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

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。

Abstract:

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.