高致病性猪繁殖与呼吸综合征病毒感染性克隆的构建

吕健; 孙志; 张建武; 刘维全; 袁世山

微生物与感染 ›› 2007, Vol. 2 ›› Issue (4) : 219-227.

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微生物与感染 ›› 2007, Vol. 2 ›› Issue (4) : 219-227.
论著

高致病性猪繁殖与呼吸综合征病毒感染性克隆的构建

  • 吕健; 孙志; 张建武; 刘维全; 袁世山
作者信息 +

ruction of a full-length infectious clone of PRRSV from

  • LV Jian; SUN Zhi; ZHANG Jian-wu; LIU Wei-quan; YUAN Shi-shan
Author information +
文章历史 +

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。

Abstract

Objective To determine if 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 eDNA clone and to On insight into the structure and function of the PRRSV genome and the mechanism of it's pathology, which will ultimately provide a is 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 T7 promoter was introduced immediately upstream of the 5'-end.A molecular marker, Mlu I restriction enzyme site, was introduced in the ORFS coding region. This process resulted in the successful construction of two infectious 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 vines 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

感染性克隆 / 高致病性 / 动物回归试验

Key words

Porcine reproductive and respiratory syndrome virus / Full length cDNA clone / High-pathogencity / Regression text

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吕健; 孙志; 张建武; 刘维全; 袁世山. 高致病性猪繁殖与呼吸综合征病毒感染性克隆的构建
[J]. 微生物与感染. 2007, 2(4): 219-227
LV Jian; SUN Zhi; ZHANG Jian-wu; LIU Wei-quan; YUAN Shi-shan. ruction of a full-length infectious clone of PRRSV from[J]. Journal of Microbes and Infections. 2007, 2(4): 219-227

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