Construction of eukaryotic expression vector of human telomerase reverse transcriptase in immortalized hepatocytes

AIM: Through constructing eukaryotic expression vectors containing human telomerase reverse transcriptase, to provide an effective and fast method for evaluating transfection of human telomerase reverse transcriptase. METHODS: The experiment was conducted at the Virus Research Room, the 302 Hospital of Chinese PLA from June 2005 to March 2006. ①Human telomerase reverse transcriptase cDNA and green fluorescent protein cDNA were cloned by polymerase chain reaction (PCR). ②The recombinant vectors of VR1012-GFP-hTERT and pEGFP-C1-hTERT were formed with gene recombination technique. The positive clones were checked up with enzymes digestion, PCR and sequencing. ③Transfection HepG2, the location and expression of human telomerase reverse transcriptase and green fluorescent protein were studied under fluorescent microscope. RESULTS: ①PCR amplification goal gene: Electrophoresis showed that the special goal strap appeared at 3.5 kb human telomerase reverse transcriptase DNA fragment and 750 bp green fluorescent protein DNA fragment. ②Construction and identification of plasmid VR1012-GFP-hTERT and pEGFP-C1-hTERT: The consequences of enzymes digestion, PCR and sequencing were match to the anticipation. ③Transfection of HepG2 cell: The fusion protein of human telomerase reverse transcriptase and green fluorescent protein distributed mainly in the nuclei of HepG2 under fluorescent microscope. CONCLUSION: ①The construction of recombinant eukaryotic expression vectors of VR1012-GFP-hTERT and pEGFP-C1-hTERT is verified successfully. These two plasmids can express fusion protein of human telomerase reverse transcriptase DNA fragment and green fluorescent protein at the same time. Transfection efficiency and result are observed timely and rapidly by observing the expression of green fluorescent protein. ②The successful construction of recombinant eukaryotic expression vectors of VR1012-GFP-hTERT and pEGFP-C1-hTERT provides a effective and fast method for evaluating stable transfection of human telomerase reverse transcriptase and a basis for studying immortalized hepatocytes.