Evaluation of Physicochemical Activity of Anticancer Fusion Proteins; Enterocin A- R type pyocin-Lactocin-Ligand Against Gastric Cancer Cell Line by Real-Time RT PCR Technique

Abstract

Gastric cancer treatment remains a major challenge. There are many reports on the positive efficacy of Bacteriocins associated with anti-cancer sequences. The main purpose of this study was to determine and design a fusion gene contraceptive containing anticancer Enterocin A, R-type pyocin, Lactocin, ligand against AGS gastric cancer cell line using Real Time_ RT_ PCR technique. This study was designed EntA-PynR-Lac recombinant proteins containing ligand and anticancer using bioinformatics software. Escherichia coli BL-21 was used to express cloned genes in expression vectors that have a T7 bacteriophage promoter. The specific ligand of the AGS cell line was designed and added to the recombinant construct sequence, and its three-dimensional structures, conformity, and stability were evaluated. Synthesis of fusion gene concentrate was performed in the expression vector, the transformation of the recombinant plasmid containing anticancer fusion gene construct in the respective host. Confirmation and purification of the recombinant protein by Western blotting and nickel column chromatography through the His tag, cytotoxicity determination against AGS cell line via the MTT technique, and finally, Real-time PCR was performed. Protein fusion at a concentration of 80 μg/ml in 24 h kills 83% of the cells tested and treatment of the cells with this concentration leads to increased expression of caspase3 and bax genes and decreased expression of bcl2. The results show that the treatment of gastric cancer cells with recombinant protein at a concentration of 80 μg/ml induces apoptosis in this cell line.