To construct a mutant enhanced green fluorescence protein (pEGFP) human telomerase reverse transcriptase (hTERT) gene expression vector (pEGFP-hTERT), to observe its expression in transfected human bladder cancer cell line T24 and its role in the molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer therapy.
MATERIALS AND METHODS
Polymerase chain reaction (PCR) amplification was performed using primers based on the gene sequence of hTERT. The PCR product was cloned into plasmid pGEMT-T Easy and the sequence of mutant hTERT gene analysed. A recombinant mutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sal I sites of the pEGFP-C1 vector. After transfecting the fusion gene into T24 cells by the method of calcium phosphate-DNA co-precipitation, we detected steady expression of the GFP-hTERT fusion protein by fluorescent-light microscopy. Changes in the proliferation of T24 cells were detected by light microscopy, and β-galactosidase staining correlated with senescence.
Identification of pEGFP-hTERT by enzyme digestion showed that the mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. Steady expression of GFP-hTERT fusion protein was located in the nucleus of transfected cells. Positive expression senescence-associated β-galactosidase staining in transfected cells increased gradually with extended cultured time, and their growth was suppressed.
The recombinant mutant vector (pEGFP-hTERT) was successfully constructed and expressed steadily in T24 cells. The mutant-type hTERT gene suppresses the proliferation of T24 cells by a competitive effect on telomerase activity. This suggests that the hTERT gene might be a suitable gene target for bladder cancer therapy.