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首页> 外文期刊>Applied and Environmental Microbiology >A Plasmid Selection System in Lactococcus lactis and Its Use for Gene Expression in L. lactis and Human Kidney Fibroblasts
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A Plasmid Selection System in Lactococcus lactis and Its Use for Gene Expression in L. lactis and Human Kidney Fibroblasts

机译：乳酸乳球菌的质粒选择系统及其在乳酸乳球菌和人肾成纤维细胞中的基因表达

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摘要

We report the development of a nonantibiotic and nonpathogenic host-plasmid selection system based on lactococcal genes and threonine complementation. We constructed an auxotrophic Lactococcus lactis MG1363Δthr strain which carries deletions in two genes encoding threonine biosynthetic enzymes. To achieve plasmid-borne complementation, we then constructed the minimal cloning vector, pJAG5, based on the genes encoding homoserine dehydrogenase-homoserine kinase (the hom-thrB operon) as a selective marker. Using strain MG1363Δthr, selection and maintenance of cells carrying pJAG5 were obtained in threonine-free defined media. Compared to the commonly used selection system based on erythromycin resistance, the designed complementation system offers a competitive and stable plasmid selection system for the production of heterologous proteins in L. lactis. The potential of pJAG5 to deliver genes for expression in eukaryotes was evaluated by insertion of a mammalian expression unit encoding a modified green fluorescent protein. The successful delivery and expression of genes in human kidney fibroblasts indicated the potential of the designed nonantibiotic host-plasmid system for use in genetic immunization.
机译：我们报告了基于乳球菌基因和苏氨酸互补的非抗生素和非致病性宿主质粒选择系统的发展。我们构建了一种营养缺陷型乳酸乳球菌MG1363Δthr菌株，该菌株在编码苏氨酸生物合成酶的两个基因中带有缺失。为了实现质粒介导的互补，我们然后基于编码高丝氨酸脱氢酶-高丝氨酸激酶（hom-thrB操纵子）作为选择标记的基因构建了最小的克隆载体pJAG5。使用菌株MG1363Δthr，在无苏氨酸的特定培养基中获得了携带pJAG5的细胞的选择和维持。与常用的基于红霉素抗性的选择系统相比，设计的互补系统为生产乳酸乳球菌中的异源蛋白提供了竞争性和稳定的质粒选择系统。通过插入编码修饰的绿色荧光蛋白的哺乳动物表达单位来评估pJAG5传递基因以在真核生物中表达的潜力。基因在人肾成纤维细胞中的成功传递和表达表明，所设计的非抗生素宿主质粒系统可用于基因免疫。

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