Preliminary Research Of 6His-VP3 Fusion Protein Preying Its Related Proteins In Tumor Cells

VP3, a 13.6 ku protein of 121 amino acids, is one of proteins encoded by the Chicken Anemia virus (CAV). Because VP3 can specifically induce apoptosis in transformed cells or tumor cells, but not in normal cells, it is also called Apoptin.It was discovered that the tumor specificity of VP3 relates to its subcellular localization. In transformed cells or tumor cells, VP3 migrates to the nuclei, whereas in non-transformed cells, it remains mainly within the cytoplasm. Meanwhile, the tumor-specific apoptotic pathway of VP3 induced does not depend on functional p53, and can't be inhibited by overexpression of Bcl-2. This distinctive characteristic of VP3 makes it the focus of the research on tumor selective therapy in recent years.VP3 is phosphorylated by some unknown kinase in tumor cells or transformed cells, then translocates into nuclei, mainly colocalizes with heterochromatin and nucleoli. And VP3 cooperatively forms distinct superstructures with DNA, which might affect cellular transcription. On the other hand, VP3 also bind and reacted with some other factors to trigger the mitochondrial pathway and induce the apoptosis of tumor cells or transformed cells.However, so far the mechanism of VP3 induced apoptosis is not very clear. Recent studies mainly focus on the proteins reacted with VP3 and elucidate the each member on the pathway of VP3 induced apoptosis to reveal the mechanism of VP3. Therefore, the study on those VP3-associated proteins is of great significance.The research is based on the construction of prokaryotic expression recombinant of pET-28a(+)-vp3 by routine molecular cloning. The expression of 6His-VP3 fusion protein was induced by IPTG and the purified protein was obtained by Ni-NTA His·Bind Resins through chromatography. After analysis on SDS-PAGE gel, 6His-VP3 obtained proved to be pure. Taking this pure protein as the bait, the preyed VP3-assocaited proteins by pull-down assay in tumor cells HepG2 were shown difference from that in normal cells L-02. And these different proteins might responsible for the VP3 tumor specificity. The further identification and analysis of these proteins will lay the foundation for discovery of mechanism of VP3.