LUYin—hua, HUA Xiu—guo, CUI Li, TAN Guo—lei, XU Li—hua ’, HUANG Wei-jian and CHEN De.sheng .CHEN Pu—yan. M olecular Cloning of the Infectious Genomic DNA of Porcine circovirus Type 2[J]. Virologica Sinica, 2003, 18(4): 371-375.
Citation: LUYin—hua, HUA Xiu—guo, CUI Li, TAN Guo—lei, XU Li—hua ’, HUANG Wei-jian, CHEN De.sheng .CHEN Pu—yan. M olecular Cloning of the Infectious Genomic DNA of Porcine circovirus Type 2 .VIROLOGICA SINICA, 2003, 18(4) : 371-375.

感染性猪圆环病毒2型基因组DNA的分子克隆

  • 摘要:本研究通过PCR扩增出猪圆环病毒2型(PCV一2)的全基因组(1 768bp),克隆入pcDNA3载体的EcoR I 酶切应点,获得含有PCV.2全基因组的重组质粒,命名为pcDNApcv2。将重组质粒大量扩增后,用EcoR I切出 l 768bp的PCV一2全基因组,在体外用T4 DNA连接酶使其连接环化。用脂质体法将体外连接产物转染无PCV污 染的PK一15细胞,经4次连续传代,用间接免疫荧光实验(IFA)及电镜观察证实已获得复制能力的PCV一2病毒。 由此可见,本试验构建的环化的PCV.2全基因组DNA具有感染性。

M olecular Cloning of the Infectious Genomic DNA of Porcine circovirus Type 2

  • The complete genome of type 2 Porcine circovirus(pcv一2)was amplified by polymerase chain reaction(PCR and cloned directly into me EcoRI sites of plasmid pcDNA3,and recominant plasmid carrying the complete genome was constructed,designated pcDNApcv2.The entire genome of PCV一2 was purified and recycled from pcDNApcv2 with the digestion of EcoRI enzyme an d then circular genomic DNA was generated by self-ligating with T4 DNA ligase in vitro.Th e non—infected PK一1 5 cells were transfected with the PCV一2 circular genome using Lipofectin Reagent.After four continuous passages,PCV一2 virus and specific antigens were visualized by electron microscopy an d IFA,respectively.Th us,the cloned circular PC V一2 genomi c DNA generated in this study was infectious in vitro.

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    M olecular Cloning of the Infectious Genomic DNA of Porcine circovirus Type 2

    • 1. Key laboratory of Animal Disease Diagnostic and Immunology,Ministry of Agriculture,Nanjing Agricultural University,Nanjing 210095,China
    • 2. Colle·. P ofAgriculture,Shanghai Jiaotong University,Shanghai 200030,China
    • 3. Anima l Science Department ofAgricultural College。Ningxia University,Hnchuan 750105,China

    Abstract: The complete genome of type 2 Porcine circovirus(pcv一2)was amplified by polymerase chain reaction(PCR and cloned directly into me EcoRI sites of plasmid pcDNA3,and recominant plasmid carrying the complete genome was constructed,designated pcDNApcv2.The entire genome of PCV一2 was purified and recycled from pcDNApcv2 with the digestion of EcoRI enzyme an d then circular genomic DNA was generated by self-ligating with T4 DNA ligase in vitro.Th e non—infected PK一1 5 cells were transfected with the PCV一2 circular genome using Lipofectin Reagent.After four continuous passages,PCV一2 virus and specific antigens were visualized by electron microscopy an d IFA,respectively.Th us,the cloned circular PC V一2 genomi c DNA generated in this study was infectious in vitro.

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