摘要
为构建高致病性猪繁殖与呼吸综合征病毒(PRRSV)感染性克隆,本实验根据 PRRSV HuN4 株基因组序列设计并合成 PRRSV 特异性引物,应用 RT-PCR 技术分6段扩增了 PRRS VHuN4 株全基因组 cDNA。将扩增的各个cDNA部分重叠片段分别克隆于 pBlueScriptⅡSK(+)载体中构建了感染性重组质粒 pHuN4。并在病毒 cDNA5'末端引入 sp6 启动子序列便于后期的体外转录获得病毒的转录本,在3'末段 Poly(A)尾引入 NotⅠ酶切位点用于线性化 pHuN4;此外,将 HuN4 基因组第14680位的A沉默突变为G产生一个 MluⅠ酶切位点作为鉴定拯救病毒的分子标记。pHuN4 通过酶切线性化后经体外转录及转染BHK细胞,并在 Marc-145 细胞中救获病毒。结果显示:救获的病毒能够在 Marc145 细胞引起明显的细胞病变;间接免疫荧光检测以及分子标记验证结果表明病毒拯救成功,而且拯救的病毒与亲本强毒生长曲线没有显著差异。利用拯救的第5代克隆病毒株对本动物进行致病性试验,结果显示实验猪在感染后第3d开始出现体温升高、厌食、消瘦等临床表现,发病率达100%,在感染后20d内陆续死亡,表明拯救的病毒保持了与亲本病毒株相一致的致病特征,以上研究证实我们成功的构建并获得 PRRSV 强毒 HuN4 株感染性克隆,为从基因水平上研究 PRRSV 的致病机制提供了技术平台。
In this study, a infectious cDNA clone of highly pathogenic porcine reproductive and respiratory syndrome virus （PRRSV） strain HuN4 was generated by cloning of the 6 overlapped RT-PCR fragments into pBlueScript Ⅱ SK（＋）, designated pHuN4. In addition, sp6 promoter at 5＇-terminus and a NotⅠsite in Poly（A） tail at 3＇-terminus were introduced into the cDNA of PRRSV for viral RNA transcription in vitro and the pHuN4 linearization, respectively. Furthmore, the A at 14,680 nt was replaced by G （silent mutation） to generate restriction enzyme MluⅠsite as molecular marker for identification of rescued virus. The pHuN4 was linearized and transcribed in vitro, the transcripts were transfected primarily into BHK cells and then Marc145 cells, the virus was successfully rescued from Marc145 cells identified by indirect immofluorescent assay and MluⅠdigestion. The rescued virus showed similar growth curve to its parental virus HuN4 strain. All the piglets infected with the fifth passage of the rescued virus developed clinical signs at 3 day-past-infection and died within 20 days. The results showed that the rescued virus remained similar biological characterizations as its parental virus. The pHuN4 developed in the study by reverse genetic system might provide a platform for further study on pathogenesis mechanism of highly pathogenic PRRSV.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2011年第7期497-502,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
NSFC-广联合基金项目(U0931003)
上海市科技人才计划项目(09XD1405400)
国际合作项目(2010DFB33920)
国家生猪现代产业技术体系建设项目(NYCYTX-009)
中央科研院所公益性基础科研业务费项目(2011JB03)
关键词
猪繁殖与呼吸综合征病毒
HuN4株
感染性分子克隆
porcine reproductive and respiratory syndrome virus HuN4 strain infectious cDNA clone
