【作者】 陈小云；

【导师】 杨汉春；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2004， 博士

【摘要】 猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus,PRRSV）引起的一种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。本研究利用反向遗传学操作技术，构建了PRRSV BJ-4株的全长cDNA克隆，并对全长cDNA克隆中存在的23个突变位点进行了回复突变，继而对突变后的全长cDNA克隆体外转录体的感染性进行了研究。为将来实现在DNA水平上对PRRSV基因组的人工操作，从而在分子水平上研究PRRSV的复制和致病机理、基因产物的功能等提供技术手段，并为构建新型的病毒载体和发展安全有效的活病毒疫苗奠定基础。 依据PRRSV BJ-4株全基因组序列，分6段设计引物，覆盖PRRSV基因组的cDNA重叠片段A1、A2、B1、B2、C和D的预期大小分别为1,294bp、3,452bp、3,376bp、1,823bp、3,484bp和2,887bp。通过优化RT-PCR反应条件，最终扩增出其全基因组cDNA片段。并将它们分别克隆入pGEM-T Easy载体或pBluescript SK+载体。然后将获得的6个克隆依次连接并克隆到低拷贝质粒载体pWSK29中，获得该毒株全长cDNA克隆pWSK DCBA。在全长cDNA克隆的两端和中间引入了病毒序列中不存在的单酶切位点，并在序列的5′端引入了SP6 RNA聚合酶启动子序列以及一个外源的“G”，在3′端引入了poly（T）₈₃序列。 对扩增出的覆盖PRRSV全基因组cDNA的6个片段分别进行序列测定，并与其原始病毒BJ-4株以及其它GenBank中已有的PRRSV序列进行比对，发现其中存在23个碱基的变异。用定向点突变方法，对这些碱基进行了回复突变，得到包含PRRSV各cDNA重叠片段的6个克隆，经序列测定证实，这6个克隆在成功恢复23个变异碱基的同时，未引入新的突变。将它们依次连接并克隆到低拷贝质粒载体pWSK29中，获得了该毒株定点突变后的全长cDNA克隆P1P2mP3P4-63。 将病毒的全长cDNA克隆P1P2mP3P4-63大量培养后，按低拷贝质粒提取方法提取质粒，用限制性内切酶Sph Ⅰ酶切线性化后，作为体外转录模板。转录出的RNA用DNase Ⅰ消化模板质粒DNA，再经脂质体DMRIE-C转染BHK-21细胞，转染后24h的培养上清接种Marc-145细胞后，连续培养3代，在传至第2代时能产生典型的细胞病变。经RT-PCR和间接免疫荧光鉴定，证实获得了有感染性的恢复病毒，表明成功地构建出了具有感染性的PRRSV全长cDNA克隆。更多还原

【Abstract】 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. The present article describes the establishment of an infectious full-length cDNA clone of PRRSV BJ-4 by utilizing reverse genetic manipulation technology. Reverse mutation of 23-nucleoitide mutations within the full-length cDNA clone were achieved. Infectious of the in vitro transcripts from the full-length cDNA clone after mutation were studied. 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 product. The infectious clone of BJ-4 provided fundamental materials for the development of novel viral vector, as well as for the development of a safe and effective live vaccine for use in pigs.Six sets of primers were designed according to the full-length genomic sequence of PRRSV BJ-4. By using the RT-PCR, 6 verlapping cDNA fragments covering the complete genome were amplified. These cDNA overlapping fragments Al, A2, Bl, B2, C, D with a size of 1,294 bp, 3,452 bp, 3,376 bp, 1,823 bp, 3,484 bp and 2,887 bp were cloned into pGEM-T Easy vector or pBluescript SK+ vector. Then the full-length cDNA clone pWSK DCBA of PRRSV BJ-4 were obtained when the 6 fragments were ligated in order and cloned into low-copy-number vector pWSK29. Unique restriction sites, which were absent from the viral sequence, were introduced at both end and within the full-length cDNA clone. At the 5’ end, the transcription initiation sequence of the SP6 RNA polymerase and a nonviral G were introducd. A poly(T)g3 sequence was introduced to the 3’ end of the cDNA clone.Six overlapping cDNA fragments covering the complete genome of PRRSV BJ-4 were sequenced, and compared with its parental virus and all other PRRSV sequence within the GenBank. All together 23-nucleoitide mutations were founded. Reverse mutation of these nucleoitide were achieved by using site-directed mutagenesis kit. Successful reverse of these 23-nucleoitide mutations was confirmed by sequencing. Sequencing analysis also showed that no other mutation was introduced into the six de novo clones. The post-mutated full-length cDNA clone PlP2mP3P4-63 of PRRSV BJ-4 were obtained whlie the 6 fragments were ligated in order and cloned into low-copy-number vector pWSK29.Full-length cDNA clone PlP2mP3P4-63 was cultured in mass volume, and plasmid DNA was purified according to the protocol of very low-copy-number plasmid. The plasmid DNA was linearized by cleavage with Vspl, then the linearized plasmids was used for in vitro transcription. Template DNA was digested by DNase I , then, BHK-21 cells were transfectd with in vrtro-transcribed RNA using transfection reagent DMRIE-C. Supernatants from cells at 24 h posttransfection were serially passaged on Marc-145 cells for three passages. Obvious cytopathiceffect was observed in the second passage. The infectious rescue viruses were obtained by RT-PCR and an indirect immuno fluorescence assay with the monoclonal antibody against N protein of PRRSV.更多还原