The Study On The Effects Of Apoptosis Of HepG₂ Cells Induced By VP3 Inducted By TAT Cell-penetrating Peptides

In 1988,Green and Frankel found respectively that protein HIV-TAT has the function of permeating into cells through membrane. Protein TAT consists of 86 amino acids in its full length. The domain which has transmembrane function is named as protein transduction domain (PTD). It consists of 11 amino acids. The research indicated that PTD is a wonderful transduction vector for protein, polypeptide even gene. The sequences of PTD with 11 amino acids is the shortest protein transduction domain by far.Apoptosis is an initiative programmed physiological process for clearing excrescent, abberance or tumor cells. Disorder of this process was found closely relation to the development of tumor. Recently, the regulating roles in cells by products of virus gene CAV(chicken anemia virus) is a new virus which can induce the apoptosis of tumor cells. The chicken anemia virus (CAV)-derived protein VP3 (Apoptin) which consists of 121 amino acids has been shown to induce apoptosis in various tumor cells, but not in normal human cells and murine diploid cells which interest us mostly. Apoptin is a powerful apoptosis-inducing protein with a potential for cancer therapy.The research is based on the constructing of the prokaryotic expression recombinants of pET28a-TAT-vp3, pET28a-TAT-GFP and pET28a-TAT-GFP- vp3 by routine molecular cloning. The expression of three kinds of fusion proteins were induced by IPTG and the purified protein was obtained by nickel affinity gel column chromatography. The transmembrane function and the induced-apoptosis effects on tumor cells were observed by protein transduction assay. The result showed that fusion proteins TAT-GFP and TAT-GFP-VP3 can permeating into cells cultured in vitro and green fluorescences can be found under fluorescence microscope. Fusion proteins TAT-VP3 and TAT-GFP-VP3 could induce apoptosis of HepG2 cells cultured in vitro.