Abstract：Bacteriocins are peptides or proteins with antimicrobial activities, and produced by some bacteria through the ribosome way. Because of their harmless and low drug resistance, they are welcomed in food and health safety. In this study, a putative bacteriocin gene AOI-3 was predicted from a whole-genome sequenced Bacillus thuringiensis (Bt) BRC-ZYR2 and amplified by PCR with total DNA. Then the 231 bp PCR product was purified and ligated to pET32a expression vector by seamless cloning technology, and transformed into Escherichia coli JM109. The selected clones were digested and sequenced. Sequence alignment showed that nucleotide homology between the gene AOI-3 and a gene (GenBank No. CP003763.1) in Bt HD-789 whole-genome was 99%, while the amino acid sequence homology with a putative Bt bacteriocin biosynthesis protein(GenBank No. WP033699510.1) was 100%. Furthermore, the conserved domain of AOI-3 bacteriocin belonged to DUF2762 (domain of unknown function, DUF) superfamily, which were annotated as holin-like proteins BhlA. Amino acid composition analysis showed that the gene sequence encoded 76 amino acids, the molecular weight was 8 813.62 Da, the isoelectric point was 4.82, without signal peptide, containing a transmembrane domain. A recombinant plasmid was transformed into BL21 (DE3) and induced with isopropyl-β-D-1-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the relative molecular weight of the expressed peptide was about 8.8 kD in supernatants, which was completely coincided with the forecasted result. The expressed peptide was furtherly purified by Ni-NTA affinity chromatography. Herein, the cloning, expression and purification of the putative bacteriocin gene AOI-3 provide basic data for evaluation of its anti-microbial activities, and elucidation of its struture and mechanism.
