Crescent Journal of Medical and Biological Sciences (Jul 2020)
Production Design of Efficient Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor Under a Gene-Specific Promoter in Prokaryotic System
Abstract
Objectives: Recombinant products play an important role in improving health conditions. In addition, human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is considered a cytokine which stimulates many differentiated myeloid cells in order to produce granulocytes, macrophages, and monocytes. Considering the clinical application of the human GM-CSF, the current study aimed to produce the recombinant human GM-CSF (rhGM-CSF) in the prokaryotic system and then evaluated its biological activity. Materials and Methods: In this experimental study, the hGM-CSF was synthesized under a specific promoter. Then, it was cloned in HindIII restriction enzyme sites of the pcDNA3.1 (+). The hGM-CSF gene cloning was assessed by polymerase chain reaction, restriction enzyme digestion, and sequencing. Subsequently, recombinant plasmids were transformed in Escherichia coli and the expression of recombinant hGM-CSF was analyzed by electrophoresis and immunoblotting. Then, the rhGM-CSF was purified using S-tag affinity chromatography and the concentration of the purified rhGM-CSF was determined by ELISA. Finally, the biological function of the rhGM-CSF on TF-1 cells was performed by MTT proliferation assay. Results: The cloned fragment on gel agarose was detected. Further, the restriction enzyme digestion and recombinant plasmid sequencing results confirmed pcDNA3.1 (+)/hGM-CSF cloning. Furthermore, the results of the expression analysis of rhGMCSF by SDS-PAGE and western blot showed a specific protein band. The concentration of the purified protein was 0.54 μg/ mL. Moreover, the proliferation index demonstrated that the treated cells were proliferated (P<0.05). The mean values of the proliferation index were 7.8. Conclusions: In general, the production of recombinant hGM-CSF protein in the prokaryotic system was simple, rapid, and inexpensive. Therefore, the functional rhGM-CSF can be expressed under gene-specific promoter without any need for the chemical inducer.
