Serving the Society Since 1986
高级搜索
Volume 22 Issue 1
February 2007

    Shun-lin HU, Qin SUN, Qu-zhi WANG, Yu-liang LIU, Yan-tao WU and Xiu-fan LIU. Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene[J]. Virologica Sinica, 2007, 22(1): 34-40.
    Citation: Shun-lin HU, Qin SUN, Qu-zhi WANG, Yu-liang LIU, Yan-tao WU, Xiu-fan LIU. Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene .VIROLOGICA SINICA, 2007, 22(1) : 34-40.
    shu

    重组新城疫病毒表达绿色荧光蛋白基因*

    • 扬州大学 农业部畜禽传染病学重点开放实验室，江苏扬州 225009

    • 通讯作者： 刘秀梵*, xfliu@yzu.edu.cn
    • 收稿日期： 2006-08-14
      录用日期： 2006-10-17
    • 摘要：将新城疫病毒 ZJI株基因组cDNA全长分成七个片段，依次连接并克隆至TVT7R转录载体中，构建了含ZJI株全基因组cDNA的转录载体 (pNDV/ZJI)， pNDV/ZJI与三个辅助表达质粒pCI-NP、pCI-P和pCI-L共转染BSR-T7/5 细胞，成功拯救出了具有感染性的新城疫病毒粒子。根据新城疫病毒的基因组结构特点，设计用于扩增绿色荧光蛋白 (GFP)基因的一对引物，利用引物两端添加的酶切位点将含有GFP基因的片段引入到 ZJI株基因组中，构建了转录载体pNDV/ZJIGFP，将pNDV/ZJIGFP与三个辅助表达质粒共转染BSR-T7/5 细胞，成功拯救出了重组的新城疫病毒，命名为NDV/ZJIGFP。重组病毒感染BSR-T7/5和鸡胚成纤维细胞48h后，在荧光显微镜下能见到明显的绿色荧光，表明GFP基因在病毒的增殖过程中得到了较好的表达，这为进一步利用该病毒进行其它外源基因的表达及新型重组疫苗的研制打下了基础。

    Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene

    • Key Laboratory for Animal Infectious Diseases of Ministry of Agriculture,Yangzhou University,Yangzhou 225009,China

    • Corresponding author: Xiu-fan LIU, xfliu@yzu.edu.cn
    • Received Date: 14 August 2006
      Accepted Date: 17 October 2006

      Fund Project: Key program of National Natural Sciences Foundation of China No.30630048

    • Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF)cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.
    • 加载中

      1. Alexander D J. 1998. Newcastle disease virus and other avian paramyxoviruses[M]. Kennett Square.:America Association of Avian Pathologists,156-163

      2. Czegledi A,Ujvari D,Somogyi E,et al. 2006. Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications[J]. Virus Res,120(1-2): 36-48.
          doi: 10.1016/j.virusres.2005.11.009

      3. Engel-Herbert I,Werner O,Teifke J P,et al. 2003. Characterization of a recombinant Newcastle disease virus expres-sing the green fluorescent protein[J]. J Virol Methods,108(1): 19-28.
          doi: 10.1016/S0166-0934(02)00247-1

      4. Hu S L,Wu Y T,Liu W B,et al. 2005. Development of monoclonal antibodies against NDV isolated from goose and their reactions with NDV isolates[J]. Chin J Veter Sci Technol,35(5): 341-345.(in Chinese)

      5. Huang Y,Wan H Q,Liu H Q,et al. 2004. Genomic sequence of an isolate of Newcastle disease virus isolated from an outbreak in geese: a novel six nucleotide insertion in the non-coding region of the nucleoprotein gene. Brief Report[J]. Arch Virol,149(7): 1445-1457.

      6. Huang Z,Krishnamurthy S,Panda A,et al. 2001. High-level expression of a foreign gene from the most 3′-proximal locus of a recombinant Newcastle disease virus[J]. J Gen Virol,82(Pt 7): 1729-1736.

      7. Johnson K N,Zeddam J L,Ball L A. 2000. Characterization and construction of functional cDNA clones of Pariacoto virus,the first Alphanodavirus isolated outside Australasia[J]. J Virol,74(11): 5123-5132.
          doi: 10.1128/JVI.74.11.5123-5132.2000

      8. Krishnamurthy S,Huang Z,Samal S K. 2000. Recovery of a virulent strain of newcastle disease virus from cloned cDNA: expression of a foreign gene results in growth retardation and attenuation[J]. Virology,278(1): 168-182.
          doi: 10.1006/viro.2000.0618

      9. Liu Y l,Wu Y T,Huang Y,et al. 2004. Cloning of NP,P and L gene of Newcastle disease virus of goose origin and identification of P gene expression.[J]. Microbiology,31(2): 37-40.(in Chinese)

      10. Liu Y L,Zhang Y M,Hu S L,et al. 2005. Generation of Newcastle disease virus strain ZJI isolated from an outbreak in geese using reverse genetics technique[J]. Acta Microbiology Sinica,5: 780-783.(in Chinese)

      11. Mayo M A. 2002. A summary of taxonomic changes recently approved by ICTV[J]. Arch Virol,147(8): 1655-1663.
          doi: 10.1007/s007050200039

      12. Park M S,Steel J,Garcia-Sastre A,et al. 2006. Engineered viral vaccine constructs with dual specificity: avian influenza and Newcastle disease[J]. Proc Natl Acad Sci U S A,103(21): 8203-8208.
          doi: 10.1073/pnas.0602566103

      13. Peeters B P,de Leeuw O S,Koch G,et al. 1999. Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence[J]. J Virol,73(6): 5001-5009.

      14. Philips R J,Samson A C R,Emmerson P T. 1998. Nucleotide sequence of the 5′-terminus of Newcastle disease virus and assembly of the complete genomic sequence: agreement with the ′rule of six′[J]. Archive of Virology,143: 1993-2002
          doi: 10.1007/s007050050435

      15. Romer-Oberdorfer A,Mundt E,Mebatsion T,et al. 1999. Generation of recombinant lentogenic Newcastle disease virus from cDNA[J]. J Gen Virol,80 (Pt 11): 2987-2995.

      16. Zhao H,Peeters B P. 2003. Recombinant Newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication[J]. J Gen Virol,84(Pt 4): 781-788.

    • 加载中

    Figures(3)

    PDF

    Article Metrics

    Article views(7780) PDF downloads(28) Cited by(0)

    Related
    Proportional views
      通讯作者: 陈斌, bchen63@163.com
      • 1. 

        沈阳化工大学材料科学与工程学院 沈阳 110142

      1. 本站搜索
      2. 百度学术搜索
      3. 万方数据库搜索
      4. CNKI搜索

      Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene

        Corresponding author: Xiu-fan LIU, xfliu@yzu.edu.cn
      • Key Laboratory for Animal Infectious Diseases of Ministry of Agriculture,Yangzhou University,Yangzhou 225009,China
      • Received Date: 14 August 2006
      • Accepted Date: 17 October 2006
      Fund Project:  Key program of National Natural Sciences Foundation of China No.30630048

      Abstract: Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF)cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.

        HTML