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Related Experiment Videos

Development of a bovine adenovirus type 3-based expression vector

S K Mittal1, L Prevec, F L Graham

  • 1Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Canada.

The Journal of General Virology
|January 1, 1995
PubMed
Summary

We created a bovine adenovirus type 3 recombinant (BAd3-Luc) expressing luciferase. This new BAd3-based vector shows potential for cattle vaccines and gene transfer applications.

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Area of Science:

  • * Virology
  • * Molecular Biology
  • * Gene Therapy

Background:

  • * Adenoviruses are widely used as gene delivery vectors.
  • * Bovine adenovirus type 3 (BAd3) is a promising candidate for veterinary and human gene therapy.
  • * Development of efficient and safe BAd3-based vectors is crucial.

Purpose of the Study:

  • * To construct and characterize a non-defective BAd3 recombinant vector (BAd3-Luc) expressing firefly luciferase.
  • * To evaluate the expression and replication properties of BAd3-Luc in cell culture.
  • * To assess the potential of BAd3-based vectors for recombinant vaccines and gene transfer.

Main Methods:

  • * Construction of BAd3-Luc by inserting the firefly luciferase gene into the E3 region of the BAd3 genome.
  • * Confirmation of gene insertion and deletion using Southern blot analysis.

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  • * Measurement of luciferase expression via enzymatic assays and Western blotting in infected cells.
  • * Analysis of luciferase expression dependence on viral DNA replication.
  • * Assessment of viral replication efficiency and infectious virus yields.

    • Main Results:

    • * Successful construction and confirmation of the BAd3-Luc recombinant.
    • * Luciferase expression was detected in Madin-Darby bovine kidney cells and 293 cells.
    • * Approximately 70-75% of luciferase expression was dependent on viral DNA replication, indicating late promoter control.
    • * BAd3-Luc showed relatively efficient replication, though yields were 10-fold lower than wild-type BAd3.
    • * Luciferase protein of the expected molecular weight was detected.

    Conclusions:

    • * BAd3-Luc is a functional recombinant bovine adenovirus vector expressing luciferase.
    • * The vector demonstrates efficient replication and gene expression, with potential for late promoter-driven transcription.
    • * BAd-based expression vectors hold significant promise for developing recombinant vaccines for cattle.
    • * These vectors may also be suitable for gene transfer applications in human cells.