Construction of eukaryotic expression vector PcDNA3.0-AIF△1-480 and its apoptosis promoting activity

OBJECTIVE:To investigate pro-apoptotic activity of truncated human AIF and its ocular mechanisms,by constructing a novel eukaryotic expression vector which only contains promote apoptosis structure domain.METHODS:A novel eukaryotic expression vector was constructed by using polymerase chain reaction(PCR).The vector were transfected into MCF-7 cells by Lipofectamine~TM 2000 and then the pro-apoptotic activity of the eukaryotic expression vector PcDNA3.0-AIF△1-480 was detected by flow cytometry technology and at the same time through JC-1 flow cytometry technology,the mitochondrial membrane potential situation were learned.In addition,the pro-apoptotic activity of the eukaryotic expression vector were detected in vivo by constructing a tumor-burdened nude mouse model.RESULTS:We successfully constructed a novel eukaryotic expression vector.In vitro,compared to control group,the vector could induce MCF7 cells apoptosis,the apoptosis rate was 0.01%,16.01%,28.55% respectively.And then the mitochondrial membrane potential of MCF7 cells transfected by Lipofectamine~TM 2000 was depolarized,and the apoptosis rate was 2.26%,60.37%,95.78% respectively.For the tumor-burdened nude mouse model,the vector could inhibit tumor growth.The median tumor size was 1 187.2 mm~3,1 000 mm~3 and 609.4 mm~3 respectively.CONCLUSION:It confirms eukaryotic expression vector PcDNA3.0-AIF△1-480 have apoptosis activity,it may not play the active role of promoting the apoptosis in the nuclei,but play the role by changing the mitochondrial membrane potential.