Apyrase Protein (PsSP42) Expression in DNA Construct Transfected COS-7 Cells

Authors
Samira Hosseinpour Jahednia 1
Hossein Rezvan 2
Alireza Nourian 2
Tahereh Taheri 3
Negar Seyed 3
Zahra Eghbali 4
Elham Gholami 3
Sima Rafati* 3
1 1 Department of Pathobiology, Faculty of Veterinary Medicine, Bu-Ali Sina University, Hamedan, Iran 2 Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran
2 1 Department of Pathobiology, Faculty of Veterinary Medicine, Bu-Ali Sina University, Hamedan, Iran
3 2 Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran
4 Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran 3 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
10.61882/ibj.5055
Abstract
Background: DNA vaccines offer dual humoral and cellular immunity, showing potential effectiveness against intracellular parasites, including Leishmania. However, challenges in delivering large DNA constructs has been already existed, which influence protein expression and immune efficacy. This study evaluated the expression of P. sergenti salivary protein PsSP42 in COS-7 cells. Confirming the expression of PsSP42 is crucial before its use as a DNA vaccine in preclinical experiments.

Methods: The expression of the PsSP42 protein was achieved using two plasmids: the small, antibiotic-free plasmid NTC9385R and the conventional plasmid VR1020. Both recombinant vectors were transfected into COS-7 cells using electroporation and PEI-mediated transfection methods. Ni-NTA beads were utilized to enrich proteins from the supernatants collected from transfected cells, and expression was confirmed by Western blotting.

Results: Successful expression of the PsSP42 protein was affirmed from both VR1020-PsSP42 and NTC-PsSP42 constructs in the transfected COS-7 cells, regardless of the transfection method employed (PEI or electroporation). A 39.6 kDa band corresponding to the PsSP42 protein was detected, indicating its secretion into the supernatants of COS-7 cells transfected with both plasmids.

Conclusion: Plasmids VR1020-PsSP42 and NTC-PsSP42 demonstrated similar protein expression levels in vitro, regardless of the transfection method used. Performing these evaluations is recommended to thoroughly assess construct expression levels before conducting in vivo studies.
Keywords
Electroporation
Gene expression
Leishmania tropica
Polyethylenimine