食品级分泌表达载体的构建及报告蛋白在乳酸乳球菌中的表达

Construction of a food-grade secretion expression vector and use it for reporter protein expression in Lactococcus lactis

为构建乳酸乳球菌食品级分泌表达载体,通过PCR扩增质粒pMG36e的p32启动子片段及乳酸乳球菌MG1363未知分泌蛋白(Usp45)基因的核糖体结合位点、分泌信号肽和成熟肽前11个氨基酸的编码序列(SPusp45),克隆到食品级载体pSH91中,构建食品级分泌性表达载体pSQ;克隆报告基因金黄色葡萄球菌核酸酶(NucA)成熟肽的编码序列nucA到pSQ中分泌信号后,转化乳酸乳球菌MBP71,构建了乳酸乳球菌食品级分泌性表达系统Llactis/pSQ-nucA;通过TB-D法和酶谱法检测Llactis/pSQ-nucA的表达形式、表达量并与以前构建的Llactis/pSQZ-nucA系统表达能力进行比较,结果发现Llactis/pSQ-nucA能够分泌性表达NucA,分泌性表达的NucA量大约是胞内NucA的10倍;Llactis/pSQ-nucA的表达量高于lactis/pSQZ-nucA。为进一步目的蛋白的的分泌性表达及食品级疫苗的研制奠定了基础。

We constructed a food-grade secretion expression vector and used it for reporter protein expression in live delivery vehicle L lactisMBP71. The p32 fragment, which containing the stronger p32 promoter,was amplified by polymerase chain reaction（PCR） with the plasmid pMG36e as template. After being purified, the p32 fragment was ligated with SPusp45 fragment amplified from genomic DNA of L lactis MG1363. The fusion fragment p32-SPusp45 was inserted into the food-grade vector pSH91 to construct a secretion expression vector, pSQ. The coding sequence of NucA （nucA） was also amplified from Staphylococcus aureus chromosome and inserted into pSQ under the control of p32 promoter to construct a recombinant plasmid pSQ-nucA. Nuclease plate activity assay and zymograme assay demonstrate that NucA was secretion expressed from L lactis harboring the recombinant plasmid pSQ- nucA, and the quantity of NucA secreted into supernamant was about ten times more than which in cell lysate. Results also indicate that expression efficiency of L lactis/pSQ-nucA was higher than that of L lactis/pSQZ-nucA, constructed by us earlier.