Cloning Of Human TRAP1Gene And Mechanisms Of Thioredoxin Reductase Inhibitor IG3-induced Death In Cervical Carcinoma Cells

Part I：Cloning of Human TRAP1GeneObjective:1．To construct the recombinant expression plasmid of TRAP1, and express recombinant humanTRAP1protein in Escherichia coli.2．To optimize the induction conditions of recombinant pET28a(+)-TRAP1for the highestexpression level of recombinant TRAP1protein.Methods:1．TRAP1gene cDNA was amplified by RT-PCR from K562/ADR cells. The TRAP1cDNAwas cloned into pET28a(+) plasmid, and the restriction enzyme cutting site of sense primerand antisense primer was NdeⅠand XhoⅠrespectively. The recombinant plasmid wastransformed into competent Escherichia coli BL21(DE3) to express recombinant humanTRAP1protein.2．Through detecting the expression level of TRAP1prtotein by SDS-PAGE after therecombinant BL21(DE3)/pET28a(+)-TRAP1was induced at different temperature and OD600,with different IPTG concentration, for different time, the induction conditions wereoptimized for the highest expression level of recombinant human TRAP1protein.Results:1．Recombinant plasmid pET28a(+)-TRAP1was successfully constrcted. After being inducedby IPTG, recombinant protein was expressed in Escherichia coli BL21(DE3). And therecombinant protein was identified as human TRAP1protein by Western Blot.2．when OD600was0.8, after being induced with0.1mM/L IPTG at39℃for6hours, theexpression level of recombinant protein was highest.Part II：Mechanisms of thioredoxin reductase inhibitor IG3-induced death in cervicalcarcinoma cellsObjective: To preliminary investigate the mechanisms of TrxR inhibitor IG3-induced cell deathin CaSki and SiHa cells.Methods: After treated with IG3, the effects of IG3on cell proliferation, cell apoptosis, cellcycle, the level of ROS, mitochondrial transmembrane potential and the expressin level of TrxRin CaSki and SiHa cells, were analyzed by MTT, AnnexinV/PI staining-flow cytometry, PI staining-flow cytometry, DCFH-DA fluorescence probe, JC-1staining-flow cytometry andWestern Blot, respectively. And the effects of IG3on expression of apoptotic protein in SiHacells was analyzed by Human Apoptosis Antibody Array kit.Results: MTT results revealed that IG3could significantly inhibit cell proliferation of CaSki andSiHa cells in vitro in a concentration-dependent and time-dependent manner. The flow cytometryanalysis demonstrated that IG3-induced cell necrosis and S cell cycle arrest in CaSki and SiHacells in a concentration-dependent manner, and significantly elevated the level of ROS, andreduced mitochondrial transmembrane potential in a time-dependent manner. The HumanApoptosis Antibody Array results suggested that IG3had no effect on the expression of apoptoticprotein in SiHa cells. Western Blot analysis showed that IG3had no effect on the expression ofTrxR in CaSki and SiHa cells.