Xu Qing-yuan,et al.Development of a reverse genetics system for epizootic hemor rhagic disease virus and evaluation of novel strains containing duplicative gene rearrangements[J].J Gen Virol,2015,96:2714-2720.
[10]Cristina C C,Meredith S,Kerstin W,et al.Replication-deficient particles:New insights into the next generation of bluetongue virus vaccines[J].J Virol,2017,91(1):e01892-16.
[2]Pretorius J M,Huismans H,Theron J.Establishment of an entirely plasmid-based reverse genetics system for Bluetongue virus[J].Virology,2015,486:71-77.
[3]Qin Yong-li,Sun En-cheng,Liu Ni-hong,et al.Identification of a linear B-cell epitope within the Bluetongue virus serotype 8NS2 protein using a phage-displayed random peptide library[J].Vet Immunol Immunop,2013,154:93-101.
[4]Attoui H,Billoir F,Cantaloube J F,et al.Strategies for the sequence determination of viral dsR NA genomes[J].J Virol Methods,2000,89:147-158.
[5]Maan S,Rao S,Maan N S,et al.Rapid cD NA synthesis and sequencing techniques for the genetic study of bluetongue and other dsR NA viruses[J].J Virol Methods,2007,143:132-139.
[6]Boyce M,Celma C C P,Roy P.Development of reverse genetics systems for bluetongue virus:Recovery of infectious virus from synthetic RNA transcripts[J].J Virol,2008,82(17):8339-8348.
[7]Kaname Y,Celma C C,Kanai Y,et al.Recovery of African horse sickness virus from synthetic RNA[J].J Gen Virol,2013,94:2259-2265.
[8]杨涛,徐青元,孙恩成,等.蓝舌病病毒反向遗传操作系统的建立[J].中国预防兽医学报,2014,36(2):85-89.
[9]Yang Tao,Zhang Ji-kai,Xu Qing-yuan,et al.Development of a reverse genetics system for epizootic hemor rhagic disease virus and evaluation of novel strains containing duplicative gene rearrangements[J].J Gen Virol,2015,96:2714-2720.
[10]Cristina C C,Meredith S,Kerstin W,et al.Replication-deficient particles:New insights into the next generation of bluetongue virus vaccines[J].J Virol,2017,91(1):e01892-16.