Research Of Universal DNA Vaccine Encoding Human Telomerase Reverse Transeriptase(hTERT)

Background and Objective:Human telomerase reverse transcriptase (hTERT),the catalytic subunit of telomerase, is sufficient to bestow telomerase activity.hTERT is an interesting example of universal TAA because it is the first tumor antigen identified to have a global expression in more than 85% of human cancer cells and its continuing expression is necessary to the oncogenic process.Together with its universal expression, hTERT represents an ideal target for cancer therapy.In this study,hTERT gene was specifically amplified and cloned into the eukaryotic expression plasmid, and a stably transfected B16 cell line highly expressing hTERT was established,which will provide a solid foundation for research of universal DNA vaccine encoding hTERT gene in the tumor immunotherapy.Methods:The gene of hTERT was amplified by RT-PCR from human prostate cancer cell line PC-3M,and then cloned into pIRES-neo eukaryotic expression vector to construct the pIRES-neo-hTERT plasmid. Under the mediation of lipofectamine,the plasmid was transfected into B16 cells,after screening culture by G418,a stably transfected cell line was established,the transcription and expression of the hTERT gene were identified by Western blot,immunofluorescence and flow cytometry assay.eTERT (1609bp-2628bp) fragment was amplified by PCR,then insened into a eukaryotic expression vector pVAX1-IRES,the recombinant plasmid pVAX1-sig-eTERT-Fc-GPI-GM/B7 (pVAX1-eTERTFcGB) was transfected to the 293T cells,and the expression was detected by immunofluorescence and flow cytometry assay.Results:The target genes hTERT and eTERT were obtained and identical with the sequences of GenBank.The eukaryotic expression vector pIRES-neo-hTERT was successfully constructed. The pIRES-neo-hTERT plasmid was successfully transfected B16 cells and a stably transfected cell line was established;PCR and enzyme digestion analysis showed that the recombinant plasmid pVAX1-eTERTFcGB was constructed successfully. The expression of this plasmid had been demonstrated by FACS and IMF.Conclusion:The target genes were obtained and the eukaryotic expression plasmids were constructed successfully. The established cell line can highly express hTERT stably;the universal DNA vaccine pVAX1-eTERTFcGB was constructed successfully, which will contribute to the research of hTERT gene in the tumor immunotherapy.