Transformation of BCG with plasmid DNA

Pasteur 1173P2 and Japanese 172 BCG substrains were transformed with plasmid pAL5000::plJ666 by electroporation and assessed by the growth of kanamycin-resistant colonies. From the transformants pYUP plasmids were isolated containing plJ666 inserted into pAL5000. The introduction, persistence and the identity of the plasmid were monitored by reisolation from consecutive subcultures and restriction analysis. The effect of variables--age, viability, glycine pretreatment of BCG cultures, electroporation parameters--on transformation frequencies were analyzed. Consecutive transformation of BCG with plasmid DNA isolated from a BCG transformant increased the efficiency from the level of 10(1)-10(2) obtained with the initial library to 10(3)-10(4) colonies/micrograms DNA with the pYUB plasmids. Maintenance of plasmid and expression of kanamycin resistance in continuous subcultures were stable for 100 generations from the first successful transformation to the present. The introduction and stable expression of foreign DNA in BCG on a plasmid vector establishes a basis for the construction of polyvalent recombinant BCG vaccine vehicles expressing not only putative protective mycobacterial antigens, but also antigens for other infectious and malignant diseases.