RESUMEN
Curcin can inhibit the proliferation of tumor cells and promote tumor cell apoptosis, but the cytotoxicity of curcin is not selective for tumors or normal cells. In order to enhance the targeting of the anti-tumor ability of curcin, a transferrin receptor (TfR) binding peptide, TfRBP9, was fused with curcin. The curcin-TfRBP9 gene was cloned into pQE-30 and the recombinant vector pQE-30-curcin-TfRBP9 was established. Then the recombinant vector pQE-30-curcin-TfRBP9 was transferred into Escherichia coli M15. After being induced by 0.5mM IPTG for 6h at 37°C, the expressed quantity of the recombinant protein was about 30% of the total protein. Recombinant curcin-TfRBP9 was expressed in the form of an inclusion body. After dissolution, purification and renaturation, the purity of the recombinant curcin-TfRBP9 reached 95%. Immunofluorescence analysis showed that the TfRBP9 significantly enhanced the ability of the curcin binding to HepG2, and was enriched in the cytoplasm. The curcin-TfRBP9 fusion protein had significant proliferation inhibition effects on the HepG2 cells that over-expressed transferrin receptors, had lower inhibitory effects on the SKBR-3 cells that expressed low transferrin receptors, and had the lowest inhibitory effects on the LO-2 cells that were normal human liver cells. Compared with curcin, the curcin-TfRBP9 induced higher apoptosis rates in the HepG2 cells.
Asunto(s)
Antineoplásicos/metabolismo , Jatropha/genética , Péptidos/genética , Proteínas de Plantas/genética , Receptores de Transferrina/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Vectores Genéticos/genética , Células Hep G2 , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Péptidos/química , Péptidos/farmacocinética , Péptidos/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/farmacocinética , Proteínas de Plantas/farmacología , Plásmidos/genética , Replegamiento Proteico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/química , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacocinética , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacologíaRESUMEN
This paper is to report the study of the pharmacokinetics of a fusion protein TAT-haFGF(14-154) for human acidic fibroblast growth factor and transcriptional activator protein in rat plasma, and the investigation of their penetration across blood-brain barrier in mice and rats, in order to provide a basis for clinical development and treatment of Alzheimer's disease. Enzyme-linked immunosorbent assay (ELISA) was used to determine concentration of TAT-haFGF(14-154) in rat plasma and in mouse brain homogenate; and immunohistochemistry was used to analyze the distribution in brain. The concentration-time curve fitted two-compartment open model which was linear kinetics elimination after a single intravenous injection of TAT-haFGF(14-154) in rat at the dose of 300 microg x kg(-1). The half life time was 0.049 +/- 0.03 h for distribution phase and 0.55 +/- 0.05 h for elimination phase, and the weight was 1/C2. The result showed that TAT-haFGF(14-154) could be detected in the brain by ELISA and immunohistochemistry, the elimination of TAT-haFGF(14-154) in rat was swift, and TAT-haFGF(14-154) could penetrate across the blood-brain barrier, distribute in pallium and hippocampus and locate in the nucleus.
