高致病性猪繁殖与呼吸综合征病毒湖南株全长感染性cDNA克隆的构建
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摘要
猪繁殖与呼吸综合征(PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种主要以母猪繁殖障碍和仔猪呼吸道疾病为特征的传染病,给养猪业带来了巨大的经济损失。本研究以2006年夏季以来我国南方部分地区陆续发生的高致病性PRRS疫情为背景,用流行的PRRSV湖南株为亲本毒株构建全长感染性cDNA克隆,为实现在DNA水平上对PRRSV基因组进行人工操作和研究PRRSV的复制和致病机理、基因功能等提供技术手段,并为构建新型病毒载体和发展安全有效的疫苗搭建平台。
根据已发表的高致病性PRRSV毒株全基因组序列及酶切位点分布,将病毒基因组分9段设计特异性引物,预期扩增覆盖全基因组的重叠cDNA片段为173、174、175、176、177、177p、178、179和215,其大小分别为486bp、2437bp、2059bp、2718bp、2406bp、1450bp、3306bp、2249bp和645bp;同时在序列5'端起始位点引入T7 RNA聚合酶启动子序列和3'端引入含30个A的poly(A)尾及单一线性化酶切位点。首先通过优化的RT-PCR反应体系扩增出目的cDNA片段,并将它们分别克隆入pMD18-T载体进行序列测定和比对分析,获取序列完全正确的cDNA克隆;然后将9个cDNA片段按构建策略进行亚克隆,在2个高拷贝质粒载体pMCS1.1和pMCS2.1的基础上构建4个高拷贝重组质粒A、B1N、B2、C,并将A、B1N、B2、C依次亚克隆到低拷贝质粒pAC1.1中,最终获得含全长cDNA克隆的重组质粒pAC-HPPRRS-F。
按低拷贝质粒提取方法大量制备含全长cDNA克隆的重组质粒pAC-HPPRRS-F,随机选取病毒基因组中的5组酶切位点,用限制性内切酶进行酶切鉴定。然后将pAC-HPPRRS-F用MluⅠ线性化后作为模板进行RNA体外转录,经脂质体Lipofectin Reagent转染Marc-145细胞以获得体外拯救病毒。转染48h后取细胞上清接种Marc-145细胞进行传代培养,盲传至第3代时产生了明显的细胞病变,经RT-PCR和免疫组织化学染色检测证实获得了PRRSV,结果表明该研究构初步建出了PRRSV湖南株全长感染性cDNA克隆。
Porcine reproductive and respiratory syndrome virus(PRRSV) is the causative agent of PRRS.It causes an important disease in pigs characterized by reproductive failure in sows and gilts and respiratory dieases in piglets,resulting in great economic losses to the swine industry.This thesis describes the construction of a full-length cDNA clone of high pathogenic PRRSV HuNan strain from the tissue of infectious porcine suffering swine high fever syndrome in the pig farm of HuNan province,during the unparalleled large-scale outbreak of swine high fever syndrome late in 2006.The availability of such cDNA clones offers an opportunity for analysis and modification of viral genomes of PRRSV at the molecular level and has greatly aided research on virus replication,pathogenesis,and function of gene.The full-length cDNA clone of HuNan strain provided fundamental materials for the development of novel viral vector,as well as for the development of a safe and effective vaccine for pigs in future.
Nine sets of primers were designed according to the full-length genomic sequence of published high pathogenic PRRSV strain.The T7 RNA polymerase promoter was introduced immediately upstream of 5'-terminal,and the ploy(A) with 30 As and a single restriction enzyme site were introduced immediately 3'-terminal of the full-length genomic sequence.By using the RT-PCR nine overlapping cDNA fragments covering the complete genome were amplified and were cloned into pMD18-T vector.These cDNA fragments 173,174,175,176,177,177p,178,179and 215 with a size of 486bp,2437bp,2059bp,2718bp,2406bp,1450bp,3306bp,2249bp,and 645bp respectively were determined by sequence and analysis.Four recombinant high-copy-number plasmids were constructed with nine cDNA fragments subcloned into two plasmid vectors pMCS1.1 or pMCS2.1,modified from plasmid pGEM-T using artifical multiple cloning sites(MCS).Finally,the full-length cDNA clone, pAC-HPPRRS-F of PRRSV HuNan strain were obtained when these fragments were transferred into a low-copy-number plasmid vector pAC1.1.
The recombinant plasmid pAC-HPPRRS-F were cultured and purified according to the protocol of the low-copy-number plasmid.The plasmid DNA was digested by five groups of restriction endonuclease which were selected randomly on the restriction enzyme sites of the genomic sequence of PRRSV Hunan strain.The pAC-HPPRRS-F was linearized by cleavage with MluⅠ,and used for RNA preparation in vitro with Transcription Kit.Then Marc-145 cells were transfectd with in vitro-transcribed RNA using Lipofectin Reagent.Supernatants from cells at 48h posttransfection were serially passaged on Marc-145 cells.Obvious cytopathic effect was observed in the third passage.The infectious rescue viruses were obtained by RT-PCR and immunohistochemical detection with monoclonal antibody against N protein of PRRSV.These results show that the full-length infectious cDNA clone for HuNan Strain of high pathogenic PRRSV was constructed successfully.
根据已发表的高致病性PRRSV毒株全基因组序列及酶切位点分布,将病毒基因组分9段设计特异性引物,预期扩增覆盖全基因组的重叠cDNA片段为173、174、175、176、177、177p、178、179和215,其大小分别为486bp、2437bp、2059bp、2718bp、2406bp、1450bp、3306bp、2249bp和645bp;同时在序列5'端起始位点引入T7 RNA聚合酶启动子序列和3'端引入含30个A的poly(A)尾及单一线性化酶切位点。首先通过优化的RT-PCR反应体系扩增出目的cDNA片段,并将它们分别克隆入pMD18-T载体进行序列测定和比对分析,获取序列完全正确的cDNA克隆;然后将9个cDNA片段按构建策略进行亚克隆,在2个高拷贝质粒载体pMCS1.1和pMCS2.1的基础上构建4个高拷贝重组质粒A、B1N、B2、C,并将A、B1N、B2、C依次亚克隆到低拷贝质粒pAC1.1中,最终获得含全长cDNA克隆的重组质粒pAC-HPPRRS-F。
按低拷贝质粒提取方法大量制备含全长cDNA克隆的重组质粒pAC-HPPRRS-F,随机选取病毒基因组中的5组酶切位点,用限制性内切酶进行酶切鉴定。然后将pAC-HPPRRS-F用MluⅠ线性化后作为模板进行RNA体外转录,经脂质体Lipofectin Reagent转染Marc-145细胞以获得体外拯救病毒。转染48h后取细胞上清接种Marc-145细胞进行传代培养,盲传至第3代时产生了明显的细胞病变,经RT-PCR和免疫组织化学染色检测证实获得了PRRSV,结果表明该研究构初步建出了PRRSV湖南株全长感染性cDNA克隆。
Porcine reproductive and respiratory syndrome virus(PRRSV) is the causative agent of PRRS.It causes an important disease in pigs characterized by reproductive failure in sows and gilts and respiratory dieases in piglets,resulting in great economic losses to the swine industry.This thesis describes the construction of a full-length cDNA clone of high pathogenic PRRSV HuNan strain from the tissue of infectious porcine suffering swine high fever syndrome in the pig farm of HuNan province,during the unparalleled large-scale outbreak of swine high fever syndrome late in 2006.The availability of such cDNA clones offers an opportunity for analysis and modification of viral genomes of PRRSV at the molecular level and has greatly aided research on virus replication,pathogenesis,and function of gene.The full-length cDNA clone of HuNan strain provided fundamental materials for the development of novel viral vector,as well as for the development of a safe and effective vaccine for pigs in future.
Nine sets of primers were designed according to the full-length genomic sequence of published high pathogenic PRRSV strain.The T7 RNA polymerase promoter was introduced immediately upstream of 5'-terminal,and the ploy(A) with 30 As and a single restriction enzyme site were introduced immediately 3'-terminal of the full-length genomic sequence.By using the RT-PCR nine overlapping cDNA fragments covering the complete genome were amplified and were cloned into pMD18-T vector.These cDNA fragments 173,174,175,176,177,177p,178,179and 215 with a size of 486bp,2437bp,2059bp,2718bp,2406bp,1450bp,3306bp,2249bp,and 645bp respectively were determined by sequence and analysis.Four recombinant high-copy-number plasmids were constructed with nine cDNA fragments subcloned into two plasmid vectors pMCS1.1 or pMCS2.1,modified from plasmid pGEM-T using artifical multiple cloning sites(MCS).Finally,the full-length cDNA clone, pAC-HPPRRS-F of PRRSV HuNan strain were obtained when these fragments were transferred into a low-copy-number plasmid vector pAC1.1.
The recombinant plasmid pAC-HPPRRS-F were cultured and purified according to the protocol of the low-copy-number plasmid.The plasmid DNA was digested by five groups of restriction endonuclease which were selected randomly on the restriction enzyme sites of the genomic sequence of PRRSV Hunan strain.The pAC-HPPRRS-F was linearized by cleavage with MluⅠ,and used for RNA preparation in vitro with Transcription Kit.Then Marc-145 cells were transfectd with in vitro-transcribed RNA using Lipofectin Reagent.Supernatants from cells at 48h posttransfection were serially passaged on Marc-145 cells.Obvious cytopathic effect was observed in the third passage.The infectious rescue viruses were obtained by RT-PCR and immunohistochemical detection with monoclonal antibody against N protein of PRRSV.These results show that the full-length infectious cDNA clone for HuNan Strain of high pathogenic PRRSV was constructed successfully.
引文
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