Radiation-inducible expression of human perforin N-terminal in lung cancer cells / 中国免疫学杂志

ABSTRACT

Objective:To construct an eukaryotic radiation-inducible expressing vector of the human perforin N-terminal(hPFN-N),and to investigate the distribution and the killing effect of human perforin N-terminal truncated 118 amino acid polypeptide (rhPFN-N,22-139aa) on tumor cells.Methods:The gene hPFN-N was amplified by PCR from the plasmid pcDNA3.1(+)/hPFN and an enkaryotic radiation-inducible expression vector pcDNA3.1(+)/ Egr-hPFN-N was constructed after DNA recombination.After transfecting SPC-A1 cells with this recombination vector via liposome mediation,the expression of the hPFN-N protein was detected by RT-PCR and Immunocytochemical method and the killing effect of hPFN-N protein was assessed by standard MTT chromatometry.Results:DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic radiation-inducible expressing vector pcDNA3.1(+)/ Egr-hPFN-N had been constructed successfully.After the recombinant plasmid being transfected into SPC-A1 cells and being irradiated by X ray,RT-PCR verified the expression of hPFN-N mRNA.The result of Immunocytochemical assay was positive and in MTT assay the killing activity of rhPFN-N on target cells was 29.2%.Conclusion:After being irradiated the hPFN-N gene is expressed on the cell membrane and the killing activity of rhPFN-N on target cells is 29.2%.