乳酸乳球菌食品级分泌性表达载体pSQZ的构建及报告蛋白的表达  

Construction of a food-grade secretion expression vector and the expression of reporter protein in Lactococcus lactis

作　　者：孙强正[1] 熊衍文[1] 叶长芸[1] 徐建国[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所

出　　处：《中国人兽共患病学报》2008年第3期224-228,275,共6页Chinese Journal of Zoonoses

摘　　要：目的构建乳酸乳球菌(Lactococcus lactis,L lactis)食品级分泌表达载体,表达报告蛋白核酸内切酶(NucA)方法PCR扩增乳酸乳球菌未知分泌蛋白(Usp45)编码基因的启动子、核糖体结合位点、分泌信号和成熟肽前11个氨基酸编码序列,克隆到载体pSH91中,构建食品级分泌性表达载体pSQZ;克隆报告蛋白金黄色葡萄球菌NucA成熟肽的编码序列nucA到pSQZ中分泌信号后,转化乳酸乳球菌MBP71,构建乳酸乳球菌分泌性表达系统L lactis MBP71/pSQZ-nucA;采用TB-D法和酶谱法检测乳酸乳球菌分泌性表达的NucA活性。结果PCR扩增得到usp45基因片段,酶切、连接到载体pSH91中构建了食品级表达载体pSQZ;将扩增的nucA基因片段克隆入载体pSQZ并转化乳酸乳球菌,得到乳酸乳球菌分泌性表达系统L lactis MBP71/pSQZ-nucA。在TB-D平板上,L lactis MBP71/pSQZ-nucA克隆子周围出现橙色光晕;在酶谱检测中,L lactis MBP71/pSQZ-nucA上清和菌体在18kDa位置均有橙色条带,但上清条带亮度和宽度大于菌体。结论成功构建了乳酸乳球菌食品级分泌表达系统,实现了报告蛋白NucA在乳酸乳球菌中分泌性表达,为进一步保护性抗原的分泌性表达研究奠定了基础。To construct a food-grade secretion expression vector and its use in the expression of reporter protein nuclease(NucA),a fragment,Usp45,containing the coding sequence of promoter,ribosomal binding site,secretion signal and the coding sequence for 11 pre-amino acids was amplified by PCR using the genomic DNA of L. lactis MG1363,and then inserted into the food-grade vector pSH91,thus to construct a secretion expression vector pSQZ. In addition,the coding sequence of NucA(nucA)was also amplified from Staphyloc...

关 键 词：乳酸乳球菌 食品级载体 分泌性表达 报告蛋白 

分 类 号：Q78[生物学—分子生物学]