Construction Of Recombinant BCG Secreting TRAIL And Immunotherapeutic Effects On Bladder Cancer In Mouse

OBJECTIVE To construct recombinant BCG secreting TRAIL and investigate the immunotherapeutic effects on bladder cancer in mouse. METHODS (1) Construction and identification of recombinant shuttle plasmid. BCG Ag85B signal sequence was amplified from the genome of BCG by using polymerase chain reaction (PCR). The cDNA fragment encoding TRAIL was extracted from the spleens of mouse by using reverse transcriptase PCR (RT-PCR) and inserted into the E.coli-BCG shuttle-vector pMV261 to get pMV261-TRAIL . A new recombinant plasmid pMV261-Ag85B-TRAIL was constructed by inserting BCG Ag85B signal sequence into pMV261-TRAIL. The recombinant plasmid pMV261-Ag85B-TRAIL was identified by restriction endonuclease digestion, PCR amplification and nucleotide sequencing respectively. (2) Construction of recombinant BCG secreting TRAIL. BCG was transformed with pMV261-Ag85B-TRAIL by eletroporation, and designated as rBCG-TRAIL. The DNA and protein expressions of TRAIL gene in rBCG-TRAIL were determined by PCR and Western blotting respectively. TRAIL in the culture supernatant of rBCG-TRAIL was detected by enzyme linked immunosorbent assay (ELISA). (3) The inhibition effects of rBCG-TRAIL on transplanted bladder tumor in T739 mouse. RESULTS (1) By partial nucleotide sequencing, the DNA were consistent with the reported TRAIL and Ag85B in the GeneBank, and then were inserted into the shuttle expression vector pMV261 toconstruct recombinant plasmid pMV261-Ag85B-TRAIL respectively. The pMV261-Ag85B-TRAIL was successfully transformed into BCG by eletroporation and was capable of synthesizing and secreting TRAIL. Western blotting revealed that the secretive proteins could specially combine with antibody against TRAIL. The level of TRAIL (638.61 pg/ml) in the culture supernatant of rBCG-TRAIL was higher than control group by ELISA. (2) At the base of successfully established the BTT739 bladder tumor model in T739 mouse, the tumor inhibition effect of rBCG-TRAIL on the tumor bearing mouse was studied by intratumoral injecting. The results revealed that tumor weight and volume inhibition effects of rBCG-TRAIL treatment group were significantly higher than that of BCG + TRAIL, BCG, TRAIL and PBS control groups. The living time of rBCG-TRAIL treatment mouse was significantly longer than that of BCG + TRAIL, BCG, TRAIL and PBS control groups (3) Observed under light microscope, the bladder tumor tissue treated by rBCG-TRAIL presented obviously inflammatory cells infiltrating, neoplasm necrosis, hemorrhage and so on. CONCLUSIONS These results suggested that the rBCG-TRAIL strain reconstructed in our investigation has enhanced irnrnunotherapeutic effects and might offer new opportunities in the treatment of bladder cancer.