高致病性猪繁殖与呼吸综合征病毒感染性克隆的构建  

Construction of a full-length infectious clone of PRRSV from"Porcine High Fever Syndrome"

作　　者：吕健[1] 孙志[1] 张建武[1] 刘维全[2] 袁世山[1] 

机构地区：[1]中国农业科学院上海兽医研究所动物传染病防治研究室农业部动物寄生虫病重点实验室,上海200232 [2]中国农业大学生物学院,北京100094

出　　处：《微生物与感染》2007年第4期219-227,共9页Journal of Microbes and Infections

基　　金：国家自然科学基金重点项目(No.30530580);上海市科技兴农重点攻关项目(2006B4);科技部重大基础研究"973"专项(2005CB523200)

摘　　要：目的确定"猪高热综合征"的主要病原是否为猪繁殖与呼吸综合征病毒(PRRSV),为探寻高致病性病毒基因组结构和功能、高致病性蓝耳病发病机制,以及进一步研制针对后者的基因工程疫苗奠定基础。方法根据PRRSV基因组序列设计特异性引物,分5段扩增了高致病性PRRSV JX143株的全长cDNA,将各cDNA片段克隆到pBlueScriptⅡSK(+)载体中,进而利用重叠区中的单酶切位点将其连接成JX143株的全长cDNA克隆pJX143和含有遗传标记MluⅠ限制性内切酶的基因组全长cDNA克隆pJX143-M。结果全长cDNA克隆经体外合成RNA后转染MA-104细胞,均获得稳定的拯救病毒。通过比较亲本病毒与拯救病毒的病毒滴度、多步生长曲线和利用拯救病毒vJX143进行动物回归试验,结果发现病毒学及生物学特性没有显著差异。结论PRRSV的JX143株病毒确实是引起"猪高热综合征"的主要病原;建立了首个高致病性PRRSV的感染性cDNA克隆,证明拯救病毒与亲本病毒生物学特性相同。Objective To determine ff the JX143 strain of the porcine reproductive and respiratory syndrome virus （PRRSV） is the etiological agent of the ＂Porcine High Fever Syndrome＂ outbreak in China. We also sought to test the genetic stability of the cDNA clone and to gain insight into the structure and function of the PRRSV genome and the mechanism of it＇ s pathology, which will ultimately provide a basis for vaccine development using genetic technologies. Methods Specific primers were designed according to the PRRSV genome. The five cDNA fragments covering the PRRSV genome were amplified and cloned into pBlueScript ⅡSK （ ＋ ） vector.A 37 promoter was introduced immediately upstream of the 5＇-end. A molecular marker, Mlu Ⅰ restriction enzyme site, was introduced in the ORF5 coding region. This process resulted in the successful construction of two infections clones.Results Full-length genomic clones of PRRSV were rescued successfully after transfection into MA104 cells. We then compared the growth properties of the rescued virus and the parental virus using a multi-step growth curve.Animal experiments were executed by inoculating the rescued virus into pigs.All results indicated that there was no difference between the parental and rescued viruses in either their virological and biological properties. Conclusion From this study, we concluded that the etiological agent of the ＂Porcine High Fever Syndrome＂ outbreak in China is, in fact, a PRRSV, such as the JX143 strain. This is the first report showing a high virulence virus infectious clone of PRRSV in China.

关 键 词：猪繁殖与呼吸综合征病毒 感染性克隆 高致病性 动物回归试验 

分 类 号：S858.28[农业科学—临床兽医学]