RAAV2-TRAIL Construction And Apoptosis Effect On Malignant Melanoma Cells

ObjectiveTo investigate the adeno-associated virus (adeno-associated virus, AAV)-mediated apoptosis of tumor necrosisfactor-related apoptosis inducing ligand (Tumor necrosis factor-related apoptosis inducing ligand, TRAIL) gene for malignant melanoma cells.MethodsPCR method was employed to amplify the extracellular region of TRAILfragment encoding sTRAIL,with TRAIL plasmid as a template,this inserted into theeukaryotic expression vector was constructed shuttle plasmid pSNAV2.0-sTRAIL,theuse of plasmid DNA in cells homologous recombination method sTRAIL fragmentinserted into the adeno-associated virus vector carrying sTRAIL constructedrecombinant adeno-associated virus rAAV2-sTRAIL.The virus in vitro transfectionof human malignant melanoma cells A375(experimental group)and humanembryonic kidney cells HEK293(control group),ELISA,Western blot analysis wasemployed to detect the expression of the target gene in cells,MTT assay its effect oncell proliferation,electron microscopy and flow cytometry was employed to analyzethe mechanism of in vitro against human malignant melanoma cell on apoptosisA375.ResultsSuccessfully constructed a virus rAAV2-sTRAIL,the target gene bands can besignificant detected by PCR;TRAIL bands can be detected significant intracellular byWestern blot method;48hours after transfection cells,TRAIL were detected byELISA, medium concentration was190.6±24.1ng/ml in culture medium ofHEK293,and medium concentration was56.7±12.4ng/ml in culture medium ofA375;rAAV2-sTRAIL can have a significant effect on A375proliferation by MTTassay,while had no significant effect on HEK293; AAV2-sTRAIL can induce apoptosis in malignant melanoma cells A375confirmed by electron microscopy andflow cytometry.ConclusionrAAV2-sTRAIL can highly expressed in normal HEK293cells and malignanthuman melanoma A375cells，and secreted into the extracellular, and had significantinduce apoptosis effect on A375cells but HEK293cells.