Effect On Human Telomerase Reverse Transcriptate Promoter In Targeting Cancer Gene Therapy

OBJECTIVE:To establish the eukaryotic expression vector containing the Human Telomerase Reverse Transcriptase promoter(hTERTp) and to observe its expression in eukaryotic cell.METHODS:1) Human genome DNA was extraed from whole blood samples with anticoagulants.2) The hTERTp gene was amplified by PCR.The amplified product was ligated with pGEM-T Easy vector and examined by electrophoresis and sequence determination.3) The CMV promoter of fluorescent vector pEGFP-C3 was replaced by hTERTp,which was amplified by PCR.The recombinant plasmid was transformed into E.coli. DH5a competent and examined by PCR,restriction enzyme digestion and DNA sequencing.4) The recombined plasmid pEGFP-C3-hTERTp was transfected into the HEK 293A cells with lipofectamine. The expressed EGFP was observed by fluorescent microscope after 48-72h.RESULTS:1) Agarose gel electrophoresis demonstrateed that 276bp hTERTp was amplified successfully,and DNA sequencing showed a sequence the same as the hTERTp registered in GenBank.2) The recombinant plasmid pEGFP-C3-hTERTp was confirmed to be right by PCR,restriction enzyme digestion and DNA sequencin. 3) Green fluorescent can be seen from HEK293A cells transfected with pEGFP-C3-hTERTp plasmid.CONCLUSION:The recombinant expression vector containing hTERTp has been successfully constructed and can be expressed in eukaryotic cell, and this study lays a foundation for further research into the function of hTERTp in tumor gene therapy.