Construction And Expression In Human Cell Of HTERT Mammalian Expression Vector

Telomeres is a specific repetitive DNA sequences of linear chromosomes, which is shorten with cell division in highly-differentiated normal cells and is supposed to be the reflection of cell replication and replication potential. Telomerase, which is a reverse transcriptase enzyme, allows stem and germ cells and some other highly proliferating cells to divide indefinitely by extend telomers. The telomerase consists of human telomerase RNA component(hTR), human telomerase related protein(hTP) and human telomerase reverse transcriptase(hTERT),and the expression level of hTERT is positively correlated to telomerase activity. Studies have showed that hTERT has a good application prospect in tumor immunotherapy.Objective:To provide a sound basis for further research on bone neoplasm immunotherapy, pCIneo-hTERT,a recombinant plasmid, was constructed, and its expression in K562 was observed.Methods and Results:1. Construction and identification of recombinant plasmidTo construct mammalian recombinant plasmid of human telomerase reverse transcription(hTERT), total RNA was extracted from the human osteosarcoma cell line HOS, and the fragment of hTERT was amplified by reverse transcription and polymerase chain reaction(RT-PCR), then linked to the NoT I and EcoR I sites of pCI-neo Mammalian Expression Vector after double digested by NoT I and EcoR I. The target fragment can be found in the transformed competent cells E.coli DH5?, and be identified by restriction endonuclease, PCR and sequences analysis.The sequence of hTERT fragment was determined and analyzed by BLAST. Gene sequencing results: the hTERT fragment was 3399 bp in length. The homology of the hTERT fragment was 100%, compared with the hTERT gene reported in Gen Bank.2. Identification of the expression of recombinant plasmidThe recombinant plasmid was transfected into the cell line K562 by electroporation, and the monoclonal cell lines were selected by G418. The result showed the expression level of hTERT in transfected cells was higher than that in non-transfected cells using the real-time PCR method.3. The effect of hTERT on proliferationFlow cytometry was used to detect the proliferation, which showed no difference before and after the transfectionConclusion: The successful construction of pCI-neo-hTERT mammalian expression plasmid and its expression in K562 cells provide an experimental basis for the further study of bone tumor.