乳酸乳球菌食品级载体的构建及Mn-SOD基因的克隆和表达

Construction of the food-grade vector for Lactococcus lactis and its use for cloning and expression of Mn-superoxide dismutase gene

目的构建乳酸乳球菌(L.lactis)食品级载体,并利用其表达锰超氧化物歧化酶(Mn SOD)。方法PCR方法扩增L.lactissubsp.cremorisWg2的隐性质粒pWV01的复制子、L.lactisMG1363的thyA基因及质粒pUC18的多克隆位点,PCR方法连接后构建食品级载体pSH91,CaCl2法转化大肠杆菌X51菌株。提取质粒pSH91电转化L.lactisMBP71(△thyA),检测转化子在改良SA培养基上的生长能力。PCR扩增L.lactisMG1363菌株的sodA基因(含自身启动子),克隆到质粒pSH91中,构建食品级表达载体pSH91∶SOD,电转化L.lactisMBP71,黄嘌呤氧化酶法测定转化子L.lactis MBP71/pSH91∶SOD的SOD酶活性。结果经PCR扩增、连接、鉴定,食品级载体pSH91构建成功;转化L.lactisMBP71后,突变株回复了在改良SA培养基上生长能力,并且其生长速率和酸化能力与野生株相似。PCR扩增得到sodA基因并克隆入pSH91,构建了食品级表达载体pSH91∶SOD。重组菌L.lactisMBP71/pSH91∶SOD的SOD酶活性较出发菌株L.lac tisMBP71高10余倍。结论成功构建了食品级载体pSH91;利用该载体可在L.lactis中表达Mn SOD等外源蛋白。

A replicon of the cryptic plasmid pWV01 for Lactococcus lactis subsp, cremoris Wg2, a selective marker of thyA gene from L. lactis MG1363 as well as the multiple cloning site of plasmid pUC 18, was amplified and ligated with PCR to construct the food-grade vector, and then transformed into E. coli X51 strain by CaCl2 method. The extracted plasmid from the X51 strain was then tramsformed into L. lactis MBP71（AthyA） mutant by electronic perforation, and the growth ability of the transformants was detected on modified SA medium. In addition, the soda gene with its own promotor from L. lactis MG1363 strain was cloned to the plasmid,thus constructing the recombinant plasmid pSH91 ： SOD,and the SOD enzymatic activity of the transformant L. lactis MBP71/pSH91 ： SOD was determined by xanthine oxidase（XOD） assay. The food-grade vector named as pSH91 was thus constructed successfully after PCR amplification, lingation and identification. When plasmid pSH91 was transformed into L. lactis A thyA mutant MBP71 by electonic perforation, the mutant strain could restore its growth ability, grow and acidify with the similar rate as those of the wide type of L. lactis CHCC373 on SA medium. The SOD enzymatic activity of the transformant L. lactis MBP71/pSH91. SOD was 10 times higher than that of the parent strain. Because all the DNA components of vector pSH91 are obtained from the food-grade bacteria and none of any antibiotic genes except a multiple cloning site is included, so this vector can be regarded as the food-grade vector and the Mn-SOD expression with the food-grade vector pSH91 is available in L. lactis MBP71 .