鼠李糖乳杆菌细菌素WO-A/2的表达及其抗菌特性  

Expression and Antibacterial Properties of Bacteriocin WO-A/2 from Lactobacillus rhamnosus

作　　者：韩岳峰 焦明 马慧君 李松建[1] 金山[1,2] HAN Yuefeng;JIAO Ming;MA Huijun;LI Songjian;JIN Shan(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease,Ministry of Agriculture and Rural Affairs,Hohhot 010018,China;Alashan Left Banner Bayanhot Town Comprehensive Support and Technology Extension Center,Bayanhot 750325,China)

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]农业农村部动物临床诊疗技术重点实验室,内蒙古呼和浩特010018 [3]阿拉善左旗巴彦浩特镇综合保障和技术推广中心,内蒙古巴彦浩特750325

出　　处：《食品与生物技术学报》2024年第5期19-25,共7页Journal of Food Science and Biotechnology

基　　金：国家重点研发计划项目(2023YFE0100400);内蒙古自治区直属高校基本科研业务项目(BR22-11-17)。

摘　　要：为建立鼠李糖乳杆菌细菌素替抗、防腐应用平台,探究鼠李糖乳杆菌细菌素的抗菌特性,利用DNASTAR软件对鼠李糖乳杆菌细菌素进行全基因序列分析,确定了鼠李糖乳杆菌细菌素的主要调控基因为wo-a/2,利用原核表达的方法表达细菌素WO-A/2,并对WO-A/2的可溶性、诱导表达条件、免疫原性、溶血活性、抑菌特性进行了分析。结果表明,作者成功构建了pET-32a-wo-a/2质粒,表达了鼠李糖乳杆菌细菌素WO-A/2,其主要以包涵体的形式存在。当表达pET-32a-wo-a/2质粒的菌株接种于含Amp^(+)的LB液体培养基中培养,以终浓度1.0 mmol/L的IPTG诱导8 h后,细菌素WO-A/2的表达量最大。通过双层牛津杯血琼脂平板法验证表明,不同浓度的鼠李糖乳杆菌细菌素WO-A/2均无溶血活性。采用双荧光染色法,通过激光共聚焦显微镜观察到细菌素WO-A/2可促使金黄色葡萄球菌凋亡。该研究为鼠李糖乳杆菌细菌素成为安全可靠的生物替抗剂提供了依据。In order to establish the application platform for antibiotic and antiseptic application of Lactobacillus rhamnosus bacteriocin,the author explored its antibacterial properties.The main regulatory gene of Lactobacillus rhamnosus was found to be wo-a/2,which was determined by DNASTAR as the whole gene sequence analysis software.The wo-a/2 gene was expressed by prokaryotic expression method and its solubility,induced expression conditions,immunogenicity,hemolytic activity and antibacterial characteristics were analyzed.The results showed that pET-32a-wo-a/2 plasmid was successfully constructed and expressed Lactobacillus rhamnosus bacteriocin WO-A/2,which mainly existed in the form of inclusion body.When pET-32a-wo-a/2 plasmid was inoculated in LB liquid medium containing Amp^(+)and induced with IPTG at a final concentration of 1.0 mmol/L for 8 h,the expression of bacteriocin WO-A/2 reached the maximum.It was verified from the double-layer Oxford cup blood agar plate method that Lactobacillus rhamnosus bacteriocin WO-A/2 had no hemolytic characteristics either at different concentrations.Bacteriocin WO-A/2 was found to promote the apoptosis of Staphylococcus aureus,as observed by dual fluorescence staining and laser confocal microscopy.This study provides a reference for Lactobacillus rhamnosus bacterin to be a safe and reliable antibiotic agent.

关 键 词：鼠李糖乳杆菌 细菌素 抗菌肽 感受态细胞 原核表达 

分 类 号：Q939.92[生物学—微生物学]