Cloning of protective antigen gene from Bacillus anthracis into Bacillus subtilis

Introduction: Protective antigen (PA) of Bacillus anthracis is used as anthrax vaccine. Cloning and expression of the PA gene in various strain such as E.coli and Bacillus subtilis was reported that most expression was in B. subtilis up to 160 μg/ml. The objectives of this study were: to clone the gene of PA in an expression vector (pWB980) and then transformation into the B. subtilis WB600 strain. Materials and Methods: The pXO1 plasmid was separated from the strain stern of B. anthracis with alcalin method and the PA gene with 2.4kb sequence amplified by PCR. Then the amplified fragment was directly cloned into pTZ57R plasmid as T-vector and transferred into E.coli DH5α using CaCl2 method. After that the gene was separated from the T-vector by enzymatic digestion ( SalI and KpnI ). Ligation between the purified gene fragment of the PA and the vector was carried out. Then it was transferred into B. subtilis WB600 by electroporation method in 1000 V. Results: In this study we isolated PA gene from B. anthracis strain stern with PCR and was cloned into pTZ57R plasmid. The Presence of the gene was confirmed by restriction analysis, PCR and sequencing. Then the PA gene was cloned into pWB980 and B. subtilis and the presence of the gene in two kanamycin resistant colonies (AMN1 and AMN3) was confirmed by restriction analysis and PCR. Conclusion: We may conclude that by making modification in the methods used and using pWB980 expression vector, we were able to clone the PA gene into B. subtilis. This is the first research project in Iran that the PA gene is isolated and cloned and B. subtilis is used as host.