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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。
Bacteriocin (Bacteriocin) is a class of antibacterial activity of peptides or proteins produced by certain bacteria through the ribosome pathway. Due to its non-deformability and resistance to bacteria, Bacteriocin attracts much attention in food and hygiene safety. In this study, a putative bacteriocin gene AOI-3 was predicted from a whole genome sequencing Bacillus thuringiensis (Bt) BRC-ZYR2 strain. PCR amplification of 231 bp target gene fragment, the use of seamless cloning technology will be connected to the target gene fragment p ET32a vector, transformed into Escherichia coli (Escherichia coli) JM109, screening of clones for digestion and sequencing validation. Gene sequence alignment showed that this gene shared 99% homology with an unannotated nucleic acid sequence (Gen Bank Accession No. CP003763.1) in the genome of Bt HD-789. Amino acid sequence alignment revealed 100% homology to a putative Bt bacteriocin biosynthesis protein (Gen Bank Accession No. WP_033699510.1). In addition, the conserved domain of the bacteriocin belongs to the DUF2762, an unknown protein of the domain of unknown function (DUF), and may have a similar role as the toxin Bhl A. Amino acid composition analysis showed that the gene sequence encoded 76 amino acids with a molecular weight of 8 813.62 Da, an isoelectric point of 4.82, no signal peptide and 1 transmembrane region. The recombinant expression vector was transformed into E. coli BL21 (DE3) Propyl-β-D-thiogalactopyranoside (IPTG) induced expression. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the expected 8.8 kD polypeptide expression was detected in the supernatant. The His-tag affinity chromatography was used to purify the target protein. In this study, the putative bacteriocin gene AOI-3 was cloned, expressed and purified to provide the basis for determining the antibacterial spectrum and understanding the structure and mechanism of bacteriocin.