Cloning, fusion and expression of recombinant Bacillus anthracis protective antigen domain 4 with cholera toxin B-subunit in E. coli

The combination or genetic connection of the antigens with cholera toxin B (ctB) subunit creates a strong mucosal antibody response. The aim of this study was to connect ctB to protein in domain 4 encoding gene of Protein arginine Domainases (PaD4) to express chimeric protein as a candidate vaccine against anthrax. In this experimental study، polymerase chain reaction (PCR) using specific primers for genes ctB and PaD4 was performed and amplified genes were cloned separately in PGEM-T easy vector. Then، PaD4 gene was connected to the 3'' end of ctB by the enzymatic digestion method and then، ctB-PaD4 fusion genes were sub-cloned into the pET28a. The strain BL21 of E. coli bacteria was transformed by the recombinant vector. The expressed chimeric protein was induced by Isopropyl β-D-1-thiogalactopyranoside and evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot techniques. CtB-PaD4 fusion gene was constructed، confirmed by PCR techniques، and sequencing. This gene was expressed in the E. coli bacteria of strain BL21 at optimum temperature of 37°C، and the chimeric protein was produced successfully. The bond in this protein was confirmed by Western blot technique and SDS. PAGE. After immunogenicity assay، this recombinant protein can be used as a new and effective chimeric subunit vaccine against Bacillus anthraces for oral or nasal consumption in future studies.