Primary Study To The Mechanism Of Spodoptera Litura Cell Spli Apaf-1Protein In Apoptosis

Study on the apoptosis pathway is widely concerned by scientists, and has made numerous achievements. At present the pathway of apoptosis in nematode, Drosophila and humans has been studied thoroughly. Because of the evolutionary conservation the apoptotic events in these three model organisms have many similarities, but there are also a lot of differences. The endogenous mitochondrial apoptosis pathway is conservative, in mammals for example, as a response to apoptotic stimuli cytochrome C releases from the mitochondria to cytosol, binds to Apaf-1and finally form a proteolytic machine termed apoptosome which will then recrut initiative procaspases and activate them. The activated initiative caspase would cleave the effective caspase and induce the apoptosis cascade. Study on the apoptosis pathway of Lepidoptera which is evolutionary similar to Drosophila has found some different phenomenons, and especially whether the endogenous pathway initiation factor cytochrome C participates in the apoptosis pathway Apoptosome is the the center of endogenous apoptosis pathway, and the subunit Apaf-1is considered the core protein of this pathway. Research on its role in lepidopteran apoptosis is advantageous to study and reveal the molecular mechanism systematically. Polyclonal antibody to spodoptera litura Apaf-1synthesized in this paper, and the preliminary study of Apaf-1function based on it.According to the SNP sequence of Apaf-1was obtained, the conserved sequence encoding144amino acids was selected as the objective fragments, constructed the prokaryotic expression vector of pET22b-Apaf1N147, expressed and purified the fusion protein His-Apaf1N147, immuned rabbit to prepare polyclonal antiserum and purified by affinity chromatography, then assayed by Western blot. The results showed that, the antibody prepared can hybridizate the exogenous Apaf-1antigen fragment and the endogenous Spodoptera litura Apaf-1protein. This provided a powerful support for the further studies on the structure and function of Apaf-1in Lepidoptera insects.By immunofluorescence and making use of the prepared Apaf-1polyclonal antibody and anti-antibody with fluorescence tag FITC, we indirectly identified the positioning of Apaf-1in Spodoptera litura cells with BSA as control, and the result showed it located in the cytoplasm. In order to further study what the role did Apaf-1play in the apoptosis pathway of lepidopteran insects we need to amplify the ORF correctly and express the protein in vitro. By varying the concentration of Mg2+and annealing temperature in the amplification system we successfully acqured the Apaf-1ORF full-length for using the high fidelity KOD enzyme and the method of double PCR. We connected it to the prokaryotic expression vectors but failed to obtain complete and correct Apaf-1. Then eukaryotic expression vector pIEl-Apaf-1-EGFP was constructed and transfected into Spodoptera litura cells which were in logarithmic growth phase. Observing with the fluorescence microscope the results showed that the fusion protein Apaf-1-GFP was expressed and located in the cytoplasm, which was consistent with the immunofluorescence experiment above.Because of the interaction between antigen and antibody, antibody and Protein G, we captured Apaf-1from Spodoptera litura cytoplasmic protein by co-immunoprecipitation which could be settled down by the complex of Cyto C and anti-Cyto C, then assayed by western blot. By pull-down technology the fusion protein his-Cyto C which combinated with Ni-NTA resin could also pull Apaf-1down when cytoplasmic protein flew through the column, and these two results suggested that there was interaction between Apaf-1and Cyto C in Spodoptera litura cell endogenous mitochondrial pathway of apoptosis. Constructed the eukaryotic expression vectors pIEl-EGFP-Cyto C and pIE2-Apaf-1-Cherry and transfected them into spli cells at the same time then observed with confocal laser scanning microscope, the results showed the phenomenon of basic co-localization of these two proteins. These studies all affirmed the existence of endogenous mitochondrial pathway similar to mammals in lepidoptera Spodoptera litura.Preparation of Apaf-1polyclonal antibody, ORF cloning and interaction with cytochrome C laid the foundation in clarifying the mechanism of insect cell apoptosis pathways in Lepidoptera insect cells.