Tumor Necrosis Factor Apoptosis Ligand Gene Cloning, Expression, Purification And Biological Activity Of A Preliminary Study

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the several members of the tumor necrosis factor (TNF) gene superfamily that induce apoptosis through engagement of death receptors (DRs).TRAIL interacts with an unusually complex receptor system of two DRs and three decoys at tumor cell membrane. This protein has garnered intense interest as a potential candidate for cancer therapy because as a trimer it selectively induces apoptosis in many transformed cells but not in normal cells.Using peripheral blood mononuclear cell total RNA of Chinese people as template, RT-PCR amplified soluble TRAIL fragment genes which were then inserted into expression vector. After transformation of this recombinant vector into bacterial, the sTRAIL protein was successfully expressed and purified to 98%. The potential use as a cancer theraputic drug of this protein was functionally tested in human osteosarcoma cell line under culture condition and in nude mouse body. The whole experiment reported as following.1. sing peripheral blood mononuclear cell total RNA of Chinese people as template, RT-PCR amplified soluble TRAIL fragment genes which were then inserted into expression vector.2. Using PCR technique, we designed two pair of PCR primers with endonuclear enzyme of EcoR I and BamH I for pBV220 expression vector, Ndel and BamHI for pRSET-A. We amplified the sTRAIL5gene fragments and successfully constructed two expression vector.3. TRAIL was successfully expressed by these two different vector system using either temperature or IPTG induction. After purification, this protein was further identified as sTRAIL by western blot.4. In vitro experiment with MTT method showed that expressed and purified sTRAIL could effectively kill human osteosarcoma cells with no side effect on normal human cells of HHCC.5. The nude mouse bearing human osteosarcoma SOSP-9607 and SOSP-9901 was made by injection of these two tumor cells into the mouse back and then the animal models were used in the in vivo tumor growth inhibition experiment. Results showed that purified sTRAIL could inhibit osteosarcoma by 61% and 63.3% respectively.