摘要
为了实现该细菌素的外源表达,本实验首先利用聚合酶链式反应从乳酸片球菌PAF中扩增出乳酸片球菌素PA-1的结构和免疫基因,然后克隆到表达载体p GEX-6p-1,构建了N端含有GST-His-DDDDK标签的重组质粒p GEX/his-ped AB,然后转化进入大肠杆菌Rosetta(DE3)感受态细胞,经异丙基硫代半乳糖苷诱导,重组乳酸片球菌素PA-1在大肠杆菌胞内成功表达。表达的融合蛋白先经过镍亲合层析柱纯化,然后注入谷胱甘肽S-转移酶亲和色谱柱用肠激酶处理,释放出成熟的乳酸片球菌素PA-1。利用高效液相色谱和质谱技术检测乳酸片球菌素PA-1纯度。以单核细胞增生李斯特氏菌CMCC54004为指示菌,利用琼脂扩散法检验乳酸片球菌素PA-1活性。结果表明,携带GST-His-DDDDK标签的融合蛋白无活性,标签切除后其抑菌活性恢复,且其纯度达90%以上。
Pediocin PA-1 is a representative class IIa bacteriocin produced by Pediococci with the potential to serve as a food-grade preservative for controlling Listeria contamination. To obtain its soluble expression, Pediocin PA-1 structural and immunity gene ped AB was amplified by polymerase chain reaction(PCR) from Pediococcus acidilactici PAF, and cloned into the vector p GEX-6p-1 to construct the p GEX/his-ped AB encoding the recombinant pediocin PA-1 with GST-His-DDDDK in the N-terminal of pediocin PA-1. The plasmid p GEX/his-ped AB was then transformed into Escherichia coli Rosetta(DE3). After induction with isopropyl-β-D-thiogalactopyranoside, the recombinant pediocin PA-1 was successfully expressed in E. coli cytoplasm. The expressed fusion protein was purifi ed by Ni-NTA metal affi nity chromatography, subsequently loaded to GST affi nity column and treated with recombinant bovine enterokinase. Mature pediocin PA-1 was liberated from the recombinant protein. The purity of pediocin PA-1 was detected by high-performance liquid chromatography and mass spectrometry. The antibacterial activity of pediocin PA-1 was determined by agar diffusion method using Listeria monocytogenes CMCC54004 as an indicator. The results showed that fusion pediocin PA-1 did not have bactericidal activity against Listeria monocytogenes, but when the GST-His-DDDDK in the N-terminal was removed, the cleaved pediocin PA-1 restored its bactericidal activity. The purifi ed pediocin PA-1 had a purity above 90%.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2016年第3期97-102,共6页
Food Science
基金
国家自然科学基金面上项目(31271930)
江苏省研究生培养创新工程项目(KYLX_1167)
江苏高校优势学科建设工程资助项目(PAPD)
江苏省普通高校研究生科研创新计划项目(CXZZ13_0636)
关键词
细菌素
乳酸片球菌素
原核表达
蛋白纯化
bacteriocin
pediocin
prokaryotic expression
protein purifi cation