乳酸片球菌R-4细菌素PA-1原核表达及其理化特性

Prokaryotic Expression and Physicochemical Properties of Bacteriocin PA-1 from Pediococcus acidilactici R-4

摘要:

为实现原核表达产出细菌素并检测其理化特性，作者将乳酸片球菌 R-4细菌素pedA基因进行扩增回收，与pMD19-T载体连接后转入E.coli DH5α感受态细胞进行克隆。提取克隆后的pedA基因与表达载体pET-32a（+）连接，形成重组质粒pET-32a-pedA并转入E.coli BL21(DE3)感受态细胞，经异丙基硫代半乳糖苷诱导，乳酸片球菌R-4细菌素PA-1在大肠杆菌细胞进行表达。表达蛋白质经Ni-NTA柱纯化后，以金黄色葡萄球菌为指示菌检测其理化特性。结果表明，在E.coli BL21(DE3)细胞中成功表达相对分子质量为26 000的乳酸片球菌R-4细菌素PA-1并完成纯化。纯化后的乳酸片球菌R-4细菌素PA-1在40~121 ℃作用20 min、在pH 2~12、紫外线照射0~10 h、过氧化氢酶作用2 h后，其抑菌范围分别为14.7~15.6 mm、14.0~16.5 mm、15.1~15.8 mm和14.9 mm，而分别经胃蛋白酶和胰蛋白酶作用2 h均失去抑菌作用。这表明乳酸片球菌R-4细菌素PA-1对高温、强酸强碱、紫外线和过氧化氢酶均具有较好的稳定性，而胃蛋白酶和胰蛋白酶会使其失活。

Abstract:

In order to produce bacteriocin in large quantity through prokaryotic expression and investigate its physicochemical properties, this study amplified and recycled the Pediococcus acidilactici R-4 bacteriocin pedA gene. The gene was subsequently ligated into the pMD19-T vector and transferred into E. coli DH5α receptor cells for cloning. The cloned pedA gene was extracted and connected to the expression vector pET-32a(+) to form a recombinant plasmid pET-32a-pedA, which was then transferred into E. coli BL21(DE3) receptor cells. Following induction with isopropyl-β-D- thiogalactoside(IPTG), Pediococcus acidilactici R-4 bacteriocin PA-1 was expressed within E. coli BL21 (DE3) cells. The expressed protein was purified by a Ni-NTA column and its physicochemical properties were determined using Staphylococcus aureus as an indicator. The results demonstrated successful expression and purification of the 26 000 of Pediococcus acidilactici R-4 bacteriocin PA-1 in E. coli BL21 (DE3) cells. The purified Pediococcus acidilactici R-4 bacteriocin PA-1 exhibited antibacterial activity within the ranges of 14.7~15.6 mm, 14.0~16.5 mm, 15.1~15.8 mm, and 14.9 mm, respectively, under the treatment of 40~121 ℃ for 20 min, pH 2 to 12, 0~10 h ultraviolet irradiation, and 2 h catalase. However, the antibacterial activity was lost after treatment with pepsin and trypsin for 2 hours. This indicates that Pediococcus acidilactici R-4 bacteriocin PA-1 has good stability to high temperature, strong acid and alkali, UV radiation, and catalase. However, it could be deactivated by pepsin and trypsin.