The Development Of A TRAIL Fusion Protein Targeting Claudin Promote Cancer Apoptosis Induction

TRAIL(TNF-relatedapoptosis-inducing ligand) is a cytokine which can induce apoptosis in cancer cells but has no detectable cytotoxic effects on normal cells. By interacting with the death receptors DR4and/or DR5, TRAIL induce apoptosis in cacer cells. There are some receptors in the plasma, the effect of TRAIL are neutralized. Thus it is very important for increasing TRAIL targeting property. In this study, we generated a fusion protein by adding CPE30to the N terminal of TRAIL114-281in E.coli, and used to human breast cancer cells MDA-MB-231and MDA-MB-435S, as to study the promoting apoptosis in cancer cells.The CPE30Trail114-281gene was designed and synthesized, by constructed the recombinant vector of CPE30Trail114-281/pGEX-6P-2. The gene was transfected into/.coli(DE3). The expression of protein was induced by IPTG, which contained the GST-tag and express soluble protein efficiently. The recombinant soluble protein was purified by Glutathione Sepharose chromatography packing. The prurity of protein was cutted sites by PreScission protease more than90%. Western boltting proved that the23kDa protein purified were CPE30Trail114-281protein. We used the Trail114-281as a control, in the expression Claudin-4of human breast cancer cells MDA-MB-231and don't express Claudin-4MDA-MB-435S on the cell apoptosis induction experiment. We observed cell apoptosis morphological changes by using inverted microscope and fluorescence microscope. Antitumor activity of TRAIL protein was measured by CCK-8method.The cell apoptosis rate was detected by Annexin V/PI method.Exposure of the MDA-MB-231cells to0.5μg/ml and1μg/ml CPE30Trail114-281. Resveratrol for24h increased inhibitory effect on cell proliferation which were (26.50±1.77)%,(48.17±2.36)%respectively, as well as enhanced the apoptotic percentage which were (16.43±0.81)%and (29.47±0.9)%separately, compared with that of the cells incubated with Trail alone(P<0.05). And for MDA-MB-435S group of two proteins no significant difference. CPE30Trail114-281protein nduced in the expression of Claudin-4the MDA-MB-231cell apoptosis enhanced.This experiment discussed the fusion protein CPE30Trail114-281promote apoptosis effect, and the preliminary evidence CPE30Trail114-281protein targeting property. In this paper, we will explore new candidate target molecules and provide certain theoretical basis in development of new cancer drugs.