| Popis: |
Background: Insulin-like growth factor binding protein-3 receptor (IGFBP-3R) (Transmembrane protein 219 [TMEM219]) binds explicitly to IGFBP-3 and exerts its apoptotic and autophagy signalling pathway. Constructing a Henrietta Lacks (HeLa) h6-TMEM219 cell characterize the therapeutic potent of TMEM219 that could interrupt the IGFBP-3/TMEM219 pathway, in cancer treatment and destructive cell illnesses such as diabetes and Alzheimer's. Materials and Methods: First, to develop stable overexpressed HeLa h6-TMEM219 cells, and Escherichia coli BL21 (DE3) with high IGFBP-3R expression, the purchased pcDNA3.1-h6-TMEM219 plasmid was transformed and integrated using CaCl2 and chemical transfection reagents, respectively. The pcDNA3.1-h6-TMEM219 transfection and protein expression was evaluated by the polymerase chain reaction (PCR), western blotting, and flow cytometry. Following the induction of h6-TMEM219 expression, a protein was purified using Ni-NTA chromatography and evaluated by the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Results: The 606 base pairs sequence in PCR outcomes confirmed successful pcDNA3.1-h6-TMEM219 transformation in E. coli BL21 and integration into the HeLa genome. The analysis of protein samples from induced E. coli BL21 and purified protein demonstrate a band of approximately 22 kDa on SDS-PAGE. Moreover, besides western blot analysis, flow cytometry findings illustrate approximately 84% of transfected HeLa cells (HeLa h6-TMEM219) overexpressed h6-TMEM219 on their surface. Conclusion: We designed a new experiment in the h6-TMEM219 expression procedure in both eukaryotic and prokaryotic hosts. All of our results confirm appropriate transformation and transfection and importantly, approve h6-TMEM 219 membrane expression. Finally, the HeLa h6-TMEM219 cells and the newly purified h6-TMEM219 leverage new studies for molecular diagnostic studies and characterize the therapeutic agents against IGFBP-3/TMEM219 signalling pathway in devastating illnesses in vitro and in vivo. |
