中文摘要：

细菌素（Bacteriocin）是由某些细菌通过核糖体途径产生的一类具有抗菌活性的多肽或蛋白质,因其无致畸变作用,也不易产生耐药性,在食品及卫生安全中备受关注。本研究以经过全基因组测序的苏云金芽胞杆菌（Bacillus thuringiensis,Bt）BRC-ZYR2为出发菌株,预测得到一个假定的细菌素基因AOI-3。PCR扩增获得231 bp目的基因片段,利用无缝克隆技术将目的基因片段连接到p ET32a载体,转入大肠杆菌（Escherichia coli）JM109,筛选克隆子进行酶切以及测序验证。基因序列比对结果显示,该基因与Bt HD-789全基因组中一段未被注释的核酸序列（Gen Bank登录号：CP003763.1）同源性达99%。氨基酸序列比对结果显示,与一个假定的Bt细菌素（Bt bacteriocin biosynthesis protein）（Gen Bank登录号：WP_033699510.1）的同源性达100%。此外,该细菌素的保守结构域属于未知功能域家族蛋白（domain of unknown function,DUF）亚族蛋白DUF2762,可能具有类似穿孔毒素Bhl A的作用。氨基酸组成分析表明,该基因序列编码76个氨基酸,分子量为8 813.62 Da,等电点为4.82,无信号肽,含有1个跨膜区,将重组表达载体转入大肠杆菌BL21（DE3）,异丙基-β-D-硫代吡喃半乳糖苷（isopropyl-β-D-1-thiogalactopyranoside,IPTG）诱导表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE）结果表明,在上清中检测到预期的8.8 k D多肽表达。采用His-tag亲和层析技术纯化目的蛋白。本研究通过对假定的细菌素基因AOI-3克隆、表达和获得多肽纯品,为测定其抑菌谱并了解细菌素结构及作用机理奠定了基础。

英文摘要：

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 sequencedBacillus thuringiensis（Bt） BRC-ZYR2 and amplified by PCR with total DNA. Then the 231 bp PCR product was purified and ligated to p ET32 a 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（Gen Bank No. CP003763.1） in Bt HD- 789 wholegenome was 99%, while the amino acid sequence homology with a putative Bt bacteriocin biosynthesis protein（Gen Bank 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 Bhl A. 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 k D 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.