Acta Pharmacologica Sinica 2005 May; 26 (5): 629-634; doi: 10.1111/j.1745-7254.2005.00094.x

Aim: To construct a Bifidobacterium infantis/CD targeting gene therapy system and observe the antitumor effect of cytosine deaminase/5-fluorocytosine (CD/5-FC) suicide gene therapy system mediated by Bifidobacterium infantis on melanoma in vitro and in vivo.

Methods: A recombinant CD/pGEX-1LamdaT plasmid was transfected into Bifidobacterium infantis by electroporation. Bifidobac-terium infantis transfected by recombinant CD/pGEX-1LamdaT plasmid was incubated with 5-FC anaerobically. Then the supernatant fluid was collected and added to melanoma B16-F10 cells to observe the killing effect for B16-F10 cells. Mice were inoculated with melanoma B16-F10 cells to establish animal models. The mice were then injected with 5-FC and Bifidobacterium infantis transfected by recombinant CD/pGEX-1LamdaT plasmid.

Results: Two segments of approximate 4.9 kb and 1.3 kb were extracted from the 6.2 kb recombinant plasmid, which were equal to the size of the pGEX-1LamdaT plasmid and CD gene, respectively. Sequencing results showed that the full length and sequence of nucleotide acid of the inserted gene in extracted recombinant plasmid was completely identical to the CD gene. In vitro, B16-F10 cells treated by supernatant fluid were remarkably damaged morphologically, and the cell growth was significantly inhibited. Experiments on the mice melanoma model showed that after treatment with a combination of transfected Bifidobacterium infantis and 5-FC, the tumor volume was significantly inhibited compared with controls.

Conclusion: The foreign gene, CD gene, was correctly inserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium infantis. CD/5-FC suicide gene therapy system mediated by Bifidobacterium infantis demonstrated a good antitumor effect on melanoma in vitro and in vivo.

Keywords: Bifidobacterium infantis; cytosine deaminase; gene therapy