摘要
Objective: To obtain the recombinant fusion AIF genes inserted into the eukaryotic expression vectorpIRES2-EGFP, to observe the expression and location ofthe fusion AIF genes (3NE: PE(280-358)-AIF1-120, and4NE: PE(280-364)-AIF1-120), and to detect and compare their apoptosis inducing effects on the transfected HeLacells. Methods: Full-length human AIF gene was clonedby RT-PCR, and its N-terminal mitochondrial localization sequence (MLS) was replaced by part sequence ofPsuedomonas exotoxin A (PE) translocation domain(PEII(280-358/364)), then the recombinant fusion geneswere inserted into the pIRES2-EGFP eukaryotic expression vector. After these genes were transiently transfected into HeLa cells with LipofectAmine, the expression of therecombinant fusion AIF genes and their effects on HeLacells were detected by fluorescent microscopy, laser confocal microscopy and electron microscopy. Results: Theeukaryotic expression vectors containing the recombinant fusion AIF genes (pIRES2-EGFP-PEII(280-358/364)-AIF1- 120) were constructed successfully. It wasdemonstrated that the fusion AIF protein genes wereexpressed effectively in the transfected cells, with the GFP co-expressed in cells by indirect immunofluorescence staining analysis. After transfection, expression of the genes could induce HeLa cells to exhibit the typical apoptosis features: such as plasma membrane blebbing and peripheral chromatin condensation. As compared with control groups, the untreated cells and the void vector transfected cells, the living cell number of the AIF gene transfected cells reduced distinctly. Conclusion: Our data prove that the expression of the recombinant human AIF fusion genes could induce apoptosis in transfected HeLa cells, which provides new strategy for cancer killing.
Objective: To obtain the recombinant fusion AIF genes inserted into the eukaryotic expression vectorpIRES2-EGFP, to observe the expression and location ofthe fusion AIF genes (3NE: PE(280-358)-AIF1-120, and4NE: PE(280-364)-AIF1-120), and to detect and compare their apoptosis inducing effects on the transfected HeLacells. Methods: Full-length human AIF gene was clonedby RT-PCR, and its N-terminal mitochondrial localization sequence (MLS) was replaced by part sequence ofPsuedomonas exotoxin A (PE) translocation domain(PEII(280-358/364)), then the recombinant fusion geneswere inserted into the pIRES2-EGFP eukaryotic expression vector. After these genes were transiently transfected into HeLa cells with LipofectAmine, the expression of therecombinant fusion AIF genes and their effects on HeLacells were detected by fluorescent microscopy, laser confocal microscopy and electron microscopy. Results: Theeukaryotic expression vectors containing the recombinant fusion AIF genes (pIRES2-EGFP-PEII(280-358/364)-AIF1- 120) were constructed successfully. It wasdemonstrated that the fusion AIF protein genes wereexpressed effectively in the transfected cells, with the GFP co-expressed in cells by indirect immunofluorescence staining analysis. After transfection, expression of the genes could induce HeLa cells to exhibit the typical apoptosis features: such as plasma membrane blebbing and peripheral chromatin condensation. As compared with control groups, the untreated cells and the void vector transfected cells, the living cell number of the AIF gene transfected cells reduced distinctly. Conclusion: Our data prove that the expression of the recombinant human AIF fusion genes could induce apoptosis in transfected HeLa cells, which provides new strategy for cancer killing.
基金
This work was supported by grants from The National Natural Sciences Foundation of China for Outstanding Youth Scholars (No.39925036)
The Military Research Fundation of China for Outstanding Youth Scholars (No.98J009)
and State 863 High Technology R
