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Volume 22 Issue 3
June 2007
    Citation: Jin-shan HUANG, Bi-fang HAO, Xiu-lian SUN, Fei DENG, Hua-lin WANG, Zhi-hong HU. Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru .VIROLOGICA SINICA, 2007, 22(3) : 218-225.
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    Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

    • 1.

      State Key Laboratory of Virology and Joint-lab of Invertebrate Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China

    • 2.

      Northwest A & F University, Yangling 712100, China

    • 3.

      Graduate School of the Chinese Academy of Sciences, Beijing 100039, China

    • Corresponding author: Zhi-hong HU, huzh@wh.iov.cn
    • Received Date: 13 November 2006
      Accepted Date: 30 January 2007
      Available online: 01 June 2007

      Fund Project: Programme Strategic Scientific Alliances between China and the Netherlands 2004CB720404National Natural Fundation of China project 30630002973 2003CB114202

    • To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae.
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      Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

        Corresponding author: Zhi-hong HU, huzh@wh.iov.cn
      • 1. State Key Laboratory of Virology and Joint-lab of Invertebrate Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
      • 2. Northwest A & F University, Yangling 712100, China
      • 3. Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
      • Received Date: 13 November 2006
      • Accepted Date: 30 January 2007
      Fund Project:  Programme Strategic Scientific Alliances between China and the Netherlands 2004CB720404National Natural Fundation of China project 30630002973 2003CB114202

      Abstract: To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae.

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