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[Rapid disruption of Bombyx mori nucleopolyhedrovirus orf60 by red recombination system]

Qiang Wang1, Zhong-Jian Guo, Qin Yao

  • 1Institute of Life Science, Jiangsu University, Zhenjiang 212013, China.

Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology
|December 7, 2007
PubMed

Insights

Researchers successfully deleted the Bombyx mori nucleopolyhedrovirus (BmNPV) orf60 gene using a Red recombinant system in Escherichia coli (E. coli). This gene knockout prevents orf60 expression in BmN cells, aiding BmNPV research.

Area of Science:

  • Molecular Biology
  • Virology
  • Genetics

Background:

  • Bombyx mori nucleopolyhedrovirus (BmNPV) is a significant insect pathogen.
  • Understanding viral gene function is crucial for developing control strategies.
  • Efficient gene manipulation systems are needed for baculovirus research.

Purpose of the Study:

  • To develop a rapid method for deleting the orf60 gene of BmNPV.
  • To confirm the successful deletion and absence of orf60 expression.
  • To provide a tool for further functional studies of BmNPV genes.

Main Methods:

  • Construction of a BmNPV bacmid in Escherichia coli (E. coli) BW25113.
  • Utilizing the Red/ET (recombineering) system for homologous recombination.
  • PCR amplification of a DNA fragment containing the chloramphenicol acetyltransferase (cat) gene.
  • Electroporation of the fragment into E. coli BW25113-Bac for gene replacement.
  • Confirmation of gene deletion using specific primer pairs and Western blot analysis.

Main Results:

  • Successful deletion of the BmNPV orf60 gene was achieved in E. coli.
  • The replacement of orf60 with the cat gene was confirmed by PCR.
  • Western blot analysis demonstrated no expression of the orf60 protein in BmN cells infected with the knockout bacmid.

Conclusions:

  • The Red recombinant system provides an efficient method for BmNPV gene deletion.
  • The generated knockout bacmid is suitable for studying the function of orf60.
  • This technique facilitates rapid genetic manipulation of BmNPV.

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