Construction of Recombinant Telomerase Reverse Transcriptase Gene Lentiviral Expression Vector and Virus Packaging

Objective: The aim is to construct lentiviral vector containing GFP reporter gene driven by human telomerase reverse transcriptase(hTERT)gene and to establish high-titer lentiviral packaging system,then observe the expression of hTERT gene.Methods: Digested the plasmid PCI-neo-hTERT with double enzyme digestion and subcloned hTERT into the lentiviral vector,pCDH-CMV-MCS-EF1-copGFP,to generate the lentiviral expression vector,pCDH-hTERT.The accuracy of hTERT fragment was confirmed by double enzyme digestion and DNA sequencing analysis.Recombinant lentiviruses were produced by 293T cells following the co-transfection of pCDH-hTERT,with the packaging plasmids pCDH-PACK-GAG、pCDH-PACK-REV and VSV-G which are the 3rd generation of lentiviral vector systems containing green fluorescent protein(GFP)gene.Virus supernatant was collected and concentrated,then the titer of virus was tested.The resulting recombinant lentiviruses which carrying hTERT were then used to infect target cell lines.GFP,hTERT mRNA and telomerase expression in 293T and hUVEC were detected by fluorescent microscope,RT-PCR,Western blotting and TRAP-PCR-ELISA.Result: Plasmid pCDH-hTERT carried the correct hTERT gene.The recombinant lentiviruses pCDH-hTERT which carried hTERT could be produced by co-transfection of pCDH-hTERT and packaging plasmids to 293T;The recombinant lentiviruses which carried hTERT could infect and deliver hTERT gene to 293T and hUVEC,and hTERT mRNA and protein expression were remarkably increased in infected cells.Conclusion: The study successfully constructed recombinant plasmid pCDH-hTERT,the recombinant lentiviruses can deliver target gene hTERT and have high infection efficiency.Extrinsic hTERT gene can reconstruct telomerase and settle a basis for establishing immortalized fibroblast of cells.