[1]张善瑞,周艳君,朱建平,等.高致病性猪繁殖与呼吸综合征病毒HuN4株感染性分子克隆的建立及拯救病毒的鉴定[J].中国预防兽医学报,2011,(07):497-502.
 ZHANG Shan-rui,ZHOU Yan-jun,ZHU Jian-ping,et al.Generation of a infectious cDNA clone of highly pathogenic porcine reproductive and respiratory syndrome virus HuN4[J].Chinese journal of preventive veterinary medicine,2011,(07):497-502.
点击复制

高致病性猪繁殖与呼吸综合征病毒HuN4株感染性分子克隆的建立及拯救病毒的鉴定
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2011年07期
页码:
497-502
栏目:
病原生物学
出版日期:
2011-07-15

文章信息/Info

Title:
Generation of a infectious cDNA clone of highly pathogenic porcine reproductive and respiratory syndrome virus HuN4
作者:
张善瑞;周艳君;朱建平;童武;姜一峰;童光志;
中国农业科学院上海兽医研究所/猪传染病研究室;
Author(s):
ZHANG Shan-rui ZHOU Yan-jun ZHU Jian-ping TONG Wu JIANG Yi-feng TONG Guang-zhi
Division of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China
关键词:
猪繁殖与呼吸综合征病毒HuN4株感染性分子克隆
Keywords:
porcine reproductive and respiratory syndrome virus HuN4 strain infectious cDNA clone
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。
Abstract:
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

参考文献/References:

[1] 童光志,周艳君,郝晓芳,田志军,仇华吉,彭金美,安同庆. 高致病性猪繁殖与呼吸综合征病毒的分离鉴定及其分子流行病学分析[J]. 中国预防兽医学报. 2007年05期
[2] 周艳君,薛强,安同庆,仇华吉,童光志,王云峰,田志军,王玫,蔡雪辉. 猪繁殖与呼吸综合征病毒CH-1a株核衣壳蛋白单克隆抗体的制备[J]. 中国预防兽医学报. 2005年06期

相似文献/References:

[1]周艳君,田志军,刘金霞,等.猪繁殖与呼吸综合征病毒M蛋白B细胞抗原表位的鉴定[J].中国预防兽医学报,2009,(04):251.
 ZHOU Yan-jun,TIAN Zhi-jun,LIU Jin-xia,et al.Identification of a B cell epitope on matrix protein of porcine reproductive and respiratory syndrome virus[J].Chinese journal of preventive veterinary medicine,2009,(07):251.
[2]曹宗喜,潘全会,张原江,等.猪繁殖与呼吸综合征病毒各结构蛋白的融合表达[J].中国预防兽医学报,2009,(02):85.
 CAO Zong-xi,PAN Quan-hui,ZHANG Yuan-jiang,et al.Fusion expression of structural proteins of porcine reproductive and respiratory syndrome virus[J].Chinese journal of preventive veterinary medicine,2009,(07):85.
[3]田志军,安同庆,周艳君,等.致弱过程中高致病性猪繁殖与呼吸综合征病毒HuN4株部分生物学特性的研究[J].中国预防兽医学报,2009,(06):424.
 TIAN Zhi-jun,AN Tong-qing,ZHOU Yan-jun,et al.Characterization of highly pathogenic porcinereproductive and respiratory syndrome virusHuN4 strain in the course of attenuation [J].Chinese journal of preventive veterinary medicine,2009,(07):424.
[4]马 平,蔡雪辉*,刘永刚,等.猪繁殖与呼吸综合征病毒GD3株的分离鉴定及其分子特征 [J].中国预防兽医学报,2009,(01):28.
 MA Ping,CAI Xue-hui*,LIU Yong-gang,et al.Isolation and characterization of PRRSV GD3 strain isolated in China [J].Chinese journal of preventive veterinary medicine,2009,(07):28.
[5]宋艳华,王海燕,李文良,等.PRRSV NSP2基因在重组杆状病毒中的表达与鉴定 [J].中国预防兽医学报,2009,(07):509.
 SONG Yan-hua,WANG Hai-yan,LI Wen-liang,et al.Expression and identification of NSP2 ofPRRSV in recombinant baculovirus [J].Chinese journal of preventive veterinary medicine,2009,(07):509.
[6]李宏宇,孙 元,常天明,等.共表达猪繁殖与呼吸综合征病毒GP5基因和猪瘟病毒E2基因的重组腺病毒的构建及其在小鼠模型上的免疫原性分析[J].中国预防兽医学报,2010,(03):161.
 LI Hong-yu,SUN Yuan,CHANG Tian-ming,et al.Immunogenicity of a recombinant adenovirus co-expressing porcine reproduction and respiratory syndrome virus GP5 protein and classical swine fever virus E2 protein in mice[J].Chinese journal of preventive veterinary medicine,2010,(07):161.
[7]庞海洋,赵焕云,张文东,等.云南高致病性猪繁殖与呼吸综合征病毒Nsp2基因变异分析[J].中国预防兽医学报,2008,(12):934.
 PANG Hai-yang,ZHAO Huang-yun,ZHANG Wen-dong,et al.Gentic variation of Nsp2 genes of highly pathogenic porcine reproductive and respiratory syndrome viruses in Yunnan province[J].Chinese journal of preventive veterinary medicine,2008,(07):934.
[8]徐明明,蔡雪辉*,刘永刚,等.高致病性PRRSV ORF3基因在昆虫细胞中的表达与免疫原性研究[J].中国预防兽医学报,2010,(06):455.
 XU Ming-ming,CAI Xue-hui*,LIU Yong-gang,et al.Expression of highly pathogenic PRRSV ORF3 gene in insect cells[J].Chinese journal of preventive veterinary medicine,2010,(07):455.
[9]邓雨修#,招丽婵#,苏润环,等.猪繁殖与呼吸综合征病毒TM 株的分离鉴定及其Nsp2序列分析[J].中国预防兽医学报,2010,(07):503.
 DENG Yu-xiu#,ZHAO Li-chan#,SU Run-huan,et al.Isolation and Nsp2 gene sequence analysis of the PRRSV TM isolate[J].Chinese journal of preventive veterinary medicine,2010,(07):503.
[10]石文达,刘永刚,王洪峰,等.猪繁殖与呼吸综合征病毒CH-1R株全基因组序列测定及遗传变异分析[J].中国预防兽医学报,2010,(07):517.
 SHI Wen-da,LIU Yong-gang,WANG Hong-feng,et al.Sequencing analysis of full length genome of porcine reproductive and respiratory syndrome virus CH-1R strain[J].Chinese journal of preventive veterinary medicine,2010,(07):517.

备注/Memo

备注/Memo:
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)
更新日期/Last Update: 2011-09-29