Construction of recombinant vector of a mutant hTERT and its expression in human bladder carcinoma cell line T24

AIM To construct a mutant pEGFP hTERT expression vector, to observe its steady expression in transfected human bladder carcinoma cell line T24 and its role in further study of molecular regulatory mechanisms of telomerase and a new target gene for bladder cancer. METHODS PCR amplification was performed by using primers based on the known gene sequence of hTERT, PCR product was cloned into plasmid pGEMT T easy and the sequence of mutant hTERT gene was analyzed. A recombinant mutant hTERT vector (pEGFP hTERT) was constructed at its EcoR I and Sal I sites. After transfecting it into bladder carcinoma cell T24 by the method of calcium phosphate DNA coprecipitation, we had detected the steady expression of GFP hTERT fusion protein by fluorescent light microscopy. The proliferation changes of bladder carcinoma cell line T24 were detected by light microscopy and β galactosidase stain correlated with senescence. RESULTS Identification of pEGFP hTERT by enzyme digestion showed that mutant hTERT fragment had been cloned into Eco RI and Sal I sites of pEGFP C1 vector. The steady expression of GFP hTERT fusion protein was localized in the nuclear of transfected cells. Positive expression senescence associated β galactosidase stain in transfected cells was gradully increased with extended cultured time and their growth was suppressed. CONCLUSION Recombinant mutant vector (pEGFP hTERT) is successfully constructed and expressed steadily in human bladder carcinoma cell T24. The mutant type hTERT gene suppresses the proliferation of bladder carcinoma cell T24 by competitive effect on telomerase activity.