The Cloning, Expression Of Human Truncated AIF Molecules And Their Apoptosis Induction On Tumor Cells

Apoptosis is programmed ceil death triggered by various physiological or pathological stimuli. Apotosis inducing factor (AIF) is a mammalian, caspase-independent death effector, which were found in 1996. In heathlty cells, AIF protein is normally confined to mitochondrial intermembrane space. However, after apoptosis is induced, AIF protein can release from mitochondria and translocates to the nucleus leading by its nuclear localization signal. Once in the nucleus, AIF causes chromatin condensation and large-scale DNA fragmentation to fragments of ~50kb.The pro-apoptotic activity of AIF is not affected by over expression of Bcl-2, and is in caspases-independent fashion. Moreover, AIF is involved in apoptosis process throughout eukaryotic kingdom and is essential for the first wave of caspase-independent programmed cell death during mammaliandevelopment. Compared with caspases and other apoptosis effectors, AIF could directly induce apoptotic cell death, beyond the inhibitory regulation from anti-apoptotic factor (eg.Bcl-2) in cytosol. Thus, it is significant and promising for AIF to be applied to induce tumor cell apoptosis in gene therapy.The research done by Susin A indicates that the molecular weight of human AIF precursor protein is 67KD. It contains several structure domains that is (1) mitochondria location sequence (MLS); (2) Spacer sequence; (3) nuclear location sequence(NLS); (4) C-terminal. The mature AIF consists of three parts: FAD binding domain(122aa-262aa and 400aa-477aa) NADH binding domain and C-terminal.The mature AIF was cloned successfully by Dr.Yu Cui Juan of our lab, at the same time, she determined its pro-apoptotic activity. However, the molecular weight of mature AIF is 57KD, which limits its application. So, the aim of our research is to clone truncated AIF genes so that we can obtain one kind AIF gene that is shorter, easier to use and its pro-apoptotic function remains unaffected. In this paper, four truncated human AIF genes coined were cloned by PCR, which were named as AIFAl-300(301aa-613aa), AIFAl-352(353aa-613aa), AIFAl-400(401aa-613aa), AIFAl-480(481aa-613aa) respectively. Then, we inserted four truncated AIF genes into the eukaryotic expressing vector pIRES2-EGFP. The sequence of genes we cloned was proved completely right by DNA sequencing. After four truncated AIF genes were transient transfected into HeLa cells, we demonstrated the expression of truncated AIF genes by Western blot and immunohistochemical staining assay. Using fluorescence microscope , the truncated AIF genes were found in cytosol and cells transfected with truncated AIF genes become round and shrink compared to the cells transfected with pIRES2-EGFP vector. Those cells morphology changes were also confirmed by indrect immuno-fluorescence labeled assay and cell skelecton staining. From the result of immuno-fluorescence labeled assay, we observed that AIF gene induces Cyt c releasing from mitochondria. Using cell skelecton staining and DAPI staining, we found that cell skelecton and nuclear was ruined badly after four truncated AIF genes were transfected. So, we concluded that...