Construction of recombnant retrovirus carrying fragment of human telomerase reverse transcriptase gene

Objective To construct recombinant retrovirus carrying fragment of human telomerase reverse transcriptase(hTERT) gene. Methods A 951 bp cDNA fragment of hTERT(1590～2540bp) in K-562 cell line was amplified by RT-PCR and inserted into pMD18T plasmid. After the recombinant plasmid was cleaved by restriction endonuclease Hind III and BamH I, the hTERT fragment was subcloned into retroviral expression vector pLXSN to form pLXSN-hTERT, which was transfected into PT67 packing cell line by lipofectamine. Using G-418 selection, resistant cell clones which carried recombinant retrovirus was obtained. The recombinant retrovirus carrying fragment of hTERT was purified by filtering supernate of cultured resistant cell clone. Expression of fragment of hTERT was detected by Western blot. Results Compared with hTERT cDNA reported in GenBank, only two nucleotide acids(2171bp, T-C, 2220bp, C-A) had changed in amplified fragment of hTERT, and the homology was 100% in amino acid. The titer of the recombinant retrovirus carrying fragment of hTERT was 2.85×105PFU/ml. The recombinant retrovirus could express one polypeptide with the molecular mass of 37 kD. Conclusion The recombinant retrovirus for expressing fragment of hTERT has been successfully constructed. It will lay a solid foundation for the succedent study on hTERT as a tumor associated antigen in denritic cell-based immunotherapy.