Background and Objectives: Bladder cancer is the second common genitourinary tract cancer in the world. Immuno therapy with intravesical Mycobacterium bovis BCG is the selective treatment for superficial bladder cancer. CD/5FC Enzyme-Prodrug system of gene therapy is an alternative technique for controlling bladder cancer. This research is aimed at the design and construction of mycobacterial shuttle vector containing hsp60 promoter, alpha antigen signal sequence and cytosine deaminase (CD) and consequent transformation to M. bovis BCG by electroporation. It is assumed that converting of 5-FC to 5-FU via CD will kill more tumor cells and increase the efficacy of this therapeutic method.
Material and Method: After amplification of mycobacterial shuttle vector gene fragments containing hsp60 promoter, alpha antigen signal sequence and Saccharomyces cerevisiaeCD gene by PCR and enzymatic digestion, they were cloned and subcloned in pBGGT and pVN2 respectively. The final plasmid was transformed to M. bovis BCG by electroporation method.
Results: Sequencing results confirmed the integrity and correctness of final vector which called pHARA. The cells which received pHARA, were cultured on Middlebrook 7H10 agar with 20µg/ml final concentration of kanamycin, and grown during 17 days. Conclusion:Mentioned recombinant BCG (rBCG) secreting CD enzyme in addition to adjuvant property and stimulation of local immunity responses against tumor cells, can also destroy more tumor cells through converting 5-FC to 5-FU via CD enzyme that intensifies BCG efficacy in treatment of superficial bladder cancer.

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