Expression of rsTRAIL and its apoptoic effect on A549 and H460~(wt) cells / 吉林大学学报(医学版)

ABSTRACT

Objective To construct prokaryotic expression plasmid of human recombinant soluble TNF-related apoptosis inducing ligand(rsTRAIL),then to investigate the effects of rsTRAIL on apoptosis in non-small cell lung carcinoma(NSCLC).Methods The encoding sequence for rsTRAIL was amplified with RT-PCR and cloned into PQE30 vector to establish the prokaryotic expression system.The competent cells of host strain of M15 were transformed by the recombinant plasmid.The expression of the target protein was induced with IPTG and purified by Ni2+-NTA agarose column.rsTRAIL was added in the media of A549 and H460wt cells,then the viability was examined by MTT assay.Apoptosis of H460wt cells was observed under fluorescence microscopy.The apoptotic rate of tumor cells was examined by FACS.Results The cloned fragment of rsTRAIL was 100% consistent with that reported in GenBank.The expressed protein with molecular weight of 21 000 in SDS-PAGE as expected was obtained and recognized by a commercial McAb.The apoptotic rate of H460wt cells after treated with rsTRAIL for 24 h was 43.2%.Cislatin enhanced the effect of rsTRAIL on A549 cells.Conclusion The rsTRAIL is obtained after Ni affinity chromatograph.rsTRAIL has a strong cytotoxic activity against NSCLC and cisplatin may enhance the antitumor effect of rsTRAIL.