Genetic transformation of BCG.

Area of Science:

• Microbiology
• Molecular Biology
• Vaccinology

Background:

• Bacillus Calmette–Guérin (BCG) is a widely used vaccine, but its efficacy can be limited.
• Developing effective recombinant BCG (rBCG) vaccines requires efficient genetic manipulation techniques.

Purpose of the Study:

• To establish a method for transforming BCG with shuttle plasmids.
• To assess the stability and expression of foreign DNA in BCG.
• To lay the groundwork for creating polyvalent recombinant BCG vaccines.

Main Methods:

• Construction of E. coli-mycobacteria shuttle plasmids (pYUB13, pYUB14) with kanamycin and chloramphenicol resistance markers.
• Transformation of BCG (Pasteur and Japanese substrains) using electroporation.
• Monitoring plasmid introduction, persistence, and expression via re-isolation, restriction analysis, and antibiotic resistance selection.
• Optimization of BCG culture conditions and electroporation parameters.

Main Results:

• Successful transformation of BCG with shuttle plasmids, yielding kanamycin-resistant colonies.
• Identification of M. fortuitum sequences essential for replication in BCG.
• Demonstrated genetic stability of hybrid plasmids, maintaining kanamycin resistance for over 250 generations.
• Increased transformation efficiency through consecutive transformations with BCG transformant DNA.

Conclusions:

• Established a robust method for BCG transformation using E. coli-mycobacteria shuttle plasmids.
• Confirmed the genetic stability and expression of foreign DNA in BCG.
• Provided a foundation for developing novel polyvalent rBCG vaccines expressing antigens for various diseases.